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Sample records for avium subespecie avium

  1. The Mycobacterium avium complex.

    PubMed Central

    Inderlied, C B; Kemper, C A; Bermudez, L E

    1993-01-01

    Mycobacterium avium complex (MAC) disease emerged early in the epidemic of AIDS as one of the common opportunistic infections afflicting human immunodeficiency virus-infected patients. However, only over the past few years has a consensus developed about its significance to the morbidity and mortality of AIDS. M. avium was well known to mycobacteriologists decades before AIDS, and the MAC was known to cause disease, albeit uncommon, in humans and animals. The early interest in the MAC provided a basis for an explosion of studies over the past 10 years largely in response to the role of the MAC in AIDS opportunistic infection. Molecular techniques have been applied to the epidemiology of MAC disease as well as to a better understanding of the genetics of antimicrobial resistance. The interaction of the MAC with the immune system is complex, and putative MAC virulence factors appear to have a direct effect on the components of cellular immunity, including the regulation of cytokine expression and function. There now is compelling evidence that disseminated MAC disease in humans contributes to both a decrease in the quality of life and survival. Disseminated disease most commonly develops late in the course of AIDS as the CD4 cells are depleted below a critical threshold, but new therapies for prophylaxis and treatment offer considerable promise. These new therapeutic modalities are likely to be useful in the treatment of other forms of MAC disease in patients without AIDS. The laboratory diagnosis of MAC disease has focused on the detection of mycobacteria in the blood and tissues, and although the existing methods are largely adequate, there is need for improvement. Indeed, the successful treatment of MAC disease clearly will require an early and rapid detection of the MAC in clinical specimens long before the establishment of the characteristic overwhelming infection of bone marrow, liver, spleen, and other tissue. Also, a standard method of susceptibility testing

  2. Reducing human exposure to Mycobacterium avium.

    PubMed

    Falkinham, Joseph O

    2013-08-01

    In light of the increasing prevalence of Mycobacterium avium pulmonary disease and the challenges of treating patients with M. avium infection, consideration of measures to reduce exposure is warranted. Because M. avium inhabits water and soil, humans are surrounded by that opportunistic pathogen. Because infection has been linked to the presence of M. avium in household plumbing, increasing hot water temperature, reducing aerosol (mist) exposures in bathrooms and showers, and installing filters that prevent the passage of mycobacteria will likely reduce M. avium exposure. Granular activated carbon (charcoal) filters support the growth of M. avium and should be avoided. When gardening, avoid the inhalation of soil dusts by using a mask or wetting the soil because peat-rich potting soils have high numbers of mycobacteria.

  3. Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Wang, Joyce; Moolji, Jalal; Dufort, Alex; Staffa, Alfredo; Domenech, Pilar; Reed, Michael B.

    2015-01-01

    ABSTRACT Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSPP15. We obtained a transposon (Tn) mutant with a disruption in the LSPP15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSPP15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen. IMPORTANCE Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth

  4. MYCOBACTERIUM AVIUM AND DRINKING WATER WHAT ARE THE CONNECTIONS?

    EPA Science Inventory

    Background: Human Mycobacterium avium infections are only known to be acquired from environmental sources such as water and soil. We compared M. avium isolates from clinical and drinking water sources using molecular tools. Methods: M. avium was isolated from water samples colle...

  5. Detection of Mycobacterium avium in pet birds

    PubMed Central

    Godoy, Silvia Neri; Sakamoto, Sidnei Miyoshi; de Paula, Cátia Dejuste; Catão-Dias, José Luiz; Matushima, Eliana Reiko

    2009-01-01

    The present study is a report on the presence of Mycobacterium avium in four birds of the psittaciform order kept as pets. Anatomopathological diagnosis showed lesions suggestive of the agent and presence of alcohol-acid resistant bacilli (AARB) shown by the Ziehl-Neelsen staining. The identification of Mycobacterium avium was performed by means of PRA (PCR Restriction Analysis). DNA was directly extracted from tissue of the lesions and blocked in paraffin. The role of this agent in pet bird infection is discussed, as well as its zoonotic potential. PMID:24031356

  6. ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

    EPA Science Inventory

    The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms were measured. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V-1s-1, and the EPMs of fifteen environmental isolates ranged from -1...

  7. ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

    EPA Science Inventory

    The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms isolated from clinical and environmental sources were measured in 9.15 mM KH2PO4 buffered water. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V-1 ...

  8. Experimental inoculation of BFDV-positive budgerigars (Melopsittacus undulatus) with two Mycobacterium avium subsp. avium isolates.

    PubMed

    Ledwoń, Aleksandra; Sapierzyński, Rafał; Augustynowicz-Kopeć, Ewa; Szeleszczuk, Piotr; Kozak, Marcin

    2014-01-01

    Beak and feather disease virus- (BFDV-) positive (naturally infected) but clinically healthy budgerigars (Melopsittacus undulatus) were inoculated with two isolates of Mycobacterium avium subsp. avium isolated from naturally infected golden pheasant (Chrysolophus pictus) and peafowl (Pavo cristatus). During a period of more than two months after inoculation, samples of cloacal and crop swabs, faeces, and blood were obtained for BFDV and Mycobacterium avium testing with PCR. Birds were euthanized nine weeks after inoculation. All infected budgerigars developed signs typical of mycobacteriosis, but more advanced clinical and pathological changes were visible in the group infected with the pheasant isolate. Only a few cloacal and crop swab samples were positive for Mycobacterium avium subsp. avium despite advanced pathological changes in the internal organs. In the groups infected with mycobacterium isolates the frequency of BFDV-positive samples was higher than in the control group. In the infected groups the frequency of BFDV was substantially higher in the cloacal swabs of birds inoculated with the pheasant isolate than in the peafowl-isolate-infected group. PMID:24738057

  9. Mycobacterium avium subsp. avium found in raptors exposed to infected domestic fowl.

    PubMed

    Kriz, Petr; Kaevska, Marija; Bartejsova, Iva; Pavlik, Ivo

    2013-09-01

    We report a case of a falcon breeding facility, where raptors (both diurnal and nocturnal) were raised in contact with domestic fowl (Gallus gallus f. domesticus) infected by Mycobacterium avium subsp. avium. Fecal and environmental samples from 20 raptors and four common ravens (Corvus corax) were collected. Mycobacterium a. avium DNA was detected in feces of four raptors (bald eagle [Haliaeetus leucocephalus], eagle owl [Bubo bubo], barn owl [Tyto alba], and little owl [Athene noctua]) using triplex quantitative real-time PCR. As both the flock of domestic fowl and one of the infected raptors had the same origin (zoological collection), they might have had a common source of colonization/infection. However, the detection of M. a. avium in feces of three other raptors may point at transmission of the agent between the birds in the facility. Contact of raptors with domestic fowl infected by M. a. avium may pose a risk for transmission of the infection for them; however, raptors from the falcon breeding facility seemed to be relatively resistant to the infection.

  10. Experimental inoculation of BFDV-positive budgerigars (Melopsittacus undulatus) with two Mycobacterium avium subsp. avium isolates.

    PubMed

    Ledwoń, Aleksandra; Sapierzyński, Rafał; Augustynowicz-Kopeć, Ewa; Szeleszczuk, Piotr; Kozak, Marcin

    2014-01-01

    Beak and feather disease virus- (BFDV-) positive (naturally infected) but clinically healthy budgerigars (Melopsittacus undulatus) were inoculated with two isolates of Mycobacterium avium subsp. avium isolated from naturally infected golden pheasant (Chrysolophus pictus) and peafowl (Pavo cristatus). During a period of more than two months after inoculation, samples of cloacal and crop swabs, faeces, and blood were obtained for BFDV and Mycobacterium avium testing with PCR. Birds were euthanized nine weeks after inoculation. All infected budgerigars developed signs typical of mycobacteriosis, but more advanced clinical and pathological changes were visible in the group infected with the pheasant isolate. Only a few cloacal and crop swab samples were positive for Mycobacterium avium subsp. avium despite advanced pathological changes in the internal organs. In the groups infected with mycobacterium isolates the frequency of BFDV-positive samples was higher than in the control group. In the infected groups the frequency of BFDV was substantially higher in the cloacal swabs of birds inoculated with the pheasant isolate than in the peafowl-isolate-infected group.

  11. Experimental Inoculation of BFDV-Positive Budgerigars (Melopsittacus undulatus) with Two Mycobacterium avium subsp. avium Isolates

    PubMed Central

    Sapierzyński, Rafał; Szeleszczuk, Piotr

    2014-01-01

    Beak and feather disease virus- (BFDV-) positive (naturally infected) but clinically healthy budgerigars (Melopsittacus undulatus) were inoculated with two isolates of Mycobacterium avium subsp. avium isolated from naturally infected golden pheasant (Chrysolophus pictus) and peafowl (Pavo cristatus). During a period of more than two months after inoculation, samples of cloacal and crop swabs, faeces, and blood were obtained for BFDV and Mycobacterium avium testing with PCR. Birds were euthanized nine weeks after inoculation. All infected budgerigars developed signs typical of mycobacteriosis, but more advanced clinical and pathological changes were visible in the group infected with the pheasant isolate. Only a few cloacal and crop swab samples were positive for Mycobacterium avium subsp. avium despite advanced pathological changes in the internal organs. In the groups infected with mycobacterium isolates the frequency of BFDV-positive samples was higher than in the control group. In the infected groups the frequency of BFDV was substantially higher in the cloacal swabs of birds inoculated with the pheasant isolate than in the peafowl-isolate-infected group. PMID:24738057

  12. Mycobacterium avium subsp. avium found in raptors exposed to infected domestic fowl.

    PubMed

    Kriz, Petr; Kaevska, Marija; Bartejsova, Iva; Pavlik, Ivo

    2013-09-01

    We report a case of a falcon breeding facility, where raptors (both diurnal and nocturnal) were raised in contact with domestic fowl (Gallus gallus f. domesticus) infected by Mycobacterium avium subsp. avium. Fecal and environmental samples from 20 raptors and four common ravens (Corvus corax) were collected. Mycobacterium a. avium DNA was detected in feces of four raptors (bald eagle [Haliaeetus leucocephalus], eagle owl [Bubo bubo], barn owl [Tyto alba], and little owl [Athene noctua]) using triplex quantitative real-time PCR. As both the flock of domestic fowl and one of the infected raptors had the same origin (zoological collection), they might have had a common source of colonization/infection. However, the detection of M. a. avium in feces of three other raptors may point at transmission of the agent between the birds in the facility. Contact of raptors with domestic fowl infected by M. a. avium may pose a risk for transmission of the infection for them; however, raptors from the falcon breeding facility seemed to be relatively resistant to the infection. PMID:24283140

  13. Genetic IS901 RFLP diversity among Mycobacterium avium subsp. avium isolates from four pheasant flocks.

    PubMed

    Moravkova, Monika; Lamka, Jiri; Slany, Michal; Pavlik, Ivo

    2013-01-01

    IS901 RFLP analysis of 36 Mycobacterium avium subsp. avium (MAA) isolates from 15 pheasants (Phasianus colchicus) and two goshawks (Accipiter gentilis) from four pheasant farms was performed. Using this method, six different IS901 RFLP types (E, F, G, M, Q, and V) were identified. The distribution of IS901 RFLP profiles was tightly linked to individual flocks. Matching IS901 RFLP profiles observed in the present study indicate MAA transmission between pheasants and goshawks in the same locality. In two flocks, different pheasants within a flock as well as in various organs of five individual pheasants were found to have two distinct IS901 RFLP profiles.

  14. Mycobacterium avium subspecies impair dendritic cell maturation.

    PubMed

    Basler, Tina; Brumshagen, Christina; Beineke, Andreas; Goethe, Ralph; Bäumer, Wolfgang

    2013-10-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a chronic, granulomatous enteritis of ruminants. Dendritic cells (DC) of the gut are ideally placed to combat invading mycobacteria; however, little is known about their interaction with MAP. Here, we investigated the interaction of MAP and the closely related M. avium ssp. avium (MAA) with murine DC and the effect of infected macrophages on DC maturation. The infection of DC with MAP or MAA induced DC maturation, which differed to that of LPS as maturation was accompanied by higher production of IL-10 and lower production of IL-12. Treatment of maturing DC with supernatants from mycobacteria-infected macrophages resulted in impaired DC maturation, leading to a semi-mature, tolerogenic DC phenotype expressing low levels of MHCII, CD86 and TNF-α after LPS stimulation. Though the cells were not completely differentiated they responded with an increased IL-10 and a decreased IL-12 production. Using recombinant cytokines we provide evidence that the semi-mature DC phenotype results from a combination of secreted cytokines and released antigenic mycobacterial components of the infected macrophage. Our results indicate that MAP and MAA are able to subvert DC function directly by infecting and indirectly via the milieu created by infected macrophages.

  15. Identification of Mycobacterium avium subsp. hominissuis Isolated From Drinking Water

    EPA Science Inventory

    Mycobacterium avium (MA) is divided into four subspecies based primarily on host-range and consists of MA subsp. avium (birds), MA subsp. silvaticum (wood pigeons), MA subsp. paratuberculosis (broad, poorly-defined host range), and the recently described MA subsp. hominissuis (hu...

  16. Avian mycobacteriosis caused by Mycobacterium avium subspecies avium in four ornamental birds and in vitro drug sensitivity testing of isolates.

    PubMed

    Stepień-Pyśniak, Dagmara; Puk, Krzysztof; Guz, Leszek; Wawrzyniak, Agata; Marek, Agnieszka; Kosikowska, Urszula

    2016-01-01

    Avian tuberculosis, one of the most important diseases affecting various species of birds, is most often caused by Mycobacterium (M.) avium. This report describes cases of M. avium subsp. avium (MAA) infection in a white-crested Holland dwarf rooster, a male and a female golden pheasant and a male peacock. We also investigated the prevalence of mycobacteria in 60 other birds and 40 alpacas. Tissue samples of necropsied birds were cultured for mycobacteria. From non-necropsied 60 other birds and alpacas only faecal samples were collected. Clinical signs in the affected white-crested Holland cock included gradual loss of body weight and hoarse attempts at crowing during its last 3 weeks, with a dramatic loss of body condition and depression over the final week. Only slight weakening was observed in the peacock just before its death, and the golden pheasants died suddenly. Diagnosis was confirmed by microbiological, molecular and pathological results. Mycobacterium avium subsp. avium strains were isolated from the internal organs of the affected birds. Only one faecal sample from 60 other birds was culture- and PCR-positive for M. avium subsp. avium, while another one was only PCR-positive for M. chelonae. We did not isolate any Mycobacterium spp. from faecal samples of alpacas and all of them were PCR-negative. All 18 isolated M. avium strains were resistant to rifampicin, isoniazid, ethambutol, ethionamide, capreomycin and ofloxacin, and susceptible to cycloserine and streptomycin.

  17. Avian mycobacteriosis caused by Mycobacterium avium subspecies avium in four ornamental birds and in vitro drug sensitivity testing of isolates.

    PubMed

    Stepień-Pyśniak, Dagmara; Puk, Krzysztof; Guz, Leszek; Wawrzyniak, Agata; Marek, Agnieszka; Kosikowska, Urszula

    2016-01-01

    Avian tuberculosis, one of the most important diseases affecting various species of birds, is most often caused by Mycobacterium (M.) avium. This report describes cases of M. avium subsp. avium (MAA) infection in a white-crested Holland dwarf rooster, a male and a female golden pheasant and a male peacock. We also investigated the prevalence of mycobacteria in 60 other birds and 40 alpacas. Tissue samples of necropsied birds were cultured for mycobacteria. From non-necropsied 60 other birds and alpacas only faecal samples were collected. Clinical signs in the affected white-crested Holland cock included gradual loss of body weight and hoarse attempts at crowing during its last 3 weeks, with a dramatic loss of body condition and depression over the final week. Only slight weakening was observed in the peacock just before its death, and the golden pheasants died suddenly. Diagnosis was confirmed by microbiological, molecular and pathological results. Mycobacterium avium subsp. avium strains were isolated from the internal organs of the affected birds. Only one faecal sample from 60 other birds was culture- and PCR-positive for M. avium subsp. avium, while another one was only PCR-positive for M. chelonae. We did not isolate any Mycobacterium spp. from faecal samples of alpacas and all of them were PCR-negative. All 18 isolated M. avium strains were resistant to rifampicin, isoniazid, ethambutol, ethionamide, capreomycin and ofloxacin, and susceptible to cycloserine and streptomycin. PMID:26904899

  18. Real-time quantitative PCR detection of Mycobacterium avium subspecies in meat products.

    PubMed

    Klanicova, B; Slana, I; Vondruskova, H; Kaevska, M; Pavlik, I

    2011-04-01

    The aim of this work was to examine various purchased meat products and to find out if any traces of Mycobacterium avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis could be detected in these samples. Analysis of the meat products (raw, cooked, and fermented) was performed using a real-time quantitative PCR (qPCR) method for the detection of specific insertion sequences: duplex qPCR for the detection of IS900 specific for M. avium subsp. paratuberculosis, and triplex qPCR for the detection of IS901 specific for Mycobacterium avium subsp. avium and IS 1245 specific for M. avium subsp. hominissuis. Of the 77 analyzed meat samples, 17 (22%) were found to contain M. avium subsp. paratuberculosis DNA, 4 (5%) samples contained Mycobacterium avium subsp. avium DNA, and in 12 (16%) samples M. avium subsp. hominissuis DNA was detected. The concentration of M. avium subsp. paratuberculosis and M. avium subsp. hominissuis DNA in some meat products exceeded 10(4) genomes per g. Culture examination of these mycobacterial subspecies was negative. By analyzing a range of meat products, we have provided evidence to support the hypothesis that M. avium is present in everyday commodities sold to the general public.

  19. Interaction of Mycobacterium avium with environmental amoebae enhances virulence.

    PubMed Central

    Cirillo, J D; Falkow, S; Tompkins, L S; Bermudez, L E

    1997-01-01

    Environmental mycobacteria are a common cause of human infections. Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases. Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought. In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii. We found that M. avium enters and replicates in A. castellanii. In addition, similar to that shown for mycobacteria within macrophages, M. avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae. In order to determine whether growth of M. avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence. We found that growth of M. avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth. Furthermore, amoeba-grown M. avium was also more virulent in the beige mouse model of infection. These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M. avium. PMID:9284149

  20. Urinary mycobacterium avium presenting as sterile pyuria

    PubMed Central

    Yang, Kai; Samplaski, Mary; Mazzulli, Tony; Lo, Kirk; Grober, Ethan; Jarvi, Keith Allen

    2016-01-01

    A 65-year-old healthy woman presented with persistent, asymptomatic sterile pyuria detected by her family physician. While she did not have symptoms, the patient recounts that she has had cloudy urine for years. Cultures of the urine for bacteria showed no growth and no fungi were identified. First-morning urine samples were sent for both tuberculosis and nontuberculosis mycobacterium species testing. The culture grew genotypically identified Mycobaterium avium complex (MAC). Mantoux skin testing was positive. No urological abnormalities were detected by ultrasound and computed tomography (CT) imaging of the urinary tract.

  1. Multidrug therapy of Mycobacterium avium subsp. avium infection in experimentally inoculated budgerigars (Melopsittacus undulatus).

    PubMed

    Ledwoń, A; Dolka, I; Dolka, B; Cegiełkowska, M; Czopowicz, M; Szeleszczuk, P

    2015-01-01

    The aim of the study was to determine whether the four-month experimental therapy of mycobacteriosis in budgerigars may cause a complete recovery. A group of nine budgerigars was infected with a Mycobacterium avium subsp. avium isolate with proven pathogenicity for budgerigars. Five weeks post-inoculation, multidrug therapy was started. Another group comprising six birds received the same treatment but no infection, and the third group also comprising six birds was kept without infection or treatment as a control. The adopted antibiotic regimen included clarithromycin 61 mg/kg b.w., moxifloxacin 25 mg/kg b.w. and ethambutol 60 mg/kg b.w. administered by crop gavage every 12 h for 18 weeks. Despite a significant improvement in the condition of the infected, treated birds, the four-month therapy was not sufficient for the complete recovery of all. PMID:26364975

  2. Characterization of a Mycobacterium avium subsp. avium Operon Associated with Virulence and Drug Detoxification

    PubMed Central

    Viale, Mariana Noelia; Imperiale, Belén; Gioffre, Andrea Karina; Colombatti Olivieri, María Alejandra; Moyano, Roberto Damián; Morcillo, Nora; Santangelo, María de la Paz; Davis, William; Romano, María Isabel

    2014-01-01

    The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo. PMID:24967408

  3. Cryptosporidium avium n. sp. (Apicomplexa: Cryptosporidiidae) in birds.

    PubMed

    Holubová, Nikola; Sak, Bohumil; Horčičková, Michaela; Hlásková, Lenka; Květoňová, Dana; Menchaca, Sarah; McEvoy, John; Kváč, Martin

    2016-06-01

    The morphological, biological, and molecular characteristics of Cryptosporidium avian genotype V are described, and the species name Cryptosporidium avium is proposed to reflect its specificity for birds under natural and experimental conditions. Oocysts of C. avium measured 5.30-6.90 μm (mean = 6.26 μm) × 4.30-5.50 μm (mean = 4.86 μm) with a length to width ratio of 1.29 (1.14-1.47). Oocysts of C. avium obtained from four naturally infected red-crowned parakeets (Cyanoramphus novaezealandiae) were infectious for 6-month-old budgerigars (Melopsittacus undulatus) and hens (Gallus gallus f. domestica). The prepatent periods in both susceptible bird species was 11 days postinfection (DPI). The infection intensity of C. avium in budgerigars and hens was low, with a maximum intensity of 5000 oocysts per gram of feces. Oocysts of C. avium were microscopically detected at only 12-16 DPI in hens and 12 DPI in budgerigars, while PCR analyses revealed the presence of specific DNA in fecal samples from 11 to 30 DPI (the conclusion of the experiment). Cryptosporidium avium was not infectious for 8-week-old SCID and BALB/c mice (Mus musculus). Naturally or experimentally infected birds showed no clinical signs of cryptosporidiosis, and no pathology was detected. Developmental stages of C. avium were detected in the ileum and cecum using scanning electron microscopy. Phylogenetic analyses based on small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. avium is genetically distinct from previously described Cryptosporidium species. PMID:26905074

  4. GENETIC FINGERPRINTING OF MYCOBACTERIUM AVIUM COMPLEX (MAC) ORGANISMS ISOLATED FROM HOSPITAL PATIENTS AND THE ENVIRONMENT

    EPA Science Inventory

    A particularly pathogenic group of mycobacteria belong to the Mycobacterium avium complex (MAC), which includes M. avium and M. intracellulare. MAC organisms cause disease in children, the elderly, and immuno-compromised individuals. A critical step in preventing MAC infections...

  5. Apoptosis of human monocytes and macrophages by Mycobacterium avium sonicate.

    PubMed Central

    Hayashi, T; Catanzaro, A; Rao, S P

    1997-01-01

    Mycobacterium avium is an intracellular organism which multiplies predominantly within human macrophages. This organism has previously been shown to induce apoptosis in human macrophages. With a view to identifying M. avium components that induce cell death in infected host cells, sonicated extracts of M. avium as well as individual components isolated from the M. avium sonicate were tested in various assays with a human monocytic cell line (THP-1). THP-1 cells incubated with M. avium sonicate showed significantly reduced viability after a 2-day exposure compared to control cells incubated with media alone. This effect was dose dependent, with only 6.6% +/- 5.2% and 48.8% +/- 10.3% of the cells being viable by trypan blue exclusion at 600 and 300 microg/ml, respectively. Control cells, on the other hand, exhibited a viability of 98.8% +/- 1.0%. In addition, an 80% ammonium sulfate fraction of the M. avium sonicate and the previously characterized 68-kDa protein were found to have similar effects on THP-1 cells. In both cases, the reduction in viability was due to apoptosis characterized by chromatin condensation, DNA fragmentation by agarose gel electrophoresis, or terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) and release of nuclear matrix protein (NMP) into the culture medium. M. avium sonicate-induced apoptosis of THP-1 cells was completely inhibited by the commonly used antioxidants pyrrolidinedithiocarbamate (PDTC) and butylated hydroxyanisole (BHA), indicating that the generation of free oxygen radicals may be responsible for inducing cell death. M. avium sonicate was found to induce apoptosis of monocyte-derived macrophages (MDMs) as well. This effect was not reversed in the presence of PDTC and was not accompanied with DNA fragmentation when determined by agarose gel electrophoresis, as seen in the case of THP-1 cells. However, these MDMs were found to contain fragmented DNA by TUNEL. These findings suggest that the mechanism

  6. A Gene Specific to Mycobacterium avium subsp. paratuberculosis, But Only at the Transcription-translation Level

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is no known antibody that detects M. avium subsp paratuberculosis and does not cross react with other M. avium subspecies. In the present study, a monoclonal antibody was identified from mice immunized with a cell membrane fraction of M. avium subsp paratuberculosis strain K-10. This antibod...

  7. Characterization of biofilm formation by clinical isolates of Mycobacterium avium.

    PubMed

    Carter, George; Wu, Martin; Drummond, Daryl C; Bermudez, Luiz E

    2003-09-01

    Mycobacterium avium is an environmental organism encountered in natural and urban water sources as well as soil. M. avium biofilm has recently been identified on sauna walls and in city water pipes and might have a role in the survival of virulent strains in the environment and in the host. To characterize the M. avium biofilm, an in vitro model was adapted wherein biofilm develops on a PVC surface. Biofilm was detected by staining with crystal violet and visualization by optical microscopy and quantified by A(570). M. avium strains MAC 101, MAC 100, MAC 104, MAC 109, MAC A5 and MAC 5501 (all isolated from the blood of AIDS patients) were used in the assays. Biofilm formation was dependent on the presence of Ca(2+), Mg(2+) or Zn(2+) ions in the water, with the maximal effect seen at a concentration of 1 micro M. The presence of 2 % glucose and peptone as sources of carbon increased the formation of biofilm, while this was partially inhibited by humic acid. Since sliding motility has been associated with the amount of glycopeptidolipid (GPL), TLC was used to determine the presence of GPL. The supernatant of a biofilm-forming culture induced formation of a stable biofilm and amikacin blocked the establishment of biofilm by M. avium strains at subinhibitory concentrations. Bacteria in the biofilm were more resistant to chlorine as well as to exposure to potassium monopersulfate and chloroheximide acetate than were planktonic bacteria. Identification of M. avium genes involved in biofilm formation and further studies of the effect of antimicrobials on the establishment of biofilm may identify approaches for inhibiting M. avium biofilm formation and colonization.

  8. Induction of Mycobacterium avium proteins upon infection of human macrophages.

    PubMed

    Brunori, Lara; Giannoni, Federico; Bini, Luca; Liberatori, Sabrina; Frota, Cristiane; Jenner, Peter; Thoresen, Ove Fredrik; Orefici, Graziella; Fattorini, Lanfranco

    2004-10-01

    Induction of Mycobacterium avium proteins labelled with [35S]methionine and mRNAs upon infection of the human macrophage cell line THP-1 was investigated by two-dimensional gel electrophoresis-mass spectrometry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. M. avium overexpressed proteins within the macrophages that are involved in fatty acids metabolism (FadE2, FixA), cell wall synthesis (KasA), and protein synthesis (EF-tu). The correlation of differential protein and mRNA expression varied between good and no correlation. Overall, these four proteins may be involved in the adaptation and survival of M. avium within human macrophages. PMID:15378697

  9. Experimental infection of Eurasian wild boar with Mycobacterium avium subsp. avium.

    PubMed

    Garrido, J M; Vicente, J; Carrasco-García, R; Galindo, R C; Minguijón, E; Ballesteros, C; Aranaz, A; Romero, B; Sevilla, I; Juste, R; de la Fuente, J; Gortazar, C

    2010-07-29

    The Eurasian wild boar (Sus scrofa) is increasingly relevant as a host for several pathogenic mycobacteria. We aimed to characterize the first experimental Mycobacterium avium subsp. avium (MAA) infection in wild boar in order to describe the lesions and the immune response as compared to uninfected controls. Twelve 1-4-month-old wild boar piglets were housed in class III bio-containment facilities. Four concentrations of MAA suspension were used: 10, 10(2) and 10(4) mycobacteria (2 animals each, oropharyngeal route) and 2.5 x 10(6) mycobacteria (2 animals each by the oropharyngeal and nasal routes). No clinical signs were observed and pathology evidenced a low pathogenicity of this MAA strain for this particular host. Bacteriological and pathological evidence of successful infection after experimental inoculation was found for the group challenged with 2.5 x 10(6) mycobacteria. These four wild boar showed a positive IFN-gamma response to the avian PPD and the real-time RT-PCR data revealed that three genes, complement component C3, IFN-gamma and RANTES, were significantly down regulated in infected animals. These results were similar to those found in naturally and experimentally M. bovis-infected wild boar and may constitute biomarkers of mycobacterial infection in this species.

  10. Mechanism of Mycobacterium avium complex pathogenesis.

    PubMed

    Reddy, V M

    1998-06-08

    Mycobacterium avium complex (MAC) group of microorganisms are the most common opportunistic bacterial pathogens causing disseminated disease in HIV infected patients. These microorganisms are ubiquitous in nature, and are acquired by respiratory and oral routes. Pathogenesis of MAC depends on the ability of the organisms to colonize intestinal/respiratory mucosa, penetrate the protective barriers and resist intracellular killing by macrophages. Transient and reversible variation of colony morphology is one the characteristic feature of MAC that plays a significant role in the virulence and pathogenesis of these microorganisms. Isogenic colony variants of MAC differ in their virulence, susceptibility to antibiotics, stimulation of oxygen radicals and cytokines. The virulent smooth transparent colony variants are more frequently isolated from AIDS patients, more efficient in mucosal colonization, and adhere more efficiently to epithelial cells as compared to the less virulent smooth opaque variants. However, both the isogenic variants bind to the mucosal epithelial cells through the same multiple receptors. In addition, both the isogenic variants of MAC also bind to intestinal mucus through a single receptor. Study of the interaction of MAC with the host cells and characterization of MAC adhesins and host cell receptors facilitates the elucidation of the mechanisms involved in MAC pathogenesis.

  11. Cavitary pulmonary infection with Mycobacterium avium observed by bronchoscopy.

    PubMed

    Uruga, Hironori; Suzuki, Aika; Hanada, Shigeo; Takaya, Hisashi; Miyamoto, Atsushi; Morokawa, Nasa; Fujii, Takeshi; Kurosaki, Atsuko; Kishi, Kazuma

    2012-10-01

    A 58-year-old man was admitted to our hospital because of fever and loss of appetite. He had undergone surgery for esophageal cancer. A chest radiography 12 years after the surgery revealed cavitary lesions in the right upper lobe of the lung. The patient was then diagnosed as having Mycobacterium avium infection. The cavitary lesions worsened 2 years after clarithromycin monotherapy. Bronchoscopy was performed to observe the interior of the cavity. Gray debris adhering to the cavitary wall decreased after intensive treatment with Streptomycin, rifabutin, levofloxacin, and ethambutol. This is a rare case in which treatment efficacy of M. avium infection was directly observed by serial bronchoscopy. PMID:23207537

  12. High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages

    PubMed Central

    Ichimura, Naoya; Sato, Megumi; Yoshimoto, Akira; Yano, Kouji; Ohkawa, Ryunosuke; Kasama, Takeshi

    2016-01-01

    High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity. PMID:27516907

  13. Fibronectin Attachment Protein Homologue Mediates Fibronectin Binding by Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Secott, T. E.; Lin, T. L.; Wu, C. C.

    2001-01-01

    Attachment of Mycobacterium avium subsp. paratuberculosis to host tissue and penetration of mucosal surfaces are pivotal events in the pathogenesis of Johne's disease. Fibronectin (FN) binding is required for attachment and internalization of several mycobacteria by epithelial cells in vitro. The objective of this study was to further characterize the FN binding activity of M. avium subsp. paratuberculosis. Although the bacteria bound FN poorly at pH above 7, brief acid pretreatment greatly enhanced FN binding within the pH range (3 to 10) studied. A 4.6-kbp fragment from an M. avium subsp. paratuberculosis genomic library was found to contain a 1,107-bp open reading frame that shows very high nucleotide sequence identity with that of the FN attachment protein (FAP) gene of M. avium subsp. avium. Pretreatment of FN with an FN-binding peptide from M. avium subsp. avium FAP abolished FN binding, indicating that M. avium subsp. paratuberculosis binds FN in a FAP-dependent manner. Pretreatment of M. avium subsp. paratuberculosis with anti-FAP immunoglobulin G did not abrogate FN binding; blocking occurred only when anti-FAP was added together with FN. FAP was detected by immunofluorescence only in lipid-extracted M. avium subsp. paratuberculosis. Western blotting and immunoelectron microscopy revealed that FAP is located near the interior of the cell envelope of M. avium subsp. paratuberculosis. The results indicate that a FAP homologue mediates the attachment of FN to M. avium subsp. paratuberculosis. Further, given the subcellular location of FAP, it is considered that this protein operates at the terminus of a coordinated FN binding system in the cell envelope of M. avium subsp. paratuberculosis. PMID:11254560

  14. Cellular Interactions in Mycobacterium avium subsp. paratuberculosis Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study of host immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) is complicated by a number of factors, including the protracted nature of the disease and the stealthy nature of the pathogen. Noted as one of the more fastidious mycobacteria, infection with MAP is often chara...

  15. Tuberculosis in Birds: Insights into the Mycobacterium avium Infections

    PubMed Central

    Dhama, Kuldeep; Mahendran, Mahesh; Tiwari, Ruchi; Dayal Singh, Shambhu; Kumar, Deepak; Singh, Shoorvir; Sawant, Pradeep Mahadev

    2011-01-01

    Tuberculosis, a List B disease of World Organization for Animal Health, caused by M. avium or M. genavense predominantly affects poultry and pet or captive birds. Clinical manifestations in birds include emaciation, depression and diarrhea along with marked atrophy of breast muscle. Unlike tuberculosis in animals and man, lesions in lungs are rare. Tubercular nodules can be seen in liver, spleen, intestine and bone marrow. Granulomatous lesion without calcification is a prominent feature. The disease is a rarity in organized poultry sector due to improved farm practices, but occurs in zoo aviaries. Molecular techniques like polymerase chain reaction combined with restriction fragment length polymorphism and gene probes aid in rapid identification and characterization of mycobacteria subspecies, and overcome disadvantages of conventional methods which are slow, labour intensive and may at times fail to produce precise results. M. avium subsp. avium with genotype IS901+ and IS1245+ causes infections in animals and human beings too. The bacterium causes sensitivity in cattle to the tuberculin test. The paper discusses in brief the M. avium infection in birds, its importance in a zoonotic perspective, and outlines conventional and novel strategies for its diagnosis, prevention and eradication in domestic/pet birds and humans alike. PMID:21776352

  16. Genomic and Transcriptomic Studies in Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarray technology is an important tool in functional genomic research. It has enabled a deeper analysis of genomic diversity among bacteria belonging to the Mycobacterium avium Complex (MAC). In addition, the expression of thousands of genes can be studied simultaneously in a single experiment...

  17. Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium

    PubMed Central

    Jeevarajah, Dharshini; Patterson, John H.; Taig, Ellen; Sargeant, Tobias; McConville, Malcolm J.; Billman-Jacobe, Helen

    2004-01-01

    Several species of mycobacteria express abundant glycopeptidolipids (GPLs) on the surfaces of their cells. The GPLs are glycolipids that contain modified sugars including acetylated 6-deoxy-talose and methylated rhamnose. Four methyltransferases have been implicated in the synthesis of the GPLs of Mycobacterium smegmatis and Mycobacterium avium. A rhamnosyl 3-O-methytransferase and a fatty acid methyltransferase of M. smegmatis have been previously characterized. In this paper, we characterize the methyltransferases that are responsible for modifying the hydroxyl groups at positions 2 and 4 of rhamnose and propose the biosynthetic sequence of GPL trimethylrhamnose formation. The analysis of M. avium genes through the creation of specific mutants is technically difficult; therefore, an alternative approach to determine the function of putative methyltransferases of M. avium was undertaken. Complementation of M. smegmatis methyltransferase mutants with M. avium genes revealed that MtfC and MtfB of the latter species have 4-O-methyltransferase activity and that MtfD is a 3-O-methyltransferase which can modify rhamnose of GPLs in M. smegmatis. PMID:15466031

  18. Mycobacterium avium subsp. paratuberculosis infection, immunology and pathology of livestock

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) infection in ruminants leads to a chronic and progressive enteric disease (Johne’s disease) that results in loss of intestinal function, poor body condition, and eventual death. Transmission is primarily through a fecal-oral route in neonates but con...

  19. [Mycobacterium avium complex in water buffaloes slaughtered for consumption].

    PubMed

    Freitas, J; Panetta, J C; Curcio, M; Ueki, S Y

    2001-06-01

    Two mycobacterium strains isolated from lung tissue a apical lymph nodes of slaughtered water buffaloes were biochemically analyzed and identified as Mycobacterium avium complex strains. Association between these microorganisms and the acquired immunodeficiency syndrome, and the potential risk posed by eating infected animals and their products, was discussed.

  20. Skin Test Reactivity and Cellular Immune Responses to Mycobacterium avium Sensitin in AIDS Patients at Risk for Disseminated M. avium Infection

    PubMed Central

    von Reyn, C. Fordham; Williams, Paige L.; Lederman, Howard M.; McCutchan, J. Allen; Koletar, Susan L.; Murphy, Robert L.; Cohn, Susan E.; Evans, Thomas; Heald, Alison E.; Colquhoun, Dodi; Bassily, Ehab L.; Currier, Judith S.

    2001-01-01

    Skin tests and lymphocyte proliferation assays (LPA) were performed with Mycobacterium avium sensitin on patients with AIDS. Among 139 subjects, 13% had positive skin test results and 32% had positive LPA results. The LPA may be a more sensitive indicator of prior M. avium infection in this population. PMID:11687476

  1. Mannosylated lipoarabinomannans from Mycobacterium avium subsp. paratuberculosis alters the inflammatory response by bovine macrophages and suppresses killing of Mycobacterium avium subsp. avium organisms.

    PubMed

    Souza, Cleverson; Davis, William C; Eckstein, Torsten M; Sreevatsan, Srinand; Weiss, Douglas J

    2013-01-01

    Analysis of the mechanisms through which pathogenic mycobacteria interfere with macrophage activation and phagosome maturation have shown that engagement of specific membrane receptors with bacterial ligands is the initiating event. Mannosylated lipoarabinomannan (Man-LAM) has been identified as one of the ligands that modulates macrophage function. We evaluated the effects of Man-LAM derived from Mycobacterium avium subsp. paratuberculosis (MAP) on bovine macrophages. Man-LAM induced a rapid and prolonged expression of IL-10 message as well as transient expression of TNF-α. Preincubation with Man-LAM for up to 16 h did not suppress expression of IL-12 in response to interferon-γ. Evaluation of the effect of Man-LAM on phagosome acidification, phagosome maturation, and killing of Mycobacterium avium subsp. avium (MAA) showed that preincubation of macrophages with Man-LAM before addition of MAA inhibited phagosome acidification, phagolysosome fusion, and reduced killing. Analysis of signaling pathways provided indirect evidence that inhibition of killing was associated with activation of the MAPK-p38 signaling pathway but not the pathway involved in regulation of expression of IL-10. These results support the hypothesis that MAP Man-LAM is one of the virulence factors facilitating survival of MAP in macrophages.

  2. Genotyping of Mycobacterium avium subsp. avium isolates from naturally infected lofts of domestic pigeons in Ahvaz by IS901 RFLP

    PubMed Central

    Parvandar-Asadollahi, Kaveh; Mosavari, Nader; Mayahi, Mansoor

    2015-01-01

    Background and Objectives: Avian tuberculosis is one of the most important infections affecting most species of birds. Mycobacterium avium can not only infect all species of birds, but also infect some domesticated mammals. The most crucial aspect of control and eradication scheme is identification of infection sources and transmission routs. Molecular techniques such as restriction fragment length polymorphism and pulse field gel electrophoresis have been shown to be much more discriminatory and suitable for use in the epidemiological study. Materials and Methods: Eighty suspected pigeons to avian tuberculosis based on their clinical signs, were subjected to the study. Forty Mycobacterium avium subsp. avium isolates out of a total of 51 identified isolates were subjected to the test. Results: IS901-RFLP using Pvu II was successfully conducted and produced 7 patterns. The majority of isolates (60%) were RFLP type PI.1. This type was the most similar type to standard strain. However, all the patterns obtained in this study were different from the standard strain. Conclusion: The result of this study indicate that these isolates probably are limited to Khuzestan region. We recommend DNA fingerprinting differentiation of non tuberculous Mycobacteria particularly Mycobacterium avium complex isolated from infected birds and human to possibly find source of infections. PMID:26719782

  3. Thermal Inactivation of Mycobacterium avium subsp. paratuberculosis in Artificially Contaminated Milk by Direct Steam Injection

    PubMed Central

    Butot, Sophie; Jagadeesan, Balamurugan; Bakker, Douwe; Donaghy, John

    2016-01-01

    ABSTRACT The efficiency of direct steam injection (DSI) at 105°C for 3 s to inactivate Mycobacterium avium subsp. paratuberculosis in milk at a pilot-plant scale was investigated. Milk samples were artificially contaminated with M. avium subsp. paratuberculosis and also with cow fecal material naturally infected with M. avium subsp. paratuberculosis. We also tested milk artificially contaminated with Mycobacterium smegmatis as a candidate surrogate to compare thermal inactivation between M. smegmatis and M. avium subsp. paratuberculosis. Following the DSI process, no viable M. avium subsp. paratuberculosis or M. smegmatis was recovered using culture methods for both strains. For pure M. avium subsp. paratuberculosis cultures, a minimum reduction of 5.6 log10 was achieved with DSI, and a minimum reduction of 5.7 log10 was found with M. smegmatis. The minimum log10 reduction for wild-type M. avium subsp. paratuberculosis naturally present in feces was 3.3. In addition, 44 dairy and nondairy powdered infant formula (PIF) ingredients used during the manufacturing process of PIF were tested for an alternate source for M. avium subsp. paratuberculosis and were found to be negative by quantitative PCR (qPCR). In conclusion, the results obtained from this study indicate that a >7-fold-log10 reduction of M. avium subsp. paratuberculosis in milk can be achieved with the applied DSI process. IMPORTANCE M. avium subsp. paratuberculosis is widespread in dairy herds in many countries. M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle, and infected animals can directly or indirectly (i.e., fecal contamination) contaminate milk. Despite much research and debate, there is no conclusive evidence that M. avium subsp. paratuberculosis is a zoonotic bacterium, i.e., one that causes disease in humans. The presence of M. avium subsp. paratuberculosis or its DNA has been reported in dairy products, including pasteurized milk, cheese, and infant formula

  4. Characterization of mouse models of Mycobacterium avium complex infection and evaluation of drug combinations.

    PubMed

    Andréjak, Claire; Almeida, Deepak V; Tyagi, Sandeep; Converse, Paul J; Ammerman, Nicole C; Grosset, Jacques H

    2015-04-01

    The Mycobacterium avium complex is the most common cause of nontuberculous mycobacterial lung disease worldwide; yet, an optimal treatment regimen for M. avium complex infection has not been established. Clarithromycin is accepted as the cornerstone drug for treatment of M. avium lung disease; however, good model systems, especially animal models, are needed to evaluate the most effective companion drugs. We performed a series of experiments to evaluate and use different mouse models (comparing BALB/c, C57BL/6, nude, and beige mice) of M. avium infection and to assess the anti-M. avium activity of single and combination drug regimens, in vitro, ex vivo, and in mice. In vitro, clarithromycin and moxifloxacin were most active against M. avium, and no antagonism was observed between these two drugs. Nude mice were more susceptible to M. avium infection than the other mouse strains tested, but the impact of treatment was most clearly seen in M. avium-infected BALB/c mice. The combination of clarithromycin-ethambutol-rifampin was more effective in all infected mice than moxifloxacin-ethambutol-rifampin; the addition of moxifloxacin to the clarithromycin-containing regimen did not increase treatment efficacy. Clarithromycin-containing regimens are the most effective for M. avium infection; substitution of moxifloxacin for clarithromycin had a negative impact on treatment efficacy. PMID:25624335

  5. Isolation of Mycobacterium avium from waterfowl with polycystic livers.

    USGS Publications Warehouse

    Roffe, Thomas J.

    1989-01-01

    An unusual gross appearance of avian tuberculosis, where fluid-filled thin-walled cysts are produced and grossly apparent in preference to granulomas, is presented. Histopathology confirmed the granulomatous nature of the lesions and the presence of intracellular acid-fast organisms. Mycobacterium avium complex was cultured from affected organs. The unusual gross presentation in these cases indicates the need to consider tuberculosis in the differential of cystic diseases of avian livers.

  6. Molecular identification of Mycobacterium avium subsp. silvaticum by duplex high-resolution melt analysis and subspecies-specific real-time PCR.

    PubMed

    Rónai, Zsuzsanna; Csivincsik, Ágnes; Dán, Ádám

    2015-05-01

    Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.

  7. Roles for Cell Wall Glycopeptidolipid in Surface Adherence and Planktonic Dispersal of Mycobacterium avium

    EPA Science Inventory

    The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. aviu...

  8. Antigenic Profiles of Recombinant Proteins from Mycobacterium avium subsp paratuberculosis in Sheep with Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to improve the ELISA test to detect Mycobacterium avium subsp paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne’s disease. In the present study, antibo...

  9. Detection of quantification of Mycobacterium avium complex organisms in drinking water

    EPA Science Inventory

    The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and others. MAC are listed on the U. S. Environmental Protection Agency’s Contaminant Candidate List 2 (CCL2) due to their association with human disease and occurrence in public dr...

  10. REAL-TIME QUANTITATIVE PCR DETECTION OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS IN DRINKING WATER

    EPA Science Inventory

    The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and others. MAC are listed on the U.S. Environmental Protection Agency's Contaminant Candidate List (CCL) due to their association with human disease and occurrence in public drinkin...

  11. Complete genome sequence of Mycobacterium avium subsp. paratuberculosis, isolated from human breast milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis is the etiologic agent of Johne’s disease. We report the draft genome sequences of six M. avium subsp paratuberculosis isolates obtained from diverse hosts including bison, cattle and sheep. These sequences will deepen our understanding of host association ...

  12. Characterization of clinical and environmental Mycobacterium avium spp. isolates and their interaction with human macrophages

    EPA Science Inventory

    Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies tha...

  13. Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne’s disease (JD). To fill this gap in JD research, monoclonal antibodies (mAbs) against M. avium subsp. paratuberculosis were produced fr...

  14. Draft genome sequence of a Mycobacterium avium complex isolate from a broadbill bird

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the draft genome sequences of ten Mycobacterium avium complex isolates obtained from diverse hosts. This collection includes isolates obtained from deer, pig, elephant, ruddy duck and Red-tailed hawk species. The type strain of Mycobacterium avium subspecies silvaticum (ATCC 49884) is also...

  15. Genomic Comparison of PE and PPE Genes in the Mycobacterium avium Complex▿ †

    PubMed Central

    Mackenzie, Nick; Alexander, David C.; Turenne, Christine Y.; Behr, Marcel A.; De Buck, Jeroen M.

    2009-01-01

    The Mycobacterium avium complex (MAC) comprises genomically similar but phenotypically divergent bacteria that inhabit diverse environments and that cause disease in different hosts. In this study, a whole-genome approach was used to examine the polymorphic PE (Pro-Glu) and PPE (Pro-Pro-Glu) gene families, implicated in immunostimulation and virulence. The four major groups of MAC organisms were examined, including the newly sequenced type strains of M. intracellulare and M. avium subsp. avium, plus M. avium subsp. paratuberculosis and M. avium subsp. hominissuis, for the purpose of finding genetic differences that could be exploited to design diagnostic tests specific to these groups and that could help explain their divergence in pathogenesis and host specificity. Unique and missing PPE genes were found in all MAC members except M. avium subsp. avium. Only M. intracellulare had a unique PE gene. Apart from this, most PE and PPE sequences were conserved, with average nucleotide sequence identities of 99.1 and 98.1%, respectively, among the M. avium subspecies, but only 82.9 and 79.7% identities with the PE and PPE sequences of M. intracellulare, respectively. A detailed analysis of the amino acid sequences was performed between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis. Most differences were detected in the PPE proteins, with amino acid substitutions and frame shifts leading to unique amino acid sequences. In conclusion, several unique PPE proteins were identified in MAC organisms next to numerous polymorphisms in both the PE and PPE gene families. These substantial differences could help explain the divergence in phenotypes within the MAC and could lead to diagnostic tests with better discriminatory abilities. PMID:19144814

  16. Systems Analysis of Early Host Gene Expression Provides Clues for Transient Mycobacterium avium ssp avium vs. Persistent Mycobacterium avium ssp paratuberculosis Intestinal Infections

    PubMed Central

    Khare, Sangeeta; Drake, Kenneth L.; Lawhon, Sara D.; Nunes, Jairo E. S.; Figueiredo, Josely F.; Rossetti, Carlos A.; Gull, Tamara; Everts, Robin E.; Lewin, Harris. A.; Adams, Leslie Garry

    2016-01-01

    It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer’s patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum

  17. Infection of Eurasian badgers (Meles meles) with Mycobacterium avium complex (MAC) bacteria.

    PubMed

    Balseiro, Ana; Merediz, Isabel; Sevilla, Iker A; García-Castro, Carmen; Gortázar, Christian; Prieto, José M; Delahay, Richard J

    2011-05-01

    There are few reports of infection with Mycobacterium avium complex (MAC) bacteria in badgers. In this study archive data relating to the isolation of MAC organisms from badgers in the UK is presented, and information derived from recent cases of such infection in Spain is used to illustrate the associated pathology and to characterise strain types. Tissue samples were cultured for mycobacteria and, in the case of Spanish badgers, were examined both histopathologically and using immunohistochemistry, and DNA typing of M. avium isolates was also carried out. A total of 5 (7.35%) and 281 (0.51%) isolates of M. avium spp. were recovered from badgers from the studies in Spain and the UK, respectively. DNA typing of the isolates from Spain identified the sub-species M. avium hominissuis and M. avium avium. These findings provide new information on the prevalence of MAC organisms in badgers in the UK and Spain. The extent to which infected badgers may be involved in the epidemiology of M. avium in other wild or domestic hosts remains unknown.

  18. Phenotypic and Genomic Analyses of the Mycobacterium avium Complex Reveal Differences in Gastrointestinal Invasion and Genomic Composition

    PubMed Central

    McGarvey, Jeffery A.; Bermudez, Luiz E.

    2001-01-01

    Mycobacterium avium and Mycobacterium intracellulare are closely related organisms and comprise the Mycobacterium avium complex. These organisms share many common characteristics, including the ability to cause life-threatening respiratory infections in people with underlying lung pathology or immunological defects and occasionally in those with no known predisposing conditions. However, the ability to invade the mucosa of the gastrointestinal tract and cause disseminated disease in AIDS patients has not been epidemiologically linked to M. intracellulare and appears to be unique to M. avium. We compared the abilities of M. avium and M. intracellulare to tolerate the acidic conditions of the stomach, to resist the membrane-disrupting activity of cationic peptides, and to invade intestinal epithelial cells in vitro and in vivo. We observed that M. avium and M. intracellulare were both tolerant to the acidic conditions encountered in the stomach and resistant to cationic peptides. However, when strains of M. avium and M. intracellulare were examined for their ability to enter cultured human intestinal cells or mouse intestinal mucosa, we observed that M. avium could invade more efficiently than M. intracellulare. To elucidate the basis of this pathogenic difference and identify genes involved in the invasion of the intestinal mucosa, we performed chromosomal DNA subtractive hybridization using M. avium and M. intracellulare chromosomal DNAs. In all, 21 genes that were present in M. avium but absent in M. intracellulare were identified, including some that may be associated with the ability of M. avium to invade the intestinal mucosa. PMID:11705893

  19. Mycobacterium avium subspecies induce differential expression of pro-inflammatory mediators in a murine macrophage model: evidence for enhanced pathogenicity of Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Basler, Tina; Geffers, Robert; Weiss, Siegfried; Valentin-Weigand, Peter; Goethe, Ralph

    2008-01-01

    Mycobacterium avium subspecies (ssp.) paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic, non-treatable granulomatous enteritis of ruminants. MAP is the only mycobacterium affecting the intestinal tract, which is of interest since it is presently the most favoured pathogen linked to Crohn's disease (CD) in humans due to its frequent detection in CD tissues. MAP is genetically closely related to other M. avium ssp. such as M. avium ssp. avium (MAA) and M. avium ssp. hominissuis (MAH) which can cause mycobacteriosis in animals and immunocompromised humans. We have recently shown that murine macrophage cell lines represent suitable systems to analyse M. avium ssp. patho-mechanisms and could show that MAP, but not MAA, specifically inhibited the antigen-specific stimulatory capacity for CD4(+) T-cells. In the present study, we compared gene expression profiles of murine RAW264.7 macrophages in response to infections with MAP or MAA using murine high-density oligonucleotide Affymetrix microarrays. A comparison of MAP and MAA infection revealed 17 differentially expressed genes. They were expressed at a much lower level in MAP-infected macrophages than in MAA-infected macrophages. Among these were the genes for IL-1beta, IL-1alpha, CXCL2, PTGS2 (COX2), lipocalin (LCN2) and TNF, which are important pro-inflammatory factors. The microarray data were confirmed for selected genes by quantitative real-time reverse transcription PCR and, by protein array analyses and ELISA. Similar to MAA, infection with MAH also showed robust induction of IL-1beta, CXCL2, COX2, LCN2 and TNF. Taken together, our results from M. avium ssp.-infected murine macrophages provide evidence that MAP in contrast to MAA and MAH specifically suppresses the pro-inflammatory defence mechanisms of infected macrophages.

  20. Clinical outbreak of Bordetella avium infection in two turkey breeder flocks.

    PubMed

    Kelly, B J; Ghazikhanian, G Y; Mayeda, B

    1986-01-01

    An acute upper respiratory disease was observed in two broad-breasted white (BBW) turkey primary breeder flocks. Associated clinical signs included sneezing, depression, and a deep dry cough originating from large conducting airways. Morbidity reached approximately 15-20% of the hens in an affected house. None of the turkeys died, and total feed consumption was not affected. A minimal effect upon egg production was noticed. Sera from an acutely affected flock exhibited a marked rise in titer to Bordetella avium compared with preinfection sera samples. In Case 1, B. avium was isolated in pure culture from affected birds. In Case 2, B. avium was diagnosed by serological results and clinical signs; bacteriological examination was not attempted. The findings presented here are consistent with an acute clinical outbreak of B. avium-induced turkey rhinotracheitis (turkey coryza) in BBW turkey breeder hens. PMID:3729868

  1. AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF MYCOBACTERIUM AVIUM COMPLEX ISOLATES RECOVERED FROM SOUTHERN CALIFORNIA

    EPA Science Inventory

    Fine-scale genotyping methods are necessary in order to identify possible sources of human exposure to opportunistic pathogens belonging to the Mycobacterium avium complex (MAC). In this study, amplified fragment length polymorphism (AFLP) analysis was evaluated for fingerprintin...

  2. Epidemiology and Ecology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa

    EPA Science Inventory

    BACKGROUND: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa are opportunistic premise plumbing pathogens (OPPPs) that persist and grow in household plumbing, habitats they share with humans. Infections caused by these OPPPs involve individuals with preexis...

  3. Trypanosoma avium incidence, pathogenicity and response to melarsomine in falcons from Kuwait.

    PubMed

    Tarello, W

    2005-03-01

    Epidemiological and clinical studies on Trypanosoma avium are lacking in the Middle East. The aims of this study were to determine the T. avium incidence in falcons from Kuwait, report clinical signs and find an effective therapy. Blood smears from 921 diseased and 56 healthy falcons were examined between May 2003 and April 2004. 12 birds 11.3%) were found infected by T. avium and ten of these were treated with melarsomine (Cymelarsan) at a dosage of 0.25 mg/kg intramuscularly for four days. All affected birds presented clinical signs, including incapacity of flying high, poor appetite, lethargy, loosing weight, weakness, dyspnoea and death. Signs disappeared within 1-7 days after administration of melarsomine. Trypomastigotes were not detected in blood smears made 1-7 days after the end of therapy. This study suggests that T. avium induces disease in falcons and that melarsomine can be an effective therapy eliminating both clinical signs and circulating trypomastigotes.

  4. Clinical outbreak of Bordetella avium infection in two turkey breeder flocks.

    PubMed

    Kelly, B J; Ghazikhanian, G Y; Mayeda, B

    1986-01-01

    An acute upper respiratory disease was observed in two broad-breasted white (BBW) turkey primary breeder flocks. Associated clinical signs included sneezing, depression, and a deep dry cough originating from large conducting airways. Morbidity reached approximately 15-20% of the hens in an affected house. None of the turkeys died, and total feed consumption was not affected. A minimal effect upon egg production was noticed. Sera from an acutely affected flock exhibited a marked rise in titer to Bordetella avium compared with preinfection sera samples. In Case 1, B. avium was isolated in pure culture from affected birds. In Case 2, B. avium was diagnosed by serological results and clinical signs; bacteriological examination was not attempted. The findings presented here are consistent with an acute clinical outbreak of B. avium-induced turkey rhinotracheitis (turkey coryza) in BBW turkey breeder hens.

  5. Composition and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain ATCC 19698. Traditional production consists of floating culture incubation at 37oC, organism inactivation by autoclaving, coarse filtrat...

  6. Flow Cytometric Detection of Mycobacterium avium subsp. paratuberculosis-Specific Antibodies in Experimentally Infected and Naturally Exposed Calves

    PubMed Central

    Bridger, P. S.; Bulun, H.; Fischer, M.; Akineden, Ö.; Seeger, T.; Barth, S.; Henrich, M.; Doll, K.; Bülte, M.; Menge, C.; Bauerfeind, R.

    2013-01-01

    A desirable test to diagnose infections with Mycobacterium avium subsp. paratuberculosis facilitates identification of infected cattle prior to the state of M. avium subsp. paratuberculosis shedding. This study aimed at adjusting a flow cytometry (FC)-based assay, using intact M. avium subsp. paratuberculosis bacteria as the antigen, for diagnosis of M. avium subsp. paratuberculosis infections in calves. Serum samples were collected from experimentally infected (n = 12) and naturally exposed (n = 32) calves. Samples from five calves from positive dams were analyzed to determine the dynamics of maternal antibodies. Samples from adult cattle with defined infection status served as the standard (18 M. avium subsp. paratuberculosis shedders, 22 M. avium subsp. paratuberculosis free). After preadsorption with Mycobacterium phlei, sera were incubated with M. avium subsp. paratuberculosis and M. avium subsp. avium bacterial suspensions, respectively, followed by the separate detection of bovine IgG, IgG1, IgG2, and IgM attached to the bacterial surface. M. avium subsp. paratuberculosis-specific sample/positive (S/P) ratios were compared to enzyme-linked immunosorbent assay (ELISA) S/P ratios. In adult cattle, the FC assay for IgG1 had a sensitivity of 78% at a specificity of 100%. Maternally acquired antibodies could be detected in calves up to 121 days of life. While all but two sera taken at day 100 ± 10 postnatum from naturally exposed calves tested negative, elevated S/P ratios (IgG and IgG1) became detectable from 44 and 46 weeks postinoculation onwards in two calves infected experimentally. Even with the optimized FC assay, M. avium subsp. paratuberculosis-specific antibodies can only occasionally be detected in infected calves less than 12 months of age. The failure to detect such antibodies apparently reflects the distinct immunobiology of M. avium subsp. paratuberculosis infections rather than methodological constraints. PMID:23885032

  7. Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection.

    PubMed

    Huntley, J F; Stabel, J R; Paustian, M L; Reinhardt, T A; Bannantine, J P

    2005-10-01

    Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization. PMID:16177367

  8. Mycobacterium avium serovars 2 and 8 infections elicit unique activation of the host macrophage immune responses.

    PubMed

    Cebula, B R; Rocco, J M; Maslow, J N; Irani, V R

    2012-12-01

    Mycobacterium avium is an opportunistic pathogen whose pathogenesis is attributed to its serovar-specific glycopeptidolipid (ssGPL), which varies among its 31 serovars. To determine if the presence and type of ssGPLs contribute to M. avium pathogenesis, we infected murine macrophages (mφs) with two M. avium wild type (wt) serovars (2 and 8) and their serovar-null strains. We examined the influence of ssGPL (presence and type) on cytokine production in non-activated (-IFN-γ) and activated (+IFN-γ) mφs, and the bacterial intra-mφ survival over a 6-day infection process. Serovar-2 infections activated TNF-α production that increased over the 6 day period and was capable of controlling the intra-mφ serovar-2 null strain. In contrast, the serovar-8 infection stimulated a strong pro-inflammatory response, but was incapable of removing the invading pathogen, maybe through IL-10 production. It was clear that the intracellular growth of serovar-null in contrast to the wt M. avium strains was easily controlled. Based on our findings and the undisputed fact that M. avium ssGPL is key to its pathogenesis, we conclude that it is not appropriate to dissect the pathogenesis of one M. avium serovar and apply those findings to other serovars. PMID:22991047

  9. Mycobacterium avium subspecies paratuberculosis, Crohn's disease and the Doomsday scenario.

    PubMed

    Hermon-Taylor, John

    2009-01-01

    Johne's disease is chronic inflammation of the intestine caused by Mycobacterium avium subspecies paratuberculosis. Infection and disease are mainly in domestic livestock but can affect many species including primates. Johne's is a new disease which emerged at the turn of the 19th and 20th centuries and principally involved Europe and North America. It has since spread to former low incidence regions to become a global problem. Crohn's disease is a chronic inflammation of the intestine in humans which emerged in Europe and North America mid 20th century and increased to become a major healthcare problem. It has now spread to former low incidence regions. Infected animals shed Mycobacterium avium subspecies paratuberculosis in milk and into the environment. Human populations are widely exposed. Outcomes maybe influenced by microbial phenotype. Exposure to extracellular forms of these pathogens may confer some natural protection; exposure to intracellular forms which have passaged through milk macrophages or environmental protists may pose a greater threat to humans particularly individuals with an inherited or acquired susceptibility. Hot spots of human disease such as in Winnipeg which sits on rock at the junction of two rivers may result from local exposure to high levels of waterborne pathogens brought down from farmland. When appropriate methods are used most people with Crohn's disease are found to be infected. There are no data which demonstrate that these pathogens are harmless to humans. An overwhelming balance of probability and Public health risk favours the conclusion that Mycobacterium avium subspecies paratuberculosis is also pathogenic for people. A two tier co-operative pathogenic mechanism is proposed in Crohn's disease. Intracellular infection with the primary pathogen widely distributed throughout the gut causes an immune dysregulation and a specific chronic enteric neuropathy with loss of mucosal integrity. Segments of gross inflammatory disease

  10. Influence of cultivar and processing on cherry (Prunus avium) allergenicity.

    PubMed

    Primavesi, L; Brenna, O V; Pompei, C; Pravettoni, V; Farioli, L; Pastorello, E A

    2006-12-27

    Oral allergy syndrome is an immediate food allergic event that affects lips, mouth, and pharynx, is often triggered by fruits and vegetables, and may be associated with pollinosis. Here, we report on the allergenic pattern of different varieties of cherry (Prunus avium) and results obtained by applying several technological processes to the selected varieties. Whole cherries were submitted to chemical peeling, thermal treatment, and syruping processes, and the relative protein extracts were analyzed by in vitro (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis) and in vivo tests (skin prick test). Electrophoretic analyses demonstrated that there was no marked difference among cherry cultivars. Chemical peeling successfully removed Pru av 3, a lipid transfer protein (LTP) responsible for oral allergy syndrome in patients without pollinosis, leading to the industrial production of cherry hypoallergenic derivatives. Furthermore, the syruping process removed almost all allergenic proteins to whom patients with pollinosis are responsive. In vivo tests confirmed electrophoretic results.

