Previous work in small animal laboratory models of tuberculosis have shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacille Calmette-Guerin (BCG) to prime and Modified Vaccinia Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad8...
Vaccination of cattle with a CpG oligodeoxynucleotide-formulated mycobacterial protein vaccine and Mycobacterium bovis BCG induces levels of protection against bovine tuberculosis superior to those induced by vaccination with BCG alone.
Wedlock, D Neil; Denis, Michel; Skinner, Margot A; Koach, Jessica; de Lisle, Geoffrey W; Vordermeier, H Martin; Hewinson, R Glyn; van Drunen Littel-van den Hurk, Sylvia; Babiuk, Lorne A; Hecker, Rolf; Buddle, Bryce M
The development of a subunit protein vaccine for bovine tuberculosis which could be used either in combination with Mycobacterium bovis BCG (to improve the efficacy of that vaccine) or alone would offer significant advantages over currently available strategies. A study was conducted with cattle to determine the protective efficacy of a strategy based on concurrent immunization with an M. bovis culture filtrate (CFP) vaccine and BCG compared to vaccination with either vaccine alone. One group of calves (10 animals per group) was vaccinated subcutaneously with CFP formulated with Emulsigen and combined with a CpG oligodeoxynucleotide (ODN). A second group was vaccinated with both the CFP vaccine and BCG injected at adjacent sites (CFP-BCG). One further group was vaccinated subcutaneously with BCG, while another group served as nonvaccinated control animals. Vaccination with CFP-BCG induced levels of antigen-specific gamma interferon (IFN-gamma) and interleukin-2 (IL-2) in whole-blood cultures that were higher than those induced by vaccination with BCG alone. The combination of CFP and BCG did not enhance the production of antibodies to M. bovis CFP compared to vaccination with CFP alone. Vaccination with CFP alone led to delayed antigen-specific IFN-gamma and IL-2 responses. Vaccination with CFP-BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared with the nonvaccinated group. In contrast, vaccination with BCG alone induced a significant enhancement of protection in only one parameter, while CFP alone induced no protection. These results suggest that a combination of a CpG ODN-formulated protein vaccine and BCG offers better protection against bovine tuberculosis than does BCG alone.
Netea, Mihai G; van Crevel, Reinout
The Bacille Calmette-Guerin (BCG) vaccine is the only vaccine proved to be effective against tuberculosis and it remains the most commonly used vaccine worldwide. In addition to its effects on mycobacterial diseases, an increasing body of epidemiological evidence accumulated since its introduction in 1921 shows that BCG also exerts beneficial non-specific effects ranging from protection against non-mycobacterial diseases, decreased incidence of allergic diseases, and treatment of certain malignancies. The biological substrate of these effects is mediated partly by heterologous effects on adaptive immunity, but also on the potentiation of innate immune responses through epigenetic mechanisms, a process termed 'trained immunity'. The process of trained immunity may also play a role in the beneficial effects of BCG against tuberculosis and Mycobacterium tuberculosis infection, and this could have important consequences for our quest for improving vaccination strategies. Copyright © 2014 Elsevier Ltd. All rights reserved.
Singhal, Amit; Mathys, Vanessa; Kiass, Mehdi; Creusy, Colette; Delaire, Baptiste; Aliouat, El Moukhtar; Dartois, Véronique; Kaplan, Gilla; Bifani, Pablo
Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccination in this model. Upon Mtb challenge, BCG vaccinated rats controlled growth of the bacilli earlier than unvaccinated rats. Histopathology analysis of infected lungs demonstrated a reduced number of granulomatous lesions and lower parenchymal inflammation in vaccinated animals. Vaccine-mediated protection correlated with the rapid accumulation of antigen specific CD4+ and CD8+ T cells in the infected lungs. Immunohistochemistry further revealed higher number of CD8+ cells in the pulmonary granulomas of vaccinated animals. Evaluation of pulmonary immune responses in vaccinated and Mtb infected rats by real time PCR at day 15 post-challenge showed reduced expression of genes responsible for negative regulation of Th1 immune responses. Thus, early protection observed in BCG vaccinated rats correlated with a similarly timed shift of immunity towards the Th1 type response. Our data support the importance of (i) the Th1-Th2 balance in the control of mycobacterial infection and (ii) the value of the Wistar rats in understanding the biology of TB. PMID:22162757
Nandakumar, Subhadra; Kannanganat, Sunil; Posey, James E; Amara, Rama Rao; Sable, Suraj B
Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live attenuated vaccine. However, the correlates of protection and waning of its immunity against tuberculosis is poorly understood. In this study, we correlated the longitudinal changes in the magnitude and functional quality of CD4(+) and CD8(+) T-cell response over a period of two years after mucosal or parenteral BCG vaccination with the strength of protection against Mycobacterium tuberculosis in mice. The BCG vaccination-induced CD4(+) and CD8(+) T cells exhibited comparable response kinetics but distinct functional attributes in-terms of IFN-γ, IL-2 and TNF-α co-production and CD62L memory marker expression. Despite a near life-long BCG persistence and the induction of enduring CD4(+) T-cell responses characterized by IFN-γ and/or TNF-α production with comparable protection, the protective efficacy waned regardless of the route of vaccination. The progressive decline in the multifactorial functional abilities of CD4(+) and CD8(+) T cells in-terms of type-1 cytokine production, proliferation and cytolytic potential corresponded with the waning of protection against M. tuberculosis infection. In addition, simultaneous increase in the dysfunctional and terminally-differentiated T cells expressing CTLA-4, KLRG-1 and IL-10 during the contraction phase of BCG-induced response coincided with the loss of protection. Our results question the empirical development of BCG-booster vaccines and emphasize the pursuit of strategies that maintain superior T-cell functional capacity. Furthermore, our results underscore the importance of understanding the comprehensive functional dynamics of antigen-specific T-cell responses in addition to cytokine polyfunctionality in BCG-vaccinated hosts while optimizing novel vaccination strategies against tuberculosis.
Kaveh, Daryan A; Garcia-Pelayo, M Carmen; Webb, Paul R; Wooff, Esen E; Bachy, Véronique S; Hogarth, Philip J
Boosting BCG using heterologous prime-boost represents a promising strategy for improved tuberculosis (TB) vaccines, and adenovirus (Ad) delivery is established as an efficacious boosting vehicle. Although studies demonstrate that intranasal administration of Ad boost to BCG offers optimal protection, this is not currently possible in cattle. Using Ad vaccine expressing the mycobacterial antigen TB10.4 (BCG/Ad-TB10.4), we demonstrate, parenteral boost of BCG immunised mice to induce specific CD8(+) IFN-γ producing T cells via synergistic priming of new epitopes. This induces significant improvement in pulmonary protection against Mycobacterium bovis over that provided by BCG when assessed in a standard 4week challenge model. However, in a stringent, year-long survival study, BCG/Ad-TB10.4 did not improve outcome over BCG, which we suggest may be due to the lack of additional memory cells (IL-2(+)) induced by boosting. These data indicate BCG-prime/parenteral-Ad-TB10.4-boost to be a promising candidate, but also highlight the need for further understanding of the mechanisms of T cell priming and associated memory using Ad delivery systems. That we were able to generate significant improvement in pulmonary protection above BCG with parenteral, rather than mucosal administration of boost vaccine is critical; suggesting that the generation of effective mucosal immunity is possible, without the risks and challenges of mucosal administration, but that further work to specifically enhance sustained protective immunity is required.
Chavez-Galan, Leslie; Vesin, Dominique; Uysal, Husnu; Blaser, Guillaume; Benkhoucha, Mahdia; Ryffel, Bernhard; Quesniaux, Valérie F. J.; Garcia, Irene
Pleural tuberculosis (TB) is a form of extra-pulmonary TB observed in patients infected with Mycobacterium tuberculosis. Accumulation of myeloid-derived suppressor cells (MDSC) has been observed in animal models of TB and in human patients but their role remains to be fully elucidated. In this study, we analyzed the role of transmembrane TNF (tmTNF) in the accumulation and function of MDSC in the pleural cavity during an acute mycobacterial infection. Mycobacterium bovis BCG-induced pleurisy was resolved in mice expressing tmTNF, but lethal in the absence of tumor necrosis factor. Pleural infection induced MDSC accumulation in the pleural cavity and functional MDSC required tmTNF to suppress T cells as did pleural wild-type MDSC. Interaction of MDSC expressing tmTNF with CD4 T cells bearing TNF receptor 2 (TNFR2), but not TNFR1, was required for MDSC suppressive activity on CD4 T cells. Expression of tmTNF attenuated Th1 cell-mediated inflammatory responses generated by the acute pleural mycobacterial infection in association with effective MDSC expressing tmTNF and interacting with CD4 T cells expressing TNFR2. In conclusion, this study provides new insights into the crucial role played by the tmTNF/TNFR2 pathway in MDSC suppressive activity required during acute pleural infection to attenuate excessive inflammation generated by the infection. PMID:28890718
Chavez-Galan, Leslie; Vesin, Dominique; Uysal, Husnu; Blaser, Guillaume; Benkhoucha, Mahdia; Ryffel, Bernhard; Quesniaux, Valérie F J; Garcia, Irene
Pleural tuberculosis (TB) is a form of extra-pulmonary TB observed in patients infected with Mycobacterium tuberculosis. Accumulation of myeloid-derived suppressor cells (MDSC) has been observed in animal models of TB and in human patients but their role remains to be fully elucidated. In this study, we analyzed the role of transmembrane TNF (tmTNF) in the accumulation and function of MDSC in the pleural cavity during an acute mycobacterial infection. Mycobacterium bovis BCG-induced pleurisy was resolved in mice expressing tmTNF, but lethal in the absence of tumor necrosis factor. Pleural infection induced MDSC accumulation in the pleural cavity and functional MDSC required tmTNF to suppress T cells as did pleural wild-type MDSC. Interaction of MDSC expressing tmTNF with CD4 T cells bearing TNF receptor 2 (TNFR2), but not TNFR1, was required for MDSC suppressive activity on CD4 T cells. Expression of tmTNF attenuated Th1 cell-mediated inflammatory responses generated by the acute pleural mycobacterial infection in association with effective MDSC expressing tmTNF and interacting with CD4 T cells expressing TNFR2. In conclusion, this study provides new insights into the crucial role played by the tmTNF/TNFR2 pathway in MDSC suppressive activity required during acute pleural infection to attenuate excessive inflammation generated by the infection.
Saqib, Mohd; Khatri, Rahul; Singh, Bindu; Gupta, Ananya; Kumar, Arvind; Bhaskar, Sangeeta
BCG, the only approved vaccine protects against severe form of childhood tuberculosis but its protective efficacy wanes in adolescence. BCG has reduced the incidence of infant TB considerably in endemic areas; therefore prime-boost strategy is the most realistic measure for control of tuberculosis in near future. Mycobacterium indicus pranii (MIP) shares significant antigenic repertoire with Mtb and BCG and has been shown to impart significant protection in animal models of tuberculosis. In this study, MIP was given as a booster to BCG vaccine which enhanced the BCG mediated immune response, resulting in higher protection. MIP booster via aerosol route was found to be more effective in protection than subcutaneous route of booster immunization. Pro-inflammatory cytokines like IFN-γ, IL-12 and IL-17 were induced at higher level in infected lungs of 'BCG-MIP' group both at mRNA expression level and in secretory form when compared with 'only BCG' group. BCG-MIP groups had increased frequency of multifunctional T cells with high MFI for IFN-γ and TNF-α in Mtb infected mice. Our data demonstrate for the first time, potential application of MIP as a booster to BCG vaccine for efficient protection against tuberculosis. This could be very cost effective strategy for efficient control of tuberculosis. Copyright © 2016 Elsevier Ltd. All rights reserved.
Gopal, Radha; Lin, Yinyao; Obermajer, Nataša; Slight, Samantha; Nuthalapati, Nikhil; Ahmed, Mushtaq; Kalinski, Pawel; Khader, Shabaana A
Generation of effective T helper cell type 1 (Th1) responses are required for immunity against intracellular bacteria. However, some intracellular bacteria require Interleukin (IL)-17 to drive Th1 immunity and subsequent protective host immunity. Here, in a model of Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccination in mice, we demonstrate that the dependence on IL-17 to drive Th1 responses is a host mechanism to overcome bacteria-induced IL-10 inhibitory effects. We show that BCG-induced Prostaglandin-E2 (PGE2) promotes the production of IL-10 which limits Th1 responses, while simultaneously inducing IL-23 and Th17 differentiation. The ability of IL-17 to down-regulate IL-10 and induce IL-12 production allows the generation of subsequent Th1 responses. Accordingly, BCG-induced Th17 responses precedes generation of Th1 responses in vivo, while absence of IL-23 pathway decreases BCG vaccine-induced Th17 and Th1 immunity and subsequent vaccine-induced protection upon M.tuberculosis challenge. Importantly, in the absence of IL-10, BCG-induced Th1 responses occurs in an IL-17-independent manner. These novel data project the IL-23/IL-17 pathway in driving Th1 responses, specifically, to overcome IL-10 mediated inhibition and that in absence of IL-10, the generation of BCG induced Th1 immunity is IL-17-independent. PMID:22101830
Pérez de Val, Bernat; Vidal, Enric; Villarreal-Ramos, Bernardo; Gilbert, Sarah C.; Andaluz, Anna; Moll, Xavier; Martín, Maite; Nofrarías, Miquel; McShane, Helen; Vordermeier, H. Martin; Domingo, Mariano
The “One world, one health” initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG. PMID:24278420
Garrido, Joseba M.; Sevilla, Iker A.; Beltrán-Beck, Beatriz; Minguijón, Esmeralda; Ballesteros, Cristina; Galindo, Ruth C.; Boadella, Mariana; Lyashchenko, Konstantin P.; Romero, Beatriz; Geijo, Maria Victoria; Ruiz-Fons, Francisco; Aranaz, Alicia; Juste, Ramón A.; Vicente, Joaquín; de la Fuente, José; Gortázar, Christian
Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines. PMID:21935486
Clark, Simon; Cross, Martin L; Nadian, Allan; Vipond, Julia; Court, Pinar; Williams, Ann; Hewinson, R Glyn; Aldwell, Frank E; Chambers, Mark A
Increased incidence of bovine tuberculosis (TB) in the United Kingdom caused by infection with Mycobacterium bovis is a cause of considerable economic loss to farmers and the government. The Eurasian badger (Meles meles) represents a wildlife source of recurrent M. bovis infections of cattle in the United Kingdom, and its vaccination against TB with M. bovis bacillus Calmette-Guérin (BCG) is an attractive disease control option. Delivery of BCG in oral bait holds the best prospect for vaccinating badgers over a wide geographical area. Using a guinea pig pulmonary challenge model, we evaluated the protective efficacy of candidate badger oral vaccines, based on broth-grown or ball-milled BCG, delivered either as aqueous suspensions or formulated in two lipids with differing fatty acid profiles (one being animal derived and the other being vegetable derived). Protection was determined in terms of increasing body weight after aerosol challenge with virulent M. bovis, reduced dissemination of M. bovis to the spleen, and, in the case of one oral formulation, restricted growth of M. bovis in the lungs. Only oral BCG formulated in lipid gave significant protection. These data point to the potential of the BCG-lipid formulation for further development as a tool for controlling tuberculosis in badgers.
Tirado, Yanely; Puig, Alina; Alvarez, Nadine; Borrero, Reinier; Aguilar, Alicia; Camacho, Frank; Reyes, Fatima; Fernández, Sonsire; Pérez, José Luis; Espinoza, Dulce Mata; Payán, Jorge Alberto Barrios; Sarmiento, María Elena; Norazmi, Mohd-Nor; Hernández-Pando, Rogelio; Acosta, Armando
Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb. PMID:25671612
Tirado, Yanely; Puig, Alina; Alvarez, Nadine; Borrero, Reinier; Aguilar, Alicia; Camacho, Frank; Reyes, Fatima; Fernández, Sonsire; Pérez, José Luis; Espinoza, Dulce Mata; Payán, Jorge Alberto Barrios; Sarmiento, María Elena; Norazmi, Mohd-Nor; Hernández-Pando, Rogelio; Acosta, Armando
Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb.
Dudek, Katarzyna; Bednarek, Dariusz; Ayling, Roger D; Kycko, Anna; Szacawa, Ewelina; Karpińska, Teresa A
Mycoplasma bovis is a major pathogen affecting cattle causing bronchopneumonia, mastitis, and other disorders. Only autogenous vaccines made specifically for individual farms are available in parts of Europe and the United States. A novel experimental vaccine composed of a field M. bovis isolate combined with saponin and Emulsigen(®) adjuvants was evaluated. Eighteen 3-4 week old calves were placed in three equal groups: vaccinated (Vac), positive control (PC) and negative control (NC). The Vac calves were subcutaneously injected with 8ml of the vaccine; the PC and NC calves received phosphate buffered saline (PBS). Three weeks later the Vac and PC calves were challenged with a virulent M. bovis strain, the NC group received PBS. Throughout the study clinical observations, microbiology and immunological tests were carried out. Three weeks post challenge two calves from each group were euthanased for necropsy and histopathological examination. The vaccine effectively stimulated the humoral immune response, with high titres of anti-M. bovis specific antibodies and total Ig concentration. This vaccine also intensified the IgA response. A clinically protective effect of the vaccine was demonstrated as it also reduced the gross pathological lung lesions and nasal shedding of M. bovis. Copyright © 2016 Elsevier Ltd. All rights reserved.
Méndez-Samperio, P; Garcia-Martinez, E; Hernandez-Garay, M; Solis-Cardona, M
In this study, we found evidence that the interleukin-10 (IL-10) protein is functionally relevant in Mycobacterium bovis BCG-induced cytokine synthesis, as neutralization of endogenously synthesized IL-10 in human cells activated with BCG resulted in a two- to threefold increase in the level of IL-1 beta. When exogenous recombinant human IL-10 was added to human mononuclear cells, a significant reduction of BCG-induced IL-1 beta secretion was observed. This inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in the presence of IL-10, and it was specific, as it was completely abolished in the presence of anti-IL-10 neutralizing monoclonal antibody while an irrelevant antibody used as a control had no effect. Taken together, these are the first studies that demonstrate that the depletion of endogenous IL-10 via anti-IL-10 antibody results in a very significantly enhanced BCG-induced IL-1 beta secretion and that the addition of exogenous IL-10 to human mononuclear cells stimulated with BCG inhibits IL-1 beta production. Further experimental work is needed to determine if the neutralization of IL-10 activity via anti-IL-10 antibody indeed enhances cytokine synthesis in vivo. However, the present results may be of importance, since the use of anti-IL-10 antibody could presumably contribute to the protective immunity induced by BCG against tuberculosis via an increase in cytokine synthesis that would amplify antimicrobial systems.
Zhao, W; Schorey, J S; Bong-Mastek, M; Ritchey, J; Brown, E J; Ratliff, T L
Intravesical Mycobacterium bovis bacillus Calmette-Gu*erin (BCG) is the treatment of choice for superficial bladder cancer. Previous studies showed that attachment of BCG to fibronectin within the bladder was necessary for mediation of the antitumor response. Further studies identified a bacterial receptor, fibronectin attachment protein (FAP), as an important mediator of BCG attachment to fibronectin. In vitro studies showed that a stable BCG/fibronectin interaction was dependent on FAP binding to fibronectin; however, no role for FAP in the attachment of BCG in vivo has been characterized. We now report the cloning of the M. bovis BCG FAP (FAP-B) and demonstrate an important role for FAP in the in vivo attachment of BCG to the bladder wall and in the induction of BCG-mediated antitumor activity. The predicted amino acid sequence for FAP-B shows 61% and 71% homology, respectively, with Mycobacterium avium FAP (FAP-A) and Mycobacterium leprae FAP (FAP-L). Rabbit polyclonal antibodies against Mycobacterium vaccae FAP (FAP-V) reacted with all 3 recombinant FAP proteins on Western blots. Functional studies show FAP-B to bind fibronectin via the highly conserved attachment regions previously identified for FAP-A and FAP-L and also to competitively inhibit attachment of BCG to matrix fibronectin. In vivo studies show FAP to be a necessary protein for the stable attachment of BCG to the bladder wall. Moreover, stable binding of BCG via FAP was shown to be necessary for the expression of BCG-induced antitumor activity. Our results demonstrate a biological role for FAP in the mediation of BCG-induced antitumor activity.
Lesellier, Sandrine; Corner, Leigh; Costello, Eamon; Lyashchenko, Konstantin; Greenwald, Rena; Esfandiari, Javan; Singh, Mahavir; Hewinson, R Glyn; Chambers, Mark; Gormley, Eamonn
European badgers (Meles meles) are a reservoir host of Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. The development of a vaccine for use in badgers is considered a key element of any campaign to eradicate the disease in livestock in both countries. In this study we have vaccinated groups of badgers with approximately 5 x 10(5)cfu of the BCG vaccine delivered via two alternative routes, subcutaneous and mucosal (intranasal/conjunctival). Following experimental endobronchial infection with approximately 10(4)cfu of M. bovis, all badgers were euthanised at 12 weeks post-infection. At post-mortem examination both vaccinated groups had significantly reduced severity of disease compared with the non-vaccinated controls. The analysis of immune responses throughout the study showed that vaccination with BCG did not generate any detectable immunological responses as measured by IFN-gamma production in antigen-stimulated peripheral blood mononuclear cells (PBMC) and IgG serological responses. However, the levels of the responses increased following M. bovis infection, and the kinetic profiles corresponded to the severity of lesions recorded post-mortem. Significant differences were observed in the timing of development of the immune responses between vaccinates and controls. The results suggest that the immunological responses are associated with the levels of protective immunity and could be used as markers to monitor control of disease in badgers following vaccination.
Sternick, J L; Schrier, D J; Moore, V L
The genetics of BCG-induced pulmonary granulomatous inflammation (PGI) and splenomegaly was studied by breeding experiments and by the use of BXD recombinant inbred (RI) and allotype-congenic mice. Breeding studies indicated that the genetic control was multifactorial; this observation was confirmed using BXD RI mice. In addition, studies with BXD RI mice suggested that genes linked to the Igh complex influence responsiveness. The influence of the Igh-linked genes was studied further using allotype-congenic mice; SJL mice (Ighb) developed significantly greater PGI than their congenic partner, SJA/9 (Igha). Data from BALB.Igb, CB-20, and BAB-14 mice suggested that Igh-linked genes influencing PGI were a considerable distance from Igh-1. Igh-linked genes that influence BCG-induced splenomegaly were located on the centrometric side of the Igh-1 locus. This was shown by data in which splenomegaly in BALB.Igb and CB-20, but not BAB-14, mice was significantly augmented over BALB/c mice. Further studies are necessary to understand the significance of these observations.
Rizzi, Caroline; Bianco, María Verónica; Blanco, Federico Carlos; Soria, Marcelo; Gravisaco, María José; Montenegro, Valeria; Vagnoni, Lucas; Buddle, Bryce; Garbaccio, Sergio; Delgado, Fernando; Leal, Karen Silva; Cataldi, Angel Adrián; Dellagostin, Odir Antônio; Bigi, Fabiana
Mycobacterium bovis is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with leuD. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with M. bovis, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur. PMID:23251517
Suazo, Feliciano Milian; Escalera, Ana María Anaya; Torres, Ruth M Gallegos
Bovine tuberculosis (TB) causes severe economic losses in livestock due to low production, animal deaths and condemnation of carcasses. It is also an important constraint in international trade of animals and animal products. A scientific committee in Great Britain in 1997 concluded that the development of a cattle vaccine would be the best option for long-term control of TB. However, vaccination of cattle currently is not accepted because the vaccine interferes with the skin reaction to the tuberculin test in the field. Efficacy of M. bovis BCG in protecting bovine and other animal species against tuberculous infection has received much study. Vaccination of cattle prevents the spread of the disease in populations by reducing the number and size of the lesions, and the load of bacteria (rather than by preventing infection). We review the literature about the efficacy of BCG in protecting cattle and other animal species against infection with field strains of M. bovis and discusses its potential use in programs of TB control in high-prevalence populations.
Capinos Scherer, Charles F; Endsley, Janice J; de Aguiar, Juliana B; Jacobs, William R; Larsen, Michelle H; Palmer, Mitchell V; Nonnecke, Brian J; Ray Waters, W; Mark Estes, D
The development of improved vaccines against tuberculosis (TB) is directly linked to the investigation of new and better correlates of protection after vaccination against TB. Cloning and characterization of bovine homologues of the antimicrobial protein granulysin (Bo-lysin) and perforin by our group could be used as potential biomarkers for TB vaccination efficacy. In the present study, we examined the kinetics of granulysin, perforin, IFNgamma and Fas-L responses to Mycobacterium bovis purified protein derivative (PPD) stimulation by peripheral blood mononuclear cells from M. bovisDeltaRD1-, BCG- and non-vaccinated cattle. Gene expression profiles following PPD stimulation showed significant increases in transcripts for granulysin and IFNgamma in both CD4(+) and CD8(+) T cells in BCG-vaccinated as compared with non-vaccinated animals. Perforin and IFNgamma examined by flow cytometry, showed a difference of 1-2% more PPD-specific cells in BCG-vaccinated than non-vaccinated animals. In the vaccine trial, granulysin and perforin were significantly increased in both vaccine groups as compared with control after vaccination and challenge. IFNgamma expression was increased only after vaccination and secretion was higher in the control, non-protected group as compared with both vaccine groups demonstrating no correlation with protection upon vaccination. In summary, results shown here provide evidence that granulysin and perforin are prospective candidates as biomarkers of protection after vaccination against TB.
Aguilo, Nacho; Toledo, Ana Maria; Lopez-Roman, Eva Maria; Perez-Herran, Esther; Gormley, Eamonn; Rullas-Trincado, Joaquin; Angulo-Barturen, Iñigo
Worldwide, the Mycobacterium bovis BCG vaccine is one of the most widely used vaccines. However, it appears to be ineffective in preventing pulmonary tuberculosis. Here, we show that pulmonary BCG vaccination of mice with a broad dose range provides superior protection against Mycobacterium tuberculosis challenge compared to that of subcutaneous vaccination. PMID:24501340
Méndez-Samperio, P; Garcia-Martinez, E; Hernandez-Garay, M; Solis-Cardona, M
In this study, we found evidence that the interleukin-10 (IL-10) protein is functionally relevant in Mycobacterium bovis BCG-induced cytokine synthesis, as neutralization of endogenously synthesized IL-10 in human cells activated with BCG resulted in a two- to threefold increase in the level of IL-1 beta. When exogenous recombinant human IL-10 was added to human mononuclear cells, a significant reduction of BCG-induced IL-1 beta secretion was observed. This inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in the presence of IL-10, and it was specific, as it was completely abolished in the presence of anti-IL-10 neutralizing monoclonal antibody while an irrelevant antibody used as a control had no effect. Taken together, these are the first studies that demonstrate that the depletion of endogenous IL-10 via anti-IL-10 antibody results in a very significantly enhanced BCG-induced IL-1 beta secretion and that the addition of exogenous IL-10 to human mononuclear cells stimulated with BCG inhibits IL-1 beta production. Further experimental work is needed to determine if the neutralization of IL-10 activity via anti-IL-10 antibody indeed enhances cytokine synthesis in vivo. However, the present results may be of importance, since the use of anti-IL-10 antibody could presumably contribute to the protective immunity induced by BCG against tuberculosis via an increase in cytokine synthesis that would amplify antimicrobial systems. PMID:9067646
Chambers, Mark A.; Williams, Ann; Gavier-Widén, Dolores; Whelan, Adam; Hall, Graham; Marsh, Philip D.; Bloom, Barry R.; Jacobs, William R.; Hewinson, R. Glyn
Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals. PMID:11083835
Tenland, Erik; Håkansson, Gisela; Alaridah, Nader; Lutay, Nataliya; Rönnholm, Anna; Hallgren, Oskar; Westergren-Thorsson, Gunilla; Godaly, Gabriela
Mycobacterium bovis bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung. PMID:27723804
Garrido, Joseba M.; Aranaz, Alicia; Sevilla, Iker; Villar, Margarita; Boadella, Mariana; Galindo, Ruth C.; Pérez de la Lastra, José M.; Moreno-Cid, Juan A.; Fernández de Mera, Isabel G.; Alberdi, Pilar; Santos, Gracia; Ballesteros, Cristina; Lyashchenko, Konstantin P.; Minguijón, Esmeralda; Romero, Beatriz; de Juan, Lucía; Domínguez, Lucas; Juste, Ramón; Gortazar, Christian
Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar. PMID:24842853
Lopez-Valencia, G; Renteria-Evangelista, T; Williams, J de Jesús; Licea-Navarro, A; Mora-Valle, A De la; Medina-Basulto, G
The protective efficacy of Mycobacterium bovis BCG (1 x 10(6) single dose) was evaluated under field conditions. A total of 140 male Holstein Friesian calves, one to two week-old were selected. Two groups of 70 each were formed, one group was vaccinated and the other was injected with a placebo during their second week of age and followed until 12 months of age. The study considered a positive case of tuberculosis to be an animal that had a positive reaction to the three following tests in a row: tuberculin, IFNgamma PPD-B and IFNgamma ESAT6-CFP10 during the 12 months of exposure. The results showed a 59.4% efficacy (IC95%: 47.64-71.16). The non-vaccinated calves were 2.4 times more at risk of becoming infected (IC95%: 1.07-5.68) compared to vaccinated animals. As a complementary test a PCR test was performed using nasal exudates in some animals from both groups using a Mycobacterium complex detection kit. All the positive PCR reactions (5/44) were found in the non-vaccinated animals. These findings suggest that the use of the BCG vaccine, even though it is not capable of protecting 100%, does prevent TB vaccinated animals from excreting bacilli in their nasal secretions at their first year of age. Copyright 2009 Elsevier Ltd. All rights reserved.