  11. Endobronchial avium mycobacteria infection in an immunocompetent child.

    PubMed

    Perisson, Caroline; Nathan, Nadia; Thierry, Briac; Corvol, Harriet

    2013-01-01

    A 12-month-old boy, with no medical history, was admitted for dyspnoea with no cough or fever. Chest auscultation revealed an expiratory wheezing with decreased right-sided breath sounds. Chest imaging revealed subcarinal adenopathy and a nodule in the right principal bronchus (RB). Bronchoscopy showed a major obstruction of the RB by a granuloma, and a smaller granuloma in the left principal bronchus. The granulation tissue was removed by laser section. Histological examination revealed a necrotising granulomatous inflammation, culture showed a Mycobacterium avium complex (MAC). Tests to rule out tuberculosis and immunodeficiency were negative. The diagnosis of an MAC endobronchial granuloma was ascertained and a multidrug therapy associating clarithromycin, rifampin and ethambutol was started. The clinical outcome was good after 3 months of treatment and the bronchoscopy normalised after 1 year. Although rare, the frequency of MAC respiratory infections in immunocompetent children can increase. Reporting these cases should help to optimise diagnosis and treatment. PMID:24252838

  12. Pulmonary Mycobacterium avium infection demonstrating unusual lobar caseous pneumonia.

    PubMed

    Okuzumi, Shinichi; Minematsu, Naoto; Sasaki, Mamoru; Ohsawa, Kazuma; Murakami, Marohito

    2016-09-01

    Mycobacterium avium complex (MAC) infection is a major medical concern in Japan because of its increased prevalence and associated mortality. A common radiological feature in pulmonary MAC infection is a mixture of two basic patterns: fibrocavitary and nodular bronchiectatic; however, lobar consolidation is rare. We report an 83-year-old man with lobar caseous pneumonia caused by pulmonary MAC infection. Radiological findings were predominantly composed of dense lobar consolidation and ground-glass opacity. A diagnosis was made in accordance with the clinical and microbiological criteria set by the American Thoracic Society. A histological examination of lung specimens obtained by using a bronchoscope revealed a caseous granulomatous inflammation with an appearance of Langhans cells. The patient was treated using combined mycobacterium chemotherapy with an initial positive response for 6 months; however, the disease progressed later. We suggest that an awareness of lobar pneumonic consolidation as a rare radiological finding in pulmonary MAC infection is important. PMID:27516892

  13. Emphysematous pyometra secondary to Enterococcus avium infection in a dog.

    PubMed

    Chang, An-Chi; Cheng, Ching-Chang; Wang, Hsien-Chi; Lee, Wei-Ming; Shyu, Ching-Lin; Lin, Cheng-Chung; Chen, Kuan-Sheng

    2016-06-16

    A 5-year-old female intact Mastiff dog was presented with a history of vaginal discharge for 1 day. Physical examination revealed a sanguineo-purulent vaginal discharge and systemic inflammatory response syndrome. Abdominal radiographs showed several dilated and gas- filled tubular loops. The differential diagnoses included emphysematous pyometra or small intestinal mechanical ileus. Surgical exploration of the abdomen demonstrated a severely dilated and gas-filled uterus, and emphysematous pyometra was confirmed. The patient's clinical signs resolved after ovariohysterectomy. Histopathology revealed mild endometrial cystic hyperplasia with infiltration of inflammatory cells in the superficial endometrial epithelia. Enterococcus avium, an α-hemolytic gram-positive coccus, was isolated from the uterus. This paper highlights the radiographic features of emphysematous pyometra and a pathogen that has never been reported to be associated with canine pyometra previously. PMID:27111397

  14. Binding of Mycobacterium avium-Mycobacterium intracellulare to human leukocytes.

    PubMed Central

    Catanzaro, A; Wright, S D

    1990-01-01

    We examined nonopsonic binding of Mycobacterium avium-Mycobacterium intracellulare (MAI) by human leukocytes. Macrophages (M phi) avidly bound fluorescently labeled MAI in the absence of serum proteins. Binding appeared to be mediated by a lineage-specific, proteinaceous receptor on M phi, since (i) binding of labeled bacteria could be competitively inhibited by unlabeled MAI, (ii) treatment of M phi with trypsin ablated the ability of M phi to bind MAI, and (iii) the capacity to bind MAI was observed on monocytes, M phi, and stimulated polymorphonuclear cells but not on lymphocytes or unstimulated polymorphonuclear cells. The receptor for MAI appeared mobile in the plane of the membrane, since spreading of M phi on a carpet of immobilized, unlabeled MAI down modulated binding of labeled MAI added in suspension. The receptor required neither calcium nor magnesium for activity and appeared different from other known receptors for intracellular pathogens. Images PMID:2387629

  15. Mycobacterium avium MAV_2941 mimics Phosphoinositol-3-Kinase to interfere with macrophage phagosome maturation

    PubMed Central

    Danelishvili, Lia; Bermudez, Luiz E.

    2015-01-01

    Mycobacterium avium subsp hominissuis (M. avium) is a pathogen that infects and survives in macrophages. Previously, we have identified the M. avium MAV_2941 gene encoding a 73 amino acid protein exported by the oligopeptide transporter OppA to the macrophage cytoplasm. Mutations in MAV_2941 were associated with significant impairment of M. avium growth in THP-1 macrophages. In this study, we investigated the molecular mechanism of MAV_2941 action and demonstrated that MAV_2941 interacts with the vesicle trafficking proteins syntaxin-8 (STX8), adaptor-related protein complex 3 (AP-3) complex subunit beta-1 (AP3B1) and Archain 1 (ARCN1) in mononuclear phagocytic cells. Sequencing analysis revealed that the binding site of MAV_2941 is structurally homologous to the human phosphatidylinositol 3-kinase (PI3K) chiefly in the region recognized by vesicle trafficking proteins. The β3A subunit of AP-3, encoded by AP3B1, is essential for trafficking cargo proteins, including lysosomal-associated membrane protein 1 (LAMP-1), to the phagosome and lysosome-related organelles. Here, we show that while the heat-killed M. avium when ingested by macrophages co-localizes with LAMP-1 protein, transfection of MAV_2941 in macrophages results in significant decrease of LAMP-1 co-localization with the heat-killed M. avium phagosomes. Mutated MAV_2941, where the amino acids homologous to the binding region of PI3K were changed, failed to interact with trafficking proteins. Inactivation of the AP3B1 gene led to alteration in the trafficking of LAMP-1. These results suggest that M. avium MAV_2941 interferes with the protein trafficking within macrophages altering the maturation of phagosome. PMID:26043821

  16. ISOLATION OF THE GENOME SEQUENCE STRAIN MYCOBACTERIUM AVIUM 104 FROM MULTIPLE PATIENTS OVER A 17-YEAR PERIOD

    EPA Science Inventory

    The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated form an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genoty...

  17. Assessing the effectiveness of low-pressure ultraviolet light for inactivating Mycobacterium avium complex (MAC) micro-organisms

    EPA Science Inventory

    Aims: To assess low-pressure ultraviolet light (LP-UV) inactivation kinetics of Mycobacterium avium complex (MAC) strains in a water matrix using collimated beam apparatus. Methods and Results: Strains of M. avium (n = 3) and Mycobacterium intracellulare (n = 2) were exposed t...

  18. Characterization to species level of Mycobacterium avium complex strains from human immunodeficiency virus-positive and -negative patients.

    PubMed Central

    Kyriakopoulos, A M; Tassios, P T; Matsiota-Bernard, P; Marinis, E; Tsaousidou, S; Legakis, N J

    1997-01-01

    Forty human clinical Mycobacterium avium-M. intracellulare complex strains isolated in Greece were characterized to the species level by PCR with three sets of primers specific for one or both species. M. avium predominated in both human immunodeficiency virus-positive and -negative patients, but the frequency of M. intracellulare isolation appeared to be higher in the latter. PMID:9350780

  19. Concomitant Mycobacterium avium infection and Hodgkin's disease in a lymph node from an HIV-negative child.

    PubMed

    de Armas, Yaxsier; Capó, Virginia; González, Ida; Mederos, Lilian; Díaz, Raúl; de Waard, Jacobus H; Rodríguez, Alberto; García, Yarmila; Cabanas, Ricardo

    2011-03-01

    We report a case of an immunocompetent child with simultaneously an infection with Mycobacterium avium and Hodgkin's disease in a cervical lymph node. A positive PCR result for M. avium on a biopsy of the lymph node directed the definitive diagnosis for both etiologies and avoided a possible dissemination of this infection after chemotherapy was started.

  20. The persistence of Mycobacterium avium in a drinking water system, what is the risk to human health?

    EPA Science Inventory

    Drinking water is believed to be a major source of human exposure to nontuberculous mycobacteria (NTM) such as Mycobacterium avium. We monitored the prevalence of M. avium in a drinking water system during the addition of filtration treatment. Our goal was to determine if the pre...

  1. Survival of Mycobacterium avium in drinking water biofilms as affected by water flow velocity, availability of phosphorus, and temperature.

    PubMed

    Torvinen, Eila; Lehtola, Markku J; Martikainen, Pertti J; Miettinen, Ilkka T

    2007-10-01

    Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.

  2. The mechanism of cytoskeleton protein β-actin and cofilin-1 of macrophages infected by Mycobacterium avium

    PubMed Central

    Wang, Jianjun; Yao, Yongliang; Wu, Jianhong; Deng, Zhiyong; Gu, Tao; Tang, Xin; Cheng, Yang; Li, Guangxin

    2016-01-01

    Cytoskeleton proteins and their regulation proteins could be influenced seriously in Mycobacterium tuberculosis infection host cells leading to the apoptosis of host cells. Macrophages infected by Mycobacterium avium were detected from cell morphology and genome levels to analyze changes of the cytoskeleton of M. avium infection macrophages. Then the expression of β-actin, cofilin-1 proteins in M. avium infected macrophages were analyzed by western blotting, and the apoptosis of M. avium infection macrophages were tested by flow cytometry. Results indicated that the morphology and genomic DNA of M. avium infection macrophages were not damaged significantly. Meanwhile, β-actin gene and its proteins in M. avium infection macrophages were both decreased, but its regulatory protein cofilin-1 was expressed conversely. Furthermore, macrophages could be induced to apoptosis due to M. avium infection by cytoskeleton changes. These findings contributed us to understand that macrophages infected by M. avium could be lead to apoptosis by regulating cytoskeleton protein β-actin or its regulatory protein cofilin-1. PMID:27158391

  3. Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria.

    PubMed

    Schorey, J S; Holsti, M A; Ratliff, T L; Allen, P M; Brown, E J

    1996-07-01

    Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Mycobacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette-Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.

  4. Estimation of Mycobacterium avium subsp. paratuberculosis Growth Parameters: Strain Characterization and Comparison of Methods▿

    PubMed Central

    Elguezabal, Natalia; Bastida, Felix; Sevilla, Iker A.; González, Nuria; Molina, Elena; Garrido, Joseba M.; Juste, Ramón A.

    2011-01-01

    The growth rate of Mycobacterium avium subsp. paratuberculosis was assessed by different methods in 7H9 medium supplemented with OADC (oleic acid, albumin, dextrose, catalase), Tween 80, and mycobactin J. Generation times and maximum specific growth rates were determined by wet weight, turbidometric measurement, viable count, and quantitative PCR (ParaTB-Kuanti; F57 gene) for 8 M. avium subsp. paratuberculosis strains (K10, 2E, 316F, 81, 445, 764, 22G, and OVICAP 49). Strain-to-strain differences were observed in growth curves and calculated parameters. The quantification methods gave different results for each strain at specific time points. Generation times ranged from an average of 1.4 days for viable count and qPCR to approximately 10 days for wet weight and turbidometry. The wet-weight, turbidometry, and ParaTB-Kuanti qPCR methods correlated best with each other. Generally, viability has been assessed by viable count as a reference method; however, due to M. avium subsp. paratuberculosis clumping problems and the presence of noncultivable M. avium subsp. paratuberculosis cells, we conclude that qPCR of a single-copy gene may be used reliably for rapid estimation of M. avium subsp. paratuberculosis bacterial numbers in a sample. PMID:22003015

  5. Genetic Analysis of Mycobacterium avium Complex Strains Used for Producing Purified Protein Derivatives

    PubMed Central

    Semret, Makeda; Bakker, Douwe; Smart, Nonie; Olsen, Ingrid; Haslov, Kaare; Behr, Marcel A.

    2006-01-01

    For over a century, purified protein derivatives (PPD) have been used to detect mycobacterial infections in humans and livestock. Among these, reagents to detect infections by Mycobacterium avium complex organisms have been produced, but the utility of these reagents has not been clearly established due in part to limited biologic and immunologic standardization. Because there is little information about the strains used to produce these reagents (avian PPD, intracellulare PPD, scrofulaceum PPD, and Johnin), we have performed genetic characterizations of strains used to produce these products. Sequence analysis of 16S rRNA and the hsp65 gene provided results concordant with species designations provided for M. avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum organisms. For M. avium strains, comparative genomic hybridization was performed on a whole-genome DNA microarray, revealing one novel 7.9-kilobase genomic deletion in certain Johnin-producing strains, in addition to genomic variability inherent to the particular M. avium subspecies. Our findings indicate that considerable genomic differences exist between organisms used for reagents and the infecting organism being studied. These results serve as a baseline for potency studies of different preparations and should aid in comparative studies of newly discovered antigens for the diagnosis of infection and disease by M. avium complex organisms. PMID:16960109

  6. Adhesion of Campylobacter jejuni and Mycobacterium avium onto polyethylene terephtalate (PET) used for bottled waters.

    PubMed

    Tatchou-Nyamsi-König, Josiane-Aurore; Dague, Etienne; Mullet, Martine; Duval, Jérôme F L; Gaboriaud, Fabien; Block, Jean-Claude

    2008-12-01

    Adhesion of the bacteria Campylobacter jejuni and Mycobacterium avium onto polyethylene terephtalate (PET), a polymer widely used within the bottled water industry was measured in two different groundwater solutions. From this, it was found that whilst the percentage cell adhesion for a given strain did not change between groundwater types, substantial variation was obtained between the two bacterial species tested: M. avium (10-30% adhered cells) and C. jejuni (1-2%) and no major variations were measured as a function of groundwater composition for a given strain. To explain this, the interfacial electro-hydrodynamic properties of the bacteria were investigated by microelectrophoresis, with the resultant data analysed on the basis of electrokinetic theory for soft biocolloidal particles. The results obtained showed that M. avium carries a significant volume charge density and that its peripheral layer exhibits limited hydrodynamic flow permeation compared to that of C. jejuni. It was also demonstrated that steric hindrance to flow penetration and the degree of hydrophobicity within/of the outer bacterial interface are larger for M. avium cells. In line with this, the larger amount of M. avium cells deposited onto PET substrates as compared to that of C. jejuni can be explained by hydrophobic attraction and chemical binding between hydrophobic PET and outer soft surface layer of the bacteria. Hydrophobicity of PET was addressed by combining contact angle analyses and force spectroscopy using CH(3)-terminated AFM tip. PMID:18929388

  7. Investigation of Spa Pools Associated with Lung Disorders Caused by Mycobacterium avium Complex in Immunocompetent Adults

    PubMed Central

    Lumb, Richard; Stapledon, Richard; Scroop, Andrew; Bond, Peter; Cunliffe, David; Goodwin, Allan; Doyle, Robyn; Bastian, Ivan

    2004-01-01

    Three cases of Mycobacterium avium complex-related lung disorders were associated with two poorly maintained spa pools by genotypic investigations. Inadequate disinfection of the two spas had reduced the load of environmental bacteria to less than 1 CFU/ml but allowed levels of M. avium complex of 4.3 × 104 and 4.5 × 103 CFU/ml. Persistence of the disease-associated genotype was demonstrated in one spa pool for over 5 months until repeated treatments with greater than 10 mg of chlorine per liter for 1-h intervals eliminated M. avium complex from the spa pool. A fourth case of Mycobacterium avium complex-related lung disease was associated epidemiologically but not genotypically with another spa pool that had had no maintenance undertaken. This spa pool contained low numbers of mycobacteria by smear and was culture positive for M. avium complex, and the nonmycobacterial organism count was 5.2 × 106 CFU/ml. Public awareness about the proper maintenance of private (residential) spa pools must be promoted by health departments in partnership with spa pool retailers. PMID:15294830

  8. Fate of Mycobacterium avium subsp. paratuberculosis in Swiss hard and semihard cheese manufactured from raw milk.

    PubMed

    Spahr, U; Schafroth, K

    2001-09-01

    Raw milk was artificially contaminated with declumped cells of Mycobacterium avium subsp. paratuberculosis at a concentration of 10(4) to 10(5) CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains of M. avium subsp. paratuberculosis were tested, and for each strain, two model hard and semihard cheeses were produced. The survival of M. avium subsp. paratuberculosis cells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese. D values were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death of M. avium subsp. paratuberculosis in cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, the D values found indicate that 10(3) to 10(4) cells of M. avium subsp. paratuberculosis per g will be inactivated.

  9. Fate of Mycobacterium avium subsp. paratuberculosis in Swiss Hard and Semihard Cheese Manufactured from Raw Milk

    PubMed Central

    Spahr, U.; Schafroth, K.

    2001-01-01

    Raw milk was artificially contaminated with declumped cells of Mycobacterium avium subsp. paratuberculosis at a concentration of 104 to 105 CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains of M. avium subsp. paratuberculosis were tested, and for each strain, two model hard and semihard cheeses were produced. The survival of M. avium subsp. paratuberculosis cells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese. D values were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death of M. avium subsp. paratuberculosis in cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, the D values found indicate that 103 to 104 cells of M. avium subsp. paratuberculosis per g will be inactivated. PMID:11526024

  10. Disseminated mycobacteriosis due to Mycobacterium avium in captive Bengal tiger (Panthera tigris).

    PubMed

    Cho, Ho-Seong; Kim, Yong-Hwan; Park, Nam-Yong

    2006-05-01

    A 2-year-old captive female Bengal Tiger (Panthera tigris) died after prolonged anorexia in the Gwangju Uchi Park Zoo, Gwangju, Republic of Korea. Necropsy revealed multiple nodules of varying sizes in the lung, liver, kidney, and spleen. Histopathologic examination revealed a typical granuloma composed of caseous necrotic areas surrounded by lymphocytes with a few giant cells and foamy macrophages. Periodic acid-Schiff stain and Gomori methenamine silver stain did not reveal any fungal bodies. The Ziehl-Neelsen acid-fast stain revealed few acid-fast organisms in the lung, liver, kidney, and spleen. A polymerase chain reaction assay of the lung, liver, kidney, and spleen yielded a positive result for Mycobacterium avium subsp. avium. This is an unusual case of disseminated infection of a wild mammal with avian mycobacteriosis, and is believed to be most likely associated with the feeding of tigers with culled chickens infected with M. avium.

  11. Disseminated mycobacteriosis due to Mycobacterium avium in captive Bengal tiger (Panthera tigris).

    PubMed

    Cho, Ho-Seong; Kim, Yong-Hwan; Park, Nam-Yong

    2006-05-01

    A 2-year-old captive female Bengal Tiger (Panthera tigris) died after prolonged anorexia in the Gwangju Uchi Park Zoo, Gwangju, Republic of Korea. Necropsy revealed multiple nodules of varying sizes in the lung, liver, kidney, and spleen. Histopathologic examination revealed a typical granuloma composed of caseous necrotic areas surrounded by lymphocytes with a few giant cells and foamy macrophages. Periodic acid-Schiff stain and Gomori methenamine silver stain did not reveal any fungal bodies. The Ziehl-Neelsen acid-fast stain revealed few acid-fast organisms in the lung, liver, kidney, and spleen. A polymerase chain reaction assay of the lung, liver, kidney, and spleen yielded a positive result for Mycobacterium avium subsp. avium. This is an unusual case of disseminated infection of a wild mammal with avian mycobacteriosis, and is believed to be most likely associated with the feeding of tigers with culled chickens infected with M. avium. PMID:16789727

  12. Description of a Novel Adhesin of Mycobacterium avium Subsp. paratuberculosis

    PubMed Central

    Viale, Mariana Noelia; Echeverria-Valencia, Gabriela; Romasanta, Pablo; Mon, María Laura; Fernandez, Marisa; Malchiodi, Emilio; Romano, María Isabel; Gioffré, Andrea Karina; Santangelo, María de la Paz

    2014-01-01

    The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding. PMID:25136616

  13. Development of vaccines to Mycobacterium avium subsp. paratuberculosis infection

    PubMed Central

    2016-01-01

    Johne's disease or paratuberculosis is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. There are no effective control measures to eradicate the disease because there are no appropriate diagnostic methods to detect subclinically infected animals. Therefore, it is very difficult to control the disease using only test and cull strategies. Vaccination against paratuberculosis has been considered as an alternative strategy to control the disease when combined with management interventions. Understanding host-pathogen interactions is extremely important to development of vaccines. It has long been known that Th1-mediated cellular immune responses are play a crucial role in protection against MAP infection. However, recent studies suggested that innate immune responses are more closely related to protective effects than adaptive immunity. Based on this understanding, several attempts have been made to develop vaccines against paratuberculosis. A variety of ideas for designing novel vaccines have emerged, and the tests of the efficacy of these vaccines are conducted constantly. However, no effective vaccines are commercially available. In this study, studies of the development of vaccines for MAP were reviewed and summarized. PMID:27489800

  14. Description of a novel adhesin of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Viale, Mariana Noelia; Echeverria-Valencia, Gabriela; Romasanta, Pablo; Mon, María Laura; Fernandez, Marisa; Malchiodi, Emilio; Romano, María Isabel; Gioffré, Andrea Karina; Santangelo, María de la Paz

    2014-01-01

    The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding. PMID:25136616

  15. Development of vaccines to Mycobacterium avium subsp. paratuberculosis infection.

    PubMed

    Park, Hong-Tae; Yoo, Han Sang

    2016-07-01

    Johne's disease or paratuberculosis is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. There are no effective control measures to eradicate the disease because there are no appropriate diagnostic methods to detect subclinically infected animals. Therefore, it is very difficult to control the disease using only test and cull strategies. Vaccination against paratuberculosis has been considered as an alternative strategy to control the disease when combined with management interventions. Understanding host-pathogen interactions is extremely important to development of vaccines. It has long been known that Th1-mediated cellular immune responses are play a crucial role in protection against MAP infection. However, recent studies suggested that innate immune responses are more closely related to protective effects than adaptive immunity. Based on this understanding, several attempts have been made to develop vaccines against paratuberculosis. A variety of ideas for designing novel vaccines have emerged, and the tests of the efficacy of these vaccines are conducted constantly. However, no effective vaccines are commercially available. In this study, studies of the development of vaccines for MAP were reviewed and summarized. PMID:27489800

  16. Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis.

    PubMed

    Manning, E J; Collins, M T

    2001-04-01

    Johne's disease, or paratuberculosis, is a chronic intestinal infection caused by Mycobacterium avium subsp. paratuberculosis. The usually fatal disease is characterised by cachexia, and in some species diarrhoea, after a long pre-clinical phase. Treatment is ineffective and economically impracticable. The infection primarily affects domestic and free-ranging ruminants, but has also been reported in primates, rabbits, stoats and foxes. Since paratuberculosis is often subclinical, under-reporting is suspected, even though the disease is notifiable in numerous countries. Herd prevalence of bovine paratuberculosis in Europe ranges from 7% to 55%. In the United States of America, herd prevalence is strongly associated with herd size; 40% of herds of more than 300 head were found to be infected. In Australia, reported dairy herd infection rates range between 9% and 22%. Paratuberculosis in domestic livestock entails significant economic losses due to several factors (e.g. reduced production, premature culling and increased veterinary costs). Free-ranging and captive wildlife are also at risk from paratuberculosis.

  17. Complete Remission of Minimal Change Disease Following an Improvement of Lung Mycobacterium avium Infection.

    PubMed

    Yamashiro, Aoi; Uchida, Takahiro; Ito, Seigo; Oshima, Naoki; Oda, Takashi; Kumagai, Hiroo

    2016-01-01

    A 46-year-old woman suddenly developed peripheral edema. Her massive proteinuria, hypoproteinemia, and renal biopsy findings yielded the diagnosis of minimal change disease (MCD). In addition, lung Mycobacterium avium infection was diagnosed according to a positive culture of her bronchoalveolar lavage fluid. The lung lesion was improved by anti-nontuberculous mycobacteria therapy. Surprisingly, her proteinuria also gradually decreased and she attained complete remission of MCD without any immunosuppressive therapy. She has subsequently remained in complete remission. We herein report an interesting case of MCD with lung Mycobacterium avium infection, suggesting a causal relationship among infection, immune system abnormality, and MCD/nephrotic syndrome.

  18. Complete Remission of Minimal Change Disease Following an Improvement of Lung Mycobacterium avium Infection.

    PubMed

    Yamashiro, Aoi; Uchida, Takahiro; Ito, Seigo; Oshima, Naoki; Oda, Takashi; Kumagai, Hiroo

    2016-01-01

    A 46-year-old woman suddenly developed peripheral edema. Her massive proteinuria, hypoproteinemia, and renal biopsy findings yielded the diagnosis of minimal change disease (MCD). In addition, lung Mycobacterium avium infection was diagnosed according to a positive culture of her bronchoalveolar lavage fluid. The lung lesion was improved by anti-nontuberculous mycobacteria therapy. Surprisingly, her proteinuria also gradually decreased and she attained complete remission of MCD without any immunosuppressive therapy. She has subsequently remained in complete remission. We herein report an interesting case of MCD with lung Mycobacterium avium infection, suggesting a causal relationship among infection, immune system abnormality, and MCD/nephrotic syndrome. PMID:27629965

  19. Tuberculosis in swine co-infected with Mycobacterium avium subsp. hominissuis and Mycobacterium bovis in a cluster from Argentina.

    PubMed

    Barandiaran, S; Pérez, A M; Gioffré, A K; Martínez Vivot, M; Cataldi, A A; Zumárraga, M J

    2015-04-01

    SUMMARY In Argentina little is known about the epidemiology of tuberculosis (TB) infection in swine. We characterized the epidemiological dynamics of Mycobacterium avium complex (MAC) infection in a swine population of Argentina using molecular tools and spatial analysis techniques. Isolates (n = 196) obtained from TB-like lesions (n = 200) were characterized by polymerase chain reaction. The isolates were positive to either M. bovis (IS6110) (n = 160) or M. avium (IS1245) (n = 16) while the remaining 20 (10.2%) isolates were positive to both M. bovis and M. avium. The detection of both bacteria together suggests co-infection at the animal level. In addition, MAC-positive isolates (n = 36) were classified as M. avium subsp. avium (MAA) (n = 30) and M. avium subsp. hominissuis (MAH) (n = 6), which resulted in five genotypes when they were typed using mycobacterial interspersed repetitive unit, variable number of tandem repeats (MIRU-VNTR). One significant (P = 0.017) spatial clustering of genotypes was detected, in which the proportion of MAH isolates was larger than expected under the null hypothesis of even distribution of genotypes. These results show that in Argentina the proportion of TB cases in pigs caused by M. avium is larger than that reported in earlier studies. The proportion of M. bovis-MAC co-infections was also higher than in previous reports. These results provide valuable information on the epidemiology of MAC infection in swine in Argentina.

  20. PD-L2 induction on dendritic cells exposed to Mycobacterium avium downregulates BCG-specific T cell response.

    PubMed

    Mendoza-Coronel, Elizabeth; Camacho-Sandoval, Rosa; Bonifaz, Laura C; López-Vidal, Yolanda

    2011-01-01

    The exposure to certain species of Nontuberculous Mycobacteria (NTM) can modulate the immune response induced by Mycobacterium bovis BCG. Mycobacterium avium has been postulated as a weak inducer of dendritic cell (DC) maturation. However, how the DC exposure to M. avium could contribute to the modulation of a BCG-specific CD4+ T cell response and the molecules involved remain unknown. Here, we exposed bone marrow-derived DCs (BMDCs) to M. avium either prior to exposure to BCG or as a unique stimulus. We found that M. avium induces high expression of PD-L2 (B7-DC) in BMDCs. This was dependent on IL-10 production through the TLR2-p38 MAPK signaling pathway. Exposure to M. avium prior to BCG results in BMDCs that do not express co-stimulatory molecules and pro-inflammatory cytokines, while the expression of PD-L2 and IL-10 was maintained. BMDCs exposed to M. avium impaired the activation of BCG-specific T cells through the PD-1: PD-L interaction. This suggests that a M. avium-induced phenotype in DCs might be implicated in the induction of mechanisms of tolerance that could impact the T cell response induced by BCG vaccination.

  1. Association of ISMav6 with the Pattern of Antibiotic Resistance in Korean Mycobacterium avium Clinical Isolates but No Relevance between Their Genotypes and Clinical Features

    PubMed Central

    Kim, Su-Young; Jeong, Byeong-Ho; Park, Hye Yun; Jeon, Kyeongman; Han, Seung Jung; Shin, Sung Jae; Koh, Won-Jung

    2016-01-01

    The aim of this study was to genetically characterize clinical isolates from patients diagnosed with Mycobacterium avium lung disease and to investigate the clinical significance. Multi-locus sequencing analysis (MLSA) and pattern of insertion sequence analysis of M. avium isolates from 92 Korean patients revealed that all isolates were M. avium subspecies hominissuis. In hsp65 sequevar analysis, codes 2, 15, and 16 were most frequently found (88/92) with similar proportions among cases additionally two isolates belonging to code N2 and an unreported code were identified, respectively. In insertion element analysis, all isolates were IS1311 positive and IS900 negative. Four of the M. avium subsp. hominissuis isolates did not harbor IS1245 and 1 of the M. avium isolates intriguingly harbored DT1, which is thought to be a M. intracellulare-specific element. M. avium subsp. hominissuis harboring ISMav6 is prevalent in Korea. No significant association between clinical manifestation and treatment response has been found in patients with the hsp65 code type and ISMav6, indicating that no specific strain/genotype among M. avium subsp. hominissuis organisms was a major source of M. avium lung disease. Interestingly, the presence of ISMav6 was correlated with greater resistance to moxifloxacin. Conclusively, the genotype of Korean M. avium subsp. hominissuis isolates is not a disease determinant responsible for lung disease and specific virulent factors of M. avium subsp. hominissuis need to be investigated further. PMID:26859598

  2. Opinions Differ by Expertise in Mycobacterium avium Complex Disease

    PubMed Central

    Prevots, D. Rebecca; Jamieson, Frances B.; Winthrop, Kevin L.

    2014-01-01

    Rationale: Pulmonary Mycobacterium avium complex treatment guidelines rely largely on expert opinion. The extent to which nonexperts agree with recommendations of experts in this clinical area is unknown. Objectives: We sought to compare practices and perceptions of prognosis between experts and nonexperts. Methods: We surveyed respirologists (Ontario, Canada, “nonexperts”) and experts from nontuberculous mycobacterial disease centers of excellence (Canada and United States). Measurements and Main Results: Forty-six Ontario respirologists (29% of 160) and 19 experts (73% of 26) participated. There was agreement between nonexperts and experts regarding disease duration before diagnosis (2 yr), likelihood of spontaneous remission (7–15%), typical duration of treatment (18 mo), first choice of therapy (guideline regimens), a subgroup of patients for whom less-intensive regimens are favored (10% after recurrence), likelihood of recurrence (30%), and median survival (10 yr in most patients). Noted differences were that nonexperts estimated fewer patients with a positive culture had disease (30% vs. 50%, P = 0.02), used intensive guidelines therapy less often in new cases (50% vs. 79%, P = 0.02), and perceived a slightly lower success rate with guidelines therapy (65% vs. 75%, P = 0.047). Response ranges were wider for nonexperts, significantly so for selection of intensive guidelines therapy in new (P = 0.01) and recurrent (P = 0.04) cases. Conclusion: Experts and nonexperts agreed on many issues. However, nonexperts perceived lower rates of disease among patients with isolates, tended to use less aggressive treatment approaches, and perceived lower success rates. Significant variability was observed in responses—often wider among nonexperts. Although these results are likely biased by referral, they may identify important areas for targeted education. PMID:24083335

  3. Thioridazine as Chemotherapy for Mycobacterium avium Complex Diseases

    PubMed Central

    Deshpande, Devyani; Srivastava, Shashikant; Musuka, Sandirai

    2016-01-01

    Mycobacterium avium-intracellulare complex (MAC) causes an intractable intracellular infection that presents as chronic pulmonary disease. Currently, therapy consists of ethambutol and macrolides and takes several years to complete. The neuroleptic phenothiazine thioridazine kills mycobacteria by inhibiting the electron transport chain. In several experiments with bacterial populations of up to 1012 CFU/ml, we failed to isolate any bacteria resistant to 3 times the MIC of thioridazine, suggesting the absence of resistant mutants at bacterial burdens severalfold higher than those encountered in patients. In the hollow-fiber model of intracellular MAC (HFS-MAC), thioridazine achieved an extracellular half-life of 16.8 h and an intracellular half-life of 19.7 h. Thioridazine concentrations were >28,000-fold higher inside infected macrophages than in the HFS-MAC central compartment (equivalent to plasma). Thioridazine maximal kill was 5.20 ± 0.75 log10 CFU/ml on day 7 (r2 = 0.96) and 7.19 ± 0.31 log10 CFU/ml on day 14 (r2 = 0.99), the highest seen with any drug in the system. Dose fractionation studies revealed that thioridazine efficacy and acquired drug resistance were driven by the peak concentation-to-MIC ratio, with a 50% effective concentration (EC50) of 2.78 ± 0.44 for microbial killing. Acquired drug resistance was encountered by day 21 with suboptimal doses, demonstrating that fluctuating drug concentrations drive evolution faster than static concentrations in mutation frequency studies. However, the thioridazine EC50 changed 16.14-fold when the concentration of fetal bovine serum was changed from 0% to 50%, suggesting that intracellular potency could be heavily curtailed by protein binding. Efficacy in patients will depend on the balance between trapping of the drug in the pulmonary system and the massive intracellular concentrations versus very high protein binding of thioridazine. PMID:27216055

  4. Transcriptional profiling of ileocecal valve of holstein dairy cows infected with mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advan...

  5. Detection of Mycobacterium avium subsp. paratuberculosis in Drinking Water and Biofilms Using Quantitative PCR

    EPA Science Inventory

    Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease in domestic animals and has been implicated in Crohn’s disease in humans. Cows infected with Johne’s disease shed large quantities of MAP into soil. Further, MAP has been isolated from surface water, is resi...

  6. Unraveling the Host Response to Mycobacterium avium subsp. paratuberculosis: One Thread at a Time

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study of host immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) is complicated by a number of factors, including the protracted nature of the disease and the stealthy nature of the pathogen. Improved tools for the measurement of immunologic responses in ruminant species, par...

  7. THE EFFECT OF TEMPERATURE ON THE GROWTH OF MYCOBACTERIUM AVIUM COMPLEX (MAC) ORGANISMS

    EPA Science Inventory

    MAC organisms are able to grow, persist, and colonize in water distribution systems and may amplify in hospital hot water systems. This study examined the response of MAC organisms (M. avium, M. intracellulare, and MX) to a range of temperatures commonly associated with drinking...

  8. Detection of Mycobacterium avium subspecies paratuberculosis in Drinking Water and Biofilms Using Quantitative PCR

    EPA Science Inventory

    Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in domestic animals and has been implicated in Crohn’s disease in humans. This bacterium is a slow growing, gram-positive, acid-fast organism which can be difficult to culture from the environment. For ...

  9. Lymphoproliferative and gamma interferon responses to stress-regulated Mycobacterium avium subsp. paratuberculosis recombinant proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease in ruminants is a chronic infection of the intestines caused by Mycobacterium avium subsp. paratuberculosis. Economic losses associated with Johne’s disease arise due to premature culling, reduced production of milk and wool and mortalities. The disease is characterised by a long inc...

  10. Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offer insights into host association

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systemat...

  11. Immunlogic responses to Mycobacterium avium subsp. paratuberculosis protein cocktail vaccines in a mouse model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease is a chronic granulomatous enteritis characterized by severe diarrhea, wasting, and a decline in milk production caused by the bacterium Mycobacterium avium subsp. paratuberculosis (MAP). The vaccine currently on the market has some limitations including a severe injection site react...

  12. Synthesis and release of sulfolipid by Mycobacterium avium during growth andcell division.

    PubMed Central

    McCarthy, C

    1976-01-01

    Mycobacterium avium exhibits a life cycle wherein small cells elongate to form filaments. The life cycle is unique in that elongated cells will undergo rapid division by fragmentation only if fatty acid is present. The utilization of [14C]palmitic acid and [3H]oleic acid by M. avium during the life cycle was assessed. Four glycolipids, identifiable by elution patterns from hydroxylapatite columns, were associated with postfission cells and contained isotope from the precursor fatty acid. The incorporation of 3H from oleic acid into the cellular glycolipids was maximal during cell division, but as much as 73% of the radioactivity was lost to the lipids from cells in the postfission status. Three of the glycolipids were sulfatides into which 36S was incorporated by M. avium. The [35]sulfatides were synthesized by cells undergoing fragmentation and were recovered from the medium at the termination of cell fission. These results demonstrated that the isotope was not lost to the cells because of turnover, but rather that the labeled compounds were released, intact, from the cells after fission. Because of the facile release of the sulfolipids, it was suggested that they were part of the cell envelope of M. avium cells during the division process. Images PMID:977128

  13. From mouth to macrophage: mechanisms of innate immune subversion by Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts...

  14. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  15. Shared Mycobacterium avium genotypes observed among unlinked clinical and environmental isolates*

    EPA Science Inventory

    Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this a...

  16. COMPARISON OF MYCOBACTERIUM AVIUM ISOLATES FROM DRINKING WATER AND FROM THE POPULATION SERVED BY THE SYSTEM

    EPA Science Inventory

    Background: Current evidence suggests that drinking water, soil, and produce are potential sources of Mycobacterium avium infections, a pathogen not known to be transmitted person-to-person.

    Methods: We sampled water during 2000 - 2002 from a large municipal drinking wate...

  17. Optimization of hexadecylpyridinium chloride decontamination for culture of Mycobacterium avium subsp. paratuberculosis from milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cows in advanced stages of Johne’s disease shed Mycobacterium avium subsp. paratuberculosis (MAP) into both their milk and feces, allowing for transmission of the bacteria between animals. The objective of this study was to formulate an optimized protocol for the isolation of MAP from milk and colos...

  18. Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the first step of a comprehensive large-scale antigen discovery project, 651 Mycobacterium avium subspecies paratuberculosis proteins were produced in Escherichia coli. All of these were purified by affinity chromatography, dialyzed in phosphate buffered saline, and analyzed on SDS-PAGE gels. C...

  19. Shared Mycobacterium avium genotypes observed among unlinked clinical and environmental isolates

    EPA Science Inventory

    Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this, ...

  20. Environmental contamination with Mycobacterium avium subsp. paratuberculosis in endemically infected dairy herds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be the primary source of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-se...

  1. Mycobacterium avium subspecies paratuberculosis recombinant proteins modulate antimycobacterial functions of bovine macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinan...

  2. Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Goldstone, Robert J.; McLuckie, Joyce; Smith, David G. E.

    2015-01-01

    Typing of Mycobacterium avium subspecies paratuberculosis strains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole-genome sequencing have extended possibilities for the characterization of Mycobacterium avium subspecies paratuberculosis, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133 Mycobacterium avium subspecies paratuberculosis isolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPs Mycobacterium avium subspecies paratuberculosis isolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit–variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing of Mycobacterium avium subspecies paratuberculosis isolates. PMID:26677250

  3. Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Leão, Célia; Goldstone, Robert J; Bryant, Josephine; McLuckie, Joyce; Inácio, João; Smith, David G E; Stevenson, Karen

    2016-03-01

    Typing of Mycobacterium avium subspecies paratuberculosis strains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole-genome sequencing have extended possibilities for the characterization of Mycobacterium avium subspecies paratuberculosis, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133 Mycobacterium avium subspecies paratuberculosis isolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPs Mycobacterium avium subspecies paratuberculosis isolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit-variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing of Mycobacterium avium subspecies paratuberculosis isolates.

  4. Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

    PubMed Central

    Begg, Douglas J.; Purdie, Auriol C.; Bannantine, John P.; Whittington, Richard J.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced. PMID:24132604

  5. Extracellular ATP is cytotoxic to mononuclear phagocytes but does not induce killing of intracellular Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Woo, Seng-Ryong; Barletta, Raúl G; Czuprynski, Charles J

    2007-09-01

    Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic granulomatous enteritis in ruminants. ATP has been reported to induce cell death of macrophages and killing of Mycobacterium species in human and murine macrophages. In this study we investigated the short-term effect of ATP on the viability of M. avium subsp. paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. Addition of 5 mM ATP to M. avium subsp. paratuberculosis-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of 2'(3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP), which is a longer-lived ATP homologue and purinergic receptor agonist, significantly increased the uptake of YO-PRO, which is a marker for membrane pore activation by P2X receptors. Addition of Bz-ATP also stimulated lactate dehydrogenase release and caspase-3 activity in infected bovine monocytes. Neither ATP nor Bz-ATP reduced the survival of M. avium subsp. paratuberculosis in bovine mononuclear phagocytes. Likewise, addition of ATP or Bz-ATP was cytotoxic to murine macrophage cell lines (RAW 264.7 and J774A.1 cells) but did not affect the intracellular survival of M. avium subsp. paratuberculosis, nor were the numbers of viable Mycobacterium avium subsp. avium or Mycobacterium bovis BCG cells altered in bovine mononuclear phagocytes or J774A.1 cells following ATP or Bz-ATP treatment. These data suggest that extracellular ATP does not induce the killing of intracellular M. avium subsp. paratuberculosis in bovine mononuclear phagocytes.

  6. Contrasting results of culture-dependent and molecular analyses of Mycobacterium avium subsp. paratuberculosis from wood bison.

    PubMed

    Forde, Taya; De Buck, Jeroen; Elkin, Brett; Kutz, Susan; van der Meer, Frank; Orsel, Karin

    2013-07-01

    Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas or expose animals to pathogens in their new environment. Mycobacterium avium subsp. paratuberculosis, a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the M. avium subsp. paratuberculosis infection status of wood bison herds in Canada and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n = 267) in nine herds using direct PCR for three M. avium subsp. paratuberculosis-specific genetic targets with different copy numbers within the M. avium subsp. paratuberculosis genome. Restriction enzyme analysis (REA) and sequencing of IS1311 were performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support M. avium subsp. paratuberculosis growth from feces and tissues of direct-PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of M. avium subsp. paratuberculosis from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different M. avium subsp. paratuberculosis strain types exist in Canadian wood bison. The presence of different M. avium subsp. paratuberculosis strains among wood bison herds should be considered in the planning of translocations.

  7. Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific.

    PubMed

    Castellanos, Elena; Aranaz, Alicia; de Juan, Lucia; Alvarez, Julio; Rodríguez, Sabrina; Romero, Beatriz; Bezos, Javier; Stevenson, Karen; Mateos, Ana; Domínguez, Lucas

    2009-07-01

    Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

  8. Immune-Enhancing Effects of Taishan Pinus massoniana Pollen Polysaccharides on DNA Vaccine Expressing Bordetella avium ompA

    PubMed Central

    Zhu, Fujie; Liu, Xiao; Sun, Zhenhong; Yu, Cuilian; Liu, Liping; Yang, Shifa; Li, Bing; Wei, Kai; Zhu, Ruiliang

    2016-01-01

    Bordetella avium is the causative agent of bordetellosis, which remains to be the cause of severe losses in the turkey industry. Given the lack of vaccines that can provide good protection, developing a novel vaccine against B. avium infection is crucial. In this study, we constructed a eukaryotic expression plasmid, which expressed the outer membrane protein A (ompA) of B. avium, to prepare a B. avium recombinant ompA-DNA vaccine. Three concentrations (low, middle, and high) of Taishan Pinus massoniana pollen polysaccharides (TPPPS), a known immunomodulator, were used as adjuvants, and their immune conditioning effects on the developed DNA vaccine were examined. The pure ompA-DNA vaccine, Freund’s incomplete adjuvant ompA-DNA vaccine, and the empty plasmid served as the controls. The chickens in each group were separately inoculated with these vaccines three times at 1, 7, and 14 days old. Dynamic changes in antibody production, cytokine secretion, and lymphocyte count were then determined from 7 to 49 days after the first inoculation. Protective rates of the vaccines were also determined after the third inoculation. Results showed that the pure DNA vaccine obviously induced the production of antibodies, the secretion of cytokines, and the increase in CD4+ and CD8+ T lymphocyte counts in peripheral blood, as well as provided a protective rate of 50% to the B. avium-challenged chickens. The chickens inoculated with the TPPPS adjuvant ompA-DNA vaccine and Freund’s adjuvant ompA-DNA vaccine demonstrated higher levels of immune responses than those inoculated with pure ompA-DNA vaccine, whereas only the ompA-DNA vaccine with 200 mg/mL TPPPS completely protected the chickens against B. avium infection. These findings indicate that the B. avium ompA-DNA vaccine combined with TPPPS is a potentially effective B. avium vaccine. PMID:26870023

  9. MIRU-VNTR genotype diversity and indications of homoplasy in M. avium strains isolated from humans and slaughter pigs in Latvia.

    PubMed

    Kalvisa, Adrija; Tsirogiannis, Constantinos; Silamikelis, Ivars; Skenders, Girts; Broka, Lonija; Zirnitis, Agris; Jansone, Inta; Ranka, Renate

    2016-09-01

    Diseases which are caused by non-tuberculous mycobacteria (NTM) are an increasing problem in the developed countries. In Latvia, one of the most clinically important members of NTM is Mycobacterium avium (M. avium), an opportunistic pathogen which has been isolated from several lung disease patients and tissue samples of slaughter pigs. This study was designed to characterize the genetic diversity of the M. avium isolates in Latvia and to compare the distribution of genotypic patterns among humans and pigs. Eleven (Hall and Salipante, 2010) clinical M. avium samples, isolated from patients of Center of Tuberculosis and Lung Diseases (years 2003-2010), and 32 isolates from pig necrotic mesenterial lymph nodes in different regions (years 2003-2007) were analyzed. The majority (42 of 43) of samples were identified as M. avium subsp. hominissuis; one porcine isolate belonged to M. avium subsp. avium. MIRU-VNTR genotyping revealed 13 distinct genotypes, among which nine genotype patterns, including M. avium subsp. avium isolate, were newly identified. IS1245 RFLP fingerprinting of 25 M. avium subsp. hominissuis samples yielded 17 different IS1245 RFLP patterns, allowing an efficient discrimination of isolates. Clusters of identical RFLP profiles were observed within host species, geographical locations and time frame of several years. Additional in silico analysis on simulated MIRU-VNTR genotype population datasets showed that the MIRU-VNTR pattern similarity could partly arise due to probabilistic increase of acquiring homoplasy among subpopulations, thus the similar MIRU-VNTR profiles of M. avium strains even in close geographical proximity should be interpreted with caution. PMID:27178993

  10. Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America.

    PubMed

    Romano, M I; Amadio, A; Bigi, F; Klepp, L; Etchechoury, I; Llana, M Noto; Morsella, C; Paolicchi, F; Pavlik, I; Bartos, M; Leão, S C; Cataldi, A

    2005-10-31

    All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.

  11. Effect of soil slope on the appearance of Mycobacterium avium subsp. paratuberculosis in water running off grassland soil after application of contaminated slurry.

    PubMed

    Salgado, M; Alfaro, M; Salazar, F; Troncoso, E; Mitchell, R M; Ramirez, L; Naguil, A; Zamorano, P; Collins, M T

    2013-06-01

    The study assessed the effect of soil slope on Mycobacterium avium subsp. paratuberculosis transport into rainwater runoff from agricultural soil after application of M. avium subsp. paratuberculosis-contaminated slurry. Under field conditions, 24 plots of undisturbed loamy soil 1 by 2 m(2) were placed on platforms. Twelve plots were used for water runoff: 6 plots at a 3% slope and 6 plots at a 15% slope. Half of the plots of each slope were treated with M. avium subsp. paratuberculosis-contaminated slurry, and half were not treated. Using the same experimental design, 12 plots were established for soil sampling on a monthly basis using the same spiked slurry application and soil slopes. Runoff following natural rainfall was collected and analyzed for M. avium subsp. paratuberculosis, coliforms, and turbidity. M. avium subsp. paratuberculosis was detected in runoff from all plots treated with contaminated slurry and one control plot. A higher slope (15%) increased the likelihood of M. avium subsp. paratuberculosis detection but did not affect the likelihood of finding coliforms. Daily rainfall increased the likelihood that runoff would have coliforms and the coliform concentration, but it decreased the M. avium subsp. paratuberculosis concentration in the runoff. When there was no runoff, rain was associated with increased M. avium subsp. paratuberculosis concentrations. Coliform counts in runoff were related to runoff turbidity. M. avium subsp. paratuberculosis presence/absence, however, was related to turbidity. Study duration decreased bacterial detection and concentration. These findings demonstrate the high likelihood that M. avium subsp. paratuberculosis in slurry spread on pastures will contaminate water runoff, particularly during seasons with high rainfall. M. avium subsp. paratuberculosis contamination of water has potential consequences for both animal and human health.

  12. Disseminated Mycobacterium avium subsp. paratuberculosis infection in two wild Eurasian otters (Lutra lutra L.) from Portugal.

    PubMed

    Matos, Ana Cristina; Figueira, Luis; Martins, Maria Helena; Matos, Manuela; Alvares, Sofia; Pinto, Maria Lurdes; Coelho, Ana Cláudia

    2013-03-01

    Disseminated Mycobacterium avium subsp. paratuberculosis (MAP) infections were found in two Eurasian otters (Lutra lutra, L. 1758) killed by vehicular trauma in February and March 2010 in Castelo Branco, Portugal. At postmortem examination, the organs showed no significant gross alterations; however, microscopically, both animals had diffuse lymphadenitis with macrophage infiltration and deposition of hyaline material in the center of the lymphoid follicles. Acid-fast organisms were isolated from gastrointestinal tissue samples via bacteriologic culture. These organisms were identified as M. avium subsp. paratuberculosis by IS900 polymerase chain reaction (PCR). Additionally, direct IS900 PCR-positive results were obtained for multiple organs of both animals. This is the first report of MAP infection of otters in Portugal.

  13. Polypoid bronchial lesions due to Scedosporium apiospermum in a patient with Mycobacterium avium complex pulmonary disease.

    PubMed

    Murayama, T; Amitani, R; Tsuyuguchi, K; Watanabe, I; Kimoto, T; Suzuki, K; Tanaka, E; Kamei, K; Nishimura, K

    1998-09-01

    A 69 yr old female was hospitalized for further examination of abnormal shadows on chest radiographs. She had a history of tuberculous pleurisy, rheumatoid arthritis and gold-induced interstitial pneumonia. On admission she still suffered from rheumatoid arthritis. A chest computed tomography scan on admission showed clusters of small nodules in subpleural regions of both lungs combined with bronchiectasis. Mycobacterium avium complex was cultured repeatedly from the sputum. Bronchoscopic examination disclosed white-yellow polypoid lesions in the orifice of the left B4 bronchus. Cultures of the brushing specimen of the polypoid lesions and bronchial aspirates from the B4 bronchus yielded smoky-grey mycelial colonies that were later identified as Scedosporium apiospermum. It was concluded that the polypoid bronchial lesions due to Scedosporium apiospermum were formed in the preexisting dilated bronchus caused by Mycobacterium avium complex pulmonary disease. PMID:9762808

  14. Disseminated mycobacteriosis manifesting as paraplegia in two Parma wallabies (Macropus parma) naturally exposed to Mycobacterium avium.

    PubMed

    Robveille, Cynthia; Albaric, Olivier; Gaide, Nicolas; Abadie, Jérome

    2015-11-01

    Two captive female Parma wallabies (Macropus parma) died after a history of flaccid paraplegia. On postmortem examination, granulomatous and suppurative osteomyelitis involving the left ischium and the lumbosacral region, with meningeal extension at the cauda equina, and caseonecrotic mastitis were the most significant changes. Multiple small nodules in the liver and spleen, and an enlargement of some lymph nodes with central caseous necrosis were also observed. Microscopically, a disseminated granulomatous inflammation with numerous multinucleate giant cells was seen. Numerous acid-fast bacilli were detected in macrophages, in multinucleated giant cells, and free in the central necrosis and suppurative exudate. After culture, polymerase chain reaction assays were carried out to detect the 65-kDa heat shock protein (Hsp65) and insertion sequences (IS)1245 and IS900. The causative agent was identified as Mycobacterium avium subsp. avium. PMID:26450834

  15. Cathepsin L maturation and activity is impaired in macrophages harboring M. avium and M. tuberculosis.

    PubMed

    Nepal, Rajeev M; Mampe, Stephanie; Shaffer, Brian; Erickson, Ann H; Bryant, Paula

    2006-06-01

    Mycobacterium tuberculosis-infected macrophages demonstrate diminished capacity to present antigens via class II MHC molecules. Since successful class II MHC-restricted antigen presentation relies on the actions of endocytic proteases, we asked whether the activities of cathepsins (Cat) B, S and L-three major lysosomal cysteine proteases-are modulated in macrophages infected with pathogenic Mycobacterium spp. Infection of murine bone marrow-derived macrophages with either Mycobacterium avium or M. tuberculosis had no obvious effect on Cat B or Cat S activity. In contrast, the activity of Cat L was altered in infected cells. Specifically, whereas the 24-kDa two-chain mature form of active Cat L predominated in uninfected cells, we observed an increase in the steady-state activity of the precursor single-chain (30 kDa) and 25-kDa two-chain forms of the enzyme in cells infected with either M. avium or M. tuberculosis. Pulse-chase analyses revealed that maturation of nascent, single-chain Cat L into the 25-kDa two-chain form was impaired in infected macrophages, and that maturation into the 24-kDa two-chain form did not occur. Consistent with these data, M. avium infection inhibited the IFNgamma-induced secretion of active two-chain Cat L by macrophages. Viable bacilli were not required to disrupt Cat L maturation, suggesting that a constitutively expressed mycobacterial component was responsible. The absence of the major active form of lysosomal Cat L in M. avium- and M. tuberculosis-infected macrophages may influence the types of T cell epitopes generated in these antigen-presenting cells, and/or the rate of class II MHC peptide loading. PMID:16636015

  16. Characterization of a Novel Plasmid, pMAH135, from Mycobacterium avium Subsp. hominissuis

    PubMed Central

    Uchiya, Kei-ichi; Takahashi, Hiroyasu; Nakagawa, Taku; Yagi, Tetsuya; Moriyama, Makoto; Inagaki, Takayuki; Ichikawa, Kazuya; Nikai, Toshiaki; Ogawa, Kenji

    2015-01-01

    Mycobacterium avium complex (MAC) causes mainly two types of disease. The first is disseminated disease in immunocompromised hosts, such as individuals infected by human immunodeficiency virus (HIV). The second is pulmonary disease in individuals without systemic immunosuppression, and the incidence of this type is increasing worldwide. M. avium subsp. hominissuis, a component of MAC, causes infection in pigs as well as in humans. Many aspects of the different modes of M. avium infection and its host specificity remain unclear. Here, we report the characteristics and complete sequence of a novel plasmid, designated pMAH135, derived from M. avium strain TH135 in an HIV-negative patient with pulmonary MAC disease. The pMAH135 plasmid consists of 194,711 nucleotides with an average G + C content of 66.5% and encodes 164 coding sequences (CDSs). This plasmid was unique in terms of its homology to other mycobacterial plasmids. Interestingly, it contains CDSs with sequence homology to mycobactin biosynthesis proteins and type VII secretion system-related proteins, which are involved in the pathogenicity of mycobacteria. It also contains putative conserved domains of the multidrug efflux transporter. Screening of isolates from humans and pigs for genes located on pMAH135 revealed that the detection rate of these genes was higher in clinical isolates from pulmonary MAC disease patients than in those from HIV-positive patients, whereas the genes were almost entirely absent in isolates from pigs. Moreover, variable number tandem repeats typing analysis showed that isolates carrying pMAH135 genes are grouped in a specific cluster. Collectively, the pMAH135 plasmid contains genes associated with M. avium’s pathogenicity and resistance to antimicrobial agents. The results of this study suggest that pMAH135 influence not only the pathological manifestations of MAC disease, but also the host specificity of MAC infection. PMID:25671431

  17. [Identification of Mycobacterium avium-intracellulare complex by PCR of AIDS and disseminated mycobacteriosis].

    PubMed

    García-Elorriaga, Guadalupe; Degollado-Estrada, Edgar; Villagómez-Ruiz, Alfredo; Cortés-Torres, Nancy; Arreguín-Reséndiz, Lilián; Del Rey-Pineda, Guillermo; González-Bonilla, César

    2016-01-01

    Introducción: el objetivo de este artículo es Identificar y diferenciar el complejo MAC por PCR en pacientes con SIDA y micobacteriosis diseminada. Métodos: se llevó a cabo un estudio transversal para identificar MAC por biología molecular. Se sintetizaron dos conjuntos de iniciadores: MAV y MIN, para M. avium y M. intracellulare, respectivamente. El ADN total de células obtenidas de 29 aislados clínicos y muestras de suero de otros 24 pacientes con SIDA e infección micobacteriana diseminada fue extraído y se amplificó por PCR con los iniciadores MAV y MIN. Cada uno de los iniciadores MAV y MIN amplificó un segmento altamente específico de 1.3 kb del ADN homólogo, respectivamente. Resultados: veintinueve ADN de los aislados clínicos de MAC identificadas por Gen-Probe AccuProbes se amplificaron con los iniciadores MAV (M. avium). De las 24 muestras clínicas, 3 fueron positivas para M. avium y 6 para M. tuberculosis. Conclusiones: nuestros resultados demostraron que la técnica de PCR se puede aplicar para la diferenciación de M. avium y M. intracellulare por iniciadores específicos 16S rRNA. En pacientes con estadio avanzado de SIDA y en quienes se sospecha micobacteriosis diseminada, la presencia de anemia (incluso con cultivos negativos) fosfatasa alcalina elevada y una mediana de CD4 de 15.9/ml, se debe considerar seriamente el diagnóstico de infección por MAC; sugerimos que, de acuerdo con nuestros resultados, se justifica una estratificación más precisa de los pacientes en términos de sus recuentos de células T CD4.

  18. Loci of Mycobacterium avium ser2 gene cluster and their functions.

    PubMed Central

    Mills, J A; McNeil, M R; Belisle, J T; Jacobs, W R; Brennan, P J

    1994-01-01

    The highly antigenic glycopeptidolipids present on the surface of members of the Mycobacterium avium complex serve to distinguish these bacteria from all others and to define the various serovars that compose this complex. Previously, the genes responsible for the biosynthesis of the disaccharide hapten [2,3-di-O-methyl-alpha-L-fucopyranosyl-(1-->3)-alpha-L-rhamnopyranose] of serovar 2 of the M. avium complex were isolated, localized to a contiguous 22- to 27-kb fragment of the M. avium genome, and designated the ser2 gene cluster (J. T. Belisle, L. Pascopella, J. M. Inamine, P. J. Brennan, and W. R. Jacobs, Jr., J. Bacteriol. 173:6991-6997, 1991). In the present study, transposon saturation mutagenesis was used to map the specific genetic loci within the ser2 gene cluster required for expression of this disaccharide. Four essential loci, termed ser2A, -B, -C, and -D, constituting a total of 5.7 kb within the ser2 gene cluster, were defined. The ser2B and ser2D loci encode the methyltransferases required to methylate the fucose at the 3 and 2 positions, respectively. The rhamnosyltransferase was encoded by ser2A, whereas either ser2C or ser2D encoded the fucosyltransferase. The ser2C and ser2D loci are also apparently involved in the de novo synthesis of fucose. Isolation of the truncated versions of the hapten induced by the transposon insertions provides genetic evidence that the glycopeptidolipids of M. avium serovar 2 are synthesized by an initial transfer of the rhamnose unit to the peptide core followed by fucose and finally O methylation of the fucosyl unit. PMID:8050992

  19. Loci of Mycobacterium avium ser2 gene cluster and their functions.

    PubMed

    Mills, J A; McNeil, M R; Belisle, J T; Jacobs, W R; Brennan, P J

    1994-08-01

    The highly antigenic glycopeptidolipids present on the surface of members of the Mycobacterium avium complex serve to distinguish these bacteria from all others and to define the various serovars that compose this complex. Previously, the genes responsible for the biosynthesis of the disaccharide hapten [2,3-di-O-methyl-alpha-L-fucopyranosyl-(1-->3)-alpha-L-rhamnopyranose] of serovar 2 of the M. avium complex were isolated, localized to a contiguous 22- to 27-kb fragment of the M. avium genome, and designated the ser2 gene cluster (J. T. Belisle, L. Pascopella, J. M. Inamine, P. J. Brennan, and W. R. Jacobs, Jr., J. Bacteriol. 173:6991-6997, 1991). In the present study, transposon saturation mutagenesis was used to map the specific genetic loci within the ser2 gene cluster required for expression of this disaccharide. Four essential loci, termed ser2A, -B, -C, and -D, constituting a total of 5.7 kb within the ser2 gene cluster, were defined. The ser2B and ser2D loci encode the methyltransferases required to methylate the fucose at the 3 and 2 positions, respectively. The rhamnosyltransferase was encoded by ser2A, whereas either ser2C or ser2D encoded the fucosyltransferase. The ser2C and ser2D loci are also apparently involved in the de novo synthesis of fucose. Isolation of the truncated versions of the hapten induced by the transposon insertions provides genetic evidence that the glycopeptidolipids of M. avium serovar 2 are synthesized by an initial transfer of the rhamnose unit to the peptide core followed by fucose and finally O methylation of the fucosyl unit. PMID:8050992

  20. Radioprotection of Swiss albino mice by Prunus avium with special reference to hematopoietic system.

    PubMed

    Sisodia, Rashmi; Singh, Smita; Mundotiya, Chaturbhuj; Meghnani, Ekta; Srivastava, Preeti

    2011-01-01

    Prunus avium (family Rosaceae) has been used ethnomedicinally for the treatment of many diseases,but its radioprotective efficacy has hardly been explored. Presence of high anthocyanin content and phenolic compound with good antioxidative capacity has been reported by researchers. Its radioprotective effect against 5, 7, 10, and 12 Gygamma radiation was evaluated by 30 day survival assay. Regression analysis yielded LD(50/30) 5.81 and 9.43Gy for irradiated only and (P. avium fruit extract) PAE + radiation groups, respectively. The dose reduction factor was computed as 1.62. For biochemical and hematological studies, Swiss albino mice were divided into four groups: (i) control (vehicle treated), (ii) PAE treated (450 mg kg/day for 15 consequetive days), (iii) irradiated (5 Gy), and (4) PAE + irradiated. The irradiation of animals resulted in a significant elevation of lipid peroxidation and depletion in glutathione and protein levels in blood serum and spleen, which could be significantly checked by administration of PAE. Radiation-induced deficit in blood sugar, cholesterol, and hematological constituents could also be modulated by supplementation of PAE before and after irradiation. The possible prophylactic and therapeutic action noted by P. avium against radiation induced metabolic disorders may be due to synergistic action of various antioxidants, minerals, vitamins, etc., present in the fruit. Further mechanistic studies aimed at identifying the role of major ingredients in the extract are needed. PMID:21609316

  1. Genetic relationships between diploid and allotetraploid cherry species (Prunus avium, Prunus x gondouinii and Prunus cerasus).

    PubMed

    Tavaud, M; Zanetto, A; David, J L; Laigret, F; Dirlewanger, E

    2004-12-01

    Prunus avium L. (diploid, AA, 2n=2x=16), Prunus cerasus L. (allotetraploid, AAFF, 2n=4x=32) species, and their hybrid Prunus x gondouinii Rehd., constitute the most widely cultivated cherry tree species. P. cerasus is supposed to be an hybrid species produced by the union of unreduced P. avium gametes and normal P. fruticosa gametes. A continuum of morphological traits between these three species makes their assignation difficult. The aim of this paper is to study the genetic relationships between tetraploid and diploid cherry species. In all, 114 genotypes belonging to these species were analyzed using 75 AFLP markers. The coordinates of these genotypes on the first axis of a correspondence analysis allowed us to clearly distinguish each species, to identify misclassifications and to assign unknown genotypes to one species. We showed that there are specific alleles in P. cerasus, which are not present in the A genome of P. avium and which probably come from the F genome of P. cerasus. The frequencies of each marker in the A and the F genomes were estimated in order to identify A and F specific markers. We discuss the utility of these specific markers for finding the origin of the A and F genomes in the allopolyploid species. PMID:15354194

  2. Prunus avium: nuclear DNA study in wild populations and sweet cherry cultivars.

    PubMed

    Guarino, Carmine; Santoro, Simona; De Simone, Luciana; Cipriani, Guido

    2009-04-01

    The PCR-SSR technique was used to detect nuclear DNA diversity in five wild populations of Prunus avium from deciduous forests in Italy, Slovenia, and Croatia and 87 sweet cherry accessions from different geographical areas that have been maintained in the sweet cherry collection in Italy. This sweet cherry collection includes local accessions from the Campania Region as well as accessions from different countries. Twenty-eight microsatellites, previously developed in this species, generated polymorphic amplification products. Between 2 and 14 alleles were revealed for the polymorphic loci studied, with the expected heterozygosity ranging from 0.045 to 0.831. The total probability of identity was 56.94 x 10-18. A model-based Bayesian clustering analysis identified nine distinct gene pools in cultivated P. avium. The probability that wild populations were assigned to cultivated gene pools indicated that three gene pools accounted for the genomic origin of 53% of P. avium sampled. A dendrogram was generated using UPGMA (unweighted pair group method with arithmetic averages) based on Nei genetic distance analysis. This dendrogram classified most of the genotypes into one major group with an additional group of five accessions. The results indicate that this set of SSRs is highly informative, and they are discussed in terms of the implications for sweet cherry characterization.

  3. Antimicrobial synergism against Mycobacterium avium complex strains isolated from patients with acquired immune deficiency syndrome.

    PubMed Central

    Yajko, D M; Kirihara, J; Sanders, C; Nassos, P; Hadley, W K

    1988-01-01

    Pairs of 11 antimicrobial agents were tested in vitro for their ability to act synergistically against three strains of Mycobacterium avium complex isolated from patients with acquired immune deficiency syndrome. From the combinations tested, four drugs (ethambutol, rifampin, ciprofloxacin, and erythromycin) were selected for more extensive study against 20 strains of M. avium complex. The inhibitory and killing synergism obtained with combinations of two, three, or four drugs was assessed by determining the fractional inhibitory concentration index and fractional bactericidal concentration index. Inhibitory synergism occurred against 90 to 100% of the strains for all drug combinations in which ethambutol was included. Killing synergism occurred against 85 to 95% of the strains when ethambutol was used in combinations which included either rifampin or ciprofloxacin. However, killing synergism occurred against only 45% of the strains when drugs were tested at concentrations that can be obtained in patient serum. In other experiments, rifabutin (Ansamycin) gave results that were comparable to those obtained with rifampin. Clofazimine did not show synergistic killing activity at a concentration that is achievable in serum for any of the drugs tested. Our results indicate that there is considerable variability in the antimicrobial susceptibility of M. avium isolates obtained from patients with acquired immune deficiency syndrome. This variability could have significant impact on the clinical response to various therapies. PMID:3196000

  4. Intermittent azithromycin for treatment of Mycobacterium avium infection in beige mice.

    PubMed Central

    Klemens, S P; Cynamon, M H

    1994-01-01

    The activity of azithromycin (AZI) was evaluated in the beige mouse model of disseminated Mycobacterium avium infection. Mice were infected intravenously with approximately 10(7) viable avium ATCC 49601. AZI at 50, 100, or 200 mg/kg of body weight or clarithromycin (CLA) at 200 mg/kg was given by gavage 5 days per week for 4 weeks. Groups of treated mice were compared with untreated control animals. A dose-related reduction in cell counts in organs was observed with AZI treatment. AZI at 200 mg/kg was more active than CLA at 200 mg/kg against organisms in spleens. The activities of these two agents at 200 mg/kg were comparable against organisms in lungs. In a second study, AZI at 200 mg/kg was given daily for 5 days; this was followed by intermittent AZI treatment for the next 3 weeks. The activities of AZI given on a three-times- and five-times-per-week basis in the continuation phase were comparable. AZI given on a once-weekly basis was less active. The regimen of AZI given in combination with rifapentine on a once-weekly basis for 8 weeks showed promising activity. Clinical evaluation of AZI and rifapentine will help to define the roles of these agents in the treatment of disseminated M. avium complex infection. PMID:7986001

  5. Mycobacterium avium subsp. paratuberculosis Antibody Response, Fecal Shedding, and Antibody Cross-Reactivity to Mycobacterium bovis in M. avium subsp. paratuberculosis-Infected Cattle Herds Vaccinated against Johne's Disease

    PubMed Central

    Hovingh, Ernest; Linscott, Rick; Martel, Edmond; Lawrence, John; Wolfgang, David; Griswold, David

    2014-01-01

    Vaccination for Johne's disease with killed inactivated vaccine in cattle herds has shown variable success. The vaccine delays the onset of disease but does not afford complete protection. Johne's disease vaccination has also been reported to interfere with measurements of cell-mediated immune responses for the detection of bovine tuberculosis. Temporal antibody responses and fecal shedding of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, were measured in 2 dairy cattle herds using Johne's disease vaccine (Mycopar) over a period of 7 years. Vaccination against Johne's disease resulted in positive serum M. avium subsp. paratuberculosis antibody responses in both herds, and the responses persisted in vaccinated cattle up to 7 years of age. Some vaccinated animals (29.4% in herd A and 36.2% in herd B) showed no serological reactivity to M. avium subsp. paratuberculosis. M. avium subsp. paratuberculosis-specific antibody responses were also detected in milk from Johne's disease-vaccinated animals, but fewer animals (39.3% in herd A and 49.4% in herd B) had positive results with milk than with serum samples. With vaccination against M. avium subsp. paratuberculosis, fecal shedding in both dairy herds was reduced significantly (P < 0.001). In addition, when selected Johne's disease-vaccinated and -infected animals were investigated for serological cross-reactivity to Mycobacterium bovis, no cross-reactivity was observed. PMID:24623626

  6. THE ISOLATION AND IDENTIFICATION OF MYCOBACTERIUM AVIUM COMPLEX (MAC) RECOVERED FROM LOS ANGELES POTABLE WATER, A POSSIBLE SOURCE OF INFECTION IN AIDS PATIENTS

    EPA Science Inventory

    Los Angeles water was investigated as a possible source of Mycobacterium avium complex (MAC) infection in patients with AIDS. MAC consists of M.avium (MA), M. intracellulare (MI) and Mycobacterium X (MX)(positive for MAC by DNA probe but not MA or MI). The study included 13 reser...

  7. Complete Genome Sequence of Mycobacterium avium, Isolated from Commercial Domestic Pekin Ducks (Anas platyrhynchos domestica), Determined Using PacBio Single-Molecule Real-Time Technology.

    PubMed

    Song, Xiao-Heng; Chen, Hong-Xi; Zhou, Wang-Shu; Wang, Jiang-Bo; Liu, Ma-Feng; Wang, Ming-Shu; Cheng, An-Chun; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue; Zhu, De-Kang

    2016-01-01

    Mycobacterium avium is an important pathogenic bacterium in birds and has never, to our knowledge, reported to be isolated from domestic ducks. We present here the complete genome sequence of a virulent strain of Mycobacterium avium, isolated from domestic Pekin ducks for the first time, which was determined by PacBio single-molecule real-time technology. PMID:27587804

  8. Highly Specific and Quick Detection of Mycobacterium avium subsp. paratuberculosis in Feces and Gut Tissue of Cattle and Humans by Multiple Real-Time PCR Assays▿

    PubMed Central

    Imirzalioglu, Can; Dahmen, Heinrich; Hain, Torsten; Billion, Andre; Kuenne, Carsten; Chakraborty, Trinad; Domann, Eugen

    2011-01-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle and may be associated with Crohn's disease (CD) in humans. It is the slowest growing of the cultivable mycobacteria, and culture from clinical, veterinary, food, or environmental specimens can take 4 months or even longer. Currently, the insertion element IS900 is used to detect M. avium subsp. paratuberculosis DNA. However, closely related IS900 elements are also present in other mycobacteria, thus limiting its specificity as a target. Here we describe the use of novel primer sets derived from the sequences of two highly specific single copy genes, MAP2765c and MAP0865, for the quantitative detection of M. avium subsp. paratuberculosis within 6 h by using real-time PCR. Specificity of the target was established using 40 M. avium subsp. paratuberculosis isolates, 67 different bacterial species, and two intestinal parasites. Using the probes and methods described, we detected 27 (2.09%) M. avium subsp. paratuberculosis-positive stool specimens from 1,293 individual stool samples by the use of either IS900 or probes deriving from the MAP2765c and MAP0865 genes described here. In general, bacterial load due to M. avium subsp. paratuberculosis was uniformly low in these samples and we estimated 500 to 5,000 M. avium subsp. paratuberculosis bacteria per gram of stool in assay-positive samples. Thus, the methods described here are useful for rapid and specific detection of M. avium subsp. paratuberculosis in clinical samples. PMID:21430100

  9. Complete Genome Sequence of Mycobacterium avium, Isolated from Commercial Domestic Pekin Ducks (Anas platyrhynchos domestica), Determined Using PacBio Single-Molecule Real-Time Technology

    PubMed Central

    Song, Xiao-Heng; Chen, Hong-Xi; Zhou, Wang-Shu; Wang, Jiang-Bo; Liu, Ma-Feng; Wang, Ming-Shu; Cheng, An-Chun; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue

    2016-01-01

    Mycobacterium avium is an important pathogenic bacterium in birds and has never, to our knowledge, reported to be isolated from domestic ducks. We present here the complete genome sequence of a virulent strain of Mycobacterium avium, isolated from domestic Pekin ducks for the first time, which was determined by PacBio single-molecule real-time technology. PMID:27587804

  10. Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms.

    PubMed

    Chern, Eunice C; King, Dawn; Haugland, Richard; Pfaller, Stacy

    2015-03-01

    Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

  11. Boosting Effect of 2-Phenylquinoline Efflux Inhibitors in Combination with Macrolides against Mycobacterium smegmatis and Mycobacterium avium.

    PubMed

    Machado, Diana; Cannalire, Rolando; Santos Costa, Sofia; Manfroni, Giuseppe; Tabarrini, Oriana; Cecchetti, Violetta; Couto, Isabel; Viveiros, Miguel; Sabatini, Stefano

    2015-12-11

    The identification of efflux inhibitors to be used as adjuvants alongside existing drug regimens could have a tremendous value in the treatment of any mycobacterial infection. Here, we investigated the ability of four 2-(4'-propoxyphenyl)quinoline Staphylococcus aureus NorA efflux inhibitors (1-4) to reduce the efflux activity in Mycobacterium smegmatis and Mycobacterium avium strains. All four compounds were able to inhibit efflux pumps in both mycobacterial species; in particular, O-ethylpiperazinyl derivative 2 showed an efflux inhibitory activity comparable to that of verapamil, the most potent mycobacterial efflux inhibitor reported to date, and was able to significantly reduce the MIC values of macrolides against different M. avium strains. The contribution of the M. avium efflux pumps MAV_1406 and MAV_1695 to clarithromycin resistance was proved because they were found to be overexpressed in two M. avium 104 isogenic strains showing high-level clarithromycin resistance. These results indicated a correlation between increased expression of efflux pumps, increased efflux, macrolide resistance, and reduction of resistance by efflux pump inhibitors such as compound 2. Additionally, compound 2 showed synergistic activity with clarithromycin, at a concentration below the cytotoxicity threshold, in an ex vivo experiment against M. avium 104-infected macrophages. In summary, the 2-(4'-propoxyphenyl)quinoline scaffold is suitable to obtain compounds endowed with good efflux pump inhibitory activity against both S. aureus and nontuberculous mycobacteria. PMID:27623057

  12. Effects of gamma interferon, interleukin-10, and transforming growth factor beta on the survival of Mycobacterium avium subsp. paratuberculosis in monocyte-derived macrophages from naturally infected cattle.

    PubMed

    Khalifeh, M S; Stabel, J R

    2004-04-01

    Gamma interferon (IFN-gamma) plays a significant role in the control of mycobacterial infections, including Mycobacterium avium subsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. avium subsp. paratuberculosis on the production of IFN-gamma, IL-10, and TGF-beta by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-gamma, IL-10, and TGF-beta on M. avium subsp. paratuberculosis survival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-beta in culture supernatants in response to stimulation with live M. avium subsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-beta, but after stimulation with live M. avium subsp. paratuberculosis, TGF-beta levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. avium subsp. paratuberculosis recovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. avium subsp. paratuberculosis. The addition of exogenous IL-10 and TGF-beta to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. avium subsp. paratuberculosis recovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-beta during infection with M. avium subsp. paratuberculosis that may be directly related to their effects on macrophage activation and killing of M. avium subsp. paratuberculosis.

  13. Mycobacterium avium-induced SOCS contributes to resistance to IFN-gamma-mediated mycobactericidal activity in human macrophages.

    PubMed

    Vázquez, Nancy; Greenwell-Wild, Teresa; Rekka, Sofia; Orenstein, Jan M; Wahl, Sharon M

    2006-11-01

    Mycobacterium avium is an opportunistic pathogen that commonly infects individuals colonized with HIV-1, although it is less frequent in the post-HAART era. These microorganisms invade macrophages after interacting with TLR2 and/or CD14 co-receptors, but signaling pathways promoting survival in macrophages are not well defined. Although IFN-gamma plays an important role in protective immunity against bacterial infections, IFN-gamma responses are compromised in AIDS patients and evidence suggests that exogenous IFN-gamma is inadequate to clear the mycobacteria. To determine the mechanism by which M. avium survives intracellularly, even in the presence of IFN-gamma, we studied the effect of mycobacteria infection in macrophages during early IFN-gamma signaling events. M. avium infected cells exhibited a reduced response to IFN-gamma, with suppressed phosphorylation of STAT-1 compared with uninfected cells. Interaction of M. avium with macrophage receptors increased gene expression of the suppressors of cytokine signaling (SOCS) to diminish IFN responsiveness. Specifically, we observed an increase in mRNA for both SOCS-3 and SOCS-1, which correlates with elevated levels of SOCS protein and positive immunostaining in M. avium/HIV-1 co-infected tissues. We also linked the p38 MAPK signaling pathway to mycobacterial-induced SOCS gene transcription. The induction of SOCS may be part of the strategy that allows the invader to render the macrophages unresponsive to IFN-gamma, which otherwise promotes clearance of the infection. Our data provide new insights into the manipulation of the host response by this opportunistic pathogen and the potential for modulating SOCS to influence the outcome of M. avium infection in immunocompromised hosts.

  14. Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga.

    PubMed

    Mura, Manuela; Bull, Tim J; Evans, Hugh; Sidi-Boumedine, Karim; McMinn, Liz; Rhodes, Glenn; Pickup, Roger; Hermon-Taylor, John

    2006-01-01

    Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.

  15. High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

    PubMed Central

    Waldron, Anna M.; Galea, Francesca; Whittington, Ann-Michele; Saunders, Vanessa F.; Begg, Douglas J.; de Silva, Kumudika; Purdie, Auriol C.; Whittington, Richard J.

    2014-01-01

    Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and

  16. PRESENCE OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN ALPACAS (LAMA PACOS) INHABITING THE CHILEAN ALTIPLANO.

    PubMed

    Salgado, Miguel; Sevilla, Iker; Rios, Carolina; Crossley, Jorge; Tejeda, Carlos; Manning, Elizabeth

    2016-03-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis. The organism causes disease in both domestically managed and wild ruminant species. South American camelids have a long, shared history with indigenous people in the Andes. Over the last few decades, increasing numbers of alpacas were exported to numerous countries outside South America. No paratuberculosis surveillance has been reported for these source herds. In this study, individual fecal samples from 85 adult alpacas were collected from six separate herds in the Chilean Altiplano. A ParaTB mycobacterial growth indicator tube (MGIT) liquid culture of each individual fecal sample, followed by real-time polymerase chain reaction (PCR) protocol was used for confirmation. DNA extracts from a subset of confirmed MAP isolates were subjected to mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. Fifteen alpaca were fecal culture test-positive. Five false-positive culture samples were negative on PCR analysis for Mycobacterium avium subsp. avium (MAA), Mycobacterium bovis (M. bovis), and the 16 S rDNA gene. Three MAP isolates subset-tested belonged to the same MIRU-VNTR type, showing four repeats for TR292 (locus 1) in contrast to the three repeats typical of the MAP reference strain K10. The number of repeats found in the remaining loci was identical to that of the K10 strain. It is not known how nor when MAP was introduced into the alpaca population in the Chilean Altiplano. The most plausible hypothesis to explain the presence of MAP in these indigenous populations is transmission by contact with infected domestic small ruminant species that may on occasion share pastures or range with alpacas. Isolation of this mycobacterial pathogen from such a remote region suggests that MAP has found its way beyond the confines of intensively managed domestic agriculture premises.

  17. Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth.

    PubMed

    Strahl, E D; Gillaspy, G E; Falkinham, J O

    2001-10-01

    Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.

  18. Molecular identification of Mycobacterium avium subspecies paratuberculosis in oral biopsies of Crohn's disease patients.

    PubMed

    Molicotti, Paola; Scanu, Antonio M; Lumbau, Aurea; Cannas, Sara; Bua, Alessandra; Lugliè, Pietrina; Zanetti, Stefania

    2013-07-10

    Oral lesions may be found in patients with Crohn's disease (CD), in a percentage up to 20%. The aim of this study was to investigate a possible relationship between Mycobacterium avium subsp. paratuberculosis (MAP) and oral lesions in CD patients. 23 oral biopsies were examined performing IS900 Nested PCR; 9 of them were positive: 8 from CD patients and 1 from a control. Our purpose is to go on with this study, amplifying the number of subjects examined and testing subjects with oral lesions related to diseases other than CD to verify the specific association between MAP and oral lesions in CD patients.

  19. Facts, myths and hypotheses on the zoonotic nature of Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Atreya, Raja; Bülte, Michael; Gerlach, Gerald-F; Goethe, Ralph; Hornef, Mathias W; Köhler, Heike; Meens, Jochen; Möbius, Petra; Roeb, Elke; Weiss, Siegfried

    2014-10-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (Johne's disease [JD]), a chronic granulomatous enteritis in ruminants. JD is one of the most widespread bacterial diseases of domestic animals with significant economic impact. The histopathological picture of JD resembles that of Crohn's disease (CD), a human chronic inflammatory bowel disease of still unresolved aetiology. An aetiological relevance of MAP for CD has been proposed. This and the ambiguity of other published epidemiological findings raise the question whether MAP represents a zoonotic agent. In this review, we will discuss evidence that MAP has zoonotic capacity.

  20. Mycobacterium avium lung disease combined with a bronchogenic cyst in an immunocompetent young adult.

    PubMed

    Kwon, Yong Soo; Han, Joungho; Jung, Ki Hwan; Kim, Je Hyeong; Koh, Won-Jung

    2013-01-01

    We report a very rare case of a bronchogenic cyst combined with nontuberculous mycobacterial pulmonary disease in an immunocompetent patient. A 21-year-old male was referred to our institution because of a cough, fever, and worsening of abnormalities on his chest radiograph, despite anti-tuberculosis treatment. Computed tomography of the chest showed a large multi-cystic mass over the right-upper lobe. Pathological examination of the excised lobe showed a bronchogenic cyst combined with a destructive cavitary lesion with granulomatous inflammation. Microbiological culture of sputum and lung tissue yielded Mycobacterium avium. The patient was administered anti-mycobacterial treatment that included clarithromycin. PMID:23346002

  1. Cutaneous gallium uptake in patients with AIDS with mycobacterium avium-intracellulare septicemia

    SciTech Connect

    Allwright, S.J.; Chapman, P.R.; Antico, V.F.; Gruenewald, S.M.

    1988-07-01

    Gallium imaging is increasingly being used for the early detection of complications in patients with AIDS. A 26-year-old homosexual man who was HIV antibody positive underwent gallium imaging for investigation of possible Pneumocystis carinii pneumonia. Widespread cutaneous focal uptake was seen, which was subsequently shown to be due to mycobacterium avium-intracellulare (MAI) septicemia. This case demonstrates the importance of whole body imaging rather than imaging target areas only, the utility of gallium imaging in aiding the early detection of clinically unsuspected disease, and shows a new pattern of gallium uptake in disseminated MAI infection.

  2. Identification and comparative analysis of a genomic island in Mycobacterium avium subsp. hominissuis.

    PubMed

    Lahiri, Annesha; Sanchini, Andrea; Semmler, Torsten; Schäfer, Hubert; Lewin, Astrid

    2014-11-01

    Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacterium causing opportunistic infections. The objective of this study was to identify flexible genome regions in MAH isolated from different sources. By comparing five complete and draft MAH genomes we identified a genomic island conferring additional flexibility to the MAH genomes. The island was absent in one of the five strains and had sizes between 16.37 and 84.85kb in the four other strains. The genes present in the islands differed among strains and included phage- and plasmid-derived genes, integrase genes, hypothetical genes, and virulence-associated genes like mmpL or mce genes.

  3. Chemical decontamination with N-acetyl-L-cysteine-sodium hydroxide improves recovery of viable Mycobacterium avium subsp. paratuberculosis organisms from cultured milk.

    PubMed

    Bradner, L; Robbe-Austerman, S; Beitz, D C; Stabel, J R

    2013-07-01

    Mycobacterium avium subsp. paratuberculosis is shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. avium subsp. paratuberculosis between animals. The objective of this study was to formulate an optimized protocol for the isolation of M. avium subsp. paratuberculosis in milk. The parameters investigated included chemical decontamination with N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para-JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 10(2) to 10(8) CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected.

  4. Chemical Decontamination with N-Acetyl-l-Cysteine–Sodium Hydroxide Improves Recovery of Viable Mycobacterium avium subsp. paratuberculosis Organisms from Cultured Milk

    PubMed Central

    Bradner, L.; Robbe-Austerman, S.; Beitz, D. C.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis is shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. avium subsp. paratuberculosis between animals. The objective of this study was to formulate an optimized protocol for the isolation of M. avium subsp. paratuberculosis in milk. The parameters investigated included chemical decontamination with N-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para-JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 102 to 108 CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected. PMID:23637290

  5. Divergent cellular responses during asymptomatic subclinical and clinical states of disease in cows naturally infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infection of the host with Mycobacterium avium subsp. paratuberculosis (MAP) results in a chronic and progressive enteritis that traverses both subclinical and clinical stages. The mechanism(s) for the shift from asymptomatic subclinical disease state to advanced clinical disease are not fully under...

  6. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized...

  7. Gamma-delta T cell responses in subclinical and clinical stages of Bovine Mycobacterium Avium Paratuberculosis infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The early immune response to Mycobacterium avium subsp. paratuberculosis (MAP) in cattle is characterized by a Th1-like immune response effective in controlling bacterial proliferation during the subclinical stage of infection. In young calves nearly 60% of circulating lymphocytes are gamma delta T ...

  8. Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberculosis in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live va...

  9. COMPARISON OF MYCOBACTERIUM AVIUM ISOLATES FROM A DRINKING WATER DISTRIBUTION SYSTEM AND FROM THE POPULATION SERVED BY THE SYSTEM

    EPA Science Inventory

    Background: Current evidence suggests that drinking water, soil, and produce are potential sources of Mycobacterium avium infections, a pathogen not known to be transmitted person-to-person.

    Methods: We sampled water during 2000-2002 from a large municipal drinking water ...

  10. Development and Evaluation of a Variable-number Tandem Repeat (VNTR) Method for Subtyping of Mycobacterium avium subspecies paratuberculosis Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne’s disease in cattle and other ruminants. Because of the apparent ease by which Map can spread to susceptible animals within a dairy herd, a better understanding of the epidemiology of Map infections is required. In thi...

  11. Middle-aged to elderly women have a higher asymptomatic infection rate with Mycobacterium avium complex, regardless of body habitus.

    PubMed

    Nishimura, Tomoyasu; Fujita-Suzuki, Yukiko; Mori, Masaaki; Carpenter, Stephen M; Fujiwara, Hiroshi; Uwamino, Yoshifumi; Tamizu, Eiko; Yano, Ikuya; Kawabe, Hiroshi; Hasegawa, Naoki

    2016-04-01

    Mycobacterium avium complex (MAC) pulmonary disease is prevalent in middle-aged to elderly women with a thin body habitus. By comparing the rate of serologically diagnosed asymptomatic MAC infection and body mass index among 1033 healthy subjects, we find that middle-aged to elderly women became infected with MAC, regardless of their body habitus.

  12. Clinical Evaluation of COBAS TaqMan PCR for the Detection of Mycobacterium tuberculosis and M. avium Complex

    PubMed Central

    Ikegame, Satoshi; Sakoda, Yoritake; Fujino, Nao; Taguchi, Kazuhito; Kawasaki, Masayuki; Kajiki, Akira

    2012-01-01

    A retrospective observational study was performed to determine the sensitivity and limitation of PCR test for the detection of Mycobacterium tuberculosis and M. avium complex. We obtained clinical specimens collected from the respiratory tract, cultured M. tuberculosis or M. avium complex, and performed PCR analysis. A total of 299 samples (M. tuberculosis, 177; M. avium, 35; M. intracellulare, 87) were analyzed by COBAS TaqMan PCR from April 2007 to March 2011. The PCR positivity rates were 50–55%, 70–100%, 88–98%, and 100% in smear-negative, smear 1+, 2+, and 3+ groups, respectively. The PCR positivity of tuberculosis in smear 1+ was 80.6%, which was statistically significantly (P < 0.001) lower than that of smear 2+ (97.3%). From January 2005 to March 2007, we collected an additional 138 samples (M. tuberculosis, 74; M. avium, 21; M. intracellulare, 43), which were analyzed by COBAS Amplicor PCR. The PCR positivity rates obtained using COBAS TaqMan PCR and COBAS Amplicor PCR were not significantly different. The sensitivity of PCR test for mycobacteria is not sufficient in case of smear 1+. Careful consideration must be given to the interpretation of negative PCR test results in smear 1+, because smear-positive tuberculosis is the criterion for isolation. PMID:23029612

  13. Evaluation of survival of Mycobacterium avium subsp. paratuberculosis (Map) in ciliates isolated from Johne’s positive cow.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Persistence of Mycobacterium avium subsp. paratuberculosis (Map) in farm environments is not well understood. Previously we examined the ability of amoebae from a cow’s watering trough to sequester and enhance growth of Map and found that one amoeba species released vesicles containin...

  14. A Novel Cell Wall Lipopeptide Is Important for Biofilm Formation and Pathogenicity of Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm formation by pathogenic bacteria plays a key role in their pathogenesis. Previously, the pstA gene was shown to be involved in the virulence of Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease in cattle and a potential risk factor for Crohn's d...

  15. Isolation of Mycobacterium avium subspecies paratuberculosis Reactive T-cells from Intestinal Biopsies of Crohn's Disease Patients

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crohn’s disease (CD) is a chronic granulomatous inflammation of the intestine. The etiology is still unknown. One hypothesis is that CD is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP) in genetically predisposed individuals. MAP causes a similar disease in ruminants,...

  16. CD4 T Cells From Intestinal Biopsies of Crohn's Disease Patients React to Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The role of Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn’s disease (CD) remains controversial. One issue that has been raised is the lack of data showing a cellular immune response to MAP. Earlier studies have mostly focused on responses in peripheral blood which have several limit...

  17. Mediation of host immune responses after immunization of neonatal calves with a heat-killed Mycobacterium avium subsp. paratuberculosis vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis(MAP) is the interference with diagnostic tests for bovine tuberculosis and paratuberculosis. The current study was designed to explore effects of immunization with a heat-killed whole cell vaccine (Mycop...

  18. ZAP-70, CTLA-4 and proximal T cell receptor signaling in cows infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paratuberculosis is a chronic intestinal disease of ruminant animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). A hallmark of paratuberculosis is a transition from a cell-mediated Th1 type response to a humoral Th2 response with the progression of disease from a subclinical to clin...

  19. Analysis of Mycobacterium avium subspecies paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies to novel mutant discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of Johne’s disease, is one of the most important bacterial pathogens in ruminants. The lack of efficacious control measures demands a thorough understanding of MAP pathogenesis to develop new vaccines and diagnostic tests. The ge...

  20. NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While intense research is being conducted to develop faster and more reliable methods for diagnosis of Johne’s disease, there are still significant knowledge gaps concerning the molecular function of Mycobacterium avium subspecies paratuberculosis proteins. Therefore, we describe atomic resolution ...

  1. Genome Sequence of Mycobacterium avium subspecies paratuberculosis Sheep Isolate 397 Reveals Striking Differences Among a Genetically Homogeneous Subspecies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genome of Mycobacterium avium subspecies paratuberculosis is remarkably homogeneous among the bovine, human and wildlife isolates. However previous work in our labs using genomic DNA hybridizations with the bovine K-10 DNA microarray has revealed substantial differences in sheep isolates. A dr...

  2. Optimization of methods for the detection of Mycobacterium avium subsp. paratuberculosis in milk and colostrum of naturally infected dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two decontamination chemicals, hexadecylpyridinium choride (HPC) and N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH), were compared for their efficacy of reducing the growth of non-specific microorganisms in milk while minimally affecting the recovery of Mycobacterium avium subsp. paratuberculosis ...

  3. Evaluation of Control Points in Youngstock and Adult Dairy Cow Management to Control Transmission of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Goal Complete a series of controlled on-farm trials to critically evaluate the efficacy and cost-benefit of commonly recommended management practices for reducing the transmission of Mycobacterium avium subsp. paratuberculosis (Map) in infected herds. Objective 1. Evaluate the effect of maternity...

  4. Shedding of Mycobacterium avium subsp. paratuberculosis into milk and colostrum of naturally infected dairy cows over complete lactation cycles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The primary mode of transmission of Mycobacterium avium subsp. paratuberculosis (MAP) is fecal-oral. However, MAP is also shed into the milk and colostrum of infected cows. The objective of this study was to identify if an association exists between stage of MAP infection and days in lactation with ...

  5. Longitudinal data collection of Mycobacterium avium subspecies Paratuberculosis infections in dairy herds. Collection and use of observational data

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Longitudinal infection data on Mycobacterium avium subspecies paratuberculosis (MAP) was collected on three dairy farms in Northeastern United States during approximately 10 years. Precise data on animal characteristics and animal location within farm were collected on these farms. Cows were followe...

  6. Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the pathogenic mechanisms and host responses to Johne’s disease, a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), is complicated by the multifaceted disease progression, late-onset host reaction, and the lack of ex vivo infection models ...

  7. ZAP-70, CTLA-4, and proximal T cell receptor signaling in cows infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paratuberculosis is a chronic intestinal disease of ruminant animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). A hallmark of paratuberculosis is a transition from a cell-mediated Th1 type response to a humoral Th2 response with the progression of disease from a subclinical to clin...

  8. The effects of progressing and nonprogressing Mycobacterium avium ssp. paratuberculosis infection on milk production in dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Longitudinal data from three commercial dairy herds in the northeast United States, collected from 2004 to 2011, were analyzed to determine the effect of Johne’s disease status and path on milk production. Disease status, as indicated by Mycobacterium avium subsp. paratuberculosis test results, was ...

  9. Differences in intermittent and continuous fecal shedding patterns between natural and experimental Mycobacterium avium subspecies paratuberculosis infections in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this paper is to study shedding patterns of cows infected with Mycobacterium avium subsp. paratuberculosis (MAP). While multiple single farm studies of MAP dynamics were reported, there is not large-scale meta-analysis of both natural and experimental infections. Large difference...

  10. Septic arthritis and granulomatous synovitis caused by infection with Mycobacterium avium complex in a horse.

    PubMed

    Hewes, Christina A; Schneider, Robert K; Baszler, Timothy V; Oaks, J Lindsay

    2005-06-15

    A 12-year-old American Saddlebred gelding was referred to a veterinary teaching hospital for evaluation of a chronic lameness problem in the right radiocarpal joint. The horse had been treated for osteoarthritis of the right radiocarpal joint with multiple injections of cortisone during the past 3 years. The horse was severely lame on the right forelimb at a trot. Radiography and computed tomography revealed a 3 x 2-cm lytic defect in the distal portion of the radius and periarticular bone proliferation around the right radiocarpal joint. Ultrasonography of the distal portion of the radius revealed a soft tissue mass in the palmarolateral aspect of the joint. Proliferative synovium with a large amount of fibrin was observed in the dorsal and palmar aspects of the joint via arthroscopic examination of the right radiocarpal joint. Histologic examination of synovial biopsy specimens revealed proliferative granulomatous synovitis with giant cells. Mycobacterium avium complex was cultured from the synovial fluid. Infection with M avium complex should be considered in horses with chronic recurring arthritis associated with granulomatous synovitis.

  11. Relatedness of Mycobacterium avium subspecies hominissuis clinical isolates of human and porcine origins assessed by MLVA.

    PubMed

    Leão, Célia; Canto, Ana; Machado, Diana; Sanches, Ilda Santos; Couto, Isabel; Viveiros, Miguel; Inácio, João; Botelho, Ana

    2014-09-17

    Mycobacterium avium subsp. hominissuis (MAH) is an important opportunistic pathogen, infecting humans and animals, notably pigs. Several methods have been used to characterize MAH strains. RFLP and PFGE typing techniques have been used as standard methods but are technically demanding. In contrast, the analysis of VNTR loci is a simpler, affordable and highly reliable PCR-based technique, allowing a numerical and reproductive digitalization of typing data. In this study, the analysis of Mycobacterium avium tandem repeats (MATRs) loci was adapted to evaluate the genetic diversity of epidemiological unrelated MAH clinical strains of human (n=28) and porcine (n=69) origins, collected from diverse geographical regions across mainland Portugal. These MAH isolates were found to be genetically diverse and genotypes are randomly distributed across the country. Some of the human strains shared identical VNTR profiles with porcine isolates. Our study shows that the VNTR genotyping using selected MATR loci is a useful analysis technique for assessing the genetic diversity of MAH isolates from Portugal. This typing method could be successfully applied in other countries toward the implementation of a worldwide open-access database of MATR-VNTR profiles of MAH isolates, allowing a better assessment of the global epidemiology traits of this important pathogenic species.

  12. Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil.

    PubMed

    Carvalho, I A; Pietralonga, P A G; Schwarz, D G G; Faria, A C S; Moreira, M A S

    2012-12-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohn's disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohn's disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil.

  13. Mycobacterium avium subsp. hominissuis infection in swine associated with peat used for bedding.

    PubMed

    Johansen, Tone Bjordal; Agdestein, Angelika; Lium, Bjørn; Jørgensen, Anne; Djønne, Berit

    2014-01-01

    Mycobacterium avium subsp. hominissuis is an environmental bacterium causing opportunistic infections in swine, resulting in economic losses. Additionally, the zoonotic aspect of such infections is of concern. In the southeastern region of Norway in 2009 and 2010, an increase in condemnation of pig carcasses with tuberculous lesions was seen at the meat inspection. The use of peat as bedding in the herds was suspected to be a common factor, and a project examining pigs and environmental samples from the herds was initiated. Lesions detected at meat inspection in pigs originating from 15 herds were sampled. Environmental samples including peat from six of the herds and from three peat production facilities were additionally collected. Samples were analysed by culture and isolates genotyped by MLVA analysis. Mycobacterium avium subsp. hominissuis was detected in 35 out of 46 pigs, in 16 out of 20 samples of peat, and in one sample of sawdust. MLVA analysis demonstrated identical isolates from peat and pigs within the same farms. Polyclonal infection was demonstrated by analysis of multiple isolates from the same pig. To conclude, the increase in condemnation of porcine carcasses at slaughter due to mycobacteriosis seemed to be related to untreated peat used as bedding.

  14. Shared Mycobacterium avium Genotypes Observed among Unlinked Clinical and Environmental Isolates

    PubMed Central

    Weigel, Kris M.; Yakrus, Mitchell A.; Becker, Annie L.; Chen, Hui-Ling; Fridley, Gina; Sikora, Arthur; Speake, Cate; Hilborn, Elizabeth D.; Pfaller, Stacy

    2013-01-01

    Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this assumption, a high-resolution PCR-based genotyping approach, large-sequence polymorphism (LSP)-mycobacterial interspersed repetitive unit–variable-number tandem repeat (MIRU-VNTR), was selected and used to analyze clinical and environmental isolates of M. avium from geographically diverse sources. Among 127 clinical isolates from seven locations in North America, South America, and Europe, 42 genotypes were observed. Among 12 of these genotypes, matches were seen in isolates from apparently unlinked patients in two or more geographic locations. Six of the 12 were also observed in environmental isolates. A subset of these isolates was further analyzed by alternative strain genotyping methods, pulsed-field gel electrophoresis and MIRU-VNTR, which confirmed the existence of geographically dispersed strain genotypes. These results suggest that caution should be exercised in interpreting high-resolution genotypic matches as evidence for an acquisition event. PMID:23851084

  15. Environmental survival of Mycobacterium avium subsp. paratuberculosis in different climatic zones of eastern Australia.

    PubMed

    Eppleston, Jeffrey; Begg, Douglas J; Dhand, Navneet K; Watt, Bruce; Whittington, Richard J

    2014-04-01

    The duration of survival of both the S and C strains of Mycobacterium avium subsp. paratuberculosis in feces was quantified in contrasting climatic zones of New South Wales, Australia, and detailed environmental temperature data were collected. Known concentrations of S and C strains in feces placed on soil in polystyrene boxes were exposed to the environment with or without the provision of shade (70%) at Bathurst, Armidale, Condobolin, and Broken Hill, and subsamples taken every 2 weeks were cultured for the presence of M. avium subsp. paratuberculosis. The duration of survival ranged from a minimum of 1 week to a maximum of 16 weeks, and the provision of 70% shade was the most important factor in extending the survival time. The hazard of death for exposed compared to shaded samples was 20 and 9 times higher for the S and C strains, respectively. Site did not affect the survival of the C strain, but for the S strain, the hazard of death was 2.3 times higher at the two arid zone sites (Broken Hill and Condobolin) than at the two temperate zone sites (Bathurst and Armidale). Temperature measurements revealed maximum temperatures exceeding 60°C and large daily temperature ranges at the soil surface, particularly in exposed boxes.

  16. Environmental Survival of Mycobacterium avium subsp. paratuberculosis in Different Climatic Zones of Eastern Australia

    PubMed Central

    Begg, Douglas J.; Dhand, Navneet K.; Watt, Bruce; Whittington, Richard J.

    2014-01-01

    The duration of survival of both the S and C strains of Mycobacterium avium subsp. paratuberculosis in feces was quantified in contrasting climatic zones of New South Wales, Australia, and detailed environmental temperature data were collected. Known concentrations of S and C strains in feces placed on soil in polystyrene boxes were exposed to the environment with or without the provision of shade (70%) at Bathurst, Armidale, Condobolin, and Broken Hill, and subsamples taken every 2 weeks were cultured for the presence of M. avium subsp. paratuberculosis. The duration of survival ranged from a minimum of 1 week to a maximum of 16 weeks, and the provision of 70% shade was the most important factor in extending the survival time. The hazard of death for exposed compared to shaded samples was 20 and 9 times higher for the S and C strains, respectively. Site did not affect the survival of the C strain, but for the S strain, the hazard of death was 2.3 times higher at the two arid zone sites (Broken Hill and Condobolin) than at the two temperate zone sites (Bathurst and Armidale). Temperature measurements revealed maximum temperatures exceeding 60°C and large daily temperature ranges at the soil surface, particularly in exposed boxes. PMID:24463974

  17. Survival of Mycobacterium avium and Mycobacterium tuberculosis in Acidified Vacuoles of Murine Macrophages

    PubMed Central

    Gomes, Maria Salomé; Paul, Simon; Moreira, Andre L.; Appelberg, Rui; Rabinovitch, Michel; Kaplan, Gilla

    1999-01-01

    Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the phagosome and inhibition of phagosome-lysosome fusion. In contrast to mycobacteria, Coxiella burnetii, the etiologic agent of Q fever, inhabits a spacious acidified intracellular vacuole which is prone to fusion with other vacuoles of the host cell, including phagosomes containing mycobacteria. The Coxiella-infected cell thus provides a unique model for investigating the survival of mycobacteria in an acidified phagosome-like compartment. In the present study, murine bone marrow-derived macrophages were infected with either Mycobacterium avium or Mycobacterium tuberculosis and then coinfected with C. burnetii. We observed that the majority of phagocytosed mycobacteria colocalized to the C. burnetii-containing vacuole, which maintained its acidic properties. In coinfected macrophages, the growth of M. avium was not impaired following fusion with the acidified vacuole. In contrast, the growth rate of M. tuberculosis was reduced in acidified vacuoles. These results suggest that although both species of mycobacteria inhibit phagosome-lysosome fusion, they may be differentially susceptible to the toxic effects of the acidic environment in the mature phagolysosome. PMID:10377091

  18. Coexistent Pseudogout and Mycobacterium avium-intracellulare Septic Arthritis in a Patient with HIV and ESRD

    PubMed Central

    Wali, Omer M.; Cervellione, Kelly L.; Singh, Bhupinder B.; Bagheri, Farshad

    2016-01-01

    Pseudogout is a crystal-induced arthropathy characterized by the deposition of calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluid, menisci, or articular cartilage. Although not very common, this entity can be seen in patients with chronic kidney disease (CKD). Septic arthritis due to Mycobacterium avium-intracellulare (MAI) is a rare entity that can affect immunocompromised patients such as those with acquired immunodeficiency syndrome (AIDS) or those who are on immunosuppressive drugs. Here, we describe a 51-year-old female who presented with fever, right knee pain, swelling, warmth, and decreased range of motion for several days. The initial assessment was consistent with pseudogout, with negative bacterial and fungal cultures. However, due to high white blood cell (WBC) count in the synovial fluid analysis, she was empirically started on intravenous (IV) vancomycin and piperacillin-tazobactam and discharged on IV vancomycin and cefepime, while acid-fast bacilli (AFB) culture was still in process. Seventeen days later, AFB culture grew Mycobacterium avium-intracellulare (MAI), and she was readmitted for relevant management. This case illustrates that septic arthritis due to MAI should be considered in the differential diagnosis of septic arthritis in immunocompromised patients. PMID:27803833

  19. Detection of Mycobacterium avium subsp. paratuberculosis in Infected Tissues by New Species-Specific Immunohistological Procedures

    PubMed Central

    Coetsier, Christophe; Havaux, Xavier; Mattelard, Francois; Sadatte, Sanaa; Cormont, Francoise; Buergelt, Klaus; Limbourg, Bernard; Latinne, Dominique; Bazin, Herve; Denef, Jean-Francois; Cocito, Carlo

    1998-01-01

    We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein of Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne’s disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930–4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947–954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne’s disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp. paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity. PMID:9665946

  20. In vitro anti-Mycobacterium avium activities of quinolones: predicted active structures and mechanistic considerations.

    PubMed

    Klopman, G; Li, J Y; Wang, S; Pearson, A J; Chang, K; Jacobs, M R; Bajaksouzian, S; Ellner, J J

    1994-08-01

    The relationship between the structures of quinolones and their anti-Mycobacterium avium activities has been previously derived by using the Multiple Computer-Automated Structure Evaluation program. A number of substructural constraints required to overcome the resistance of most of the strains have been identified. Nineteen new quinolones which qualify under these substructural requirements were identified by the program and subsequently tested. The results show that the substructural attributes identified by the program produced a successful a priori prediction of the anti-M. avium activities of the new quinolones. All 19 quinolones were found to be active, and 4 of them are as active or better than ciprofloxacin. With these new quinolones, the updated multiple computer-automated structure evaluation program structure-activity relationship analysis has helped to uncover additional information about the nature of the substituents at the C5 and C7 positions needed for optimal inhibitory activity. A possible explanation of drug resistance based on the observation of suicide inactivation of bacterial cytochrome P-450 by the cyclopropylamine moiety has also been proposed and is discussed in this report. Furthermore, we confirm the view that the amount of the uncharged form present in a neutral pH solution plays a crucial role in the drug's penetration ability.

  1. Co-infection by Cryptococcus neoformans and Mycobacterium avium intracellulare in AIDS.

    PubMed

    Arastéh, K; Cordes, C; Futh, U; Grosse, G; Dietz, E; Staib, F

    In the observation of various opportunistic pathogens in HIV-positive persons, co-infection by Cryptococcus neoformans together with Mycobacterium avium intracellulare was found if there was a CD4 lymphocyte count as low as 3-20 microl. In 1540 HIV-positive patients under treatment at a Berlin hospital (Auguste-Viktoria-Krankenhaus) during 1985-1994, all AIDS-relevant diseases were examined in a multivariate analysis as variables of influence on the manifestation of a systemic Mycobacterium avium complex (MAC) infection. The analysis involved data on 36 cases of cryptococcosis and 202 cases with a typical clinical course in whom MAC had been detected at sterile body sites. As significant and independent factors of influence, the following were identified: C. neoformans infection, wasting syndrome, lower age, low CD4 lymphocyte count and preceding Pneumocystis carinii pneumonia (PcP) prophylaxis. Cryptococcosis ranged first with an odds ratio of 2.75. The concomitant manifestation of cryptococcosis and systemic MAC infection in six patients is shown. Because both opportunists, C. neoformans and avian mycobacteria, may have their common habitat in droppings of defined species of pet birds, a common source of infection deserves further clinical and epidemiological attention.

  2. Co-infection by Cryptococcus neoformans and Mycobacterium avium intracellulare in AIDS. Clinical and epidemiological aspects.

    PubMed

    Arastéh, K; Cordes, C; Futh, U; Grosse, G; Dietz, E; Staib, F

    In the observation of various opportunistic pathogens in HIV-positive persons, co-infection by Cryptococcus neoformans together with Mycobacterium avium intracellulare was found if there was a CD4 lymphocyte count as low as 3-20/microliters. In 1540 HIV-positive patients under treatment at a Berlin hospital (Auguste-Viktoria-Krankenhaus) during 1985-1994, all AIDS-relevant diseases were examined in a multivariate analysis as variables of influence on the manifestation of a systemic Mycobacterium avium complex (MAC) infection. The analysis involved data on 36 cases of cryptococcosis and 202 cases with a typical clinical course in whom MAC had been detected at sterile body sites. As significant and independent factors of influence, the following were identified: C. neoformans infection, wasting syndrome, lower age, low CD4 lymphocyte count and preceding Pneumocystis carinii pneumonia (PcP) prophylaxis. Cryptococcosis ranged first with an ods ratio of 2.75. The concomitant manifestation of cryptococcosis and systemic MAC infection in six patients is shown. Because both opportunists, C. neoformans and avian mycobacteria, may have their common habitat in droppings of defined species of pet birds, a common source of infection deserves further clinical and epidemiological attention.

  3. Detection of Mycobacterium tuberculosis and Mycobacterium avium Complexes by Real-Time PCR in Bovine Milk from Brazilian Dairy Farms.

    PubMed

    Bezerra, André Vinícius Andrade; Dos Reis, Emily Marques; Rodrigues, Rogério Oliveira; Cenci, Alexander; Cerva, Cristine; Mayer, Fabiana Quoos

    2015-05-01

    Foodborne diseases are a public health problem worldwide. The consumption of contaminated raw milk has been recognized as a major cause of transmission of bovine tuberculosis to humans. Other mycobacteria that may be present in raw milk and may cause diseases are those belonging to the Mycobacterium avium complex. In this study, molecular biology tools were applied to investigate raw milk contamination with Mycobacterium spp. in family dairy farms from Rio Grande do Sul, southern Brazil. Furthermore, different variables related to the source of the milk, herd characteristics, and management were evaluated for their effect on milk contamination. Five hundred and two samples were analyzed, of which 354 were from the Northwest region (102 farms with samples from 93 bulk tanks and 261 animals) and 148 from the South region of the state (22 farms with samples from 23 bulk tanks and 125 animals). Among them, 10 (1.99%) and 7 (1.39%) were positive for Mycobacterium tuberculosis (9 confirmed as Mycobacterium bovis) and M. avium complexes, respectively. There was no difference in the frequencies of positive samples between the regions or the sample sources. Of the positive samples, 4 were collected from a bulk tank (1 positive for M. avium and 3 for M. tuberculosis). Moreover, 1 sample was positive concomitantly for M. tuberculosis and M. avium complexes. On risk analysis, no variable was associated with raw milk contamination by M. tuberculosis complex species. However, washing the udders of all animals and drying them with paper towels were weakly classified as risk factors for M. avium contamination. Positive samples were obtained from both animals and bulk tanks, which emphasizes the importance of tuberculosis control programs and provides evidence that milk monitoring can be used as a control practice. Moreover, the findings of this study reinforce the need for awareness of the problems of raw milk consumption among the general population. PMID:25951404

  4. Detection of Mycobacterium tuberculosis and Mycobacterium avium Complexes by Real-Time PCR in Bovine Milk from Brazilian Dairy Farms.

    PubMed

    Bezerra, André Vinícius Andrade; Dos Reis, Emily Marques; Rodrigues, Rogério Oliveira; Cenci, Alexander; Cerva, Cristine; Mayer, Fabiana Quoos

    2015-05-01

    Foodborne diseases are a public health problem worldwide. The consumption of contaminated raw milk has been recognized as a major cause of transmission of bovine tuberculosis to humans. Other mycobacteria that may be present in raw milk and may cause diseases are those belonging to the Mycobacterium avium complex. In this study, molecular biology tools were applied to investigate raw milk contamination with Mycobacterium spp. in family dairy farms from Rio Grande do Sul, southern Brazil. Furthermore, different variables related to the source of the milk, herd characteristics, and management were evaluated for their effect on milk contamination. Five hundred and two samples were analyzed, of which 354 were from the Northwest region (102 farms with samples from 93 bulk tanks and 261 animals) and 148 from the South region of the state (22 farms with samples from 23 bulk tanks and 125 animals). Among them, 10 (1.99%) and 7 (1.39%) were positive for Mycobacterium tuberculosis (9 confirmed as Mycobacterium bovis) and M. avium complexes, respectively. There was no difference in the frequencies of positive samples between the regions or the sample sources. Of the positive samples, 4 were collected from a bulk tank (1 positive for M. avium and 3 for M. tuberculosis). Moreover, 1 sample was positive concomitantly for M. tuberculosis and M. avium complexes. On risk analysis, no variable was associated with raw milk contamination by M. tuberculosis complex species. However, washing the udders of all animals and drying them with paper towels were weakly classified as risk factors for M. avium contamination. Positive samples were obtained from both animals and bulk tanks, which emphasizes the importance of tuberculosis control programs and provides evidence that milk monitoring can be used as a control practice. Moreover, the findings of this study reinforce the need for awareness of the problems of raw milk consumption among the general population.

  5. Bacteremia with an iliopsoas abscess and osteomyelitis of the femoral head caused by Enterococcus avium in a patient with end-stage kidney disease.

    PubMed

    Okada, Akira; Hangai, Mika; Oda, Toshimi

    2015-01-01

    A 70-year-old man on hemodialysis for end-stage kidney disease due to polycystic kidney disease presented with hip pain on extension and a high C-reactive protein level. Further examinations revealed an iliopsoas abscess and femoral head osteomyelitis caused by Enterococcus avium (E. avium) detected in blood and pus cultures. Complete resolution of the infection with ampicillin-resistant E. avium required six months of vancomycin therapy and two surgical drainage procedures. There have been no previous case reports in which both blood and abscess cultures confirmed E. avium infection. Careful attention should be paid to the detection of non-specific symptoms in patients on hemodialysis, with blood cultures being essential in such cases.

  6. Treatment with antibiotics is detrimental to the recovery of viable Mycobacterium avium subsp. paratuberculosis cultured from milk and colostrum of dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibiotic cocktails are frequently used as secondary decontaminants prior to the culture of Mycobacterium avium subsp. paratuberculosis (MAP). This study investigated whether secondary incubation with an antibiotic cocktail containing vancomycin, nalidixic acid, and amphotericin B after primary exp...

  7. Efficacy of various pasteurization time-temperature conditions in combination with homogenization on inactivation of Mycobacterium avium subsp. paratuberculosis in milk.

    PubMed

    Grant, Irene R; Williams, Alan G; Rowe, Michael T; Muir, D Donald

    2005-06-01

    The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 10(1) to 10(5) M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in2, applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or "miniclump" status by homogenization at 2,500 lb/in2. Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization.

  8. Key Role for the Alternative Sigma Factor, SigH, in the Intracellular Life of Mycobacterium avium subsp. paratuberculosis during Macrophage Stress

    PubMed Central

    Ghosh, Pallab; Wu, Chia-wei

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell, M. avium subsp. paratuberculosis is known to survive harsh microenvironments, especially those inside activated macrophages. To improve our understanding of M. avium subsp. paratuberculosis pathogenesis, we examined phagosome maturation associated with transcriptional responses of M. avium subsp. paratuberculosis during macrophage infection. Monitoring cellular markers, only live M. avium subsp. paratuberculosis bacilli were able to prevent phagosome maturation and reduce its acidification. On the transcriptional level, over 300 M. avium subsp. paratuberculosis genes were significantly and differentially regulated in both naive and IFN-γ-activated macrophages. These genes include the sigma factor H (sigH) that was shown to be important for M. avium subsp. paratuberculosis survival inside gamma interferon (IFN-γ)-activated bovine macrophages. Interestingly, an sigH-knockout mutant showed increased sensitivity to a sustained level of thiol-specific oxidative stress. Large-scale RNA sequence analysis revealed that a large number of genes belong to the sigH regulon, especially following diamide stress. Genes involved in oxidative stress and virulence were among the induced genes in the sigH regulon with a putative consensus sequence for SigH binding that was recognized in a subset of these genes (n = 30), suggesting direct regulation by SigH. Finally, mice infections showed a significant attenuation of the ΔsigH mutant compared to its parental strain, suggesting a role for sigH in M. avium subsp. paratuberculosis virulence. Such analysis could identify potential targets for further testing as vaccine candidates against Johne's disease. PMID:23569115

  9. Mycobacterium avium infection in HIV-1-infected subjects increases monokine secretion and is associated with enhanced viral load and diminished immune response to viral antigens.

    PubMed Central

    Denis, M; Ghadirian, E

    1994-01-01

    The complex interaction between HIV-1 infection and Mycobacterium avium was studied. Viral burden was assessed, as well as immune response to HIV-1 in the context of Myco. avium infections. We also examined serum cytokine levels and cytokine release by blood mononuclear cells in HIV-1-infected subjects, infected or not with Myco. avium. Undetectable serum levels of IL-1, tumour necrosis factor-alpha (TNF-alpha) and IL-6 were found in normal controls and in groups I, II and III of HIV-1-infected subjects. Moderate levels of TNF-alpha, IL-1 and IL-6 were found in the sera of group IV patients. When group IV was subdivided into subjects with and without Myco. avium infections, subjects with Myco, avium infections were shown to have higher serum levels of TNF-alpha, IL-1 beta and IL-6 than those with other infections. Blood mononuclear cells from controls and HIV subjects were stimulated with bacterial lipopolysaccharide, and cytokine levels assessed. Cells from group II patients were shown to secrete normal levels of TNF-alpha and IL-6, and lower levels of IL-1 beta; group III subjects released higher levels of IL-6. Patients in group IV had blood cells that released elevated levels of IL-6 and TNF-alpha, and lower levels of IL-1 beta. Group IV subjects with Myco. avium infections had blood cells that released higher levels of TNF-alpha, IL-6 and IL-1 than group IV subjects with other infections. Assessment of viral burden in cells of HIV-1-infected subjects revealed that Myco. avium-infected subjects had a higher level of virus burden and a lower level of lymphoproliferative response to an inactivated gp120-depleted HIV-1 antigen than AIDS subjects with other infections. These data suggest that Myco. avium infections in HIV-1-infected subjects hasten the progression of viral disease, enhance cytokine release and contribute to the anergy to viral antigens. PMID:8033423

  10. Mycobacterium avium infection in mice is associated with time-related expression of Th1 and Th2 CD4+ T-lymphocyte response.

    PubMed Central

    Azouaou, N; Petrofsky, M; Young, L S; Bermudez, L E

    1997-01-01

    Disseminated infection caused by organisms of Mycobacterium avium complex is common in acquired immune deficiency syndrome (AIDS) patients. M. avium is an intracellular bacterium that multiplies within macrophages. We examined the effect of M. avium infection on the T-helper cell response in C57/BL/6 black mice. At weekly intervals, CD4+ T-cells were isolated from spleens and lines were created. T-cell lines were exposed to sonicated M. avium in the presence of feeder cells and macrophages and the supernatant were collected to measure the concentrations of interferon-gamma (IFN-gamma and interleukin-10 (IL-10). Production of IFN-gamma in CD4+ T-cells obtained from uninfected mice did not vary significantly during the 5 weeks. Levels of IFN-gamma produced by T-cell lines of infected mice were similar to the control mice during the first 2 weeks but significantly reduced (approximately 30 ng/ml) thereafter. In contrast, production of IL-10 by T-cell lines of infected mice was in a range of 190 to 342 pg/ml in weeks 1, 2 and 3, but increased to an average of 1300 pg/ml at weeks 4 and 5. Pre-immunized mice, when infected with M. avium strain 101, showed a different profile of T-cell cytokines, with high IFN-gamma and low IL-10 production. Proteins purified from a number of disease-associated (D-A) and non-D-A strains of M. avium were tested for the ability to induce IL-10. 65,000 MW and 60,000 MW proteins of M. avium induced significantly more IL-10 than 45,000 MW, 33,000 MW and 27,000 MW proteins. These results showed that M. avium predominantly stimulates either Th1 or Th2 T-helper cells according to the phase of the infection. PMID:9301531

  11. Intracellular fate of Mycobacterium avium subspecies paratuberculosis in monocytes from normal and infected, interferon-responsive cows as determined by a radiometric method.

    PubMed

    Zhao, B Y; Czuprynski, C J; Collins, M T

    1999-01-01

    The ability of Mycobacterium avium subsp. paratuberculosis to survive in bovine monocytes was studied using radiometric (BACTEC) culture, standard plate counting and microscopic counting of acid-fast stained monocyte monolayers. Results of microscopic counts sharply contrasted with results of viable counts determined both by plate counting and radiometric counting. We observed an early phase (the first 6 d after in vitro infection) of intracellular bacillary growth, followed by a later phase of mycobacteriostasis or killing (up to 12 d after in vitro infection) in monocytes from non-infected cows. The data suggest that multiplication and death of M. avium subsp. paratuberculosis occur simultaneously in bovine monocytes infected in vitro. Using the BACTEC method, we compared the ability of bovine monocytes from normal cows and cows infected with M. avium subsp. paratuberculosis and showing evidence of a strong Thl-like cellular immune response to ingest and inhibit the intracellular growth of M. avium subsp. paratuberculosis. There was a trend toward greater phagocytosis and faster killing of Mycobacterium avium subsp. paratuberculosis by monocytes from the infected, immune responder cows. However, the observed numbers of viable M. avium subsp. paratuberculosis at each time after monocyte infection were not significantly different between normal and infected cows.

  12. Clofazimine Prevents the Regrowth of Mycobacterium abscessus and Mycobacterium avium Type Strains Exposed to Amikacin and Clarithromycin

    PubMed Central

    Ferro, Beatriz E.; Meletiadis, Joseph; Wattenberg, Melanie; de Jong, Arjan; van Soolingen, Dick; Mouton, Johan W.

    2015-01-01

    Multidrug therapy is a standard practice when treating infections by nontuberculous mycobacteria (NTM), but few treatment options exist. We conducted this study to define the drug-drug interaction between clofazimine and both amikacin and clarithromycin and its contribution to NTM treatment. Mycobacterium abscessus and Mycobacterium avium type strains were used. Time-kill assays for clofazimine alone and combined with amikacin or clarithromycin were performed at concentrations of 0.25× to 2× MIC. Pharmacodynamic interactions were assessed by response surface model of Bliss independence (RSBI) and isobolographic analysis of Loewe additivity (ISLA), calculating the percentage of statistically significant Bliss interactions and interaction indices (I), respectively. Monte Carlo simulations with predicted human lung concentrations were used to calculate target attainment rates for combination and monotherapy regimens. Clofazimine alone was bacteriostatic for both NTM. Clofazimine-amikacin was synergistic against M. abscessus (I = 0.41; 95% confidence interval [CI], 0.29 to 0.55) and M. avium (I = 0.027; 95% CI, 0.007 to 0.048). Based on RSBI analysis, synergistic interactions of 28.4 to 29.0% and 23.2 to 56.7% were observed at 1× to 2× MIC and 0.25× to 2× MIC for M. abscessus and M. avium, respectively. Clofazimine-clarithromycin was also synergistic against M. abscessus (I = 0.53; 95% CI, 0.35 to 0.72) and M. avium (I = 0.16; 95% CI, 0.04 to 0.35), RSBI analysis showed 23.5% and 23.3 to 53.3% at 2× MIC and 0.25× to 0.5× MIC for M. abscessus and M. avium, respectively. Clofazimine prevented the regrowth observed with amikacin or clarithromycin alone. Target attainment rates of combination regimens were >60% higher than those of monotherapy regimens for M. abscessus and M. avium. The combination of clofazimine with amikacin or clarithromycin was synergistic in vitro. This suggests a potential role for clofazimine in treatment regimens that warrants further

  13. Detection of Mycobacterium avium subsp. paratuberculosis by a Direct In Situ PCR Method

    PubMed Central

    Delgado, Fernando; Aguilar, Diana; Garbaccio, Sergio; Francinelli, Gladys; Hernández-Pando, R.; Romano, María Isabel

    2011-01-01

    In situ detection of Mycobacterium avium subsp. paratuberculosis is useful for diagnosis and research of paratuberculosis. The aim of this paper was to detect this agent in formalin-fixed, paraffin-embedded tissue samples by a direct in situ PCR. The technique was performed on ileum or ileocaecal lymph node samples from 8 naturally infected cattle and 1 healthy calf, by using p89 and p92 primers for amplification of IS900 sequence. Moderate positive signal was detected in all positive samples and not in negative control, but tissues resulted were affected in many cases due to the enzymatic treatment and the high temperature exposition. Although the technique was useful for Map detection, the signal was lower than immunohistochemistry probably because of the fixation process. In one case, signal was higher, which might be due to the detection of spheroplasts. Thus, the described method should be recommended when others resulted negative or for spheroplasts detection. PMID:21772965

  14. Opportunistic Pathogens Mycobacterium Avium Complex (MAC) and Legionella spp. Colonise Model Shower.

    PubMed

    Whiley, Harriet; Giglio, Steven; Bentham, Richard

    2015-07-24

    Legionella spp. and Mycobacterium avium complex (MAC) are opportunistic pathogens of public health concern. Hot water systems, including showers, have been identified as a potential source of infection. This paper describes the colonization of Legionella and MAC on the flexible tubing within a model potable shower system, utilizing thermostatic mixing and a flexible shower head. A MAC qPCR method of enumeration was also developed. MAC and Legionella spp. were detected within the biofilm at maximum concentrations of 7.0 × 104 and 2.0 × 103 copies/cm2 PVC tubing respectively. No significant changes were observed between sample of the flexible shower tubing that dried between uses and those that remained filled with water. This suggested the "unhooking" showerheads and allowing them to dry is not an effective method to reduce the risk of Legionella or MAC colonisation.

  15. Characterisation of an ELISA detecting immunoglobulin G to Mycobacterium avium subsp. paratuberculosis in bovine colostrum.

    PubMed

    Zervens, Lisa Marie-Louise; Nielsen, Søren Saxmose; Jungersen, Gregers

    2013-09-01

    Although colostrum has been used to detect specific immunoglobulin (Ig) G to Mycobacterium avium subsp. paratuberculosis (MAP) in cattle, confounding, non-specific reactions can be a problem. The objectives of this study were to determine the proportion of non-specific ELISA reactions in samples of colostrum taken between 0 and 4 days-in-milk (DIM), and to assess the probability of an animal testing positive for MAP specific IgG over this time-period. Non-specific reactions were found in 3/365 (0.8%) of samples. The odds of an animal testing positive on day of calving were 130 times higher than at 4 DIM. The findings suggest colostral samples may have enhanced diagnostic potential over milk samples in determining if cattle have been exposed to or infected with MAP. PMID:23611487

  16. Association between cattle herd Mycobacterium avium subsp. paratuberculosis (MAP) infection and infection of a hare population.

    PubMed

    Salgado, Miguel; Monti, Gustavo; Sevilla, Iker; Manning, Elizabeth

    2014-10-01

    Paratuberculosis has long been considered a disease of domestic and wild ruminants only. The known host range of Mycobacterium avium subsp. paratuberculosis (MAP) was recently extended to include non-ruminant wildlife species believed to be exposed to spillover of MAP from infected domestic cattle herds. The aim of the present study was to assess the association between cattle herd MAP infection pressure level and the infection level of a hare population in two dairy farms of southern Chile. Fifty hares from a herd A and 42 hares from herd B were captured and sampled for MAP culture. The results showed a statistically significant association between the cattle herds' infection prevalence and the hare infection prevalence.

  17. Mycobacterium avium complex (MAC) osteomyelitis and septic arthritis in an immunocompetent host.

    PubMed

    Jones, A R; Bartlett, J; McCormack, J G

    1995-01-01

    We describe a case of Mycobacterium avium complex (MAC) osteomyelitis and septic arthritis in an immunocompetent man. Infection was derived from a chainsaw injury sustained on the lateral aspect of the ankle 13 years earlier, and had spread through the bone, joint and soft tissue emerging at the medial aspect. This was successfully treated with surgical debridement, drainage, arthrodesis and 18 months of chemotherapy consisting of clarithromycin, rifampicin, ethambutol, and ciprofloxacin with an initial 2 weeks of amikacin. Infections with this organisms are generally associated with immunocompromised states, particularly advanced AIDS. However, our patient illustrates that atypical mycobacterial infections must also be considered in immunocompetent patients who have a prolonged clinical course and an appropriate potential source of infection.

  18. Genetic diversity in wild sweet cherries (Prunus avium) in Turkey revealed by SSR markers.

    PubMed

    Ercisli, S; Agar, G; Yildirim, N; Duralija, B; Vokurka, A; Karlidag, H

    2011-06-21

    Wild sweet cherry (Prunus avium) trees are abundant in the northern part of Turkey, including the Coruh Valley. We analyzed 18 wild sweet cherry genotypes collected from diverse environments in the upper Coruh Valley in Turkey to determine genetic variation, using 10 SSR primers. These SSR primers generated 46 alleles; the number of alleles per primer ranged from 3 to 7, with a mean of 4.6. The primer PS12A02 gave the highest number of polymorphic bands (N = 7), while CPSCT010, UDAp-401 and UDAp-404 gave the lowest number (N = 3). Seven groups were separated in the dendrogram, although most of the genotypes did not cluster according to phenological and morphological traits. This level of genetic diversity in these wild sweet cherry genotypes is very high and therefore these trees would be useful as breeders for crosses between cultivated sweet cherry and wild genotypes.

  19. Rapid susceptibility testing of Mycobacterium avium complex and Mycobacterium tuberculosis isolated from AIDS patients

    NASA Technical Reports Server (NTRS)

    Dhople, Arvind M.

    1994-01-01

    In ominous projections issued by both U.S. Public Health Service and the World Health Organization, the epidemic of HIV infection will continue to rise more rapidly worldwide than predicted earlier. The AIDS patients are susceptible to diseases called opportunistic infections of which tuberculosis and Mycobacterium avium complex (MAC) infection are most common. This has created an urgent need to uncover new drugs for the treatment of these infections. In the seventies, NASA scientists at Goddard Space Flight Center, Greenbelt, MD, had adopted a biochemical indicator, adenosine triphosphate (ATP), to detect presence of life in extraterrestrial space. We proposed to develop ATP assay technique to determine sensitivity of antibacterial compounds against MAC and M. tuberculosis.