Corner, Leigh A L; Costello, Eamon; O'Meara, Damien; Lesellier, Sandrine; Aldwell, Frank E; Singh, Mahavir; Hewinson, R Glyn; Chambers, Mark A; Gormley, Eamonn
Eurasian badgers (Meles meles) are a reservoir host of Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. The development of a vaccine for use in badgers is considered a key element of any long-term sustainable campaign to eradicate the disease from livestock in both countries. The aim of this study was to investigate the protective response of badgers vaccinated orally with Bacille Calmette-Guérin (BCG) encapsulated in a lipid formulation, followed by experimental challenge with M. bovis. A group of badgers was vaccinated by inoculating the BCG-lipid mixture containing approximately 10(8)colony forming units (cfu) of BCG into the oesophagus. The control group was sham inoculated with the lipid formulation only. Thirteen weeks after vaccination all the badgers were challenged with approximately 10(4)cfu of M. bovis delivered by endobronchial inoculation. Blood samples were taken throughout the study and the cell mediated immune (CMI) responses in peripheral blood were monitored by the IFN-gamma ELISA and ELISPOT assay. At 17 weeks after infection all the badgers were examined post-mortem to assess the pathological and bacteriological responses to challenge. All badgers in both groups were found to be infected. However, a significant protective effect of BCG vaccination was measured as a decrease in the number and severity of gross lesions, lower bacterial load in the lungs, and fewer sites of infection. The analysis of immune responses showed that vaccination with BCG did not generate any detectable CMI immunological responses, however the levels of the responses increased in both groups following M. bovis infection. The results of the study showed that vaccination with oral BCG in the lipid formulation generated a protective effect in the badgers.
Clark, Simon; Cross, Martin L; Smith, Alan; Court, Pinar; Vipond, Julia; Nadian, Allan; Hewinson, R Glyn; Batchelor, Hannah K; Perrie, Yvonne; Williams, Ann; Aldwell, Frank E; Chambers, Mark A
Bovine tuberculosis (bTB) caused by infection with Mycobacterium bovis is causing considerable economic loss to farmers and Government in the United Kingdom as its incidence is increasing. Efforts to control bTB in the UK are hampered by the infection in Eurasian badgers (Meles meles) that represent a wildlife reservoir and source of recurrent M. bovis exposure to cattle. Vaccination of badgers with the human TB vaccine, M. bovis Bacille Calmette-Guérin (BCG), in oral bait represents a possible disease control tool and holds the best prospect for reaching badger populations over a wide geographical area. Using mouse and guinea pig models, we evaluated the immunogenicity and protective efficacy, respectively, of candidate badger oral vaccines based on formulation of BCG in lipid matrix, alginate beads, or a novel microcapsular hybrid of both lipid and alginate. Two different oral doses of BCG were evaluated in each formulation for their protective efficacy in guinea pigs, while a single dose was evaluated in mice. In mice, significant immune responses (based on lymphocyte proliferation and expression of IFN-gamma) were only seen with the lipid matrix and the lipid in alginate microcapsular formulation, corresponding to the isolation of viable BCG from alimentary tract lymph nodes. In guinea pigs, only BCG formulated in lipid matrix conferred protection to the spleen and lungs following aerosol route challenge with M. bovis. Protection was seen with delivery doses in the range 10(6)-10(7) CFU, although this was more consistent in the spleen at the higher dose. No protection in terms of organ CFU was seen with BCG administered in alginate beads or in lipid in alginate microcapsules, although 10(7) in the latter formulation conferred protection in terms of increasing body weight after challenge and a smaller lung to body weight ratio at necropsy. These results highlight the potential for lipid, rather than alginate, -based vaccine formulations as suitable delivery
Sander, Peter; Clark, Simon; Petrera, Agnese; Vilaplana, Cristina; Meuli, Michael; Selchow, Petra; Zelmer, Andrea; Mohanan, Deepa; Andreu, Nuria; Rayner, Emma; Dal Molin, Michael; Bancroft, Gregory J; Johansen, Pål; Cardona, Pere-Joan; Williams, Ann; Böttger, Erik C
Having demonstrated previously that deletion of zinc metalloprotease zmp1 in Mycobacterium bovis BCG increased immunogenicity of BCG vaccines, we here investigated the protective efficacy of BCG zmp1 deletion mutants in a guinea pig model of tuberculosis infection. zmp1 deletion mutants of BCG provided enhanced protection by reducing the bacterial load of tubercle bacilli in the lungs of infected guinea pigs. The increased efficacy of BCG due to zmp1 deletion was demonstrated in both BCG Pasteur and BCG Denmark indicating that the improved protection by zmp1 deletion is independent from the BCG sub-strain. In addition, unmarked BCG Δzmp1 mutant strains showed a better safety profile in a CB-17 SCID mouse survival model than the parental BCG strains. Together, these results support the further development of BCG Δzmp1 for use in clinical trials.
de Klerk, Lin-Mari; Michel, Anita L; Bengis, Roy G; Kriek, Nicolaas P J; Godfroid, Jacques
Vaccination has been discussed as a practical option to control bovine tuberculosis in countries where a wildlife reservoir of the disease is present. African buffaloes (Syncerus caffer) are the main wildlife reservoir of Mycobacterium bovis in certain South African game parks and vaccination is not only the most promising but the only ethically acceptable control measure currently available. The use of Bacillus Calmette-Guérin vaccine (Pasteur strain) to vaccinate fourteen African buffalo yearlings and their reactions to subsequent intratonsilar challenge with a field strain of M. bovis are described. The BCG vaccine was administered twice intramuscularly, six weeks apart. All vaccinates and thirteen control buffaloes were euthanized and necropsies performed 9 months after the challenge. Standard sets of lymph nodes from the head, the thoracic cavity and abdomen were cultured and examined histopathologically. No significant reduction in number of lesions or severity of disease was noted, concluding that the BCG vaccine did not induce sufficient protection able to limit the shedding of organisms. The age of the buffaloes, route of vaccination and prior exposure to environmental mycobacteria are among the possible reasons for vaccination failure.
Callis, A H; Schrier, D J; David, C S; Moore, V L
We previously reported that BCG-induced anergy in mice (evaluated by delayed hypersensitivity to sheep erythrocytes) is unigenic and influenced by genes linked to the immunoglobulin heavy chain allotype (Igh). Using congenic mice (either H-2k or H-2b), we could not detect H-2-linked control of anergy. The current study re-examines this issue by using both BXD (H-2b or H-2d) and BXH (H-2b or H-2k) recombinant inbred (RI) mice as well as H-2 recombinant mice of different haplotypes. BXD RI (H-2b) mice were more anergic than BXD RI (H-2d) animals. Also, BXD RI (Ighb animals were more anergic than BXD RI (Ighc) mice. By evaluating combinations of H-2 haplotypes and Igh allotypes, we found the most anergic animals to be H-2b, Ighb. BCG-induced anergy then appears to be influenced by genes linked to both the H-2 and Igh complexes. BCG-induced anergy developed in H-2 recombinant mice (C57BL/10 background) that were either H-2b or H-2k, but not in H-2d animals. Experiments in the B10.A mouse suggested that genes within the H-2K through H-2I were influential. A more definitive map is presented of Igh-linked genes influencing anergy, suggesting that these genes are approximately 23 recombination units on the centromeric side of Igh-1 between Igh-Src and Lyb-7.
Callis, A H; Schrier, D J; David, C S; Moore, V L
We previously reported that BCG-induced anergy in mice (evaluated by delayed hypersensitivity to sheep erythrocytes) is unigenic and influenced by genes linked to the immunoglobulin heavy chain allotype (Igh). Using congenic mice (either H-2k or H-2b), we could not detect H-2-linked control of anergy. The current study re-examines this issue by using both BXD (H-2b or H-2d) and BXH (H-2b or H-2k) recombinant inbred (RI) mice as well as H-2 recombinant mice of different haplotypes. BXD RI (H-2b) mice were more anergic than BXD RI (H-2d) animals. Also, BXD RI (Ighb animals were more anergic than BXD RI (Ighc) mice. By evaluating combinations of H-2 haplotypes and Igh allotypes, we found the most anergic animals to be H-2b, Ighb. BCG-induced anergy then appears to be influenced by genes linked to both the H-2 and Igh complexes. BCG-induced anergy developed in H-2 recombinant mice (C57BL/10 background) that were either H-2b or H-2k, but not in H-2d animals. Experiments in the B10.A mouse suggested that genes within the H-2K through H-2I were influential. A more definitive map is presented of Igh-linked genes influencing anergy, suggesting that these genes are approximately 23 recombination units on the centromeric side of Igh-1 between Igh-Src and Lyb-7. PMID:6409803
Whelan, Adam O; Wright, D Craig; Chambers, Mark A; Singh, Mahavir; Hewinson, R Glyn; Vordermeier, H Martin
Development of cattle vaccines against bovine tuberculosis is a GB research priority. Recently, it has been shown that formalin-killed Bacille Calmette-Guérin (BCG) delivered with the liposomal adjuvant NAX687 imparted significant protection against Mycobacterium bovis infection in the guinea pig aerosol infection model. Extending these studies, we inoculated calves with live BCG, formalin-killed BCG and formalin-killed BCG formulated in NAX687. Live and killed BCG vaccine formulations induced primary effector T-cell populations comparably, both killed BCG formulations also induced potent humoral immune responses. In contrast, live BCG generated enhanced central memory responses against the protective antigen Ag85A whilst killed BCG-induced such responses only poorly. However, the poor capacity of killed BCG to generate central memory could be partially overcome by formulation with NAX687. Measurement of central memory responses induced by TB vaccine candidates in cattle may provide a useful correlate of protection and warrants further investigation in challenge experiments.
Corner, Leigh A L; Costello, Eamon; Lesellier, Sandrine; O'Meara, Damien; Gormley, Eamonn
Mycobacterium bovis is endemic in badger (Meles meles) populations of Ireland and the United Kingdom and infected badgers are a potential source of infection for cattle. In domestic livestock tuberculosis causes economic losses from lost production and the costs associated with eradication programmes, and in addition there is a risk of zoonotic infection. Whereas culling is currently used to control tuberculous badger populations in Ireland, vaccination, if it were available, would be preferred. A study was undertaken to examine the protective responses of badgers vaccinated either by the subcutaneous or mucosal (intranasal and conjunctival) routes with bacille Calmette-Guérin (BCG), when challenged with M. bovis by the endobronchial route. Three groups of badgers were used. The first group (n=4) was vaccinated with approximately 5 x 10(5) colony forming units (cfu) of BCG by subcutaneous injection. In the second group (n=5) badgers were vaccinated via the mucosal route by instilling 1.0 x 10(5)cfu into each conjunctival sac and spraying 1.0 x 10(5)cfu into each nostril (final vaccine dose of 4 x 10(5)cfu). The control (n=5) badgers served as a non-vaccinated group. Twelve weeks post-vaccination all badgers in the three groups were challenged with approximately 10(4)cfu of M. bovis by endobronchial inoculation. At 12 weeks post-infection all badgers were examined post-mortem to assess the pathological and bacteriological responses to challenge. Gross and histological lesions of tuberculosis were seen in all challenged badgers and M. bovis was recovered from all challenged badgers. However, across six of the eight parameters used to measure disease severity, the infection in the vaccinated badgers was significantly less severe than in the control group. The BCG vaccine induced a significant protective effect in the badgers and the protective immunity was generated by subcutaneous and mucosal vaccination.
Background Pneumonitis is a rare complication of bacillus Calmette-Guerin (BCG) immunotherapy seen in patients with urothelial cancer following the repeated administration of BCG. However, no case of BCG-induced pleurisy has been reported. Case presentation We here report the first case of pneumonitis with lymphocytic pleurisy following bacillus Calmette-Guerin (BCG) immunotherapy. Although marked T helper cell alveolitis was found by bronchoalveolar lavage and transbronchial biopsies, no acid-fast bacillus could be identified in recovered BALF or pleural effusion. The lymphocyte stimulation test of BCG was strongly positive. However, levels of serum and bronchoalveolar lavage fluid KL-6, a useful marker for hypersensitivity pneumonitis (HP), were within normal ranges. Conclusion We speculate that the pathogenesis of our case may be a hypersensitive reaction to the proteic component of BCG entering the lung and pleural space, which is different from the etiology of the common type of HP. PMID:24593234
Parlane, Natalie A; Shu, Dairu; Subharat, Supatsak; Wedlock, D Neil; Rehm, Bernd H A; de Lisle, Geoffrey W; Buddle, Bryce M
In both humans and animals, controversy exists concerning the duration of protection induced by BCG vaccine against tuberculosis (TB) and whether revaccination enhances protection. A long-term study was undertaken to determine whether BCG-vaccinated calves would be protected against challenge with Mycobacterium bovis 2½ years after vaccination and to determine the effect of revaccination after 2 years. Seventy-nine calves were divided into five groups (n = 15-17 calves/group) with four of the groups vaccinated subcutaneously with 105 CFU of BCG Danish at 2-4 weeks of age and the fifth group serving as non-vaccinated controls. Three of the four BCG-vaccinated groups were revaccinated 2 years after the initial vaccination. One BCG-vaccinated group was revaccinated with BCG. A second group was vaccinated subcutaneously with a TB protein vaccine consisting of biopolyester particles (Biobeads) displaying two mycobacterial proteins, ESAT-6 and Antigen 85A, mixed with an adjuvant. A third group was vaccinated with TB proteins from M. bovis culture filtrate, mixed with an adjuvant. Twenty-three weeks after the BCG revaccination, all animals were challenged endotracheally with virulent M. bovis and a further 13 weeks later, animals were killed and necropsied to determine protection against TB. The BCG-vaccinated animals produced positive tuberculin caudal fold intradermal (15 of 62 animals) and IFN-γ TB test responses (six of 62 animals) at 6 months after vaccination, but not at subsequent time-points compared to the non-vaccinated animals. Calves receiving a single vaccination with BCG vaccine 2½ years prior to challenge were not protected against TB, while those revaccinated with BCG 2 years after the initial vaccination displayed significant reductions in lung and pulmonary lymph node lesion scores compared to the non-vaccinated animals. In contrast, no reduction in lesion scores was observed in the animals revaccinated with the TB protein vaccines with their immune
Background Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain. Results BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge. Conclusions Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions. PMID:24766746
Hogarth, Philip J; Logan, Karen E; Vordermeier, H Martin; Singh, Mahavir; Hewinson, R Glyn; Chambers, Mark A
In a number of clinical studies the current TB vaccine, Mycobacterium bovis bacille Calmette-Guerin (BCG), has provided little or no protection against pulmonary tuberculosis in cattle and man. A new generation of vaccines is therefore required to replace or supplement BCG. Safety concerns surrounding a number of strategies make protein subunits an attractive approach. Moreover, novel prime-boost strategies based on primary immunisations with BCG are not only showing promise but also present a clear strategy for testing new TB vaccines in clinical studies. We report the evaluation of six protein vaccine candidates for their ability to induce protective immunity in a murine virulent M. bovis challenge model. One protein (Rv3019c) induced reproducibly significant protection in the spleen and lungs approaching that induced by BCG. Detailed analysis of antigen-specific T cell responses revealed that despite robust responses in the spleen and lungs of vaccinated mice, there was no correlation between these responses and the protective efficacy of the vaccine. Significantly, Rv3019c also stimulated IFN-gamma responses in PBMC from BCG vaccinated cattle, indicating its potential for use in a heterologous prime-boost strategy in conjunction with BCG in the target species.
Hedhli, Dorsaf; Denis, Olivier; Barkan, Daniel; Daffé, Mamadou; Glickman, Michael S.; Huygen, Kris
The existing vaccine against tuberculosis (M. bovis BCG) exerts some protection against the extrapulmonary forms of the disease, particularly in young children, but is not very effective against the pulmonary form of TB, which often results from the reactivation of a latent M. tuberculosis (M.tb)infection. Among the new approaches in TB vaccine development, live attenuated M.tb mutants are a promising new avenue. Here we report on the vaccine potential of two highly attenuated M.tb mutants, MGM1991 and M.tbhma::hyg (HMA), lacking all oxygenated mycolates in their cell wall. In C57BL/6 mice, stronger Th1 (IFN-γ, IL-2 and TNF-α) and IL-17 responses could be induced following subcutaneous vaccination with either of the two mutants, than following vaccination with M. bovis BCG. Significantly more mycobacteria specific IFN-γ producing CD4+ and particularly CD8+ T cells could be detected by intracellular cytokine staining in mice vaccinated with the M.tb mutants. Finally, vaccination with either of the two mutants conferred stronger protection against intratracheal M.tb challenge than vaccination with BCG, as indicated by reduced bacterial replication in lungs at 4 to 12 weeks after challenge. Protection against M. tb dissemination, as indicated by reduced bacterial numbers in spleen, was comparable for both mutants to protection conferred by BCG. PMID:24146869
Sarfas, C.; West, K.; Sibley, L. S.; Wareham, A. S.; Clark, S.; Dennis, M. J.; Williams, A.; Marsh, P. D.; Sharpe, S. A.
Nine million cases of tuberculosis (TB) were reported in 2013, with a further 1.5 million deaths attributed to the disease. When delivered as an intradermal (i.d.) injection, the Mycobacterium bovis BCG vaccine provides limited protection, whereas aerosol delivery has been shown to enhance efficacy in experimental models. In this study, we used the rhesus macaque model to characterize the mucosal and systemic immune response induced by aerosol-delivered BCG vaccine. Aerosol delivery of BCG induced both Th1 and Th17 cytokine responses. Polyfunctional CD4 T cells were detected in bronchoalveolar lavage (BAL) fluid and peripheral blood mononuclear cells (PBMCs) 8 weeks following vaccination in a dose-dependent manner. A similar trend was seen in peripheral gamma interferon (IFN-γ) spot-forming units measured by enzyme-linked immunosorbent spot (ELISpot) assay and serum anti-purified protein derivative (PPD) IgG levels. CD8 T cells predominantly expressed cytokines individually, with pronounced tumor necrosis factor alpha (TNF-α) production by BAL fluid cells. T-cell memory phenotype analysis revealed that CD4 and CD8 populations isolated from BAL fluid samples were polarized toward an effector memory phenotype, whereas the frequencies of peripheral central memory T cells increased significantly and remained elevated following aerosol vaccination. Expression patterns of the α4β1 integrin lung homing markers remained consistently high on CD4 and CD8 T cells isolated from BAL fluid and varied on peripheral T cells. This characterization of aerosol BCG vaccination highlights features of the resulting mycobacterium-specific immune response that may contribute to the enhanced protection previously reported in aerosol BCG vaccination studies and will inform future studies involving vaccines delivered to the mucosal surfaces of the lung. PMID:26108288
Tolderlund, Knud; Bunch-Christensen, Kirsten; Waaler, Hans
In assessing the biological activity of BCG vaccine by tuberculin testing of vaccinated guinea-pigs, it is necessary to take into account the rates of development and waning of allergy, and also the boosting effect on waning allergy caused by the tuberculin test itself. Using Danish liquid vaccine (in approximately standard dose), the authors have carried out two series of tests, involving more than six hundred guinea-pigs, to evaluate the significance of these factors. Post-vaccination tuberculin sensitivity was found to reach a maximum within 1-2 months. Three months after vaccination the BCG-induced allergy began to wane, and after 12 months it had dropped almost to the level observed in non-vaccinated guinea-pigs. The tuberculin test had a strong boosting effect, however, and even 12 months after vaccination the waning sensitivity could be considerably increased by a single injection of 10 TU of tuberculin. An analysis of the results showed that the waning of the level of allergy takes place gradually over several months. This is not an effect of aging, however, as the response to vaccination was found to be independent of the age of the animals. The indications of this study are that tuberculin testing of guinea-pigs used for the laboratory control of BCG vaccine is best performed about 6 weeks after vaccination. PMID:20604065
Wedlock, D Neil; Aldwell, Frank E; Vordermeier, H Martin; Hewinson, R Glyn; Buddle, Bryce M
Mycobacterium bovis bacille Calmette-Guérin (BCG) delivered to calves by the oral route in a formulated lipid matrix has been previously shown to induce protection against bovine tuberculosis. A study was conducted in cattle to determine if a combination of a low dose of oral BCG and a protein vaccine could induce protective immunity to tuberculosis while not sensitising animals to tuberculin. Groups of calves (10 per group) were vaccinated by administering 2 × 10(7)colony forming units (CFU) of BCG orally or a combination of 2 × 10(7)CFU oral BCG and a protein vaccine comprised of M. bovis culture filtrate proteins (CFP) formulated with the adjuvants Chitin and Gel 01 and delivered by the intranasal route, or CFP formulated with Emulsigen and the TLR2 agonist Pam(3)CSK(4) and administered by the subcutaneous (s.c.) route. Two further groups were vaccinated with the CFP/Chitin/Gel 01 or CFP/Emulsigen/Pam(3)CSK(4) vaccines alone. Positive control groups were given 10(8)CFU oral BCG or 10(6)CFU s.c. BCG while a negative control group was non-vaccinated. All animals were challenged with M. bovis 15 weeks after vaccination and euthanized and necropsied at 16 weeks following challenge. Groups of cattle vaccinated with s.c. BCG, 10(8)CFU or 2 × 10(7)CFU oral BCG showed significant reductions in seven, three and four pathological or microbiological disease parameters, respectively, compared to the results for the non-vaccinated group. There was no evidence of protection in calves vaccinated with the combination of oral BCG and CFP/Emulsigen/Pam(3)CSK(4) or oral BCG and CFP/Chitin/Gel 01 or vaccinated with the protein vaccines alone. Positive responses in the comparative cervical skin test at 12 weeks after vaccination were only observed in animals vaccinated with s.c. BCG, 10(8)CFU oral BCG or a combination of 2 × 10(7)CFU oral BCG and CFP/Chitin/Gel 01. In conclusion, co-administration of a protein vaccine, administered by either systemic or mucosal routes with oral
Vicente, Belén; López-Abán, Julio; Rojas-Caraballo, José; Pérez del Villar, Luis; Hillyer, George V; Martínez-Fernández, Antonio R; Muro, Antonio
Several efforts have been made to identify anti-schistosomiasis vaccine candidates and new vaccination systems. The fatty acid binding protein (FAPB) has been shown to induce a high level of protection in trematode infection. The adjuvant adaptation (ADAD) vaccination system was used in this study, including recombinant FABP, a natural immunomodulator and saponins. Mice immunised with the ADAD system were able to up-regulate proinflammatory cytokines (IL-1 and IL-6) and induce high IgG2a levels. Moreover, there was a significant reduction in worm burden, egg liver and hepatic lesion in vaccinated mice in two independent experiments involving Schistosoma bovis infected mice. The foregoing data shows that ADAD system using FABP provide a good alternative for triggering an effective immune response against animal schistosomiasis. Copyright © 2014 Elsevier Inc. All rights reserved.
Moliva, Juan I.; Turner, Joanne; Torrelles, Jordi B.
Mycobacterium tuberculosis (M.tb) infection leads to active tuberculosis (TB), a disease that kills one human every 18 seconds. Current therapies available to combat TB include chemotherapy and the preventative vaccine Mycobacterium bovis Bacille Calmette et Guérin (BCG). Increased reporting of drug resistant M.tb strains worldwide indicates that drug development cannot be the primary mechanism for eradication. BCG vaccination has been used globally for protection against childhood and disseminated TB, however, its efficacy at protecting against pulmonary TB in adult and aging populations is highly variable. In this regard, the immune response generated by BCG vaccination is incapable of sterilizing the lung post M.tb infection as indicated by the large proportion of individuals with latent TB infection that have received BCG. Although many new TB vaccine candidates have entered the development pipeline, only a few have moved to human clinical trials; where they showed no efficacy and/or were withdrawn due to safety regulations. These trials highlight our limited understanding of protective immunity against the development of active TB. Here, we discuss current vaccination strategies and their impact on the generation and sustainability of protective immunity against TB. PMID:26319069
Moliva, Juan I; Turner, Joanne; Torrelles, Jordi B
Mycobacterium tuberculosis (M.tb) infection leads to active tuberculosis (TB), a disease that kills one human every 18s. Current therapies available to combat TB include chemotherapy and the preventative vaccine Mycobacterium bovis Bacille Calmette et Guérin (BCG). Increased reporting of drug resistant M.tb strains worldwide indicates that drug development cannot be the primary mechanism for eradication. BCG vaccination has been used globally for protection against childhood and disseminated TB, however, its efficacy at protecting against pulmonary TB in adult and aging populations is highly variable. In this regard, the immune response generated by BCG vaccination is incapable of sterilizing the lung post M.tb infection as indicated by the large proportion of individuals with latent TB infection that have received BCG. Although many new TB vaccine candidates have entered the development pipeline, only a few have moved to human clinical trials; where they showed no efficacy and/or were withdrawn due to safety regulations. These trials highlight our limited understanding of protective immunity against the development of active TB. Here, we discuss current vaccination strategies and their impact on the generation and sustainability of protective immunity against TB.
Xu, Zheng Zhong; Chen, Xiang; Hu, Ting; Meng, Chuang; Wang, Xiao Bo; Rao, Yan; Zhang, Xiao Ming; Yin, Yue Lan; Pan, Zhi Ming; Jiao, Xin An
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is currently the only vaccine available for preventing tuberculosis (TB), however, BCG has varying success in preventing pulmonary TB. In this study, a recombinant BCG (rBCG::Ag85A) strain overexpressing the immunodominant Ag85A antigen was constructed, and its immunogenicity and protective efficacy were evaluated. Our results indicated that the Ag85A protein was successfully overexpressed in rBCG::Ag85A, and the Ag85A peptide-MHC complexes on draining lymph node dendritic cells of C57BL/6 mice infected with rBCG::Ag85A were detectable 4 h post-infection. The C57BL/6 mice infected with this strain had stronger antigen-specific interferon-gamma (IFN-γ) responses and higher antibody titers than those immunized with BCG, and the protective experiments showed that rBCG::Ag85A can enhance protection against Mycobacterium tuberculosis (M. tuberculosis) H37Rv infection compared to the BCG vaccine alone. Our results demonstrate the potential of rBCG::Ag85A as a candidate vaccine against TB.
Gengenbacher, Martin; Nieuwenhuizen, Natalie; Vogelzang, Alexis; Liu, Haipeng; Kaiser, Peggy; Schuerer, Stefanie; Lazar, Doris; Wagner, Ina; Mollenkopf, Hans-Joachim
ABSTRACT The current tuberculosis (TB) vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), provides insufficient protection against pulmonary TB. Previously, we generated a listeriolysin-expressing recombinant BCG strain, which to date has successfully completed phase I and phase IIa clinical trials. In an attempt to further improve efficacy, we deleted the antiapoptotic virulence gene nuoG, encoding NADH dehydrogenase 1 subunit G, from BCG ΔureC::hly. In vitro, deletion of nuoG unexpectedly led to strongly increased recruitment of the autophagosome marker LC3 to the engulfed vaccine, suggesting that nuoG also affects xenophagic pathways. In mice, BCG ΔureC::hly ΔnuoG vaccination was safer than BCG and improved protection over that of parental BCG ΔureC::hly, significantly reducing TB load in murine lungs, ameliorating pulmonary pathology, and enhancing immune responses. Transcriptome analysis of draining lymph nodes after vaccination with either BCG ΔureC::hly or BCG ΔureC::hly ΔnuoG demonstrated earlier and stronger induction of immune responses than that with BCG SSI and suggested upregulation of inflammasome activation and interferon-induced GTPases. In summary, BCG ΔureC::hly ΔnuoG is a promising next-generation TB vaccine candidate with excellent efficacy and safety. PMID:27222470
Shkurupii, V A; Kim, L B; Potapova, O V; Sharkova, T V; Putyatina, A N; Nikonova, I K
Generalized BCG-induced granulomatous was simulated in BALB/c male mice. The number of tuberculous granulomas in the liver and their size as well as the number of hepatocytes showing vacuolar degeneration increased from day 3 to 180 postinfection. Necrotic changes in hepatocytes were most pronounced at the acute phase of inflammation (days 3 to 30). Proliferative processes in the liver parenchyma in the experimental group were less marked than in the control. Increased content of collagen fibers in the liver was determined by excessive collagen synthesis in necrotic areas as well as increased amount of granulomas and fibroblasts. Enhanced proliferative and fibroplastic activity of fibroblasts in granulomas and liver parenchyma was evidently determined by activated granuloma macrophages. These shifts determined changes in the liver content of hydroxyproline during the acute and chronic periods of the disease.
Kim, L B; Shkurupy, V A; Putyatina, A N
Experimental BCG-induced granulomatosis in mice was used to study changes in the dynamics of individual liver proteoglycan components reflecting phasic extracellular matrix remodeling, determined by the host-parasite interaction and associated with granuloma development. In the early BCG-granulomatosis period, the increase in individual proteoglycan components promotes granuloma formation, providing conditions for mycobacteria adhesion to host cells, migration of phagocytic cells from circulation, and cell-cell interaction leading to granuloma development and fibrosis. Later, reduced reserve capacity of the extracellular matrix, development of interstitial fibrosis and granuloma fibrosis can lead to trophic shortage for cells within the granulomas, migration of macrophages out of them, and development of spontaneous necrosis and apoptosis typical of tuberculosis.
Induction of Unconventional T Cells by a Mutant Mycobacterium bovis BCG Strain Formulated in Cationic Liposomes Correlates with Protection against Mycobacterium tuberculosis Infections of Immunocompromised Mice
Yabe, Idalia; Morris, Sheldon; Cowley, Siobhan
Earlier studies aimed at defining protective immunity induced by Mycobacterium bovis BCG immunization have largely focused on the induction of antituberculosis CD4+ and CD8+ T cell responses. Here we describe a vaccine consisting of a BCGΔmmaA4 deletion mutant formulated in dimethyl dioctadecyl-ammonium bromide (DDA) with d-(+)-trehalose 6,6′-dibehenate (TDB) (DDA/TDB) adjuvant (A4/Adj) that protected TCRδ−/− mice depleted of CD4+, CD8+, and NK1.1+ T cells against an aerosol challenge with M. tuberculosis. These mice were significantly protected relative to mice immunized with a nonadjuvanted BCGΔmmaA4 (BCG-A4) mutant and nonvaccinated controls at 2 months and 9 months postvaccination. In the absence of all T cells following treatment with anti-Thy1.2 antibody, the immunized mice lost the ability to control the infection. These results indicate that an unconventional T cell population was mediating protection in the absence of CD4+, CD8+, NK1.1+, and TCRγδ T cells and could exhibit memory. Focusing on CD4− CD8− double-negative (DN) T cells, we found that these cells accumulated in the lungs postchallenge significantly more in A4/Adj-immunized mice and induced significantly greater frequencies of pulmonary gamma interferon (IFN-γ)-producing cells than were seen in the nonvaccinated or nonadjuvanted BCG control groups. Moreover, pulmonary DN T cells from the A4/Adj group exhibited significantly higher IFN-γ integrated median fluorescence intensity (iMFI) values than were seen in the control groups. We also showed that enriched DN T cells from mice immunized with A4/Adj could control mycobacterial growth in vitro significantly better than naive whole-spleen cells. These results suggest that formulating BCG in DDA/TDB adjuvant confers superior protection in immunocompromised mice and likely involves the induction of long-lived memory DN T cells. PMID:27226281
Goulart, Cibelly; Rodriguez, Dunia; Kanno, Alex I; Converso, Thiago Rojas; Lu, Ying-Jie; Malley, Richard; Leite, Luciana C C
Pneumococcal diseases remain a substantial cause of mortality in young children in developing countries. The development of potentially serotype-transcending vaccines has been extensively studied; ideally, such a vaccine should include antigens that are able to induce protection against colonization (likely mediated by interleukin-17A [IL-17A]) and invasive disease (likely mediated by antibody). The use of strong adjuvants or alternative delivery systems that are able to improve the immunological response of recombinant proteins has been proposed but poses potential safety and practical concerns in children. We have previously constructed a recombinant Mycobacterium bovis BCG strain expressing a pneumococcal surface protein A (PspA)-PdT fusion protein (rBCG PspA-PdT) that was able to induce an effective immune response and protection against sepsis in a prime-boost strategy. Here, we constructed two new rBCG strains expressing the pneumococcal proteins SP 0148 and SP 2108, which confer IL-17A-dependent protection against pneumococcal colonization in mouse models. Immunization of mice with rBCG 0148 or rBCG 2108 in a prime-boost strategy induced IL-17A and gamma interferon (IFN-γ) production. The combination of these rBCG strains with rBCG PspA-PdT (rBCG Mix), followed by a booster dose of the combined recombinant proteins (rMix) induced an IL-17A response against SP 0148 and SP 2108 and a humoral response characterized by increased levels of IgG2c against PspA and functional antibodies against pneumolysin. Furthermore, immunization with the rBCG Mix prime/rMix booster (rBCG Mix/rMix) provides protection against pneumococcal colonization and sepsis. These results suggest the use of combined rBCG strains as a potentially serotype-transcending pneumococcal vaccine in a prime-boost strategy, which could provide protection against pneumococcal colonization and sepsis. Copyright © 2017 American Society for Microbiology.
Tokunaga, T; Yano, O; Kuramoto, E; Kimura, Y; Yamamoto, T; Kataoka, T; Yamamoto, S
Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.
Derrick, Steven C.; Dao, Dee; Yang, Amy; Kolibab, Kris; Jacobs, William R.; Morris, Sheldon L.
A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) – D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe
Sharma, Deepika; Tiwari, Brijendra Kumar; Mehto, Subhash; Antony, Cecil; Kak, Gunjan; Singh, Yogendra; Natarajan, Krishnamurthy
The prevalence of Mycobacterium tuberculosis (M. tb) strains eliciting drug resistance has necessitated the need for understanding the complexities of host pathogen interactions. The regulation of calcium homeostasis by Voltage Gated Calcium Channel (VGCCs) upon M. tb infection has recently assumed importance in this area. We previously showed a suppressor role of VGCC during M. tb infections and recently reported the mechanisms of its regulation by M. tb. Here in this report, we further characterize the role of VGCC in mediating defence responses of macrophages during mycobacterial infection. We report that activation of VGCC during infection synergistically downmodulates the generation of oxidative burst (ROS) by macrophages. This attenuation of ROS is regulated in a manner which is dependent on Toll like Receptor (TLR) and also on the route of calcium influx, Protein Kinase C (PKC) and by Mitogen Activation Protein Kinase (MAPK) pathways. VGCC activation during infection increases cell survival and downmodulates autophagy. Concomitantly, pro-inflammatory responses such as IL-12 and IFN-γ secretion and the levels of their receptors on cell surface are inhibited. Finally, the ability of phagosomes to fuse with lysosomes in M. bovis BCG and M. tb H37Rv infected macrophages is also compromised when VGCC activation occurs during infection. The results point towards a well-orchestrated strategy adopted by mycobacteria to supress protective responses mounted by the host. This begins with the increase in the surface levels of VGCCs by mycobacteria and their antigens by well-controlled and regulated mechanisms. Subsequent activation of the upregulated VGCC following tweaking of calcium levels by molecular sensors in turn mediates suppressor responses and prepare the macrophages for long term persistent infection. PMID:27723836
Brynjolfsson, Siggeir F; Bjarnarson, Stefania P; Mori, Elena; Del Giudice, Giuseppe; Jonsdottir, Ingileif
Mycobacterium bovis BCG is administered to human neonates in many countries worldwide. The objective of the study was to assess if BCG could act as an adjuvant for polysaccharide-protein conjugate vaccines in newborns and thereby induce protective immunity against encapsulated bacteria in early infancy when susceptibility is high. We assessed whether BCG could enhance immune responses to a meningococcal C (MenC) conjugate vaccine, MenC-CRM(197), in mice primed as neonates, broaden the antibody response from a dominant IgG1 toward a mixed IgG1 and IgG2a/IgG2b response, and increase protective efficacy, as measured by serum bactericidal activity (SBA). Two-week-old mice were primed subcutaneously (s.c.) with MenC-CRM(197). BCG was administered concomitantly, a day or a week before MenC-CRM(197). An adjuvant effect of BCG was observed only when it was given concomitantly with MenC-CRM(197), with increased IgG response (P = 0.002) and SBA (8-fold) after a second immunization with MenC-CRM(197) without BCG, indicating increased T-cell help. In neonatal mice (1 week old) primed s.c. with MenC-CRM(197) together with BCG, MenC-polysaccharide (PS)-specific IgG was enhanced compared to MenC-CRM(197) alone (P = 0.0015). Sixteen days after the second immunization with MenC-CRM(197), increased IgG (P < 0.05), IgG1 (P < 0.05), IgG2a (P = 0.06), and IgG2b (P < 0.05) were observed, and only mice primed with MenC-CRM(197) plus BCG showed affinity maturation and detectable SBA (SBA > 128). Thus, vaccination with a meningococcal conjugate vaccine (and possibly with other conjugates) may benefit from concomitant administration of BCG in the neonatal period to accelerate and enhance production of protective antibodies, compared to the current infant administration of conjugate which follows BCG vaccination at birth.
Ni, Weihua; Wang, Fang; Liu, Guomu; Zhang, Nannan; Yuan, Hongyan; Jie, Jing; Tai, Guixiang
Our previous study demonstrated that maltose-binding protein (MBP) combined with BCG induced synergistic mouse Th1 activation in vivo. Here, to explore the mechanism of MBP combined with BCG on Th1 activation, mouse purified CD4(+) T cells were stimulated with MBP and BCG in vitro. The results showed that MBP combined with BCG synergistically increased IFN-γ production, accompanied with the upregulation of TLR2/9 expressions, suggesting that TLR2/9 were involved in the combination-induced Th1 activation. Next, TLR2 antibodies and TLR9 inhibitor were used to further analyze the effects of TLRs in Th1 activation. Results showed TLR2 antibody partly decreased MBP combined with BCG-induced IFN-γ production, MyD88 expression and IκB phosphorylation, indicating that TLR2-mediated MyD88-dependent pathway was involved in the MBP combined with BCG-induced Th1 activation. Moreover, MBP combined with BCG-induced Th1 activation was completely abrogated by TLR9 inhibitor, suggesting that TLR9-mediated MyD88-dependent pathway played a more important role than TLR2 in the combination-induced Th1 activation. Further study showed that TLR9 inhibitor downregulated TLR2 expression, suggesting that TLR9 signaling regulated TLR2 activation to favor Th1 resonse induced by MBP combined with BCG. Collectively, we demonstrated for the first time that the cross-talk of TLR2 and TLR9 triggered Th1 activation collaboratively and our findings provided valuable information about designing more effective adjuvant for cancer therapy.
Méndez-Samperio, Patricia; Alba, Laura; Trejo, Artemisa
Induction of human beta defensin-2 (HBD-2) by mycobacteria has been reported. However, the molecular mechanism(s) by which mycobacteria up-regulates HBD-2 gene expression in epithelial cells remains poorly understood. In this work, we provide evidence that the induction of HBD-2 mRNA in response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) was inhibited in a dose-dependent manner by pretreatment with a cell-permeable BAPTA-AM, which chelates intracellular calcium. Our data also demonstrate that HBD-2 mRNA induction by M. bovis in A549 lung epithelial cells requires activation of calmodulin. Interestingly, HBD-2 mRNA expression in response to M. bovis BCG was attenuated by pretreatment with SB203580 (an inhibitor of p38 mitogen-activated protein kinase [MAPK]), but not by an inhibitor of extracellular signal-regulated kinase (ERK): PD98059. Furthermore, we found that a second p38 MAPK inhibitor (SB202190) significantly blocked M. bovis BCG-mediated HBD-2 induction in A549 lung epithelial cells. Together, these data suggest that M. bovis BCG induces HBD-2 mRNA expression in A549 lung epithelial cells at least in part mediated through intracellular calcium flux as well as activation of signaling protein of p38MAPK, but not ERK.
Acosta, Fermín; Chernyaeva, Ekatherina; Mendoza, Libardo; Sambrano, Dilcia; Correa, Ricardo; Rotkevich, Mikhail; Tarté, Miroslava; Hernández, Humberto; Velazco, Bredio; de Escobar, Cecilia; de Waard, Jacobus H.
Panama remains free of zoonotic tuberculosis caused by Mycobacterium bovis. However, DNA fingerprinting of 7 M. bovis isolates from a 2013 bovine tuberculosis outbreak indicated minimal homology with strains previously circulating in Panama. M. bovis dispersion into Panama highlights the need for enhanced genotype testing to track zoonotic infections. PMID:25988479
Guerrero, G. G.; Rangel-Moreno, J.; Islas-Trujillo, S.; Rojas-Espinosa, Ó.
Leprosy caused by Mycobacterium leprae primarily affects the skin and peripheral nerves. As a human infectious disease, it is still a significant health and economic burden on developing countries. Although multidrug therapy is reducing the number of active cases to approximately 0.5 million, the number of cases per year is not declining. Therefore, alternative host-directed strategies should be addressed to improve treatment efficacy and outcome. In this work, using murine leprosy as a model, a very similar granulomatous skin lesion to human leprosy, we have found that successive IFN-alpha boosting protects BCG-vaccinated mice against M. lepraemurium infection. No difference in the seric isotype and all IgG subclasses measured, neither in the TH1 nor in the TH2 type cytokine production, was seen. However, an enhanced iNOS/NO production in BCG-vaccinated/i.m. IFN-alpha boosted mice was observed. The data provided in this study suggest a promising use for IFN-alpha boosting as a new prophylactic alternative to be explored in human leprosy by targeting host innate cell response. PMID:26484351
Guerrero, G G; Rangel-Moreno, J; Islas-Trujillo, S; Rojas-Espinosa, Ó
Leprosy caused by Mycobacterium leprae primarily affects the skin and peripheral nerves. As a human infectious disease, it is still a significant health and economic burden on developing countries. Although multidrug therapy is reducing the number of active cases to approximately 0.5 million, the number of cases per year is not declining. Therefore, alternative host-directed strategies should be addressed to improve treatment efficacy and outcome. In this work, using murine leprosy as a model, a very similar granulomatous skin lesion to human leprosy, we have found that successive IFN-alpha boosting protects BCG-vaccinated mice against M. lepraemurium infection. No difference in the seric isotype and all IgG subclasses measured, neither in the TH1 nor in the TH2 type cytokine production, was seen. However, an enhanced iNOS/NO production in BCG-vaccinated/i.m. IFN-alpha boosted mice was observed. The data provided in this study suggest a promising use for IFN-alpha boosting as a new prophylactic alternative to be explored in human leprosy by targeting host innate cell response.
Moura, A C; Werneck-Barroso, E; Rosas, E C; Henriques, M G; Cordeiro, R S
Mycobacteria as intracellular pathogens have evolved mechanisms to survive within macrophages. Our previous data showed that M. leprae (ML), unlike M. bovis BCG, did not induce an inflammatory response in the mice subcutaneous tissue. Further, ML inhibited BCG-induced foot pad oedema and seemed to transform macrophages in epithelioid cells. Since these mycobacteria share common antigens, here we seeked to compare the acute and chronic cellular response evoked by ML and BCG in pleurisy of a mycobacteria-susceptible mice (BALB/c). The total leukocytes, the cell type that migrated to the pleural cavity and macrophage activation assayed by nitric oxide release were determined. Live or dead BCG Moreau recruited the same extent of cells, essentially monocytes and neutrophils, dose-dependently, in both acute and chronic pleurisy. BCG-induced eosinophilia was observed only in the acute response (after 24 h of injection). A significant nitric oxide release by pleural macrophages was triggered by BCG Moreau without previous activation. Nevertheless, ML failed to recruit leukocytes to the pleural space or to lead to nitric oxide production despite the number of bacilli used and the time studied (1, 7 or 14 days after injection). Although these mycobacteria have common antigens that cross-react, these data show a distinct ability of ML or BCG to recruit cells to the pleural space and to activate pleural macrophage for nitric oxide production in vivo.
Mycoplasma bovis in bison is a newly emerging and potentially devastating threat to the bison industry. This bacterium is increasingly being identified, both in the United States and Canada, as the cause of severe respiratory disease outbreaks with devastating consequences for the health of the ani...
Research conducted at the USDA/ARS/National Animal Disease Center in Ames, Iowa, reveals that ELISAs designed for detection of M. bovis-specific serum IgG in cattle may not be optimal for identification of seropositive bison, particularly those with low to moderate levels of antibody. In a study so...
Buddle, B M; Aldwell, F E; Keen, D L; Parlane, N A; Hamel, K L; de Lisle, G W
. In Study 2, oral administration of Danish BCG induced protection against challenge with M. bovis, which persisted for at least 54 weeks after vaccination. Some protection was observed in possums challenged 54 weeks after vaccination, but this protection was significantly less than that observed in groups vaccinated 29 or 8 weeks prior to challenge. There was a strong relationship between the proportion of animals producing positive lymphocyte proliferation responses to M. bovis antigens and protection against challenge with M. bovis. Factors considered potentially capable of interfering with vaccination, including feeding dead BCG to possums prior to feeding live BCG, feeding multiple doses of BCG at one time, and changing strains of BCG, were shown not to interfere with the acquisition of protective immune responses in possums. Protection against tuberculosis was undiminished up to 29 weeks after vaccination with BCG administered orally. It is concluded that vaccination of possums by feeding pellets containing BCG is a robust and efficient approach to enhance the resistance of these animals to tuberculosis.
Martino, Angelo; Casetti, Rita; Poccia, Fabrizio
Since drug-activated gammadelta T cells promote dendritic cell (DC) maturation, we analyzed the effect of combining gammadelta T cell specific drugs with BCG in vitro. BCG-induced DC maturation was increased by bromohydrin-pirophosphate (BrHPP) or zoledronate (Zol)-activated gammadelta T cells. Specifically, the co-culture with activated Vgamma9Vdelta2 T cells with BCG-infected DC resulted in a significant increase of the expression of CD80, CD86, CD40 and CD25 molecules on DC. Moreover, DC were able to produce increased levels of TNF-alpha and synthesize ex novo IL-15 without altering the IL-10/IL-12 immunoregulatory pathway. Finally, the Th1 immunity induced by BCG-infected DC on naïve CD4 T cells was increased by gammadelta T cell activation with BrHpp or Zol. These data indicate that gammadelta T cell triggering drugs could be used to enhance the BCG induced Th1 immunity.
Menezes-de-Lima-Júnior, O; Werneck-Barroso, E; Cordeiro, R S; Henriques, M G
In this work we characterize the Mycobacterium bovis bacillus Calmette-Guerin (BCG) -induced pleurisy and investigate the role of chemical mediators and cytokines in BCG-induced granulocyte accumulation at 24 h. Intrathoracic injection of BCG in C57B1/6 mice induces a biphasic inflammatory reaction with intense leukocyte accumulation at 24 h and 15 days. Neutrophils were observed in the pleural cavity at 4-24 h, mononuclear cells and eosinophils after 24 h. A new wave of mononuclear cells and neutrophils were observed after 15 days. Pretreatments with dexamethasone, BW 755C, BW A4C, WEB 2170, L-NAME, and monoclonal antibody (mAb) anti-interleukin-5 (IL-5; TRFK-5) had inhibited the eosinophil accumulation. On the other hand, only the pretreatments with dexamethasone, L-NAME, or mAb anti-tumor necrosis factor alpha (TNF-alpha; MP6-XT3) had inhibited the neutrophil influx. These results suggest the involvement of leukotrienes, platelet-activating factor, nitric oxide, and IL-5 in the eosinophil accumulation, and a role for nitric oxide and TNF-alpha in the neutrophil influx induced by BCG.
Garcia-Pelayo, M Carmen; Bachy, Véronique S; Kaveh, Daryan A; Hogarth, Philip J
It is generally assumed that the inbred mouse strains BALB/c (H-2(d)) and C57BL/6 (H-2(b)) respond to mycobacterial infection with distinct polarisation of T helper responses, with C57BL/6 predisposed to Th1 and BALB/c to Th2. We investigated this in a BCG-immunisation, Mycobacterium bovis challenge model. Following immunisation, lung and spleen cell cytokine responses to in vitro re-stimulation with a cocktail of seven secreted, immunogenic, recombinant mycobacterial proteins were determined. In both lung and spleen, BALB/c cells produced at least 2-fold more IFN-γ, and up to 7-fold more IL-2 and IL-17 than C57BL/6 cells, whereas IL-10 production was reciprocally increased in C57BL/6 mice. These data suggest that, contrary to reports in the literature, specific mycobacterial antigens are able to induce strong Th1 and Th17 responses in BALB/c mice following BCG vaccination, whilst in C57BL/6 mice, the Th1 response is partly counterbalanced by IL-10. After subsequent M. bovis low dose challenge, protection, as measured in the lungs and dissemination to the spleen, was equivalent in BALB/c and C57BL/6 mice, indicating that BCG-induced immunity was equivalent in both strains. Thus, the differential immune responses do not appear to have a role in protection, but further, as yet unidentified, specific immune responses play a significant role. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.
Kim, L B; Shkurupy, V A; Putyatina, A N
Experiments on the model of mouse BCG-induced granulomatous showed that the content of glycosaminoglycans and proteoglycans in the extracellular matrix of the liver and lungs are changed at the early stages of inflammation (days 3 and 30 postinfection) before cell destruction in the organs begins. This is related to degradation of extracellular matrix structures. Their high content in the blood and interstitium probably contributes to the formation of granulomas, fibroblast proliferation and organ fibrosis. These processes depend on the infection route that determines different conditions for generalization of the inflammation process. Intravenous method of vaccine injection is preferable to use when designing the experiments simulating tuberculosis granulomatosis, especially for the analysis of its early stages.
Song, Tengfei; Dong, Chunsheng; Xiong, Sidong
Tuberculosis, which is caused by intracellular mycobacterium Mycobacterium tuberculosis (Mtb), remains one of the most serious global public health concerns. The mechanisms by which innate immunity regulates the inflammatory responses and affects mycobacterial infection remain unclear. In this study, signaling lymphocyte-activation molecule family 1 (SLAMF1) was significantly upregulated in Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected RAW264.7 cells. Overexpression of SLAMF1 significantly increased the production of inflammatory factors TNF-α and IL-1β, as well as chemokine MCP-1, both in vitro and in vivo upon mycobacteria BCG infection. By contrast, knockdown of SLAMF1 significantly decreased the production of TNF-α, IL-1β, and MCP-1. Western blot analysis indicated that the NF-κB signaling pathway may contribute to the elevated inflammatory response promoted by SLAMF1, as evidenced by higher levels of phosphorylated p65 and IκBα detected with SLAMF1 overexpression. Furthermore, SLAMF1 upregulation facilitated bacterial clearance in infected RAW264.7 cells and in the lungs of infected mice. In conclusion, we demonstrated that BCG infection significantly upregulated SLAMF1, which enhanced inflammatory response by activating the NF-κB signaling pathway and facilitated bacterial clearance in BCG-infected RAW264.7 cells and mice.
Carlétti, Dyego; Morais da Fonseca, Denise; Gembre, Ana Flávia; Masson, Ana Paula; Weijenborg Campos, Lívia; Leite, Luciana C C; Rodrigues Pires, Andréa; Lannes-Vieira, Joseli; Lopes Silva, Célio; Bonato, Vânia Luiza Deperon; Horn, Cynthia
Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6'-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB.
Carlétti, Dyego; Morais da Fonseca, Denise; Gembre, Ana Flávia; Masson, Ana Paula; Weijenborg Campos, Lívia; Leite, Luciana C. C.; Rodrigues Pires, Andréa; Lannes-Vieira, Joseli; Lopes Silva, Célio; Bonato, Vânia Luiza Deperon
Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6′-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB. PMID:23740922
Shibata, Yoshimi; Gabbard, Jon; Yamashita, Makiko; Tsuji, Shoutaro; Smith, Mike; Nishiyama, Akihito; Henriksen, Ruth Ann; Myrvik, Quentin N
Previous studies have shown that prostaglandin E(2) (PGE(2)) release by splenic F4/80(+) cyclooxygenase (COX)-2(+) macrophages (MØ) isolated from mice, treated with mycobacterial components, plays a major role in the regulation of immune responses. However, splenic MØ, isolated from untreated mice and treated in vitro with lipopolysaccharide and interferon-gamma, express COX-1 and COX-2 within 1 day but release only minimal amounts of PGE(2) following elicitation with calcium ionophore A23187. For further characterization of in vivo requirements for development of PGE(2)-releasing MØ (PGE(2)-MØ), C57Bl/6 [wild-type (WT)], and interleukin (IL)-10-deficient (IL-10(-/-)) mice were treated intraperitoneally with heat-killed Mycobacterium bovis bacillus Calmette-Guerin (HK-BCG). One day following injection, COX-2 was induced in splenic MØ of both mouse strains. However, PGE(2) biosynthesis by these MØ was not increased. Thus, expression of COX-2 is not sufficient to induce PGE(2) production in vivo or in vitro. In sharp contrast, 14 days after HK-BCG treatment, PGE(2) release by COX-2(+) splenic MØ increased as much as sevenfold, and a greater increase was seen in IL-10(-/-) cells than in WT cells. To further determine whether the 14-day splenic PGE(2)-MØ could be derived from bone marrow precursors, we established a chimera in which bone marrow cells were transfused from green fluorescent protein (GFP)-transgenic donors to WT mice. Donors and recipients were treated with HK-BCG simultaneously, and marrow transfusion was performed on Days 1 and 2. On Day 14 after BCG treatment, a significant number of spleen cells coexpressed COX-2 and GFP, indicating that bone marrow-derived COX-2(+) MØ may be responsible for the increased PGE(2) production.
Palavecino, Christian E; Céspedes, Pablo F; Gómez, Roberto S; Kalergis, Alexis M; Bueno, Susan M
Along with the human respiratory syncytial virus (hRSV), the human metapneumovirus (hMPV) is one of the leading causes of childhood hospitalization and a major health burden worldwide. Unfortunately, owing to an inefficient immunological memory, hMPV infection provides limited immune protection against reinfection. Furthermore, hMPV can induce an inadequate Th2 type immune response that causes severe lung inflammation, leading to airway obstruction. Similar to hRSV, it is likely that an effective clearance of hMPV would require a balanced Th1 type immunity by the host, involving the activation of IFN-γ-secreting T cells. A recognized inducer of Th1 immunity is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which has been used in newborns for many decades and in several countries as a tuberculosis vaccine. We have previously shown that immunization with BCG strains expressing hRSV Ags can induce an efficient immune response that protects against this virus. In this study, we show that immunization with rBCG strains expressing the phosphoprotein from hMPV also can induce protective Th1 immunity. Mice immunized with rBCG were protected against weight loss, airway inflammation, and viral replication in the lungs after hMPV infection. Our rBCG vaccine also induced the activation of hMPV-specific T cells producing IFN-γ and IL-2, which could protect from hMPV infection when transferred to recipient mice. These data strongly support the notion that rBCG induces protective Th1 immunity and could be considered as an efficient vaccine against hMPV.
Jiang, LiNa; Liu, GuoMu; Ni, WeiHua; Zhang, NanNan; Jie, Jing; Xie, Fei
To explore whether TLR2/TLR4 could be involved in the maturation of dendritic cells and polarization of CD4+ T cells induced by dendritic cells stimulated with MBP and BCG, in vitro and in vivo experiments using TLR2−/− or TLR4−/− mice were employed. MBP and BCG elevated CD80, CD86 and MHC class II expressed on dendritic cells and increased IL-12 protein, induced DC maturation, and indirectly promoted Th1 activation. Moreover, MBP and BCG upregulated costimulatory molecules on DCs in a TLR2- and TLR4-dependent manner. The levels of IFN-γ, IL-4, and IL-10 in CD4+ T cells cocultured with dendritic cells from different types of mice were determined with ELISPOT or ELISA method. TLR2/TLR4 is important in the maturation and activation of dendritic cells and the activation of Th1 cells induced by stimulation with MBP and BCG. In conclusion, TLR2 and TLR4 play an important role in the upregulation of costimulatory molecules and MHC class II molecules on dendritic cells and the activation of Th1 cells induced by stimulation with MBP and BCG. The results above indicate that the combination of MBP and BCG induced the maturation and activation of dendritic cells and promoted Th1 activation via TLR2/TLR4. PMID:28293065
Spencer, Charles Thomas; Abate, Getahun; Blazevic, Azra; Hoft, Daniel F.