  20. Opportunistic Pathogens Mycobacterium Avium Complex (MAC) and Legionella spp. Colonise Model Shower.

    PubMed

    Whiley, Harriet; Giglio, Steven; Bentham, Richard

    2015-01-01

    Legionella spp. and Mycobacterium avium complex (MAC) are opportunistic pathogens of public health concern. Hot water systems, including showers, have been identified as a potential source of infection. This paper describes the colonization of Legionella and MAC on the flexible tubing within a model potable shower system, utilizing thermostatic mixing and a flexible shower head. A MAC qPCR method of enumeration was also developed. MAC and Legionella spp. were detected within the biofilm at maximum concentrations of 7.0 × 104 and 2.0 × 103 copies/cm2 PVC tubing respectively. No significant changes were observed between sample of the flexible shower tubing that dried between uses and those that remained filled with water. This suggested the "unhooking" showerheads and allowing them to dry is not an effective method to reduce the risk of Legionella or MAC colonisation. PMID:26213977

  1. Mycobacterium avium-related epizootic in free-ranging lesser flamingos in Kenya.

    PubMed

    Kock, N D; Kock, R A; Wambua, J; Kamau, G J; Mohan, K

    1999-04-01

    An epizootic in free-ranging lesser flamingos (Phoeniconaias minor) in Kenya resulted in more than 18,500 deaths from August through mid-November 1993. Disease was concentrated along the shores of Rift Valley Lakes Bogoria and Nakuru (Kenya) and did not involve any of the other avian or mammalian species frequenting the lakes. Coincidental to the outbreak was a bloom of algae on Lake Bogoria, toxins from which were first suspected to be causative. Discrete necrotic and granulomatous lesions were often noted in spleen and liver, and Mycobacterium avium serovar I was isolated from both organs. Escherichia coli and Pseudomonas aeruginosa also were often recovered in pure culture from liver. Gross and histopathological evaluation of the cases disclosed signs of acute sepsis and also chronic, potentially life-threatening lesions of mycobacteriosis, primarily involving the spleen and liver. Lesions typical for algae toxicosis were not seen in any birds. Deaths were attributed to septicemia complicated in those affected, by mycobacteriosis.

  2. Opportunistic Pathogens Mycobacterium Avium Complex (MAC) and Legionella spp. Colonise Model Shower

    PubMed Central

    Whiley, Harriet; Giglio, Steven; Bentham, Richard

    2015-01-01

    Legionella spp. and Mycobacterium avium complex (MAC) are opportunistic pathogens of public health concern. Hot water systems, including showers, have been identified as a potential source of infection. This paper describes the colonization of Legionella and MAC on the flexible tubing within a model potable shower system, utilizing thermostatic mixing and a flexible shower head. A MAC qPCR method of enumeration was also developed. MAC and Legionella spp. were detected within the biofilm at maximum concentrations of 7.0 × 104 and 2.0 × 103 copies/cm2 PVC tubing respectively. No significant changes were observed between sample of the flexible shower tubing that dried between uses and those that remained filled with water. This suggested the “unhooking” showerheads and allowing them to dry is not an effective method to reduce the risk of Legionella or MAC colonisation. PMID:26213977

  3. Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level.

    PubMed

    Stephan, R; Schumacher, S; Tasara, T; Grant, I R

    2007-08-01

    A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn's disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.

  4. Disseminated mycobacterium avium complex as protein-losing enteropathy in a non-HIV patient.

    PubMed

    Konjeti, Venkata Rajesh; Paluri, Sravanthi

    2014-01-01

    Disseminated mycobacterium avium complex (MAC) causing protein-losing enteropathy (PLE) due to intestinal lymphangiectasia (IL) in a non-HIV immunocompromised state is extremely rare. We present a case of 56-year-old male who was evaluated for worsening dyspnea and found to have right-sided chylous pleural effusion as well as worsening abdominal and retroperitoneal lymphadenopathy. He had a history of psoriasis for which hewas on etanercept and alefacept which were stopped two years prior to the presentation. The evaluation revealed a MAC infection in his lymph nodes--a low CD4 count but negative for HIV. He was started on MAC therapy. He subsequently developed noninfectious diarrhea, Hypoalbuminemia, recurrentpleural effusions, ascites, and Pneumocystis jiroveci pneumonia (PJP). Despite appropriate antibiotics and management--including total parental nutrition (TPN) with a medium-chain triglyceride enriched low fat diet--the patient's clinical condition deteriorated rapidly resulting in death. PMID:25672059

  5. Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle.

    PubMed

    Holbert, Sébastien; Branger, Maxime; Souriau, Armel; Lamoureux, Bérénice; Ganneau, Christelle; Richard, Gaëlle; Cochard, Thierry; Tholoniat, Christophe; Bay, Sylvie; Winter, Nathalie; Moyen, Jean Louis; Biet, Franck

    2015-10-01

    After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture.

  6. Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle.

    PubMed

    Holbert, Sébastien; Branger, Maxime; Souriau, Armel; Lamoureux, Bérénice; Ganneau, Christelle; Richard, Gaëlle; Cochard, Thierry; Tholoniat, Christophe; Bay, Sylvie; Winter, Nathalie; Moyen, Jean Louis; Biet, Franck

    2015-10-01

    After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture. PMID:26412530

  7. Infection of Eurasian badgers (Meles meles) with Mycobacterium bovis and Mycobacterium avium complex in Spain.

    PubMed

    Balseiro, Ana; Rodríguez, Oscar; González-Quirós, Pablo; Merediz, Isabel; Sevilla, Iker A; Davé, Dipesh; Dalley, Deanna J; Lesellier, Sandrine; Chambers, Mark A; Bezos, Javier; Muñoz, Marta; Delahay, Richard J; Gortázar, Christian; Prieto, José M

    2011-11-01

    The prevalence, distribution and pathology related to infection with Mycobacterium bovis and other mycobacteria were determined in trapped (n=36) and road-killed (n=121) badgers in Spain from 2006 to 2010. The prevalence of M. bovis based on bacteriological culture from road-killed badgers was 8/121 (6.6%) and from trapped badgers was 0/36 (0%). Tuberculosis/M. bovis infection was evident in 15/121 (12.4%) road-killed badgers when bacteriology and histopathology were combined. Mycobacterium avium complex was isolated by culture from the tracheal aspirate of 1/36 (2.8%) trapped badgers and from tissue pools from 8/121 (6.6%) road-killed badgers.

  8. Induction of murine macrophage TNF-alpha synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid.

    PubMed

    Irani, Vida R; Maslow, Joel N

    2005-05-15

    We studied whether complement receptor (CR) mediated Mycobacterium avium interaction modulated macrophage TNF-alpha expression. Compared to control conditions, infections performed with C3-depletion yielded significantly higher TNF-alpha levels. Blockage of the CR4 iC3b site yielded increases in TNF-alpha for all morphotypic variants of a virulent serovar-8 strain (smooth transparent (SmT), smooth opaque (SmO), serovar-specific glycopeptidolipid (ssGPL) deficient knockout mutant) whereas CR3 blockage increased TNF-alpha only for SmT and ssGPL-deficient strains. Thus, complement-mediated binding of M. avium to CR3 and CR4 was shown to modulate TNF-alpha expression. The differential activation of morphotypic and isogenic variants of a single strain provides an excellent model system to delineate signaling pathways. PMID:15899409

  9. A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples.

    PubMed

    Kumanan, Vijayarani; Nugen, Sam R; Baeumner, Antje J; Chang, Yung-Fu

    2009-03-01

    A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (10(1) to 10(6)) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortutitum, M. scrofalaceum, M. intracellular, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR. PMID:19255522

  10. Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics

    PubMed Central

    Rose, Sasha J.; Babrak, Lmar M.; Bermudez, Luiz E.

    2015-01-01

    Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections. PMID:26010725

  11. Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics.

    PubMed

    Rose, Sasha J; Babrak, Lmar M; Bermudez, Luiz E

    2015-01-01

    Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.

  12. [Rapid diagnosis of mycobacteria, especially Mycobacterium tuberculosis and Mycobacterium avium complex, using MB Check system].

    PubMed

    Saito, H; Tomioka, H; Sato, K; Inoue, K; Shigeto, E

    1992-07-01

    Fourty-five sputum specimens were subjected to isolation for mycobacteria either MB Check system (MB method; F. Hoffmann-La Roche Ltd., Basel, Switzerland) or 3% Ogawa egg medium (Ogawa method). Test sputum was treated with 4 volumes of 4% NaOH for 1-2 min and 0.1 ml of the resulting mixture was inoculated onto 3% Ogawa egg medium. The remaining portion of the mixture was neutralized with IN HCl, diluted with 1/15 M phosphate buffer (PB; pH 6.8), and subsequently centrifuged at 3,000 rpm for 20 min. The sediment was suspended in 1.5 ml of PB and 0.5 ml each was inoculated into MB Check M bottle (20 ml) supplemented with M supplement (1 ml). In MB method, bacterial growth was measured on Middlebrook 7H11 agar medium and Middlebrook 7H11 agar medium containing NAP (p-nitro-alpha-acetylamino-beta-hydroxy-propiophenone). Among 45 sputum specimens, the number of positive specimens for mycobacterial growth in the above two cultivation methods and time required for growth were as follows: 3% Ogawa egg medium; 12 specimens (26.7%) gave positive growth, including 7 of M. tuberculosis complex strains on 14-35 days (average 22 days) and 5 of M. avium complex strains on 14-21 days (average 18 days); MB method; 15 of specimens (33.3%) gave positive growth, including 8 of M. tuberculosis complex strains on 7-21 days (average 15 days), 6 of M. avium complex strains on 7-14 days (average 11 days) and 1 of M. scrofulaceum strain on 28 days. There was no specimen which was positive for mycobacterial growth on 3% Ogawa egg medium but negative on MB medium. PMID:1434318

  13. Azithromycin Dose To Maximize Efficacy and Suppress Acquired Drug Resistance in Pulmonary Mycobacterium avium Disease

    PubMed Central

    Deshpande, Devyani; Pasipanodya, Jotam G.

    2016-01-01

    Mycobacterium avium complex is now the leading mycobacterial cause of chronic pneumonia in the United States. Macrolides and ethambutol form the backbone of the regimen used in the treatment of pulmonary disease. However, therapy outcomes remain poor, with microbial cure rates of 4% in cavitary disease. The treatment dose of azithromycin has mostly been borrowed from that used to treat other bacterial pneumonias; there are no formal dose-response studies in pulmonary M. avium disease and the optimal dose is unclear. We utilized population pharmacokinetics and pharmacokinetics/pharmacodynamics-derived azithromycin exposures associated with optimal microbial kill or resistance suppression to perform 10,000 patient Monte Carlo simulations of dose effect studies for daily azithromycin doses of 0.5 to 10 g. The currently recommended dose of 500 mg per day achieved the target exposures in 0% of patients. Exposures associated with optimal kill and resistance suppression were achieved in 87 and 54% of patients, respectively, only by the very high dose of 8 g per day. The azithromycin susceptibility breakpoint above which patients failed therapy on the very high doses of 8 g per day was an MIC of 16 mg/liter, suggesting a critical concentration of 32 mg/liter, which is 8-fold lower than the currently used susceptibility breakpoint of 256 mg/liter. If the standard dose of 500 mg a day were used, then the critical concentration would fall to 2 mg/liter, 128-fold lower than 256 mg/liter. The misclassification of resistant isolates as susceptible could explain the high failure rates of current doses. PMID:26810646

  14. GENETIC DIVERSITY OF SOME IRANIAN SWEET CHERRY (PRUNUS AVIUM) CULTIVARS USING MICROSATELLITE MARKERS AND MORPHOLOGICAL TRAITS.

    PubMed

    Farsad, A; Esna-Ashari, M

    2016-01-01

    The aim of this study was to characterize 23 important Iranian sweet cherry (Prunus avium) cultivars collected from different provinces of Iran and 1 foreign cultivar, which was used as control, considered for breeding programs by using 21 microsatellite markers and 27 morphological traits. In sweet cherry (Prunus avium) accessions, leaf, fruit, and stone morphological characters were evaluated during two consecutive years. The study revealed a high variability in the set of evaluated sweet cherry accessions. The majority of important correlations were determined among variables representing fruit and leaf size and variables related to color. Cluster analysis distinguished sweet cherry accessions into two distinct groups. Principal component analysis (PCA) of qualitative and quantitative morphological parameters explained over 86.59% of total variability in the first seven axes. In PCA, leaf traits such as leaf length and width, and fruit traits such as length, width, and weight, and fruit flesh and juice color were predominant in the first two components, indicating that they were useful for the assessment of sweet cherry germplasm characterization. Out of 21 SSR markers, 16 were polymorphic, producing 177 alleles that varied from 4 to 16 alleles (9.35 on average) with a mean heterozygosity value of 0.82 that produced successful amplifications and revealed DNA polymorphisms. Allele size varied from 95 to 290 bp. Cluster analyses showed that the studied sweet cherry genotypes were classified intofive main groups based mainly on their species characteristics and SSR data. In general, our results did not show a clear structuring of genetic variability within the Iranian diffusion area of sweet cherry, so it was not possible to draw any indications on regions of provenance delimitation. The results of this study contribute to a better understanding of sweet cherry genetic variations in Iran, thus making for more efficient programs aimed at preserving biodiversity and

  15. Host responses to the pathogen Mycobacterium avium subsp. paratuberculosis and beneficial microbes exhibit host sex specificity.

    PubMed

    Karunasena, Enusha; McMahon, K Wyatt; Chang, David; Brashears, Mindy M

    2014-08-01

    Differences between microbial pathogenesis in male and female hosts are well characterized in disease conditions connected to sexual transmission. However, limited biological insight is available on variances attributed to sex specificity in host-microbe interactions, and it is most often a minimized variable outside these transmission events. In this work, we studied two gut microbes-a pathogen, Mycobacterium avium subsp. paratuberculosis, and a probiotic, Lactobacillus animalis NP-51-and the interaction between each agent and the male and female gastrointestinal systems. This trial was conducted in BALB/c mice (n=5 per experimental group and per sex at a given time point), with analysis at four time points over 180 days. Host responses to M.avium subsp. paratuberculosis and L. animalis were sensitive to sex. Cytokines that were significantly different (P ≤ 0.05) betweenthe sexes included interleukin-1α/β (IL-1α/β), IL-17, IL-6, IL-10, IL-12, and gamma interferon (IFN-) and were dependent on experimental conditions. However, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and IL-13/23 showed no sex specificity. A metabolomics study indicated a 0.5- to 2.0-fold (log2 scale) increase in short-chain fatty acids (butyrate and acetate) in males and greater increases in o-phosphocholine or histidine from female colon tissues; variances distinct to each sex were observed with age or long-term probiotic consumption. Two genera, Staphylococcus and Roseburia, were consistently overrepresented in females compared to males; other species were specific to one sex but fluctuated depending on experimental conditions. The differences observed suggest that male and female gut tissues and microbiota respond to newly introduced microorganisms differently and that gut-associated microorganisms with host immune system responses and metabolic activity are supported by biology distinct to the host sex.

  16. GENETIC DIVERSITY OF SOME IRANIAN SWEET CHERRY (PRUNUS AVIUM) CULTIVARS USING MICROSATELLITE MARKERS AND MORPHOLOGICAL TRAITS.

    PubMed

    Farsad, A; Esna-Ashari, M

    2016-01-01

    The aim of this study was to characterize 23 important Iranian sweet cherry (Prunus avium) cultivars collected from different provinces of Iran and 1 foreign cultivar, which was used as control, considered for breeding programs by using 21 microsatellite markers and 27 morphological traits. In sweet cherry (Prunus avium) accessions, leaf, fruit, and stone morphological characters were evaluated during two consecutive years. The study revealed a high variability in the set of evaluated sweet cherry accessions. The majority of important correlations were determined among variables representing fruit and leaf size and variables related to color. Cluster analysis distinguished sweet cherry accessions into two distinct groups. Principal component analysis (PCA) of qualitative and quantitative morphological parameters explained over 86.59% of total variability in the first seven axes. In PCA, leaf traits such as leaf length and width, and fruit traits such as length, width, and weight, and fruit flesh and juice color were predominant in the first two components, indicating that they were useful for the assessment of sweet cherry germplasm characterization. Out of 21 SSR markers, 16 were polymorphic, producing 177 alleles that varied from 4 to 16 alleles (9.35 on average) with a mean heterozygosity value of 0.82 that produced successful amplifications and revealed DNA polymorphisms. Allele size varied from 95 to 290 bp. Cluster analyses showed that the studied sweet cherry genotypes were classified intofive main groups based mainly on their species characteristics and SSR data. In general, our results did not show a clear structuring of genetic variability within the Iranian diffusion area of sweet cherry, so it was not possible to draw any indications on regions of provenance delimitation. The results of this study contribute to a better understanding of sweet cherry genetic variations in Iran, thus making for more efficient programs aimed at preserving biodiversity and

  17. Host Responses to the Pathogen Mycobacterium avium subsp. paratuberculosis and Beneficial Microbes Exhibit Host Sex Specificity

    PubMed Central

    McMahon, K. Wyatt; Chang, David; Brashears, Mindy M.

    2014-01-01

    Differences between microbial pathogenesis in male and female hosts are well characterized in disease conditions connected to sexual transmission. However, limited biological insight is available on variances attributed to sex specificity in host-microbe interactions, and it is most often a minimized variable outside these transmission events. In this work, we studied two gut microbes—a pathogen, Mycobacterium avium subsp. paratuberculosis, and a probiotic, Lactobacillus animalis NP-51—and the interaction between each agent and the male and female gastrointestinal systems. This trial was conducted in BALB/c mice (n = 5 per experimental group and per sex at a given time point), with analysis at four time points over 180 days. Host responses to M. avium subsp. paratuberculosis and L. animalis were sensitive to sex. Cytokines that were significantly different (P ≤ 0.05) between the sexes included interleukin-1α/β (IL-1α/β), IL-17, IL-6, IL-10, IL-12, and gamma interferon (IFN-γ) and were dependent on experimental conditions. However, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and IL-13/23 showed no sex specificity. A metabolomics study indicated a 0.5- to 2.0-fold (log2 scale) increase in short-chain fatty acids (butyrate and acetate) in males and greater increases in o-phosphocholine or histidine from female colon tissues; variances distinct to each sex were observed with age or long-term probiotic consumption. Two genera, Staphylococcus and Roseburia, were consistently overrepresented in females compared to males; other species were specific to one sex but fluctuated depending on experimental conditions. The differences observed suggest that male and female gut tissues and microbiota respond to newly introduced microorganisms differently and that gut-associated microorganisms with host immune system responses and metabolic activity are supported by biology distinct to the host sex. PMID:24814797

  18. Superoxide dismutase activity of Mycobacterium avium, M. intracellulare, and M. scrofulaceum.

    PubMed Central

    Mayer, B K; Falkinham, J O

    1986-01-01

    Superoxide dismutase (EC 1.15.1.1) (SOD) activity has been detected in crude cell extracts of representative strains of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group. Polyacrylamide gel electrophoresis demonstrated a single SOD activity band for each of the MAIS strains, though there were differences in mobility. All M. avium and M. intracellulare and two of five M. scrofulaceum strains demonstrated a single activity band of identical mobility (Rf = 0.83), while the SOD activity band for the three remaining M. scrofulaceum strains migrated farther (Rf = 0.85). The differences in mobility correlated with differences in sensitivity to NaN3 and H2O2. The SOD activities of the majority of the MAIS strains which displayed the slower-migrating activity band were inhibited 22 to 81% after 15 min of exposure to 5 mM H2O2, suggesting that both iron and manganese may be present in a single enzyme. The SOD activities of the three M. scrofulaceum strains which had the faster-migrating activity band were inhibited 100% after only 5 min of exposure to 5 mM H2O2 and exhibited greater sensitivity to 5 and 10 mM NaN3, characteristics of an iron-containing SOD. A concentration of 1 mM KCN did not cause inhibition of enzyme activity in any of the MAIS strains tested. Extracellular SOD activity was detected in four of six MAIS strains and was shown to be identical in mobility to the SOD activity of the crude extracts. Images PMID:3744555

  19. Further studies on the GS element. A novel mycobacterial insertion sequence (IS1612), inserted into an acetylase gene (mpa) in Mycobacterium avium subsp. silvaticum but not in Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Bull, T J; Sheridan, J M; Martin, H; Sumar, N; Tizard, M; Hermon-Taylor, J

    2000-12-20

    We have recently described the GS element, found in Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. silvaticum (MAS) and some isolates of Mycobacterium avium subsp. avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids. Here we describe a further gene of low GC% content (mpa), within the GS element in MAP. mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri. Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB). In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence. This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E. coli) and IS1415 (R. erythropolis). Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element. Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster. This cluster has a high GC% content which suggests a 'border' for the GS element. A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element. This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature. Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.

  20. Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

    PubMed Central

    Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

    1992-01-01

    Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the

  1. Mycobacterium avium subspecies paratuberculosis infection of cattle does not diminish peripheral blood-derived macrophage mycobactericidal activity.

    PubMed

    Hostetter, J; Zhang, W; Simutis, F

    2006-09-15

    Ruminants infected with Mycobacterium avium subspecies paratuberculosis consistently develop a multibacillary form of disease that is centered on the ileum. Mechanisms responsible for failure of macrophage function during multibacillary disease are incompletely characterized. Our data suggest that mycobactericidal functions are present, and potentially enhanced, in monocyte-derived macrophages from M. avium subsp. paratuberculosis infected cattle. Addition of CD4(+) T cells from infected animals to autologous in vitro infected macrophages did not increase bacterial killing. In contrast, CD4(+) T cells from non-infected animals did increase bacterial killing in autologous macrophages. In macrophages from both infected and non-infected cattle, bacterial killing appeared to be independent of interferon-gamma (IFN-gamma) and nitric oxide production.

  2. Use of Specific rRNA Oligonucleotide Probes for Microscopic Detection of Mycobacterium avium Complex Organisms in Tissue

    PubMed Central

    Amand, Allison L. St.; Frank, Daniel N.; De Groote, Mary Ann; Pace, Norman R.

    2005-01-01

    Members of the Mycobacterium avium complex (MAC) are important environmental pathogens that are implicated in several chronic, idiopathic diseases. Diagnosis of MAC-based diseases is compromised by the need to cultivate these fastidious and slowly growing organisms in order to identify which mycobacterial species are present. Detection is particularly difficult when MAC is intracellular or embedded within mammalian tissues. We report on the development of culture-independent, in situ hybridization (ISH) assays for the detection of MAC in culture, sputum, and tissue. This assay includes a highly reliable technique for the permeabilization of mycobacterial cells within culture and tissues. We describe a set of rRNA-based oligonucleotide probes that specifically detect either M. intracellulare, the two M. avium subspecies associated with human disease, or all members of MAC. The results call into question the validity of ISH results derived by the use of other gene loci, such as IS900. PMID:15814959

  3. Immune response of poults following intranasal inoculation with Artvax vaccine and a formalin-inactivated Bordetella avium bacterin.

    PubMed

    Hofstad, M S; Jeska, E L

    1985-01-01

    Poults 3 weeks and older developed temporary tracheal resistance to intranasal challenge following inoculation of either Artvax vaccine or formalin-inactivated Bordetella avium bacterin by the intranasal and eyedrop routes. Resistance usually persisted for 3-4 weeks after B. avium challenge. However, with constant exposure to infected controls, the vaccinated birds eventually developed tracheal infection. Day-old poults did not respond to either the Artvax or the bacterin and were completely susceptible to challenge. Two-week-old poults responded to some degree, but poults 3 weeks old and older responded best. Poults inoculated with bacterin by aerosol or by drinking water did not respond as well as those inoculated by the intranasal and eyedrop routes. When poults were given a single subcutaneous injection at 3 weeks of age and challenged 2 weeks later, three of five resisted infection for 18 days.

  4. Localization of Mycobacterium avium-intracellulare within a skin lesion of bacillary angiomatosis in a patient with AIDS.

    PubMed

    Sagerman, P M; Relman, D A; Niroomand, F; Niedt, G W

    1992-09-01

    We report a 39-year-old man who had AIDS and who presented with an unusual cutaneous vascular lesion, which was clinically thought to be Kaposi's sarcoma. Histologically, the lesion was characterized by capillary proliferation and a mixed inflammatory infiltrate that included numerous histiocytes. The lesion was found to contain slender intracellular acid-fast bacilli, as well as plump extracellular Warthin-Starry-positive bacilli. The acid-fast bacilli were confirmed to be Mycobacterium avium-intracellulare by subsequent positive blood cultures for this organism. To further investigate the lesion, polymerase chain reaction DNA amplification and sequencing was performed, and the lesion was found to contain DNA sequences identical to those previously established for the agent of bacillary angiomatosis. The lesion is thought to represent a lesion of bacillary angiomatosis with secondary involvement by M. avium-intracellulare.

  5. An unusual presentation of Mycobacterium avium spp. paratuberculosis infection in a captive tundra reindeer (Rangifer tarandus tarandus).

    PubMed

    Del-Pozo, J; Girling, S; McLuckie, J; Abbondati, E; Stevenson, K

    2013-07-01

    This report describes an unusual presentation of paratuberculosis in a captive, 4-year-old female tundra reindeer (Rangifer tarandus tarandus). The gross and histological presentation was consistent with clinical paratuberculosis as previously reported for other ruminants, with poor body condition, subcutaneous oedema, granulomatous ileitis (multibacillary), mesenteric lymphadenitis and hepatitis. However, this animal also presented with unusual lung lesions, with necrosis and mineralization similar to that reported for Mycobacterium bovis in other wild and domestic ruminants. The presence of DNA of Mycobacterium avium subsp. paratuberculosis was confirmed by polymerase chain reaction (PCR) in intestine and lung tissue (IS900, Hsp65) and PCR tests for the detection of Mycobacterium tuberculosis complex and other members of the M. avium complex were negative.

  6. Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay.

    PubMed

    Sevilla, Iker A; Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M; Juste, Ramón A

    2015-03-01

    Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.

  7. Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

    PubMed Central

    Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M.

    2015-01-01

    Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses. PMID:25588660

  8. Cloning, sequencing, and expression of the mig gene of Mycobacterium avium, which codes for a secreted macrophage-induced protein.

    PubMed Central

    Plum, G; Brenden, M; Clark-Curtiss, J E; Pulverer, G

    1997-01-01

    Mycobacterium avium is an intracellular pathogen that has evolved to be a frequent cause of disseminated infection in immunocompromised patients. Although these bacilli are readily phagocytized, they are able to survive and even multiply within human macrophages. The process whereby mycobacteria circumvent the lytic functions of the macrophages is currently not well understood, but this is a key aspect in the pathogenicity of all pathogenic mycobacteria. Previously, we identified a gene in M. avium, designated mig (for macrophage-induced gene), the expression of which is induced when the bacilli grow in human macrophages (G. Plum and J. E. Clark-Curtiss, Infect. Immun. 62:476-483, 1994). In the present study we show that (i) the nucleotide sequence of the mig gene has an open reading frame of 295 amino acids with a strong bias for mycobacterial codon usage, (ii) the mig gene also codes for a putative signal peptide of 19 amino acid residues, (iii) mig is induced by acidity to be expressed as an early-secreted 30-kDa protein, and (iv) the Mig protein exhibits an AMP-binding domain signature. However, beyond this motif which is common to enzymes that activate a large variety of substrates, no homologies to known sequences are found. We also show that (v) Mycobacterium smegmatis strains expressing the Mig protein have a limited advantage for survival in macrophages. These findings may be concordant with a role of the mig gene in the virulence of M. avium. PMID:9353032

  9. Apa antigen of Mycobacterium avium subsp. paratuberculosis as a target for species-specific immunodetection of the bacteria in infected tissues of cattle with paratuberculosis.

    PubMed

    Souza, Giliane S; Rodrigues, Ana Bárbara F; Gioffré, Andrea; Romano, Maria I; Carvalho, Eulógio C Q; Ventura, Thatiana L B; Lasunskaia, Elena B

    2011-09-15

    Comparative genomics of Mycobacterium spp. have revealed conservative genes and respective proteins differently expressed in mycobacteria that could be used as targets for the species-specific immunodiagnostics. The alanine and proline-rich antigen Apa is a mycobacterial protein that present significant variability in primary sequence length and composition between members of M. avium and M. tuberculosis complexes. In this study, the recombinant Apa protein encoded by the MAP1569/ModD gene of M. avium subsp. paratuberculosis (Map) was used to generate a panel of monoclonal antibodies which were shown to recognize the most important veterinary pathogens of the M. avium complex, specifically Map and M. avium subsp. hominissuis, and which did not cross-react with M. bovis or M. tuberculosis. The produced antibodies were demonstrated to be a useful tool for the species-specific immunofluorescence or immunohistochemical detection of Map in experimentally infected cell cultures or intestinal tissues from cattle with bovine paratuberculosis and, additionally, they may be employed for the discrimination of pathogenic M. avium subspecies via Western blotting.

  10. A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk.

    PubMed

    Rodríguez-Lázaro, David; Lloyd, Joy; Herrewegh, Arnold; Ikonomopoulos, John; D'Agostino, Martin; Pla, Maria; Cook, Nigel

    2004-08-01

    A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.

  11. Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction.

    PubMed Central

    Oh, Y K; Straubinger, R M

    1996-01-01

    Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages. We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion. M. avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M. avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes. CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios. Surface labeling of M. avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line. By fluorescence spectroscopy, live M. avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M. avium encountered an environmental pH of < 5.0. Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection. pH became more homogeneous 6 h after infection. M. avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate. Opsonized, unopsonized, and IgG-coated M. avium cells entered compartments of similar pH. Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared. Only 18% of live fluorescent M. avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized. Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M. avium. PMID:8557358

  12. Epidemiology and Ecology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa

    PubMed Central

    Hilborn, Elizabeth D.; Arduino, Matthew J.; Pruden, Amy; Edwards, Marc A.

    2015-01-01

    Background Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa are opportunistic premise plumbing pathogens (OPPPs) that persist and grow in household plumbing, habitats they share with humans. Infections caused by these OPPPs involve individuals with preexisting risk factors and frequently require hospitalization. Objectives The objectives of this report are to alert professionals of the impact of OPPPs, the fact that 30% of the population may be exposed to OPPPs, and the need to develop means to reduce OPPP exposure. We herein present a review of the epidemiology and ecology of these three bacterial OPPPs, specifically to identify common and unique features. Methods A Water Research Foundation–sponsored workshop gathered experts from across the United States to review the characteristics of OPPPs, identify problems, and develop a list of research priorities to address critical knowledge gaps with respect to increasing OPPP-associated disease. Discussion OPPPs share the common characteristics of disinfectant resistance and growth in biofilms in water distribution systems or premise plumbing. Thus, they share a number of habitats with humans (e.g., showers) that can lead to exposure and infection. The frequency of OPPP-infected individuals is rising and will likely continue to rise as the number of at-risk individuals is increasing. Improved reporting of OPPP disease and increased understanding of the genetic, physiologic, and structural characteristics governing the persistence and growth of OPPPs in drinking water distribution systems and premise plumbing is needed. Conclusions Because broadly effective community-level engineering interventions for the control of OPPPs have yet to be identified, and because the number of at-risk individuals will continue to rise, it is likely that OPPP-related infections will continue to increase. However, it is possible that individuals can take measures (e.g., raise hot water heater temperatures and filter

  13. Effects of elevated CO(2) on growth and chloroplast proteins in Prunus avium.

    PubMed

    Wilkins, D.; Van Oosten, J.-J.; Besford, R. T.

    1994-01-01

    To predict the future carbon sequestering capacity of trees, we need information about the possible acclimatory mechanisms of plant growth and photosynthesis in rising atmospheric CO(2) under a variety of environmental conditions. We have, therefore, studied the growth response of a tree species (Prunus avium L. Stella (wild cherry)) to elevated CO(2) and characterized the associated changes in photosynthetic machinery of the leaf tissue. Self-pollinated seedlings and mature cuttings (clones) from the same parent plant of P. avium were grown for two consecutive growing seasons (about 60 days each) in ambient CO(2) (350 micro mol mol(-1) CO(2)) or elevated CO(2) (700 micro mol mol(-1) CO(2)) with a high or low nutrient supply. The degree of acclimation of leaf biochemistry and growth response to elevated CO(2) was dependent on the plant material (seedling or mature cutting) and nutrient supply. There was little or no growth response to elevated CO(2) in seedlings or cuttings in the low nutrient supply treatments, whereas, in both seasons, there was a strongly positive growth response to elevated CO(2) in seedlings and cuttings in the high nutrient supply regimes, resulting in increases in the root/shoot ratio and in carbon allocation to the roots. In contrast, the protein content and activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39) were down regulated in elevated CO(2). The loss of Rubisco on an area basis in plants in the elevated CO(2) treatments was compensated for at the canopy level by increased leaf area. The loss of Rubisco protein was accompanied by decreases in the contents of chlorophyll and the thylakoid membrane proteins D(1), D(2) and cytochrome f, which are involved in light harvesting and photo-electron transport. We conclude that, in the medium- to long-term, the initial stimulation of biomass production by elevated CO(2) may be increasingly offset by a lower photosynthetic capacity per unit leaf area in perennial

  14. TGF-β-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates

    PubMed Central

    L'Abbate, Carolina; Cipriano, Ivone; Pérez-Hurtado, Elizabeth Cristina; Leão, Sylvia Cardoso; Carneiro, Célia Regina Whitaker; Machado, Joel

    2011-01-01

    Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth. PMID:21731758

  15. Direct identification and discernment of Mycobacterium avium and Mycobacterium intracellulare using a real-time RNA isothermal amplification and detection method.

    PubMed

    Cui, Zhenling; Li, Yuanyuan; Cheng, Song; Yang, Hua; Lu, Junmei; Zhu, Honglei; Hu, Zhongyi

    2015-12-01

    The purpose of this work was to establish a real-time simultaneous amplification and testing method for identification and discernment of Mycobacterium avium and Mycobacterium intracellulare (SAT-MAC assay) and to evaluate the efficiency with which this method can detect isolated strains and clinical sputum specimens. The specific 16S rRNA sequences of M. avium and M. intracellulare were used as targets to design RNA probes and a reverse transcription primer containing T7 promoter. RNA isothermal amplification and real-time fluorescence detection were performed at 42 °C. SAT-MAC assay, culture tests on Lowenstein-Jensen (L-J) culture medium and PCR-sequencing were used to test the clinical isolated strains and sputum specimens. The limit of detection (LOD) of M. avium and M. intracellulare by SAT-MAC was found to be 30 CFU/mL and 20 CFU/mL. SAT-MAC showed high specificity in 21 species of mycobacteria standard strains and 5 species of non-mycobacteria bacteria. Using PCR-sequencing as the reference method, both rates of SAT-MAC assay for identifying M. avium and M. intracellulare from clinical isolates were 100% (259/259). Consistent with the results of L-J culture combined PCR-sequencing, the coincidence rate of SAT-MAC assay in clinical sputum specimens was 100% (369/369) for M. avium and 99.19% (366/369) for Mycobacterium intracellular. The SAT-MAC assay can identify and distinguish M. avium and M. intracellulare rapidly and accurately. It may be suitable for use in clinical microbiology laboratories.

  16. The Environment of “Mycobacterium avium subsp. hominissuis” Microaggregates Induces Synthesis of Small Proteins Associated with Efficient Infection of Respiratory Epithelial Cells

    PubMed Central

    Babrak, Lmar; Danelishvili, Lia; Rose, Sasha J.; Kornberg, Tiffany

    2014-01-01

    “Mycobacterium avium subsp. hominissuis” is an opportunistic environmental pathogen that causes respiratory illness in immunocompromised patients, such as those with cystic fibrosis as well as other chronic respiratory diseases. Currently, there is no efficient approach to prevent or treat M. avium subsp. hominissuis infection in the lungs. During initial colonization of the airways, M. avium subsp. hominissuis forms microaggregates composed of 3 to 20 bacteria on human respiratory epithelial cells, which provides an environment for phenotypic changes leading to efficient mucosal invasion in vitro and in vivo. DNA microarray analysis was employed to identify genes associated with the microaggregate phenotype. The gene encoding microaggregate-binding protein 1 (MBP-1) (MAV_3013) is highly expressed during microaggregate formation. When expressed in noninvasive Mycobacterium smegmatis, MBP-1 increased the ability of the bacteria to bind to HEp-2 epithelial cells. Using anti-MBP-1 immune serum, microaggregate binding to HEp-2 cells was significantly reduced. By far-Western blotting, and verified by coimmunoprecipitation, we observed that MBP-1 interacts with the host cytoskeletal protein vimentin. As visualized by confocal microscopy, microaggregates, as well as MBP-1, induced vimentin polymerization at the site of bacterium-host cell contact. Binding of microaggregates to HEp-2 cells was inhibited by treatment with an antivimentin antibody, suggesting that MBP-1 expression is important for M. avium subsp. hominissuis adherence to the host cell. MBP-1 immune serum significantly inhibited M. avium subsp. hominissuis infection throughout the respiratory tracts of mice. This study characterizes a pathogenic mechanism utilized by M. avium subsp. hominissuis to bind and invade the host respiratory epithelium, suggesting new potential targets for the development of antivirulence therapy. PMID:25422262

  17. Genetic Structure of Mycobacterium avium subsp. paratuberculosis Population in Cattle Herds in Quebec as Revealed by Using a Combination of Multilocus Genomic Analyses

    PubMed Central

    Sohal, Jagdip Singh; Arsenault, Julie; Labrecque, Olivia; Fairbrother, Julie-Hélène; Roy, Jean-Philippe; Fecteau, Gilles

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis, a granulomatous enteritis affecting a wide range of domestic and wild ruminants worldwide. A variety of molecular typing tools are used to distinguish M. avium subsp. paratuberculosis strains, contributing to a better understanding of M. avium subsp. paratuberculosis epidemiology. In the present study, PCR-based typing methods, including mycobacterial interspersed repetitive units/variable-number tandem repeats (MIRU-VNTR) and small sequence repeats (SSR) in addition to IS1311 PCR-restriction enzyme analysis (PCR-REA), were used to investigate the genetic heterogeneity of 200 M. avium subsp. paratuberculosis strains from dairy herds located in the province of Quebec, Canada. The majority of strains were of the “cattle type,” or type II, although 3 strains were of the “bison type.” A total of 38 genotypes, including a novel one, were identified using a combination of 17 genetic markers, which generated a Simpson's index of genetic diversity of 0.876. Additional analyses revealed no differences in genetic diversity between environmental and individual strains. Of note, a spatial and spatiotemporal cluster was evidenced regarding the distribution of one of the most common genotypes. The population had an overall homogeneous genetic structure, although a few strains stemmed out of the consensus cluster, including the bison-type strains. The genetic structure of M. avium subsp. paratuberculosis populations within most herds suggested intraherd dissemination and microevolution, although evidence of interherd contamination was also revealed. The level of genetic diversity obtained by combining MIRU-VNTR and SSR markers shows a promising avenue for molecular epidemiology investigations of M. avium subsp. paratuberculosis transmission patterns. PMID:24829229

  18. Genetic structure of Mycobacterium avium subsp. paratuberculosis population in cattle herds in Quebec as revealed by using a combination of multilocus genomic analyses.

    PubMed

    Sohal, Jagdip Singh; Arsenault, Julie; Labrecque, Olivia; Fairbrother, Julie-Hélène; Roy, Jean-Philippe; Fecteau, Gilles; L'Homme, Yvan

    2014-08-01

    Mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis, a granulomatous enteritis affecting a wide range of domestic and wild ruminants worldwide. A variety of molecular typing tools are used to distinguish M. avium subsp. paratuberculosis strains, contributing to a better understanding of M. avium subsp. paratuberculosis epidemiology. In the present study, PCR-based typing methods, including mycobacterial interspersed repetitive units/variable-number tandem repeats (MIRU-VNTR) and small sequence repeats (SSR) in addition to IS1311 PCR-restriction enzyme analysis (PCR-REA), were used to investigate the genetic heterogeneity of 200 M. avium subsp. paratuberculosis strains from dairy herds located in the province of Quebec, Canada. The majority of strains were of the "cattle type," or type II, although 3 strains were of the "bison type." A total of 38 genotypes, including a novel one, were identified using a combination of 17 genetic markers, which generated a Simpson's index of genetic diversity of 0.876. Additional analyses revealed no differences in genetic diversity between environmental and individual strains. Of note, a spatial and spatiotemporal cluster was evidenced regarding the distribution of one of the most common genotypes. The population had an overall homogeneous genetic structure, although a few strains stemmed out of the consensus cluster, including the bison-type strains. The genetic structure of M. avium subsp. paratuberculosis populations within most herds suggested intraherd dissemination and microevolution, although evidence of interherd contamination was also revealed. The level of genetic diversity obtained by combining MIRU-VNTR and SSR markers shows a promising avenue for molecular epidemiology investigations of M. avium subsp. paratuberculosis transmission patterns. PMID:24829229

  19. Bovine PGLYRP1 polymorphisms and their association with resistance to Mycobacterium avium ssp. paratuberculosis.

    PubMed

    Pant, S D; Verschoor, C P; Schenkel, F S; You, Q; Kelton, D F; Karrow, N A

    2011-08-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes a chronic, granulomatous inflammatory condition of the intestines in ruminants and wild-type species. It causes significant economic losses to the dairy and beef industries owing to reduced productivity, premature culling and mortality. Bovine peptidoglycan recognition protein 1 is an important pattern recognition molecule that is capable of directly killing microorganisms. The goal of this study was to identify single nucleotide polymorphisms (SNPs) in the gene encoding bovine peptidoglycan recognition protein 1 and to assess their association with susceptibility to MAP infection in dairy cattle. Blood and milk samples were collected from Holsteins in Southwestern and Eastern Ontario and tested for MAP infection using blood and milk ELISAs. A resource population consisting of 197 infected (S/P > 0.25) and 242 healthy (S/P < 0.10) cattle was constructed. Sequencing of pooled DNA was used to identify three SNPs (c.102G>C, c.480G>A and c.625C>A) that were genotyped in the resource population. Statistical analysis was performed using a logistic regression model fitting the additive and dominance effects of each SNP in the model. SNP c.480G>A (P = 0.054) was found to be associated with susceptibility to MAP infection. Cows with a copy of the major allele 'G' at this locus had an odds ratio of 1.51 (95% CI: 0.99-2.31) for being infected with MAP.

  20. The Association of Mycobacterium avium subsp. paratuberculosis with Inflammatory Bowel Disease

    PubMed Central

    Timms, Verlaine J.; Daskalopoulos, George; Mitchell, Hazel M.; Neilan, Brett A.

    2016-01-01

    The association of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) with Crohn’s disease is a controversial issue. M. paratuberculosis is detected by amplifying the IS900 gene, as microbial culture is unreliable from humans. We determined the presence of M. paratuberculosis in patients with Crohn’s disease (CD) (n = 22), ulcerative colitis (UC) (n = 20), aphthous ulcers (n = 21) and controls (n = 42) using PCR assays validated on bovine tissue. Culture from human tissue was also performed. M. paratuberculosis prevalence in the CD and UC groups was compared to the prevalence in age and sex matched non-inflammatory bowel disease controls. Patients and controls were determined to be M. paratuberculosis positive if all three PCR assays were positive. A significant association was found between M. paratuberculosis and Crohn’s disease (p = 0.02) that was not related to age, gender, place of birth, smoking or alcohol intake. No significant association was detected between M. paratuberculosis and UC or aphthous ulcers; however, one M. paratuberculosis isolate was successfully cultured from a patient with UC. We report the resistance of this isolate to ethambutol, rifampin, clofazamine and streptomycin. Interestingly this isolate could not only survive but could grow slowly at 5°C. We demonstrate a significant association between M. paratuberculosis and CD using multiple pre-validated PCR assays and that M. paratuberculosis can be isolated from patients with UC. PMID:26849125

  1. High concentrations of anthocyanins in genuine cherry-juice of old local Austrian Prunus avium varieties.

    PubMed

    Schüller, Elisabeth; Halbwirth, Heidi; Mikulic-Petkovsek, Maja; Slatnar, Ana; Veberic, Robert; Forneck, Astrid; Stich, Karl; Spornberger, Andreas

    2015-04-15

    Antioxidant activity and polyphenols were quantified in vapour-extracted juice of nine Austrian, partially endemic varieties of sweet cherry (Prunus avium): cv. 'Spätbraune von Purbach', cv. 'Early Rivers', cv. 'Joiser Einsiedekirsche', cv. 'Große Schwarze Knorpelkirsche' and four unidentified local varieties. Additionally the effect of storage was evaluated for six of the varieties. A variety showing the highest antioxidant capacity (9.64 μmol Trolox equivalents per mL), total polyphenols (2747 mg/L) and total cyanidins (1085 mg/L) was suitable for mechanical harvest and its juice did not show any losses of antioxidant capacity and total anthocyanin concentration during storage. The juice of cv. 'Große Schwarze Knorpelkirsche' had also high concentrations of total anthocyanins (873 mg/L), but showed substantial losses through storage. The local Austrian sweet cherry varieties from the Pannonian climate zone are particularly suitable for the production of processed products like cherry juice with high content of anthocyanins and polyphenols. PMID:25466109

  2. LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Trangoni, Marcos D.; Gioffré, Andrea K.; Cerón Cucchi, María E.; Caimi, Karina C.; Ruybal, Paula; Zumárraga, Martín J.; Cravero, Silvio L.

    2015-01-01

    In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries. PMID:26273282

  3. Search for Mycobacterium avium Subspecies paratuberculosis Antigens for the Diagnosis of Paratuberculosis

    PubMed Central

    Mon, María Laura; Viale, Mariana; Baschetti, Guido; Alvarado Pinedo, Fiorella; Gioffre, Andrea; Travería, Gabriel; Willemsen, Peter; Bakker, Douwe; Romano, María Isabel

    2012-01-01

    The aim of this study was to evaluate a wide panel of antigens of Mycobacterium avium subsp. paratuberculosis (MAP) to select candidates for the diagnosis of paratuberculosis (PTB). A total of 54 recombinant proteins were spotted onto nitrocellulose membranes and exposed to sera from animals with PTB (n = 25), healthy animals (n = 10), and animals experimentally infected with M. bovis (n = 8). This initial screening allowed us to select seven antigens: MAP 2513, MAP 1693, MAP 2020, MAP 0038, MAP 1272, MAP 0209c, and MAP 0210c, which reacted with sera from animals with PTB and showed little cross-reactivity with sera from healthy animals and animals experimentally infected with M. bovis. The second step was to evaluate the antigen cocktail of these seven antigens by ELISA. For this evaluation, we used sera from animals with PTB (n = 25), healthy animals (n = 26), and animals experimentally infected with M. bovis (n = 17). Using ELISA, the cocktail of the seven selected MAP antigens reacted with sera from 18 of the 25 animals with PTB and did not exhibit cross-reactivity with healthy animals and only low reactivity with animals with bovine tuberculosis. The combined application of these antigens could form part of a test which may help in the diagnosis of PTB. PMID:22792514

  4. Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle.

    PubMed

    Ahlstrom, Christina; Barkema, Herman W; Stevenson, Karen; Zadoks, Ruth N; Biek, Roman; Kao, Rowland; Trewby, Hannah; Haupstein, Deb; Kelton, David F; Fecteau, Gilles; Labrecque, Olivia; Keefe, Greg P; McKenna, Shawn L B; Tahlan, Kapil; De Buck, Jeroen

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne's disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six "Bison type" isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale. PMID:26871723

  5. Persistence of Mycobacterium avium subspecies paratuberculosis in endangered Florida Key deer and Key deer habitat.

    PubMed

    Murray, Heidi L; Yabsley, Michael J; Keel, M Kevin; Manning, Elizabeth J B; Wilmers, Thomas J; Corn, Joseph L

    2014-04-01

    Mycobacterium avium subsp. paratuberculosis (MAP) was first reported in the endangered Key deer (Odocoileus virginianus clavium) in 1996 on Big Pine Key, Florida, USA. By 2008, eight additional MAP-positive Key deer had been identified on Big Pine Key and the nearby Newfound Harbor Keys. This study was conducted to determine if MAP was still present in Key deer and whether natural or man-made freshwater sources were contaminated with MAP. Between November 2009 and September 2012, MAP was isolated from 36/369 (10%) fecal samples collected from the ground throughout the Key deer range on Big Pine Key and the Newfound Harbor Keys, but all 36 positive samples were from Little Palm Island (36/142 [25%]). Only 1/729 (0.1%) environmental samples was positive; this was from the garden fountain on Little Palm Island (1/81 [1%]). In addition, MAP was detected in 3/43 (7%) necropsied Key deer, all from Little Palm Island (3/3 [100%]). Of these three Key deer, pooled samples from the ileum, cecum, and ileocecal lymph node from two were MAP-culture positive and feces from one of these were culture-positive. The third deer was only PCR-positive. Evidence of MAP was only detected on Little Palm Island during this sampling period and environmental contamination was limited.

  6. Composition and Potency Characterization of Mycobacterium avium subsp. paratuberculosis Purified Protein Derivatives

    PubMed Central

    Capsel, Randal T.; Thoen, Charles O.; Reinhardt, Timothy A.; Lippolis, John D.; Olsen, Renee; Stabel, Judith R.; Bannantine, John P.

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain. Traditional production consists of floating culture incubation at 37°C, organism inactivation by autoclaving, coarse filtration, and protein precipitation. Three traditional production PPDs were used in this study including lot 9801, which served as a reference and has been used in the field for decades. Alternative production PPDs (0902A and 0902B), in which the autoclaving step was removed, were also analyzed in this study. SDS-PAGE analysis revealed protein smearing in traditional PPDs, but distinct bands were observed in the alternative PPD preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, whereas an immunoreactive smear was observed with the traditional PPDs. Mass spectrometry identified 194 proteins among three PPD lots representing the two different production methods, ten of which were present in all PPDs examined. Selected proteins identified by mass spectrometry were recombinantly expressed and purified from E. coli and evaluated by the guinea pig potency test. Seven recombinant proteins showed greater erythema as compared to the reference PPD lot 9801 in paired guinea pigs and were able to stimulate interferon-gamma production in blood from Johne’s positive animals. These results suggest that autoclaving culture suspensions is not a necessary step in PPD production and specific proteins could supplant the PPD antigen for intradermal skin testing procedures and for use as in-vitro assay reagents. PMID:27136199

  7. Diuretic effect of powdered Cerasus avium (cherry) tails on healthy volunteers.

    PubMed

    Hooman, Nakysa; Mojab, Faraz; Nickavar, Bahman; Pouryousefi-Kermani, Pouran

    2009-10-01

    In this study, the diuretic activity of powdered cherry stalk was evaluated in 13 healthy volunteers by means of their water balance. In addition to biochemical parameters, such as urinary electrolyte concentration, osmolality and any adverse reaction were determined. The capsules of cherry stalks were administered at an equivalent dose of 2.0 grams of the plant per person. Urinary biochemical determination was made of concentration of electrolytes (sodium, potassium, chloride and calcium), urinary volume and osmolality by standard laboratory procedures. Statistical evaluation was performed by Student's-t and Wilcoxon rank tests. After administration of cherry stalk, the mean of urine calcium, sodium, chloride, and urine volume increased, but the amount of urine potassium and urine osmolality did not change. No adverse reaction was observed. Powdered C. avium stalk increased mild urine volume confirming thus the claimed diuretic effect of the herb. Administration of cherry stalk caused urinary sodium and chloride rising less than loop diuretics but higher than the others. Because of rising calcium excretion, it should be used with cautious in those with urolithiasis. PMID:19783515

  8. Short communication: effect of homogenization on heat inactivation of Mycobacterium avium subspecies paratuberculosis in milk.

    PubMed

    Hammer, P; Kiesner, C; Walte, H-G C

    2014-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) can be present in cow milk and low numbers may survive high-temperature, short-time (HTST) pasteurization. Although HTST treatment leads to inactivation of at least 5 log10 cycles, it might become necessary to enhance the efficacy of HTST by additional treatments such as homogenization if the debate about the role of MAP in Crohn's disease of humans concludes that MAP is a zoonotic agent. This study aimed to determine whether disrupting the clumps of MAP in milk by homogenization during the heat treatment process would enhance the inactivation of MAP. We used HTST pasteurization in a continuous-flow pilot-plant pasteurizer and evaluated the effect of upstream, downstream, and in-hold homogenization on inactivation of MAP. Reduction of MAP at 72°C with a holding time of 28s was between 3.7 and 6.9 log10 cycles, with an overall mean of 5.5 log10 cycles. None of the 3 homogenization modes applied showed a statistically significant additional effect on the inactivation of MAP during HTST treatment.

  9. Association of TLR4 polymorphisms with Mycobacterium avium subspecies paratuberculosis infection status in Canadian Holsteins.

    PubMed

    Sharma, B S; Abo-Ismail, M K; Schenkel, F S; You, Q; Verschoor, C P; Pant, S D; Karrow, N A

    2015-10-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic enteritis in cattle that results in substantial financial losses to the cattle industry worldwide. Given that susceptibility to MAP infection is determined in part by genetics, marker-assisted selection may help in the breeding of animals that are more resistant to MAP infection. The toll-like receptor 4 gene (TLR4) was selected as a potential candidate gene because of its role in innate immunity and its involvement in MAP recognition and infection. The objective of this study, therefore, was to identify associations between TLR4 polymorphisms and susceptibility to MAP infection in Canadian Holstein cows. Two biologically relevant SNPs, including c.-226G>C in the 5'-untranslated region and the non-synonymous SNP c.2021C>T in the potential TIR domain, were selected for an association analysis with MAP infection status in 409 Canadian Holsteins. The haplotype C-T from these combined SNPs yielded significant association with susceptibility to MAP infection, supporting the involvement of TLR4 in susceptibility to MAP infection.

  10. Impact of the shedding level on transmission of persistent infections in Mycobacterium avium subspecies paratuberculosis (MAP).

    PubMed

    Slater, Noa; Mitchell, Rebecca Mans; Whitlock, Robert H; Fyock, Terry; Pradhan, Abani Kumar; Knupfer, Elena; Schukken, Ynte Hein; Louzoun, Yoram

    2016-01-01

    Super-shedders are infectious individuals that contribute a disproportionate amount of infectious pathogen load to the environment. A super-shedder host may produce up to 10,000 times more pathogens than other infectious hosts. Super-shedders have been reported for multiple human and animal diseases. If their contribution to infection dynamics was linear to the pathogen load, they would dominate infection dynamics. We here focus on quantifying the effect of super-shedders on the spread of infection in natural environments to test if such an effect actually occurs in Mycobacterium avium subspecies paratuberculosis (MAP). We study a case where the infection dynamics and the bacterial load shed by each host at every point in time are known. Using a maximum likelihood approach, we estimate the parameters of a model with multiple transmission routes, including direct contact, indirect contact and a background infection risk. We use longitudinal data from persistent infections (MAP), where infectious individuals have a wide distribution of infectious loads, ranging upward of three orders of magnitude. We show based on these parameters that the effect of super-shedders for MAP is limited and that the effect of the individual bacterial load is limited and the relationship between bacterial load and the infectiousness is highly concave. A 1000-fold increase in the bacterial contribution is equivalent to up to a 2-3 fold increase in infectiousness.

  11. Mycobacterium avium subspecies paratuberculosis in the etiology of Crohn’s disease, cause or epiphenomenon?

    PubMed Central

    Liverani, Elisa; Scaioli, Eleonora; Cardamone, Carla; Dal Monte, Paola; Belluzzi, Andrea

    2014-01-01

    The origin of inflammatory bowel disease is unknown. Attempts have been made to isolate a microorganism that could explain the onset of inflammation, but no pathological agent has ever been identified. Johne’s disease is a granulomatous chronic enteritis of cattle and sheep caused by Mycobacterium avium subspecies paratuberculosis (MAP) and shows some analogies with Crohn’s disease (CD). Several studies have tried to clarify if MAP has a role in the etiology of CD. The present article provides an overview of the evidence in favor and against the “MAP-hypothesis”, analyzing the methods commonly adopted to detect MAP and the role of antimycobacterial therapy in patients with inflammatory bowel disease. Studies were identified through the electronic database, MEDLINE, and were selected based on their relevance to the objective of the review. The presence of MAP was investigated using multiple diagnostic methods for MAP detection and in different tissue samples from patients affected by CD or ulcerative colitis and in healthy controls. On the basis of their studies, several authors support a close relationship between MAP and CD. Although increasing evidence of MAP detection in CD patients is unquestionable, a clear etiological link still needs to be proven. PMID:25278700

  12. Short communication: Viable Mycobacterium avium subspecies paratuberculosis in retail artisanal Coalho cheese from Northeastern Brazil.

    PubMed

    Faria, A C S; Schwarz, D G G; Carvalho, I A; Rocha, B B; De Carvalho Castro, K N; Silva, M R; Moreira, M A S

    2014-07-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis and it potentially plays a role in Crohn's disease. In humans, the main route of transmission of MAP might be the intake of contaminated milk and dairy products. Considering that MAP has already been detected in many types of cheese in different counties, and that Coalho cheese is an important dairy product in northeastern Brazil, the aim of this study was to report the first detection of MAP in retail Coalho cheese in Brazil by PCR and culture. Of 30 retail Coalho cheese samples, 3 (10%) amplified fragments of a similar size to that expected (626 bp) were obtained and viable MAP was recovered by culture from 1 (3.3%) sample. The DNA from the positive culture sample was sequenced and showed 99% identity with the insertion sequence IS900 deposited in GenBank. It was possible to identify the presence of MAP-specific DNA in the analyzed samples for the first time in Brazil, and to recover viable cells from retail Coalho cheese.

  13. Mycobacterium avium complex--the role of potable water in disease transmission.

    PubMed

    Whiley, H; Keegan, A; Giglio, S; Bentham, R

    2012-08-01

    Mycobacterium avium complex (MAC) is a group of opportunistic pathogens of major public health concern. It is responsible for a wide spectrum of disease dependent on subspecies, route of infection and patients pre-existing conditions. Presently, there is limited research on the incidence of MAC infection that considers both pulmonary and other clinical manifestations. MAC has been isolated from various terrestrial and aquatic environments including natural waters, engineered water systems and soils. Identifying the specific environmental sources responsible for human infection is essential in minimizing disease prevalence. This paper reviews current literature and case studies regarding the wide spectrum of disease caused by MAC and the role of potable water in disease transmission. Potable water was recognized as a putative pathway for MAC infection. Contaminated potable water sources associated with human infection included warm water distribution systems, showers, faucets, household drinking water, swimming pools and hot tub spas. MAC can maintain long-term contamination of potable water sources through its high resistance to disinfectants, association with biofilms and intracellular parasitism of free-living protozoa. Further research is required to investigate the efficiency of water treatment processes against MAC and into construction and maintenance of warm water distribution systems and the role they play in MAC proliferation.

  14. Rapid susceptibility testing of mycobacterium avium complex and mycobacterium tuberculosis isolated from AIDS patients

    NASA Technical Reports Server (NTRS)

    Dhople, Arvind M.

    1993-01-01

    In ominous projections issued by both U.S. Public Health Service and the World Health Organization, the epidemic of the Human Immunodeficiency Virus (HIV) infection will continue to rise more rapidly worldwide than predicted earlier. The Acquired Immunodeficiency Syndrome (AIDS) patients are susceptible to diseases called opportunistic infections of which tuberculosis and M. avium Complex (MAC) infection are most common. This has created an urgent need to uncover new drugs for the treatment of these infections. In the seventies, NASA scientists at Goddard Space Flight Center, Greenbelt, Maryland, had adopted a biochemical indicator, adenosine triphosphate (ATP), to detect presence of life in extraterrestrial space. Therefore, we proposed to develop ATP assay technique to determine sensitivity of antibacterial compounds against MAC and M. tuberculosis. The work was initiated in June 1992. In the last report, we described our efforts in developing ATP assay method using MAC. Studies were continued further, and during the period of this report, we established the relationship between colony forming units and ATP levels of these organisms during the growth cycle. Also, we evaluated the effects of standard antimycobacterial drugs using ATP assay technique and compared the results with those obtained with conventional tube dilution proportional method.

  15. Mycobacterium avium Subspecies paratuberculosis: Human Exposure through Environmental and Domestic Aerosols

    PubMed Central

    Rhodes, Glenn; Richardson, Hollian; Hermon-Taylor, John; Weightman, Andrew; Higham, Andrew; Pickup, Roger

    2014-01-01

    Mycobacterium avium subspecies paratuberculosis (Map) causes Johne’s disease in animals and is significantly associated with Crohn’s disease (CD) in humans. Our previous studies have shown Map to be present in U.K. rivers due to land deposition from chronic livestock infection and runoff driven by rainfall. The epidemiology of CD in Cardiff showed a significant association with the River Taff, in which Map can be detected on a regular basis. We have previously hypothesized that aerosols from the river might influence the epidemiology of CD. In this preliminary study, we detected Map by quantitative PCR in one of five aerosol samples collected above the River Taff. In addition, we examined domestic showers from different regions in the U.K. and detected Map in three out of 30 independent samples. In detecting Map in river aerosols and those from domestic showers, this is the first study to provide evidence that aerosols are an exposure route for Map to humans and may play a role in the epidemiology of CD. PMID:25438013

  16. Experimental Infection of Goats with Mycobacterium avium subsp. hominissuis: a Model for Comparative Tuberculosis Research.

    PubMed

    Schinköthe, J; Möbius, P; Köhler, H; Liebler-Tenorio, E M

    2016-01-01

    Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic pathogen that causes infections in man and animals. In this study, 18 goat kids were inoculated orally with a high dose of MAH. One group of goats (n = 9) developed severe clinical disease for up to 2-3 months post inoculation (mpi). At necropsy examination, there were ulcerative and granulomatous lesions in gut-associated lymphoid tissue and granulomas with extensive necrosis in the lymph nodes (LNs) of the cranial mesenteric lymphocentre (CMLNs). Culture revealed growth of MAH in all lesions with systemic spread. A second group of goats were healthy at the end of the trial (13 mpi); however, all had extensive granulomas in the CMLNs, but no extra-intestinal spread of bacteria. Moderate faecal shedding occurred in all goats up to 2 mpi. Microscopical characterization of the granulomas revealed solid non-necrotic, necrotic, calcified and fibrocalcified granulomas with resemblance to those seen in human and bovine tuberculosis. The two different courses of disease, with highly heterogenic lesions, systemic spread in goats with severe clinical disease and the development of granulomas of all stages in the surviving goats, makes the experimental infection of goats with MAH a valuable model for tuberculosis research. This model might allow new insights into host-pathogen interaction and anti-mycobacterial compound testing. PMID:27426001

  17. Mycobacterium avium ss. paratuberculosis Zoonosis - The Hundred Year War - Beyond Crohn's Disease.

    PubMed

    Sechi, Leonardo A; Dow, Coad Thomas

    2015-01-01

    The factitive role of Mycobacterium avium ss. paratuberculosis (MAP) in Crohn's disease has been debated for more than a century. The controversy is due to the fact that Crohn's disease is so similar to a disease of MAP-infected ruminant animals, Johne's disease; and, though MAP can be readily detected in the infected ruminants, it is much more difficult to detect in humans. Molecular techniques that can detect MAP in pathologic Crohn's specimens as well as dedicated specialty labs successful in culturing MAP from Crohn's patients have provided strong argument for MAP's role in Crohn's disease. Perhaps more incriminating for MAP as a zoonotic agent is the increasing number of diseases with which MAP has been related: Blau syndrome, type 1 diabetes, Hashimoto thyroiditis, and multiple sclerosis. In this article, we debate about genetic susceptibility to mycobacterial infection and human exposure to MAP; moreover, it suggests that molecular mimicry between protein epitopes of MAP and human proteins is a likely bridge between infection and these autoimmune disorders.

  18. Persistence of Mycobacterium avium subspecies paratuberculosis in endangered Florida Key deer and Key deer habitat.

    PubMed

    Murray, Heidi L; Yabsley, Michael J; Keel, M Kevin; Manning, Elizabeth J B; Wilmers, Thomas J; Corn, Joseph L

    2014-04-01

    Mycobacterium avium subsp. paratuberculosis (MAP) was first reported in the endangered Key deer (Odocoileus virginianus clavium) in 1996 on Big Pine Key, Florida, USA. By 2008, eight additional MAP-positive Key deer had been identified on Big Pine Key and the nearby Newfound Harbor Keys. This study was conducted to determine if MAP was still present in Key deer and whether natural or man-made freshwater sources were contaminated with MAP. Between November 2009 and September 2012, MAP was isolated from 36/369 (10%) fecal samples collected from the ground throughout the Key deer range on Big Pine Key and the Newfound Harbor Keys, but all 36 positive samples were from Little Palm Island (36/142 [25%]). Only 1/729 (0.1%) environmental samples was positive; this was from the garden fountain on Little Palm Island (1/81 [1%]). In addition, MAP was detected in 3/43 (7%) necropsied Key deer, all from Little Palm Island (3/3 [100%]). Of these three Key deer, pooled samples from the ileum, cecum, and ileocecal lymph node from two were MAP-culture positive and feces from one of these were culture-positive. The third deer was only PCR-positive. Evidence of MAP was only detected on Little Palm Island during this sampling period and environmental contamination was limited. PMID:24506424

  19. FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis

    PubMed Central

    Eckelt, Elke; Meißner, Thorsten; Meens, Jochen; Laarmann, Kristin; Nerlich, Andreas; Jarek, Michael; Weiss, Siegfried; Gerlach, Gerald-F.; Goethe, Ralph

    2015-01-01

    The ferric uptake regulator A (FurA) is known to be involved in iron homeostasis and stress response in many bacteria. In mycobacteria the precise role of FurA is still unclear. In the presented study, we addressed the functional role of FurA in the ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) by construction of a furA deletion strain (MAPΔfurA). RNA deep sequencing revealed that the FurA regulon consists of repressed and activated genes associated to stress response or intracellular survival. Not a single gene related to metal homeostasis was affected by furA deletion. A decisive role of FurA during intracellular survival in macrophages was shown by significantly enhanced survival of MAPΔfurA compared to the wildtype, indicating that a principal task of mycobacterial FurA is oxidative stress response regulation in macrophages. This resistance was not associated with altered survival of mice after long term infection with MAP. Our results demonstrate for the first time, that mycobacterial FurA is not involved in the regulation of iron homeostasis. However, they provide strong evidence that FurA contributes to intracellular survival as an oxidative stress sensing regulator. PMID:25705205

  20. Host Transcriptional Profiles and Immunopathologic Response following Mycobacterium avium subsp. paratuberculosis Infection in Mice

    PubMed Central

    Shin, Min-Kyoung; Park, Hongtae; Shin, Seung Won; Jung, Myunghwan; Lee, Su-Hyung; Kim, Dae-Yong; Yoo, Han Sang

    2015-01-01

    Paratuberculosis or Johne’s disease is a chronic granulomatous enteropathy in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. In the present study, we examined the host response to MAP infection in spleens of mice in order to investigate the host immunopathology accompanying host-pathogen interaction. Transcriptional profiles of the MAP-infected mice at 3 and 6 weeks p.i. showed severe histopathological changes, whereas those at 12 weeks p.i. displayed reduced lesion severity in the spleen and liver. MAP-infected mice at 3 and 6 weeks p.i. showed up-regulation of interferon-related genes, scavenger receptor, and complement components, suggesting an initial innate immune reaction, such as macrophage activation, bactericidal activity, and macrophage invasion of MAP. Concurrently, MAP-infected mice at 3 and 6 weeks p.i. were also suggested to express M2 macrophage phenotype with up-regulation of Mrc1, and Marco and down-regulation of MHC class II, Ccr7, and Irf5, and canonical pathways related to the T cell response including ICOS-ICOSL signaling in T helper cells, calcium-induced T lymphocyte apoptosis, and CD28 signaling in T helper cell. These results provide information which furthers the understanding of the immunopathologic response to MAP infection in mice, thereby providing insights valuable for research into the pathogenesis for MAP infection. PMID:26439498

  1. Isolation and characterization of a novel bacteriophage against Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Basra, Simone; Anany, Hany; Brovko, Lioubov; Kropinski, Andrew M; Griffiths, Mansel W

    2014-10-01

    Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, has a doubling time of 24 hours, making rapid detection very difficult. Mycobacteriophages can be used in the detection of disease-causing mycobacteria such as MAP. Isolation and sequencing the genomes of lytic MAP bacteriophages are important preliminary steps towards designing phage-based rapid detection assays for this bacterium. A simple optimized protocol was developed to allow reproducible production of confluent growth of MAP on plates within four to six weeks of incubation at 30 °C. This protocol was applied to the screening of environmental and fecal samples for bacteriophages inhibiting the growth of MAP. As a result, a lytic phage, vB_MapS_FF47, was isolated from bovine feces. FF47 contains a double-stranded DNA genome ~48 kb in length with 73 protein coding sequences. It does not carry temperate or known virulence genes. This phage was shown to be most closely related to Mycobacterium phage Muddy, isolated in South Africa, and Gordonia phage GTE2; however, it could not infect any of the tested Gordonia, Rhodococcus, or Nocardia spp. that GTE2 could. The protocols that were developed for growth and phage isolation have potential applications in a high-throughput screening for compounds inhibiting the growth of MAP. This work describes the first time that a phage was isolated against M. paratuberculosis.

  2. Ecology and genomic features of infection with Mycobacterium avium subspecies paratuberculosis in Egypt.

    PubMed

    Amin, Adel S; Hsu, Chung-Yi; Darwish, Samah F; Ghosh, Pallab; AbdEl-Fatah, Eman M; Behour, Tahani S; Talaat, Adel M

    2015-04-01

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, or Johne's disease, in cattle, with potential involvement in cases of Crohn's disease in humans. Johne's disease is found worldwide and is economically important for both beef and dairy industries. In an effort to characterize this important infection in Egypt, we analysed the ecological and genomic features of recent isolates of M. paratuberculosis. In this report, we examined 26 Holstein dairy herds distributed throughout Egypt, from 2010 to 2013. Using PCR analysis of faecal samples, we estimated a mean herd-level prevalence of 65.4 %, with animal-level infection that reached a mean of 13.6 % among animals suffering from diarrhoea. Whole genome sequencing of field isolates identified numerous single nucleotide polymorphisms among field isolates relative to the standard M. paratuberculosis K10 genome. Interestingly, the virulence of M. paratuberculosis isolates from Egypt revealed diverse virulence phenotypes in the murine model of paratuberculosis, with significant differences in tissue colonization, particularly during the chronic stage of infection. Overall, our analysis confirmed that Johne's disease is a newly identified problem in Egypt and indicated that M. paratuberculosis has potentially diverse genotypes that impact its virulence. Further ecological mapping and genomic analysis of M. paratuberculosis will enhance our understanding of the transmission and evolutionary dynamics of this pathogen under natural field conditions.

  3. Isolation of Mycobacterium avium complex from bone marrow aspirates of AIDS patients in Brazil.

    PubMed

    Barreto, J A; Palaci, M; Ferrazoli, L; Martins, M C; Suleiman, J; Lorenço, R; Ferreira, O C; Riley, L W; Johnson, W D; Galvão, P A

    1993-09-01

    Mycobacterium avium complex (MAC) infection has not been reported as a major opportunistic infection among patients with AIDS in Latin America or Africa. In this study, 125 AIDS patients who had persistent fever, anemia, and leukopenia were examined among 2628 AIDS patients admitted to Instituto de Infectologia Emilio Ribas between May 1990 and April 1992. From the bone marrow aspirates of the 125 patients, MAC was isolated from 23 (18.4%) and Mycobacterium tuberculosis was isolated from 9 (7.2%). Between 1985 and 1990, only 11 MAC isolations among 60,000 cultures obtained from human immunodeficiency virus-seronegative patients were documented in São Paulo. Hence, the minimal estimated rate of MAC infection in AIDS patients in this city was 23/2628, or 0.88%. These findings suggest that MAC infection is an important opportunistic infection, especially among a subset of patients with AIDS in Brazil who have clinical characteristics and risk activities similar to those associated with MAC infections in North America and Europe.

  4. Assessment of food as a source of exposure to Mycobacterium avium subspecies paratuberculosis (MAP).

    PubMed

    2010-07-01

    The National Advisory Committee on Microbiological Criteria for Foods assessed the importance of food as a source of exposure to Mycobacterium avium subspecies paratuberculosis (MAP). MAP is the causative agent of Johne's disease, which affects primarily the small intestine of all ruminants. The significance of MAP as a human pathogen is unknown and is being investigated by several research groups. This document also reviews the efficacy of current detection methods, processing interventions, and MAP inactivation. Research needs related to MAP are provided. The Committee reached the following conclusions: current methods for detection of MAP have significant limitations, and a standard method for the detection of viable MAP cells is needed. Aside from MAP-infected domestic ruminant animals, the organism is found infrequently. If MAP in cattle is controlled, the source of MAP in other animals, food, and water may largely be eliminated. Milk, particularly raw milk, may be a likely food source for human exposure to MAP. Given the prevalence of MAP in U.S. cattle herds, ground beef may be a potential source of MAP. Although humans may be exposed to MAP through a variety of routes, including food and the environment, the frequency and amount of exposure will require additional research. PMID:20615354

  5. Mycobacterium avium ss. paratuberculosis Zoonosis – The Hundred Year War – Beyond Crohn’s Disease

    PubMed Central

    Sechi, Leonardo A.; Dow, Coad Thomas

    2015-01-01

    The factitive role of Mycobacterium avium ss. paratuberculosis (MAP) in Crohn’s disease has been debated for more than a century. The controversy is due to the fact that Crohn’s disease is so similar to a disease of MAP-infected ruminant animals, Johne’s disease; and, though MAP can be readily detected in the infected ruminants, it is much more difficult to detect in humans. Molecular techniques that can detect MAP in pathologic Crohn’s specimens as well as dedicated specialty labs successful in culturing MAP from Crohn’s patients have provided strong argument for MAP’s role in Crohn’s disease. Perhaps more incriminating for MAP as a zoonotic agent is the increasing number of diseases with which MAP has been related: Blau syndrome, type 1 diabetes, Hashimoto thyroiditis, and multiple sclerosis. In this article, we debate about genetic susceptibility to mycobacterial infection and human exposure to MAP; moreover, it suggests that molecular mimicry between protein epitopes of MAP and human proteins is a likely bridge between infection and these autoimmune disorders. PMID:25788897

  6. A population genetic analysis of chloroplast DNA in wild populations of Prunus avium L. in Europe.

    PubMed

    Mohanty, A; Martín, J P; Aguinagalde, I

    2001-10-01

    A population genetic study of chloroplast DNA was carried out in 23 wild populations of Prunus avium sampled from several European deciduous forests. An analysis of approx. 9% of the chloroplast genome detected mostly insertion-deletion mutations and one point mutation. In all, 16 haplotypes were detected. Six haplotypes were shared by two or more populations and 10 were unique. One haplotype was present in 21 of the 23 populations and 161 of 211 individuals, which probably indicates its ancient origin. The level of population subdivision, using unordered and ordered alleles, was low, GSTC=0.29 and NSTC=0.33, respectively. The difference between GSTC and NSTC is nonsignificant, indicating an absence of correlation between haplotype phylogeny and geographical distribution. The absence of phylogeographic structure in wild cherry may be attributed to long distance gene flow among populations by birds, animals and anthropogenic activities. The minimum-length spanning tree depicting the phylogenetic relationships between the haplotypes indicates the possible existence of two lineages represented by the haplotypes H3 and H4. The information about homogeneity or heterogeneity of populations in terms of haplotype constitution and detection of rare haplotypes in some populations will be useful for formulation of conservation and management strategies of wild cherry.

  7. Survey of Mycobacterium avium subspecies paratuberculosis in road-killed wild carnivores in Portugal.

    PubMed

    Matos, Ana Cristina; Figueira, Luis; Martins, Maria Helena; Loureiro, Filipa; Pinto, Maria Lurdes; Matos, Manuela; Coelho, Ana Cláudia

    2014-12-01

    A survey to determine the occurrence of Mycobacterium avium subsp. paratuberculosis (MAP) in wild carnivores in Portugal was conducted by testing samples from road-killed animals between 2009 and 2012. Postmortem examinations were performed and tissues were collected from wild carnivores representing four families and six different species, with a total of 74 animals analyzed. Cultures were performed by using Löwenstein-Jensen and Middlebrook 7H11 solid media and acid-fast isolates were identified by polymerase chain reaction (PCR) and mycobactin dependency characteristics. Tissues were also screened for MAP by directly extracting DNA and testing for the MAP-specific sequences. The occurrence of infected animals (an animal had at least one tissue that was positive for culture or direct PCR) was 27.0% (n = 20). MAP was isolated from culture of 25 tissue samples (3.8%) and was detected by direct PCR in 40 (6.0%) samples. Infection was recorded in 5/6 studied species: 7/49 (14.3%) red foxes (Vulpes vulpes), 3/3 (100%) beech martens (Martes foina), 2/4 (50.0%) Eurasian otters (Lutra lutra), 7/15 (46.7%) Egyptian mongooses (Herpestes ichneumon), and 1/1 (100%) European badger (Meles meles). These species represent three different taxonomic families: Canidae (14.3% were positive), Mustelidae (75.0% were positive), and Herpestidae (46.7% were positive). The results of this study confirm the presence of MAP infection in wild carnivores in Portugal. PMID:25632662

  8. Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

    PubMed Central

    Johnston, Christopher D.; Bannantine, John P.; Govender, Rodney; Endersen, Lorraine; Pletzer, Daniel; Weingart, Helge; Coffey, Aidan; O'Mahony, Jim; Sleator, Roy D.

    2014-01-01

    It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease. PMID:25237653

  9. Effect of temperature on pollen tube kinetics and dynamics in sweet cherry, Prunus avium (Rosaceae).

    PubMed

    Hedhly, A; Hormaza, J I; Herrero, M

    2004-04-01

    Prevailing ambient temperature during the reproductive phase is one of several important factors for seed and fruit set in different plant species, and its consequences on reproductive success may increase with global warming. The effect of temperature on pollen performance was evaluated in sweet cherry (Prunus avium L.), comparing as pollen donors two cultivars that differ in their adaptation to temperature. 'Sunburst' is a cultivar that originated in Canada with a pedigree of cultivars from Northern Europe, while 'Cristobalina' is a cultivar native to southeast Spain, adapted to warmer conditions. Temperature effects were tested either in controlled-temperature chambers or in the field in a plastic cage. In both genotypes, an increase in temperature reduced pollen germination, but accelerated pollen tube growth. However, a different genotypic response, which reflected the overall adaptation of the pollen donor, was obtained for pollen tube dynamics, expressed as the census of the microgametophyte population that successfully reached the base of the style. While both cultivars performed similarly at 20°C, the microgametophyte population was reduced at 30°C for Sunburst and at 10°C for Cristobalina. These results indicate a differential genotypic response to temperature during the reproductive phase, which could be important in terms of the time needed for a plant species to adapt to rapid temperature changes.

  10. FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis.

    PubMed

    Eckelt, Elke; Meißner, Thorsten; Meens, Jochen; Laarmann, Kristin; Nerlich, Andreas; Jarek, Michael; Weiss, Siegfried; Gerlach, Gerald-F; Goethe, Ralph

    2015-01-01

    The ferric uptake regulator A (FurA) is known to be involved in iron homeostasis and stress response in many bacteria. In mycobacteria the precise role of FurA is still unclear. In the presented study, we addressed the functional role of FurA in the ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) by construction of a furA deletion strain (MAPΔfurA). RNA deep sequencing revealed that the FurA regulon consists of repressed and activated genes associated to stress response or intracellular survival. Not a single gene related to metal homeostasis was affected by furA deletion. A decisive role of FurA during intracellular survival in macrophages was shown by significantly enhanced survival of MAPΔfurA compared to the wildtype, indicating that a principal task of mycobacterial FurA is oxidative stress response regulation in macrophages. This resistance was not associated with altered survival of mice after long term infection with MAP. Our results demonstrate for the first time, that mycobacterial FurA is not involved in the regulation of iron homeostasis. However, they provide strong evidence that FurA contributes to intracellular survival as an oxidative stress sensing regulator.

  11. Macrophage polarization in cattle experimentally exposed to Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Thirunavukkarasu, Shyamala; de Silva, Kumudika; Begg, Douglas J; Whittington, Richard J; Plain, Karren M

    2015-12-01

    Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in cattle, has significant impacts on the livestock industry and has been implicated in the etiology of Crohn's disease. Macrophages play a key role in JD pathogenesis, which is driven by the manipulation of host immune mechanisms by MAP. A change in the macrophage microenvironment due to pathogenic or host-derived stimuli can lead to classical (M1) or alternative (M2) polarization of macrophages. In addition, prior exposure to antigenic stimuli has been reported to alter the response of macrophages to subsequent stimuli. However, macrophage polarization in response to MAP exposure and its possible implications have not been previously addressed. In this study, we have comprehensively examined monocyte/macrophage polarization and responsiveness to antigens from MAP-exposed and unexposed animals. At 3 years post-exposure, there was a heterogeneous macrophage activation pattern characterized by both classical and alternate phenotypes. Moreover, subsequent exposure of macrophages from MAP-exposed cattle to antigens from MAP and other mycobacterial species led to significant variation in the production of nitric oxide, interleukin-10 and tumour necrosis factor α. These results indicate the previously unreported possibility of changes in the activation state and responsiveness of circulating monocytes/macrophages from MAP-exposed cattle.

  12. On deaf ears, Mycobacterium avium paratuberculosis in pathogenesis Crohn’s and other diseases

    PubMed Central

    Davis, William C

    2015-01-01

    The historic suggestion that Mycobacterium avium subsp. paratuberculosis (Map) might be a zoonotic pathogen was based on the apparent similarity of lesions in the intestine of patients with Crohn’s disease (CD) with those present in cattle infected with Map, the etiological agent of Johne’s disease. Reluctance to fully explore this possibility has been attributed to the difficulty in demonstrating the presence of Map in tissues from patients with CD. Advances in technology have resolved this problem and revealed the presence of Map in a significant proportion of patients with CD and other diseases. The seminal finding from recent investigations, however, is the detection of Map in healthy individuals with no clinical signs of disease. The latter observation indicates all humans are susceptible to infection with Map and lends support to the thesis that Map is zoonotic, with a latent stage of infection similar to tuberculosis, where infection leads to the development of an immune response that controls but does not eliminate the pathogen. This clarifies one of the reasons why it has been so difficult to document that Map is zoonotic and associated with the pathogenesis of CD and other diseases. As discussed in the present review, a better understanding of the immune response to Map is needed to determine how infection is usually kept under immune control during the latent stage of infection and elucidate the triggering events that lead to disease progression in the natural host and pathogenesis of CD and immune related diseases in humans. PMID:26730151

  13. Host gene expression for Mycobacterium avium subsp. paratuberculosis infection in human THP-1 macrophages.

    PubMed

    Shin, Min-Kyoung; Shin, Seung Won; Jung, Myunghwan; Park, Hongtae; Park, Hyun-Eui; Yoo, Han Sang

    2015-07-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, which causes considerable economic loss in the dairy industry and has a possible relationship to Crohn's disease (CD) in humans. As MAP has been detected in retail pasteurized milk samples, its transmission via milk is of concern. Despite its possible role in the etiology of CD, there have been few studies examining the interactions between MAP and human cells. In the current study, we applied Ingenuity Pathway Analysis to the transcription profiles generated from a murine model with MAP infection as part of a previously conducted study. Twenty-one genes were selected as potential host immune responses, compared with the transcriptional profiles in naturally MAP-infected cattle, and validated in MAP-infected human monocyte-derived macrophage THP-1 cells. Of these, the potential host responses included up-regulation of genes related to immune response (CD14, S100A8, S100A9, LTF, HP and CHCIL3), up-regulation of Th1-polarizing factor (CCL4, CCL5, CXCL9 and CXCL10), down-regulation of genes related to metabolism (ELANE, IGF1, TCF7L2 and MPO) and no significant response of other genes (GADD45a, GPNMB, HMOX1, IFNG and NQO1) in THP-1 cells infected with MAP. PMID:25877879

  14. Detection of Mycobacterium avium subspecies paratuberculosis in several herds of Arctic Caribou (Rangifer tarandus ssp.).

    PubMed

    Forde, Taya; Orsel, Karin; De Buck, Jeroen; Côté, Steeve D; Cuyler, Christine; Davison, Tracy; Elkin, Brett; Kelly, Allicia; Kienzler, Martin; Popko, Richard; Taillon, Joëlle; Veitch, Alasdair; Kutz, Susan

    2012-10-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is a common pathogen in domestic ruminants that causes granulomatous inflammation of the small intestine leading to emaciation and wasting. Clinical disease (Johne's disease) is also reported for several wild ruminant species. Between 2007 and 2009 we collected 561 fecal samples from caribou (Rangifer tarandus ssp.) representing 10 herds of migratory caribou, two herds of caribou from Greenland, and three populations of boreal woodland caribou. Feces were tested for MAP by bacterial culture and PCR targeting the IS900 insertion sequence. In total, 31 samples from eight different populations representing all three ecotypes were found positive for MAP by PCR, with one sample from the Rivière-aux-Feuilles herd also being culture positive for the type II (cattle) strain. The proportion of positive animals was particularly high in the Akia-Maniitsoq herd in Greenland, and Rivière-aux-Feuilles and Riviè re-George herds in northeastern Canada (23.4, 11.5, and 10.0%, respectively). Our results indicate that MAP is present in several caribou herds of different ecotypes in northern Canada and Greenland and that MAP circulates within wildlife populations that do not have ongoing contact with domestic livestock. The epidemiology, pathogenicity, and effects on the health of caribou in northern ecosystems remain unknown.

  15. Seroprevalence and risk factors of Mycobacterium avium subspecies paratuberculosis infection in domestic sika deer in China.

    PubMed

    Meng, Qing-Feng; Li, Ying; Yang, Fan; Yao, Gui-Zhi; Qian, Ai-Dong; Wang, Wei-Li; Cong, Wei

    2015-06-01

    Paratuberculosis or Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic infectious granulomatous enteritis of ruminants and other animals, which has a worldwide occurrence, but little is known of MAP infection in domestic sika deer in Jilin Province, China. The objective of the present investigation was to examine seroprevalence and risk factors of MAP infection in Jilin Province. Serum samples collected from 1400 sika deer from 16 sika deer herds were collected in the 4 districts of the province between May 2013 and August 2014 and were tested independently for the presence of antibodies against MAP. A total of 247 (17.64 %) sika deer tested positive for MAP antibodies using a commercially available enzyme immunoassay kit. The management level of farm and collecting region of sika deer was the main risk factor associated with MAP infection. The present study revealed the seroprevalence of MAP infection in sika deer in Jilin Province, China, which provided the baseline data for taking comprehensive countermeasures and measures in effectively preventing and controlling MAP infection in sika deer.

  16. Seroprevalence and risk factors of Mycobacterium avium subspecies paratuberculosis infection in domestic sika deer in China.

    PubMed

    Meng, Qing-Feng; Li, Ying; Yang, Fan; Yao, Gui-Zhi; Qian, Ai-Dong; Wang, Wei-Li; Cong, Wei

    2015-06-01

    Paratuberculosis or Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic infectious granulomatous enteritis of ruminants and other animals, which has a worldwide occurrence, but little is known of MAP infection in domestic sika deer in Jilin Province, China. The objective of the present investigation was to examine seroprevalence and risk factors of MAP infection in Jilin Province. Serum samples collected from 1400 sika deer from 16 sika deer herds were collected in the 4 districts of the province between May 2013 and August 2014 and were tested independently for the presence of antibodies against MAP. A total of 247 (17.64 %) sika deer tested positive for MAP antibodies using a commercially available enzyme immunoassay kit. The management level of farm and collecting region of sika deer was the main risk factor associated with MAP infection. The present study revealed the seroprevalence of MAP infection in sika deer in Jilin Province, China, which provided the baseline data for taking comprehensive countermeasures and measures in effectively preventing and controlling MAP infection in sika deer. PMID:25904509

  17. Mycobacterium avium subsp. paratuberculosis is not discerned in diabetes mellitus patients in Hyderabad, India.

    PubMed

    Rani, Pittu Sandhya; Doddam, Sankara Narayana; Agrawal, Sashank; Hasnain, Seyed E; Sechi, Leonardo A; Kumar, Ashutosh; Ahmed, Niyaz

    2014-07-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is an obligate intracellular pathogen. It causes chronic intestinal inflammation in ruminants known as Johne's disease and is associated with human Crohn's disease. Furthermore, association of MAP with other autoimmune diseases, such as type-1 diabetes, has been established in patients from Sardinia (Italy) which is a MAP endemic and genetically isolated region. Due to largest livestock population and consequently high MAP prevalence amidst a very high diabetes incidence in India, we sought to test this association on a limited number of patient samples from Hyderabad. Our results of ELISA with MAP lysate and MAP-specific protein MAP3738c as well as PCR/real-time PCR of MAP-specific sequences IS900 and/or f57 indicated that, in contrast to Sardinian diabetic patients, MAP infection in blood is not discerned in diabetic patients in Hyderabad. The association of a mycobacterial trigger with diabetes therefore could well be a population-specific phenomenon, highly dependent on genetic repertoire and the environment of susceptible populations. However, a larger study is needed in order to confirm this.

  18. Genomic variations associated with attenuation in Mycobacterium avium subsp. paratuberculosis vaccine strains

    PubMed Central

    2013-01-01

    Background Mycobacterium avium subspecies paratuberculosis (MAP) whole cell vaccines have been widely used tools in the control of Johne’s disease in animals despite being unable to provide complete protection. Current vaccine strains derive from stocks created many decades ago; however their genotypes, underlying mechanisms and relative degree of their attenuation are largely unknown. Results Using mouse virulence studies we confirm that MAP vaccine strains 316 F, II and 2e have diverse but clearly attenuated survival and persistence characteristics compared with wild type strains. Using a pan genomic microarray we characterise the genomic variations in a panel of vaccine strains sourced from stocks spanning over 40 years of maintenance. We describe multiple genomic variations specific for individual vaccine stocks in both deletion (26–32 Kbp) and tandem duplicated (11–40 Kbp) large variable genomic islands and insertion sequence copy numbers. We show individual differences suitable for diagnostic differentiation between vaccine and wild type genotypes and provide evidence for functionality of some of the deleted MAP-specific genes and their possible relation to attenuation. Conclusions This study shows how culture environments have influenced MAP genome diversity resulting in large tandem genomic duplications, deletions and transposable element activity. In combination with classical selective systematic subculture this has led to fixation of specific MAP genomic alterations in some vaccine strain lineages which link the resulting attenuated phenotypes with deficiencies in high reactive oxygen species handling. PMID:23339684

  19. Overexpression of RORγt Enhances Pulmonary Inflammation after Infection with Mycobacterium Avium

    PubMed Central

    Matsuyama, Masashi; Ishii, Yukio; Sakurai, Hirofumi; Ano, Satoshi; Morishima, Yuko; Yoh, Keigyou; Takahashi, Satoru; Ogawa, Kenji; Hizawa, Nobuyuki

    2016-01-01

    Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial disease in humans. The role of Th17 immunity in the pathogenesis of intracellular bacteria, such as MAC, is not currently understood. Transcription factor RAR-related orphan receptor gamma t (RORγt) is known as the master regulator for Th17 cell development. Here, we investigated the role of RORγt in host responses against MAC infection. Wild-type (WT) mice and RORγt-overexpressing mice were infected with MAC via intratracheal inoculation. Systemic MAC growth was not different between WT mice and RORγt-overexpressing mice. However, neutrophilic pulmonary inflammation following MAC infection was enhanced in RORγt-overexpressing mice compared with that in WT mice. The cytokine expression shifted toward a Th17 phenotype in the lungs of RORγt-overexpressing mice following MAC infection; the levels of IL-6 and IL-17 were significantly higher in the lung of these mice than in WT mice. In addition to the increase in IL-17 single-positive T cells, T cells producing both IL-17 and interferon-γ were elevated in the lung of RORγt-overexpressing mice following MAC infection. These findings suggest that RORγt overexpression-mediated Th17 bias contributes to local inflammation rather than systemic responses, by regulating neutrophil recruitment into the sites of infection during MAC infection. PMID:26784959

  20. Ulcerative colitis and Crohn's disease: is Mycobacterium avium subspecies paratuberculosis the common villain?

    PubMed Central

    2010-01-01

    Mycobacterium avium, subspecies paratuberculosis (MAP) causes a chronic disease of the intestines in dairy cows and a wide range of other animals, including nonhuman primates, called Johne's ("Yo-knee's") disease. MAP has been consistently identified by a variety of techniques in humans with Crohn's disease. The research investigating the presence of MAP in patients with Crohn's disease has often identified MAP in the "negative" ulcerative colitis controls as well, suggesting that ulcerative colitis is also caused by MAP. Like other infectious diseases, dose, route of infection, age, sex and genes influence whether an individual infected with MAP develops ulcerative colitis or Crohn's disease. The apparently opposite role of smoking, increasing the risk of Crohn's disease while decreasing the risk of ulcerative colitis, is explained by a more careful review of the literature that reveals smoking causes an increase in both diseases but switches the phenotype from ulcerative colitis to Crohn's disease. MAP as the sole etiologic agent of both ulcerative colitis and Crohn's disease explains their common epidemiology, geographic distribution and familial and sporadic clusters, providing a unified hypothesis for the prevention and cure of the no longer "idiopathic" inflammatory bowel diseases. PMID:21167058

  1. Survey of Mycobacterium avium subspecies paratuberculosis in road-killed wild carnivores in Portugal.

    PubMed

    Matos, Ana Cristina; Figueira, Luis; Martins, Maria Helena; Loureiro, Filipa; Pinto, Maria Lurdes; Matos, Manuela; Coelho, Ana Cláudia

    2014-12-01

    A survey to determine the occurrence of Mycobacterium avium subsp. paratuberculosis (MAP) in wild carnivores in Portugal was conducted by testing samples from road-killed animals between 2009 and 2012. Postmortem examinations were performed and tissues were collected from wild carnivores representing four families and six different species, with a total of 74 animals analyzed. Cultures were performed by using Löwenstein-Jensen and Middlebrook 7H11 solid media and acid-fast isolates were identified by polymerase chain reaction (PCR) and mycobactin dependency characteristics. Tissues were also screened for MAP by directly extracting DNA and testing for the MAP-specific sequences. The occurrence of infected animals (an animal had at least one tissue that was positive for culture or direct PCR) was 27.0% (n = 20). MAP was isolated from culture of 25 tissue samples (3.8%) and was detected by direct PCR in 40 (6.0%) samples. Infection was recorded in 5/6 studied species: 7/49 (14.3%) red foxes (Vulpes vulpes), 3/3 (100%) beech martens (Martes foina), 2/4 (50.0%) Eurasian otters (Lutra lutra), 7/15 (46.7%) Egyptian mongooses (Herpestes ichneumon), and 1/1 (100%) European badger (Meles meles). These species represent three different taxonomic families: Canidae (14.3% were positive), Mustelidae (75.0% were positive), and Herpestidae (46.7% were positive). The results of this study confirm the presence of MAP infection in wild carnivores in Portugal.

  2. Proteome-scale identification and characterization of mitochondria targeting proteins of Mycobacterium avium subspecies paratuberculosis: Potential virulence factors modulating host mitochondrial function.

    PubMed

    Rana, Aarti; Kumar, Devender; Rub, Abdur; Akhter, Yusuf

    2015-07-01

    Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's Disease among ruminants. During the course of infection, it expresses a number of proteins for its successful persistence inside the host that cause variety of physiological abnormalities in the host. Mitochondrion is one of the attractive targets for pathogenic bacteria. Employing a proteome-wide sequence and structural signature based approach we have identified 46 M. avium subsp. paratuberculosis proteins as potential targets for the host mitochondrial targeting. These may act as virulence factors modulating mitochondrial physiology for bacterial survival and immune evasion inside the host cells.

  3. In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae to ticarcillin in combination with clavulanic acid.

    PubMed Central

    Casal, M J; Rodriguez, F C; Luna, M D; Benavente, M C

    1987-01-01

    The in vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae (M. chelonei) to ticarcillin in combination with calvulanic acid (CA) was studied by the agar dilution method. All the M. tuberculosis, M. bovis, and M. africanum strains were inhibited at a ticarcillin concentration of 32 micrograms/ml or lower in combination with 5 micrograms of CA. M. chelonae and M. avium strains proved resistant to more than 128 micrograms of ticarcillin plus 5 micrograms of CA per ml. M. fortuitum strains needed 128 micrograms of ticarcillin plus 5 micrograms of CA to inhibit approximately 30% of the isolates. PMID:3105441

  4. Sensitive and specific enzyme-linked immunosorbent assay for detecting serum antibodies against Mycobacterium avium subsp. paratuberculosis in fallow deer.

    PubMed

    Prieto, José M; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2014-08-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations.

  5. Serologic tests for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa).

    PubMed

    Boadella, Mariana; Lyashchenko, Konstantin; Greenwald, Reena; Esfandiari, Javan; Jaroso, Raquel; Carta, Tania; Garrido, Joseba M; Vicente, Joaquín; de la Fuente, José; Gortázar, Christian

    2011-01-01

    New tools to detect exposure of free-range Eurasian wild boar (Sus scrofa scrofa) to pathogenic mycobacteria would be valuable for improved disease surveillance and wildlife management. Two hundred sera from wild boar of known Mycobacterium bovis infection status were used to evaluate test suitability for the detection of antibodies against M. bovis and Mycobacterium avium subsp. paratuberculosis (or cross-reacting members of the M. avium complex). Two traditional enzyme-linked immunosorbent assays were evaluated using M. bovis purified protein derivative (bPPD) and paratuberculosis protoplasmatic antigen 3 (PPA3) as antigens, respectively, and a new point-of-care test format for bovine tuberculosis (bTB) that uses the innovative dual-path platform (DPP TB) test. The effect of individual factors (sex, age, lesions) on the diagnostic performance of the serologic tests was also determined. Although the DPP had a sensitivity of 89.6% and a specificity of 90.4%, for bPPD, the sensitivity was 79.2% and the specificity 100%. Both tests had a kappa agreement of 0.80. Sixty-five of 68 (95.6%) wild boar sera with antibodies against the PPA3 antigen corresponded to known M. bovis-infected wild boar. Significant differences were not observed in the bPPD and DPP readings among lesion categories or between age classes. A slight sex-related difference in sensitivity toward males in the DPP was found, but it was not detected in the bPPD enzyme-linked immunosorbent assay. The results support the use of antibody-based diagnostic tests for both large-scale and individual bTB testing of Eurasian wild boar and suggest that wild boar cannot be used as sentinels for infections caused by M. avium complex members.

  6. Cytokine Gene Expression in Peripheral Blood Mononuclear Cells and Tissues of Cattle Infected with Mycobacterium avium subsp. paratuberculosis: Evidence for an Inherent Proinflammatory Gene Expression Pattern

    PubMed Central

    Coussens, Paul M.; Verman, Nitin; Coussens, Marc A.; Elftman, Michael D.; McNulty, Amanda M.

    2004-01-01

    In cattle and other ruminants, infection with the intracellular pathogen Mycobacterium avium subsp. paratuberculosis results in a granulomatous enteritis (Johne's disease) that is often fatal. The key features of host immunity to M. avium subsp. paratuberculosis infection include an appropriate early proinflammatory and cytotoxic response (Th1-like) that eventually gives way to a predominant antibody-based response (Th2-like). Clinical disease symptoms often appear subsequent to waning of the Th1-like immune response. Understanding why this shift in the immune response occurs and the underlying molecular mechanisms involved is critical to future control measures and diagnosis. Previous studies have suggested that M. avium subsp. paratuberculosis may suppress gene expression in peripheral blood mononuclear cells (PBMCs) from infected cows, despite a continued inflammatory reaction at sites of infection. In the present study, we tested the hypothesis that exposure to M. avium subsp. paratuberculosis suppresses a proinflammatory gene expression pattern in PBMCs from infected cows. To do this, we examined expression of genes encoding interleukin-1α (IL-1α), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-16, and IL-18, as well as genes encoding gamma interferon (IFN-γ), transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α), in PBMCs, intestinal lesions, and mesenteric lymph nodes of cattle naturally infected with M. avium subsp. paratuberculosis. Cytokine gene expression in these cells and tissues was compared to expression in similar cells and tissues from control uninfected cattle. Our comprehensive results demonstrate that for most cytokine genes, including the genes encoding IFN-γ, TGF-β, TNF-α, IL-1α, IL-4, IL-6, IL-8, and IL-12p35, differential expression in PBMCs from infected and control cattle did not require stimulation with M. avium subsp. paratuberculosis. In fact, stimulation with M. avium subsp. paratuberculosis tended

  7. Thermal inactivation profiles of Mycobacterium avium subsp. paratuberculosis in lamb skeletal muscle homogenate fluid.

    PubMed

    Whittington, Richard J; Waldron, Anna; Warne, Darian

    2010-01-31

    Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in livestock and there is a debate about its role in humans in chronic inflammatory bowel disorders such as Crohn's disease, but the relationship remains unproven. Nevertheless livestock health authorities in many countries aim to lower the prevalence of this infection to reduce potential contamination of the human food supply. MAP may occur in bovine milk and data on thermal inactivation suggest pasteurisation is an effective process. Recently MAP has been identified in skeletal muscle of cattle and sheep but there are no data on its thermal inactivation in these substrates. In this study the inactivation of MAP was studied in a fluid homogenate of lamb skeletal muscle at temperatures previously identified as being relevant to cooking processes applied by domestic consumers. A PCR thermocycler was used to ensure accurate temperatures and rapid heat exchange, while radiometric culture was used to ensure sensitive detection of viable MAP for determination of D and z values. Among the two predominant strains of MAP, S and C, D(55) ranged from 56 to 89 min, D(60) was 8 to 11 min, D(65) was 26 to 35s while D(70) was 1.5 to 1.8s. Values for z were 4.21C degrees for the S strain and 4.51C degrees for the C strain. At temperatures of 65-70 degrees C, MAP appeared to be less heat tolerant in skeletal muscle fluid than in previous reports using milk as the medium. The total thermal exposure of MAP during baking of a sample of 16 leg-of-lamb roasts in domestic ovens was determined to result in more than 20 log reductions in most cases, that is the product was microbiologically safe. Based on the models used in this study, there is a low probability of survival of MAP provided that red meat is cooked to recommended standards. PMID:19896745

  8. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

  9. SNP genotyping of animal and human derived isolates of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Wynne, James W; Beller, Christie; Boyd, Victoria; Francis, Barry; Gwoźdź, Jacek; Carajias, Marios; Heine, Hans G; Wagner, Josef; Kirkwood, Carl D; Michalski, Wojtek P

    2014-08-27

    Mycobacterium avium subsp. paratuberculosis (MAP) is the aetiological agent of Johne's disease (JD), a chronic granulomatous enteritis that affects ruminants worldwide. While the ability of MAP to cause disease in animals is clear, the role of this bacterium in human inflammatory bowel diseases remains unresolved. Previous whole genome sequencing of MAP isolates derived from human and three animal hosts showed that human isolates were genetically similar and showed a close phylogenetic relationship to one bovine isolate. In contrast, other animal derived isolates were more genetically diverse. The present study aimed to investigate the frequency of this human strain across 52 wild-type MAP isolates, collected predominantly from Australia. A Luminex based SNP genotyping approach was utilised to genotype SNPs that had previously been shown to be specific to the human, bovine or ovine isolate types. Fourteen SNPs were initially evaluated across a reference panel of isolates with known genotypes. A subset of seven SNPs was chosen for analysis within the wild-type collection. Of the seven SNPs, three were found to be unique to paediatric human isolates. No wild-type isolates contain these SNP alleles. Interestingly, and in contrast to the paediatric isolates, three additional adult human isolates (derived from adult Crohn's disease patients) also did not contain these SNP alleles. Furthermore we identified two SNPs, which demonstrate extensive polymorphism within the animal-derived MAP isolates. One of which appears unique to ovine and a single camel isolate. From this study we suggest the existence of genetic heterogeneity between human derived MAP isolates, some of which are highly similar to those derived from bovine hosts, but others of which are more divergent.

  10. Short communication: Evaluation of sampling socks for detection of Mycobacterium avium ssp. paratuberculosis on dairy farms.

    PubMed

    Wolf, R; Orsel, K; De Buck, J; Kanevets, U; Barkema, H W

    2016-04-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a production-limiting disease in cattle. Detection of infected herds is often done using environmental samples (ES) of manure, which are collected in cattle pens and manure storage areas. Disadvantages of the method are that sample accuracy is affected by cattle housing and type of manure storage area. Furthermore, some sampling locations (e.g., manure lagoons) are frequently not readily accessible. However, sampling socks (SO), as used for Salmonella spp. testing in chicken flocks, might be an easy to use and accurate alternative to ES. The objective of the study was to assess accuracy of SO for detection of MAP in dairy herds. At each of 102 participating herds, 6 ES and 2 SO were collected. In total, 45 herds had only negative samples in both methods and 29 herds had ≥1 positive ES and ≥1 positive SO. Furthermore, 27 herds with ≥1 positive ES had no positive SO, and 1 herd with no positive ES had 1 positive SO. Bayesian simulation with informative priors on sensitivity of ES and MAP herd prevalence provided a posterior sensitivity for SO of 43.5% (95% probability interval=33-58), and 78.5% (95% probability interval=62-93) for ES. Although SO were easy to use, accuracy was lower than for ES. Therefore, with improvements in the sampling protocol (e.g., more SO per farm and more frequent herd visits), as well as improvements in the laboratory protocol, perhaps SO would be a useful alternative for ES. PMID:26851860

  11. Where Are All the Mycobacterium avium Subspecies paratuberculosis in Patients with Crohn's Disease?

    PubMed Central

    Pierce, Ellen S.

    2009-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic granulomatous inflammation of the intestines, Johne's disease, in dairy cows and every other species of mammal in which it has been identified. MAP has been identified in the mucosal layer and deeper bowel wall in patients with Crohn's disease by methods other than light microscopy, and by direct visualization in small numbers by light microscopy. MAP has not been accepted as the cause of Crohn's disease in part because it has not been seen under the microscope in large numbers in the intestines of patients with Crohn's disease. An analysis of the literature on the pathology of Crohn's disease and on possible MAP infection in Crohn's patients suggests that MAP might directly infect endothelial cells and adipocytes and cause them to proliferate, causing focal obstruction within already existing vessels (including granuloma formation), the development of new vessels (neoangiogenesis and lymphangiogenesis), and the “creeping fat” of the mesentery that is unique in human pathology to Crohn's disease but also occurs in bovine Johne's disease. Large numbers of MAP might therefore be found in the mesentery attached to segments of intestine affected by Crohn's disease rather than in the bowel wall, the blood and lymphatic vessels running through the mesentery, or the mesenteric fat itself. The walls of fistulas might result from the neoangiogenesis or lymphangiogenesis that occurs in the bowel wall in Crohn's disease and therefore are also possible sites of large numbers of MAP. The direct visualization of large numbers of MAP organisms in the tissues of patients with Crohn's disease will help establish that MAP causes Crohn's disease. PMID:19325887

  12. Transcriptional Profiling of Ileocecal Valve of Holstein Dairy Cows Infected with Mycobacterium avium subsp. Paratuberculosis.

    PubMed

    Hempel, Randy J; Bannantine, John P; Stabel, Judith R

    2016-01-01

    Johne's disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and provides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection.

  13. A rational framework for evaluating the next generation of vaccines against Mycobacterium avium subspecies paratuberculosis

    PubMed Central

    Bannantine, John P.; Hines, Murray E.; Bermudez, Luiz E.; Talaat, Adel M.; Sreevatsan, Srinand; Stabel, Judith R.; Chang, Yung-Fu; Coussens, Paul M.; Barletta, Raúl G.; Davis, William C.; Collins, Desmond M.; Gröhn, Yrjö T.; Kapur, Vivek

    2014-01-01

    Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johne's disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne's disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development

  14. Volatile Emissions from Mycobacterium avium subsp. paratuberculosis Mirror Bacterial Growth and Enable Distinction of Different Strains

    PubMed Central

    Trefz, Phillip; Koehler, Heike; Klepik, Klaus; Moebius, Petra; Reinhold, Petra; Schubert, Jochen K.; Miekisch, Wolfram

    2013-01-01

    Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold’s egg yolk medium in dilutions of 10-0, 10-2, 10-4 and 10-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP

  15. Shedding patterns of dairy calves experimentally infected with Mycobacterium avium subspecies paratuberculosis

    PubMed Central

    2014-01-01

    Although substantial fecal shedding is expected to start years after initial infection with Mycobacterium avium subspecies paratuberculosis (MAP), the potential for shedding by calves and therefore calf-to-calf transmission is underestimated in current Johne’s disease (JD) control programs. Shedding patterns were determined in this study in experimentally infected calves. Fifty calves were challenged at 2 weeks or at 3, 6, 9 or 12 months of age (6 calves served as a control group). In each age group, 5 calves were inoculated with a low and 5 with a high dose of MAP. Fecal culture was performed monthly until necropsy at 17 months of age. Overall, 61% of inoculated calves, representing all age and dose groups, shed MAP in their feces at least once during the follow-up period. Although most calves shed sporadically, 4 calves in the 2-week and 3-month high dose groups shed at every sampling. In general, shedding peaked 2 months after inoculation. Calves inoculated at 2 weeks or 3 months with a high dose of MAP shed more frequently than those inoculated with a low dose. Calves shedding frequently had more culture-positive tissue locations and more severe gross and histological lesions at necropsy. In conclusion, calves inoculated up to 1 year of age shed MAP in their feces shortly after inoculation. Consequently, there is potential for MAP transfer between calves (especially if they are group housed) and therefore, JD control programs should consider young calves as a source of infection. PMID:25224905

  16. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

  17. Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank

    PubMed Central

    Rathnaiah, Govardhan; Lamont, Elise A.; Harris, N. Beth; Fenton, Robert J.; Zinniel, Denise K.; Liu, Xiaofei; Sotos, Josh; Feng, Zhengyu; Livneh-Kol, Ayala; Shpigel, Nahum Y.; Czuprynski, Charles J.; Sreevatsan, Srinand; Barletta, Raúl G.

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated. PMID:25360421

  18. Thermal inactivation profiles of Mycobacterium avium subsp. paratuberculosis in lamb skeletal muscle homogenate fluid.

    PubMed

    Whittington, Richard J; Waldron, Anna; Warne, Darian

    2010-01-31

    Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in livestock and there is a debate about its role in humans in chronic inflammatory bowel disorders such as Crohn's disease, but the relationship remains unproven. Nevertheless livestock health authorities in many countries aim to lower the prevalence of this infection to reduce potential contamination of the human food supply. MAP may occur in bovine milk and data on thermal inactivation suggest pasteurisation is an effective process. Recently MAP has been identified in skeletal muscle of cattle and sheep but there are no data on its thermal inactivation in these substrates. In this study the inactivation of MAP was studied in a fluid homogenate of lamb skeletal muscle at temperatures previously identified as being relevant to cooking processes applied by domestic consumers. A PCR thermocycler was used to ensure accurate temperatures and rapid heat exchange, while radiometric culture was used to ensure sensitive detection of viable MAP for determination of D and z values. Among the two predominant strains of MAP, S and C, D(55) ranged from 56 to 89 min, D(60) was 8 to 11 min, D(65) was 26 to 35s while D(70) was 1.5 to 1.8s. Values for z were 4.21C degrees for the S strain and 4.51C degrees for the C strain. At temperatures of 65-70 degrees C, MAP appeared to be less heat tolerant in skeletal muscle fluid than in previous reports using milk as the medium. The total thermal exposure of MAP during baking of a sample of 16 leg-of-lamb roasts in domestic ovens was determined to result in more than 20 log reductions in most cases, that is the product was microbiologically safe. Based on the models used in this study, there is a low probability of survival of MAP provided that red meat is cooked to recommended standards.

  19. Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle

    PubMed Central

    Ahlstrom, Christina; Barkema, Herman W.; Stevenson, Karen; Zadoks, Ruth N.; Biek, Roman; Kao, Rowland; Trewby, Hannah; Haupstein, Deb; Kelton, David F.; Fecteau, Gilles; Labrecque, Olivia; Keefe, Greg P.; McKenna, Shawn L. B.; Tahlan, Kapil; De Buck, Jeroen

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale. PMID:26871723

  20. Evidence for genes associated with the ability of Mycobacterium avium subsp. hominissuis to escape apoptotic macrophages

    PubMed Central

    Bermudez, Luiz E.; Danelishvili, Lia; Babrack, Lmar; Pham, Tuan

    2015-01-01

    Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacteria that infects immunocompromised humans. MAH cases are increasing in incidence, making it crucial to gain knowledge of the pathogenic mechanisms associated with the bacterium. MAH infects macrophages and after several days the infection triggers the phagocyte apoptosis. Many of the intracellular MAH escape the cell undergoing apoptosis leading to infection of neighboring macrophages. We screened a transposon bank of MAH mutants in U937 mononuclear phagocytes for the inability to escape macrophages undergoing apoptosis. Mutations in genes; MAV_2235, MAV_2120, MAV_2410, and MAV_4563 resulted in the inability of the bacteria to exit macrophages upon apoptosis. Complementation of the mutations corrected the phenotype either completely or partially. Testing for the ability of the mutants to survive in macrophages compared to the wild-type bacterium revealed that the mutant clones were not attenuated up to 4 days of infection. Testing in vivo, however, demonstrated that all the MAH clones were attenuated compared with the wild-type MAC 104 in tissues of mice. Although the mechanism associated with the bacterial inability to leave apoptotic macrophages is unknown, the identification of macrophage cytoplasm targets for the MAH proteins suggest that they interfere either with protein degradation machinery or post-translation mechanisms. The identification of tatC as a MAH protein involved in the ability of MAH to leave macrophages, suggests that secreted effector(s) are involved in the process. The study reveals a pathway of escape from macrophages, not shared with Mycobacterium tuberculosis. PMID:26380226

  1. Dysbiosis of the Fecal Microbiota in Cattle Infected with Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Vecchiarelli, Bonnie; Indugu, Nagaraju; Kumar, Sanjay; Gallagher, Susan C.; Fyock, Terry L.; Sweeney, Raymond W.

    2016-01-01

    Johne's disease (JD) is a chronic, intestinal infection of cattle, caused by Mycobacterium avium subsp. paratuberculosis (MAP). It results in granulomatous inflammation of the intestinal lining, leading to malabsorption, diarrhea, and weight loss. Crohn’s disease (CD), a chronic, inflammatory gastrointestinal disease of humans, has many clinical and pathologic similarities to JD. Dysbiosis of the enteric microbiota has been demonstrated in CD patients. It is speculated that this dysbiosis may contribute to the intestinal inflammation observed in those patients. The purpose of this study was to investigate the diversity patterns of fecal bacterial populations in cattle infected with MAP, compared to those of uninfected control cattle, using phylogenomic analysis. Fecal samples were selected to include samples from 20 MAP-positive cows; 25 MAP-negative herdmates; and 25 MAP-negative cows from a MAP-free herd. The genomic DNA was extracted; PCR amplified sequenced on a 454 Roche platform, and analyzed using QIIME. Approximately 199,077 reads were analyzed from 70 bacterial communities (average of 2,843 reads/sample). The composition of bacterial communities differed between the 3 treatment groups (P < 0.001; Permanova test). Taxonomic assignment of the operational taxonomic units (OTUs) identified 17 bacterial phyla across all samples. Bacteroidetes and Firmicutes constituted more than 95% of the bacterial population in the negative and exposed groups. In the positive group, lineages of Actinobacteria and Proteobacteria increased and those of Bacteroidetes and Firmicutes decreased (P < 0.001). Actinobacteria was highly abundant (30% of the total bacteria) in the positive group compared to exposed and negative groups (0.1–0.2%). Notably, the genus Arthrobacter was found to predominate Actinobacteria in the positive group. This study indicates that MAP-infected cattle have a different composition of their fecal microbiota than MAP-negative cattle. PMID:27494144

  2. Isolation of Mycobacterium avium subsp. paratuberculosis from muscle tissue of naturally infected cattle.

    PubMed

    Alonso-Hearn, Marta; Molina, Elena; Geijo, Marivi; Vazquez, Patricia; Sevilla, Iker; Garrido, Joseba M; Juste, Ramon A

    2009-05-01

    Johne's disease or paratuberculosis is a chronic granulomatous inflammation of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Recent studies suggest an association between MAP and Crohn's disease in humans. MAP can become widely distributed within the tissues of infected animals, and meat may be a possible route of exposure of MAP to humans. In this study, 47 dairy and beef cattle were examined for the occurrence of viable MAP in diaphragm muscle. At the slaughterhouse, gut tissues, diaphragm muscle, blood, and feces of the 47 animals were collected for bacteriological culture, as well as gut samples for histopathological analysis. MAP was detected by bacteriological culture and conventional and real-time IS900 polymerase chain reaction in the diaphragm muscle of six infected cattle at slaughter (13%). The six animals showing evidence of MAP in diaphragm muscle had diffuse lesions and severe granulomatous inflammation in ileocecal lymph nodes, jejunal lymph nodes, ileocecal valve, and ileum. All six had heavy bacterial load in mesenteric lymph nodes, ileocecal valve, ileum, and jejunum, and four showed clinical signs of paratuberculosis. Two animals did not show clinical signs but had viable MAP in intestinal tissues and in diaphragm muscle as well. MAP was found in blood of only one of the six animals showing evidence of MAP in diaphragm muscle and in feces of three of them. In general, there was a positive association between enteric lesion severity, clinical signs of paratuberculosis, heavy bacterial load in intestinal tissues, fecal shedding of MAP, and the presence of disseminated MAP infection in diaphragm muscle. The results of this study demonstrated that MAP can be detected and cultured from muscle of MAP-infected cattle destined for human consumption and suggest a possible risk of exposure of humans to MAP via contaminated meat.

  3. Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies paratuberculosis Infection

    PubMed Central

    Fisher, Colleen A.; Bhattarai, Eric K.; Osterstock, Jason B.; Dowd, Scot E.; Seabury, Paul M.; Vikram, Meenu; Whitlock, Robert H.; Schukken, Ynte H.; Schnabel, Robert D.; Taylor, Jeremy F.; Womack, James E.; Seabury, Christopher M.

    2011-01-01

    Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several

  4. Microaggregate-associated protein involved in invasion of epithelial cells by Mycobacterium avium subsp. hominissuis

    PubMed Central

    Babrak, Lmar; Danelishvili, Lia; Rose, Sasha J; Bermudez, Luiz E

    2015-01-01

    The environmental opportunistic pathogen Mycobacterium avium subsp hominissuis (MAH), a member of the nontuberculous mycobacteria (NTM) cluster, causes respiratory as well as disseminated disease in patients such as those with chronic respiratory illnesses or AIDS. Currently, there is no effective method to prevent NTM respiratory infections. The formation of mycobacterial microaggregates comprises of phenotypic changes that lead to efficient adherence and invasion of the respiratory mucosa in vitro and in vivo. Microaggregate adhesion to the respiratory epithelium is mediated in part through the mycobacterial protein, MAV_3013 (MBP-1). Through DNA microarray analysis, the small hypothetical gene MAV_0831 (Microaggregate Invasion Protein-1, MIP-1) was identified as being upregulated during microaggregate formation. When MIP-1 was overexpressed in poorly-invasive Mycobacterium smegmatis, it provided the bacterium the ability to bind and enter epithelial cells. In addition, incubating microaggregates with recombinant MIP-1 protein enhanced the ability of microaggregates to invade HEp-2 cells, and exposure to anti-MIP-1 immune serum reduced the invasion of the host epithelium. Through protein-protein interaction assays, MIP-1 was found to bind to the host protein filamin A, a cytoskeletal actin-binding protein integral to the modulation of host cell shape and migration. As visualized by immunofluorescence, filamin A was able to co-localize with microaggregates and to a lesser extent planktonic bacteria. Invasion of HEp-2 cells by microaggregates and planktonic bacteria was also inhibited by the addition of anti-filamin A antibody suggesting that filamin A plays an important role during infection. In addition, at earlier time points binding and invasion assay results suggest that MBP-1 participates significantly during the first interactions with the host cell while MIP-1 becomes important once the bacteria adhere to the host epithelium. In summary, we have unveiled

  5. Transcriptional Profiling of Ileocecal Valve of Holstein Dairy Cows Infected with Mycobacterium avium subsp. Paratuberculosis

    PubMed Central

    Hempel, Randy J.; Bannantine, John P.

    2016-01-01

    Johne’s disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and provides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection. PMID:27093613

  6. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival.

    PubMed

    Kugadas, Abirami; Lamont, Elise A; Bannantine, John P; Shoyama, Fernanda M; Brenner, Evan; Janagama, Harish K; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc(2) 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

  7. A survey to detect the presence of Mycobacterium avium subspecies paratuberculosis in Kangaroo Island macropods.

    PubMed

    Cleland, P C; Lehmann, D R; Phillips, P H; Cousins, D V; Reddacliff, L A; Whittington, R J

    2010-10-26

    The aim of this study was to determine whether Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) infection was present in macropods grazing with infected sheep on Kangaroo Island in 2001-2002, and to assess the likely role of such infection in the epidemiology of ovine paratuberculosis. Ileum and associated lymphatics from 482 macropods were examined using radiometric culture followed by PCR for IS900 and restriction endonuclease analysis (REA) for species identification, and isolates were strain typed using PCR for IS1311 and REA. Ileum and mesenteric lymph nodes from animals with positive tissue cultures or gross lesions suggestive of paratuberculosis were examined histologically. Faeces from a total of 840 animals were cultured in pools of 20, and individual faecal cultures were done from tissue culture positive animals, from those with microscopic lesions, and from selected animals with gross lesions. Eight animals (1.7%) yielded positive tissue cultures, and all isolates were the sheep (S) strain. Two animals that were tissue culture positive also had histopathological evidence of paratuberculosis. Twelve culture negative animals had microscopic lesions consistent with mycobacterial infection, and M. genavense was identified by PCR from a paraffin block from one of these animals. All faecal cultures were negative. These results indicate that a small proportion of macropods can become infected with M. a. paratuberculosis when grazing with infected sheep. However, excretion of large numbers of viable organisms is rare in macropods, and it is unlikely that macropods provide a wildlife reservoir of infection that would seriously compromise control efforts for paratuberculosis in sheep. PMID:20400245

  8. Transcriptional Profiling of Ileocecal Valve of Holstein Dairy Cows Infected with Mycobacterium avium subsp. Paratuberculosis.

    PubMed

    Hempel, Randy J; Bannantine, John P; Stabel, Judith R

    2016-01-01

    Johne's disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and provides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection. PMID:27093613

  9. The Significance of Mycobacterium abscessus Subspecies abscessus Isolation During Mycobacterium avium Complex Lung Disease Therapy

    PubMed Central

    Philley, Julie V.; Brown-Elliott, Barbara A.; Benwill, Jeana L.; Shepherd, Sara; York, Deanna; Wallace, Richard J.

    2015-01-01

    BACKGROUND: Isolation of Mycobacterium abscessus subspecies abscessus (MAA) is common during Mycobacterium avium complex (MAC) lung disease therapy, but there is limited information about the clinical significance of the MAA isolates. METHODS: We identified 53 of 180 patients (29%) treated for MAC lung disease who had isolation of MAA during MAC lung disease therapy. Patients were divided into those without (group 1) and those with (group 2) MAA lung disease. RESULTS: There were no significant demographic differences between patients with and without MAA isolation or between groups 1 and 2. Group 1 and 2 patients had similar total sputum cultures obtained (P = .7; 95% CI, −13.4 to 8.6) and length of follow-up (P = .8; 95% CI, −21.5 to 16.1). Group 2 patients had significantly more total positive cultures for MAA (mean±SD, 15.0 ± 11.1 vs 1.2 ± 0.4; P < .0001; 95% CI, −17.7 to −9.9), were significantly more likely to develop new or enlarging cavitary lesions while on MAC therapy (P > .0001), and were significantly more likely to meet all three American Thoracic Society diagnostic criteria for nontuberculous mycobacterial disease (21 of 21 [100%] vs 0 of 32 [0%]; P < .0001) compared with group 1 patients. Group 1 patients were significantly more likely to have single, positive MAA cultures than group 2 patients (25 of 31 vs 0 of 21; P < .0001). CONCLUSIONS: Microbiologic and clinical follow-up after completion of MAC lung disease therapy is required to determine the significance of MAA isolated during MAC lung disease therapy. Single MAA isolates are not likely to be clinically significant. PMID:25357074

  10. Modeling of Mycobacterium avium subsp. paratuberculosis dynamics in a dairy herd: An individual based approach.

    PubMed

    Al-Mamun, Mohammad A; Smith, Rebecca L; Schukken, Ynte H; Gröhn, Yrjö T

    2016-11-01

    In the dairy industry, Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP) is one of the major investigated diseases. To date, researchers have suggested some control strategies for JD, such as test-and-cull based herd management, isolated calf rearing management, and vaccinations. Due to the slow progressing nature of MAP, tests with low diagnostic test sensitivity and specificity, and economic limitations, implementing these strategies has not resulted in elimination of MAP from farms. To date, no study has integrated detailed dairy herd dynamics with different MAP transmission routes. We have developed an individual-based dairy herd model by incorporating basic herd dynamics in a closed herd environment where no new animals have been bought from outside. The model considered three age groups of animals: calves, heifers and adults. It includes sequential life events of a dairy animal and such key dynamic processes of the dairy herd as lactation cycle, calving, voluntary waiting period, insemination, pregnancy, dry-off period and calf and heifer rearing. After initially validating that the model reproduced typical herd dynamics, it was extended by incorporating MAP infection dynamics, where each individual adult animal belonged to one of four infection compartments: susceptible, latent, low shedding and high shedding. The model includes two disease transmission routes: horizontal transmission (i.e., fecal-oral) and vertical transmission (i.e., in utero infection). The results confirm that this model can simulate a realistic dairy herd and that inclusion of the above-mentioned dynamic processes provides useful information about individual infected animals to farmers. Access to the individual animal information offers more validity to assessment of appropriate control strategies for an endemically MAP infected herd. This model can serve as an accurate and novel tool not only to better understand MAP dynamics, but is also valuable as an

  11. Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models.

    PubMed

    Colavecchia, Silvia B; Fernández, Bárbara; Jolly, Ana; Minatel, Leonardo; Hajos, Silvia E; Paolicchi, Fernando A; Mundo, Silvia L

    2016-08-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.

  12. Mycobacterium avium Subspecies paratuberculosis Infection Modifies Gut Microbiota under Different Dietary Conditions in a Rabbit Model

    PubMed Central

    Arrazuria, Rakel; Elguezabal, Natalia; Juste, Ramon A.; Derakhshani, Hooman; Khafipour, Ehsan

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) the causative agent of paratuberculosis, produces a chronic granulomatous inflammation of the gastrointestinal tract of ruminants. It has been recently suggested that MAP infection may be associated with dysbiosis of intestinal microbiota in ruminants. Since diet is one of the key factors affecting the balance of microbial populations in the digestive tract, we intended to evaluate the effect of MAP infection in a rabbit model fed a regular or high fiber diet during challenge. The composition of microbiota of the cecal content and the sacculus rotundus was studied in 20 New Zealand white female rabbits. The extracted DNA was subjected to paired-end Illumina sequencing of the V3-V4 hypervariable region of the 16S rRNA gene for microbiota analysis. Microbial richness (Chao1) in the cecal content was significantly increased by MAP infection in regular diet rabbits (p = 0.0043) and marginally increased (p = 0.0503) in the high fiber group. Analysis of beta-diversity showed that MAP infection produces deeper changes in the microbiota of sacculus rotundus than in the cecal content. A lower abundance of Proteobacteria in the cecal content of infected animals fed the high fiber diet and also lower abundance of Bacteroidetes in the sacculus rotundus of infected animals fed the regular diet were observed. Based on OPLS-DA analysis, we observed that some bacteria repeatedly appear to be positively associated with infection in different samples under different diets (families Dehalobacteriaceae, Coriobacteriaceae, and Mogibacteriaceae; genus Anaerofustis). The same phenomenon was observed with some of the bacteria negatively associated with MAP infection (genera Anaerostipes and Coprobacillus). However, other groups of bacteria (Enterobacteriaceae family and ML615J-28 order) were positively associated with infection in some circumstances and negatively associated with infection in others. Data demonstrate that MAP infection

  13. In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Bergmann, Andreas; Trefz, Phillip; Fischer, Sina; Klepik, Klaus; Walter, Gudrun; Steffens, Markus; Ziller, Mario; Schubert, Jochen K.; Reinhold, Petra; Köhler, Heike; Miekisch, Wolfram

    2015-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests. PMID:25915653

  14. Modulation of Innate Host Factors by Mycobacterium avium Complex in Human Macrophages Includes Interleukin 17

    PubMed Central

    Vázquez, Nancy; Rekka, Sofia; Gliozzi, Maria; Feng, Carl G.; Amarnath, Shoba; Orenstein, Jan M.; Wahl, Sharon M.