Mycobacterium tuberculosis and M. bovis-BCG induce potent expansions of human memory Vγ9+Vδ2+ T cells capable of IFN-γ production, cytolytic activity and mycobacterial growth inhibition. Certain phosphoantigens expressed by mycobacteria can stimulate γ9δ2 T cell expansions, suggesting that purified or synthetic forms of these phosphoantigens may be useful alone or as components of new vaccines or immunotherapeutics. However, we show that while mycobacteria-activated γ9δ2 T cells potently inhibit intracellular mycobacterial growth, phosphoantigen-activated γ9δ2 T cells fail to inhibit mycobacteria, although both develop similar effector cytokine and cytolytic functional capacities. γ9δ2 T cells receiving TLR-mediated co-stimulation during phosphoantigen activation also failed to inhibit mycobacterial growth. We hypothesized that mycobacteria express antigens, other than the previously identified phosphoantigens, that induce protective subsets of γ9δ2 T cells. Testing this hypothesis, we compared the TCR sequence diversity of γ9δ2 T cells expanded with BCG-infected versus phosphoantigen-treated DC. BCG-stimulated γ9δ2 T cells displayed a more restricted TCR diversity than phosphoantigen-activated γ9δ2 T cells. In addition, only a subset of phosphoantigen-activated γ9δ2 T cells functionally responded to mycobacteria-infected DC. Furthermore, differential inhibitory functions of BCG- and phosphoantigen-activated γ9δ2 T cells were confirmed at the clonal level and were not due to differences in TCR avidity. Our results demonstrate that BCG infection can activate and expand protective subsets of phosphoantigen responsive γ9δ2 T cells, and provide the first indication that γ9δ2 T cells can develop pathogen specificity similar to αβ T cells. Specific targeting of protective γ9δ2 T cell subsets will be important for future tuberculosis vaccines. PMID:18802050
Rizzi, Caroline; Peiter, Ana Carolina; Oliveira, Thaís Larré; Seixas, Amilton Clair Pinto; Leal, Karen Silva; Hartwig, Daiane Drawanz; Seixas, Fabiana Kommling; Borsuk, Sibele; Dellagostin, Odir Antônio
BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems. PMID:28177046
Ancelet, Lindsay R.; Aldwell, Frank E.; Rich, Fenella J.; Kirman, Joanna R.
Oral delivery of BCG in a lipid formulation (Liporale™-BCG) targets delivery of viable bacilli to the mesenteric lymph nodes and confers protection against an aerosol Mycobacterium tuberculosis challenge. The magnitude, quality and duration of the effector and memory immune response induced by Liporale™-BCG vaccination is unknown. Therefore, we compared the effector and memory CD4+ T cell response in the spleen and lungs of mice vaccinated with Liporale™-BCG to the response induced by subcutaneous BCG vaccination. Liporale™-BCG vaccination induced a long-lived CD4+ T cell response, evident by the detection of effector CD4+ T cells in the lungs and a significant increase in the number of Ag85B tetramer-specific CD4+ T cells in the spleen up to 30 weeks post vaccination. Moreover, following polyclonal stimulation, Liporale™-BCG vaccination, but not s.c. BCG vaccination, induced a significant increase in both the percentage of CD4+ T cells in the lungs capable of producing IFNγ and the number of multifunctional CD4+ T cells in the lungs at 30 weeks post vaccination. These results demonstrate that orally delivered Liporale™-BCG vaccine induces a long-lived multifunctional immune response, and could therefore represent a practical and effective means of delivering novel BCG-based TB vaccines. PMID:23049885
Hervas-Stubbs, Sandra; Majlessi, Laleh; Simsova, Marcela; Morova, Jana; Rojas, Marie-Jesus; Nouzé, Clémence; Brodin, Priscille; Sebo, Peter; Leclerc, Claude
TB10.4 is a newly identified antigen of Mycobacterium tuberculosis recognized by human and murine T cells upon mycobacterial infection. Here, we show that immunization with Mycobacterium bovis BCG induces a strong, genetically controlled, Th1 immune response against TB10.4 in mice. BALB/c and C57BL/6 strains behave as high and low responders to TB10.4 protein, respectively. The TB10.4:74-88 peptide was identified as an immunodominant CD4+ T-cell epitope for H-2d mice. Since recent results, as well as the present study, have raised interest in TB10.4 as a subunit vaccine, we analyzed immune responses induced by this antigen delivered by a new vector, the adenylate cyclase (CyaA) of Bordetella pertussis. CyaA is able to target dendritic cells and to deliver CD4+ or CD8+ T-cell epitopes to the major histocompatibility complex class II/I molecule presentation pathways, triggering specific Th1 or cytotoxic T-lymphocyte (CTL) responses. Several CyaA harboring either the entire TB10.4 protein or various subfragments containing the TB10.4:20-28 CTL epitope were shown to induce TB10.4-specific Th1 CD4+ and CD8+ T-cell responses. However, none of the recombinant CyaA, injected in the absence of adjuvant, was able to induce protection against M. tuberculosis infection. In contrast, TB10.4 protein administered with a cocktail of strong adjuvants that triggered a strong Th1 CD4+ T-cell response induced significant protection against M. tuberculosis challenge. These results confirm the potential value of the TB10.4 protein as a candidate vaccine and show that the presence of high frequencies of CD4+ T cells specific to this strong immunogen correlates with protection against M. tuberculosis infection. PMID:16714570
Pérez de Val, Bernat; Vidal, Enric; López-Soria, Sergio; Marco, Alberto; Cervera, Zoraida; Martín, Maite; Mercader, Irene; Singh, Mahavir; Raeber, Alex; Domingo, Mariano
Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed. BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination. BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible. Copyright © 2016 Elsevier Ltd. All rights reserved.
Efficacy of Oral and Parenteral Routes of Mycobacterium bovis bacille Calmette-Guerin Vaccination Against Experimental Bovine Tuberculosis in White-tailed deer (Odocoileus virginianus): A Feasibility Study
We investigated the efficacy of oral and parenteral Mycobacterium bovis bacille Calmette-Guerin Danish strain 1331 (BCG) in its ability to protect white-tailed deer (Odocoileus virginianus) against disease caused by M. bovis infection. Thirty white-tailed deer were divided into four groups. One gr...
The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In thi...
Souza, Mariana C.; Penido, Carmen; Costa, Maria F. S.; Henriques, Maria Graças
Tuberculous pleurisy is a frequent extrapulmonary manifestation characterized by accumulation of fluid and inflammatory cells in the pleural space. Here, we investigated the mechanisms of T-lymphocyte accumulation in the pleural space by using a murine model of pleurisy induced by Mycobacterium bovis BCG. Intrathoracic (i.t.) injection of BCG (4.5 × 105 bacteria/cavity) induced accumulation of T lymphocytes in the pleural cavities of C57BL/6 mice. We observed the presence of CFU in pleural washes conducted 1, 2, 3, 7, and 15 days after pleurisy induction. Pretreatment with fucoidan inhibited T-lymphocyte accumulation at 1 day, but not at 15 days, after BCG-induced pleurisy. Accordingly, adoptive transfer of fluorescein isothiocyanate-labeled blood mononuclear cells to infected mice showed that T lymphocytes migrated into the pleural cavity 1 day (but not 15 days) after BCG injection. Cell-free pleural wash fluids recovered from mice 1 day after BCG i.t. stimulation (day 1 BCG-PW), but not day 7 or day 15 BCG-PW, induced in vitro T-cell transmigration, which was dependent on L-, P-, and E-selectins. In contrast, day 7 BCG-PW (but not day 1 BCG-PW) induced in vitro T-lymphocyte proliferation via interleukin-2 (IL-2) and gamma interferon (IFN-γ). Accordingly, in vivo IL-2 or IFN-γ neutralization abolished T-lymphocyte accumulation 7 days after pleurisy induction. Our results demonstrate that pleural infection induced by BCG leads to T-lymphocyte accumulation in two waves. The acute phase depends on selectin-mediated migration, while the second wave of T-lymphocyte accumulation seems to depend on a local proliferation induced by cytokines produced in situ. PMID:18809659
Aranaz, Alicia; de Juan, Lucía; Montero, Natalia; Sánchez, Celia; Galka, Margarita; Delso, Consuelo; Álvarez, Julio; Romero, Beatriz; Bezos, Javier; Vela, Ana I.; Briones, Victor; Mateos, Ana; Domínguez, Lucas
Mycobacterium bovis infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovis through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (Cervus elaphus), fallow deer (Dama dama), wild boar (Sus scrofa), Iberian lynx (Lynx pardina), hare (Lepus europaeus), and cattle (Bos taurus). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovis strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures. PMID:15184440
Oldiges, Daiane P.; Laughery, Jacob M.; Tagliari, Nelson Junior; Leite Filho, Ronaldo Viana; Davis, William C.; da Silva Vaz, Itabajara; Termignoni, Carlos; Knowles, Donald P.; Suarez, Carlos E.
The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves
Mostowy, Serge; Inwald, Jackie; Gordon, Steve; Martin, Carlos; Warren, Rob; Kremer, Kristin; Cousins, Debby; Behr, Marcel A.
Though careful consideration has been placed towards genetic characterization of tubercle bacillus isolates causing disease in humans, those causing disease predominantly among wild and domesticated mammals have received less attention. In contrast to Mycobacterium tuberculosis, whose host range is largely specific to humans, M. bovis and “M bovis-like” organisms infect a broad range of animal species beyond their most prominent host in cattle. To determine whether strains of variable genomic content are associated with distinct distributions of disease, the DNA contents of M. bovis or M. bovis-like isolates from a variety of hosts were investigated via Affymetrix GeneChip. Consistent with previous genomic analysis of the M. tuberculosis complex (MTC), large sequence polymorphisms of putative diagnostic and biological consequence were able to unambiguously distinguish interrogated isolates. The distribution of deleted regions indicates organisms genomically removed from M. bovis and also points to structured genomic variability within M. bovis. Certain genomic profiles spanned a variety of hosts but were clustered by geography, while others associated primarily with host type. In contrast to the prevailing assumption that M. bovis has broad host capacity, genomic profiles suggest that distinct MTC lineages differentially infect a variety of mammals. From this, a phylogenetic stratification of genotypes offers a predictive framework upon which to base future genetic and phenotypic studies of the MTC. PMID:16159772
Garnier, Thierry; Eiglmeier, Karin; Camus, Jean-Christophe; Medina, Nadine; Mansoor, Huma; Pryor, Melinda; Duthoy, Stephanie; Grondin, Sophie; Lacroix, Celine; Monsempe, Christel; Simon, Sylvie; Harris, Barbara; Atkin, Rebecca; Doggett, Jon; Mayes, Rebecca; Keating, Lisa; Wheeler, Paul R.; Parkhill, Julian; Barrell, Bart G.; Cole, Stewart T.; Gordon, Stephen V.; Hewinson, R. Glyn
Mycobacterium bovis is the causative agent of tuberculosis in a range of animal species and man, with worldwide annual losses to agriculture of $3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. M. bovis was also the progenitor for the M. bovis bacillus Calmette–Guérin vaccine strain, the most widely used human vaccine. Here we describe the 4,345,492-bp genome sequence of M. bovis AF2122/97 and its comparison with the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. Strikingly, the genome sequence of M. bovis is >99.95% identical to that of M. tuberculosis, but deletion of genetic information has led to a reduced genome size. Comparison with M. leprae reveals a number of common gene losses, suggesting the removal of functional redundancy. Cell wall components and secreted proteins show the greatest variation, indicating their potential role in host–bacillus interactions or immune evasion. Furthermore, there are no genes unique to M. bovis, implying that differential gene expression may be the key to the host tropisms of human and bovine bacilli. The genome sequence therefore offers major insight on the evolution, host preference, and pathobiology of M. bovis. PMID:12788972
Stothard, J R; Lockyer, A E; Kabatereine, N B; Tukahebwa, E M; Kazibwe, F; Rollinson, D; Fenwick, A
During routine parasitological surveillance and monitoring activities within a National Control Programme for control of human schistosomiasis in Uganda, it was noted that cattle grazing in a water meadow immediately adjacent to Tonya primary school, where the prevalence of intestinal schistosomiasis in children was in excess of 90%, were unusually emaciated. To test the hypothesis that there may have been an anthropozoonotic focus of Schistosoma mansoni within the local herd, a young female heifer, clearly emaciated and c. 8 months old, was slaughtered from which schistosome worms were later recovered by dissection. As female worms inspected by microscopy were not gravid, morphological identification proved inconclusive but analysis of cytochrome oxidase subunit I (COI) and small subunit (SSU) ribosomal DNA sequences from these worms identified them as Schistosoma bovis Sonsino, 1876. This is the first substantiated report of S. bovis from Lake Albert, western Uganda. Further epidemiological surveys are needed to clarify the extent of bovine schistosomiasis within this region, particularly so since this lakeside plain has been earmarked as a future game reserve.
Jang, Hyeung-Jin; Nde, Chantal; Toghrol, Freshteh; Bentley, William E
Mycobacterium bovis BCG strain Pasteur 1173P2 responds with adaptive and protective strategies against oxidative stress. Despite advances in our understanding of the responses to oxidative stress in many specific cases, the connectivity between targeted protective genes and the rest of cell metabolism remains obscure. This study was therefore carried out to investigate the genome-wide response of M. bovis BCG to hydrogen peroxide after 10 and 60 min of treatment. ATP measurements were carried out in order to monitor the changes in M. bovis BCG growth over a 1 h period. The furA gene in Mycobacterium bovis, a pleiotropic regulator that couples iron metabolism to the oxidative stress response was involved in the response to hydrogen peroxide stress. There were also increased levels of catalase/ peroxidase (KatG) and the biosynthesis operon of mycobactin. This study revealed significant upregulation of the oxidative response group of M. bovis, amino acid transport and metabolism, defense mechanisms, DNA replication, recombination and repair, and downregulation of cell cycle control, mitosis, and meiosis, lipid transport and metabolism, and cell wall/membrane biogenesis. This study shows that the treatment of M. bovis BCG with hydrogen peroxide induces iron acquisition related genes and oxidative stress response genes within one hour of treatment.
... be consumed: although M. bovis infection in U.S. domestic cattle is substantially reduced compared to the past, ... their health care providers are aware that they work in close contact with animals. Additional Information • • U.S. ...
Brooks, B W; Barnum, D A
The biochemical and morphological characteristics of 104 strains of Corynebacterium bovis isolated from bovine milk samples and the C. bovis reference strain were found to be uniform. Valuable criteria for identification were presence of catalase and oxidase, production of acid from glucose and fructose and a requirement for enriched basal media. Six strains of human and three strains of bovine origin were found to be inconsistent with the reference strain. PMID:6722650
Badawy, Ahmed Ibrahem I; Lutz, Kathleen; Taubert, Anja; Zahner, Horst; Hermosilla, Carlos
Host immune responses conducted against antigens of Eimeria bovis are key factors for the development of protective immunity against this protozoan disease. In this study we investigated the expression of E. bovis-derived antigens on the host cell surface membrane during E. bovis first merogony in vitro. Host cells carrying E. bovis-meront I stages expressed E. bovis host cell surface antigens (EbHCSAg) on their surface membrane which were recognised by hyperimmune sera of calves and by sera from rats immunized with E. bovis merozoites I, when tested by indirect immune fluorescent antibody test (IIFAT), laser scanning confocal microscopy (LSCM) and immune electron microscopy. Expression of EbHCSAg on permissive host cells was earliest detected 7 days p. i., thus coinciding with the onset of the parasite replication. Membrane-associated EbHCSAg were removed from infected host cells by proteinase K, partially by Triton X-100, Triton X-114 and Triton X-405, but not by 1 M NaCl, CHAPS or phospholipase C treatment. Antibodies, affinity-purified on paraformaldehyde/glutardialdehyde (PAGA)-fixed E. bovis meront I-infected bovine host cells bound to the surface meront I-carrying cells and to merozoites I (IIFAT, LSCM) but, in contrast to untreated sera, not to sporozoites. When tested on methanol-fixed merozoites I and sporozoites by IIFAT, affinity-purified antibodies bound to structures in the apical complex area of merozoites I, but not to sporozoites, whilst untreated sera caused diffuse labelling of internal structures of both parasite stages. Immune electron microscopy demonstrated binding of affinity-purified antibodies to micronemes and dense granules of merozoites I. Although the function of EbHCSAg is still unknown, results of this study might suggest an involvement in the development of protective immunity against E. bovis infections.
Palmer, M V
Mycobacterium bovis is the cause of tuberculosis in animals and sometimes humans. Many developed nations have long-standing programmes to eradicate tuberculosis in livestock, principally cattle. As disease prevalence in cattle decreases these efforts are sometimes impeded by passage of M. bovis from wildlife to cattle. In epidemiological terms, disease can persist in some wildlife species, creating disease reservoirs, if the basic reproduction rate (R0) and critical community size (CCS) thresholds are achieved. Recognized wildlife reservoir hosts of M. bovis include the brushtail possum (Trichosurus vulpecula) in New Zealand, European badger (Meles meles) in Great Britain and Ireland, African buffalo (Syncerus caffer) in South Africa, wild boar (Sus scrofa) in the Iberian Peninsula and white-tailed deer (Odocoileus virginianus) in Michigan, USA. The epidemiological concepts of R0 and CCS are related to more tangible disease/pathogen characteristics such as prevalence, pathogen-induced pathology, host behaviour and ecology. An understanding of both epidemiological and disease/pathogen characteristics is necessary to identify wildlife reservoirs of M. bovis. In some cases, there is a single wildlife reservoir host involved in transmission of M. bovis to cattle. Complexity increases, however, in multihost systems where multiple potential reservoir hosts exist. Bovine tuberculosis eradication efforts require elimination of M. bovis transmission between wildlife reservoirs and cattle. For successful eradication identification of true wildlife reservoirs is critical, as disease control efforts are most effective when directed towards true reservoirs.
Mycoplasma bovis (M. bovis) has recently emerged as a significant health threat in bison and is an increasing concern and source of economic loss for producers. Clinical manifestations of infection documented in bison include pneumonia, respiratory distress and polyarthritis. The current study des...
Moura, A C; Leonardo, P S; Henriques, M G; Cordeiro, R S
The effect of mycobacterial lipids on the onset of the early acute inflammatory response in BALB/c mice pleurisy was investigated. Intact Mycobacterium leprae and Mycobacterium bovis BCG (BCG), their lipids, and delipidated mycobacteria were used to evaluate total leukocytes and cell types migrated to the pleural cavity (8 animals/experimental group). BCG Moreau (x10(-6)/cavity), delipidated BCG and its lipids gradually recruited cells leading to arrival, respectively, of neutrophils (7.8+/-1.9, 4.7+/-0.9, 1.8+/-0.25) followed by mononuclear cells (4.8+/-0.8, 3.7+/-0.7, 2.45+/-0.22) and eosinophils (0.39+/-0.08, 0.32+/-0.11, 0.41+/-0.65). BCG delipidation decreased the number of migrated total leukocytes (ANOVA, and Newman-Keuls-Student-test), whereas M. leprae delipidation accumulated neutrophils (0.85+/-0.01) and eosinophils (1.65+/-0.18). Intact M. leprae and its lipids did not incite any cell recruitment. Apolar external cell wall lipids from M. leprae and BCG induce different cellular responses. They seem to have a crucial importance at the first contact of mycobacteria with the host cell, modulating the influx of neutrophils/macrophages in the early (4/24 h) onset of the inflammatory reaction.
Nde, Chantal W; Toghrol, Freshteh; Jang, Hyeung-Jin; Bentley, William E
Tuberculosis is a leading cause of death worldwide and infects thousands of Americans annually. Mycobacterium bovis causes tuberculosis in humans and several animal species. Peracetic acid is an approved tuberculocide in hospital and domestic environments. This study presents for the first time the transcriptomic changes in M. bovis BCG after treatment with 0.1 mM peracetic acid for 10 and 20 min. This study also presents for the first time a comparison among the transcriptomic responses of M. bovis BCG to three oxidative disinfectants: peracetic acid, sodium hypochlorite, and hydrogen peroxide after 10 min of treatment. Results indicate that arginine biosynthesis, virulence, and oxidative stress response genes were upregulated after both peracetic acid treatment times. Three DNA repair genes were downregulated after 10 and 20 min and cell wall component genes were upregulated after 20 min. The devR-devS signal transduction system was upregulated after 10 min, suggesting a role in the protection against peracetic acid treatment. Results also suggest that peracetic acid and sodium hypochlorite both induce the expression of the ctpF gene which is upregulated in hypoxic environments. Further, this study reveals that in M. bovis BCG, hydrogen peroxide and peracetic acid both induce the expression of katG involved in oxidative stress response and the mbtD and mbtI genes involved in iron regulation/virulence.
Ruoff, K L; Ferraro, M J; Holden, J; Kunz, L J
Streptococci identified as Streptococcus bovis, S. bovis variant, and Streptococcus salivarius were examined with respect to physiological and serological characteristics and cellular fatty acid content. Similarities in physiological reactions and problems encountered in serological analysis were noted, suggesting that an expanded battery of physiological tests is needed to definitively identify these streptococci. Cellular fatty acid analysis provided an accurate method for distinguishing S. salivarius from S. bovis and S. bovis variant.
Mycoplasma bovis (M. bovis) is an emerging bacterial pathogen that has caused severe disease among ranched bison (Bison bison) herds in North America. Unlike cattle, M. bovis in bison seems to be a primary pathogen, affecting animals in feedlots as well as breeding-age cows on pasture. Mortality r...
Lang, Mirjam; Kann, Michael; Zahner, Horst; Taubert, Anja; Hermosilla, Carlos
Sophisticated evasion strategies of obligate intracellular parasites, in particular prevention of host cell apoptosis, are necessary to ensure successful replication. To study the ability of Eimeria bovis in this regard, in vitro experiments were performed applying bovine foetal gastrointestinal cells (BFGC), bovine umbilical vein endothelial cells (BUVEC) and African green monkey kidney cells (VERO) as host cells. BUVEC and BFGC allow maturation of sporozoites to macromeronts, in VERO cells sporozoites survive for weeks without showing further development. In highly infected BUVEC monolayers, infected cells survived until merozoite release whereas uninfected cells underwent apoptosis. Light microscopy and TUNEL assays performed 3-10 days p.i. showed that, within infected BFGC and VERO cell monolayers, uninfected cells underwent programmed cell death after application of various inducers of apoptosis, whereas infected cells survived. Incidentally, the anti-apoptotic efficacies in infected cells were independent of the drugs and the host cell type. We could not demonstrate significant differences between infected and uninfected cells after colchicin treatment in terms of translation of phosphatidylserines to the host cell surface, caspase 3 activity and cytochrome c release, probably since obtainable infection rates were too low. However, we could show by laser scanning confocal microscopy on single cell levels that the expression of the anti-apoptotic factors cellular Flice inhibitory protein (c-FLIP) and cellular inhibition of apoptosis protein 1 (c-IAP1) were enhanced in E. bovis infected cells after application of colchicin, in the latter case also in non-infected cells directly neighbouring infected ones. Our data show that E. bovis protects its host cell from apoptosis by increasing expression of c-IAP1 and c-FLIP.
Nishiyama, Akihito; Shinohara, Tsutomu; Pantuso, Traci; Tsuji, Shoutaro; Yamashita, Makiko; Shinohara, Shizuka; Myrvik, Quentin N; Henriksen, Ruth Ann; Shibata, Yoshimi
When macrophages phagocytose chitin (N-acetyl-d-glucosamine polymer) microparticles, mitogen-activated protein kinases (MAPK) are immediately activated, followed by the release of Th1 cytokines, but not IL-10. To determine whether phagocytosis and macrophage activation in response to chitin microparticles are dependent on membrane cholesterol, RAW264.7 macrophages were treated with methyl-beta-cytodextrin (MBCD) and stimulated with chitin. These results were compared with the corresponding effects of bacterial components including heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guèrin (BCG) and an oligodeoxynucleotide (ODN) of bacterial DNA (CpG-ODN). The MBCD treatment did not alter chitin binding or the phagocytosis of chitin particles 20 min after stimulation. At the same time, however, chitin-induced phosphorylation of cellular MAPK was accelerated and enhanced in an MBCD dose-dependent manner. The increased phosphorylation was also observed for chitin phagosome-associated p38 and ERK1/2. In contrast, CpG-ODN and HK-BCG induced activation of MAPK in MBCD-treated cells at levels comparable to, or only slightly more than, those of control cells. We also found that MBCD treatment enhanced the production of tumor necrosis factor-alpha (TNF-alpha) and the expression of cyclooxygenase-2 (COX-2) in response to chitin microparticles. In neither MBCD- nor saline-treated macrophages, did chitin particles induce detectable IL-10 mRNA synthesis. CpG-ODN induced TNF-alpha production, and COX-2 expression were less sensitive to MBCD treatment. Among the agonists studied, our results indicate that macrophage activation by chitin microparticles was most sensitive to cholesterol depletion, suggesting that membrane structures integrated by cholesterol are important for physiological regulation of chitin microparticle-induced cellular activation.
An attenuated Mycobacterium bovis RD1 knockout (Delta RD1) vaccine administered to calves at 2 weeks of age provided similar efficacy as M. bovis bacille Calmette Guerin (BCG) against low dose, aerosol challenge with virulent M. bovis at 3.5m of age. Approximately 4.5 months after challenge, both De...
Chastant-Maillard, S; Boulouis, H-J; Reynaud, K; Thoumire, S; Gandoin, C; Bouillin, C; Cordonnier, N; Maillard, R
Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue.
Background Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. Results The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. Conclusions Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB. PMID:21507239
Bianco, María V; Clark, Simon; Blanco, Federico C; Garbaccio, Sergio; García, Elizabeth; Cataldi, Angel A; Williams, Ann; Bigi, Fabiana
A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.
In vitro inhibition of lipopolysaccharide and mycobacterium bovis bacillus Calmette Guérin-induced inflammatory cytokines and in vivo protection from D-galactosamine/LPS -mediated liver injury by the medicinal plant Sclerocarya birrea.
Fotio, A L; Olleros, M L; Vesin, D; Tauzin, S; Bisig, R; Dimo, T; Nguelefack, T B; Dongo, E; Kamtchouing, P; Garcia, I
Sclerocarya birrea is a medicinal plant used for the treatment of inflammatory- and bacterial-related diseases. The present study investigated in vitro and in vivo the effects of the stem bark methanol extract of S. birrea. Nitrite, TNF, IL-1beta, IL-6 and IL-12p40 production by bone marrow-derived macrophages (BMDM) pre-incubated with or without S. birrea, and stimulated with Lipopolysaccharide (LPS) or infected with live Mycobacterium bovis Bacillus Calmette Guérin (BCG) was evaluated. S. birrea extract inhibited, in a concentration-dependent manner, nitrite, TNF, IL-1beta, IL-6 and IL-12p40 production by BMDM stimulated with LPS or infected with live BCG. The iNOS expression was reduced by S. birrea after stimulation of BMDM with LPS. In addition, S. birrea inhibited the nuclear factor kB (NF-kB) activation by both LPS and BCG. The effects of the plant extract were also evaluated in an in vivo model of liver injury induced by D-galactosamine/LPS (D-GalN/LPS) administration in mice. S. birrea limited D-GalN/LPS-liver injury as assessed by a reduction in transaminases and TNF, IL-1beta, IL-6 serum levels, and translocation of NF-kB to the nucleus. Taken together, our data indicate that stem bark methanol extract of S. birrea possesses anti-inflammatory properties by inhibiting NF-kB activation and cytokine release induced by inflammatory or infectious stimuli.