    2012-01-01

    Background. Although opportunistic infections due to Mycobacterium avium complex (MAC) have been less common since the introduction of highly active antiretroviral therapy, globally, human immunodeficiency virus-1 (HIV-1)–positive patients remain predisposed to these infections. Absence of a properly functioning acquired immune response allows MAC persistence within macrophages localized in lymph nodes coinfected with HIV and MAC. Although a deficiency in interferon γ appears to play a part in the ability of MAC to deflect the macrophage-associated antimicrobial attack, questions about this process remain. Our study examines the ability of MAC to regulate interleukin 17 (IL-17), a proinflammatory cytokine involved in host cell recruitment. Methods. Coinfected lymph nodes were examined for IL-17 by immunohistochemical analysis. In vitro, macrophages exposed to mycobacteria were evaluated for transcription activities, proteins, and signaling pathways responsible for IL-17 expression. Infected macrophages were also analyzed for expression of interleukin 21 (IL-21) and negative regulators of immune responses. Results. Infection of macrophages triggered synthesis of IL-17, correlating with IL-17 expression by macrophages in coinfected lymph nodes. Infected macrophages exposed to exogenous IL-17 expressed CXCL10, which favors recruitment of new macrophages as targets for infection. Blockade of nuclear factor κ-light-chain-enhancer of activated B cells and mitogen-activated protein kinase pathways suppressed mycobacteria-induced IL-17 expression. MAC triggered expression of IL-21, IRF4, and STAT3 genes related to IL-17 regulation, as well as expression of the negative immunoregulators CD274(PD-L1) and suppressors of cytokine signaling. Conclusions. MAC-infected macrophages can provide an alternative source for IL-17 that favors accumulation of new targets for perpetuating bacterial and viral infection while suppressing host antimicrobial immune responses. PMID

  15. Metabolic adaptation of Mycobacterium avium subsp. paratuberculosis to the gut environment.

    PubMed

    Weigoldt, Mathias; Meens, Jochen; Bange, Franz-Christoph; Pich, Andreas; Gerlach, Gerald F; Goethe, Ralph

    2013-02-01

    Knowledge on the proteome level about the adaptation of pathogenic mycobacteria to the environment in their natural hosts is limited. Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a chronic and incurable granulomatous enteritis of ruminants, and has been suggested to be a putative aetiological agent of Crohn's disease in humans. Using a comprehensive LC-MS-MS and 2D difference gel electrophoresis (DIGE) approach, we compared the protein profiles of clinical strains of MAP prepared from the gastrointestinal tract of diseased cows with the protein profiles of the same strains after they were grown in vitro. LC-MS-MS analyses revealed that the principal enzymes for the central carbon metabolic pathways, including glycolysis, gluconeogenesis, the tricaboxylic acid cycle and the pentose phosphate pathway, were present under both conditions. Moreover, a broad spectrum of enzymes for β-oxidation of lipids, nine of which have been shown to be necessary for mycobacterial growth on cholesterol, were detected in vivo and in vitro. Using 2D-DIGE we found increased levels of several key enzymes that indicated adaptation of MAP to the host. Among these, FadE5, FadE25 and AdhB indicated that cholesterol is used as a carbon source in the bovine intestinal mucosa; the respiratory enzymes AtpA, NuoG and SdhA suggested increased respiration during infection. Furthermore higher levels of the pentose phosphate pathway enzymes Gnd2, Zwf and Tal as well as of KatG, SodA and GroEL indicated a vigorous stress response of MAP in vivo. In conclusion, our results provide novel insights into the metabolic adaptation of a pathogenic mycobacterium in its natural host. PMID:23223439

  16. Metabolic adaptation of Mycobacterium avium subsp. paratuberculosis to the gut environment.

    PubMed

    Weigoldt, Mathias; Meens, Jochen; Bange, Franz-Christoph; Pich, Andreas; Gerlach, Gerald F; Goethe, Ralph

    2013-02-01

    Knowledge on the proteome level about the adaptation of pathogenic mycobacteria to the environment in their natural hosts is limited. Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a chronic and incurable granulomatous enteritis of ruminants, and has been suggested to be a putative aetiological agent of Crohn's disease in humans. Using a comprehensive LC-MS-MS and 2D difference gel electrophoresis (DIGE) approach, we compared the protein profiles of clinical strains of MAP prepared from the gastrointestinal tract of diseased cows with the protein profiles of the same strains after they were grown in vitro. LC-MS-MS analyses revealed that the principal enzymes for the central carbon metabolic pathways, including glycolysis, gluconeogenesis, the tricaboxylic acid cycle and the pentose phosphate pathway, were present under both conditions. Moreover, a broad spectrum of enzymes for β-oxidation of lipids, nine of which have been shown to be necessary for mycobacterial growth on cholesterol, were detected in vivo and in vitro. Using 2D-DIGE we found increased levels of several key enzymes that indicated adaptation of MAP to the host. Among these, FadE5, FadE25 and AdhB indicated that cholesterol is used as a carbon source in the bovine intestinal mucosa; the respiratory enzymes AtpA, NuoG and SdhA suggested increased respiration during infection. Furthermore higher levels of the pentose phosphate pathway enzymes Gnd2, Zwf and Tal as well as of KatG, SodA and GroEL indicated a vigorous stress response of MAP in vivo. In conclusion, our results provide novel insights into the metabolic adaptation of a pathogenic mycobacterium in its natural host.

  17. Systemic and mucosal immune reactivity upon Mycobacterium avium ssp. paratuberculosis infection in mice.

    PubMed

    Koc, Arzu; Bargen, Imke; Suwandi, Abdulhadi; Roderfeld, Martin; Tschuschner, Annette; Rath, Timo; Gerlach, Gerald F; Hornef, Mathias; Goethe, Ralph; Weiss, Siegfried; Roeb, Elke

    2014-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne's disease, an inflammatory bowel disorder of ruminants. Due to the similar pathology, MAP was also suggested to cause Crohn's disease (CD). Despite of intensive research, this question is still not settled, possibly due to the lack of versatile mouse models. The aim of this study was to identify basic immunologic mechanisms in response to MAP infection. Immune compromised C57BL/6 Rag2-/- mice were infected with MAP intraperitoneally. Such chronically infected mice were then reconstituted with CD4+ and CD8+ T cells 28 days after infection. A systemic inflammatory response, detected as enlargement of the spleen and granuloma formation in the liver, was observed in mice infected and reconstituted with CD4+ T cells. Whereby inflammation in infected and CD4+CD45RB(hi) T cell reconstituted animals was always higher than in the other groups. Reconstitution of infected animals with CD8+ T cells did not result in any inflammatory signs. Interestingly, various markers of inflammation were strongly up-regulated in the colon of infected mice reconstituted with CD4+CD45RB(lo/int) T cells. We propose, the usual non-colitogenic CD4+CD45RB(lo/int) T cells were converted into inflammatory T cells by the interaction with MAP. However, the power of such cells might be not sufficient for a fully established inflammatory response in the colon. Nevertheless, our model system appears to mirror aspects of an inflammatory bowel disease (IBD) like CD and Johne's diseases. Thus, it will provide an experimental platform on which further knowledge on IBD and the involvement of MAP in the induction of CD could be acquired.

  18. Detection of mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany.

    PubMed

    Galiero, Alessia; Fratini, Filippo; Mataragka, Antonia; Turchi, Barbara; Nuvoloni, Roberta; Ikonomopoulos, John; Cerri, Domenico

    2016-01-18

    Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto's thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms. Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×10(4)Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR.

  19. Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese.

    PubMed

    Botsaris, George; Slana, Iva; Liapi, Maria; Dodd, Christine; Economides, Constantinos; Rees, Catherine; Pavlik, Ivo

    2010-07-31

    Mycobacterium avium subsp. paratuberculosis (MAP) may have a role in the development of Crohn's disease in humans via the consumption of contaminated milk and milk products. Detection of MAP from milk and dairy products has been reported from countries on the European continent, Argentina, the UK and Australia. In this study three different methods (quantitative real time PCR, combined phage IS900 PCR and conventional cultivation) were used to detect the presence of MAP in bulk tank milk (BTM) and cheese originating from sheep, goat and mixed milks from farms and products in Cyprus. During the first survey the presence of MAP was detected in 63 (28.6%) of cows' BTM samples by quantitative real time PCR. A second survey of BTM used a new combined phage IS900 PCR assay, and in this case MAP was detected in 50 (22.2%) samples showing a good level of agreement by both methods. None of the herds tested were known to be affected by Johne's disease and the presence of viable MAP was confirmed by conventional culture in only two cases of cows BTM. This suggests that either rapid method used is more sensitive than the conventional culture when testing raw milk samples for MAP. The two isolates recovered from BTM were identified by IS1311 PCR REA as cattle and sheep strains, respectively. In contrast when cheese samples were tested, MAP DNA was detected by quantitative real time PCR in seven (25.0%) samples (n=28). However no viable MAP was detected when either the combined phage IS900 PCR or conventional culture methods were used.

  20. Evaluation of evapotranspiration estimation methods for sweet cherry trees (Prunus avium) in sub-humid climate.

    PubMed

    Denmirtas, Cigdem; Buyukcangaz, Hakan; Yazgan, Senih; Candogan, Burak Nazmi

    2007-02-01

    This study was carried out in the summer of 2001 in a 3 year old and in the summer of 2002 in a 4 year old sweet cherry trees (Prunus avium, variety Z-900) on Mazzard rootstocks in Bayramic-Canakkale which is located in the west part of Turkey. Micro-sprinkler irrigation was selected as the irrigation method. The trees were subjected to four micro-sprinkler irrigation treatments (T-1, T-2, T-3 and T-4). The water applied in treatment T-3 was considered sufficient to satisfy fully needs of the crop (100% of ETc) and to allow good rooting and tree growth. The water balance relationship was used in estimating ETc. A total of 4 climatological methods were selected for estimating reference crop evapotranspiration on a daily basis. Some of these methods are based on combination theory and others are empirical methods based primarily on solar radiation, temperature ans relative humidity. An attempt was made in the current study to develop regional relationship between the evapotranspiration measured and that estimated by the climatological methods, such as FAO-Penman, Penman-Monteith, FAO-Radiation and FAO-Blaney-Criddle. Performance of the climatological methods in estimating the ETo values as compared to the measured values was evaluated on the basis of root mean square error (RMSE). In 2001, the Penman-Monteith equation gave the best results followed by FAO-Penman, FAO-Radiation and FAO-Blaney-Criddle. In 2002, the Penman-Monteith and FAO-Blaney-Criddle equations gave same results. PMID:19069518

  1. Macrolide/Azalide Therapy for Nodular/Bronchiectatic Mycobacterium avium Complex Lung Disease

    PubMed Central

    Wallace, Richard J.; Brown-Elliott, Barbara A.; McNulty, Steven; Philley, Julie V.; Killingley, Jessica; Wilson, Rebecca W.; York, Deanna S.; Shepherd, Sara

    2014-01-01

    BACKGROUND: There is no large study validating the appropriateness of current treatment guidelines for Mycobacterium avium complex (MAC) lung disease. This is a retrospective single-center review evaluating the efficacy of macrolide/azalide-containing regimens for nodular/bronchiectatic (NB) MAC lung disease. METHODS: Patients were treated according to contemporary guidelines with evaluation of microbiologic responses. Macrolide susceptibility of MAC isolates was done at initiation of therapy, 6 to 12 months during therapy, and on the first microbiologic recurrence isolate. Microbiologic recurrence isolates also underwent genotyping for comparison with the original isolates. RESULTS: One hundred eighty patients completed > 12 months of macrolide/azalide multidrug therapy. Sputum conversion to culture negative occurred in 154 of 180 patients (86%). There were no differences in response between clarithromycin or azithromycin regimens. Treatment regimen modification occurred more frequently with daily (24 of 30 [80%]) vs intermittent (2 of 180 [1%]) therapy (P = .0001). No patient developed macrolide resistance during treatment. Microbiologic recurrences during therapy occurred in 14% of patients: 73% with reinfection MAC isolates, 27% with true relapse isolates (P = .03). Overall, treatment success (ie, sputum conversion without true microbiologic relapse) was achieved in 84% of patients. Microbiologic recurrences occurred in 74 of 155 patients (48%) after completion of therapy: 75% reinfection isolates, 25% true relapse isolates. CONCLUSIONS: Current guidelines for macrolide/azalide-based therapies for NB MAC lung disease result in favorable microbiologic outcomes for most patients without promotion of macrolide resistance. Intermittent therapy is effective and significantly better tolerated than daily therapy. Microbiologic recurrences during or after therapy are common and most often due to reinfection MAC genotypes. PMID:24457542

  2. Relationship Between Lung Cancer and Mycobacterium Avium Complex Isolated Using Bronchoscopy

    PubMed Central

    Tamura, Atsuhisa; Hebisawa, Akira; Kusaka, Kei; Hirose, Takashi; Suzuki, Junko; Yamane, Akira; Nagai, Hideaki; Fukami, Takeshi; Ohta, Ken; Takahashi, Fumiaki

    2016-01-01

    Introduction: The incidence of Mycobacterium avium complex (MAC)-positive respiratory specimen cultures and MAC lung disease (MACLD) is increasing worldwide. This retrospective study aimed to assess the association between MAC culture-positive bronchoscopy specimens and lung cancer. Materials and Methods: The medical records of 1382 untreated lung cancer patients between 2003 and 2011 were collected using our hospital database. Of them, records for 1258 that had undergone bronchoscopy together with sampling for mycobacterial culture were reviewed. Patient characteristics were compared between those with MAC-positive/other nontuberculous mycobacteria (NTM)-negative bronchial washings and those with MAC-negative/other NTM-negative bronchial washings. Patients with MAC-positive lung cancer were cross-sectionally divided into MACLD and non-MACLD groups, and their features were assessed. Follow-up data for patients with lung cancer but without MACLD were reviewed for subsequent development of MACLD. Results: Of the 1258 patients with lung cancer, 25 (2.0%) had MAC-positive/other NTM-negative bronchial washings. The proportion of women (52% vs 30%; P = 0.0274) and patient age (72 years vs 69 years; P = 0.0380) were significantly higher in the MAC-positive/other NTM-negative lung cancer group (n = 25) than in the MAC-negative/other NTM-negative lung cancer group (n = 1223). There were 10 patients with lung cancer and MACLD and 15 without MACLD; significant differences in patient characteristics were not found between the two groups, and none of the 15 patients without MACLD subsequently developed MACLD. Conclusion: MAC culture-positive bronchial washing is positively associated with lung cancer. Female sex and advanced age, but not lung cancer characteristics, were found to be associated with MAC infection in patients with lung cancer. PMID:27335625

  3. Detection of mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany.

    PubMed

    Galiero, Alessia; Fratini, Filippo; Mataragka, Antonia; Turchi, Barbara; Nuvoloni, Roberta; Ikonomopoulos, John; Cerri, Domenico

    2016-01-18

    Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto's thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms. Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×10(4)Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR. PMID:26555160

  4. Cooccurrence of free-living amoebae and nontuberculous Mycobacteria in hospital water networks, and preferential growth of Mycobacterium avium in Acanthamoeba lenticulata.

    PubMed

    Ovrutsky, Alida R; Chan, Edward D; Kartalija, Marinka; Bai, Xiyuan; Jackson, Mary; Gibbs, Sara; Falkinham, Joseph O; Iseman, Michael D; Reynolds, Paul R; McDonnell, Gerald; Thomas, Vincent

    2013-05-01

    The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.

  5. Accumulation of γ‐aminobutyric acid by E nterococcus avium 9184 in scallop solution in a two‐stage fermentation strategy

    PubMed Central

    Yang, Haoyue; Hu, Linfeng; Liu, Song

    2015-01-01

    Summary In this study, a new bacterial strain having a high ability to produce γ‐aminobutyric acid (GABA) was isolated from naturally fermented scallop solution and was identified as E nterococcus avium. To the best of our knowledge, this is the first study to prove that E . avium possesses glutamate decarboxylase activity. The strain was then mutagenized with UV radiation and was designated as E . avium 9184. Scallop solution was used as the culture medium to produce GABA. A two‐stage fermentation strategy was applied to accumulate GABA. In the first stage, cell growth was regulated. Optimum conditions for cell growth were pH, 6.5; temperature, 37°C; and glucose concentration, 10 g·L−1. This produced a maximum dry cell mass of 2.10 g·L−1. In the second stage, GABA formation was regulated. GABA concentration reached 3.71 g·L−1 at 96 h pH 6.0, 37°C and initial l‐monosodium glutamate concentration of 10 g·L−1. Thus, compared with traditional one‐stage fermentation, the two‐stage fermentation significantly increased GABA accumulation. These results provide preliminary data to produce GABA using E . avium and also provide a new approach to process and utilize shellfish. PMID:26200650

  6. PCR analysis of the presence and location of Mycobacterium avium in a constructed reed bed, with implications for avian tuberculosis control.

    PubMed

    Drewe, Julian A; Mwangi, David; Donoghue, Helen D; Cromie, Ruth L

    2009-02-01

    The potential of reed beds to act as biofilters of pathogenic and environmental mycobacteria was investigated through examination of the fate of mycobacteria in a constructed reed bed filtering effluent from a large captive wildfowl collection. Particular emphasis was placed on the presence and location of Mycobacterium avium--the causal agent of avian tuberculosis (ATB)--in an effort to clarify the potential role of reed beds in the control of this disease. Water, sediment, and stems and roots of common reed (Phragmites australis) and greater reedmace (Typha latifolia) were taken from 15 locations within the reed bed plus sites upstream and downstream. Samples were analysed for mycobacteria using PCR and specifically for M. avium using nested PCR. Environmental mycobacteria were found throughout the entire reed bed but M. avium was not found downstream of the first vegetation growth. The reed bed was found to effectively remove M. avium from the water through a combination of sedimentation and adsorption onto vegetation stems. The results of this study show that constructed reed beds composed of a settlement lagoon and one or more vegetation beds can act as valuable and ecologically friendly tools in the environmental control of ATB.

  7. Evaluation of the association between fecal excretion of Mycobacterium avium subsp paratuberculosis and detection in colostrum and on teat skin surfaces of dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective—To evaluate the association between fecal excretion of Mycobacterium avium subsp paratuberculosis (MAP) by dairy cows in the periparturient period and detection of MAP DNA in colostrum specimens and on teat skin surfaces. Design—Cross-sectional study. Animals—112 Holstein cows. Procedures—...

  8. Chemical decontamination with n-acetyl-l-cysteine-sodium hydroxide improves recovery of viable Mycobacterium avium subsp. paratuberculosis organisms from cultured milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is shed into milk and feces of cows with advanced Johne’s disease, allowing transmission of MAP among animals. The objective of this study was to formulate an optimized protocol for the isolation of MAP from milk. Parameters investigated included che...

  9. Predicting the role of IL-10 in the regulation of the adaptive immune responses in Mycobacterium avium subsp. paratuberculosis infections using mathematical models

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular bacterial pathogen that causes Johne’s disease in cattle and other animals. Infection follows ingestion of the bacteria primarily through the fecal oral route and results in the colonization of the intestine and a granulomatous en...

  10. Clinical disease and stage of lactation influences shedding of Mycobacterium avium subsp. paratuberculosis into milk and colostrum of naturally infected dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and co...

  11. Effect of feeding heat-treated colostrum on risk for infection with Mycobacterium avium subsp. paratuberculosis, milk production and longevity in Holstein dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In summer 2007, a randomized controlled clinical trial was initiated on 6 large Midwest commercial dairy farms to investigate the effect of feeding heat-treated (HT) colostrum on transmission of Mycobacterium avium subsp. paratuberculosis and on future milk production and longevity within the herd. ...

  12. [Strategies for Mycobacterium avium complex infection control in Japan: how do they improve the present situation?].

    PubMed

    Ogawa, Kenji; Sano, Chiaki

    2013-03-01

    Mycobacterium avium complex (MAC) were the most frequently isolated (about 80%) and most common cause of lung nontuberculosis. Its rate of infection is globally increasing, especially in Japan. In this situation, it is urgently needed to provide scientific evidences and develop therapeutic interventions in MAC infections. Recently, more and more patients are elderly women with no history of smoking, and they have reticulonodular infiltrates and patchy bilateral bronchiectasis. However the prognostic and intractable factors of MAC infections are poorly known. In this symposium, we address five novel strategies for MAC infection, concerning the more accurate incidence and prevalence rates compared with other countries, host defense associated with Th1/Th17 balance, route of MAC infection related soil exposure, MAC IgA antibody as a diagnosis maker, and improved chemotherapy including aminoglycoside or new quinolone. Appropriate clinical intervention may help to reduce the prolongation of MAC infection or enhance the activity of chemotherapy for the improved control of MAC. Below are the abstracts for each of the five speakers. 1. Review of current epidemiological study of pulmonary nontuberculous mycobacterial disease in Japan and the rest of the world: Kozo MORIMOTO (Respiratory Center, Fukujuji Hospital, Japan Anti-Tuberculosis Association) The studies on pulmonary nontuberculous mycobacterial (NTM) disease prevalence were started in early 1970s in Japan by the Mycobacteriosis Research Group of National Chest Hospitals. They were followed by a questionnaire survey in 1990s, by the National Tuberculosis and NTM Survey in late 1990s, and recently by the questionnaire surveys conducted by the NTM Disease Research Committee. The latest data in Japan (from 2007) indicated a morbidity rate of 5.7 per 100,000 population. Deaths from NTM disease were reported for the first time in 1970 and showed a marked, steady increase until 2007, with 912 deaths in that year. We

  13. Acidic methanolysis v. alkaline saponification in gas chromatographic characterization of mycobacteria: differentiation between Mycobacterium avium-intracellulare and Mycobacterium gastri.

    PubMed

    Larsson, L

    1983-08-01

    Mycobacterium avium-intracellulare and M.gastri were analyzed with capillary gas chromatography after each strain had been subjected to acidic methanolysis or to alkaline saponification followed by methylation. Prominent peaks of myristic, palmitoleic, palmitic, oleic, stearic and tuberculostearic acids were found in the chromatograms of both species, whereas 2-octadecanol and 2-eicosanol were detected only in M. avium-intracellulare. In initial runs, both of the derivatization principles yielded virtually identical chromatograms for a given strain. After repeated injections of extracts from alkaline saponification, however, the alcohol peaks showed pronounced tailing and finally almost disappeared from the chromatograms. This disadvantage, which was not observed when only acid methanolysis was used, could be overcome with trifluoroacetylation. Restored peak shape of the underivatized alcohols could be achieved by washing the cross-linked stationary phase in the capillary tubing with organic solvents. The study demonstrated the importance of conditions which enable separation of 2-octadecanol and 2-eicosanol when gas chromatography is used for species identification of mycobacteria.

  14. Preliminary Evaluation of a Sitafloxacin-Containing Regimen for Relapsed or Refractory Pulmonary Mycobacterium avium Complex Disease

    PubMed Central

    Fujita, Kohei; Fujita, Masaki; Ito, Yutaka; Hirai, Toyohiro; Mio, Tadashi; Watanabe, Kentaro; Mishima, Michiaki

    2016-01-01

    Although sitafloxacin (STFX) is known to have a favorable minimum inhibitory concentration for Mycobacterium avium, few studies have evaluated the clinical efficacy of an STFX-containing regimen for pulmonary M avium complex (MAC) disease. To evaluate the clinical efficacy of STFX-containing regimens for relapsed or refractory pulmonary MAC disease, we retrospectively reviewed 18 patients with pulmonary MAC disease who received STFX for at least 4 weeks for pulmonary MAC disease between January 2008 and February 2016. Of 18 patients, 10 (55.6%) showed improved radiological characteristics and 8 (44.4%) showed negative sputum cultures at 6 months. Regarding the clinical symptoms, improvements were observed in decreasing order in sputum production (77.8%), cough (72.2%), and malaise (55.6%). Common adverse events included nausea or vomiting (38.9%), followed by loose stool or diarrhea (27.8%) and sleepiness (11.1%). Although this study contained a small number of subjects, we describe a STFX-containing regimen that was effective in achieving sputum culture negative conversions and had an acceptable adverse events profile.

  15. Genetic relatedness of Mycobacterium avium-intracellulare complex isolates from patients with pulmonary MAC disease and their residential soils.

    PubMed

    Fujita, K; Ito, Y; Hirai, T; Maekawa, K; Imai, S; Tatsumi, S; Niimi, A; Iinuma, Y; Ichiyama, S; Mishima, M

    2013-06-01

    Mycobacterium avium-intracellulare complex (MAC) strains were recovered from 48.9% of residential soil samples (agricultural farms (n = 7), residential yards (n = 79), and planting pots (n = 49)) of 100 pulmonary MAC patients and 35 non-infected control patients. The frequency of MAC recovery did not differ among soil types or among patients regardless of the presence of pulmonary MAC disease, infecting MAC species or period of soil exposure. Variable numbers of tandem repeats (VNTR) analysis for MAC clinical and soil isolates revealed 78 different patterns in 47 M. avium clinical isolates and 41 soil isolates, and 53 different patterns in 18 M. intracellulare clinical isolates and 37 soil isolates. Six clinical and corresponding soil isolate pairs with an identical VNTR genotype were from case patients with high soil exposure (≥2 h per week, 37.5% (6/16) with high exposure compared with 0.0% (0/19) with low or no exposure, p <0.01), suggesting that residential soils are a likely source of pulmonary MAC infection.

  16. Molecular and quantitative signatures of biparental inbreeding depression in the self-incompatible tree species Prunus avium.

    PubMed

    Jolivet, C; Rogge, M; Degen, B

    2013-05-01

    Genetic diversity strongly influences populations' adaptability to changing environments and therefore survival. Sustainable forest management practices have multiple roles including conservation of genetic resources and timber production. In this study, we aimed at better understanding the variation in genetic diversity among adult and offspring individuals, and the effects of mating system on offspring survival and growth in wild cherry, Prunus avium. We analysed adult trees and open pollinated seed-families from three stands in Germany at eight microsatellite loci and one incompatibility system locus and conducted paternity analyses. Seed viability testing and seed sowing in a nursery allowed further testing for the effects of pollen donor diversity and genetic similarity between mates on the offspring performance at the seed and seedling stages. Our results were contrasting across stands. Loss of genetic diversity from adult to seedling stages and positive effect of mate diversity on offspring performance occurred in one stand only, whereas biparental inbreeding depression and significant decrease in fixation index from adults to seedlings was detected in two stands. We discussed the effects of stand genetic diversity on the magnitude of biparental inbreeding depression at several life-stages and its consequences on the management of genetic resources in P. avium.

  17. Concurrent infection with Mycobacterium avium subsp. hominissuis and Giardia duodenalis in a chinchilla (Chinchilla lanigera f. dom.).

    PubMed

    Barthel, Yvonne; Drews, Sandra; Fehr, Michael; Moser, Irmgard; Matz-Rensing, Kerstin; Baumgärtner, Wolfgang; Wohlsein, Peter

    2016-01-01

    A 3-year-old, female chinchilla (Chinchilla lanigera f. dom.) suffered from prolonged vaginal discharge. Sonographically, multiple nodules were detected in the uterus, and the lung showed a diffuse radiodensity. Ovario-hysterectomy was performed and histology of the uterus revealed a severe multifocal pyogranulomatous metritis with myriads of acid-fast rod-shaped bacilli. Microbiological culture of formalin-fixed uterine tissue and a native vaginal swab resulted in the growth of mycobacteria that were identified as Mycobacterium (M.) avium subsp. hominissuis. The animal was euthanized and pathomorphological examination revealed severe multifocal granulomatous inflammation of lung, mediastinal and mesenteric lymph nodes, intestine, pancreas and kidneys. In addition, an infection of the small intestine with Giardia duodenalis was confirmed immunohistochemically. This is the first report describing a concurrent infection with M. avium subsp. hominissuis and Giardia duodenalis in a chinchilla. Both pathogens represent a potential health risk especially for young or immunosuppressed persons, in particular if infected animals show unspecific clinical symptoms. PMID:27344917

  18. Broncho-Pleural Fistula with Hydropneumothorax at CT: Diagnostic Implications in Mycobacterium avium Complex Lung Disease with Pleural Involvement

    PubMed Central

    Yoon, Hyun Jung; Chung, Myung Jin; Lee, Kyung Soo; Kim, Jung Soo; Park, Hye Yun

    2016-01-01

    Objective To determine the patho-mechanism of pleural effusion or hydropneumothorax in Mycobacterium avium complex (MAC) lung disease through the computed tomographic (CT) findings. Materials and Methods We retrospectively collected data from 5 patients who had pleural fluid samples that were culture-positive for MAC between January 2001 and December 2013. The clinical findings were investigated and the radiological findings on chest CT were reviewed by 2 radiologists. Results The 5 patients were all male with a median age of 77 and all had underlying comorbid conditions. Pleural fluid analysis revealed a wide range of white blood cell counts (410–100690/µL). The causative microorganisms were determined as Mycobacterium avium and Mycobacterium intracellulare in 1 and 4 patients, respectively. Radiologically, the peripheral portion of the involved lung demonstrated fibro-bullous changes or cavitary lesions causing lung destruction, reflecting the chronic, insidious nature of MAC lung disease. All patients had broncho-pleural fistulas (BPFs) and pneumothorax was accompanied with pleural effusion. Conclusion In patients with underlying MAC lung disease who present with pleural effusion, the presence of BPFs and pleural air on CT imaging are indicative that spread of MAC infection is the cause of the effusion. PMID:26957917

  19. Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

    PubMed

    Nazareth, Nair; Magro, Fernando; Appelberg, Rui; Silva, Jani; Gracio, Daniela; Coelho, Rosa; Cabral, José Miguel; Abreu, Candida; Macedo, Guilherme; Bull, Tim J; Sarmento, Amélia

    2015-12-01

    Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn's disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.

  20. Molecular and quantitative signatures of biparental inbreeding depression in the self-incompatible tree species Prunus avium

    PubMed Central

    Jolivet, C; Rogge, M; Degen, B

    2013-01-01

    Genetic diversity strongly influences populations' adaptability to changing environments and therefore survival. Sustainable forest management practices have multiple roles including conservation of genetic resources and timber production. In this study, we aimed at better understanding the variation in genetic diversity among adult and offspring individuals, and the effects of mating system on offspring survival and growth in wild cherry, Prunus avium. We analysed adult trees and open pollinated seed-families from three stands in Germany at eight microsatellite loci and one incompatibility system locus and conducted paternity analyses. Seed viability testing and seed sowing in a nursery allowed further testing for the effects of pollen donor diversity and genetic similarity between mates on the offspring performance at the seed and seedling stages. Our results were contrasting across stands. Loss of genetic diversity from adult to seedling stages and positive effect of mate diversity on offspring performance occurred in one stand only, whereas biparental inbreeding depression and significant decrease in fixation index from adults to seedlings was detected in two stands. We discussed the effects of stand genetic diversity on the magnitude of biparental inbreeding depression at several life-stages and its consequences on the management of genetic resources in P. avium. PMID:23211795

  1. Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe.

    PubMed

    Moreira, A R; Paolicchi, F; Morsella, C; Zumarraga, M; Cataldi, A; Fabiana, B; Alicia, A; Piet, O; van Soolingen, D; Isabel, R M

    1999-12-01

    Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated 'A', 'B', 'C' and 'E') were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern 'A', was found in 46 isolates (75%). The second, pattern 'E', included 8 isolates (13%), while the third, pattern 'B', included 6 isolates (10%). Pattern 'C' was found for only one isolate. All of the deer isolates were classified as pattern 'A', while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern 'A', was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns 'B', 'C' and 'E', were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.

  2. Culture and serologic survey for Mycobacterium avium subsp. paratuberculosis infection among southeastern white-tailed deer (Odocoileus virginianus).

    PubMed

    Davidson, William R; Manning, Elizabeth J B; Nettles, Victor F

    2004-04-01

    From July 1998 through October 2002, radiometric culture (ileocecal lymph node, mesenteric lymph node, and feces) and serologic testing by enzyme-linked immunosorbent assay (ELISA) were used to survey white-tailed deer (Odocoilens virgianus) from the soutlheastern United States for infection by Mycobacterium avium subsp. paratuberculosis (Mptb), the causative agent of paratuberculosis (Johne's disease). Mycobacterium avium subsp. paratuberculosis was isolated from the ileocecal lymph node of one of 313 deer (0.3%) originating from 63 populations in Alabama, Arkansas, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, South Carolina, Tennessee, and West Virginia (USA). Six deer (2%), all from different populations, had ELISA results above a 0.25 sample-to-positive cutoff value, but none of the ELISA reactors originated from the population from which the single Mptb isolation was made. These six deer were seronegative when tested by agar gel immunodiffusion (AGID). Collectively, these data indicate that white-tailed deer currently do not constitute a broad regional reservoir for Mptb; however, further study is warranted to clarify the significance, if any, of infected deer to the epizootiology of paratuberculosis on a local scale. Adaptation and validation of an ELISA or another serologic assay for use with deer and other wildlife would markedly enhance Mptb surveillanece among wild populations and would be a powerful tool for gaining information on the role of wild species in epidemiology of paratuberculosis.

  3. Isolation and identification of Mycobacterium avium complex and other nontuberculosis mycobacteria from drinking-water in Basra governorate, Iraq.

    PubMed

    Al-Sulami, A A; Al-Taee, A M R; Wida'a, Q H

    2012-03-01

    This study aimed to determine the occurrence of Mycobacterium avium complex and other nontuberculous mycobacteria in drinking-water in Basra governorate, Iraq and their susceptibility to several antibiotics and the effect of 0.5 mg/L of chlorine on their survival. A total of 404 samples of drinking-water were collected from 33 different districts of the governorate from November 2006 to August 2007. Filtered samples were incubated for 7 days or less in a monophasic-biphasic culture setup of tuberculosis broth and Lowenstein-Jensen agar. The 252 isolates were identified as M. avium complex (21), M. marinum (15), M. kansasii (30), M. simiae (20), M. szulgai (19), M. xenopi (16), M. malmoense (11), M. fortuitum (37), M. chelonae (50) and M. abscessus (33). Isolates were tested for antibiotic susceptibility as well as their ability to tolerate chlorine at a concentration of 0.5 mg/L. The presence of these pathogenic bacteria in drinking-water renders the water unfit for human consumption.

  4. Absence of Mycobacterium intracellulare and presence of Mycobacterium chimaera in household water and biofilm samples of patients in the United States with Mycobacterium avium complex respiratory disease.

    PubMed

    Wallace, Richard J; Iakhiaeva, Elena; Williams, Myra D; Brown-Elliott, Barbara A; Vasireddy, Sruthi; Vasireddy, Ravikiran; Lande, Leah; Peterson, Donald D; Sawicki, Janet; Kwait, Rebecca; Tichenor, Wellington S; Turenne, Christine; Falkinham, Joseph O

    2013-06-01

    Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC.

  5. Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Khalifeh, M S; Al-Majali, A M; Stabel, J R

    2009-09-15

    Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) as well as the pro-inflammatory cytokine IFN-gamma were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-gamma stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-beta prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN

  6. Occurrence of Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta cow-calf operations.

    PubMed

    Pruvot, M; Kutz, S; Barkema, H W; De Buck, J; Orsel, K

    2014-11-01

    Mycobacterium avium subsp. paratuberculosis (MAP) and Neospora caninum (NC) are two pathogens causing important production limiting diseases in the cattle industry. Significant impacts of MAP and NC have been reported on dairy cattle herds, but little is known about the importance, risk factors and transmission patterns in western Canadian cow-calf herds. In this cross-sectional study, the prevalence of MAP and NC infection in southwest Alberta cow-calf herds was estimated, risk factors for NC were identified, and the reproductive impacts of the two pathogens were assessed. Blood and fecal samples were collected from 840 cows on 28 cow-calf operations. Individual cow and herd management information was collected by self-administered questionnaires and one-on-one interviews. Bayesian estimates of the true prevalence of MAP and NC were computed, and bivariable and multivariable statistical analysis were done to assess the association between the NC serological status and ranch management risk factors, and the clinical effects of the two pathogens. Bayesian estimates of true prevalence indicated that 20% (95% probability interval: 8-38%) of herds had at least one MAP-positive cow, with a within-herd prevalence in positive herds of 22% (8-45%). From the Bayesian posterior distributions of NC prevalence, the median herd-level prevalence was 66% (33-95%) with 10% (4-21%) cow-level prevalence in positive herds. Multivariable analysis indicated that introducing purchased animals in the herd might increase the risk of NC. The negative association of NC with proper carcass disposal and presence of horses on ranch (possibly in relation to herd monitoring and guarding activities), may suggest the importance of wild carnivores in the dynamics of this pathogen in the study area. We also observed an association between MAP and NC serological status and the number of abortions. Additional studies should be done to further examine specific risk factors for MAP and NC, assess the

  7. Scavenging of reactive oxygen species by a glycolipid fraction of Mycobacterium avium serovar 2.

    PubMed

    Scherer, T A; Lauredo, I T; Abraham, W M

    1997-01-01

    Previous experiments indicated that MIF-A3, a peptidoglycolipid extracted from Mycobacterium avium serovar 2 (Mycobacterium paratuberculosis 18), inhibits the killing of Candida albicans by activated bovine peripheral blood-derived macrophages and murine thioglycollate-elicited peritoneal macrophages in vitro. Subsequent in vitro data from our laboratory indicated that this reduction in killing may be related to the ability of MIF-A3 to scavenge reactive oxygen species (ROS). In this study we examined this hypothesis directly by determining if MIF-A3 reduced exogenous H2O2-induced candidacidal activity. When Candida albicans was incubated with H2O2 (4 mM) alone, colony-forming units/ml x 10(4) (CFU/ml) were 0.4 +/- 0.1 (mean +/- SE, n = 4) as compared to 11.3 +/- 2.0 CFU/ml in control (untreated) cultures (p < .05). The addition of catalase at concentrations > or = 6.8 U/ml, completely blocked the fungicidal effect of H2O2. However, reducing the amount of catalase from 6.8 U/ml to 3.4 U/ml resulted in a loss of scavenging activity, which was associated with a 50% increase in H2O2-mediated killing. Substituting MIF-A3 (400 micrograms/ml) for catalase, also reduced H2O2-induced fungicidal activity. In the absence of MIF-A3, H2O2 reduced Candida albicans to less than 10(3) CFU/ml. However, in the presence of MIF-A3 the CFU/ml of Candida albicans increased 7.5-fold. Based on concentration-response curves of H2O2 inhibition vs. increasing amounts of catalase we determined that the relative inhibitory capacity of the MIF-A3 (400 micrograms/ml) was approximately 1.0 U/ml "catalase equivalents." These findings provide direct evidence that MIF-A3 can scavenge H2O2, and reduce H2O2-induced killing of Candida albicans. PMID:8981049

  8. The potential Public Health Impact of Mycobacterium avium ssp. paratuberculosis: Global Opinion Survey of Topic Specialists.

    PubMed

    Waddell, L A; Rajić, A; Stärk, K D C; McEwen, S A

    2016-05-01

    Global research knowledge has accumulated over the past few decades, and there is reasonable evidence for a positive association between Mycobacterium avium spp. paratuberculosis and Crohn's disease in humans, although its role as a human pathogen has not been entirely accepted. For this reason, management of public health risk due to M. paratuberculosis remains an important policy issue in agri-food public health arenas in many countries. Responsible authorities must decide whether existing mitigation strategies are sufficient to prevent or reduce human exposure to M. paratuberculosis. A Web-based questionnaire was administered to topic specialists to elicit empirical knowledge and opinion on the overall public health impact of M. paratuberculosis, the importance of various routes of human exposure to the pathogen, existing mitigation strategies and the need for future strategies. The questionnaire had four sections and consisted of 20 closed and five open questions. Topic specialists believed that M. paratuberculosis is likely a risk to human health (44.8%) and, given the paucity of available evidence, most frequently ranked it as a moderate public health issue (40.1%). A significant correlation was detected between topic specialists' commitment to M. paratuberculosis in terms of the number of years or proportion of work dedicated to this topic, and the likelihood of an extreme answer (high or low) to the above questions. Topic specialists identified contact with ruminants and dairy products as the most likely routes of exposure for humans. There was consensus on exposure routes for ruminants and what commodities to target in mitigation efforts. Described mandatory programmes mainly focused on culling diseased animals and voluntary on-farm prevention programmes. Despite ongoing difficulties in the identification of subclinical infections in animals, the topic specialists largely agreed that further enhancement of on-farm programmes in affected commodities by

  9. Lactase persistence, NOD2 status and Mycobacterium avium subsp. paratuberculosis infection associations to Inflammatory Bowel Disease

    PubMed Central

    2012-01-01

    Background Inflammatory Bowel Disease (IBD), which includes both Crohn’s disease (CD) and ulcerative colitis (UC), is caused by a complex interplay involving genetic predisposition, environmental factors and an infectious agent. Mycobacterium avium subsp. paratuberculosis (MAP) is a promising pathogen candidate since it produces a chronic intestinal inflammatory disease in ruminants that resembles CD in humans. MAP is a ubiquitous microorganism, although its presence in the food chain, especially in milk from infected animals, is what made us think that there could be an association between lactase persistence (LP) and IBD. The LCT mutation has brought adaptation to dairy farming which in turn would have increased exposure of the population to infection by MAP. NOD2 gene mutations are highly associated to CD. Methods In our study, CD and UC patients and controls from the North of Spain were genotyped for the lactase gene (LCT) and for three NOD-2 variants, R702W, G908R and Cins1007fs. MAP PCR was carried out in order to assess MAP infection status and these results were correlated with LCT and NOD2 genotypes. Results As for LP, no association was found with IBD, although UC patients were less likely to present the T/T−13910 variant compared to controls, showing a higher C-allele frequency and a tendency to lactase non-persistence (LNP). NOD2 mutations were associated to CD being the per-allele risk higher for the Cins1007fs variant. MAP infection was more extended among the healthy controls (45.2%) compared to CD patients (21.38%) and UC patients (19.04%) and this was attributed to therapy. The Asturian CD cohort presented higher levels of MAP prevalence (38.6%) compared to the Basque CD cohort (15.5%), differences also attributed to therapy. No interaction was found between MAP infection and LCT or NOD2 status. Conclusions We conclude that LP is not significantly associated with IBD, but that MAP infection and NOD2 do show not mutually interacting associations

  10. A screening sampling plan to detect Mycobacterium avium subspecies paratuberculosis-positive dairy herds.

    PubMed

    Serraino, A; Arrigoni, N; Ostanello, F; Ricchi, M; Marchetti, G; Bonilauri, P; Bonfante, E; Giacometti, F

    2014-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic contagious bacterial disease primarily affecting dairy cattle. Paratuberculosis represents a dual problem for the milk production chain: in addition to economic losses to affected herds, MAP may have zoonotic potential. Infected herds must be identified in order to implement programs designed to reduce the incidence of disease within and between herds and to prevent MAP from entering the food chain. The objective of this study was to evaluate the sensitivity and specificity of a screening sampling plan (SSP) to detect MAP-positive dairy herds by repetitive analysis of bulk tank milk (BTM) samples by ELISA and in-line milk filter (ILMF) samples by PCR. Samples from BTM and ILMF were collected twice from 569 dairy herds in southern Italy. Additionally, 12,016 individual milk samples were collected: 9,509 from 102 SSP-positive herds (SSP MAP-positive) and 2,507 from 21 randomly selected SSP-negative herds (SSP MAP-negative). There was a total of 126 SSP MAP-positive herds (i.e., 21.3% SSP MAP-positive herds; 95% confidence interval=18.0-24.9); the within-herd apparent prevalence (AP) ranged between 0.00 and 22.73% (mean 6.07%). A significant difference in within-herd AP was shown between SSP MAP-positive herds and SSP MAP-negative herds. A highly significant association was shown between the median AP herd status (>5%) and positivity to at least one ILMF or BTM sample. The SSP detected a minimum of 56.25% of low AP herds (AP ≤ 2.0%) up to a maximum of 100% of herds with a within-herd AP ≥ 8.0%. Overall, the SSP detected 85.57% of herds in which at least one individual milk sample was positive by ELISA. The proposed SSP was an inexpensive and useful tool to detect MAP-positive herds with a higher risk of infection diffusion and milk contamination. Although the SSP cannot be used for MAP-free certification of herds, it could be useful to prioritize appropriate

  11. Identification of putative candidate genes involved in cuticle formation in Prunus avium (sweet cherry) fruit

    PubMed Central

    Alkio, Merianne; Jonas, Uwe; Sprink, Thorben; van Nocker, Steven; Knoche, Moritz

    2012-01-01

    Background and Aims The cuticular membrane (CM) of Prunus avium (sweet cherry) and other fleshy fruit is under stress. Previous research indicates that the resultant strain promotes microscopic cuticular cracking. Microcracks impair the function of the CM as a barrier against pathogens and uncontrolled water loss/uptake. Stress and strain result from a cessation of CM deposition during early development, while the fruit surface continues to expand. The cessation of CM deposition, in turn, may be related to an early downregulation of CM-related genes. The aims of this study were to identify genes potentially involved in CM formation in sweet cherry fruit and to quantify their expression levels. Methods Fruit growth and CM deposition were quantified weekly from anthesis to maturity and rates of CM deposition were calculated. Sequences of genes expressed in the sweet cherry fruit skin (exocarp) were generated using high-throughput sequencing of cDNA and de novo assembly and analysed using bioinformatics tools. Relative mRNA levels of selected genes were quantified in the exocarp and fruit flesh (mesocarp) weekly using reverse transcriptase-quantitative real-time PCR and compared with the calculated CM deposition rate over time. Key Results The rate of CM deposition peaked at 93 (±5) μg per fruit d−1 about 19 d after anthesis. Based on sequence analyses, 18 genes were selected as potentially involved in CM formation. Selected sweet cherry genes shared up to 100 and 98 % similarity with the respective Prunus persica (peach) and Arabidopsis thaliana genes. Expression of 13 putative CM-related genes was restricted to the exocarp and correlated positively with the CM deposition rate. Conclusions The results support the view that the cessation of CM deposition during early sweet cherry fruit development is accounted for by a downregulation of genes involved in CM deposition. Genes that merit further investigation include PaWINA, PaWINB, PaLipase, PaLTPG1, PaATT1, Pa

  12. Economic analysis of Mycobacterium avium subspecies paratuberculosis vaccines in dairy herds.

    PubMed

    Cho, J; Tauer, L W; Schukken, Y H; Gómez, M I; Smith, R L; Lu, Z; Grohn, Y T

    2012-04-01

    Johne's disease, or paratuberculosis, is a chronic infectious enteric disease of ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). Given the absence of a fail-safe method of prevention or a cure, Johne's disease can inflict significant economic loss on the US dairy industry, with an estimated annual cost of over $200 million. Currently available MAP control strategies include management measures to improve hygiene, culling MAP serologic- or fecal-positive adult cows, and vaccination. Although the 2 first control strategies have been reported to be effective in reducing the incidence of MAP infection, the changes in herd management needed to conduct these control strategies require significant effort on the part of the dairy producer. On the other hand, vaccination is relatively simple to apply and requires minor changes in herd management. Despite these advantages, only 5% of US dairy operations use vaccination to control MAP. This low level of adoption of this technology is due to limited information on its cost-effectiveness and efficacy and some important inherent drawbacks associated with current MAP vaccines. This study investigates the epidemiological effect and economic values of MAP vaccines in various stages of development. We create scenarios for the potential epidemiological effects of MAP vaccines, and then estimate economically justifiable monetary values at which vaccines become economically beneficial to dairy producers such that a net present value (NPV) of a farm's net cash flow can be higher than the NPV of a farm using no control or alternative nonvaccine controls. Any vaccination with either low or high efficacy considered in this study yielded a higher NPV compared with a no MAP control. Moreover, high-efficacy vaccines generated an even higher NPV compared with alternative controls, making vaccination economically attractive. Two high-efficacy vaccines were particularly effective in MAP control and NPV

  13. Peak Plasma Concentration of Azithromycin and Treatment Responses in Mycobacterium avium Complex Lung Disease.

    PubMed

    Jeong, Byeong-Ho; Jeon, Kyeongman; Park, Hye Yun; Moon, Seong Mi; Kim, Su-Young; Lee, Soo-Youn; Shin, Sung Jae; Daley, Charles L; Koh, Won-Jung

    2016-10-01

    Macrolides, such as azithromycin (AZM) and clarithromycin, are the cornerstones of treatment for Mycobacterium avium complex lung disease (MAC-LD). Current guidelines recommend daily therapy with AZM for cavitary MAC-LD and intermittent therapy for noncavitary MAC-LD, but the effectiveness of these regimens has not been thoroughly investigated. This study evaluated associations between microbiological response and estimated peak plasma concentrations (Cmax) of AZM. The AZM Cmax was measured in patients receiving daily therapy (250 mg of AZM daily, n = 77) or intermittent therapy (500 mg of AZM three times weekly, n = 89) for MAC-LD and daily therapy for Mycobacterium abscessus complex LD (MABC-LD) (250 mg of AZM daily, n = 55). The AZM Cmax was lower with the daily regimen for MAC-LD (median, 0.24 μg/ml) than with the intermittent regimen for MAC-LD (median, 0.65 μg/ml; P < 0.001) or daily therapy for MABC-LD (median, 0.53 μg/ml; P < 0.001). After adjusting for confounding factors, AZM Cmax was independently associated with favorable microbiological responses in MAC-LD patients receiving a daily regimen (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 1.01 to 2.48; P = 0.044) but not an intermittent regimen (aOR, 0.85; 95% CI, 0.58 to 1.23, P = 0.379). With the daily AZM-based multidrug regimen for MAC-LD, a low AZM Cmax was common, whereas a higher AZM Cmax was associated with favorable microbiologic responses. The results also suggested that the addition of rifampin may lower AZM Cmax When a daily AZM-based multidrug regimen is used for treating severe MAC-LD, such as cavitary disease, the currently recommended AZM dose might be suboptimal. (This study has been registered at ClinicalTrials.gov under identifier NCT00970801.). PMID:27480854

  14. Fate of Mycobacterium avium subsp. paratuberculosis after application of contaminated dairy cattle manure to agricultural soils.

    PubMed

    Salgado, M; Collins, M T; Salazar, F; Kruze, J; Bölske, G; Söderlund, R; Juste, R; Sevilla, I A; Biet, F; Troncoso, F; Alfaro, M

    2011-03-01

    Details regarding the fate of Mycobacterium avium subsp. paratuberculosis (basonym, Mycobacterium paratuberculosis) after manure application on grassland are unknown. To evaluate this, intact soil columns were collected in plastic pipes (lysimeters) and placed under controlled conditions to test the effect of a loamy or sandy soil composition and the amount of rainfall on the fate of M. paratuberculosis applied to the soil surface with manure slurry. The experiment was organized as a randomized design with two factors and three replicates. M. paratuberculosis-contaminated manure was spread on the top of the 90-cm soil columns. After weekly simulated rainfall applications, water drainage samples (leachates) were collected from the base of each lysimeter and cultured for M. paratuberculosis using Bactec MGIT ParaTB medium and supplements. Grass was harvested, quantified, and tested from each lysimeter soil surface. The identity of all probable M. paratuberculosis isolates was confirmed by PCR for IS900 and F57 genetic elements. There was a lag time of 2 months after each treatment before M. paratuberculosis was found in leachates. The greatest proportions of M. paratuberculosis-positive leachates were from sandy-soil lysimeters in the manure-treated group receiving the equivalent of 1,000 mm annual rainfall. Under the higher rainfall regimen (2,000 mm/year), M. paratuberculosis was detected more often from lysimeters with loamy soil than sandy soil. Among all lysimeters, M. paratuberculosis was detected more often in grass clippings than in lysimeter leachates. At the end of the trial, lysimeters were disassembled and soil cultured at different depths, and we found that M. paratuberculosis was recovered only from the uppermost levels of the soil columns in the treated group. Factors associated with M. paratuberculosis presence in leachates were soil type and soil pH (P < 0.05). For M. paratuberculosis presence in grass clippings, only manure application showed a

  15. Environmental contamination with Mycobacterium avium subsp. paratuberculosis in endemically infected dairy herds.

    PubMed

    Smith, R L; Schukken, Y H; Pradhan, A K; Smith, J M; Whitlock, R H; Van Kessel, J S; Wolfgang, D R; Grohn, Y T

    2011-10-01

    Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates

  16. Mycobacterium avium subspecies paratuberculosis causes Crohn's disease in some inflammatory bowel disease patients

    PubMed Central

    Naser, Saleh A; Sagramsingh, Sudesh R; Naser, Abed S; Thanigachalam, Saisathya

    2014-01-01

    Crohn’s disease (CD) is a chronic inflammatory condition that plagues millions all over the world. This debilitating bowel disease can start in early childhood and continue into late adulthood. Signs and symptoms are usually many and multiple tests are often required for the diagnosis and confirmation of this disease. However, little is still understood about the cause(s) of CD. As a result, several theories have been proposed over the years. One theory in particular is that Mycobacterium avium subspecies paratuberculosis (MAP) is intimately linked to the etiology of CD. This fastidious bacterium also known to cause Johne’s disease in cattle has infected the intestines of animals for years. It is believed that due to the thick, waxy cell wall of MAP it is able to survive the process of pasteurization as well as chemical processes seen in irrigation purification systems. Subsequently meat, dairy products and water serve as key vehicles in the transmission of MAP infection to humans (from farm to fork) who have a genetic predisposition, thus leading to the development of CD. The challenges faced in culturing this bacterium from CD are many. Examples include its extreme slow growth, lack of cell wall, low abundance, and its mycobactin dependency. In this review article, data from 60 studies showing the detection and isolation of MAP by PCR and culture techniques have been reviewed. Although this review may not be 100% comprehensive of all studies, clearly the majority of the studies overwhelmingly and definitively support the role of MAP in at least 30%-50% of CD patients. It is very possible that lack of detection of MAP from some CD patients may be due to the absence of MAP role in these patients. The latter statement is conditional on utilization of methodology appropriate for detection of human MAP strains. Ultimately, stratification of CD and inflammatory bowel disease patients for the presence or absence of MAP is necessary for appropriate and effective

  17. Evidence of birth seasonality and clustering of Mycobacterium avium subspecies paratuberculosis infection in US dairy herds.

    PubMed

    Zare, Y; Shook, G E; Collins, M T; Kirkpatrick, B W

    2013-11-01

    Paratuberculosis (Johne's disease) is a contagious intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). In cattle, young calves are at the highest risk for acquiring the infection which occurs mainly through ingestion of MAP from contaminated milk, colostrum and feces or environmental contacts. Data consisted of birth dates and ELISA results of 8000 mature cows from 24 Jersey herds from throughout the US and 4 Wisconsin Holstein herds. Some herds also had complete fecal culture (FC) results. The first infection (case) definition (CD1) relied on only ELISA results. A second case definition (CD2) was used in which results of both ELISA and FC tests were considered: animals testing positive to either test were considered "test-positives" and cows testing negative to ELISA or to both ELISA and FC were regarded as "test-negatives". Objective one was to assess seasonality in birth of MAP-infected animals. The effects of age, breed, herd and season of birth (expressed as the sine and cosine functions of birth days within year) were examined using logistic regression. Age was significantly associated with the MAP infection status of dairy cows for both CDs (OR=1.11; 95% CI 1.09, 1.14; P<0.0001 for CD1; OR=1.16; 95% CI 1.08, 1.24; P<0.0001 for CD2). Season of birth had a significant effect on the risk of MAP infection based on CD1 (OR=0.79; 95% CI 0.71, 0.89; P<0.001 for cosine of birth days) with a peak in summer and a trough in winter based on the fitted model. Objective two was to assess whether test-positive animals were randomly distributed or were clustered by date of birth within herds. A temporal cluster analysis approach (scan statistic) implemented in SaTScan software was used for each case definition to detect clusters of birth cohorts using birthdates. Results identified significant clustering of MAP infection cases for CD1 in multiple herds (P<0.05). These results necessitate matching cases and controls of MAP

  18. Proteomic Comparison of Fruit Ripening between 'Hedelfinger' Sweet Cherry (Prunus avium L.) and Its Somaclonal Variant 'HS'.

    PubMed

    Prinsi, Bhakti; Negri, Alfredo S; Espen, Luca; Piagnani, M Claudia

    2016-05-25

    The somaclonal variant HS, from sweet cherry (Prunus avium L.) 'Hedelfinger' (H), was previously selected for reduced tree vegetative vigor and lesser canopy density. In this work, we compared H and HS fruits at early unripe (green) and full ripe (dark red) stages by biochemical and proteomic approaches. The main biochemical parameters showed that fruit quality was not affected by somaclonal variation. The proteomic analysis identified 39 proteins differentially accumulated between H and HS fruits at the two ripening stages, embracing enzymes involved in several pathways, such as carbon metabolism, cell wall modification, stress response, and secondary metabolism. The evaluation of fruit phenolic composition by mass spectrometry showed that HS sweet cherries have higher levels of procyanidin, flavonol, and anthocyanin compounds. This work provides the first proteomic characterization of fruit ripening in sweet cherry, revealing new positive traits of the HS somaclonal variant. PMID:27144542

  19. Isolation of the Mycobacterium avium-M. intracellulare-M. scrofulaceum complex from tank water in Queensland, Australia.

    PubMed

    Tuffley, R E; Holbeche, J D

    1980-01-01

    Disease-associated serotypes of Mycobacterium intracellulare and M. avium have been isolated from 32 of 141 rainwater tanks situated in the basin of the Fitzroy River and its tributaries in central Queensland, 7 of 32 tanks situated in the hinterland of the coastal city of Rockhampton, and 2 of 32 tanks sampled repetitively in the southeastern Queensland city of Toowoomba. M. gordonae was also isolated from 23 of the river basin tanks, from 9 in the Rockhampton hinterland, and from 5 in the city of Toowoomba. One half of these isolates came from tanks which also yielded M. intracellulare. Mycobacteria of the M. terrae-M. triviale-M. nonchromogenicum complex were found in 7 tanks, usually in conjunction with M. intracellulare. The humans who consume the contaminated tank water are free of symptoms but have not been medically examined. It is suggested that mycobacteria adhering to dust particles disturbed by mechanical cultivation may be the source of contamination.

  20. Antineutrophil Cytoplasmic Antibody-associated Vasculitis Superimposed on Infection-related Glomerulonephritis Secondary to Pulmonary Mycobacterium avium Complex Infection.

    PubMed

    Asano, Shuichi; Mizuno, Shige; Okachi, Shotaro; Aso, Hiromichi; Wakahara, Keiko; Hashimoto, Naozumi; Ito, Satoru; Kozaki, Yohei; Katsuno, Takayuki; Maruyama, Shoichi; Hasegawa, Yoshinori

    2016-01-01

    A 73-year-old woman was diagnosed with pulmonary Mycobacterium avium complex (MAC) infection and received no treatment. Disease progression was evident one year later with the development of myeloperoxidase-antineutrophil cytoplasmic antibody (ANCA) titers and systemic symptoms of a fever, polyarthritis, purpura, and rapidly progressive glomerulonephritis. Her symptoms did not improve with antibiotic treatment. A renal biopsy revealed crescentic glomerulonephritis with immunodeposition. According to these findings, she was diagnosed with ANCA-associated vasculitis (AAV) superimposed on infection-related glomerulonephritis (IRGN). Although there was a risk of aggravating an underlying infection, the combination therapy of corticosteroid and antibiotics improved AAV, IRGN, and even the lung radiological findings. To the best of our knowledge, this is the first case of AAV and IRGN secondary to pulmonary MAC infection. PMID:27580547

  1. Comparison of the antimicrobial activities of gallium nitrate and gallium maltolate against Mycobacterium avium subsp. paratuberculosis in vitro.

    PubMed

    Fecteau, Marie-Eve; Aceto, Helen W; Bernstein, Lawrence R; Sweeney, Raymond W

    2014-10-01

    Johne's disease (JD) is an enteric infection of cattle and other ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This study compared the antimicrobial activities of gallium nitrate (GaN) and gallium maltolate (GaM) against two field MAP isolates by use of broth culture. The concentrations that resulted in 99% growth inhibition of isolates 1 and 2 were, respectively, 636 µM and 183 µM for GaN, and 251 µM and 142 µM for GaM. For both isolates, time to detection was significantly higher for GaM than GaN. These results suggest that GaM is more efficient than GaN in inhibiting MAP growth in vitro.

  2. The presence of opportunistic pathogens, Legionella spp., L. pneumophila and Mycobacterium avium complex, in South Australian reuse water distribution pipelines.

    PubMed

    Whiley, H; Keegan, A; Fallowfield, H; Bentham, R

    2015-06-01

    Water reuse has become increasingly important for sustainable water management. Currently, its application is primarily constrained by the potential health risks. Presently there is limited knowledge regarding the presence and fate of opportunistic pathogens along reuse water distribution pipelines. In this study opportunistic human pathogens Legionella spp., L. pneumophila and Mycobacterium avium complex were detected using real-time polymerase chain reaction along two South Australian reuse water distribution pipelines at maximum concentrations of 10⁵, 10³ and 10⁵ copies/mL, respectively. During the summer period of sampling the concentration of all three organisms significantly increased (P < 0.05) along the pipeline, suggesting multiplication and hence viability. No seasonality in the decrease in chlorine residual along the pipelines was observed. This suggests that the combination of reduced chlorine residual and increased water temperature promoted the presence of these opportunistic pathogens. PMID:26042986

  3. Cytological comparison of leaves and stems of Prunus avium L. shoots cultured on a solid medium with agar or gelrite.

    PubMed

    Franck, T; Crèvecoeur, M; Wuest, J; Greppin, H; Gaspar, T

    1998-01-01

    An axillary proliferating clone of Prunus avium L. was subcultured every four weeks on solid MS medium with agar as the gelling agent. Vitrification (hyperhydricity) of shoots was induced in one four week cycle with the same medium except that agar was replaced by gelrite. During culture on the vitrifying medium, the water content of the shoots progressively increased with a parallel decrease in chlorophyll content. Cytological differences between the leaves and stems of the vitrified and normal shoots were detected by light and electron (both transmission and scanning) microscopy. Leaves of vitrified shoots were characterized by lower number of chloroplasts in the palisade parenchyma and by a defective cuticle. The stems of vitrified shoots had a less developed and lignifled xylem tissue, lacked sclerenchymatic areas and showed hypertrophy of the cortical parenchyma. More intense vacuolar activity with evaginations of the chloroplast envelope into the vacuole was noted in cells of vitrified leaves.

  4. PCR-based detection of Mycobacterium avium subsp. paratuberculosis infection in cattle in South Korea using fecal samples

    PubMed Central

    PARK, Hong-Tae; SHIN, Min-Kyoung; PARK, Hyun-Eui; CHO, Yong-Il; YOO, Han Sang

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis (PTB). The first step in the control of PTB is the identification and isolation of sub-clinical fecal shedders from the herd. In the current study, real-time and nested PCR targeting MAP-specific genetic elements (IS900 and ISMAP02) DNA isolated from fecal samples were used to detect MAP infection in cattle. Of the 1,562 fecal samples obtained from 37 herds, regardless of diarrhea, 35 samples tested positive in both IS900-targeted real-time and ISMAP02-targeted nested PCR. At the herd level, 12 of the 37 herds were found to be positive for MAP. Detection rates of the PCR tests were similar to those reported for ELISA-based methods. These results suggest that PCR can be used to detect sub-clinical fecal shedders of MAP. PMID:27301582

  5. The role of polar auxin transport through pedicels of Prunus avium L. in relation to fruit development and retention.

    PubMed

    Else, Mark A; Stankiewicz-Davies, Anna P; Crisp, Carol M; Atkinson, Christopher J

    2004-09-01

    It was investigated whether premature fruit abscission in Prunus avium L. was triggered by a reduction in polar auxin transport (PAT). The capacity of pedicels to transport tritiated IAA ([3H]-IAA) via the PAT pathway was measured at intervals throughout flower and fruit development. The extent of passive diffusion, assessed by concurrent applications of [14C]-benzoic acid ([14C]-BA), was negligible. Transported radioactivity recovered from agar blocks eluted at the same retention time as authentic [3H]-IAA during HPLC fractionation. The capacity for PAT was already high 7 d before anthesis and increased further following the fertilization of flowers at anthesis. PAT intensity was greatest immediately following fertilization and at the beginning of the cell expansion phase of fruit growth; the transport intensity in fruitlets destined to abscind was negligible. The amount of endogenous IAA moving through the PAT pathway was greatest during the first 3 weeks after fertilization and was again high at the beginning of the fruit expansion stage. IAA export in the phloem increased following fertilization then declined below detectable levels. ABA export in the phloem increased markedly during stone formation and at the onset of fruit expansion. TIBA applied to pedicels of fruit in situ promoted fruitlet abscission in 2000 but not in 2001, despite PAT capacity being reduced by over 98% in the treated pedicels. The application of TIBA to pedicels did not affect fruit expansion. The role of PAT and IAA in relation to the development and retention of Prunus avium fruit is discussed. PMID:15310825

  6. Possible transmission of Mycobacterium avium subspecies paratuberculosis through potable water: lessons from an urban cluster of Crohn's disease

    PubMed Central

    Pierce, Ellen S

    2009-01-01

    A "cluster" of patients refers to the geographic proximity of unrelated patients with the same disease and suggests a common environmental cause for that disease. Clusters of patients with Crohn's disease have been linked to the presence of an infectious microorganism in unpasteurized milk and cheese, untreated water supplied by wells or springs, animal manure used as fertilizer for family vegetable gardens, and bodies of water contaminated by agricultural runoff. Mycobacterium avium subspecies paratuberculosis (MAP) is the suspected cause of Crohn's disease. MAP causes a disease in dairy cows and other animals that is similar to Crohn's disease, called Johne's ('Yo-knees') disease or paratuberculosis. Dairy cows with Johne's disease secrete MAP into their milk and excrete MAP into their feces. MAP is present in untreated water such as well water, in bodies of water contaminated by agricultural runoff, and in unpasteurized milk and cheese. The "treatment" of "tap" water to make it "drinkable" or "potable" by the processes of sedimentation, filtration and chlorination has little to no effect on MAP. MAP is so resistant to chlorine disinfection that such disinfection actually selects for its growth. Other subspecies of Mycobacterium avium grow in biofilms present on tap water pipes. Despite the documented presence of MAP in tap water and its probable growth on tap water pipes, clusters of Crohn's disease have not previously been described in relationship to tap water pipes supplying patients' homes. This report describes three unrelated individuals who lived on the same block along a street in a midwestern American city and developed Crohn's disease within four years of each other in the 1960's. A common tap water pipe supplied their homes. This is the first reported cluster of Crohn's disease possibly linked to fully treated drinking water, and is consistent with previously reported clusters of Crohn's disease linked to an infectious microorganism in water. PMID

  7. Survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and caliciviruses in drinking water-associated biofilms grown under high-shear turbulent flow.

    PubMed

    Lehtola, Markku J; Torvinen, Eila; Kusnetsov, Jaana; Pitkänen, Tarja; Maunula, Leena; von Bonsdorff, Carl-Henrik; Martikainen, Pertti J; Wilks, Sandra A; Keevil, C William; Miettinen, Ilkka T

    2007-05-01

    Most of the bacteria in drinking water distribution systems are associated with biofilms. In biofilms, their nutrient supply is better than in water, and biofilms can provide shelter against disinfection. We used a Propella biofilm reactor for studying the survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and canine calicivirus (CaCV) (as a surrogate for human norovirus) in drinking water biofilms grown under high-shear turbulent-flow conditions. The numbers of M. avium and L. pneumophila were analyzed with both culture methods and with peptide nucleic acid fluorescence in situ hybridization (FISH) methods. Even though the numbers of pathogens in biofilms decreased during the experiments, M. avium and L. pneumophila survived in biofilms for more than 2 to 4 weeks in culturable forms. CaCV was detectable with a reverse transcription-PCR method in biofilms for more than 3 weeks. E. coli was detectable by culture for only 4 days in biofilms and 8 days in water, suggesting that it is a poor indicator of the presence of certain waterborne pathogens. With L. pneumophila and M. avium, culture methods underestimated the numbers of bacteria present compared to the FISH results. This study clearly proved that pathogenic bacteria entering water distribution systems can survive in biofilms for at least several weeks, even under conditions of high-shear turbulent flow, and may be a risk to water consumers. Also, considering the low number of virus particles needed to result in an infection, their extended survival in biofilms must be taken into account as a risk for the consumer.

  8. Development and validation of a liquid medium (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis to replace modified Bactec 12B medium.

    PubMed

    Whittington, Richard J; Whittington, Ann-Michele; Waldron, Anna; Begg, Douglas J; de Silva, Kumi; Purdie, Auriol C; Plain, Karren M

    2013-12-01

    Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 μl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

  9. Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications.

    PubMed

    Novikov, Alexey; Shah, Nita R; AlBitar-Nehme, Sami; Basheer, Soorej M; Trento, Ilaria; Tirsoaga, Alina; Moksa, Michelle; Hirst, Martin; Perry, Malcolm B; Hamidi, Asmaa El; Fernandez, Rachel C; Caroff, Martine

    2014-08-01

    Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.

  10. A multilaboratory evaluation of a commercial enzyme-linked immunosorbent assay test for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in cattle.

    PubMed

    Dargatz, David A; Byrum, Beverly A; Collins, Michael T; Goyal, Sagar M; Hietala, Sharon K; Jacobson, Richard H; Kopral, Christine A; Martin, Barbara M; McCluskey, Brian J; Tewari, Deepanker

    2004-11-01

    Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturer's directions. The S/ P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA

  11. Integrated MicroRNA-mRNA-Analysis of Human Monocyte Derived Macrophages upon Mycobacterium avium subsp. hominissuis Infection

    PubMed Central

    Sharbati, Jutta; Lewin, Astrid; Kutz-Lohroff, Barbara; Kamal, Elisabeth; Einspanier, Ralf; Sharbati, Soroush

    2011-01-01

    Background Many efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections. Methodology/Principal Findings We performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR). The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs. Conclusions/Significance We show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response. In addition, functional

  12. Marked Differences in Mucosal Immune Responses Induced in Ileal versus Jejunal Peyer’s Patches to Mycobacterium avium subsp. paratuberculosis Secreted Proteins following Targeted Enteric Infection in Young Calves

    PubMed Central

    Facciuolo, Antonio; Gonzalez-Cano, Patricia; Napper, Scott; Griebel, Philip J.

    2016-01-01

    In cattle, Mycobacterium avium subsp. paratuberculosis infection is primarily mediated through M cells overlying Peyer’s patches (PP) in the ileum. The capacity of M. avium subsp. paratuberculosis to invade ileal PP (IPP) versus discrete PP in the jejunum (JPP) and subsequent differences in mucosal immune responses were investigated. Intestinal segments were surgically prepared in both mid-jejunum, containing two JPPs, and in terminal small intestine containing continuous IPP. M. avium subsp. paratuberculosis (109 CFU) was injected into the lumen of half of each intestinal segment when calves were 10–14 days-old and infection confirmed 1–2 months later by PCR and immunohistochemistry. Thirteen recombinant M. avium subsp. paratuberculosis proteins, previously identified as immunogenic, were used to analyze pathogen-specific B- and T-cell responses in PP and mesenteric lymph nodes. IgA plasma cell responses to 9 of 13 recombinant proteins were detected in JPP but not in IPP. Secretory IgA reacting in ELISA with 9 of the 13 recombinant proteins was detected in luminal contents from both jejunal and ileal segments. These observations support the conclusion that pathogen-specific IgA B cells were induced in JPP but not IPP early after a primary infection. The presence of secretory IgA in intestinal contents is consistent with dissemination of IgA plasma cells from the identified mucosa-associated immune induction sites. This is the first direct evidence for M. avium subsp. paratuberculosis uptake by bovine JPP and for local induction of pathogen-specific IgA plasma cell responses after enteric infection. We also provide evidence that bacterial invasion of IPP, a primary B lymphoid tissue, provides a novel strategy to evade induction of mucosal immune responses. Over 60% of PPs in the newborn calf small intestine is primary lymphoid tissue, which has significant implications when designing oral vaccines or diagnostic tests to detect early M. avium subsp

  13. Physicochemical characteristics, antioxidant activity, organic acid and sugar contents of 12 sweet cherry (Prunus avium L.) cultivars grown in Turkey.

    PubMed

    Hayaloglu, Ali Adnan; Demir, Nurullah

    2015-03-01

    Physical characteristics, antioxidant activity and chemical constituents of 12 cultivars (Prunus avium L.) of sweet cherry (Belge, Bing, Dalbasti, Durona di Cesena, Lambert, Merton Late, Starks Gold, Summit, Sweetheart, Van, Vista, and 0-900 Ziraat) were investigated. Significant differences (P < 0.05) were observed among tested cultivars for pH, total soluble solid, hardness, color parameters, antioxidant activities and pomological measurements (P < 0.05). The color parameters were important tools for the determination of fruit maturity and anthocyanin contents. Belge cultivar showed the highest levels of total phenolic and anthocyanin, while Starks Gold contained the lowest level of anthocyanins. The darker cultivars, measured by ABTS(+•) , DPPH(•) and FRAP, exhibited higher antioxidant activities than the lighter ones. Bing (42.78 g/kg) and Sweetheart (40.53 g/kg) cultivars contained higher levels of malic acid, which was the most intense organic acid in sweet cherries. Four different sugars were observed in the samples and their concentrations ordered as glucose > fructose > sucrose > xylose. Sugar alcohol in the cherries was represented by sorbitol (more than 90%) and its concentration varied between 13.93 and 27.12 g/kg. As a result significant differences were observed among the physical properties and chemical constituents of the cherry cultivars. PMID:25631389

  14. Preharvest application of oxalic acid increased fruit size, bioactive compounds, and antioxidant capacity in sweet cherry cultivars (Prunus avium L.).

    PubMed

    Martínez-Esplá, Alejandra; Zapata, Pedro Javier; Valero, Daniel; García-Viguera, Cristina; Castillo, Salvador; Serrano, María

    2014-04-16

    Trees of 'Sweet Heart' and 'Sweet Late' sweet cherry cultivars (Prunus avium L.) were treated with oxalic acid (OA) at 0.5, 1.0, and 2.0 mM at 98, 112, and 126 days after full blossom. Results showed that all treatments increased fruit size at harvest, manifested by higher fruit volume and weight in cherries from treated trees than from controls, the higher effect being found with 2.0 mM OA (18 and 30% higher weight for 'Sweet Heart' and 'Sweet Late', respectively). Other quality parameters, such as color and firmness, were also increased by OA treatments, although no significant differences were found in total soluble solids or total acidity, showing that OA treatments did not affect the on-tree ripening process of sweet cherry. However, the increases in total anthocyanins, total phenolics, and antioxidant activity associated with the ripening process were higher in treated than in control cherries, leading to fruit with high bioactive compounds and antioxidant potential at commercial harvest (≅45% more anthocyanins and ≅20% more total phenolics). In addition, individual anthocyanins, flavonols, and chlorogenic acid derivatives were also increased by OA treatment. Thus, OA preharvest treatments could be an efficient and natural way to increase the quality and functional properties of sweet cherries. PMID:24684635

  15. Fruit quality and bioactive compounds relevant to human health of sweet cherry (Prunus avium L.) cultivars grown in Italy.

    PubMed

    Ballistreri, Gabriele; Continella, Alberto; Gentile, Alessandra; Amenta, Margherita; Fabroni, Simona; Rapisarda, Paolo

    2013-10-15

    The fruit quality characteristics, phenolic compounds and antioxidant capacities of 24 sweet cherry (Prunus avium L.) cultivars grown on the mountainsides of the Etna volcano (Sicily, Italy) were evaluated. High-performance liquid chromatographic methods were used to identify and quantify sugars, organic acids and phenolics. A total of seven phenolic compounds were characterised as hydroxycinnamic acid derivatives (neochlorogenic acid, p-coumaroylquinic acid and chlorogenic acid) and anthocyanins (cyanidin 3-glucoside, cyanidin 3-rutinoside, pelargonidin 3-rutinoside and peonidin 3-rutinoside). The total anthocyanin content ranged from 6.21 to 94.20mg cyanidin 3-glucoside equivalents/100g fresh weight (FW), while the total phenol content ranged from 84.96 to 162.21mg gallic acid equivalents/100g FW. The oxygen radical absorbance capacity (ORAC) assay indicated that fruit of all genotypes possessed considerable antioxidant activity. The high level of phenolic compounds and antioxidant capacity of some sweet cherry fruits implied that they might be sources of bioactive compounds that are relevant to human health. PMID:23692746

  16. Serum BAFF levels, Methypredsinolone therapy, Epstein-Barr Virus and Mycobacterium avium subsp. paratuberculosis infection in Multiple Sclerosis patients

    PubMed Central

    Mameli, Giuseppe; Cocco, Eleonora; Frau, Jessica; Arru, Giannina; Caggiu, Elisa; Marrosu, Maria Giovanna; Sechi, Leonardo A.

    2016-01-01

    Elevated B lymphocyte activating factor BAFF levels have been reported in multiple sclerosis (MS) patients; moreover, disease-modifying treatments (DMT) have shown to influence blood BAFF levels in MS patients, although the significance of these changes is still controversial. In addition, BAFF levels were reported increased during infectious diseases. In our study, we wanted to investigate on the serum BAFF concentrations correlated to the antibody response against Mycobacterium avium subspecies paratuberculosis (MAP), Epstein-Barr virus (EBV) and their human homologous epitopes in MS and in patients affected with other neurological diseases (OND), divided in Inflammatory Neurological Diseases (IND), Non Inflammatory Neurological Diseases (NIND) and Undetermined Neurological Diseases (UND), in comparison to healthy controls (HCs). Our results confirmed a statistically significant high BAFF levels in MS and IND patients in comparison to HCs but not NIND and UND patients. Interestingly, BAFF levels were inversely proportional to antibodies level against EBV and MAP peptides and the BAFF levels significantly decreased in MS patients after methylprednisolone therapy. These results implicate that lower circulating BAFF concentrations were present in MS patients with humoral response against MAP and EBV. In conclusion MS patients with no IgGs against EBV and MAP may support the hypothesis that elevated blood BAFF levels could be associated with a more stable disease. PMID:27383531

  17. Evaluation of a Commercial Real-Time PCR Kit for the Detection of Mycobacterium avium subspecies paratuberculosis in Milk.

    PubMed

    Alajmi, Ahmad; Klein, Günter; Grabowski, Nils Th; Fohler, Svenja; Akineden, Ömer; Abdulmawjood, Amir

    2016-11-01

    There are several commercial test kits for Mycobacterium avium subspecies paratuberculosis (MAP) detection, each with different advantages, disadvantages, and applications. In the present study, a real-time PCR kit targeting the unique transposon sequence ISMAP02 was evaluated. The analytical sensitivity was determined using the type strain ATCC 19698, and the specificity was validated by testing fifteen MAP isolates, thirteen non-MAP Mycobacterium isolates, and eight non-Mycobacterium isolates. Six spiking experiments were performed using raw milk and reconstituted infant milk artificially contaminated with dilutions containing 10(0)-10(5) MAP cells mL(-1). Sensitivity and specificity were at 100 %. The detection probabilities in raw milk and reconstituted infant milk for the samples (containing 1.4 × 10(1) and 1.7 × 10(1) MAP cell 50 mL(-1)) were 16.6 and 91.6 %, respectively. Thus, the tested kit yielded satisfying results to detect MAP in milk. PMID:27502065

  18. Induction of in vivo resistance to Mycobacterium avium infection by intramuscular injection with DNA encoding interleukin-18

    PubMed Central

    Kim, S H; Cho, D; Kim, T S

    2001-01-01

    Interferon-gamma (IFN-γ) is closely associated with the generation of cell-mediated immunity and resistance to intracellular parasites. Interleukin-18 (IL-18) was known to strongly induce IFN-γ production by T cells and natural killer (NK) cells. In order to determine whether injection with DNA encoding IL-18 can stimulate the resistance to Mycobacterium avium complex (MAC) infection, the mature IL-18 cDNA with κ leader sequence was cloned under control of the cytomegalovirus (CMV) promoter (TcCMVIL-18) and its effect on MAC infection was investigated in genetically susceptible BALB/c mice. Injection with the TcCMVIL-18 DNA during intranasal infection with MAC resulted in a significant decrease in bacterial load of lung during the entire 8-week observation period, while injection with the TcCMV control DNA did not. Lung cells in mice injected with the TcCMVIL-18 DNA showed persistent production of IFN-γ throughout the 8-week period. Furthermore, immunization with the TcCMVIL-18 DNA induced and maintained significantly higher levels of cytotoxic activity and nitric oxide production by lung cells than immunization with the TcCMV control vector. This work suggests that IL-18 DNA vaccination may be useful in the immunotherapeutic or immunoprotection approaches of infections by intracellular parasites such as mycobacteria. PMID:11260329

  19. Effect of pulsed electric fields on the flavour profile of red-fleshed sweet cherries (Prunus avium var. Stella).

    PubMed

    Sotelo, Kristine Ann Gualberto; Hamid, Nazimah; Oey, Indrawati; Gutierrez-Maddox, Noemi; Ma, Qianli; Leong, Sze Ying

    2015-03-23

    The aim of this research was to study the effect of pulsed electric fields (PEF) on the flavour profile of red-fleshed sweet cherries (Prunus avium variety Stella). The cherry samples were treated at a constant pulse frequency of 100 Hz, a constant pulse width of 20 μs, different electric field strengths between 0.3 and 2.5 kV/cm and specific energy ranging from 31 to 55 kJ/kg. Volatile compounds of samples were analysed using an automated headspace solid phase microextraction (HS-SPME) method coupled with gas chromatography-mass spectrometry (GC-MS). A total of 33 volatile compounds were identified with benzaldehyde, hexanal, (E)-2-hexenal, (Z)-2-hexen-1-ol, and benzyl alcohol being the predominant volatiles in different PEF-treated samples. Aldehydes namely butanal, octanal, 2-octenal, and nonanal, and (Z)-2-hexen-1-ol increased significantly 24 h after PEF treatment at electric field strengths of more than 1.0 kV/cm. Samples incubated for 24 h after PEF treatment (S3) generated higher concentrations of volatiles than samples immediately after PEF treatments (S2). Quantitative results revealed that more flavour volatiles were released and associated with S3 samples after 24 h storage and S2 samples immediately after PEF both with the highest electric field intensities. Interestingly, this study found that the PEF treatments at the applied electric field strength and energy did not result in releasing/producing undesirable flavour compounds.

  20. High incidence of Mycobacterium avium subspecies hominissuis infection in a zoo population of bongo antelopes (Tragelaphus eurycerus).

    PubMed

    Moravkova, Monika; Mrlik, Vojtech; Parmova, Ilona; Kriz, Petr; Pavlik, Ivo

    2013-07-01

    Mycobacterium avium subsp. hominissuis (Mah) infection was diagnosed in 5 captive bongo antelopes (Tragelaphus eurycerus) originating from a collection in a zoological garden. The animals suffered from emaciation. Postmortem examination revealed nodular lesions in the lungs of all 5 examined animals. Acid-fast bacilli were observed in the lungs of 4 animals. Culture and polymerase chain reaction identification based on IS901 negativity and IS1245 positivity confirmed Mah infection in the lungs of all 5 antelopes. In 3 animals, Mah was also isolated from other organs (liver, spleen, and kidney). Molecular analysis of these isolates using IS1245 restriction fragment length polymorphism and/or mycobacterial interspersed repetitive units-variable number tandem repeat revealed that the studied antelopes were infected by 1 identical genotype. Furthermore, in 2 antelopes, other genotypes were also detected. This shows the possibility of either genetic modifications occurring during infection or polyclonal infection. Culture examination of environmental samples from the enclosures holding the bongos revealed Mah in mulch bark, peat, and soil. Genotyping of these environmental isolates determined several genotypes with 1 dominant genotype that was identical to the dominant genotype detected in antelopes.

  1. Pasteurization of milk and the heat resistance of Mycobacterium avium subsp. paratuberculosis: a critical review of the data.

    PubMed

    Lund, Barbara M; Gould, Grahame W; Rampling, Anita M

    2002-07-25

    Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) causes Johne's disease in ruminants (including cattle, sheep and goats) and other animals, and may contribute to Crohn's disease in humans. This possibility, and the fact that M. paratuberculosis may be present in raw milk, make it important to ensure that the heat treatment specified for pasteurization of milk will give acceptable inactivation of this bacterium, with an adequate margin of safety. Published studies of the heat resistance of this bacterium in milk have given widely differing results. Possible reasons for these differences, and the technical problems involved in the work, are reviewed. It is concluded that there is a need (i) for the adoption of an agreed Performance Criterion for pasteurization of milk in relation to this bacterium, (ii) a need for definitive laboratory experiments to understand and determine the heat resistance of M. paratuberculosis, and (iii) a need for an assessment of whether the minimum heat treatments specified at present for pasteurization of milk (Process Criteria) will meet the Performance Criterion for M. paratuberculosis. Measures are also required to ensure that commercial processes deliver continually the specified heat treatment, and to ensure that post-pasteurization contamination is avoided.

  2. Johne's disease: reliability of environmental sampling to characterize Mycobacterium avium subspecies paratuberculosis (MAP) infection in beef cow-calf herds.

    PubMed

    Klawonn, W; Einax, E; Pützschel, R; Schmidt, M; Donat, K

    2016-08-01

    Environmental samples are considered to be a cost-effective method of identifying Mycobacterium avium subsp. paratuberculosis (MAP)-positive dairy herds, but evidence for beef cow-calf herds is weak. This study aims at evaluating this approach in a total of 20 German herds that were characterized by individual faecal samples (n = 2545) of all cows. For 14 MAP-positive herds having at least one MAP-positive animal, the within-herd prevalence was calculated from concurrent individual faecal culture-based testing. Six herds certified as 'MAP free' based on the negative results of previous years served as MAP-negative controls. On average, six environmental samples were taken at the end of winter from areas with high cow traffic and tested for MAP by faecal culture. According to the environmental samples, nine (64·3%) out of the 14 MAP-positive cow-calf herds were infected. The percentage of positive environmental samples and the apparent within-herd prevalence (Spearman's P = 0·73, P < 0·001) as well as the herd-level test results (positive and negative) and the herd's status based on individual testing (Fisher's exact test, P = 0·014) showed a positive association. Considering limitations in low-prevalence herds, MAP-positive beef cow-calf herds are detectable by environmental samples in temperate climate zones.

  3. Post-storage cell wall metabolism in two sweet cherry (Prunus avium L.) cultivars displaying different postharvest performance.

    PubMed

    Belge, Burcu; Comabella, Eva; Graell, Jordi; Lara, Isabel

    2015-09-01

    The biochemical processes underlying firmness loss of sweet cherry (Prunus avium L.) fruit are poorly understood. Studies on cell wall metabolism of sweet cherry have been generally undertaken during on-tree development or at harvest maturity, while published reports on postharvest changes are scarce and fragmentary. In this work, cell wall modifications after storage at 0 ℃ were studied in two cherry cultivars ('Celeste' and 'Somerset') displaying different postharvest potential. Firmness was largely determined by the yields of the Na2CO3- and KOH-soluble fractions, enriched in covalently-bound pectins and in matrix glycans, respectively, and correlated well with ascorbic acid contents. The yields of these two cell wall fractions were correlated inversely with pectinmethylesterase and endo-1,4-β-d-glucanase activities, indicating a relevant role of these two enzymes in postharvest firmness changes in sweet cherry. The amount of solubilised cell wall materials was closely associated to the contents of dehydroascorbic acid, suggesting the possible involvement of oxidative mechanisms in cell wall disassembly. These data may help understanding the evolution of fruit quality during the marketing period, and give hints for the design of suitable management strategies to preserve key attributes.

  4. Epstein Barr Virus and Mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of MS patients

    PubMed Central

    Mameli, Giuseppe; Cocco, Eleonora; Frau, Jessica; Marrosu, Maria Giovanna; Sechi, Leonardo Antonio

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) and Epstein-Barr virus (EBV) epitopes elicit a consistent humoral response in serum of multiple sclerosis patients, but the cross reactivity against the homologous myelin basic protein (MBP) and human interferon regulatory factor 5 (IRF5) has not been searched within the Cerebral Spinal Fluid (CSF). We evaluated in sera and CSF of patients with MS and with other neurological diseases (OND) the humoral response against EBV/MAP peptides and the IRF5/MBP. Our data showed that EBV and MAP peptides are able to induce a specific humoral immune response in MS patients compared to OND controls both in serum and in CSF. An intrathecal specific synthesis of IgG against MBP and their EBV and MAP homologous as indicated by the antibody index was observed in MS patients. The humoral response against EBV, MAP, MBP and IRF5 was significantly higher in MS patients compared to OND both in serum and in CSF. The higher presence of antibodies against MBP and their MAP and EBV homologous in CSF during relapses suggests a possible role of the pathogens in enhancing inflammation. PMID:26956729

  5. Genetic variability of wild cherry (Prunus avium L.) seed stands in Slovenia as revealed by nuclear microsatellite loci.

    PubMed

    Jarni, Kristjan; De Cuyper, Bart; Brus, Robert

    2012-01-01

    Microsatellite markers were used to describe the genetic variability of four seed stands of wild cherry (Prunus avium L.). One hundred and thirty one individuals were genotyped at ten nuclear microsatellite loci. Total genetic diversity was high (H(E) = 0.704), while differences between stands were small but significant (F(ST) = 0.053, G'(ST) = 0.234). There was a significant amount of clonal reproduction in one stand, with only 11 genotypes identified among 36 trees. One stand showed a significant excess (F(IS) = -0.044) of heterozygosity, and one showed a deficit (F(IS) = 0.044). Our results demonstrate the importance of taking into account the biological and genetic characteristics of species in forest management, especially when determining a new seed stand. The small genetic differences found between seed stands indicate that a large number of stands are not required. However, they should be carefully selected and should possess adequate genetic variability to ensure low relatedness between seed trees.

  6. Mycobacterium avium-intracellulare pulmonary infection complicated by cutaneous leukocytoclastic vasculitis in a woman with anorexia nervosa.

    PubMed

    Walsh, T L; Baca, V; Stalling, S S; Natalie, A A; Veldkamp, P J

    2014-06-01

    A 53-year-old Caucasian woman with a history of anorexia nervosa developed a bilateral lower extremity rash comprised of palpable red to violaceous, sub-centimeter papular lesions that increased in quantity rapidly. She also noted a 2-month history of non-productive cough. Imaging modalities revealed a thin-walled cavitary lesion in the right lung apex and scattered nodular opacities. Acid fast bacilli (AFB) were found in sputum and subsequently identified by culture as Mycobacterium avium-intracellulare (MAI). Punch biopsies of her skin lesions yielded a histological diagnosis of small-to-medium vessel vasculitis. Stains and cultures for organisms were negative. Her skin lesions resolved quickly after the initiation of antimicrobial therapy for MAI. Hypersensitivity vasculitis associated with an atypical mycobacterial infection is unusual. The postulated underlying mechanism is the deposit of immune complexes and not the bacillus itself. While cutaneous leukocytoclastic vasculitis (CLV) due to MAI is certainly a rare entity, it should be entertained in patients with vasculitic skin lesions and a concomitant pulmonary disease. PMID:24363210

  7. Serum BAFF levels, Methypredsinolone therapy, Epstein-Barr Virus and Mycobacterium avium subsp. paratuberculosis infection in Multiple Sclerosis patients.

    PubMed

    Mameli, Giuseppe; Cocco, Eleonora; Frau, Jessica; Arru, Giannina; Caggiu, Elisa; Marrosu, Maria Giovanna; Sechi, Leonardo A

    2016-01-01

    Elevated B lymphocyte activating factor BAFF levels have been reported in multiple sclerosis (MS) patients; moreover, disease-modifying treatments (DMT) have shown to influence blood BAFF levels in MS patients, although the significance of these changes is still controversial. In addition, BAFF levels were reported increased during infectious diseases. In our study, we wanted to investigate on the serum BAFF concentrations correlated to the antibody response against Mycobacterium avium subspecies paratuberculosis (MAP), Epstein-Barr virus (EBV) and their human homologous epitopes in MS and in patients affected with other neurological diseases (OND), divided in Inflammatory Neurological Diseases (IND), Non Inflammatory Neurological Diseases (NIND) and Undetermined Neurological Diseases (UND), in comparison to healthy controls (HCs). Our results confirmed a statistically significant high BAFF levels in MS and IND patients in comparison to HCs but not NIND and UND patients. Interestingly, BAFF levels were inversely proportional to antibodies level against EBV and MAP peptides and the BAFF levels significantly decreased in MS patients after methylprednisolone therapy. These results implicate that lower circulating BAFF concentrations were present in MS patients with humoral response against MAP and EBV. In conclusion MS patients with no IgGs against EBV and MAP may support the hypothesis that elevated blood BAFF levels could be associated with a more stable disease. PMID:27383531

  8. Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

    PubMed

    Nazareth, Nair; Magro, Fernando; Machado, Elisabete; Ribeiro, Teresa Gonçalves; Martinho, António; Rodrigues, Pedro; Alves, Rita; Macedo, Gonçalo Nuno; Gracio, Daniela; Coelho, Rosa; Abreu, Candida; Appelberg, Rui; Dias, Camila; Macedo, Guilherme; Bull, Tim; Sarmento, Amélia

    2015-12-01

    Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

  9. Survey on the presence of Mycobacterium avium subsp. paratuberculosis in ground beef from an industrial meat plant.

    PubMed

    Savi, R; Ricchi, M; Cammi, G; Garbarino, C; Leo, S; Pongolini, S; Arrigoni, N

    2015-06-12

    Paratuberculosis of ruminants is characterised by chronic enteritis but, at advanced stages of the disease, a systemic dissemination of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues and organs can occur. MAP has been recovered from lymph nodes and muscles of clinical and sub-clinical cows. In most countries, dairy and beef cattle infected with paratuberculosis are routinely sent to slaughter and the consumption of their meat could be a possible route of human exposure to MAP. However, few studies on MAP in ground beef are currently available. During the period November 2013-March 2014 we carried out a survey on the ground beef produced in an industrial meat processing plant. One-hundred and forty samples of ground meat were analysed by IS900-qPCR and culture (VersaTrek System). The limit of detection (LOD) of qPCR was 630 MAP cells/g (107 CFU/g) while the LOD for culture was 170-230 MAP cells/g (62-115 CFU/g). No samples were positive by direct IS900 qPCR, while two samples were positive by liquid culture. Our data suggest that the presence of live MAP in raw minced meat is possible. In order to avoid exposure for humans through the consumption of contaminated meat, proper cooking of meat is recommended.

  10. Gene expression profiling of PBMCs from Holstein and Jersey cows sub-clinically infected with Mycobacterium avium ssp. paratuberculosis.

    PubMed

    Verschoor, Chris P; Pant, Sameer D; You, Qiumei; Kelton, David F; Karrow, Niel A

    2010-09-15

    Infection of calves with intracellular Mycobacterium avium ssp. paratuberculosis (MAP) commonly results in a granulomatous, chronic inflammatory bowel disease known as Johne's disease. The asymptomatic stage of this infection can persist for the entire production life of an adult cow, resulting in reduced performance and premature culling, as well as transmission of MAP to progeny and herd-mates. It has been previously shown that the gene expression profiles of peripheral blood mononuclear cells (PBMCs) of healthy cows, and those chronically infected with MAP are inherently different, and that these changes may be indicative of disease progression. Since resistance to MAP infection is a heritable trait, and has been proposed to differ amongst domestic dairy cattle breeds, the objective of the present study was to compare gene expression profiles of PBMCs from healthy adult Holstein and Jersey cows to those considered to be sub-clinically infected with MAP, as indicated by serum ELISA. Microarray analysis using a platform containing more than 10,000 probes and ontological analysis identified differences in gene expression between a) healthy and infected cows, including genes involved in the inflammatory response, and calcium binding, and b) infected Holsteins and Jerseys, including genes involved in the immune response, and antigen processing and presentation. These results suggest a mixed pro- and anti-inflammatory phenotype of PBMCs from MAP-infected as compared to healthy control animals, and inherently different levels of immune and inflammatory-related gene expression between MAP-infected Holsteins and Jerseys.

  11. Evaluation of multiple genomic targets for identification and confirmation of Mycobacterium avium subsp. paratuberculosis isolates using real-time PCR.

    PubMed

    Rajeev, Sreekumari; Zhang, Yan; Sreevatsan, Srinand; Motiwala, Alifiya S; Byrum, Beverly

    2005-02-25

    Specificity of six previously published Mycobacterium avium subsp. paratuberculosis (MAP) genomic loci, including 10, 38, 56, 93, 251, and 252 were evaluated in this study. Target 251 which was identified as MAP-specific was further evaluated in 210 MAP isolates, 14 non-MAP mycobacterial species, 7 atypical mycobacterial isolates, and 9 other bacterial species using real-time PCR. A previously published IS900 primer and probe combination was used as a positive control along with a universal ribosomal DNA gene sequence (UVA) as an internal control to evaluate PCR inhibition. All MAP isolates were positive with IS900, 251, and UVA by real-time PCR. All non-MAP mycobacterial species except one atypical mycobacterial isolate and other bacterial species used in this study were negative for IS900. All of these species were negative for 251. The atypical mycobacterial isolate, positive for IS900 and UVA, was negative for 251. A combination of IS900 and 251 PCR is ideal for sensitive and specific confirmation of MAP isolates from conventional fecal cultures. This study also evaluated the specificity of 251 real-time PCR, on broth cultures from 50 known bovine fecal samples. Acid fast staining followed by IS900 and 251 real-time PCR can be used for accurate identification and confirmation of MAP from broth cultures.

  12. Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis-specific membrane-associated antigen by Lactobacillus salivarius.

    PubMed

    Johnston, Christopher; Douarre, Pierre E; Soulimane, Tewfik; Pletzer, Daniel; Weingart, Helge; MacSharry, John; Coffey, Aidan; Sleator, Roy D; O'Mahony, Jim

    2013-06-01

    Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease. PMID:23620276

  13. Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

    PubMed Central

    Gioffré, Andrea; Correa Muñoz, Magnolia; Alvarado Pinedo, María F.; Vaca, Roberto; Morsella, Claudia; Fiorentino, María Andrea; Paolicchi, Fernando; Ruybal, Paula; Zumárraga, Martín; Travería, Gabriel E.; Romano, María Isabel

    2015-01-01

    Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents. PMID:26273274

  14. Phenotype definition is a main point in genome-wide association studies for bovine Mycobacterium avium ssp. paratuberculosis infection status.

    PubMed

    Küpper, J; Brandt, H; Donat, K; Erhardt, G

    2014-10-01

    Paratuberculosis caused by Mycobacterium avium ssp. paratuberculosis (MAP) causes economic losses and is present in dairy herds worldwide. Different studies used different diagnostic tests to detect infection status and are the basis of genome-wide association (GWA) studies with inconsistent results. Therefore, the aim of this study was to identify and compare genomic regions associated with MAP susceptibility in the same cohort of cattle using different diagnostic tests. The GWA study was performed in German Holsteins within a case-control assay using 305 cows tested for MAP by fecal culture and additional with four different commercial ELISA-tests. Genotyping was performed with the Illumina Bovine SNP50 BeadChip. The results using fecal culture or ELISA test led to the identification of different genetic loci. Two single-nucleotide polymorphisms showed significant association with the ELISA-status. However, no significant association for MAP infection could be confirmed. Our results show that the definition of the MAP-phenotype has an important impact on the outcome of GWA studies for paratuberculosis.

  15. Genetic Variability of Wild Cherry (Prunus avium L.) Seed Stands in Slovenia as Revealed by Nuclear Microsatellite Loci

    PubMed Central

    Jarni, Kristjan; De Cuyper, Bart; Brus, Robert

    2012-01-01

    Microsatellite markers were used to describe the genetic variability of four seed stands of wild cherry (Prunus avium L.). One hundred and thirty one individuals were genotyped at ten nuclear microsatellite loci. Total genetic diversity was high (HE = 0.704), while differences between stands were small but significant (FST = 0.053, G′ST = 0.234). There was a significant amount of clonal reproduction in one stand, with only 11 genotypes identified among 36 trees. One stand showed a significant excess (FIS = −0.044) of heterozygosity, and one showed a deficit (FIS = 0.044). Our results demonstrate the importance of taking into account the biological and genetic characteristics of species in forest management, especially when determining a new seed stand. The small genetic differences found between seed stands indicate that a large number of stands are not required. However, they should be carefully selected and should possess adequate genetic variability to ensure low relatedness between seed trees. PMID:22911762

  16. Induction of matrix metalloproteinases and TLR2 and 6 in murine colon after oral exposure to Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Roderfeld, Martin; Koc, Arzu; Rath, Timo; Blöcher, Sonja; Tschuschner, Annette; Akineden, Ömer; Fischer, Marta; von Gerlach, Susanne; Goethe, Ralph; Eckelt, Elke; Meens, Jochen; Bülte, Michael; Basler, Tina; Roeb, Elke

    2012-06-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in Crohn's disease. Recent evidence suggests that MAP can induce the expression of Matrix Metalloproteinases (MMPs), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within the present study, we analysed whether oral MAP exposure can induce colonic MMP expression in vivo. In MAP exposed mice MAP and spheroplasts were visualized in intramucosal leukocyte aggregates. MAP exposed mice exhibited a higher colonic expression of Mmp-2, -9, -13, -14, Timp-1, Tlr2, Tlr6, Il-1β, and Tnf-α. Cell clusters of MMP-9 positive cells adjacent to intramucosal leukocyte aggregates and CD45(+) leukocytes were identified as the major cellular sources of MMP-9. Enhanced TLR2 expression was visualized on the luminal side of colonic enterocytes. Although MAP exposure did not lead to macroscopic intestinal inflammation, the observed MAP spheroplasts in intramucosal leukocyte aggregates together with increased colonic expression of toll-like receptors, pro-inflammatory cytokines, and MMPs upon MAP exposure represents a part of the host immune response towards MAP.

  17. Interaction between Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium subspecies paratuberculosis with the enteric glia and microglial cells

    PubMed Central

    2011-01-01

    Background We investigated the interaction of Mycobacterium avium subspecies paratuberculosis, M. bovis and M. tuberculosis and different glial cells (enteric glial and microglial cells) in order to evaluate the infecting ability of these microorganisms and the effects produced on these cells, such as the evaluation of cytokines expression. Results Our experiments demonstrated the adhesion of M. paratuberculosis to the enteroglial cells and the induction of IL-1A and IL-6 expression; M. tuberculosis and M. bovis showed a good adhesive capability to the enteric cell line with the expression of the following cytokines: IL-1A and IL-1B, TNF-α, G-CSF and GM-CSF; M. bovis induced the expression of IL-6 too. The experiment performed with the microglial cells confirmed the results obtained with the enteroglial cells after the infection with M. tuberculosis and M. bovis, whereas M. paratuberculosis stimulated the production of IL-1A and IL-1B. Conclusion Enteroglial and microglial cells, could be the target of pathogenic mycobacteria and, even if present in different locations (Enteric Nervous System and Central Nervous System), show to have similar mechanism of immunomodulation. PMID:22151930

  18. Can Immune Response Mechanisms Explain the Fecal Shedding Patterns of Cattle Infected with Mycobacterium avium Subspecies paratuberculosis?

    PubMed Central

    Magombedze, Gesham; Eda, Shigetoshi; Koets, Ad

    2016-01-01

    Johne’s disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns. PMID:26808389

  19. Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods.

    PubMed

    Klanicova, B; Slana, I; Roubal, P; Pavlik, I; Kralik, P

    2012-07-01

    Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16 h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12 weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

  20. Impact of test sensitivity and specificity on pig producer incentives to control Mycobacterium avium infections in finishing pigs.

    PubMed

    van Wagenberg, Coen P A; Backus, Gé B C; Wisselink, Henk J; van der Vorst, Jack G A J; Urlings, Bert A P

    2013-09-01

    In this paper we analyze the impact of the sensitivity and specificity of a Mycobacterium avium (Ma) test on pig producer incentives to control Ma in finishing pigs. A possible Ma control system which includes a serodiagnostic test and a penalty on finishing pigs in herds detected with Ma infection was modelled. Using a dynamic optimization model and a grid search of deliveries of herds from pig producers to slaughterhouse, optimal control measures for pig producers and optimal penalty values for deliveries with increased Ma risk were identified for different sensitivity and specificity values. Results showed that higher sensitivity and lower specificity induced use of more intense control measures and resulted in higher pig producer costs and lower Ma seroprevalence. The minimal penalty value needed to comply with a threshold for Ma seroprevalence in finishing pigs at slaughter was lower at higher sensitivity and lower specificity. With imperfect specificity a larger sample size decreased pig producer incentives to control Ma seroprevalence, because the higher number of false positives resulted in an increased probability of rejecting a batch of finishing pigs irrespective of whether the pig producer applied control measures. We conclude that test sensitivity and specificity must be considered in incentive system design to induce pig producers to control Ma in finishing pigs with minimum negative effects.

  1. Paternal-specific S-allele transmission in sweet cherry (Prunus avium L.): the potential for sexual selection.

    PubMed

    Hedhly, A; Wünsch, A; Kartal, Ö; Herrero, M; Hormaza, J I

    2016-03-01

    Homomorphic self-incompatibility is a well-studied example of a physiological process that is thought to increase population diversity and reduce the expression of inbreeding depression. Whereas theoretical models predict the presence of a large number of S-haplotypes with equal frequencies at equilibrium, unequal allele frequencies have been repeatedly reported and attributed to sampling effects, population structure, demographic perturbation, sheltered deleterious mutations or selection pressure on linked genes. However, it is unclear to what extent unequal segregations are the results of gametophytic or sexual selection. Although these two forces are difficult to disentangle, testing S-alleles in the offspring of controlled crosses provides an opportunity to separate these two phenomena. In this work, segregation and transmission of S-alleles have been characterized in progenies of mixed donors and fully compatible pollinations under field conditions in Prunus avium. Seed set patterns and pollen performance have also been characterized. The results reveal paternal-specific distorted transmission of S-alleles in most of the crosses. Interestingly, S-allele segregation within any given paternal or maternal S-locus was random. Observations on pollen germination, pollen tube growth rate, pollen tube cohort size, seed set dynamics and transmission patterns strongly suggest post-pollination, prezygotic sexual selection, with male-male competition as the most likely mechanism. According to these results, post-pollination sexual selection takes precedence over frequency-dependent selection in explaining unequal S-haplotype frequencies.

  2. Growth of Mycobacterium avium subsp. paratuberculosis, Escherichia coli, and Salmonella Enteritidis during Preparation and Storage of Yogurt

    PubMed Central

    Cirone, K.; Huberman, Y.; Morsella, C.; Méndez, L.; Jorge, M.; Paolicchi, F.

    2013-01-01

    The purpose of this study was to determine the viability of Mycobacterium avium subsp. paratuberculosis (MAP), Escherichia coli (E. coli), and Salmonella Enteritidis (S. Enteritidis) during preparation and refrigerated storage of yogurt. Three yogurts were prepared using pasteurized commercial milk. Each yogurt was artificially contaminated with (1) MAP, (2) E. coli + S. Enteritidis, and (3) MAP + E. coli + S. Enteritidis. Samples were taken during and after the fermentation process until day 20 after inoculation. MAP was not detected during their preparation and short-term storage but was recuperated after starting at 180 min after inoculation storage. Live bacterial counts of E. coli, and S. Enteritidis increased during the first 24 hours, followed by a slight decrease towards the end of the study. In this study it was shown how MAP, E. coli, and S. Enteritidis resisted the acidic conditions generated during the preparation of yogurt and low storage temperatures. This work contributes to current knowledge regarding survival of MAP, E. coli, and S. Enteritidis during preparation and refrigerated storage of yogurt and emphasizes the need to improve hygiene measures to ensure the absence of these pathogenic microorganisms in dairy products. PMID:24455399

  3. Syndrome of selective IgM deficiency with severe T cell deficiency associated with disseminated cutaneous mycobacterium avium intracellulaire infection

    PubMed Central

    Gharib, Asal; Louis, Ankmalika Gupta; Agrawal, Sudhanshu; Gupta, Sudhir

    2015-01-01

    Cutaneous non-disseminated, non-tuberculous mycobacterial infections have been reported in both immunocompetent and immunocompromised subjects. Systemic Mycobacterium avium intracellulaire (MAI) have been reported in non-HIV patients with Idiopathic CD4 lymphocytopenia. We report a comprehensive immunological analysis in syndrome of selective IgM deficiency and T lymphocytopenia (both CD4+ and CD8+) with disseminated cutaneous MAI infection. Naïve (TN) and Central memory (TCM) subsets of both CD4+ and CD8+ T cells were decreased, whereas terminally differentiated effector memory (TEMRA) subset of CD4+ and CD8+ T cells were markedly increased. IFN-γ producing T cells were markedly decreased. Although CD14highCD16- proinflammatory monocytes were modestly increased, IFN-γR+ monocytes were markedly decreased. The expression of TLR3, TLR5, TLR7, and TLR9 on monocytes was decreased. Germinal center B cells (CD19+IgD-CD38+CD27lo) and B1 cells (CD20+CD27+CD43+CD70-) were markedly decreased. A role of immune alterations, including B cells and antibodies in disseminated cutaneous MAI infection is discussed. PMID:26550546

  4. A systematic review of risk factors associated with the introduction of Mycobacterium avium spp. paratuberculosis (MAP) into dairy herds

    PubMed Central

    Rangel, Saray J.; Paré, Julie; Doré, Elizabeth; Arango, Juan C.; Côté, Geneviève; Buczinski, Sebastien; Labrecque, Olivia; Fairbrother, Julie H.; Roy, Jean P.; Wellemans, Vincent; Fecteau, Gilles

    2015-01-01

    The objective of this study was to systematically collect and appraise the scientific evidence related to risk factors associated with the introduction of Mycobacterium avium spp. paratuberculosis (MAP) into a herd of cattle. An electronic search was conducted to collect relevant references addressing 2 specific questions: are i) purchasing/introduction of cattle into a herd, and ii) presence of wildlife or domestic animals, risk factors for the introduction of MAP into a herd? The screening was based on titles and abstracts and selected studies were fully analyzed. Seventeen manuscripts published between 1996 and 2011 were ultimately analyzed. Unit of interest was mainly the herd (n = 17). The specific description of the risk factors studied varied between studies. The principal study design was cross-sectional (n = 15). The review indicated that purchase/introduction of animals was an important risk factor and that the importance of wildlife or other domestic species as a mechanism for transmission into a cattle herd was not measurable. PMID:25694667

  5. First isolation of mycobacterium avium subsp. Paratuberculosis from wild guanacos (Lama guanicoe) on Tierra del Fuego Island.

    PubMed

    Salgado, M; Herthnek, D; Bölske, G; Leiva, S; Kruze, J

    2009-04-01

    The aim of this study was to search for Mycobacterium avium subsp. paratuberculosis (Map) infection in a free-ranging wild animal species in a region where Johnes's disease has yet to be reported and to classify Map isolates using a genomic typing method. Fecal samples were obtained from 501 wild guanacos (Lama guanicoe) from Tierra del Fuego Island, Chile, in August 2006. Samples were cultured using Herrold's egg yolk medium with and without mycobactin J. After 9 mo of incubation, suspected Map colonies showing mycobactin dependence were confirmed by real-time polymerase chain reaction (PCR) based on IS900 and F57. Isolates were further tested using IS1311 PCR with restriction endonuclease analysis in order to type the guanaco Map strains. Twenty-one of 501 (4.2%) animals were fecal culture-positive for Map; identity was confirmed by real-time PCR and isolates were classified as cattle-type. Most culture-positive animals were located in four contiguous geographic areas, and the infection was most commonly found among adult animals. Prevalence was higher in females (5.9%) than males (3.1%) but the difference was not statistically significant. This represents the first isolation of Map from a free-ranging wildlife species in Chile. It expands the geographic range of paratuberculosis and the diversity of wildlife species that can become infected with Map.

  6. Fruit quality and bioactive compounds relevant to human health of sweet cherry (Prunus avium L.) cultivars grown in Italy.

    PubMed

    Ballistreri, Gabriele; Continella, Alberto; Gentile, Alessandra; Amenta, Margherita; Fabroni, Simona; Rapisarda, Paolo

    2013-10-15

    The fruit quality characteristics, phenolic compounds and antioxidant capacities of 24 sweet cherry (Prunus avium L.) cultivars grown on the mountainsides of the Etna volcano (Sicily, Italy) were evaluated. High-performance liquid chromatographic methods were used to identify and quantify sugars, organic acids and phenolics. A total of seven phenolic compounds were characterised as hydroxycinnamic acid derivatives (neochlorogenic acid, p-coumaroylquinic acid and chlorogenic acid) and anthocyanins (cyanidin 3-glucoside, cyanidin 3-rutinoside, pelargonidin 3-rutinoside and peonidin 3-rutinoside). The total anthocyanin content ranged from 6.21 to 94.20mg cyanidin 3-glucoside equivalents/100g fresh weight (FW), while the total phenol content ranged from 84.96 to 162.21mg gallic acid equivalents/100g FW. The oxygen radical absorbance capacity (ORAC) assay indicated that fruit of all genotypes possessed considerable antioxidant activity. The high level of phenolic compounds and antioxidant capacity of some sweet cherry fruits implied that they might be sources of bioactive compounds that are relevant to human health.

  7. Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis.

    PubMed

    Gioffré, Andrea; Correa Muñoz, Magnolia; Alvarado Pinedo, María F; Vaca, Roberto; Morsella, Claudia; Fiorentino, María Andrea; Paolicchi, Fernando; Ruybal, Paula; Zumárraga, Martín; Travería, Gabriel E; Romano, María Isabel

    2015-06-01

    Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents. PMID:26273274

  8. Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis

    PubMed Central

    2012-01-01

    Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne’s disease, an intestinal disease of ruminants with major economic consequences. Infectious bacilli are phagocytosed by host macrophages upon exposure where they persist, resulting in lengthy subclinical phases of infection that can lead to immunopathology and disease dissemination. Consequently, analysis of the macrophage transcriptome in response to M. avium subsp. paratuberculosis infection can provide valuable insights into the molecular mechanisms that underlie Johne’s disease. Here, we investigate pan-genomic gene expression in bovine monocyte-derived macrophages (MDM) purified from seven age-matched females, in response to in vitro infection with M. avium subsp. paratuberculosis (multiplicity of infection 2:1) at intervals of 2 hours, 6 hours and 24 hours post-infection (hpi). Differentially expressed genes were identified by comparing the transcriptomes of the infected MDM to the non-infected control MDM at each time point (adjusted P-value threshold ≤ 0.10). 1050 differentially expressed unique genes were identified 2 hpi, with 974 and 78 differentially expressed unique genes detected 6 and 24 hpi, respectively. Furthermore, in the infected MDM the number of upregulated genes exceeded the number of downregulated genes at each time point, with the fold-change in expression for the upregulated genes markedly higher than that for the downregulated genes. Inspection and systems biology analysis of the differentially expressed genes revealed an enrichment of genes involved in the inflammatory response, cell signalling pathways and apoptosis. The transcriptional changes associated with cellular signalling and the inflammatory response may reflect different immuno-modulatory mechanisms that underlie host-pathogen interactions during infection. PMID:22455317

  9. Virulence and Immune Response Induced by Mycobacterium avium Complex Strains in a Model of Progressive Pulmonary Tuberculosis and Subcutaneous Infection in BALB/c Mice

    PubMed Central

    González-Pérez, Mónica; Mariño-Ramírez, Leonardo; Parra-López, Carlos Alberto; Murcia, Martha Isabel; Marquina, Brenda; Mata-Espinoza, Dulce; Rodriguez-Míguez, Yadira; Baay-Guzman, Guillermina J.; Huerta-Yepez, Sara

    2013-01-01

    The genus Mycobacterium comprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies with Mycobacterium tuberculosis. The biological differentiation between M. tuberculosis and nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not. Mycobacterium avium complex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after the M. tuberculosis complex. Here, we evaluated the virulence and immune response of M. avium subsp. avium and Mycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue. PMID:23959717

  10. Mycobacterium avium-Intracellulare Complex (MAC) Producing a Periportal Pseudotumor in a Patient With HIV and a Normal CD4 Count

    PubMed Central

    Johnson, Jessica; Driscoll, Meghan; Cohen, Michael

    2016-01-01

    Mycobacterium avium-intracellulare complex (MAC) is an opportunistic infection typically associated with profound immunosuppression, such as AIDS. The presentation of disseminated MAC can be subtle and mimic systemic symptoms associated with lymphoma; abdominal pseudotumor is an exceptionally rare presentation. In the era of highly active anti-retroviral therapy (HAART), opportunistic infections are increasingly rare, and secondary prophylaxis for MAC may be discontinued after adequate therapy and immune reconstitution. Recurrence of disseminated MAC after adequate therapy may be due to macrolide resistance, but with an adequate CD4 T-cell count and undetectable HIV viral load, recurrence raises questions of more subtle immune dysregulation. PMID:27807554

  11. Mycobacterium avium complex olecranon bursitis resolves without antimicrobials or surgical intervention: A case report and review of the literature

    PubMed Central

    Working, Selene; Tyser, Andrew; Levy, Dana

    2015-01-01

    Introduction Nontuberculous mycobacteria are an uncommon cause of septic olecranon bursitis, though cases have increasingly been described in both immunocompromised and immunocompetent hosts. Guidelines recommend a combination of surgical resection and antimicrobials for treatment. This case is the first reported case of nontuberculous mycobacterial olecranon bursitis that resolved without medical or surgical intervention. Case presentation A 67-year-old female developed a painless, fluctuant swelling of the olecranon bursa following blunt trauma to the elbow. Due to persistent bursal swelling, she underwent three separate therapeutic bursal aspirations, two involving intrabursal steroid injection. After the third aspiration, the bursa became erythematous and severely swollen, and bursal fluid grew Mycobacterium avium complex. Triple-drug antimycobacterial therapy was initiated, but discontinued abruptly due to a rash. Surgery was not performed. The patient was observed off antimicrobials, and gradually clinically improved with a compressive dressing. By 14 months after initial presentation, clinical exam revealed complete resolution of the previously erythematous bursal mass. Discussion This is the first reported case of nontuberculous mycobacterial olecranon bursitis managed successfully without surgery or antimicrobials. Musculoskeletal nontuberculous mycobacterial infections are challenging given the lack of clinical data about optimal duration and choice of antimicrobials or the role of surgery. Additionally, the potential toxicity and drug interactions of antimycobacterials are not insignificant and warrant close monitoring if treatment is pursued. Conclusion This case raises an important clinical question of whether close observation off antimicrobials is appropriate in select cases of immunocompetent patients with localized atypical mycobacterial disease of soft tissue and skeletal structures. PMID:26793457

  12. Genome-Wide Analysis of the Emerging Infection with Mycobacterium avium Subspecies paratuberculosis in the Arabian Camels (Camelus dromedarius)

    PubMed Central

    Ghosh, Pallab; Hsu, Chungyi; Alyamani, Essam J.; Shehata, Maher M.; Al-Dubaib, Musaad A.; Al-Naeem, Abdulmohsen; Hashad, Mahmoud; Mahmoud, Osama M.; Alharbi, Khalid B. J.; Al-Busadah, Khalid; Al-Swailem, Abdulaziz M.; Talaat, Adel M.

    2012-01-01

    Mycobacterium avium subspecies paratuberculosis (M. ap) is the causative agent of paratuberculosis or Johne's disease (JD) in herbivores with potential involvement in cases of Crohn's disease in humans. JD is spread worldwide and is economically important for both beef and dairy industries. Generally, pathogenic ovine strains (M. ap-S) are mainly found in sheep while bovine strains (M. ap-C) infect other ruminants (e.g. cattle, goat, deer), as well as sheep. In an effort to characterize this emerging infection in dromedary/Arabian camels, we successfully cultured M. ap from several samples collected from infected camels suffering from chronic, intermittent diarrhea suggestive of JD. Gene-based typing of isolates indicated that all isolates belong to sheep lineage of strains of M. ap (M. ap-S), suggesting a putative transmission from infected sheep herds. Screening sheep and goat herds associated with camels identified the circulation of this type in sheep but not goats. The current genome-wide analysis recognizes these camel isolates as a sub-lineage of the sheep strain with a significant number of single nucleotide polymorphisms (SNPs) between sheep and camel isolates (∼1000 SNPs). Such polymorphism could represent geographical differences among isolates or host adaptation of M. ap during camel infection. To our knowledge, this is the first attempt to examine the genomic basis of this emerging infection in camels with implications on the evolution of this important pathogen. The sequenced genomes of M. ap isolates from camels will further assist our efforts to understand JD pathogenesis and the dynamic of disease transmission across animal species. PMID:22393374

  13. Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

    PubMed Central

    O’Brien, Lorna M.; Stewart, Linda D.; Strain, Sam A. J.; Grant, Irene R.

    2016-01-01

    The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay. PMID:26815790

  14. Comprehensive Insights in the Mycobacterium avium subsp. paratuberculosis Genome Using New WGS Data of Sheep Strain JIII-386 from Germany

    PubMed Central

    Möbius, Petra; Hölzer, Martin; Felder, Marius; Nordsiek, Gabriele; Groth, Marco; Köhler, Heike; Reichwald, Kathrin; Platzer, Matthias; Marz, Manja

    2015-01-01

    Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP)—the etiologic agent of Johne’s disease—affects cattle, sheep, and other ruminants worldwide. To decipher phenotypic differences among sheep and cattle strains (belonging to MAP-S [Type-I/III], respectively, MAP-C [Type-II]), comparative genome analysis needs data from diverse isolates originating from different geographic regions of the world. This study presents the so far best assembled genome of a MAP-S-strain: Sheep isolate JIII-386 from Germany. One newly sequenced cattle isolate (JII-1961, Germany), four published MAP strains of MAP-C and MAP-S from the United States and Australia, and M. a. subsp. hominissuis (MAH) strain 104 were used for assembly improvement and comparisons. All genomes were annotated by BacProt and results compared with NCBI (National Center for Biotechnology Information) annotation. Corresponding protein-coding sequences (CDSs) were detected, but also CDSs that were exclusively determined by either NCBI or BacProt. A new Shine–Dalgarno sequence motif (5′-AGCTGG-3′) was extracted. Novel CDSs including PE-PGRS family protein genes and about 80 noncoding RNAs exhibiting high sequence conservation are presented. Previously found genetic differences between MAP-types are partially revised. Four of ten assumed MAP-S-specific large sequence polymorphism regions (LSPSs) are still present in MAP-C strains; new LSPSs were identified. Independently of the regional origin of the strains, the number of individual CDSs and single nucleotide variants confirms the strong similarity of MAP-C strains and shows higher diversity among MAP-S strains. This study gives ambiguous results regarding the hypothesis that MAP-S is the evolutionary intermediate between MAH and MAP-C, but it clearly shows a higher similarity of MAP to MAH than to Mycobacterium intracellulare. PMID:26384038

  15. Effects of 10-GHz microwaves on hematological parameters in Swiss albino mice and their modulation by Prunus avium.

    PubMed

    Sisodia, Rashmi; Rifat, Faiza; Sharma, Archana; Srivastava, Preeti; Sharma, K

    2013-01-01

    The objective of this study was to investigate the modulatory role of Prunus avium fruit extract (PAE) on several blood parameters after exposure to 10-GHz microwaves. Swiss albino mice from an inbred colony were selected and divided into 3 groups. Mice in group I served as the control; they were placed in a Plexiglas cage (without energizing the system) for 2 hours/day for 30 consecutive days. Group II mice were exposed to 10-GHz microwaves for 2 hours/day for 30 consecutive days. Mice in group III received PAE (500 mg/kg/body weight) orally once daily 1 hour before exposure to 10-GHz microwaves (2 hours/day) for 30 consecutive days. After 30 days of treatment, blood samples were collected from mice in all groups and analyzed. Hemoglobin, monocytes, packed cell volume, red blood cells, mean corpuscular hemoglobin concentration declined significantly (P ≤ 0.01), whereas white blood cells, lymphocytes, erythrocyte sedimentation rate, and mean corpuscular volume increased significantly (P ≤ 0.01) compared to the control group (group I). Cholesterol, alkaline phosphatase, and lipid peroxidation also increased significantly (P ≤ 0.01). Depletion in blood sugar, total protein, acid phosphatase, and glutathione levels was noted after microwave exposure compared with levels in the sham-exposed (control) mice. Histopathological alterations in blood cells also were seen. Signs of improvements in the hematological, biochemical, and histopathological parameters were recorded in group III, where PAE was supplemented before exposure. Exposure to microwaves influences hematological parameters, which could be ameliorated by the supplementation of PAE.

  16. The effects of progressing and nonprogressing Mycobacterium avium ssp. paratuberculosis infection on milk production in dairy cows.

    PubMed

    Smith, Rebecca L; Gröhn, Y T; Pradhan, A K; Whitlock, R H; Van Kessel, J S; Smith, J M; Wolfgang, D R; Schukken, Y H

    2016-02-01

    Longitudinal data from 3 commercial dairy herds in the northeast United States, collected from 2004 to 2011, were analyzed to determine the effect of Mycobacterium avium ssp. paratuberculosis (MAP) infection status and progression path on milk production. Disease status, as indicated by MAP test results, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues and feces at slaughter. Milk production data were collected from the Dairy Herd Information Association. Animals with positive MAP test results were categorized, based on test results over the full course of the study, as high path (at least one high-positive culture) or low path (at least one positive culture or ELISA). The cumulative numbers of positive ELISA and culture results were recorded. The effects of both MAP infection path, status, and number of positive tests on milk production were analyzed using a mixed linear model with an autocorrelation random effect structure. Low- and high-path animals produced more milk before their first positive test than always-negative animals, especially high-path animals. Although mean production decreased after a first positive test, low-path animals were shown to recover some productivity. High-path animals continued to exhibit a decrease in milk production, especially after their first high-positive fecal culture. These results show that not all animals that test positive for MAP will have long-term production losses. Milk production decreased significantly with each additional positive test. Ultimately, production loss appeared to be a function of MAP infection progression. PMID:26686721

  17. Invited review: modeling within-herd transmission of Mycobacterium avium subspecies paratuberculosis in dairy cattle: a review.

    PubMed

    Marcé, C; Ezanno, P; Weber, M F; Seegers, H; Pfeiffer, D U; Fourichon, C

    2010-10-01

    Epidemiological models have been developed to test hypotheses on Mycobacterium avium ssp. paratuberculosis (Map) transmission in a herd, and to compare different paratuberculosis control strategies and alternatives for certification-and-surveillance schemes. The models are simplified representations of existing biological processes tailored to the questions they are intended to answer. Such models depend on available knowledge about the underlying processes, notably in relation to pathogen transmission. All decisions relating to integration of specific aspects of the herd structure and transmission mechanisms as well as modeling objective will influence model behavior and simulation results. This paper examines assumptions on pathogen transmission and risk mitigation represented in 8 epidemiological models of within-herd Map transmission in dairy cattle. We describe available models' structure and examine them in the context of current knowledge about host infection and pathogen transmission pathways. We investigate how population structure and herd management are modeled with regard to their influence on contact structure and pathogen transmission. We show that assumptions about routes of transmission and their contribution within a herd vary greatly among models. Gaps of knowledge that are pivotal to defining transmission equations and parameters, such as variation of susceptibility with age and variability of pattern of shedding, are identified. Quantitative estimates of this incomplete information should be targeted by future research. Existing models could be improved by considering indirect transmission via the environment taking account of Map survival and contact structure between animals in a herd, and by including calf-to-calf transmission, which has recently been proven as being important.

  18. Systems Biology Analysis of Gene Expression during In Vivo Mycobacterium avium paratuberculosis Enteric Colonization Reveals Role for Immune Tolerance

    PubMed Central

    Khare, Sangeeta; Lawhon, Sara D.; Drake, Kenneth L.; Nunes, Jairo E. S.; Figueiredo, Josely F.; Rossetti, Carlos A.; Gull, Tamara; Everts, Robin E.; Lewin, Harris A.; Galindo, Cristi L.; Garner, Harold R.; Adams, Leslie Garry

    2012-01-01

    Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways

  19. Paradigm redux--Mycobacterium avium subspecies paratuberculosis-macrophage interactions show clear variations between bovine and human physiological body temperatures.

    PubMed

    Lamont, Elise A; Sreevatsan, Srinand

    2010-05-01

    The physiological conditions encountered by pathogenic mycobacteria inside their hosts significantly influence their adaptation, virulence, and gene expression. Current in vitro models investigating host-pathogen interactions of Mycobacterium avium subsp. paratuberculosis use 37 degrees C, the normal body temperatures of mice and humans. However since the physiological temperature of MAP's natural host is 39 degrees C, we hypothesized that host and pathogen behavior to vary considerably in comparison to 37 degrees C. Our MAP-macrophage interaction studies show striking differences in regards to velocity of cell invasion of MAP as well as bacterial and host gene regulation at 39 degrees C compared with 37 degrees C. Upregulation of host genes (nod2, tlr2, mapkp38 and il-10) follow a similar trend at 37 degrees C and 39 degrees C; however, there is over a five-fold increase as early as 0.5 and 2 h in 39 degrees C treatments. While host signaling is completed by 48 h p.i. at 39 degrees C in MDMs cultures due to early cell death, signaling and infection is sustained at 37 degrees C. Surprisingly, transcription of MAP genes did not show a set pattern and were upregulated at different time points for both temperatures. Interestingly, MAP genes encoding a lipase (lipN) and an oxidoreductase (MAP3464) are staggered at 39 degrees C, while they increase steadily at 37 degrees C. In conclusion, infection and culture at a physiologically relevant temperature influences host-pathogen interaction, which may have far reaching ramifications including for currently used animal models, in vitro culture methods, bacterial pathogenesis and host responses, and vaccine candidate design and screening.

  20. Prevalence and Association of Mycobacterium avium subspecies paratuberculosis with Disease Course in Patients with Ulcero-Constrictive Ileocolonic Disease

    PubMed Central

    Khan, Imteyaz Ahmad; Pilli, Sucharita; A, Surendranath; Rampal, Ritika; Chauhan, Sudhir Kumar; Tiwari, Veena; Mouli, Venigalla Pratap; Kedia, Saurabh; Nayak, Baibaswata; Das, Prasenjit; Makharia, Govind K.; Ahuja, Vineet

    2016-01-01

    Background Association of Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn’s disease (CD) has been controversial due to contradictory reports. Therefore, we determined the prevalence of MAP in patients with CD and intestinal tuberculosis (ITB) and its association with clinical course. Methodology Blood and intestinal biopsies were taken from 69 CD, 32 ITB patients and 41 patients with haemorrhoidal bleed who served as controls. qPCR targeting of MAP-specific IS900 gene was used to detect the presence of MAP DNA. qPCR results were further validated by sequencing. Immunohistochemistry (IHC) was used to detect the presence of MAP antigen in biopsy specimens. CD and ITB patients were followed-up for disease course and response to therapy. Principal Findings The frequency of MAP-specific DNA in biopsies by qPCR was significantly higher in CD patients (23.2%, p = 0.03) as compared to controls (7.3%). No significant difference in intestinal MAP presence was observed between ITB patients (12.5%, p = 0.6) and controls (7.3%). MAP presence in blood of CD patients was 10.1% as compared to 4.9% in controls while no patients with ITB were found to be positive (p = 0.1). Using IHC for detection of MAP antigen, the prevalence of MAP in CD was 2.9%, 12.5% in ITB patients and 2.4% in controls. However, long-term follow-up of the patients revealed no significant associations between clinical characteristics and treatment outcomes with MAP positivity. Conclusion We report significantly high prevalence of MAP in intestinal biopsies of CD patients. However, the presence of MAP does not affect the disease course and treatment outcomes in either CD or ITB patients. PMID:27019109

  1. Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. paratuberculosis in Japanese multiple sclerosis patients

    PubMed Central

    Cossu, Davide; Yokoyama, Kazumasa; Sechi, Leonardo Antonio; Otsubo, Shigeru; Tomizawa, Yuji; Momotani, Eiichi; Hattori, Nobutaka

    2016-01-01

    Several works have demonstrated the existence of a link between Mycobacterium avium subsp. paratuberculosis (MAP) and MS in Italy. In this study, we analyzed the serology of MAP in a Japanese population while looking at several markers of MAP. Fifty MS patients, 12 clinically isolated syndrome (CIS) patients, 30 other neurological disorders (OND) patients, and 50 healthy controls (HCs) were tested using ELISA for the presence of IgG antibodies toward immunodominant epitopes MAP_0106c121-132, homologues MBP85-98, homologues IRF5424-432, MAP_402718-32, and MAP_2694295-303. MAP-positive patients were also analyzed in relation to their clinical/demographic characteristics. Amongst all peptides, only antibodies against MAP_2694295-303 were more prevalent in MS patients (30%), as compared to OND patients (3%) (p = 0.009; area under roc curve (AUC) = 0.61) and HCs (2%) (p = 0.0004; AUC = 0.65) and in CIS patients (25%) compared to HCs (p = 0.023; AUC = 0.55). Logistic regression analysis showed a higher frequency of anti-MAP_2694295-303 antibodies in the sera of oligoclonal bands positive MS patients (p = 0.2; OR = 2, 95%CI: 0.55–7.7). These findings support the view that MAP could act as a risk factor or a triggering agent of MS in some Japanese patients with a genetic susceptibility to the mycobacterium. PMID:27356622

  2. Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle.