Fend, R; Geddes, R; Lesellier, S; Vordermeier, H-M; Corner, L A L; Gormley, E; Costello, E; Hewinson, R G; Marlin, D J; Woodman, A C; Chambers, M A
It is estimated that more than 50 million cattle are infected with Mycobacterium bovis worldwide, resulting in severe economic losses. Current diagnosis of tuberculosis (TB) in cattle relies on tuberculin skin testing, and when combined with the slaughter of test-positive animals, it has significantly reduced the incidence of bovine TB. The failure to eradicate bovine TB in Great Britain has been attributed in part to a reservoir of the infection in badgers (Meles meles). Accurate and reliable diagnosis of infection is the cornerstone of TB control. Bacteriological diagnosis has these characteristics, but only with samples collected postmortem. Unlike significant wild animal reservoirs of M. bovis that are considered pests in other countries, such as the brushtail possum (Trichosurus vulpecula) in New Zealand, the badger and its sett are protected under United Kingdom legislation (The Protection of Badgers Act 1992). Therefore, an accurate in vitro test for badgers is needed urgently to determine the extent of the reservoir of infection cheaply and without destroying badgers. For cattle, a rapid on-farm test to complement the existing tests (the skin test and gamma interferon assay) would be highly desirable. To this end, we have investigated the potential of an electronic nose (EN) to diagnose infection of cattle or badgers with M. bovis, using a serum sample. Samples were obtained from both experimentally infected badgers and cattle, as well as naturally infected badgers. Without exception, the EN was able to discriminate infected animals from controls as early as 3 weeks after infection with M. bovis, the earliest time point examined postchallenge. The EN approach described here is a straightforward alternative to conventional methods of TB diagnosis, and it offers considerable potential as a sensitive, rapid, and cost-effective means of diagnosing M. bovis infection in cattle and badgers.
Cunha, Mónica V; Azorín, Beatriz; Peñuela, Rocío G; Albuquerque, Teresa; Botelho, Ana
Anthropogenic activities have cumulatively led to the dramatic decline of world populations of vultures that currently face serious survival challenges in several regions of the world. In Portugal, the three resident species qualify as endangered and are under conservation efforts, mainly in the central east and south-east regions, where habitat protection and artificial feeding stations were implemented. Concurrently, the areas under protection are highly affected by tuberculosis (TB) in cattle and wild ungulates, whose potentially infected carcasses may naturally or artificially be used as feed by local vultures. In this work, we opportunistically surveyed populations of Eurasian griffon (Gyps fulvus) and Eurasian black vulture (Aegypius monachus) for the presence of Mycobacterium bovis. Nine pathogenic mycobacteria, including one M. bovis isolate, were cultured from the oropharynx of nine of the surveyed vultures (n = 55), sampled in recovery centres or in artificial feeding stations. Genotyping of the M. bovis strain indicated spoligotype SB0121, the most frequent type in Portugal, and a unique MIRU-VNTR profile that differed in two loci from the profiles of SB0121 bovine and deer strains from the same geographical area. The M. bovis-positive griffon exhibited poor clinical condition when admitted to the recovery centre; however, clinical evidence of TB was not present. Although the significance of M. bovis isolation in this vulture specimen could not be ascertained and despite the accepted notion that vultures are naturally resistant to microbial pathogens, the sanitary follow-up of Accipitridae vulture populations in TB-hotspot areas is essential to safeguard ongoing conservation efforts and also to evaluate the suitability of standing legislation on deliberate supplementary feeding schemes for menaced birds of prey.
Herrera, Paul; Kwon, Young Min; Ricke, Steven C
Streptococcus bovis is an indigenous resident in the gastrointestinal tracts of both humans and animals. S. bovis is one of the major causes of bacterial endocarditis and has been implicated in the incidence of human colon cancer, possibly due to chronic inflammatory response at the site of intestinal colonization. Certain feeding regimens in ruminants can lead to overgrowth of S. bovis in the rumen, resulting in the over-production of lactate and capsular polysaccharide causing acute ruminal acidosis and bloat, respectively. There are multiple strategies in controlling acute lactic acidosis and bloat. The incidence of the two diseases may be controlled by strict dietary management. Gradual introduction of grain-based diets and the feeding of coarsely chopped roughage decrease the incidence of the two disease entities. Ionophores, which have been used to enhance feed conversion and growth rate in cattle, have been shown to inhibit the growth of lactic acid bacteria in the rumen. Other methods of controlling lactic acid bacteria in the ruminal environment (dietary supplementation of long-chain fatty acids, induction of passive and active immune responses to the bacteria, and the use of lytic bacteriophages) have also been investigated. It is anticipated that through continued in-depth ecological analysis of S. bovis the characteristics responsible for human and animal pathogenesis would be sufficiently identified to a point where more effective control strategies for the control of this bacteria can be developed.
Aerosol and intratracheal inoculation routes are commonly used for experimental biology purposes to infect cattle with virulent Mycobacterium bovis, each resulting primarily in a respiratory tract infection including lungs and lung-associated lymph nodes. Disease severity is dose and time dependent...
Corner, L A L; Costello, E; Lesellier, S; O'Meara, D; Sleeman, D P; Gormley, E
The aim was to develop an endobronchial infection procedure for the study of Mycobacterium bovis infection in badgers. The badgers were anaesthetised and a cannula was passed per os to the tracheal bifurcation. When in place 1 ml of M. bovis suspension was inoculated. Three concentrations of M. bovis suspension were used; <10 colony forming units (cfu), approximately 10(2) cfu and approximately 3 x 10(3) cfu. The badgers were examined at three weekly intervals for clinical signs of disease and a tracheal aspirate was collected at each examination. The badgers were euthanased 17 weeks post infection (pi) and at the post mortem examination a wide range of tissues were examined for gross and histopathological lesions of tuberculosis and cultured for M. bovis. A sample of bronchial alveolar lavage (BAL) fluid was collected at post mortem for culture. At post mortem examination 17 weeks after infection, gross and histopathological lesions of tuberculosis were observed in all badgers inoculated with the high and medium dose and 1/3 inoculated with the low dose. M. bovis was recovered from all inoculated badgers. Infection in the high dose group was more widely disseminated than in the other groups. The number of sites with gross and histopathological lesions increased with increasing dose of M. bovis. All tracheal aspirates were negative on culture and only one BAL, collected from a badger of the high dose group, was positive on culture. No clinical signs due to the experimental infection were observed. The endobronchial route of inoculation is an effective route for establishing experimental infection, and could be used for studies of tuberculosis pathogenesis, immunology of M. bovis infection in badgers and for challenging badgers in vaccine protection studies. Badgers appeared to be very susceptible to infection by this procedure even with a dose of < 10 cfu but appear to control and limit the resulting infection.
Ma, Hongyue; Zhou, Jing; Jiang, Jiejun; Duan, Jinao; Xu, Huiqin; Tang, Yuping; Lv, Gaohong; Zhang, Junfeng; Zhan, Zhen; Ding, Anwei
Toad Venom, called chansu (CS) in China, is an anti-inflammatory drug used in small doses for the treatment of various types of inflammation in China. Its use is hampered by the cardiotoxicity of bufadienolides derived from Toad Venom. Bezoar Bovis is another frequently used drug in Toad Venom preparations for the treatment of inflammatory or cardiovascular diseases in Asia. We explored whether Bezoar Bovis could protect against CS-induced acute toxicity in mice. Toxicity was assessed by the general features of poisoning, electrocardiography (ECG), and levels of creatine kinase (CK), lactate dehydrogenase (LDH) and calcium ions (Ca(2+)) in cardiac tissues. Toad Venom (90 mg/kg) caused opisthotonus, ventricular arrhythmias, and increases in cardiac levels of Ca(2+), CK and LDH. Pretreatment with Bezoar Bovis (120, 240 and 480 mg/kg) significantly reduced the prevalence of opisthotonus and mortality, and prevented cardiotoxicity in CS-treated mice as evidenced by decreases in the scores of arrhythmias and cardiac levels of CK, LDH and Ca(2+). Furthermore, the bilirubin, and taurine derived from Bezoar Bovis offered marked protection against the arrhythmias induced by CS or bufalin in vivo and in vitro. An anti-inflammatory study showed that Bezoar Bovis did not compromise the anti-inflammatory activity of Toad Venom on concanavalin-A (ConA)-stimulated proliferation of human peripheral blood mononuclear cells. These results suggested that Bezoar Bovis elicited protective and anti-arrhythmic effects against Toad Venom intoxication in mice, and is a novel antidote in combination with Toad Venom therapy.
Taubert, Anja; Hermosilla, Carlos; Sühwold, Anke; Zahner, Horst
Cellular immune responses against Eimeria bovis are highly specific and a key factor for the development of protection against challenge infections. In this study we investigate the cellular immune responses of E. bovis primary and challenge infected calves stimulated in vitro by E. bovis merozoite I-antigen. Primary infection was accompanied by an increase of IFN-gamma and IL-2 gene transcription in whole blood samples, peaking during prepatency (8-12 days p.i.) and declining thereafter, whereas IL-4 gene transcription was induced predominantly in patency. IL-10 mRNA was not influenced by E. bovis infection. Both CD4+ and CD8+ T cells were identified as source of IFN-gamma gene transcripts, whilst IL-2 and IL-4 gene transcription was enhanced mainly in CD4+ T cells. Increased levels of IFN-gamma transcripts and protein were also found in lymphocytes isolated from ileocaecal lymph node biopsy 8 days p.i., and in cell culture supernatants obtained from antigen-stimulated peripheral blood mononuclear cells (PBMC) at days 8 and 12 p.i., respectively. Challenge infections of calves influenced neither IFN-gamma nor IL-2 gene transcription in peripheral blood or in lymph node-derived lymphocytes. In contrast, IL-4 gene transcription was increased in lymphocytes isolated from draining lymph nodes. Besides antigen-specific reactions we also found an infection-triggered induction of the non-specific activation state of PBMC in the course of primary infection as measured by the intracellular IFN-gamma and IL-4 content of phorbol-12-myristate-13-acetate/ionomycin-stimulated PBMC. This may represent a new mechanism of immune cells of E. bovis-infected calves contributing to ongoing immune reactions.
Lysnyansky, Inna; Ayling, Roger D.
Mycoplasma bovis is a cell-wall-less bacterium and belongs to the class Mollicutes. It is the most important etiological agent of bovine mycoplasmoses in North America and Europe, causing respiratory disease, mastitis, otitis media, arthritis, and reproductive disease. Clinical disease associated with M. bovis is often chronic, debilitating, and poorly responsive to antimicrobial therapy, resulting in significant economic loss, the full extent of which is difficult to estimate. Until M. bovis vaccines are universally available, sanitary control measures and antimicrobial treatment are the only approaches that can be used in attempts to control M. bovis infections. However, in vitro studies show that many of the current M. bovis isolates circulating in Europe have high minimum inhibitory concentrations (MIC) for many of the commercially available antimicrobials. In this review we summarize the current MIC trends indicating the development of antimicrobial resistance in M. bovis as well as the known molecular mechanisms by which resistance is acquired. PMID:27199926
Feenstra, A; Bisgaard Madsen, E; Friis, N F; Meyling, A; Ahrens, P
After an outbreak of mastitis in cattle caused by Mycoplasma bovis a study was made in 5 herds with recent cases (principal herds) and in 4 control herds. In the principal herds, M. bovis was isolated from milk samples, nasal swabs, and from one vaginal swab. M. bovis was also isolated from nasal swabs of calves in 2 of the 4 control herds, whereas all milk samples and vaginal swabs from the control herds were negative. Evaluation of serum antibody titres to M. bovis among non-mastitic animals of 3 principal herds and 1 control herd showed no difference in distribution of the titre values, which generally were low. However, cows excreting M. bovis in the milk had high antibody titres. The way of introduction to the herds and the spread of the infection within the herds could not be established by the study, which was supplemented by a DNA restriction fragment analysis of a number of M. bovis isolates.
ter Laak, E A; Wentink, G H; Zimmer, G M
The epidemiology, therapy, and prevention of M. bovis infections are briefly reviewed. In a survey begun in 1982, M. bovis was found frequently in the respiratory tract [corrected] of veal calves and beef cattle with respiratory problems. In replacement calves infected with respiratory disease in dairy herds, however, the organism has only been detected since 1986. Respiratory tract specimens collected from calves with respiratory disease were submitted for examination for M. bovis from 1986 to 1991 and originated from 83 herds. Mycoplasma bovis was detected in specimens from 59 of the herds, 20% of which were dairy herds and 80% fattening herds. Arthritis caused by M. bovis was observed in 12 herds until July 1991. Since 1976 when the first mastitis outbreak caused by M. bovis was diagnosed, M. bovis has caused 14 more outbreaks. The number of diseased cattle varied from 1 tot 16 per farm, and clinical signs of mastitis varied from mild to severe. In all instances the infection has been eradicated from the herds. Because M. bovis can cause great losses in intensively reared cattle herds, it is advisable to separate purchased veal calves and beef cattle from dairy cattle to prevent further spread of M. bovis.
Bruning-Fann, C S; Schmitt, S M; Fitzgerald, S D; Payeur, J B; Whipple, D L; Cooley, T M; Carlson, T; Friedrich, P
During a survey for tuberculosis in wild carnivores and omnivores, Mycobacterium bovis was cultured from pooled lymph nodes of three adult female coyotes (Canis latrans) harvested by hunters in Michigan (USA). No gross or histologic lesions suggestive of tuberculosis were seen in these animals. One coyote was taken from Montmorency county and two coyotes from Alcona county located in the north-eastern portion of Michigan's Lower Peninsula where free-ranging white-tailed deer (Odocoileus virginianus) have been found infected with bovine tuberculosis. It is thought that these coyotes became infected with M. bovis through the consumption of tuberculous deer. Other species included in the survey were the opossum (Didelphis virginiana), raccoon (Procyon lotor), red fox (Vulpes vulpes), bobcat (Felis rufus), and badger (Taxidea taxus).
Mycoplasma bovis arthritis and pneumonia in six-month-old calvesSudden deaths in housed suckler cows due to hypomagnesaemiaBovine respiratory syncytial virus infection in two-year-old heifersBovine abortion associated with Parachlamydia speciesFibrinous pericarditis due to Aeromonas hydrophila in weaner pigsThese are among matters discussed in the disease surveillance report for December 2016 from SAC Consulting: Veterinary Services (SAC C VS). British Veterinary Association.
Sühwold, Anke; Hermosilla, Carlos; Seeger, Torsten; Zahner, Horst; Taubert, Anja
Eimeria bovis infections commonly have clinical impact only on young animals, as homologous reinfections generally are under immunological control. So far, the nature of the immune responses delivering protection to calves has not been investigated. In this study we therefore analysed local and peripheral proliferative T cell activities of primary and challenge-infected calves and investigated the occurrence of T cell phenotypes in the peripheral blood and in mucosal gut segments isolated either by bioptic means or by necropsies.We show that lymphocytes of E. bovis-infected calves exhibit effective, transient antigen-specific proliferative responses in the course of prepatency of primary infection but fail to react after homologous reinfection suggesting early abrogation of parasite development. Whilst in primary infection an expansion of peripheral CD4+ T cells was observed, reinfection had no effect on the proportions of CD4+, CD8+ subsets or gammadeltaTCR+ T cells. In contrast, both E. bovis primary and challenge infections had an impact on local tissue T cell distribution. Primary infection was characterised by a CD4+ T cell infiltration early in prepatency in ileum and later in colon mucosa, whereas CD8+ T cells were only found accumulating in the latter gut segment. Challenge infection led to infiltration of both CD4+ and CD8+ T cells in small intestine and large intestine segments indicating protective functions of both cell types. In contrast, infiltration of ileum and colon mucosa with gammadeltaTCR+ T cells was restricted to primary infection.
Kreuger, M R; Tames, D R; Mariano, M
The microbicidal activity of macrophages in an inflammatory milieu has been related to the production of a large number of cytokins and intermediary metabolites of oxygen and nitrogen among them, nitric oxide (NO). Considering that granulomatous inflammation is predominantly composed of macrophages and epithelioid cells, we decided to investigate the participation of NO in this peculiar type of inflammation. Two models were used: glass cover slip implantation into the subcutaneous tissue of mice and, the inoculation of live bacillus Calmette-Guérin (BCG) into the footpad of the animals. Using a histochemical method for the detection of NO synthase and of the concentration of citrulin metabolized by cells obtained from cover slips implanted on different time intervals or BCG-activated peritoneal cells, it was possible to demonstrate that epithelioid cells do not produce NO. Cells from granuloma induced by BCG inoculation express NO synthase, with different degrees of reactivity with a higher intensity in the cytoplasm of cells located in the edge of the lesions. The expression of NO synthase in the cytoplasm of these cells decreases with the age of the lesions. It could also be demonstrated that in mice treated with l-name, an inhibitor of NO metabolism, the lesions induced by BCG lost the granulomatous architecture, were necrotic, and had a significant increase in the bacillary load of the lesion. These data allow us to conclude that NO production by macrophages is a determining factor in the organization of the granulomatous lesion and that it also controls the bacterial load in BCG-induced lesions in mice. Images Figure 1 Figure 2 Figure 4 Figure 6 PMID:9824487
Dan, J M; Crespo, M; Silveira, F P; Kaplan, R; Aslam, S
We present a report of extrapulmonary Mycobacterium bovis infection in a lung transplant recipient. M. bovis is acquired predominantly by zoonotic transmission, particularly from consumption of unpasteurized foods. We discuss epidemiologic exposure, especially as relates to the Mexico-US border, clinical characteristics, resistance profile, and treatment.
García-Bocanegra, I; Barranco, I; Rodríguez-Gómez, I M; Pérez, B; Gómez-Laguna, J; Rodríguez, S; Ruiz-Villamayor, E; Perea, A
We report three cases of tuberculosis in alpacas from Spain caused by Mycobacterium bovis. The animals revealed two different lesional patterns. Mycobacterial culture and PCR assay yielded positive results for M. bovis. Molecular typing of the isolates identified spoligotype SB0295 and identical variable-number tandem repeat (VNTR) allele sizes.
Jadeja, L.; Kantarjian, H.; Bolivar, R.
We describe the first patient with simultaneous S bovis septicemia and meningitis associated with chronic radiation enterocolitis. This case underlines the value of a thorough gastrointestinal evaluation of all patients with S bovis infection, and the need for a neurologic investigation even with minor neurologic manifestations.
de Souza Figueiredo, Eduardo Eustáquio; Silvestre, Flávia Galindo; Campos, Wilma Neres; Furlanetto, Leone Vinícius; Medeiros, Luciana; Lilenbaum, Walter; Fonseca, Leila Sousa; Silva, Joab Trajano; Paschoalin, Vânia Margaret Flosi
Isolates from suggestive bovine tuberculosis lesions were tested by a multiplex polymerase chain reaction (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, specific for M. bovis and Mycobacterium tuberculosis complex respectively. The m-PCR successfully identified as M. bovis 88.24% of the isolates. PMID:24031349
In the early 20th century there were large numbers of tuberculous cattle in many countries. An association was made between the number of M. bovis infected humans and the prevalence of tuberculosis in cattle. Mandatory pasteurization of milk caused the prevalence of human tuberculosis due to M. bovi...
Previous research indicated that Streptococcus bovis, a lactate producing ruminal bacterium, was similar or better than commercial silage inoculants. This study assessed the potential of two S. bovis strains, JB1 (a bacteriocin negative strain) and HC5 (a bacteriocin producing strain). Four treatmen...
Jones, B A; Muck, R E; Ricke, S C
Three strains of Streptococcus bovis, a homolactic bacterium capable of utilizing starch, were evaluated for growth kinetics and ability to decrease the pH of alfalfa silage. A selected strain was evaluated for its competitiveness as an inoculant with Enterococcus faecium, an organism used in inoculants, and for its ability to enhance the effect of a commercial inoculant. Testing was completed over three studies using wilted alfalfa (28 to 34% dry matter) ensiled into laboratory silos. Treatments were control, E. faecium, E. faecium and commercial inoculant, S. bovis, and S. bovis and commercial inoculant. Replicate silos were emptied and analyzed at 0.5, 1, 2, 4, 8, and 40 days for pH, fermentation products, and nitrogen fractions. S. bovis alone lowered the pH quicker and improved silage parameters early in the fermentation compared with E. faecium, the commercial inoculant, and control treatments. When combined with a commercial inoculant, S. bovis lowered pH more quickly than the commercial inoculant alone and E. faecium plus commercial inoculant. At 40 days, S. bovis combination had lower pH and ammonia nitrogen and acetate contents than the E. faecium combination. Starch in the silage was not utilized by S. bovis as had been anticipated. Results indicate that S. bovis was more effective than E. faecium as a silage inoculant and could enhance a commercial inoculant on low-dry-matter alfalfa. PMID:1746960
Objective. Mycobacterium bovis is the causative agent of bovine tuberculosis, a relevant zoonosis that can spread to humans through inhalation or by ingestion. M. bovis multiplies slowly, so infected animals may be sent to slaughter during the early stages of the disease before diagnosis and when ...
Mycoplasma bovis (M. bovis) is an emerging, primary pathogen of ranched bison (Bison bison) in North America. It causes severe disease among animals in feedlots as well as breeding-age cows and bulls on pasture. Mortality in adult bison is as high as 25 percent, resulting in significant economic l...
Liébana, Ernesto; Girvin, Robert M.; Welsh, Michael; Neill, Sydney D.; Pollock, John M.
Protective immunity against tuberculosis is considered to be essentially cell mediated, and an important role for CD8+ T lymphocytes has been suggested by several studies of murine and human infections. The present work, using an experimental model of infection with Mycobacterium bovis in cattle, showed that live M. bovis elicits the activation of CD8+ T cells in vitro. However, a sonic extract prepared from M. bovis (MBSE) and protein purified derivative (PPDb) also induced a considerable degree of activation of the CD8+ T cells. Analysis of proliferative responses of peripheral blood mononuclear cells, purified CD8+ T cells, and CD8+ T-cell clones to M. bovis and to soluble antigenic preparations (MBSE, PPDb) showed that the responses of all three types of cells were always superior for live mycobacteria but that strong responses were also obtained with complex soluble preparations. Furthermore, while cytotoxic capabilities were not investigated, the CD8+ T cells were found to produce and release gamma interferon in response to antigen (live and soluble), which indicated one possible protective mechanism for these cells in bovine tuberculosis. Finally, it was demonstrated by metabolic inhibition with brefeldin A and cytochalasin D at the clonal level that an endogenous pathway of antigen processing is required for presentation to bovine CD8+ cells and that presentation is also dependent on phagocytosis of the antigen. PMID:10024540
Liébana, E; Girvin, R M; Welsh, M; Neill, S D; Pollock, J M
Protective immunity against tuberculosis is considered to be essentially cell mediated, and an important role for CD8(+) T lymphocytes has been suggested by several studies of murine and human infections. The present work, using an experimental model of infection with Mycobacterium bovis in cattle, showed that live M. bovis elicits the activation of CD8(+) T cells in vitro. However, a sonic extract prepared from M. bovis (MBSE) and protein purified derivative (PPDb) also induced a considerable degree of activation of the CD8(+) T cells. Analysis of proliferative responses of peripheral blood mononuclear cells, purified CD8(+) T cells, and CD8(+) T-cell clones to M. bovis and to soluble antigenic preparations (MBSE, PPDb) showed that the responses of all three types of cells were always superior for live mycobacteria but that strong responses were also obtained with complex soluble preparations. Furthermore, while cytotoxic capabilities were not investigated, the CD8(+) T cells were found to produce and release gamma interferon in response to antigen (live and soluble), which indicated one possible protective mechanism for these cells in bovine tuberculosis. Finally, it was demonstrated by metabolic inhibition with brefeldin A and cytochalasin D at the clonal level that an endogenous pathway of antigen processing is required for presentation to bovine CD8(+) cells and that presentation is also dependent on phagocytosis of the antigen.
Sarfas, Charlotte; Knight, Gwenan M.; White, Andrew; Pathan, Ansar A.; McShane, Helen; Evans, Thomas G.; Sharpe, Sally; White, Richard G.
ABSTRACT Macaques play a central role in the development of human tuberculosis (TB) vaccines. Immune and challenge responses differ across macaque and human subpopulations. We used novel immunostimulation/immunodynamic modeling methods in a proof-of-concept study to determine which macaque subpopulations best predicted immune responses in different human subpopulations. Data on gamma interferon (IFN-γ)-secreting CD4+ T cells over time after recent Mycobacterium bovis BCG vaccination were available for 55 humans and 81 macaques. Human population covariates were baseline BCG vaccination status, time since BCG vaccination, gender, and the monocyte/lymphocyte cell count ratio. The macaque population covariate was the colony of origin. A two-compartment mathematical model describing the dynamics of the IFN-γ T cell response after BCG vaccination was calibrated to these data using nonlinear mixed-effects methods. The model was calibrated to macaque and human data separately. The association between subpopulations and the BCG immune response in each species was assessed. The macaque subpopulations that best predicted immune responses in different human subpopulations were identified using Bayesian information criteria. We found that the macaque colony and the human baseline BCG status were significantly (P < 0.05) associated with the BCG-induced immune response. For humans who were BCG naïve at baseline, Indonesian cynomolgus macaques and Indian rhesus macaques best predicted the immune response. For humans who had already been BCG vaccinated at baseline, Mauritian cynomolgus macaques best predicted the immune response. This work suggests that the immune responses of different human populations may be best modeled by different macaque colonies, and it demonstrates the potential utility of immunostimulation/immunodynamic modeling to accelerate TB vaccine development. PMID:28077441
Rhodes, Sophie J; Sarfas, Charlotte; Knight, Gwenan M; White, Andrew; Pathan, Ansar A; McShane, Helen; Evans, Thomas G; Fletcher, Helen; Sharpe, Sally; White, Richard G
Macaques play a central role in the development of human tuberculosis (TB) vaccines. Immune and challenge responses differ across macaque and human subpopulations. We used novel immunostimulation/immunodynamic modeling methods in a proof-of-concept study to determine which macaque subpopulations best predicted immune responses in different human subpopulations. Data on gamma interferon (IFN-γ)-secreting CD4(+) T cells over time after recent Mycobacterium bovis BCG vaccination were available for 55 humans and 81 macaques. Human population covariates were baseline BCG vaccination status, time since BCG vaccination, gender, and the monocyte/lymphocyte cell count ratio. The macaque population covariate was the colony of origin. A two-compartment mathematical model describing the dynamics of the IFN-γ T cell response after BCG vaccination was calibrated to these data using nonlinear mixed-effects methods. The model was calibrated to macaque and human data separately. The association between subpopulations and the BCG immune response in each species was assessed. The macaque subpopulations that best predicted immune responses in different human subpopulations were identified using Bayesian information criteria. We found that the macaque colony and the human baseline BCG status were significantly (P < 0.05) associated with the BCG-induced immune response. For humans who were BCG naïve at baseline, Indonesian cynomolgus macaques and Indian rhesus macaques best predicted the immune response. For humans who had already been BCG vaccinated at baseline, Mauritian cynomolgus macaques best predicted the immune response. This work suggests that the immune responses of different human populations may be best modeled by different macaque colonies, and it demonstrates the potential utility of immunostimulation/immunodynamic modeling to accelerate TB vaccine development.
Ramos, Daniela Fernandes; Tavares, Lucas; da Silva, Pedro Eduardo Almeida; Dellagostin, Odir Antônio
Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications. PMID:25242917
Mol, J P S; Carvalho, T F; Fonseca, A A; Sales, E B; Issa, M A; Rezende, L C; Hodon, M A; Tinoco, H P; Malta, M C C; Pessanha, A T; Pierezan, F; Mota, P M P C; Paixão, T A; Santos, R L
Tuberculosis, associated with Mycobacterium bovis, was diagnosed post mortem in an adult female capybara (Hydrochoerus hydrochaeris), kept at the Pampulha Ecological Park, Belo Horizonte, Brazil, in a large metropolitan area. On post-mortem examination, there were numerous firm white nodules scattered throughout all lobes of both lungs. Tissue samples were collected for histological and microbiological examination. Microscopically, the pulmonary nodules were multifocal to coalescing granulomas and intralesional acid-fast bacilli were evident in Ziehl-Neelsen-stained sections of the lung and spleen. Colonies with morphological features of Mycobacterium spp. were isolated from lung samples and conventional polymerase chain reaction (PCR) with genomic DNA from the isolates was positive for M. bovis; sequencing indicated 100% identity with the region of difference 4 (RD4) of M. bovis. In addition, M. bovis DNA was detected in the lung by quantitative PCR. The finding of M. bovis in a capybara indicates a potential public health risk in a zoological collection.
Mycobacterium bovis (M. bovis) causes tuberculosis in white-tailed deer (WTD). Natural infection of WTD with M. bovis is most closely mimicked by instilling inoculum into palatine tonsilar crypts. One hundred fifty days after intratonsilar inoculation, M. bovis was cultured from 30 tissues originati...
Kowalewicz-Kulbat, Magdalena; Pestel, Joël; Biet, Franck; Locht, Camille; Tonnel, André-Bernard; Druszczyńska, Magdalena; Rudnicka, Wiesława
The polarized response of T helper-2 (Th2) lymphocytes to an allergen is considered to be the main cause of the pathogenesis of asthma. In this study, we asked a question whether M. bovis BCG mycobacteria which are known for the preferential stimulation of T helper-1 (Th1) immunity, diminish the effector functions of Th2 cells from allergic patients upon stimulation with a common house dust mite Der p-1 allergen. Our results allow a positive answer to this question. We demonstrate that BCG modulates the dendritic cell-dependent allergen presentation process and switches naive T lymphocytes towards an anti-allergic Th1 profile.
Mateos Colino, Alfonso; Sousa Escandón, Manuel Alejandro; Golpe Gómez, Rafael; García Figueras, Roberto; Pérez Valcarcel, Javier; Fernández, María Armesto
To focus on the need of including tuberculosis among differential diagnoses of any epidymo-testicular mass, especially if its evolution is torpid. A 73-year-old man who presented with scrotum abscess underwent surgical drainage and antibiotic treatment, but suppuration relapsed through cutaneous fistulae. A epipidymectomy was then performed, which demonstrated tuberculous granulomas. Torax Rx showed a cystic apical pulmonary wound which was treated with 3 antituberculostatics for 12 months. Sputum culture was positive for Micobacterium Bovis. Aspirative punction under sonographic control is a valuable technique to avoid mutilating surgeries and to permit an almost always effective treatment, before the appearance of permanent lesions which lead to sterility.
Pugh, G W; Wilbur, L A; Beall, C W
Selected cultures of Moraxella bovis were studied in calves, using chambers fabricated from a semipermeable membrane supported and protected by perforated plastic golf balls. Plain balls, semipermeable membrane-covered balls, and balls that contained bags fabricated from semipermeable membranes were surgically implanted subcutaneously in calves at a site approximately 25 cm ventral to the paralumbar fossa and cranial to the prefemoral lymph node. After calves recovered from surgery, chambers were inoculated with different cultures of M bovis. Cultural examination of samples taken from inoculated chambers indicated that M bovis cultures were maintained within the chambers for variable times, depending on the characteristics of the culture inoculated. A smooth (piliated) culture dissociated into a rough (nonpiliated) culture after 4 weeks of incubation within a chamber but none of the rough cultures became smooth. The technique offers a method to study the in vivo variations in a series of cultures or strains of microorganisms under the influence of the same host factors. The selective permeability of the chamber may be controlled by using membranes of different porosities to control the flow of entering or exiting dialysate.
Kubica, Tanja; Rüsch-Gerdes, Sabine; Niemann, Stefan
The prevalence of the Mycobacterium bovis subsp. caprae and M. bovis subsp. bovis among German tuberculosis cases caused by the bovine tubercle bacillus from 1999 to 2001 was determined. Isolates from 166 patients living in Germany and 10 animals were analyzed by conventional laboratory procedures, spoligotyping, and partly by PCR-restriction fragment length polymorphism analysis of the gyrB gene. By spoligotyping, 55 of 176 isolates (31%) could be identified as M. bovis subsp. caprae, and 121 (69%) were confirmed as M. bovis subsp. bovis. In general, a low variability of spoligotypes with 59 distinct patterns and a cluster rate of 77% (136 isolates/19 clusters) was determined. About half of all isolates were grouped in the three main clusters with 29, 30, and 35 isolates, respectively. Differences in age and gender between the patient groups infected with M. bovis subsp. bovis and M. bovis subsp. caprae did not reach statistical significance. However, marked differences in the geographical prevalence of M. bovis subsp. caprae were observed, ranging from fewer than 10% of all M. bovis isolates in the north up to more than 80% of isolates in the south of Germany. In conclusion, M. bovis subsp. caprae accounts for a high ratio of human M. bovis-associated tuberculosis cases in Germany and was more frequently found in the southern part. PMID:12843046
Chambers, M A; Stagg, D; Gavier-Widén, D; Lowrie, D; Newell, D; Hewinson, R G
Nucleic acid (DNA) vaccination against tuberculosis in the European badger (Meles meles) is one approach to addressing the escalating problem of bovine tuberculosis in Great Britain. The aim of vaccination is to reduce the burden of tuberculosis within the badger population and the shedding of Mycobacterium bovis to levels that would break the transmission of infection to cattle. To this end, the vaccine would be required to limit the amount of disseminated tuberculosis in the badger, especially dissemination to the kidney from where M. bovis can be shed in the urine. A promising candidate DNA vaccine encoding a 26 kDa major antigen (MPB83) of M. bovis was evaluated in a mouse model of disseminated M. bovis infection. Using the DNA vaccine, protection against infection of the kidney was found to be greater than that achieved with the current live vaccine, Bacille Calmette-Guerin (BCG). Kidney tissue and skeletal muscle from the badger was used to derive primary cell cultures in which to examine the expression of MPB83 following transfection with the DNA vaccine. Kidney cortex gave rise to a monotypic culture of epithelial cells whilst the muscle gave rise to a mixed culture of fibroblasts and myoblasts. During culture the myoblasts differentiated into multinucleated myotubes, verified by immunofluorescent detection of mammalian desmin. Successful expression of MPB83 by transfected epithelial and myotube cells was confirmed by immunofluorescence using a monoclonal antibody specific to the protein. These observations fulfil the early requirements for the development of a DNA vaccine for badger tuberculosis.
Ameni, Gobena; Vordermeier, Martin; Aseffa, Abraham; Young, Douglas B; Hewinson, R Glyn
In developing countries, the conventional test and slaughter strategy for the control of bovine tuberculosis is prohibitively expensive, and alternative control methods such as vaccination are urgently required. In this study, the efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) for protection against bovine tuberculosis (bTB) was evaluated in Holstein calves under field conditions in Ethiopia. Thirteen neonatally vaccinated and 14 control calves were exposed for 10 to 23 months to skin test reactor cows. Gamma interferon (IFN-γ) testing, comparative intradermal tuberculin testing, postmortem examination, and bacteriological culture were used for the evaluation of BCG efficacy. The overall mean pathology score was significantly (P < 0.05) higher in control calves than in vaccinated calves. Culture positivity for Mycobacterium bovis was higher in the control calves than in the vaccinated calves, and significantly more BCG-vaccinated animals would have passed a standard meat inspection (P = 0.021). Overall, the protective efficacy of BCG was between 56% and 68%, depending on the parameters selected. Moreover, by measuring gamma interferon responses to the antigens ESAT-6 and CFP-10, which are present in M. bovis but absent from BCG, throughout the experiment, we were able to distinguish between vaccinated animals that were protected against bTB and those animals that were not protected. In conclusion, the present trial demonstrated an encouraging protective effect of BCG against bTB in a natural transmission setting in Ethiopia.
Prieto, Claudia I; Bosch, Alejandra; Zielinski, Gustavo; Cúneo, José; Yantorno, Osvaldo M
Pili are the principal antigens and virulence factors of Moraxella bovis, the etiological agent of infectious bovine keratoconjunctivitis (IBK). Although it has been reported that the low efficacy of whole cell vaccines against IBK is mainly due to the difficulties in keeping the cellular piliation level of M. bovis during the growth of bacteria in stirred bioreactors, the problem has not yet been overcome because the mechanisms involved in the loss of piliation are still not fully clarified. In this work we found that during the culture of M. bovis in liquid media, around 15% of the cells changed from piliated to non-piliated phenotypes at the end of the growth. Nevertheless, we demonstrated that the main cause of cellular piliation loss in M. bovis growing in stirred and/or sparged bioreactors is due to shear forces, which are a function of the volumetric gassed power drawn (P(g)V(-1)). Therefore, we tested here the use of bubble column bioreactors to protect M. bovis cell-bound pili from mechanical agitation damage effects. These bioreactors operated at a superficial air velocity of 0.0065 m s(-1) yielded a cellular piliation level of 25%, in contrast to 1% obtained for stirred bioreactors. The addition of carboxymethylcellulose (CMC) at 0.10% (w v(-1)) to culture medium proved to be suitable to improve the final piliation level (65%). We demonstrated by FT-IR spectroscopy and ELISA technique, that this chemical additive has a pili protective role interacting with the cells but without affecting pili antigenic properties.
Yavuz, Ahmet; İnci, Abdullah; Düzlü, Önder; Bişkin, Zuhal; Yıldırım, Alparslan
This study was carried out to determine the molecular characterization of msa-2c gene of one Babesia bovis isolate from cattle in the Aegean Region and to compare identities with similar isolates from the World and Turkey. Between 2008-2010 blood samples were collected from a total of 235 cattle localized in 9 provinces of the Marmara and Aegean Regions. Smears were prepared, genomic DNA's were extracted from the blood samples and investigated for Babesia species by RLB. PCR was performed on one sample determined as B. bovis, the obtained amplicon was purified, sequenced and deposited to GenBank. Identities with similar isolates from Turkey and the World were investigated. Bovine babesiosis was not determined in the microscopic examination. According to the RLB results there was no B. bovis positivity in cattle from the Marmara Region, while only one B. bovis positivity was detected in cattle from the Aegean Region. The molecular prevalence of B. bovis was determined as 0.42% in the total of the examined 235 cattle. The sequenced B. bovis isolate shared 91-92% and 89-96% identities with the isolates from Turkey and the World, respectively. Molecular characterization of msa-2c gene region of B. bovis detected from cattle in the Aegean Region was carried out in this study.
Lassen, Brian; Lepik, Triin; Bangoura, Berit
Eimeriosis is a disease that occurs globally and often affects cattle grazing on pastures contaminated with oocysts of the pathogenic species Eimeria bovis, Eimeria zuernii or Eimeria alabamensis, respectively. Nonetheless, little is understood regarding oocyst persistence on the pasture. The study was performed in the temperate climate zone. Soil samples were spiked with 100,000 E. bovis oocysts in July 2010 or with 50,000 oocysts in October 2010, respectively, both either with our without addition of cattle faeces. The soil samples were exposed to natural environmental conditions until April 2011. A subset of the samples was analysed immediately after spiking as positive control. The oocysts were recovered by a flotation method and counted in a reading chamber. On average, 23 % of the oocysts could be recovered from the positive control. The recovery of oocysts dropped to 0.30 % of the original level in the samples prepared in July independent of the addition of faeces, whereas the oocyst count was higher in the samples prepared in October, both without (2.05 %) and with (2.64 %) faecal material. No differences were observed between presence of oocysts or oocyst counts recovered in the presence or absence of faeces. Presence of faeces had a positive influence on oocyst integrity. During the winter season, the number of oocysts in the soil was lowered under the detection limit in most samples. On the other hand, the comparatively short 3-month summer period had a significant influence on the number of persisting oocysts too.
Beltrán-Beck, Beatriz; Romero, Beatriz; Boadella, Mariana; Casal, Carmen; Bezos, Javier; Mazariegos, María; Martín, MariPaz; Galindo, Ruth C.; Pérez de la Lastra, José M.; Villar, Margarita; Garrido, Joseba M.; Sevilla, Iker A.; Asensio, Fernando; Sicilia, Javier; Lyashchenko, Konstantin P.; Domínguez, Lucas; Juste, Ramón A.; de la Fuente, José
Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research. PMID:24920604
Britton, Gary; MacDonald, Douglas C; Brown, Jeremy S; Collins, Mary K; Goodman, Anna L
Although bacillus Calmette-Guérin (BCG) is an established vaccine with excellent efficacy against disseminated Mycobacterium tuberculosis infection in young children, efficacy in adults suffering from respiratory tuberculosis (TB) is suboptimal. Prime-boost viral vectored vaccines have been shown to induce effective immune responses and lentivectors (LV) have been shown to improve mucosal immunity in the lung. A mucosal boost to induce local immunogenicity is also referred to as a 'pull' in a prime and pull approach, which has been found to be a promising vaccine strategy. The majority of infants worldwide receive BCG immunization through current vaccine protocols. We therefore aimed to investigate the role of a boost (or pull) immunization with an LV vaccine expressing the promising TB antigen (Ag85A). We immunized BALB/c mice subcutaneously with BCG or an LV vaccine expressing a nuclear factor-κB activator vFLIP together with Ag85A (LV vF/85A), then boosted with intranasal LV vF/85A. Prime and pull immunization with LV85A induced significantly enhanced CD8(+) and CD4(+) T-cell responses in the lung, but did not protect against intranasal BCG challenge. In contrast, little T-cell response in the lung was seen when the prime vaccine was BCG, and intranasal vF/85A provided no additional protection against mucosal BCG infection. Our study demonstrates that not all LV prime and pull approaches may be successful against TB in man and careful antigen and immune activator selection is therefore required. © 2015 John Wiley & Sons Ltd.
Britton, Gary; MacDonald, Douglas C; Brown, Jeremy S; Collins, Mary K; Goodman, Anna L
Although bacillus Calmette–Guérin (BCG) is an established vaccine with excellent efficacy against disseminated Mycobacterium tuberculosis infection in young children, efficacy in adults suffering from respiratory tuberculosis (TB) is suboptimal. Prime-boost viral vectored vaccines have been shown to induce effective immune responses and lentivectors (LV) have been shown to improve mucosal immunity in the lung. A mucosal boost to induce local immunogenicity is also referred to as a ‘pull’ in a prime and pull approach, which has been found to be a promising vaccine strategy. The majority of infants worldwide receive BCG immunization through current vaccine protocols. We therefore aimed to investigate the role of a boost (or pull) immunization with an LV vaccine expressing the promising TB antigen (Ag85A). We immunized BALB/c mice subcutaneously with BCG or an LV vaccine expressing a nuclear factor-κB activator vFLIP together with Ag85A (LV vF/85A), then boosted with intranasal LV vF/85A. Prime and pull immunization with LV85A induced significantly enhanced CD8+ and CD4+ T-cell responses in the lung, but did not protect against intranasal BCG challenge. In contrast, little T-cell response in the lung was seen when the prime vaccine was BCG, and intranasal vF/85A provided no additional protection against mucosal BCG infection. Our study demonstrates that not all LV prime and pull approaches may be successful against TB in man and careful antigen and immune activator selection is therefore required. PMID:26095282
Caimi, Karina; Romano, Maria I.; Alito, Alicia; Zumarraga, Martin; Bigi, Fabiana; Cataldi, Angel
Spoligotyping is a major tool for molecular typing of Mycobacterium bovis. This technique is based on the polymorphism of spacers that separate direct repeats (DRs) in the M. tuberculosis complex DR region. Numerous M. bovis strains show a lack of several spacers which appears as a gap in the spoligotyping pattern. To determine whether these gaps contain alternative spacers not included in the spoligotyping membrane, PCRs using primers that hybridize to the spacers adjacent to the gaps were performed. Comparing the sizes of products obtained by PCR with those deduced from spoligotyping patterns, fragments were selected and sequenced to look for alternative spacers. Upon analysis of the sequences, five alternative spacers were detected, although deletions of spacers are mainly responsible for the observed gaps. The alternative spacers, which are more frequent in M. bovis than in M. tuberculosis, may contribute to increased M. bovis differentiation. PMID:11230428
Background The global epidemiology of parasitic helminths and mycobacterial infections display extensive geographical overlap, especially in the rural and urban communities of developing countries. We investigated whether co-infection with the gastrointestinal tract-restricted helminth, Trichuris muris, and the intracellular bacterium, Mycobacterium bovis (M. bovis) BCG, would alter host immune responses to, or the pathological effect of, either infection. Results We demonstrate that both pathogens are capable of negatively affecting local and systemic immune responses towards each other by modifying cytokine phenotypes and by inducing general immune suppression. T. muris infection influenced non-specific and pathogen-specific immunity to M. bovis BCG by down-regulating pulmonary TH1 and Treg responses and inducing systemic TH2 responses. However, co-infection did not alter mycobacterial multiplication or dissemination and host pulmonary histopathology remained unaffected compared to BCG-only infected mice. Interestingly, prior M. bovis BCG infection significantly delayed helminth clearance and increased intestinal crypt cell proliferation in BALB/c mice. This was accompanied by a significant reduction in systemic helminth-specific TH1 and TH2 cytokine responses and significantly reduced local TH1 and TH2 responses in comparison to T. muris-only infected mice. Conclusion Our data demonstrate that co-infection with pathogens inducing opposing immune phenotypes, can have differential effects on compartmentalized host immune protection to either pathogen. In spite of local and systemic decreases in TH1 and increases in TH2 responses co-infected mice clear M. bovis BCG at the same rate as BCG only infected animals, whereas prior mycobacterial infection initiates prolonged worm infestation in parallel to decreased pathogen-specific TH2 cytokine production. PMID:24433309
Hayashi, Daisuke; Takii, Takemasa; Mukai, Tetsu; Makino, Masahiko; Yasuda, Emi; Horita, Yasuhiro; Yamamoto, Ryuji; Fujiwara, Akiko; Kanai, Keita; Kondo, Maki; Kawarazaki, Aya; Yano, Ikuya; Yamamoto, Saburo; Onozaki, Kikuo
In order to evaluate the biochemical characteristics of 14 substrains of Mycobacterium bovis bacillus Calmette Guérin (BCG) - Russia, Moreau, Japan, Sweden, Birkhaug, Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia and Pasteur - we performed eight different biochemical tests, including those for nitrate reduction, catalase, niacin accumulation, urease, Tween 80 hydrolysis, pyrazinamidase, p-amino salicylate degradation and resistance to thiophene 2-carboxylic acid hydrazide. Catalase activities of the substrains were all low. Data for nitrate reduction, niacin accumulation, Tween 80 hydrolysis, susceptibility to hydrogen peroxide and nitrate, and optimal pH for growth were all variable among these substrains. These findings suggest that the heterogeneities of biochemical characteristics are relevant to the differences in resistance of BCG substrains to environmental stress. The study also contributes to the re-evaluation of BCG substrains for use as vaccines.
Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare antigen-specific immune responses to varied patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis), colonization without path...
Molad, T; Fleiderovitz, L; Leibovich, B; Wolkomirsky, R; Erster, O; Roth, A; Mazuz, M L; Markovics, A; Shkap, V
This study demonstrated the genetic diversity among MSA-2c, MSA-2a1 and MSA-2b proteins of Babesia bovis isolates obtained from bovine blood and Rhipicephalus annulatus tick samples. The least identities that were observed among the deduced amino acid sequences of MSA-2c, MSA-2a1 and MSA-2b were 55, 63, and 71%, respectively. During the study four B. bovis calves, aged about 1 month, were found to be infected with virulent field strains and developed babesiosis. Probably, the calves had received insufficient antibodies, or the antibodies raised against the vaccine strain did not cross-protect against virulent field isolates. The complete msa-2 locus from the Israeli B. bovis vaccine strain and two field isolates were characterized. Similarly to the Australian strains and isolates, the msa-2 loci of the examined Israeli strain and isolates had only two msa-2 genes - msa-2c and msa-2a/b - located between msa-2c and orfB. Several of the examined samples, contained different MSA-2 genotypes concurrently. No obvious geographical relationships among isolates from various regions of Israel were established. Moreover, in the phylogenetic analyses, the Israeli deduced MSA-2 amino acid sequences of the three examined genes were clustered together with sequences derived from other countries, proving that the msa-2 gene sequences of B. bovis shared the same genetic characters worldwide. The present study clearly showed that the MSA-2 proteins of B. bovis isolates from Israel were genetically distinct from the vaccine strains. Thus, further research will be needed in order to understand the genetic diversity mechanisms of B. bovis, and the immunological responses of the infected animals.
Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. Here we propose using transfected ...
An immunosensor method for diagnosis of Babesia bovis in cattle based on impedance measurement is presented in this study. The method probes the interaction between serum antibodies against B. bovis infected cattle and recombinant protein, RAP-1, with C-terminal obtained from a Portuguese B. bovis s...
... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Tapeworm cysts (cysticercus bovis) in... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of... humerus. (2) Carcasses of cattle showing one or more tapeworm lesions of cysticercus bovis but not so...
... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Tapeworm cysts (cysticercus bovis) in... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of... humerus. (2) Carcasses of cattle showing one or more tapeworm lesions of cysticercus bovis but not so...
... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Tapeworm cysts (cysticercus bovis) in... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of... humerus. (2) Carcasses of cattle showing one or more tapeworm lesions of cysticercus bovis but not so...
... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Tapeworm cysts (cysticercus bovis) in... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of... humerus. (2) Carcasses of cattle showing one or more tapeworm lesions of cysticercus bovis but not so...
... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Tapeworm cysts (cysticercus bovis) in... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of... humerus. (2) Carcasses of cattle showing one or more tapeworm lesions of cysticercus bovis but not so...
Mycobacterium bovis infection of cats is exceedingly rare in non-endemic regions for bovine tuberculosis. This case study describes the diagnosis and clinical management of pulmonary M. bovis infection in two indoor-housed cats and their association with at least one M. bovis-infected human in Texas...
Mycoplasma bovis (M. bovis) has recently emerged as a significant and costly infectious disease problem in bison, generally presenting as severe, caseonecrotic pneumonia. Here we describe three diagnostic cases in which M. bovis is strongly implicated as a causative agent of necrotic pharyngitis. ...
Brito, Luciana G; Rocha, Rodrigo B; Barbieri, Fábio da S; Ribeiro, Elisana S; Vendrami, Fabiano B; Souza, Gislaine C R; Giglioti, Rodrigo; Regitano, Luciana C A; Falcoski, Thaís O R S; Tizioto, Polyana C; Oliveira, Márcia C S
The present study provides the first epidemiological data on infection with Babesia bovis in cattle raised in the southwestern Brazilian Amazon. Blood clot samples were filtered through nylon cloth before being submitted to DNA extraction. PCR and nested-PCR were applied to assess the frequency of infection with B. bovis in calves with ages from 4 to 12 months bred in 4 microregions each in the states of Rondônia and Acre. After the DNA was extracted from the samples, the infection in cattle was investigated by amplification of the "rap1" gene from B. bovis. The DNA amplification results revealed a frequency of infection with B. bovis of 95.1% (272/286) in the samples from Rondônia and 96.1% (195/203) in those from Acre. The high frequency of B. bovis infection in the animals with ages from 4 to 12 months indicates a situation of enzootic stability in the regions studied. The infection rates are comparable to those detected by immunodiagnostic techniques in other endemic regions of Brazil. Copyright © 2012 Elsevier GmbH. All rights reserved.
Stalheim, O H; Proctor, S J
Two pregnant cows aborted 11 and 18 days after Mycoplasma agalactiae subsp bovis was inoculated into the amniotic fluids. The placentas were retained. The fetuses (approx 100 and 150 days of age) were decomposed; M agalactiae subsp bovis was recovered from several tissues of the fetuses, the placentas, and fetal fluids. The same organism was given by intraperitoneal injection to 2 other pregnant (130 and 180 days, respectively) cows. At necropsy of the latter 36 days later, placentitis was severe; M agalactiae subsp bovis was recovered from the placentas of both cows and from the fetus of 1 cow. Control cows given sterile mycoplasma cultural medium by intraamnion or intraperitoneal injection did not abort and were not infected. When first recovered from the bovine placenta and fetus, M agalactiae subsp bovis grew slowly in liquid medium and assumed bizarre colonial morphology on solidified medium. Colonies were small (0.1 to 0.5 mm) and dark and lacked halos, but they reacted specifically in the direct fluorescent antibody test with equine M agalactiae subsp bovis antiserum. After 1 or 2 subcultures, the isolates grew at a normal rate and displayed their usual colonial morphology.
Majid, A.A.; Bushera, H.O.; Saad, A.M.; Hussein, M.F.; Taylor, M.G.; Dargie, J.D.; Marshall, T.F.; Nelson, G.S.
Previous work has shown that cattle can acquire a strong resistance to Schistosoma bovis infection following repeated natural exposure. Partial resistance to a laboratory challenge with S. bovis has also been demonstrated in calves after immunization with an irradiated schistosomular or cercarial vaccine. The aim of the present study was to see whether this type of caccine could protect calves under the very different conditions of natural exposure to S. bovis in the field. Thirty 6- to 9-month-old calves were each immunized with 10,000 irradiated S. bovis schistosomula by intramuscular injection and 8 weeks later were released into an enzootic area along with 30 unvaccinated animals. The calves were followed up for 10 months, during which period protection was evidenced by a lower mortality rate, a slower rate of acquisition of infection, and lower fecal egg counts in the vaccinated calves. Necropsy of the survivors showed 60 to 70% reductions in worm and tissue egg counts of the vaccinated calves as compared to those not vaccinated.
Faurholt-Jepsen, Daniel; Lillebaek, Troels; Nielsen, Ming-Yuan; Nielsen, Susanne Dam
In Denmark, tuberculous meningitis is rare. Central nervous system (CNS) involvement with Mycobacterium bovis is even rarer and has only been seen three times since 1992. We present a case of M. bovis meningitis in a previously healthy young Nigerian-born male, who had been exposed to unpasteurized dairy products in Nigeria but had no known contact with larger mammals. Before the development of meningitis, the patient had several contacts with the health system due to fever and non-specific symptoms. Finally, upon hospital admission, the patient was diagnosed with M. tuberculosis complex meningitis and treated empirically. After 13 days he was discharged without neurological sequelae. Later, the culture revealed M. bovis and treatment was adjusted accordingly.
Irvin, R T; To, M; Costerton, J W
Alysiella bovis adheres to surfaces by means of short, ruthenium red-staining, rod-like fimbriae. The fimbriae remain associated with the cell envelope of A. bovis, even when sonicated or exposed sequentially to toluene, Triton X-100, lysozyme, ribonuclease, and deoxyribonuclease. Adhesion of outer membrane-derived cell wall ghosts of A. bovis to glass was inhibited by IO4-, sodium dodecyl sulfate, urea, pronase, and trypsin. Protease treatment digested the fimbriae from the distal end, and exposure to sodium dodecyl sulfate depolymerized the fimbriae. Exposure of ghosts to 1% sodium dodecyl sulfate preferentially solubilized a 16,500-dalton protein which was subsequently purified by gel filtration and demonstrated to be a glycoprotein (ca. 17% carbohydrate). Antibodies raised against the 16,500-dalton glycoprotein agglutinated whole cells and inhibited adhesion of ghosts to glass. Images PMID:6209260
Buddle, Bryce M; Denis, Michel; Aldwell, Frank E; Martin Vordermeier, H; Glyn Hewinson, R; Neil Wedlock, D
Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine delivered to calves by the subcutaneous (s.c.) or by the oral route in a formulated lipid matrix has been previously shown to induce similar levels of protection against bovine tuberculosis. The current study was aimed at determining whether a combination of delivering BCG by s.c. and oral routes would enhance levels of protection, compared to only one route of vaccination. Forty calves were randomly divided into four groups (10/group). Calves were vaccinated with 10(6)colony forming units (CFU) of BCG Pasteur by the s.c. route or orally with 10(9)CFU BCG incorporated into a lipid formulation. One group received a combination of BCG administered by both the s.c. and oral routes and a non-vaccinated group served as a control. The two groups of calves that received s.c. BCG produced strong IFN-gamma responses in whole blood cultures stimulated with bovine purified protein derivative (PPD) 3 weeks after vaccination. Cattle vaccinated just with oral BCG in a lipid matrix produced a strong IFN-gamma response 8 weeks after vaccination, and peaking at 11 weeks after vaccination. All calves were challenged by the intratracheal route with M. bovis 15 weeks after vaccination and were euthanized and necropsied to assess protection at 17 weeks following challenge. BCG given s.c. or orally induced significant and comparable levels of protection against the virulent challenge. Vaccination of cattle by a combination of s.c./oral routes did not enhance protection beyond that achieved by s.c. or oral vaccination alone. We conclude that vaccination of cattle with BCG by a combination of routes has no beneficial additive effects, compared to a single s.c. administration of BCG or BCG given orally in a lipid formulation.