    PubMed

    Eamens, Graeme J; Whittington, Richard J; Turner, Mark J; Austin, Susan L; Fell, Shayne A; Marsh, Ian B

    2007-11-15

    Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.

  3. Differences in the peripheral immune response between lambs and adult ewes experimentally infected with Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Delgado, Laetitia; Juste, Ramón A; Muñoz, María; Morales, Silvia; Benavides, Julio; Ferreras, M Carmen; Marín, J Francisco García; Pérez, Valentín

    2012-01-15

    The peripheral immune response, and its relationship with the outcome of the infection according to the age of the animal, has been investigated in young lambs and adult ewes experimentally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (Map). Sixteen 1.5-month-old lambs out of 24 and 23 adult ewes out of 30 were orally challenged with an ovine Map field isolate. Animals were divided into two groups: HD, infected with a higher dose of Map and LD, with a lower dose. The remaining animals were used as uninfected control groups. Animals were euthanized at 110-120 and 210-220 days post-infection (dpi). Along the experiment, the humoral response and the specific and non-specific IFN-γ production were assessed. An intradermal skin test (IDT), using avian PPD, was also performed at 90 and 195 dpi. Samples of intestine and related lymphoid tissue were taken for histological, bacteriological and PCR studies. The Ab and IFN-γ production as well as the IDT response appeared earlier and with more intensity in the adult ewes compared to the lambs. The basal non-specific IFN-γ levels increased only in the adult ewes from the HD group. Animals from the LD and HD groups were positive to PCR; however, lesions consistent with paratuberculosis were exclusively observed in the HD group, both in lambs and in adult sheep, but they only progressed to more advanced stages in the former. These results suggest that the peripheral immune response induced by Map infection in the adult ewes is more efficient to control the progression of the infection than in lambs. This could likely be due to the existence of previous contacts with Map or other mycobacteria in the adult sheep compared to the young lambs.

  4. Effects of biological and methodological factors on volatile organic compound patterns during cultural growth of Mycobacterium avium ssp. paratuberculosis.

    PubMed

    Küntzel, Anne; Fischer, Sina; Bergmann, Andreas; Oertel, Peter; Steffens, Markus; Trefz, Phillip; Miekisch, Wolfram; Schubert, Jochen K; Reinhold, Petra; Köhler, Heike

    2016-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants. Bacterial growth is still the diagnostic 'gold standard', but is very time consuming. MAP-specific volatile organic compounds (VOCs) above media could accelerate cultural diagnosis. The aim of this project was to assess the kinetics of a VOC profile linked to the growth of MAP in vitro. The following sources of variability were taken into account: five different culture media, three different MAP strains, inoculation with different bacterial counts, and different periods of incubation. Needle-trap microextraction was employed for pre-concentration of VOCs, and gas chromatography-mass spectrometry for subsequent analysis. All volatiles were identified and calibrated by analysing pure references at different concentration levels. More than 100 VOCs were measured in headspaces above MAP-inoculated and control slants. Results confirmed different VOC profiles above different culture media. Emissions could be assigned to either egg-containing media or synthetic ingredients. 43 VOCs were identified as potential biomarkers of MAP growth on Herrold's Egg Yolk Medium without significant differences between the tree MAP strains. Substances belonged to the classes of alcohols, aldehydes, esters, ketones, aliphatic and aromatic hydrocarbons. With increasing bacterial density the VOC concentrations above MAP expressed different patterns: the majority of substances increased (although a few decreased after reaching a peak), but nine VOCs clearly decreased. Data support the hypotheses that (i) bacteria emit different metabolites on different culture media; (ii) different MAP strains show uniform VOC patterns; and (iii) cultural diagnosis can be accelerated by taking specific VOC profiles into account. PMID:27604146

  5. Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq

    PubMed Central

    Casey, Maura E.; Meade, Kieran G.; Nalpas, Nicolas C.; Taraktsoglou, Maria; Browne, John A.; Killick, Kate E.; Park, Stephen D. E.; Gormley, Eamonn; Hokamp, Karsten; Magee, David A.; MacHugh, David E.

    2015-01-01

    Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP), is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne’s disease. Here, we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM) transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a 6-h infection time course with non-infected controls. We observed 245 and 574 differentially expressed (DE) genes in MAP-infected versus non-infected control samples (adjusted P value ≤0.05) at 2 and 6 h post-infection, respectively. Functional analyses of these DE genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix® microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection. PMID:25699042

  6. High herd-level prevalence of Mycobacterium avium subspecies paratuberculosis in Western Canadian dairy farms, based on environmental sampling.

    PubMed

    Wolf, R; Barkema, H W; De Buck, J; Slomp, M; Flaig, J; Haupstein, D; Pickel, C; Orsel, K

    2014-10-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive enteritis in ruminants. The pathogen is present in most countries with modern dairy production, causing substantial economic losses for the industry. The objectives of this study were to estimate dairy herd prevalence of MAP in the Western Canadian provinces of Alberta and Saskatchewan, and to determine whether herd size and housing system (tie-stall versus freestall or loose housing) affected the risk of a herd testing positive for MAP. Six environmental samples were collected on 360 Alberta farms (60% of registered producers) and on 166 Saskatchewan dairy farms (99%). In total, 47% of the sampled farms in Alberta and 53% of the sampled farms in Saskatchewan had at least one environmental sample that was MAP culture positive and were, therefore, defined as infected. Sensitivity of environmental sampling was estimated using 3 subsequent annual tests performed on 82 farms. Because laboratory protocols were continuously improved throughout the project, the sensitivity increased over time. Therefore, a mean of the sensitivity estimates weighted on sampling year was constructed; this resulted in sensitivities of 68 and 69% for Alberta and Saskatchewan, respectively. Implementing those estimates in an approximate Bayesian computation model resulted in a true herd prevalence of 68% (95% probability interval: 60-80%) for Alberta and 76% (95% probability interval: 70-85%) for Saskatchewan. Herds with >200 cows had 3.54 times higher odds of being environmental sample positive and had more positive samples than herds with <50 cows (neither province nor housing system affected those results). In conclusion, the majority of Alberta and Saskatchewan dairy farms were infected with MAP and larger herds were more often MAP positive than smaller herds.

  7. Heme Utilization in Bordetella avium Is Regulated by RhuI, a Heme-Responsive Extracytoplasmic Function Sigma Factor

    PubMed Central

    Kirby, Amy E.; Metzger, Daniel J.; Murphy, Erin R.; Connell, Terry D.

    2001-01-01

    Efficient utilization of heme as an iron (Fe) source by Bordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that rhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuI was correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal to bhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced into Escherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. coli demonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the rhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent. PMID:11598070

  8. Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry ( Prunus avium L.).

    PubMed

    Wünsch, A; Hormaza, J I

    2004-01-01

    Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease ( S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5' flanking regions of four S-RNases of sweet cherry ( Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S23, S24 and S25 present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S12 amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5'-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5'-flanking regions of S-RNases from the Maloideae. S6- and S24-RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.

  9. Optimization of methods for detecting Mycobacterium avium subsp. paratuberculosis in environmental samples using quantitative, real-time PCR.

    PubMed

    Cook, Kimberly L; Britt, Jenks S

    2007-04-01

    Detection of Johne's disease, an enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), has been impeded by the lack of rapid, reliable detection methods. The goal of this study was to optimize methodologies for detecting M. paratuberculosis in manure from an infected dairy cow or in contaminated soil samples using a quantitative, real-time PCR (QRT-PCR) based analysis. Three different nucleic acid extraction techniques, the efficiency of direct versus indirect sample extraction, and sample pooling were assessed. The limit of detection was investigated by adding dilutions of M. paratuberculosis to soil. Results show that the highest yield (19.4+/-2.3 microg(-1) DNA extract) and the highest copy number of the targeted M. paratuberculosis IS900 sequence (1.3+/-0.2x10(8) copies g(-1) manure) were obtained with DNA extracted from manure using Qbiogene's Fast DNA Spin kit for soil. Pooling ten samples of M. paratuberculosis-contaminated soil improved the limit of detection ten fold (between 20 and 115 M. paratuberculosis cells g(-1) soil). Detection was between 65% and 95% higher when samples were extracted directly using bead-beating than when using pre-treatment with cell extraction buffers. The final soil-sampling and extraction regime was applied for detection of M. paratuberculosis in pasture soil after the removal of a M. paratuberculosis culture positive dairy cow. M. paratuberculosis remained in the pasture soil for more than 200 days. Results from these studies suggest that DNA extraction method, sampling protocol and PCR conditions each critically influence the outcome and validity of the QRT-PCR analysis of M. paratuberculosis concentrations in environmental samples.

  10. Quantification of the Sensitivity of Mycobacterium avium subsp paratuberculosis and Salmonella enterica subsp enterica to Low pH and High Organic Acids using Propidium Monoazide and Quantitative PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis (Map) and Salmonella enterica subsp enterica (S. enterica) are two pathogens that are a concern to food and animal safety due to their ability to withstand harsh conditions encountered in the natural environment and within the host during pathogenesis. Acid...

  11. Prevalence of Mycobacterium avium subsp. paratuberculosis in Ileocecal Lymph Nodes and on Hides and Carcasses from Cull Cows and Fed Cattle at Commercial Beef Processing Plants in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clinical associations between Crohn’s disease in humans and Mycobacterium avium subspecies paratuberculosis (Map) have been suggested but not confirmed. Map is the causative agent for Johne’s disease in cattle. Infected cattle could be sources for Map transmission to humans via dairy and beef prod...

  12. The other way around: Probiotic lactobacillus acidophilus NP51 restricts progression of Mycobacterium avium subspecies paratuberculosis (MAP) infection in Balb/c mice through activation of CD8+ T cell-mediated immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to examine immune effects of feeding novel probiotic Lactobacillus acidophilus strain NP51 to specific pathogen-free Balb/c mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease (JD). We hypothesized that fe...

  13. Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System.

    PubMed

    Rocchetti, Talita T; Silbert, Suzane; Gostnell, Alicia; Kubasek, Carly; Widen, Raymond

    2016-06-01

    A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

  14. Short communication: Application of an N-acetyl-L-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subsp. paratuberculosis from milk of naturally infected cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne’s disease and, therefore, is a route of transmission for infection in youngstock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of ...

  15. Rapid and Sensitive Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

    PubMed Central

    Khare, Sangeeta; Ficht, Thomas A.; Santos, Renato L.; Romano, Juan; Ficht, Allison R.; Zhang, Shuping; Grant, Irene R.; Libal, Melissa; Hunter, David; Adams, L. Garry

    2004-01-01

    Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions. PMID:15004056

  16. Non-Major Histocompatibility Complex Control of Antibody Isotype and Th1 versus Th2 Cytokines during Experimental Infection of Mice with Mycobacterium avium

    PubMed Central

    Nagabhushanam, Vijaya; Cheers, Christina

    2001-01-01

    Infection of different strains of mice with Mycobacterium avium has revealed genetic control of the immunoglobulin isotype induced and of the balance between Th1 and Th2 cytokines. Female BALB/c or C57BL/10 mice were infected intranasally with 105 M. avium organisms. The antibody response was measured over 18 weeks by enzyme-linked immunosorbent assay and Western blotting, while numbers of cytokine-producing cells were assessed at 12 to 15 weeks by ELISPOT assay. Upon infection, C57BL/10 mice produced a clear Th1 response with strong gamma interferon (IFN-γ) production, no interleukin-4 (IL-4), and almost entirely immunoglobulin G2a (IgG2a) antibody. In contrast, BALB/c mice developed T cells producing IL-4, as well as those producing IFN-γ, while the antibody response was a mixture of IgG1 and IgG2a. Antibodies from BALB/c mice were also able to recognize a greater range of antigens than were C56BL/10 mice. B10D2 mice, which carry the BALB/c major histocompatibility complex haplotype on a C57BL/10 background, followed the C57BL/10 cytokine pattern. Mice infected with Listeria monocytogenes did not show a similar response dichotomy. PMID:11179347

  17. Implications of PCR and ELISA results on the routes of bulk-tank contamination with Mycobacterium avium ssp. paratuberculosis.

    PubMed

    Beaver, A; Cazer, C L; Ruegg, P L; Gröhn, Y T; Schukken, Y H

    2016-02-01

    Mycobacterium avium ssp. paratuberculosis (MAP), the etiologic agent of Johne's disease in dairy cattle, may enter the bulk tank via environmental contamination or direct excretion into milk. Traditionally, diagnostics to identify MAP in milk target either MAP antibodies (by ELISA) or the organism itself (by culture or PCR). High ELISA titers may be directly associated with excretion of MAP into milk but only indirectly linked to environmental contamination of the bulk tank. Patterns of bulk-milk ELISA and bulk-milk PCR results could therefore provide insight into the routes of contamination and level of infection or environmental burden. Coupled with questionnaire responses pertaining to management, the results of these diagnostic tests could reveal correlations with herd characteristics or on-farm practices that distinguish herds with high and low environmental bulk-tank MAP contamination. A questionnaire on hygiene, management, and Johne's specific parameters was administered to 292 dairy farms in New York, Oregon, and Wisconsin. Bulk-tank samples were collected from each farm for evaluation by real-time PCR and ELISA. Before DNA extraction and testing of the unknown samples, bulk-milk template preparation was optimized with respect to parameters such as MAP fractionation patterns and lysis. Two regression models were developed to explore the relationships among bulk-tank PCR, ELISA, environmental predictors, and herd characteristics. First, ELISA optical density (OD) was designated as the outcome in a linear regression model. Second, the log odds of being PCR positive in the bulk tank were modeled using binary logistic regression with penalized maximum likelihood. The proportion of PCR-positive bulk tanks was highest for New York and for organic farms, providing a clue as to the geographical patterns of MAP-positive bulk-tank samples and relationship to production type. Bulk-milk PCR positivity was also higher for large relative to small herds. The models

  18. The modification and evaluation of an ELISA test for the surveillance of Mycobacterium avium subsp. paratuberculosis infection in wild ruminants

    PubMed Central

    2013-01-01

    Background Enzyme-linked immunosorbent assay (ELISA) is often used to test wildlife samples for Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, commercially available kits are only validated for use with domestic ruminant species. A literature review was performed to document the current use of MAP serum ELISA in wild and semi-domestic ruminants. We then modified and evaluated a commercial ELISA kit (IDEXX Mycobacterium paratuberculosis Antibody Test Kit) for use with species for which it was not originally developed: elk (Cervus elaphus), bison (Bison bison) and caribou (Rangifer tarandus). We tested the affinity of different conjugates for immunoglobulin G (IgG) isolated from these species, performed checkerboard tests to determine the optimal dilutions of samples and conjugates, and established cut-off values using two different methods: a Receiver Operational Curve on a panel of known samples for elk, and an alternate method involving a panel of unknown serum samples for the three species. Results We found that the anti-bovine conjugate included in the IDEXX ELISA kit has limited affinity for elk, bison, and caribou IgG. Protein G showed good affinity for IgG of all three species, while anti-deer conjugate also bound elk and caribou IgG. Using Protein G with elk serum, a cut-off sample-to-positive (S/P) value of 0.22 was selected, resulting in a sensitivity and specificity of 73% and 90%, respectively, whereas, using an anti-deer conjugate with elk serum, an S/P cut-off value of 0.29 gave a sensitivity of 68%, with 100% specificity. Cut-off values for bison and caribou using the Protein G conjugate were 0.17 and 0.25 respectively. Conclusions Due to incomplete reporting and a lack of test validation, it is difficult to critically appraise results of many sero-surveys that have previously been done for MAP in wildlife. Commercial ELISA kits may have limited or no capacity to detect antibodies from species other than for which they were

  19. Genome-Wide Association Analysis and Genomic Prediction of Mycobacterium avium Subspecies paratuberculosis Infection in US Jersey Cattle

    PubMed Central

    Zare, Yalda; Shook, George E.; Collins, Michael T.; Kirkpatrick, Brian W.

    2014-01-01

    Paratuberculosis (Johne’s disease), an enteric disorder in ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), causes economic losses in excess of $200 million annually to the US dairy industry. To identify genomic regions underlying susceptibility to MAP infection in Jersey cattle, a case-control genome-wide association study (GWAS) was performed. Blood and fecal samples were collected from ∼5,000 mature cows in 30 commercial Jersey herds from across the US. Discovery data consisted of 450 cases and 439 controls genotyped with the Illumina BovineSNP50 BeadChip. Cases were animals with positive ELISA and fecal culture (FC) results. Controls were animals negative to both ELISA and FC tests that matched cases on birth date and herd. Validation data consisted of 180 animals including 90 cases (positive to FC) and 90 controls (negative to ELISA and FC), selected from discovery herds and genotyped by Illumina BovineLD BeadChip (∼7K SNPs). Two analytical approaches were used: single-marker GWAS using the GRAMMAR-GC method and Bayesian variable selection (Bayes C) using GenSel software. GRAMMAR-GC identified one SNP on BTA7 at 68 megabases (Mb) surpassing a significance threshold of 5×10−5. ARS-BFGL-NGS-11887 on BTA23 (27.7 Mb) accounted for the highest percentage of genetic variance (3.3%) in the Bayes C analysis. SNPs identified in common by GRAMMAR-GC and Bayes C in both discovery and combined data were mapped to BTA23 (27, 29 and 44 Mb), 3 (100, 101, 106 and 107 Mb) and 17 (57 Mb). Correspondence between results of GRAMMAR-GC and Bayes C was high (70–80% of most significant SNPs in common). These SNPs could potentially be associated with causal variants underlying susceptibility to MAP infection in Jersey cattle. Predictive performance of the model developed by Bayes C for prediction of infection status of animals in validation set was low (55% probability of correct ranking of paired case and control samples). PMID:24523889

  20. Exploring the role of Mycobacterium avium subspecies paratuberculosis in the pathogenesis of type 1 diabetes mellitus: a pilot study

    PubMed Central

    2013-01-01

    Background Although the etiology of Type 1 Diabetes mellitus (T1DM) has not been determined, genetic polymorphism in key genes, including SLC11A1, and association with Mycobacterium avium subspecies paratuberculosis (MAP) have been reported. We hypothesize that molecular mimicry between MAP Heat shock protein 65 K (Hsp65) and human Glutamic Acid Decarboxylase 65 K (GAD65) may be the trigger leading to autoimmune destruction of beta cells in patients exposed to MAP. Method Peptide sequences of MAP Hsp65 and human GAD65 were investigated for amino acid sequence homology and cross reactivity. A total of 18 blood samples from T1DM and controls were evaluated for the presence of MAP. Results Peptide BLAST analysis revealed a 44% overall identity between MAP Hsp65 and GAD65 with 75% positives in a 16 amino acid region. PyMOL 3D-structural analyses identified the same 16 amino acid region as a potential epitope for antibody binding. Preliminary data suggests a cross reactivity between MAP Hsp65, and a healthy rat pancreatic tissue homogenate against plasma from T1DM patients and rabbit polyclonal anti-MAP IgG. Long-term culture of human blood resulted MAP detection in 3/10 T1DM and 4/8 controls whereas MAP IgG was detected in 5/10 T1DM samples and 3/8 non-diabetic controls. Conclusion The high degree of homology between GAD65 and MAP Hsp65 in an antigenic peptide region supports a possible mycobacterial role in triggering autoimmune destruction of pancreatic cells in T1DM. Reactivity of T1DM patient sera with MAP Hsp65 supports this finding. Culture of MAP from the blood of T1DM patients is intriguing. Overall, the preliminary data are mixed and do not exclude a possible role for MAP in T1DM pathogenesis. A larger study including well-characterized controls is needed to investigate the intriguing question of whether MAP is associated with T1DM or not? PMID:23759115

  1. Measurement of antioxidant activity and antioxidant compounds under versatile extraction conditions: I. the immuno-biochemical antioxidant properties of sweet cherry (Prunus avium) extracts.

    PubMed

    Hanbali, Lama B; Ghadieh, Rana M; Hasan, Hiba A; K Nakhal, Yasmine; Haddad, John J

    2013-01-01

    Previously, we have meticulously examined the efficacy of the measurable antimicrobial activity of sweet cherry (Prunus avium) extracts on a wide spectrum of gram-positive and gram-negative bacteria, in addition to the fungus, Candida albicans, a priori. In order to further understand the biochemical constituents and antioxidant activities of a variety of extracts of sweet cherries, antioxidant compounds of immunological significance, including L-ascorbic acid (vitamin C), phenols, flavonoids, and anthocyanins, and the total antioxidant (free radical scavenging) activity were simultaneously measured under varying and versatile extraction conditions (mild heating [5, 10 and 20 min.], and brief microwave exposure [1, 2 and 5 min.]) for a variety of extracts: i) whole juice extracts (WJE), ii) methanol-extracted juice (MEJ), iii) ddH2O-extracted pomace (dPOM), and iv) methanol-extracted pomace (mPOM). The antioxidant activity under the versatile extraction conditions adopted in this study was conspicuously reduced, such that the % inhibition against 2,2- diphenyl-1-picrylhydrazyl (DPPH) followed an inverse, negative correlational trendline. Moreover, ascorbic acid content was not affected with mild to prolonged heating or microwave exposure, except tangibly with dPOM and mPOM. The total phenols content assessed showed no significant variations, as compared with control extracts. In a manner similar to ascorbic acid, total flavonoids were mildly reduced under varying conditions, an effect mimicked to a certain extent with anthocyanins. Assessment of extraction means as compared with WJE revealed sharp decrease in the antioxidant activity for dPOM and mPOM, significant increase in L-ascorbic acid, total phenol, and flavonoid contents for MEJ, dPOM, and mPOM, and mild decrease in anthocyanin contents for dPOM and mPOM. These results confirm the measurable antioxidant activities and contents of P. avium extracts under versatile conditions of mild exposure, an effect

  2. Measurement of antioxidant activity and antioxidant compounds under versatile extraction conditions: I. the immuno-biochemical antioxidant properties of sweet cherry (Prunus avium) extracts.

    PubMed

    Hanbali, Lama B; Ghadieh, Rana M; Hasan, Hiba A; K Nakhal, Yasmine; Haddad, John J

    2013-01-01

    Previously, we have meticulously examined the efficacy of the measurable antimicrobial activity of sweet cherry (Prunus avium) extracts on a wide spectrum of gram-positive and gram-negative bacteria, in addition to the fungus, Candida albicans, a priori. In order to further understand the biochemical constituents and antioxidant activities of a variety of extracts of sweet cherries, antioxidant compounds of immunological significance, including L-ascorbic acid (vitamin C), phenols, flavonoids, and anthocyanins, and the total antioxidant (free radical scavenging) activity were simultaneously measured under varying and versatile extraction conditions (mild heating [5, 10 and 20 min.], and brief microwave exposure [1, 2 and 5 min.]) for a variety of extracts: i) whole juice extracts (WJE), ii) methanol-extracted juice (MEJ), iii) ddH2O-extracted pomace (dPOM), and iv) methanol-extracted pomace (mPOM). The antioxidant activity under the versatile extraction conditions adopted in this study was conspicuously reduced, such that the % inhibition against 2,2- diphenyl-1-picrylhydrazyl (DPPH) followed an inverse, negative correlational trendline. Moreover, ascorbic acid content was not affected with mild to prolonged heating or microwave exposure, except tangibly with dPOM and mPOM. The total phenols content assessed showed no significant variations, as compared with control extracts. In a manner similar to ascorbic acid, total flavonoids were mildly reduced under varying conditions, an effect mimicked to a certain extent with anthocyanins. Assessment of extraction means as compared with WJE revealed sharp decrease in the antioxidant activity for dPOM and mPOM, significant increase in L-ascorbic acid, total phenol, and flavonoid contents for MEJ, dPOM, and mPOM, and mild decrease in anthocyanin contents for dPOM and mPOM. These results confirm the measurable antioxidant activities and contents of P. avium extracts under versatile conditions of mild exposure, an effect

  3. Studies on pathology, cytokine gene expression and molecular typing of Mycobacterium avium subsp. paratuberculosis of naturally occurring Johne's disease in bullocks.

    PubMed

    Narnaware, S D; Periasamy, S; Tripathi, B N

    2016-06-01

    Pathology of Johne's disease (JD) in bullocks (castrated, adult male cattle) is rarely studied. Here, we report the pathology and cytokine gene expression of naturally occurring JD in bullocks. The small intestine and mesenteric lymph nodes collected from 404 bullocks, aged between 5 and 10years, were examined for JD lesions and detection of Mycobacterium avium subsp. paratuberculosis (Map). A total of 8.7% bullocks exhibited JD lesions, which were classified into multibacillary-diffuse granulomatous (n=2), paucibacillary-focal granulomatous (n=18) and paucibacillary-diffuse lymphocytic (n=15) lesions. The tissue cytokine gene expression profiles in all three forms of lesions corroborated with different immuno-pathological processes of JD in bullocks. The molecular typing and gene sequencing identified Map isolates from bullocks as bison type. PMID:27234539

  4. [Disseminated Mycobacterium avium complex infections in AIDS. Apropos of 100 cases. Groupe d'Epidémiologie clinique du SIDA en Aquitaine].

    PubMed

    Lasseur, C; Maugein, J; Pellegrin, J L; Dupon, M; Ragnaud, J M; Morlat, P; Pellegrin, I; Constans, J; Monlun, E; Chene, G

    1995-01-01

    The improvement of survival of AIDS patients allowed the emergence of disseminated Mycobacterium avium Complex infections (D.MAC). Here we report the experience of the group of "Epidémiologie clinique du sida en Aquitaine (GECSA)" about 100 patients. There were no differences according to sex, age and route of acquisition of HIV. Clinical and biological characteristics of the infections were not specific. The mean TCD4+ lymphocytes count was 18/mm3. The diagnostic was generally established by systematic blood culture on Septi-Chek in patients with TCD4+ lymphocytes count below 75/mm3. The recommendations on therapy for D.MAC are to use regimen containing azithromycin or clarithromycin, ethambutol and one of the following drugs, rifabutin, clofazimine, amikacin, or ciprofloxacin. Rifabutin is recommended for prophylaxis in patients with lymphocytes TCD4+ count below 100/mm3.

  5. No evidence that wild red deer (Cervus elaphus) on the Iberian Peninsula are a reservoir of Mycobacterium avium subspecies paratuberculosis infection.

    PubMed

    Carta, T; Martin-Hernando, M P; Boadella, M; Fernández-de-Mera, I G; Balseiro, A; Sevilla, I A; Vicente, J; Maio, E; Vieira-Pinto, M; Alvarez, J; Pérez-de-la-Lastra, J M; Garrido, J; Gortazar, C

    2012-06-01

    The potential role of red deer (Cervus elaphus) as a reservoir of Mycobacterium avium subspecies paratuberculosis (MAP) infection is largely unknown. A total of 332 wild red deer were investigated using post-mortem examination, bacteriology and serology. Only three animals (1.12%) were found to have lesions on histopathological examination and no MAP bacteria were recovered on culture. The results suggest it is unlikely that wild red deer make a significant contribution to the maintenance of MAP infection in the region. The cross-reactivity of the ELISAs used indicates this diagnostic modality is ineffective in the detection of MAP infection in this species. The implications of these results for the control of this important pathogen in both livestock and wildlife are discussed.

  6. Detection of Mycobacterium avium subsp. paratuberculosis in intestinal and lymph node tissues of water buffaloes (Bubalus bubalis) by PCR and bacterial culture.

    PubMed

    Sivakumar, P; Tripathi, B N; Singh, Nem

    2005-07-01

    The efficacy of bacterial culture and IS900-specific polymerase chain reaction (PCR) was compared for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) from the intestinal and mesenteric lymph node tissues of water buffaloes (Bubalus bubalis) showing lesions of paratuberculosis (Johne's disease). Out of 20 (4.9%) animals showing histological lesions suggestive of paratuberculosis, 14 (70%) and 6 (30%) were positive in the PCR and bacterial culture, respectively. The results of this study suggested that PCR was more sensitive than bacterial culture in detection of subclinical paratuberculosis in water buffaloes. The bacterial concentration from large amount of tissues by differential and density gradient centrifugation method was found to facilitate the diagnosis by smear examination and PCR. The specificity of the PCR was confirmed by the product size and restriction digestion pattern of the amplicons. The sequence analysis of the amplified products (626bp of IS900 gene) from buffalo strain showed more than 97% homology with the published sequences.

  7. Mycobacterium Avium Complex (MAC)

    MedlinePlus

    ... MAC? The symptoms of MAC can include high fevers, chills, diarrhea , weight loss, stomach aches, fatigue, and anemia ( ... anemia. Many drug interactions. Rifampin (Rifampicin, Rifadin, Rimactane): fever, chills, muscle or bone pain; can turn urine, sweat, ...

  8. Development and application of quantitative polymerase chain reaction assay based on the ABI 7700 system (TaqMan) for detection and quantification of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Kim, Sung G; Shin, Sang J; Jacobson, Richard H; Miller, Loretta J; Harpending, Peter R; Stehman, Susan M; Rossiter, Christine A; Lein, Donald A

    2002-03-01

    Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The result of conventional PCR tests has been only qualitative, either positive or negative; it does not present any quantitative information about the number of the agents in the specimen. A quantitative PCR method (IS900 TaqMan) was developed to measure the number of M. a. paratuberculosis organisms present in field and clinical samples. The sensitivity of IS900 TaqMan was 1 colony-forming unit (CFU) for M. a. paratuberculosis ATCC 19698. The specificity of the method was determined by testing 14 mycobacterial species (M. abscessus, M. asiaticum, M. avium subsp. avium, M. bovis, M. fortuitum subsp. fortuitum, M. intracellulare, M. kansasii, M. marinum, M. phlei, M. scrofulaceum, M. simiae, M. smegmatis, M. terrae, and M. ulcerans) and 9 nonmycobacterial species (Borrelia burgdorferi, Chlamydia psittaci, Ehrlichia canis, E. equi, E. risticii, Escherichia coli, E. coli O157:H7, Streptococcus equi, and S. zooepidemicus). Even at high cell numbers (10(5) CFU/reaction), most of the organisms tested negative for the IS900 insertion element except M. marinum and M. scrofulaceum. This finding for M. scrofulaceum was consistent with previous reports that several M. scrofulaceum-like isolates were positive for IS900. Those isolates had 71-79% homology with M. a. paratuberculosis in the region of IS900. When used in conjunction with the new liquid medium-based ESP culture system II for bovine clinical fecal samples, IS900 TaqMan confirmed that the ESP II-positive samples contained 10(5)-10(6) CFU/ml of M. a. paratuberculosis. All of the 222 ESP II-positive and acid-fast bacilli-positive samples tested in this study were positive by IS900 TaqMan. IS900 TaqMan was also useful in the study of growth characteristics of 3 groups of M. a. paratuberculosis strains in bovine fecal samples

  9. Comparative Transcriptome Analysis of Genes Involved in Anthocyanin Biosynthesis in the Red and Yellow Fruits of Sweet Cherry (Prunus avium L.)

    PubMed Central

    Wei, Hairong; Chen, Xin; Zong, Xiaojuan; Shu, Huairui; Gao, Dongsheng; Liu, Qingzhong

    2015-01-01

    Background Fruit color is one of the most important economic traits of the sweet cherry (Prunus avium L.). The red coloration of sweet cherry fruit is mainly attributed to anthocyanins. However, limited information is available regarding the molecular mechanisms underlying anthocyanin biosynthesis and its regulation in sweet cherry. Methodology/Principal Findings In this study, a reference transcriptome of P. avium L. was sequenced and annotated to identify the transcriptional determinants of fruit color. Normalized cDNA libraries from red and yellow fruits were sequenced using the next-generation Illumina/Solexa sequencing platform and de novo assembly. Over 66 million high-quality reads were assembled into 43,128 unigenes using a combined assembly strategy. Then a total of 22,452 unigenes were compared to public databases using homology searches, and 20,095 of these unigenes were annotated in the Nr protein database. Furthermore, transcriptome differences between the four stages of fruit ripening were analyzed using Illumina digital gene expression (DGE) profiling. Biological pathway analysis revealed that 72 unigenes were involved in anthocyanin biosynthesis. The expression patterns of unigenes encoding phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavanone 3’-hydroxylase (F3’H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP glucose: flavonol 3-O-glucosyltransferase (UFGT) during fruit ripening differed between red and yellow fruit. In addition, we identified some transcription factor families (such as MYB, bHLH and WD40) that may control anthocyanin biosynthesis. We confirmed the altered expression levels of eighteen unigenes that encode anthocyanin biosynthetic enzymes and transcription factors using quantitative real-time PCR (qRT-PCR). Conclusions/Significance The obtained sweet cherry transcriptome and DGE profiling data

  10. Effect of feeding heat-treated colostrum on risk for infection with Mycobacterium avium ssp. paratuberculosis, milk production, and longevity in Holstein dairy cows.

    PubMed

    Godden, S M; Wells, S; Donahue, M; Stabel, J; Oakes, J M; Sreevatsan, S; Fetrow, J

    2015-08-01

    In summer 2007, a randomized controlled field trial was initiated on 6 large Midwest commercial dairy farms to investigate the effect of feeding heat-treated (HT) colostrum on transmission of Mycobacterium avium ssp. paratuberculosis (MAP) and on future milk production and longevity within the herd. On each farm, colostrum was collected daily from fresh cows, pooled, divided into 2 aliquots, and then 1 aliquot was heat-treated in a commercial batch pasteurizer at 60°C for 60min. A sample from each batch of colostrum was collected for PCR testing (MAP-positive vs. MAP-negative). Newborn heifer calves were removed from the dam within 30 to 60min of birth and systematically assigned to be fed 3.8 L of either fresh (FR; n=434) or heat-treated (HT; n=490) colostrum within 2h of birth. After reaching adulthood (>2 yr old), study animals were tested once annually for 3 yr (2010, 2011, 2012) for infection with MAP using serum ELISA and fecal culture. Lactation records describing milk production data and death or culling events were collected during the 3-yr testing period. Multivariable model logistic and linear regression was used to investigate the effect of feeding HT colostrum on risk for testing positive to MAP during the 3-yr testing period (positive/negative; logistic regression) and on first and second lactation milk yield (kg/cow; linear regression), respectively. Cox proportional hazards regression was used to investigate the effect of feeding HT colostrum on risk and time to removal from the herd. Fifteen percent of all study animals were fed PCR-positive colostrum. By the end of the 3-yr testing period, no difference was noted in the proportion of animals testing positive for MAP, with either serum ELISA or fecal culture, when comparing the HT group (10.5%) versus the FR group (8.1%). There was no effect of treatment on first- (HT=11.797kg; FR=11,671kg) or second-lactation (HT=11,013kg; FR=11,235kg) milk production. The proportion of cows leaving the herd by

  11. Long-term detection of Mycobacterium avium subspecies paratuberculosis in individual and bulk tank milk from a dairy herd with a low prevalence of Johne's disease.

    PubMed

    Khol, J L; Wassertheurer, M; Sodoma, E; Revilla-Fernández, S; Damoser, J; Osterreicher, E; Dünser, M; Kleb, U; Baumgartner, W

    2013-06-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease (JD) in ruminants and is shed into the milk of infected cows, which contributes to the controversial discussion about a possible link between MAP and Crohn's disease in humans. The aim of the study was to investigate the risk for the entry of MAP in the food chain via milk from dairy farms with subclinical JD. Therefore, the occurrence of MAP in the milk of a dairy herd with a low prevalence of JD was studied in single and bulk tank milk samples over a period of 23 mo and compared with MAP shedding into feces. Milk, fecal, and blood samples were taken from all cows older than 1.5 yr of age at the beginning and the end of the trial and analyzed for MAP or specific antibodies. In addition, 63 cows (33 MAP infected and 30 MAP noninfected) were selected for monthly sampling. Raw and pasteurized bulk tank milk samples were collected on a monthly basis. The milk samples were tested for MAP by real-time quantitative PCR (qPCR), and the fecal samples were tested for bacterial shedding by qPCR or solid culture. Based on the results of the herd investigations, the prevalence of cows shedding MAP was around 5%; no cases of clinical JD were observed during the study period. The results of the ELISA showed high variation, with 2.1 to 5.1% positive milk samples and 14.9 to 18.8% ELISA-positive blood samples. Monthly milk sampling revealed low levels of MAP shedding into the individual milk samples of both MAP-infected and noninfected cows, with only 13 cows shedding the bacterium into milk during the study period. Mycobacterium avium ssp. paratuberculosis was not detected by qPCR in any raw or pasteurized bulk tank milk sample throughout the study. A significant positive association could be found between MAP shedding into milk and feces. From the results of the present study, it can be concluded that MAP is only shed via milk in a small proportion of cows with subclinical JD for a limited period of time and

  12. Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages.

    PubMed

    Everman, Jamie L; Bermudez, Luiz E

    2015-01-01

    Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection.

  13. Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages is not affected by host infection status but depends on the infecting bacterial genotype.

    PubMed

    Gollnick, Nicole S; Mitchell, Rebecca M; Baumgart, Martin; Janagama, Harish K; Sreevatsan, Srinand; Schukken, Ynte H

    2007-12-15

    In this study we investigated the ability of different Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains to survive in bovine monocyte-derived macrophages (MDMs) of cows naturally infected with M. paratuberculosis and control cows. We tested the hypotheses that infection status of cows affects macrophage killing ability and that survival of M. paratuberculosis in macrophages is dependent on the strain. Peripheral blood mononuclear cells (PBMC) were obtained from Johne's disease-positive (n=3) and age and stage of lactation matched Johne's disease-negative (n=3) multiparious cows. Following differentiation, MDMs were challenged in vitro with four M. paratuberculosis strains of different host specificity (cattle and sheep). Two hours and 2, 4, and 7 days after infection, ingestion, and intracellular survival of M. paratuberculosis strains were determined by fluorescence microscopy. There was no effect of the origin of MDMs (Johne's disease-positive or control animals) on phagocytosis, survival of bacteria, or macrophage survival. In contrast, important strain differences were observed. These findings suggest that some M. paratuberculosis strains interfere more successfully than others with the ability of macrophages to kill intracellular pathogens which may make it important to include strain typing when designing control programs.

  14. Application of cattle slurry containing Mycobacterium avium subsp. paratuberculosis (MAP) to grassland soil and its effect on the relationship between MAP and free-living amoeba.

    PubMed

    Salgado, M; Alfaro, M; Salazar, F; Badilla, X; Troncoso, E; Zambrano, A; González, M; Mitchell, R M; Collins, M T

    2015-01-30

    Slurry from dairy farms is commonly used to fertilize crops and pastures. This mixture of manure, urine and water can harbor multiple microbial pathogens among which Mycobacterium avium subsp. paratuberculosis (MAP) is a major concern. Persistence of MAP in soil and infection of soil Acanthamoeba was evaluated by culture, real-time IS900 PCR, and by staining of amoeba with acid-fast and vital stains comparing soils irrigated with MAP-spiked or control dairy farm slurry. MAP DNA was detected in soil for the 8 month study duration. MAP was detected by PCR from more soil samples for plots receiving MAP-spiked slurry (n=61/66) than from soils receiving control slurry (n=10/66 samples). Vital stains verified that intracellular MAP in amoeba was viable. More MAP was found in amoeba at the end of the study than immediately after slurry application. There was no relationship between MAP presence in soil and in amoeba over time. Infection of amoeba by MAP provides a protected niche for the persistence and even possibly the replication of MAP in soils. As others have suggested, MAP-infected amoeba may act like a "Trojan horse" providing a means for persistence in soils and potentially a source of infection for grazing animals.

  15. Efficacy of 'indigenous vaccine' using native 'Indian bison type' genotype of Mycobacterium avium subspecies paratuberculosis for the control of clinical Johne's disease in an organized goat herd.

    PubMed

    Singh, K; Chandel, B S; Chauhan, H C; Dadawala, A; Singh, S V; Singh, P K

    2013-06-01

    Therapeutic efficacy of a new 'Indigenous vaccine' prepared from native highly pathogenic 'Indian Bison Type' genotype of Mycobacterium avium subspecies paratuberculosis (MAP) of goat origin has been evaluated with respect to control of clinical Johne's disease in naturally infected Mehsana breed of goat in North Gujarat. Fifty goats from Sheep and Goats Research Station, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, were randomly divided into 2 groups viz.,'Vaccinated'(n = 35) and 'Control'(n = 15). After vaccination, goats were monitored for physical condition, morbidity, mortality, body weights, shedding of MAP in feces, internal condition, gross lesions and humoral immune responses up to 120 days (at each interval of 30 days). At the end of 120 days trial, there was marked overall improvement in physical condition and body weights of vaccinated goats as compared to 'Control' goats. Vaccinated goats gained significantly (P < 0.05) higher body weights, hardly exhibited any lesions characteristic of JD, had significantly higher (P < 0.01) antibody titers and shedding of MAP was significantly (P < 0.01) reduced. Few of the vaccinated goats were positive for MAP DNA in faecal PCR and blood PCR before vaccination. However, all were found as negative at 120 days post vaccination (DPV). Overall vaccine exhibited effective in restriction of MAP infection and significant improvement in production parameters and reduction in mortality and morbidity due to JD. The trial in the herd will be continued.

  16. Modeling the occurrence of Mycobacterium avium subsp. paratuberculosis in bulk raw milk and the impact of management options for exposure mitigation.

    PubMed

    Boulais, Christophe; Wacker, Ron; Augustin, Jean-Christophe; Cheikh, Mohamed Hedi Ben; Peladan, Fabrice

    2011-07-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (Johne's disease) in cattle and other farm ruminants. The potential role of MAP in Crohn's disease in humans and the contribution of dairy products to human exposure to MAP continue to be the subject of scientific debate. The occurrence of MAP in bulk raw milk from dairy herds was assessed using a stochastic modeling approach. Raw milk samples were collected from bulk tanks in dairy plants and tested for the presence of MAP. Results from this analytical screening were used in a Bayesian network to update the model prediction. Of the 83 raw milk samples tested, 4 were positive for MAP by culture and PCR. We estimated that the level of MAP in bulk tanks ranged from 0 CFU/ml for the 2.5th percentile to 65 CFU/ml for the 97.5th percentile, with 95% credibility intervals of [0, 0] and [16, 326], respectively. The model was used to evaluate the effect of measures aimed at reducing the occurrence of MAP in raw milk. Reducing the prevalence of paratuberculosis has less of an effect on the occurrence of MAP in bulk raw milk than does managing clinically infected animals through good farming practices.

  17. Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages

    PubMed Central

    Everman, Jamie L.; Bermudez, Luiz E.

    2015-01-01

    Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection. PMID:26301206

  18. Descriptive analysis of the prevalence and the molecular epidemiology of Mycobacterium avium complex-infected pigs that were slaughtered on the main island of Okinawa.

    PubMed

    Hibiya, K; Kazumi, Y; Nishiuchi, Y; Sugawara, I; Miyagi, K; Oda, Y; Oda, E; Fujita, J

    2010-09-01

    Recent genetic studies have revealed that several epidemiological factors affect Mycobacterium avium complex (MAC) infection in pig populations. However, mechanisms underlying the spread of MAC infection among hog farms have not been clarified. In consideration of this situation, we cross-sectionally investigated the mechanisms underlying the spread of MAC on the island of Okinawa. Pigs slaughtered (n=706,763) and 331 hog farms on Okinawa were surveyed during the years 2002-2004. Two outbreaks of MAC infection were occurred in several farms during survey period. Bacteria were isolated from randomly selected pigs and genotype of isolates was determined by using genetic finger printing methods with the insertion sequence (IS) 1245 restriction fragment length polymorphism (RFLP). Most isolates had large numbers of IS1245 copies, while strains with low copy numbers of IS1245 and isolates without IS1245 were seen in few farms. MACs strains were repeatedly isolated from pigs of the affected farms during the survey period. Those farms with an identical pig rearing systems showed synchronic changes in the prevalence of MAC infection. An industrial farm without an outbreak had an independent pig flow, but maintained distinct MAC strains. Multivariate analysis did not reveal independent factors for the prevalence of the MAC infection. These findings suggest that there were three clusters distinguished genetically in the main island of Okinawa, which were potentially spread by common pig flow. However, the outbreaks occurred because of unspecified conditions on each farm environment.

  19. Evaluation of age-dependent susceptibility in calves infected with two doses of Mycobacterium avium subspecies paratuberculosis using pathology and tissue culture

    PubMed Central

    2013-01-01

    The longstanding assumption that calves of more than 6 months of age are more resistant to Mycobacterium avium subspecies paratuberculosis (MAP) infection has recently been challenged. In order to elucidate this, a challenge experiment was performed to evaluate age- and dose-dependent susceptibility to MAP infection in dairy calves. Fifty-six calves from MAP-negative dams were randomly allocated to 10 MAP challenge groups (5 animals per group) and a negative control group (6 calves). Calves were inoculated orally on 2 consecutive days at 5 ages: 2 weeks and 3, 6, 9 or 12 months. Within each age group 5 calves received either a high – or low – dose of 5 × 109 CFU or 5 × 107 CFU, respectively. All calves were euthanized at 17 months of age. Macroscopic and histological lesions were assessed and bacterial culture was done on numerous tissue samples. Within all 5 age groups, calves were successfully infected with either dose of MAP. Calves inoculated at < 6 months usually had more culture-positive tissue locations and higher histological lesion scores. Furthermore, those infected with a high dose had more severe scores for histologic and macroscopic lesions as well as more culture-positive tissue locations compared to calves infected with a low dose. In conclusion, calves to 1 year of age were susceptible to MAP infection and a high infection dose produced more severe lesions than a low dose. PMID:24099491

  20. Sensitivity of solid culture, broth culture, and real-time PCR assays for milk and colostrum samples from Mycobacterium avium ssp. paratuberculosis-infectious dairy cows.

    PubMed

    Laurin, Emilie; McKenna, Shawn; Chaffer, Marcelo; Keefe, Greg

    2015-12-01

    Mycobacterium avium ssp. paratuberculosis (MAP) can be shed in feces, milk, and colostrum. The goal of this study was to assess assays that detect MAP in these sample types, including effects of lactation stage or season. Understanding the performance of these assays could improve how they are used, limiting the risk of infection to calves. Forty-six previously confirmed MAP-positive cows from 7 Atlantic Canadian dairy farms were identified for colostrum sampling and monthly sampling of milk and feces over a 12-mo period. Samples were assayed for MAP using solid culture, broth culture, and direct real-time PCR (qPCR). Across assay types, test sensitivity when applied to milk samples averaged 25% of that when applied to fecal samples. For colostrum samples, sensitivity depended on assay type, with sensitivity of qPCR being approximately 46% of that in feces. Across sample types, sensitivity of qPCR was higher than that of the other assays. Sensitivity of qPCR, when applied to milk samples, was significantly higher in summer than in other seasons. Summer was also the season with highest agreement between milk and fecal samples collected within the same month. Our results suggest that qPCR would detect more cows shedding MAP in their milk and colostrum than solid or broth culture assays, particularly during the summer, thus providing better management information to limit exposure of calves to this infectious organism. PMID:26476944

  1. A comparison of cell mediators and serum cytokines transcript expression between male and female mice infected with Mycobacterium avium subspecies paratuberculosis and/or consuming probiotics.

    PubMed

    Karunasena, Enusha; McMahon, Kevin W; Kurkure, Paresh C; Brashears, Mindy M

    2014-11-01

    The gut immune system is complex, and dysregulation leads to a number of disorders including inflammatory bowel syndrome and (in livestock) Johne's disease. Previous work has demonstrated that males and females respond differently to treatment with pathologic and probiotic microorganisms, suggesting that a 'one-size-fits-all' approach to treat GIT inflammation may be inadequate. While we had observed significant differences between males and females in terms of cytokine production, it remains unclear how these changes occur. To better understand the mechanisms, transcript expression of genes important to gut immunoregulation were monitored from male and female BALB/c mice consuming the probiotic Lactobacillus animalis (1 × 10(6) CFU g(-1) ) and infected with the gut pathogen, Mycobacterium avium subspecies paratuberculosis (1 × 10(7) CFU). Expression of transcripts analyzed included those important to the immune system, intestinal cell differentiation, and/or regulation. Males generally displayed increased expression of Th 2 and B-cell mediators, and females showed repressed cytokine expression after MAP infection (IL-6, TNF-α, IL-1 among others). Additionally, regulation of pro-inflammatory mediators in female mice consuming probiotics suggests females responded positively to L. animalis when compared to males. Therefore, we speculate that studying mechanistic changes associated with sex and immunoregulation in gastrointestinal tissues could further elucidate host response to microorganisms.

  2. Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages.

    PubMed

    Everman, Jamie L; Bermudez, Luiz E

    2015-01-01

    Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection. PMID:26301206

  3. Evaluation of "Indigenous Absorbed ELISA Kit" for the Estimation of Seroprevalence of Mycobacterium avium Subspecies paratuberculosis Antibodies in Human Beings in North India.

    PubMed

    Singh, A V; Singh, S V; Verma, D K; Yadav, R; Singh, P K; Sohal, J S

    2011-01-01

    In present pilot study aimed to estimate, presence of Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in the human serum samples originating from North India using "Indigenous absorbed ELISA kit" (ELISA kit). The phase I, "ELISA kit" was optimized using protoplasmic antigen from native isolate of MAP "Indian Bison type" recovered from the biopsies of Crohn's disease patients. The phase II, sensitivity and specificity of the kit were estimated as 40.0 and 83.3%, respectively, when evaluated in 40 human serum samples (5 Crohn's disease and 22 ulcerative colitis patients and 13 healthy human subjects) with defined MAP status with respect to stool culture. Seroprevalence of MAP antibodies was higher in CD patients (80.0%) as compared to ulcerative colitis patients (4.5%) and normal human subjects (15.3%). The phase III, seroprevalence of MAP antibodies was estimated as 23.4%, on the basis of the screening of 452 human serum samples (without history) from different geographical regions of North India. Region-wise, 34.0, 33.3, 32.8, 25.0, 23.0, 17.7, and 12.5% samples were positive from the states of Punjab, Uttarakhand, New Delhi, Himachal Pradesh, Haryana, Uttar Pradesh and Jammu and Kashmir, respectively. Study reported moderately higher presence of MAP antibodies in human population, which necessitates programs to reduce the bioburden of MAP in the environment and in animal population.

  4. Soluble BAFF Level Is Not Correlated to Mycobacterium avium Subspecies Paratuberculosis Antibodies and Increases After Interferon-β Therapy in Multiple Sclerosis Patients.

    PubMed

    Mameli, Giuseppe; Cossu, Davide; Caggiu, Elisa; Arru, Giannina; Niegowska, Magdalena; Cocco, Eleonora; Frau, Jessica; Marrosu, Maria Giovanna; Sechi, Leonardo A

    2016-09-01

    B cells are being recognized as one of the major players in the pathogenesis of multiple sclerosis (MS). The B cell activating factor (BAFF) system plays an essential role in B cell homeostasis and function in the periphery. Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to MS in Sardinia. Antibodies against a MAP surface protein, MAP_2694, have been found significantly associated to MS patients, and this response was modified by interferon-β therapy. Increased BAFF levels following IFN-β therapy have been also described in MS patients. In this study, we evaluated whether soluble BAFF levels are comparable in men and women affected by MS and performed a correlation of the reported BAFF increase in MS patients under IFN-β therapy with changes of humoral response against MAP_2694. For these reasons, we investigated 44 MS patients before and after IFN-β therapy. A significant difference of BAFF levels was found between men and women with MS; moreover, we confirmed that IFN-β therapy strongly induces BAFF serum levels, but this was not related to the modification of immunological response against MAP_2694. In conclusion, our study highlights that IFN-β therapy induces the potent B cell survival factor BAFF without alterations of the humoral immune response against MAP.

  5. Type 1 Diabetes at-risk children highly recognize Mycobacterium avium subspecies paratuberculosis epitopes homologous to human Znt8 and Proinsulin

    PubMed Central

    Niegowska, Magdalena; Rapini, Novella; Piccinini, Simona; Mameli, Giuseppe; Caggiu, Elisa; Manca Bitti, Maria Luisa; Sechi, Leonardo A.

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to T1D as a putative environmental agent triggering or accelerating the disease in Sardinian and Italian populations. Our aim was to investigate the role of MAP in T1D development by evaluating levels of antibodies directed against MAP epitopes and their human homologs corresponding to ZnT8 and proinsulin (PI) in 54 T1D at-risk children from mainland Italy and 42 healthy controls (HCs). A higher prevalence was detected for MAP/ZnT8 pairs (62,96% T1D vs. 7,14% HCs; p < 0.0001) compared to MAP/PI epitopes (22,22% T1D vs. 9,52% HCs) and decreasing trends were observed upon time-point analyses for most peptides. Similarly, classical ZnT8 Abs and GADA decreased in a time-dependent manner, whereas IAA titers increased by 12%. Responses in 0–9 year-old children were stronger than in 10–18 age group (75% vs. 69,1%; p < 0.04). Younger age, female sex and concomitant autoimmune disorders contributed to a stronger seroreactivity suggesting a possible implication of MAP in multiple autoimmune syndrome. Cross-reactivity of the homologous epitopes was reflected by a high correlation coefficient (r2 > 0.8) and a pairwise overlap of positivity (>83% for MAP/ZnT8). PMID:26923214

  6. Evidence of a pro-apoptotic effect of specific antibodies in a bovine macrophage model of infection with Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Jolly, Ana; Lompardía, Silvina; Hajos, Silvia E; Mundo, Silvia L

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-α secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-α. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention. PMID:26827838

  7. Unequal allelic frequencies at the self-incompatibility locus within local populations of Prunus avium L.: an effect of population structure?

    PubMed

    Stoeckel, S; Castric, V; Mariette, S; Vekemans, X

    2008-05-01

    In this paper, we investigated the genetic structure and distribution of allelic frequencies at the gametophytic self-incompatibility locus in three populations of Prunus avium L. In line with theoretical predictions under balancing selection, genetic structure at the self-incompatibility locus was almost three times lower than at seven unlinked microsatellites. Furthermore, we found that S-allele frequencies in wild cherry populations departed significantly from the expected isoplethic distribution towards which balancing selection is expected to drive allelic frequencies (i.e. identical frequency equal to the inverse of the number of alleles in the population). To assess whether this departure could be caused either by drift alone or by population structure, we used numerical simulations to compare our observations with allelic frequency distributions expected : (1) within a single deme from a subdivided population with various levels of differentiation; and (2) within a finite panmictic population with identical allelic diversity. We also investigated the effects of sample size and degree of population structure on tests of departure from isoplethic equilibrium. Overall, our results showed that the observed allele frequency distributions were consistent with a model of subdivided population with demes linked by moderate migration rate.

  8. [Clinical features of the patients with "secondary infection" of Mycobacterium avium complex--Radiographic pattern of progressions in the patients with and without underlying pulmonary conditions].

    PubMed

    Tanaka, E; Amitani, R; Kuze, F

    1993-01-01

    We reviewed the radiographic features of 42 patients with Mycobacterium avium complex (MAC) infection. Six cases were definite "primary", 20 were "secondary", and 15 were indeterminate (probably "primary"). In the definite and probable "primary" cases, and some of the "secondary" cases, pulmonary lesions slowly progressed following a common and characteristic sequence from a cluster of small nodules or fibro-productive nodules to those with subpleural thickening, or with thickening of the draining bronchi, or with both subpleural and bronchial thickening, and to cystic bronchiectatic changes associated with collapse of the segment or the lobe, in the final stage. Cases of airspace pneumonia appeared rarely. In these cases, neither apical-subapical region prevalence, pleural effusion, nor mediastinal lymphadenopathy were found. On the contrary, in five cases of "secondary" infection, MAC lesions located at the same place with the predisposing lung disease and did not progress during more than five years of observation. In the progressive cases of "secondary" infection, however, the appearance of new lesions and the progressions thereafter showed the same pattern as in "primary" infection. These features would suggest that MAC infection would occur and progress slowly among patients who had unknown pathogenetical factor, whether they had underlying predisposing lung conditions or not.

  9. Potential role of real-time PCR for detecting Mycobacterium avium subsp. paratuberculosis in chronically diseased milking cows: a case control study.

    PubMed

    Alajmi, Ahmad; Klein, Günter; Greiner, Matthias; Grabowski, Nils Th; Fohler, Svenja; Campe, Amely; Scheu, Theresa; Hoedemaker, Martina; Abdulmawjood, Amir

    2016-01-01

    Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) is economically important to dairy operations. In the present study, a real-time PCR kit (certified for use in Germany) was used to detect MAP in bovine fecal and milk samples within a case control study examining different factors for their association with chronic disease in Northern Germany.The aim of this study was to describe the suitability of the MAP real-time PCR kit to detect MAP in feces and milk of chronically diseased dairy cows. For MAP detection a total of 928 fecal, 922 composite foremilk, and 92 bulk milk samples obtained from 58 cases and 35 control dairy herds were investigated. The real-time PCR showed MAP positive results for 11 (18.96%) and 6 (17.14%) of the case and control herds, respectively. All bulk milk samples were MAP negative. The results of fecal and milk samples were moderately correlated (kappa = 0.27). These data indicate that real-time PCR results have diagnostic value for diagnosing MAP positive animals, and that fecal samples are more suitable than milk samples for assessing the reasons for chronic disease on dairy farms. PMID:27529992

  10. Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis.

    PubMed

    Schinköthe, Jan; Köhler, Heike; Liebler-Tenorio, Elisabeth M

    2016-08-01

    Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4(+), CD8(+), γδ, B lymphocytes and plasma, CD25(+), CD68(+), MHC-II(+), Ki67(+) and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4(+) T cells, but only few epitheloid macrophages were observed in severely sick goats at 2-3mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research. PMID:27477506

  11. Mycobacterium avium Subspecies paratuberculosis and Bovine Leukemia Virus Seroprevalence and Associated Risk Factors in Commercial Dairy and Beef Cattle in Northern and Northeastern China

    PubMed Central

    Sun, Wu-Wen; Lv, Wen-Fa; Cong, Wei; Meng, Qing-Feng; Wang, Chun-Feng; Shan, Xiao-Feng; Qian, Ai-Dong

    2015-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) and bovine leukemia virus (BLV) are important pathogens, commonly responsible for economical loss to cattle farms all over the world, yet their epidemiology in commercial dairy and beef cattle in China is still unknown. Thus, from September 2013 to December 2014, a large-scale seroprevalence study was conducted to determine the seroprevalence and identify herd-level risk factors associated with MAP and BLV infection. The source sample was 3674 cattle from 113 herds in northern and northeastern China. Antibodies against MAP and BLV were detected using ELISA tests. At animal-level, the seroprevalence of antibodies against MAP and BLV was 11.79% (433/3674) and 18.29% (672/3674), respectively. At herd-level, the seroprevalence of antibodies against MAP and BLV was 20.35% and 21.24% (24/113), respectively. Herd size was identified to be associated with MAP infection while herd size and presence of cattle introduced from other farms were significantly associated with BLV infection. Further research is needed to confirm these findings and improve the knowledge of the epidemiology of these two pathogens in these regions and elsewhere in China. PMID:26504798

  12. Short communication: Herd-level prevalence of Mycobacterium avium ssp. paratuberculosis is not associated with participation in a voluntary Alberta Johne's disease control program.

    PubMed

    Ritter, C; Wolf, R; Adams, C L; Kelton, D F; Pickel, C; Mason, S; Orsel, K; De Buck, J; Barkema, H W

    2016-03-01

    Johne's disease (JD) control programs for dairy farms have the general objective of reducing both cow- and herd-level prevalence of Mycobacterium avium ssp. paratuberculosis (MAP). An important aspect of many programs is herd testing for MAP to determine the infection status of participating farms. However, it is uncertain whether MAP herd-level prevalence on farms voluntarily participating in a JD control program is different from that on nonparticipating farms. Therefore, the aim was to compare MAP infection status of participants and nonparticipants in the Alberta Johne's Disease Initiative (AJDI), a voluntary JD control program initiated in 2010 in Alberta, Canada. Between September 2012 and August 2013, environmental fecal samples were collected from 93 randomly selected farms not enrolled in the AJDI. Additionally, 81 farms that initially enrolled in the AJDI during the same time interval were also sampled. Samples were collected from 6 defined locations on each farm and cultured for MAP. Results were confirmed using conventional IS900 PCR and F 0285 quantitative PCR. Overall, 51% of participating and 51% of nonparticipating farms were identified as being MAP-infected. Furthermore, based on multivariable logistic regression, the number of MAP-positive samples was not associated with AJDI participation (taking herd size into account as a potentially modifying or confounding variable). In conclusion, there was no indication that voluntary participation in the AJDI was associated with herd-level MAP prevalence.

  13. Transcriptional dynamics of the developing sweet cherry (Prunus avium L.) fruit: sequencing, annotation and expression profiling of exocarp-associated genes.

    PubMed

    Alkio, Merianne; Jonas, Uwe; Declercq, Myriam; Van Nocker, Steven; Knoche, Moritz

    2014-01-01

    The exocarp, or skin, of fleshy fruit is a specialized tissue that protects the fruit, attracts seed dispersing fruit eaters, and has large economical relevance for fruit quality. Development of the exocarp involves regulated activities of many genes. This research analyzed global gene expression in the exocarp of developing sweet cherry (Prunus avium L., 'Regina'), a fruit crop species with little public genomic resources. A catalog of transcript models (contigs) representing expressed genes was constructed from de novo assembled short complementary DNA (cDNA) sequences generated from developing fruit between flowering and maturity at 14 time points. Expression levels in each sample were estimated for 34 695 contigs from numbers of reads mapping to each contig. Contigs were annotated functionally based on BLAST, gene ontology and InterProScan analyses. Coregulated genes were detected using partitional clustering of expression patterns. The results are discussed with emphasis on genes putatively involved in cuticle deposition, cell wall metabolism and sugar transport. The high temporal resolution of the expression patterns presented here reveals finely tuned developmental specialization of individual members of gene families. Moreover, the de novo assembled sweet cherry fruit transcriptome with 7760 full-length protein coding sequences and over 20 000 other, annotated cDNA sequences together with their developmental expression patterns is expected to accelerate molecular research on this important tree fruit crop. PMID:26504533

  14. Short communication: Herd-level prevalence of Mycobacterium avium ssp. paratuberculosis is not associated with participation in a voluntary Alberta Johne's disease control program.

    PubMed

    Ritter, C; Wolf, R; Adams, C L; Kelton, D F; Pickel, C; Mason, S; Orsel, K; De Buck, J; Barkema, H W

    2016-03-01

    Johne's disease (JD) control programs for dairy farms have the general objective of reducing both cow- and herd-level prevalence of Mycobacterium avium ssp. paratuberculosis (MAP). An important aspect of many programs is herd testing for MAP to determine the infection status of participating farms. However, it is uncertain whether MAP herd-level prevalence on farms voluntarily participating in a JD control program is different from that on nonparticipating farms. Therefore, the aim was to compare MAP infection status of participants and nonparticipants in the Alberta Johne's Disease Initiative (AJDI), a voluntary JD control program initiated in 2010 in Alberta, Canada. Between September 2012 and August 2013, environmental fecal samples were collected from 93 randomly selected farms not enrolled in the AJDI. Additionally, 81 farms that initially enrolled in the AJDI during the same time interval were also sampled. Samples were collected from 6 defined locations on each farm and cultured for MAP. Results were confirmed using conventional IS900 PCR and F 0285 quantitative PCR. Overall, 51% of participating and 51% of nonparticipating farms were identified as being MAP-infected. Furthermore, based on multivariable logistic regression, the number of MAP-positive samples was not associated with AJDI participation (taking herd size into account as a potentially modifying or confounding variable). In conclusion, there was no indication that voluntary participation in the AJDI was associated with herd-level MAP prevalence. PMID:26778309

  15. Within-herd prevalence thresholds for the detection of Mycobacterium avium subspecies paratuberculosis-positive dairy herds using boot swabs and liquid manure samples.

    PubMed

    Donat, K; Hahn, N; Eisenberg, T; Schlez, K; Köhler, H; Wolter, W; Rohde, M; Pützschel, R; Rösler, U; Failing, K; Zschöck, P M

    2016-01-01

    The control of Johne's disease requires the identification of Mycobacterium