El-Sayed, A; El-Shannat, S; Kamel, M; Castañeda-Vazquez, M A; Castañeda-Vazquez, H
Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is a serious re-emerging disease in both animals and humans. The evolution of the Multi- and Extensively drug-resistant M. bovis strains (MDR-TB and XDR-TB) represents a global threat to public health. Worldwide, the disease is responsible for great economic losses in the veterinary field, serious threat to the ecosystem, and about 3.1% of human TB cases, up to 16% in Tanzania. Only thorough investigation to understand the pathogen's epidemiology can help in controlling the disease and minimizing its threat. For this purpose, various tools have been developed for use in advanced molecular epidemiological studies of bTB, either alone or in combination with standard conventional epidemiological approaches. These techniques enable the analysis of the intra- and inter-species transmission dynamics of bTB. The delivered data can reveal detailed insights into the source of infection, correlations among human and bovine isolates, strain diversity and evolution, spread, geographical localization, host preference, tracing of certain virulence factors such as antibiotic resistance genes, and finally the risk factors for the maintenance and spread of M. bovis. They also allow for the determination of epidemic and endemic strains. This, in turn, has a significant diagnostic impact and helps in vaccine development for bTB eradication programs. The present review discusses many topics including the aetiology, epidemiology and importance of M. bovis, the prevalence of bTB in humans and animals in various countries, the molecular epidemiology of M. bovis, and finally applied molecular epidemiological techniques. © 2015 Blackwell Verlag GmbH.
Vazquez-Chacon, Carlos A; Martínez-Guarneros, Armando; Couvin, David; González-Y-Merchand, Jorge A; Rivera-Gutierrez, Sandra; Escobar-Gutierrez, Alejandro; De-la-Cruz López, Juan J; Gomez-Bustamante, Adriana; Gonzalez-Macal, Gabriela A; Gonçalves Rossi, Livia Maria; Muñiz-Salazar, Raquel; Rastogi, Nalin; Vaughan, Gilberto
Here, we describe the molecular characterization of six human Mycobacterium bovis clinical isolates, including three multidrug resistant (MDR) strains, collected in Mexico through the National Survey on Tuberculosis Drug Resistance (ENTB-2008), a nationally representative survey conducted during 2008-2009 in nine states with a stratified cluster sampling design. The genetic background of bovine M. bovis strains identified in three different states of Mexico was studied in parallel to assess molecular relatedness of bovine and human strains. Additionally, resistance to first and second line anti-tuberculosis (TB) drugs and molecular identification of mutations conferring drug resistance was also performed. All strains were characterized by spoligotyping and 24-loci MIRU-VNTRs, and analyzed using the SITVIT2 (n = 112,000 strains) and SITVITBovis (n = 25,000 strains) proprietary databases of Institut Pasteur de la Guadeloupe. Furthermore, data from this study (n = 55 isolates), were also compared with genotypes recorded for M. bovis from USA (n = 203), Argentina (n = 726), as well as other isolates from Mexico (independent from the present study; n = 147), to determine any evidence for genetic relatedness between circulating M. bovis strains. The results showed that all human M. bovis cases were not genetically related between them or to any bovine strain. Interestingly, a high degree of genetic variability was observed among bovine strains. Several autochthonous and presumably imported strains were identified. The emergence of drug-resistant M. bovis is an important public health problem that jeopardizes the success of TB control programs in the region.
Galdy, Salvatore; Nastasi, Giuseppe
A relationship between infective endocarditis and colon cancer was established in 1950, and Streptococcus bovis was successfully isolated in 1970. However, this association and its pathogenesis still remain unclear. In this paper, we describe the clinical case of a patient with a history of colon cancer and infective endocarditis caused by Streptococcus bovis. The role of S bovis as an aetiological agent in the development of colon cancer is intriguing but uncertain. S bovis infection should be considered a silent sign of gastrointestinal malignancy or hepatic disease. We believe that in order to demonstrate the presence of colon cancer, all patients with S bovis infection require an endoscopic investigation of the colon.
Shu, Q; Bir, S H; Gill, H S; Duan, E; Xu, Y; Hiliard; Rowe, J B
Recent studies have shown that immunization with Streptococcus bovis using Freund's complete adjuvant (FCA) may confer protection against lactic acidosis in sheep. The major objective of this study was to compare the antibody responses to S. bovis in a practically acceptable adjuvant (Freund's incomplete adjuvant (FIA); QuilA; dextran sulphate (Dex); Imject Alum; or Gerbu) and in FCA. Thirty-five sheep were randomly allocated to 7 treatment groups. Six groups were immunized with S. bovis in an adjuvant; the other group served as the non-immunization control. The primary immunization was administered intramuscularly on day 0. followed by a booster injection on day 28. Immunization with FCA induced the highest saliva and serum antibody responses. The saliva antibody concentrations in the FIA and QuilA groups were significantly higher than those in the Alum, Dex and Gerbu groups (p < 0.01). The serum antibody concentration in the FIA group was significantly higher than those in the QuilA, Alum. Dex and Gerbu groups (p < 0.01). Immunization enhanced the antibody level in faeces (p < 0.05), but there was no significant difference between the different adjuvant groups (p > 0.05). Seven and 14 days following booster immunization, the saliva antibody levels induced by QuilA and/or FIA were comparable with the level stimulated by FCA (p > 0.05). There was a strongly positive correlation (R2 = 0.770, p < 0.01) between the antibody concentrations in salival and serum. Compared with the controls, a higher faecal dry matter content was observed in the animals immunized with either FCA or QuilA. The change in faecal dry matter content was positively associated with the faecal antibody concentration (R2 = 0.441, p < 0.05). These results indicate that FIA and QuilA were effective at inducing high levels of antibody responses to S. bovis, and suggest that either Freund's incomplete adjuvant or QuilA may be useful for preparing a practically acceptable vaccine against lactic
Gil-Turnes, C; de Araujo, F L
Sera were produced in rabbits against nine Moraxella bovis strains isolated in Brazil and three in the United States. Antigens were prepared for double immunodiffusion tests by thawing concentrated suspensions of the strains. Sera were tested against homologous and heterologous antigen preparations by the double immunodiffusion method. Sera showing precipitin bands with heterologous antigens were absorbed. Antigenic differences were detected between the strains and a provisional grouping of strains of M. bovis was suggested on the basis of antigenic composition. Differences between isolates from different geographical locations were found and some strains appeared antigenically more complex than others. The relevance of this work to vaccine production was suggested.
Gil-Turnes, C; de Araujo, F L
Sera were produced in rabbits against nine Moraxella bovis strains isolated in Brazil and three in the United States. Antigens were prepared for double immunodiffusion tests by thawing concentrated suspensions of the strains. Sera were tested against homologous and heterologous antigen preparations by the double immunodiffusion method. Sera showing precipitin bands with heterologous antigens were absorbed. Antigenic differences were detected between the strains and a provisional grouping of strains of M. bovis was suggested on the basis of antigenic composition. Differences between isolates from different geographical locations were found and some strains appeared antigenically more complex than others. The relevance of this work to vaccine production was suggested. Images Fig. 1 PMID:6178487
Gil-Turnes, C; Ribeiro, G A
Several properties of the adhesins of eight isolates of Moraxella bovis recovered from cattle suffering from infectious keratoconjunctivitis, were studied. Adhesions were detected through autoagglutination in saline and hemagglutination. Autoagglutinating strains agglutinated red blood cells of the chicken, rabbit, sheep and swine, but not those of the guinea pig. The adhesins were not inhibited by D-mannose or D-galactose and resisted heating at 100 degrees C for 15 minutes. Magnesium chloride at a final concentration of 10% inhibited autoagglutination and hemagglutination. The value of the hemagglutination test for monitoring synthesis of fimbriae by M. bovis, is discussed. PMID:3986674
Lee, JangEun; Ling, Changying; Kosmalski, Michelle M.; Hulseberg, Paul; Schreiber, Heidi A.; Sandor, Matyas; Fabry, Zsuzsanna
To study whether cerebral mycobacterial infection induces granuloma and protective immunity similar to systemic infection, we intracerebrally infected mice with Mycobacterium bovis bacilli Calmette-Guerin. Granuloma and IFN-γ+CD4+ T cell responses are induced in the central nervous system (CNS) similar to periphery, but the presence of IFN-γIL-17 double-positive CD4+ T cells is unique to the CNS. The major CNS source of TNF-α is microglia, with modest production by CD4+ T cells and macrophage. Protective immunity is accompanied by accumulation of Foxp3+CD4+ T cells and PD-L2+ dendritic cells, suggesting that both inflammatory and anti-inflammatory responses develop in the CNS following mycobacterial infection. PMID:19535154
Nishi, John S; Shury, Todd; Elkin, Brett T
In Canada, there are two known regional foci where wildlife populations are infected with bovine tuberculosis (Mycobacterium bovis) and considered to be disease reservoirs. Free-ranging populations of wood bison (Bison bison athabascae) in and around Wood Buffalo National Park (WBNP) and wapiti (Cervus elaphus manitobensis) in and around Riding Mountain National Park (RMNP) are infected with bovine tuberculosis. In this paper, we provide an overview of these diseased wild ungulate populations and the complexities of attempting to manage issues relating to bovine tuberculosis in and around protected areas. We do not describe the quantitative science and epidemiological data in detail from these case histories, but instead compare and contrast these two cases from a broader perspective. This is achieved by reviewing the context and process by which a diverse group of stakeholders engage and develop strategies to address the controversial problems that diseased wildlife populations often present. We suggest that understanding the factors that drive the strategic-level management processes is equally important for addressing a wildlife disease problem as the tactical-level issues, such as design and implementation of technically sound field research and management programs. Understanding the experiences within the WBNP and RMNP areas, particularly the strategies that have failed or succeeded, may prove useful to understanding and improving management approaches when wildlife are infected with M. bovis. Applying this understanding is consistent with the principles of adaptive management in which we learn from previous experiences to develop better strategies for the future.
Méndez-Samperio, Patricia; Ayala, Hilda; Vázquez, Abraham
Interaction between CD40L (CD154) on activated T cells and its receptor CD40 on antigen-presenting cells has been reported to be important in the resolution of infection by mycobacteria. However, the mechanism(s) by which Mycobacterium bovis bacillus Calmette-Guérin (BCG) up-regulates membrane expression of CD40L molecules is poorly understood. This study was done to investigate the role of the nuclear factor κB (NF-κB) signaling pathway in the regulation of CD40L expression in human CD4+ T cells stimulated with BCG. Specific pharmacologic inhibition of the NF-κB pathway revealed that this signaling cascade was required in the regulation of CD40L expression on the surface of BCG-activated CD4+ T cells. These results were further supported by the fact that treatment of BCG-activated CD4+ T cells with these pharmacological inhibitors significantly down-regulated CD40L mRNA. In this study, inhibitor κBα (IκBα) and IκBβ protein production was not affected by the chemical protease inhibitors and, more importantly, BCG led to the rapid but transient induction of NF-κB activity. Our results also indicated that CD40L expression on BCG-activated CD4+ T cells resulted from transcriptional up-regulation of the CD40L gene by a mechanism which is independent of de novo protein synthesis. Interestingly, BCG-induced activation of NF-κB and the increased CD40L cell surface expression were blocked by the protein kinase C (PKC) inhibitors 1-[5-isoquinolinesulfonyl]-2-methylpiperazine and salicylate, both of which block phosphorylation of IκB. Moreover, rottlerin a Ca2+-independent PKC isoform inhibitor, significantly down-regulated CD40L mRNA in BCG-activated CD4+ T cells. These data strongly suggest that CD40L expression by BCG-activated CD4+ T cells is regulated via the PKC pathway and by NF-κB DNA binding activity. PMID:12738634
Combrink, M P; Troskie, P C; de Klerk, D G; Pienaar, R; Latif, A A; Mans, B J
The South African Babesia bovis live blood vaccine, originating from a field isolate attenuated by 23 serial syringe passages in splenectomized calves, has lost the ability to infect the natural vector Rhipicephalus (Boophilus) microplus. In this study, infection with mixed parasites from the vaccine strain and a field isolate, resulted in transmission of both genotype populations. Comparing the field isolate and transmitted combination indicated no significant difference in their virulence, while challenge of vaccinated cattle with these isolates showed the ability of the vaccine to protect against both. Limiting dilution of the transmitted combination, followed by infection of splenectomized cattle (n=34) yielded no single infections for the vaccine strain genotype, seven clonal lines of the field isolate and one mixture of vaccine strain and field isolate. Only one of two field isolate clonal lines selected for vector transmission study was transmitted. Showing that B. bovis isolates can contain both tick transmissible and non-transmissible subpopulations. The findings of this study also indicate the probability of vaccine co-infection transmission occurring in the field, which may result in new genotype populations of B. bovis. However, the impact of this recombination with field isolates is considered negligible since a genotypically diverse population of B. bovis is already present in South Africa.
Drewe, J A; Foote, A K; Sutcliffe, R L; Pearce, G P
Pathological lesions associated with Mycobacterium bovis infection (bovine tuberculosis; bTB) in free-living meerkats (Suricata suricatta) in the Kalahari Desert of South Africa are described. The pathology of bTB in meerkats was determined through detailed post-mortem examinations of 57 animals (52 meerkats showing clinical signs of bTB, and five not showing signs of disease). Lymph nodes and tissue lesions thought to be associated with bTB were cultured for mycobacteria. All 52 bTB-infected meerkats showed gross or microscopical granulomatous lesions, but M. bovis was cultured from only 42% (22/52) of these animals. The majority (96%, 50/52) of diseased meerkats had lesions in multiple sites, the pattern of which suggested haematogenous spread of M. bovis infection in this species. The histological characteristics of the tuberculous lesions, together with the gross pathology and the wide range of body systems affected, indicate that infection in meerkats is acquired principally via the respiratory and oral routes, whereas excretion is most likely via the respiratory tract and suppurating skin wounds. Urine and faeces appear to be unlikely sources of infection. The findings of this study provide information on the transmission, pathogenesis and epidemiology of bTB in meerkats that is likely to be relevant to the understanding of M. bovis infection in other social mammal species such as the European badger (Meles meles).
... required for the Reference PPD Tuberculin. Allowance should be made for deaths during the sensitization period. (2) Sensitization of guinea pigs. (i) Sensitize one group of guinea pigs to M. bovis. Inject each... the same sensitization and the same PPD tuberculin injection, then divide by the number of animals in...
Muñoz Mendoza, Marta; Juan, Lucía de; Menéndez, Santiago; Ocampo, Antón; Mourelo, Jorge; Sáez, José L; Domínguez, Lucas; Gortázar, Christian; García Marín, Juan F; Balseiro, Ana
Tuberculosis was diagnosed in three flocks of sheep in Galicia, Spain, in 2009 and 2010. Two flocks were infected with Mycobacterium bovis and one flock was infected with Mycobacterium caprae. Infection was confirmed by the comparative intradermal tuberculin test, bacteriology, molecular analysis and histopathology. Sheep have the potential to act as a reservoir for tuberculosis. Copyright © 2011 Elsevier Ltd. All rights reserved.
Three isolates of Moraxella bovis, recovered from cattle with signs of infectious bovine keratoconjunctivitis, were tested for autoagglutinating activity, hemagglutinating activity and pathogenicity in young calves. Only the autoagglutinating and hemagglutinating isolates were pathogenic in calves. Treatment of the pathogenic isolates with magnesium chloride eliminated their pathogenic effects. PMID:6667436
Lantos, Ákos; Niemann, Stefan; Mezősi, László; Sós, Endre; Erdélyi, Károly; Dávid, Sándor; Parsons, Linda M.; Kubica, Tanja; Rüsch-Gerdes, Sabine
We report the first case of pulmonary tuberculosis caused by Mycobacterium bovis subsp. caprae in a captive Siberian tiger, an endangered feline. The pathogen was isolated from a tracheal aspirate obtained by bronchoscopy. This procedure provided a reliable in vivo diagnostic method in conjunction with conventional and molecular tests for the detection of mycobacteria. PMID:14718093
Behrendt, Jan Hillern; Hermosilla, Carlos; Hardt, Martin; Failing, Klaus; Zahner, Horst; Taubert, Anja
For successful in vivo infection, Eimeria bovis sporozoites have to traverse the mucosal layer of the ileum to infect lymphatic endothelial cells and may, thereby, be exposed to the interstitial fluid and to the lymph representing potential targets for leukocytes. To mimic this situation in vitro, we exposed E. bovis sporozoites to bovine PMN and found enhanced elimination of the parasites. Addition of immune serum clearly increased these reactions, whereas neonatal calf serum had no effect, thus proposing a PMN-derived antibody-dependent cytotoxicity. Scanning and transmission electron microscopy showed PMN engulfing sporozoites or extending filopodia towards them and occasionally incorporating the parasites. PMN reacted with enhanced transcription of IL-6, MCP-1, GROalpha, TNF-alpha, and iNOS genes after exposure to sporozoites while stimulation with merozoite-antigen, in addition, upregulated IL-8, IP-10 and IL-12 gene transcription. Furthermore, enhanced in vitro oxidative burst and phagocytic activities were observed after contact of PMN with viable sporozoites. To verify the potential role of PMN in the in vivo situation, we analysed the general phagocytic and oxidative burst activities of PMN obtained ex vivo from E. bovis experimentally infected calves. Enhanced levels of both activities were found early p.i. (1-5 days) and towards the end of the first schizogony (days 13-22 p.i.) underlining the in vitro data. Our results suggest that PMN-mediated, innate immune reactions play an important role in the early immune response to E. bovis infections in calves.
McElwain, Terry F.; Ueti, Massaro W.; Scoles, Glen A.; Reif, Kathryn E.; Lau, Audrey O. T.
Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype. PMID:25114111
Sondgeroth, Kerry S; McElwain, Terry F; Ueti, Massaro W; Scoles, Glen A; Reif, Kathryn E; Lau, Audrey O T
Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Ramarokoto, H; Andrianasolo, D; Rasolonavalona, T; Ramaroson, F; Razafitsiarovana, I; Vincent, V; Ratsimba, L; Rasolofo Razanamparany, V
We report a chronic case of pulmonary tuberculosis in a Malagasy citizen from Antsohihy (West of Madagascar), who was infected with a multi-drug resistant Mycobacterium bovis strain. This is the first case reported of the isolation of such a strain in Madagascar.
Introduction. Cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis and a relevant zoonosis to humans, may be sent to slaughter before diagnosis of infection because of slow multiplication of the pathogen. Purpose. This study evaluates multiple processing interventi...
Burr, Holly N; Wolf, Felix R; Lipman, Neil S
Corynebacterium bovis is a common pathogen in athymic nude mouse colonies. Control and eradication of the organism are challenging because depopulation and restricted colony access are often not options within vivaria. We evaluated potential sources and dissemination routes of C. bovis in an enzootically infected colony. Immunocompetent mice and personnel were evaluated for their potential to carry C. bovis, and husbandry and sanitation methods were evaluated for their efficacy in preventing cross-contamination. C. bovis was detected in furred immunocompetent mice previously exposed to infected athymic nude mice and in the nasopharynx of humans. Microisolation cages were not effective in maintaining athymic nude mice C. bovis-free when they were housed in a room known to contain immunodeficient mice with C. bovis infections. A tunnel washer that provided a ≥180 °F final rinse provided effective elimination of C. bovis from cage components. Passive and active air sampling techniques showed airborne dispersal of C. bovis despite the use of individually ventilated caging systems and stringent operational standards. Bacterial growth was not observed in settle plates placed inside autoclaved individually ventilated microisolation cages on various ventilated racks for 24-h periods. C. bovis aerosolization was shown to be a means of spread of the bacterium during cage-change procedures inside a class II type A2 biosafety cabinet. Our findings indicate that C. bovis can be a pervasive environmental contaminant in infected rodent holding rooms and successful eradication strategies must include environmental decontamination and attention to air quality.
Shimada, Kayoko; Azuma, Yuko; Kawase, Masaya; Takahashi, Toshiharu; Schaffer, Stephen W; Takahashi, Kyoko
Calculus Bovis (C. Bovis) is a commonly used animal-derived therapeutic preparation. To meet the increasing clinical demand for the preparation, two artificial substitutes for Bos Taurus have been introduced in China: artificial C. Bovis and in vitro cultured C. Bovis. However, information on their efficacy and safety is inadequate. Therefore, we investigated the biological differences between the commonly used natural preparation and its two substitutes, with the aim of not only identifying the differences but also providing a procedure to distinguish between the different preparations.In the study, we prepared 9 natural C. Bovis, 2 artificial C. Bovis, and 2 in vitro cultured C. Bovis preparations for evaluation. Differences were noted between the three preparations relative to their effect on viability of cardiac fibroblasts from 1-day-old Wistar rats. Although natural C. Bovis had no effect on cell viability, 1-h treatment of the cells with 0.25 mg/ml of the substitutes significantly reduced cell viability, as detected by the MTS assay. Based on liquid chromatography and inductively coupled plasma mass spectrometry, the preparations also differed in composition. Indeed, the substitutes contained more taurine, cholic acid, iron, magnesium, and calcium than the natural preparations. They also differed spectroscopically.The present results reveal significant biological differences between natural C. Bovis and two of its substitutes. Since the substitutes appear to contain more taurine, cholic acid, and elements, these constituents may serve as markers to distinguish between natural C. Bovis and its substitutes.
White-tailed deer (Odocoileus virginianus) serve as a reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and can be a source of infection in cattle. Vaccination with M. bovis bacille Calmette-Guerin (BCG) is being considered for management of bovine tuberculosis in deer. Presently, no...
Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock. The cause for many faltering eradication programs is the presence of wildlife reservoirs of M. bovis. One approach in dealing with this wildlife reservoir is to vaccinate ...
Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa
BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.
Gobin, J; Wong, D K; Gibson, B W; Horwitz, M A
Pathogenic mycobacteria must acquire iron in the host in order to multiply and cause disease. To do so, they release abundant quantities of siderophores called exochelins, which have the capacity to scavenge iron from host iron-binding proteins and deliver it to the mycobacteria. In this study, we have characterized the exochelins of Mycobacterium bovis, the causative agent of bovine and occasionally of human tuberculosis, and the highly attenuated descendant of M. bovis, bacillus Calmette-Guérin (BCG), widely used as a vaccine against human tuberculosis. The M. bovis type strain, five substrains of M. bovis BCG (Copenhagen, Glaxo, Japanese, Pasteur, and Tice), and two strains of virulent Mycobacterium tuberculosis all produce the same set of exochelins, although the relative amounts of individual exochelins may differ. Among these mycobacteria, the total amount of exochelins produced is greatest in M. tuberculosis, intermediate in M. bovis, and smallest in M. bovis BCG.
Altuzarra, Raúl; Vidal, Enric; Moll, Xavier; Espada, Yvonne; Sevilla, Iker A.; Domingo, Mariano; Garrido, Joseba M.; Juste, Ramón A.; Prieto, Miguel; Pérez de Val, Bernat
Background/Aims Animal tuberculosis (TB) is a complex animal health problem that causes disruption to trade and significant economic losses. TB involves a multi-host system where sheep, traditionally considered a rare host of this infection, have been recently included. The aims of this study were to develop an experimental TB infection model in sheep with a Mycobacterium caprae field strain isolated from a tuberculous diseased ewe, and to use this to evaluate the safety and efficacy of two vaccines against TB in sheep, the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. Methods Eighteen 2 month-old lambs were experimentally challenged with M. caprae by the endotracheal route (1.5 × 103 CFU). They were separated per treatment group into parenterally vaccinated with a live BCG Danish strain vaccine (n = 6), orally vaccinated with a suspension of HIMB (n = 6) and unvaccinated controls (n = 6). Clinical, immunological, pathological and bacteriological parameters of infection were measured. Results All lambs were successfully infected and developed gross TB lesions in the respiratory system. The BCG vaccine conferred considerable protection against experimental TB in lambs, as measured by a reduction of the gross lesion volumes and bacterial load. However, HIMB vaccinated animals did not show protection. Conclusions This study proposes a reliable new experimental model for a better understanding of tuberculosis in sheep. BCG vaccination offers an effective prospect for controlling the disease. Moreover alternative doses and/or routes of administration should be considered to evaluate the efficacy of the HIMB vaccine candidate. PMID:28678885
Balseiro, Ana; Altuzarra, Raúl; Vidal, Enric; Moll, Xavier; Espada, Yvonne; Sevilla, Iker A; Domingo, Mariano; Garrido, Joseba M; Juste, Ramón A; Prieto, Miguel; Pérez de Val, Bernat
Animal tuberculosis (TB) is a complex animal health problem that causes disruption to trade and significant economic losses. TB involves a multi-host system where sheep, traditionally considered a rare host of this infection, have been recently included. The aims of this study were to develop an experimental TB infection model in sheep with a Mycobacterium caprae field strain isolated from a tuberculous diseased ewe, and to use this to evaluate the safety and efficacy of two vaccines against TB in sheep, the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. Eighteen 2 month-old lambs were experimentally challenged with M. caprae by the endotracheal route (1.5 × 103 CFU). They were separated per treatment group into parenterally vaccinated with a live BCG Danish strain vaccine (n = 6), orally vaccinated with a suspension of HIMB (n = 6) and unvaccinated controls (n = 6). Clinical, immunological, pathological and bacteriological parameters of infection were measured. All lambs were successfully infected and developed gross TB lesions in the respiratory system. The BCG vaccine conferred considerable protection against experimental TB in lambs, as measured by a reduction of the gross lesion volumes and bacterial load. However, HIMB vaccinated animals did not show protection. This study proposes a reliable new experimental model for a better understanding of tuberculosis in sheep. BCG vaccination offers an effective prospect for controlling the disease. Moreover alternative doses and/or routes of administration should be considered to evaluate the efficacy of the HIMB vaccine candidate.
Spositto, F L E; Campanerut, P A Z; Ghiraldi, L D; Leite, C Q F; Hirata, M H; Hirata, R D C; Siqueira, V L D; Cardoso, R Fressatti
We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.
Kim, Jae Myung; Jang, Young-Boo; Jang, Yunho; Yu, So Yoon; Kim, Jiro; Moon, Oun Kyung; Jung, Suk Chan; Lee, Min Kwon; Jeong, Tae Nam
Mycobacterium (M.) bovis causes tuberculosis and has a broad host range, including humans, livestock, and wild animals. M. bovis infection of wild boar has been reported in several European countries. We report here the first case of M. bovis infection in a domesticated wild sow in Korea. Granulomatous and necrotizing lesions with small numbers of acid-fast bacilli were observed in nodules of the lung of wild sow. Furthermore, the M. bovis isolate from the wild sow had spoligotype SB0140 and a novel MIRU-VNTR allelic profile, which is not found in cattle and deer in Korea. PMID:26726026
Sechi, Leonardo A.; Duprè, Ilaria; Leori, Guido; Fadda, Giovanni; Zanetti, Stefania
Amplification of a specific, 500-bp fragment from Mycobacterium bovis isolates and use of the fragment to differentiate between Mycobacterium tuberculosis and M. bovis was previously reported (J. G. Rodriguez, G. A. Meja, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131–2138, 1995). In the present study, 30 M. bovis isolates from Sardinian cattle were examined for the presence of this 500-bp fragment; 4 of the 30 isolates lacked the fragment. This result indicates that identification of M. bovis strains by amplification of the 500-bp sequence may lead to false-negative results. PMID:11015414
Barbier, Elodie; Rochelet, Murielle; Gal, Laurent; Boschiroli, Maria Laura; Hartmann, Alain
Mycobacterium bovis, the causative agent of the bovine tuberculosis (bTB), mainly affects cattle, its natural reservoir, but also a wide range of domestic and wild mammals. Besides direct transmission via contaminated aerosols, indirect transmission of the M. bovis between wildlife and livestock might occur by inhalation or ingestion of environmental substrates contaminated through infected animal shedding. We monitored the survival of M. bovis in two soil samples chosen for their contrasted physical and-chemical properties (i.e. pH, clay content). The population of M. bovis spiked in sterile soils was enumerated by a culture-based method after 14, 30, 60, 90, 120 and 150 days of incubation at 4°C and 22°C. A qPCR based assay targeting the IS1561' locus was also performed to monitor M. bovis in both sterile and biotic spiked soils. The analysis of survival profiles using culture-based method showed that M. bovis survived longer at lower temperature (4°C versus 22°C) whereas the impact of soil characteristics on M. bovis persistence was not obvious. Furthermore, qPCR-based assay detected M. bovis for a longer period of time than the culture based method with higher gene copy numbers observed in sterile soils than in biotic ones. Impact of soil type on M. bovis persistence need to be deepened in order to fill the gap of knowledge concerning indirect transmission of the disease.
Daly, Meighan; Diegel, Kelly L; Fitzgerald, Scott D; Schooley, Angie; Berry, Dale E; Kaneene, John B
The state of Michigan has recognized the presence of Mycobacterium bovis in its free-ranging white-tailed deer population since 1994. This endemic infection is primarily located in a 12-county area in the northeastern lower peninsula of Michigan. A statewide surveillance and eradication program of the disease has been in effect since 1994. Worldwide, Mycobacterium tuberculosis complex organisms have a known predilection toward development of antimicrobial resistance. The objective of this study was to investigate the antimicrobial susceptibility of M. bovis isolates from white-tailed deer in Michigan and detect any changes in susceptibility over time. M. bovis isolates from 2 fall hunting seasons (1999 and 2004) were used in this study. The fall season of 2004 marked the first documented case of direct transmission of M. bovis from a wild deer to a human in Michigan. Since M. bovis is a zoonotic disease, knowledge of susceptibility can expedite treatment options in humans. M. bovis isolates were obtained from 58 deer, 4 coyotes, 3 cattle, 2 raccoons, and 1 human case from the 2 years combined. Methods of susceptibility testing included 1% proportion agar plates and Bactec radiometric broth testing. M. bovis was found to be uniformly resistant to the antibiotic pyrazinamide; this resistance is common to all M. bovis isolates. No other antimicrobial resistance was found in any of the tested M. bovis isolates, which may be, in part, attributed to the lack of any significant treatment pressure in wildlife.
Zumárraga, M J; Vivot, M Martínez; Marticorena, D; Bernardelli, A; Fasán, R; Iachini, R; Cataldi, A A
In the present work, 19 Mycobacterium bovis isolates from different cats were typified by spoligotyping. We detected nine spoligotypes. There was only one cluster, which grouped 11 of the isolates (57.9%), showing the main spoligotype from cattle from Argentina. The rest of the spoligotypes presented only one isolate each. Five of them were not found in cattle, and were unique and exclusive of cats. The isolates studied show that tuberculosis of bovine origin in cats constitutes a potential public health problem in Buenos Aires region. The identification of genotypes from non-natural hosts could contribute to understand the spread of bovine tuberculosis. This is the first report showing genetic profiles of M. bovis isolates in felines from Argentina.
Barandiaran, Soledad; Martínez Vivot, Marcela; Moras, Eduardo Vicente; Cataldi, Angel Adrián; Zumárraga, Martín José
A total of 143 Mycobacterium bovis isolates of pigs, from the most productive swine area in Argentina, were typed by spoligotyping. Twenty-two different spoligotypes were identified, and 133 (93%) isolates were grouped into 12 clusters. One of them, designed SB0140, was the most frequent because it held 83 (58%) isolates. This spoligotype also grouped 362 (43%) out of 841 isolates from previously typed cattle and, thus, constitutes the most frequent in our country. In addition, 135 (94%) isolates revealed spoligotypes identical to those of cattle, showing an epidemiological link. On the other hand, there were seven novel spoligotypes, six of which were also unique since they had only one isolate each. This study aimed to identify the spoligotypes of M. bovis isolated from pigs to contribute to a better understanding of the distribution of bovine tuberculosis in the main productive area of Argentina. PMID:21547236
Nigou, J; Gilleron, M; Brando, T; Vercellone, A; Puzo, G
The structures of the hydrophilic parietal and cellular arabinomannans isolated from Mycobacterium bovis BCG cell wall [Nigou et al. (1997) J Biol Chem 272: 23094-103] were investigated. Their molecular mass as determined by MALDI-TOF mass spectrometry was around 16 kDa. Concerning cap structure, capillary electrophoresis analysis demonstrated that dimannoside (Manpalpha1-->2Manp) was the most abundant motif (65-75%). Using two-dimensional 1H-13C NMR spectroscopy, the mannan core was unambiguously demonstrated to be composed of -->6Manpalpha1--> backbone substituted at some O-2 by a single Manp unit. The branching degree was determined as 84%. Finally, arabinomannans were found to be devoid of the phosphatidyl-myo-inositol anchor and, by aminonaphthalene disulfonate tagging, the mannan core was shown to contain a reducing end. This constitutes the main difference between arabinomannans and lipoarabinomannans from Mycobacterium bovis BCG.
Ben-Chetrit, E; Wiener-Well, Y; Kashat, L; Yinnon, A M; Assous, M V
Bacteremia with Streptococcus bovis/equinus complex strains is associated with hepatobiliary disease, colorectal lesions (CL), and infective endocarditis (IE). This study addressed the clinical significance of subspecies distinction of previously designated S. bovis blood culture isolates according to the updated nomenclature. During 2002-2013, all blood culture isolates previously designated as S. bovis were recultured and identified using 16S rRNA gene sequencing and MALDI-TOF (Bruker BioTyper and Vitek MS, bioMérieux). Clinical data of patients aged ≥18 years were reviewed. A review of four recent case series was performed as well. Forty blood isolates were identified using 16S rRNA sequencing. Twenty-six bacteremic patients had S. gallolyticus ssp. pasteurianus, six had S. gallolyticus ssp. gallolyticus, two had S. gallolyticus ssp. macedonicus, and six had S. infantarius bacteremia. Species diagnosis using Vitek and bioMérieux MALDI-TOF technology was applicable in 37 and 36 samples, respectively, and was successful in all samples (100 %). Subspecies identification was confirmed in 30 (83 %) samples (as compared with 16S rRNA sequencing detection). IE was diagnosed in 22 (59 %) patients and CL in 8 (20 %) patients. Both complications were associated with all subspecies. Combining our results with those of four recent series resulted in, overall, 320 bacteremic cases, of which 88 (28 %) had CL and 66 (21 %) had IE. All 'bovis/equinus' complex subspecies were associated with CL or IE. From a clinical point of view, species diagnosis using MALDI-TOF MS should suffice to warrant consideration of transesophageal echocardiography and colonoscopy.
Öztürk, Cihadiye Elif; Şahin, İdris; Öksüz, Şükrü; Kılıç, Nida; Kılınçel, Özge; Aydın, Leyla; Atik, Dursun; Afşin, Emine
Throughout the history of mankind, tuberculosis (TB) has caused serious illness and still continues to do so. Archaeobiological studies indicated that TB in humans dates back to 4000-8000 BC, and cases were shown to be due to Mycobacterium bovis subsp.bovis rather than Mycobacterium tuberculosis. Moreover, this situation was thought to begin with domestication of animals, consumption of their milk, and living together in the same environment with them. Over time, with the consumption of boiled milk and with the establishment of separate animal shelters, M.bovis subsp. bovis infection began to be seen rarely. Today, M.bovis infection is mostly transmitted from animals to humans and very rarely from humans to other humans. The most significant means of transmission of the infection are to the gastrointestinal tract via consumption of raw milk and to the respiratory system via droplet infection from the animals with disease. In this study, it was planned to investigate the cause of occurrence of TB in cattles in Düzce in the past few years along with the presence of bovine type TB in cases of human tuberculosis. We aimed to carry out subtype determination of the M.tuberculosis complex (MTBC) strains isolated in our mycobacteriology laboratory between the years 2004-2014, and evaluate the clinical and sociodemographic data of patients in whom M.bovis subsp. bovis was detected. The strains that were selected for the study have been isolated from radiometric BACTEC™ 12B broth and/or Löwenstein-Jensen (LJ) media between 2004-2009, and BACTEC™ MGIT™ (Mycobacteria Growth Indicator Tube) and/or LJ media between 2009-2014 periods. The GenoType MTBC Kit (Hain-Lifescience GmbH, Germany) was used in the study for determination of the subspecies. Extraction and amplification of DNA and hybridizations were performed according to test procedure in order to investigate the presence of subtypes of the MTBC species in skimmed milk from collections stored at -20°C. In the
Vergnon-Miszczycha, Delphine; Suy, Florence; Robert, Florence; Carricajo, Anne; Fresard, Anne; Cazorla, Céline; Guglielminotti, Claire; Lucht, Frédéric; Botelho-Nevers, Elisabeth
Guillain-Barré syndrome (GBS) is an autoimmune disease that can be triggered by different infectious agents. Here we report the case of a 26-year-old Algerian woman who developed GBS associated with a Mycobacterium bovis cervical lymphadenitis. Following intravenous immunoglobulin therapy, the patient's neurologic state returned to normal after 3 months. The lymphadenitis responded more slowly to the antituberculous treatment and an excision of necrotic cervical lymph nodes had to be performed four times. Antibiotics were administered for 16 months: ethambutol was stopped after 2 months, and rifampicin and isoniazid pursued for 14 months. An extensive etiological investigation showed that, in this case, the only likely infectious trigger GBS was the concomitant M. bovis infection. To our knowledge, this is the first report of GBS triggered by M. bovis. We performed a literature review revealing that the association between tuberculosis and Guillain-Barré syndrome is very rare (only seven cases previously reported) but is not coincidental. Physicians should be aware that tuberculosis can be a cause of GBS.
Herrera-Sánchez, Diana Andrea; Castilla-Rodríguez, Jaisel Luz; Castrejón-Vázquez, María Isabel; Vargas-Camaño, María Eugenia; Medina-Torres, Edgar Alejandro; Blancas-Galicia, Lizbeth; Espinosa-Padilla, Sara Elva
Common variable immunodeficiency (CVID) is an heterogeneous group of disorders characterized by impaired antibody production. It shows a wide spectrum of manifestations including severe and recurrent respiratory infections (Streptococcus pneumoniae, Haemophilus) and gastrointestinal (Campylobacter jejuni, rotavirus and Giardia lamblia). Viral infections caused by herpes zoster, cytomegalovirus (CMV) and hepatitis C are rare. The opportunistic agents such as CMV, Pneumocystis jirovecii, cryptococcus and atypical mycobacteria have been reported as isolated cases. This paper reports the case of a 38-year-old female patient, who began six years before with weight loss of 7 kg in six months, fatigue, weakness, sweating, fever and abdominal pain. Furthermore, patient had intestinal obstruction and abdominal CT showed mesenteric lymph growth. The mesenteric lymph node biopsy revealed positives Mycobacterium PCR, Ziehl-Neelsen staining and culture for M. bovis. In the laparotomy postoperative period was complicated with nosocomial pneumonia, requiring mechanical ventilation and tracheostomy. Two years later, she developed right renal abscess that required surgical drainage, once again with a positive culture for Mycobacterium bovis. She was referred to highly specialized hospital and we documented panhypogammaglobulinemia and lymphopenia. Secondary causes of hypogammaglobulinemia were ruled out and common variable immunodeficiency (CVID) was confirmed, we started IVIG replacement. Four years later she developed mixed cellularity Hodgkin's lymphoma. Until today she continues with IVIG and chemotherapy. This report of a patient with CVID and Mycobacterium bovis infection, a unusual association, shows the cellular immunity susceptibility in this immunodeficiency, additional to the humoral defect.
Hermosilla, Carlos; Ruiz, Antonio; Taubert, Anja
Apicomplexan parasites are obligate intracellular protozoans and are well recognized modulators of the host cell machinery on varying levels such as host cell metabolism, MHC expression, cell cycle, or apoptosis in order to guarantee their intracellular development and survival. One of the most thoroughly examined apicomplexan pathogens demonstrating a potent manipulative capacity with respect to various host cell functions is Toxoplasma gondii, a protozoon exhibiting rapid intracellular development with small meronts in any nucleated cell, almost irrespective of the cell type or host origin. In contrast, Eimeria bovis merogony I is host- and cell type-restricted and occurs exclusively in bovine endothelial host cells. Furthermore, as a peculiarity, intracellular E. bovis meront I development is a long-lasting process (up to 3 weeks), leading to the formation of huge macromeronts of up to 300 μm in size, containing up to 120,000 merozoites I as offspring. In consequence, the necessity for intense host cell modulation to support this particular development appears even more pressing than in other apicomplexan parasite cases. Here we review the data currently available on E. bovis-host cell interactions, indicating the intriguing capacity of this protozoan to exploit and utilize its host cell for its own benefit.
Rocha, Vivianne Cambuí Figueiredo; Figueiredo, Salomão Cambuí de; Rosales, Cesar Alejandro Rodriguez; Porto, Camila Dias; Sequeira, Julio Lopes; Neto, José Soares Ferreira; Paes, Antônio Carlos; Salgado, Vanessa Riesz
Tuberculosis (TB) is a chronic disease caused by bacteria belonging to the Mycobacterium tuberculosis complex (MtbC). This disease rarely affects dogs. Canine infections are usually caused by M. tuberculosis. Mycobacterium bovis infections are rare in dogs and associated with consumption of raw milk or contaminated products. Here, we report a Boxer dog who had a M. bovis infection and was admitted to a Brazilian veterinary hospital with a presumptive diagnosis of chronic ehrlichiosis. Despite receiving treatment for chronic ehrlichiosis, it progressed to death. TB was diagnosed during post-mortem examinations using histopathological analysis. Ziehl-Neelsen staining revealed acid-fast bacilli in the kidneys, liver, mesentery, and a mass adhered to the liver. Further, PCR-restriction analysis was performed to identify mycobacteria in the samples. A restriction profile compatible with MtbC was found in the lungs. In addition, PCR-based MtbC typing deletions at different loci of chromosome 9 enabled the identification of M. bovis in the lungs. Therefore, it is very essential to perform differential diagnosis of TB in dogs with non-specific clinical signs and who do not respond to treatment, particularly those who had been in contact with TB-infected cattle or owners. Further, we highlight the use of molecular methods for the identification of bacilli, improving the diagnosis and aiding epidemiological studies.
Clarridge, J E; Attorri, S M; Zhang, Q; Bartell, J
We characterized 22 human clinical strains of Streptococcus bovis by genotypic (16S rRNA gene sequence analysis [MicroSeq]; Applied Biosystems, Foster City, Calif.) and phenotypic (API 20 Strep and Rapid ID32 Strep systems (bioMerieux Vitek, Hazelton, Mo.) methods. The strains, isolated from blood, cerebrospinal fluid (CSF), and urine, formed two distinct 16S ribosomal DNA sequence clusters. Three strains which were associated with endocarditis urinary tract infection (UTI), and sepsis clustered with the S. bovis type strain ATCC 33317 (cluster 1); other closely related type strains were S. equinus and S. infantarius. Nineteen strains clustered at a distance of about 2.5% dissimilarity to the S. bovis type strain (cluster 2) and were associated with central nervous system (CNS) disease in addition to endocarditis, UTI, and sepsis. All strains were distinct from S. gallolyticus. Within cluster 2, a single strain grouped with ATCC strain 43143 (cluster 2a) and may be phenotypically distinct. All the other strains formed a second subgroup (cluster 2b) that was biochemically similar to S. bovis biotype II/2 (mannitol negative and beta galactosidase, alpha galactosidase, beta glucuronidase, and trehalose positive). The API 20 Strep system identified isolates of cluster 2b as S. bovis biotype II/2, those of cluster 1 as S. bovis biotype II/1, and that of cluster 2a as S. bovis biotype I. There was an excellent correlation of biotype and genotype: S. bovis biotype II/2 isolates form a separate genospecies distinct from the S. bovis, S. gallolyticus, and S. infantarius type strains and are the most common isolates in adult males.
Biet, Franck; Boschiroli, Maria Laura; Thorel, Marie Françoise; Guilloteau, Laurence A
Pathogens that are transmitted between the environment, wildlife, livestock and humans represent major challenges for the protection of human and domestic animal health, the economic sustainability of agriculture, and the conservation of wildlife. Among such pathogens, the genus Mycobacterium is well represented by M. bovis, the etiological agent of bovine tuberculosis, M. avium ssp. paratuberculosis (Map) the etiological agent of Johne disease, M. avium ssp. avium (Maa) and in a few common cases by other emergent environmental mycobacteria. Epidemiologic surveys performed in Europe, North America and New Zealand have demonstrated the existence and importance of environmental and wildlife reservoirs of mycobacterial infections that limit the attempts of disease control programmes. The aim of this review is to examine the zoonotic aspects of mycobacteria transmitted from the environment and wildlife. This work is focused on the species of two main groups of mycobacteria classified as important pathogens for humans and animals: first, M. bovis, the causative agent of bovine tuberculosis, which belongs to the M. tuberculosis complex and has a broad host range including wildlife, captive wildlife, domestic livestock, non-human primates and humans; the second group examined, is the M. avium-intracellulare complex (MAC) which includes M. avium ssp. avium causing major health problems in AIDS patients and M. avium ssp. paratuberculosis the etiological agent of Johne disease in cattle and identified in patients with Crohn disease. MAC agents, in addition to a broad host range, are environmental mycobacteria found in numerous biotopes including the soil, water, aerosols, protozoa, deep litter and fresh tropical vegetation. This review examines the possible reservoirs of these pathogens in the environment and in wildlife, their role as sources of infection in humans and animals and their health impact on humans. The possibilities of control and management programmes for
Smyth, Allister J.; Welsh, Michael D.; Girvin, R. Martyn; Pollock, John M.
It is generally accepted that protective immunity against tuberculosis is generated through the cell-mediated immune (CMI) system, and a greater understanding of such responses is required if better vaccines and diagnostic tests are to be developed. γδ T cells form a major proportion of the peripheral blood mononuclear cells (PBMC) in the ruminant system and, considering data from other species, may have a significant role in CMI responses in bovine tuberculosis. This study compared the in vitro responses of αβ and γδ T cells from Mycobacterium bovis-infected and uninfected cattle. The results showed that, following 24 h of culture of PBMC with M. bovis-derived antigens, the majority of γδ T cells from infected animals became highly activated (upregulation of interleukin-2R), while a lower proportion of the αβ T-cell population showed activation. Similar responses were evident to a lesser degree in uninfected animals. Study of the kinetics of this response showed that γδ T cells remained significantly activated for at least 7 days in culture, while activation of αβ T cells declined during that period. Subsequent analysis revealed that the majority of activated γδ T cells expressed WC1, a 215-kDa surface molecule which is not expressed on human or murine γδ T cells. Furthermore, in comparison with what was found for CD4+ T cells, M. bovis antigen was found to induce strong cellular proliferation but relatively little gamma interferon release by purified WC1+ γδ T cells. Overall, while the role of these cells in protective immunity remains unclear, their highly activated status in response to M. bovis suggests an important role in antimycobacterial immunity, and the ability of γδ T cells to influence other immune cell functions remains to be elucidated, particularly in relation to CMI-based diagnostic tests. PMID:11119493
Babesia bovis is a tick-borne apicomplexan parasite that causes an acute disease in cattle. This study describes stable expression of an exogenous gfp-bsd gene in B. bovis transformed parasites. Cultured B. bovis infected erythrocytes of the biologically cloned Mo7 strain were transfected by electro...
Sanabria, Lidia; Lagrave, Lorena; Nishibe, Christiane; Ribas, Augusto C A; Zumárraga, Martín J; Almeida, Nalvo F; Araújo, Flábio R
This work reports the draft genome sequences of the Mycobacterium bovis strains M1009 and M1010, isolated from the lymph nodes of two infected cows on a beef farm in Paraguay. Comparative genomics between these strains and other regional strains may provide more insights regarding M. bovis epidemiology in South America. Copyright © 2017 Sanabria et al.
Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and is the bacterium isolated most frequently from the polymicrobial syndrome known as bovine respiratory disease complex (BRDC). Recently, M. bovis has emerged as a significant health problem in bison, causing necro...
Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and is the bacterium isolated most frequently from the polymicrobial syndrome known as bovine respiratory disease complex (BRDC). Recently, M. bovis has emerged as a significant problem in bison, causing necrotic pha...
Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) and t...
Mycobacterium bovis is the cause of tuberculosis in most animal species, including cattle and is a serious zoonotic pathogen. In humans, M. bovis infection can result in disease clinically indistinguishable from that caused by Mycobacterium tuberculosis, the cause of most tuberculosis in humans. Reg...
Recently, several serologic tests designed to detect immunodominant antibodies to M. bovis antigens (e.g., MPB83, MPB70, ESAT-6, and CFP10) have emerged for potential use with samples from cattle. Of these, a commercial ELISA to MPB83/MPB70 (M. bovis antibody ELISA) has gained approval for use in ca...
Díez-Delgado, Iratxe; Contreras, Marinela; Vicente, Joaquín; Cabezas-Cruz, Alejandro; Manrique, Marina; Tobes, Raquel; López, Vladimir; Romero, Beatriz; Domínguez, Lucas; Garrido, Joseba M.; Gortazar, Christian
Here we report the complete genome sequences of field isolates of Mycobacterium bovis and the related mycobacterial species, Mycobacterium caprae. The genomes of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different virulence, prevalence, and host distribution phenotypes were sequenced. PMID:26112781
Mycobacterium bovis is an important animal pathogen worldwide that parasitizes wild and domesticated vertebrate livestock as well as humans. A comparison of the five M. bovis complete genomes from UK, South Korea, Brazil and USA revealed four novel large-scale structural variations of at least 2,000...
Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans or animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and the other members o...
Branger, Maxime; Hauer, Amandine; Michelet, Lorraine; Karoui, Claudine; Cochard, Thierry; De Cruz, Krystel; Henault, Sylvie
Mycobacterium bovis is the etiologic agent of bovine tuberculosis, a chronic infectious disease, affecting livestock, wild animals, and sometimes humans. We report the draft genome sequence of a Mycobacterium bovis strain isolated from wild boar of spoligotype SB0120 (or BCG-like) also present in wildlife-livestock multi-host systems. PMID:27834714
Sanabria, Lidia; Lagrave, Lorena; Nishibe, Christiane; Ribas, Augusto C. A.; Zumárraga, Martín J.; Araújo, Flábio R.
ABSTRACT This work reports the draft genome sequences of the Mycobacterium bovis strains M1009 and M1010, isolated from the lymph nodes of two infected cows on a beef farm in Paraguay. Comparative genomics between these strains and other regional strains may provide more insights regarding M. bovis epidemiology in South America. PMID:28705977
Fox, Lawrence K.; Muller, Fredrick J.; Wedam, Michael L.; Schneider, Christopher S.; Biddle, Mary Kate
Findings of herd investigations of heifers with prepubertal mastitis are presented. Mycoplasma bovis was isolated from lacteal secretions and tissue samples of necropsied heifers; the same strain infected dams and herd mates. Vertical transmission is suggested in this first report of intramammary infections of M. bovis in peripubertal heifers. PMID:19183734
Sahraoui, Naima; Müller, Borna; Guetarni, Djamel; Boulahbal, Fadéla; Yala, Djamel; Ouzrout, Rachid; Berg, Stefan; Smith, Noel H; Zinsstag, Jakob
Background Bovine Tuberculosis is prevalent in Algeria despite governmental attempts to control the disease. The objective of this study was to conduct, for the first time, molecular characterization of a population sample of Mycobacterium bovis strains isolated from slaughter cattle in Algeria. Between August and November 2007, 7250 animals were consecutively screened at the abattoirs of Algiers and Blida. In 260 animals, gross visible granulomatous lesions were detected and put into culture. Bacterial isolates were subsequently analysed by molecular methods. Results Altogether, 101 bacterial strains from 100 animals were subjected to molecular characterization. M. bovis was isolated from 88 animals. Other bacteria isolated included one strain of M. caprae, four Rhodococcus equi strains, three Non-tuberculous Mycobacteria (NTM) and five strains of other bacterial species. The M. bovis strains isolated showed 22 different spoligotype patterns; four of them had not been previously reported. The majority of M. bovis strains (89%) showed spoligotype patterns that were previously observed in strains from European cattle. Variable Number of Tandem Repeat (VNTR) typing supported a link between M. bovis strains from Algeria and France. One spoligotype pattern has also been shown to be frequent in M. bovis strains from Mali although the VNTR pattern of the Algerian strains differed from the Malian strains. Conclusion M. bovis infections account for a high amount of granulomatous lesions detected in Algerian slaughter cattle during standard meat inspection at Algiers and Blida abattoir. Molecular typing results suggested a link between Algerian and European strains of M. bovis. PMID:19173726
de la Fuente, José; Díez-Delgado, Iratxe; Contreras, Marinela; Vicente, Joaquín; Cabezas-Cruz, Alejandro; Manrique, Marina; Tobes, Raquel; López, Vladimir; Romero, Beatriz; Domínguez, Lucas; Garrido, Joseba M; Juste, Ramón; Gortazar, Christian
Here we report the complete genome sequences of field isolates of Mycobacterium bovis and the related mycobacterial species, Mycobacterium caprae. The genomes of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different virulence, prevalence, and host distribution phenotypes were sequenced. Copyright © 2015 de la Fuente et al.
Manuel, Christopher; Pugazhenthi, Umarani; Spiegel, Shannon; Leszczynski, Jori
Rodent health-monitoring programs based on sampling an IVC system's exhaust air dust (EAD) has enhanced and evenreplaced traditional sentinels for some rodent pathogens. EAD testing by qPCR assay is an optimal surveillance methodfor the rapid detection of Corynebacterium bovis-infected immunodeficient mice. Here we demonstrate that an active EADsurveillance program for C. bovis can be used to maintain nude mice C. bovis-free after the transition from historically enzootically infected colonies. During 3 events over 3 y, rapid detection of infection, elimination of infected mice, aggressivequarantine measures, and local decontamination prevented the spread of C. bovis within 2 barrier rooms. In total, 4 cages ofinfected nude mice were identified and removed, preventing the spread of infection to 469 other cages of immunodeficientmice. In addition, we present data regarding a refinement to EAD testing which enables row-specific surveillance of an IVCrack. This technique systemically decreases the amount of testing required to locate an individually infected cage. Due to ourability to rapidly detect and localize an infected cage, we were able to investigate the route of C. bovis introduction into ourbarrier rooms. Our epidemiologic investigation suggested that the transmission of C. bovis occurred through contaminated,cryopreserved, patient-derived xenograft tumor tissue. This previously unknown source of C. bovis can infect mice used topropagate these tumors. Together, these data demonstrate that a remediation program that combines rapid detection, testand-cull, and local decontamination under quarantine conditions can eliminate C. bovis from a mouse colony.
Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer in order to interrupt...
Skinner, Margot A; Parlane, Natalie; McCarthy, Allison; Buddle, Bryce M
Cytotoxic T-cell responses are thought to play a significant role in the host defence against mycobacterial infections. Little is understood about such responses of cattle to Mycobacterium bovis, the causative agent of bovine tuberculosis. The work described in this report demonstrates the activity of cytotoxic cells during experimental infection of cattle with M. bovis. The cytotoxic cells were found to have the ability to specifically lyse macrophages infected with M. bovis and were detected in peripheral blood lymphocytes after in vitro re-exposure to M. bovis. Cytotoxic activity was detected 4 weeks after experimental infection with M. bovis; a similar level of activity was maintained during the infection and it was mediated by both WC1+γδ and CD8+ T cells. In addition, inhibition of the growth of M. bovis within infected macrophages was detected when they were exposed to cultures containing M. bovis-specific cytotoxic cells. The ability to detect cytotoxic cells after infection of cattle with M. bovis will allow their activity to be measured during vaccination trials. Correlation of cytotoxic activity with disease outcome may aid in the design of new vaccines and vaccination strategies. PMID:14511237
Brooks, B W; Barnum, D A; Meek, A H
An observational study of Corynebacterium bovis was conducted in 74 Ontario dairy herds. The levels of infection with C. bovis were 19.9, 36.2 and 85.6% at the quarter, cow and herd level, respectively. Teat disinfection was found to be the variable best able to distinguish between herds with a high or low C. bovis quarter infection rate. Mean total milk somatic cell counts for 1103 quarters and 107 cows infected with only C. bovis ranged between 150,000 and 200,000/mL and were significantly higher than for uninfected quarters or cows. The rate of infection with mastitis pathogens was not significantly different in quarters previously colonized with only C. bovis compared to previously uninfected quarters. PMID:6831308
Arcangioli, Marie-Anne; Duet, Arnaud; Meyer, Gilles; Dernburg, Ann; Bézille, Pierre; Poumarat, François; Le Grand, Dominique
To assess the prevalence and relative importance of Mycoplasma bovis among the pathological agents implicated in bovine respiratory disease (BRD), we surveyed 135 veal calves from nine feedlots in eastern France during naturally occurring outbreaks of respiratory disease. Occurrence of respiratory pathogens, M. bovis, bovine viral diarrhoea (BVD) virus, bovine respiratory syncytial (BRS) virus and parainfluenza-3 (PI3) virus was investigated by seroconversion and isolation of bacteria and viruses from broncho-alveolar fluids. M. bovis and pathogenic respiratory bacteria were isolated in eight of the nine feedlots, and from 106 and 32, respectively, of the 135 tested animals. Seroconversion to PI3 virus occurred in four lots. BVD and BRS viruses were detected in eight and one lot, respectively. M. bovis was the most frequently isolated aetiologic agent in these BRD outbreaks, spreading early and widely throughout the affected units (60-100% rate of isolation and seroconversion). These results stress the importance of M. bovis in the BRD complex.
Szewzyk, R; Svenson, S B; Hoffner, S E; Bölske, G; Wahlström, H; Englund, L; Engvall, A; Källenius, G
Forty-nine isolates of Mycobacterium bovis from humans and animals in Sweden were analyzed by restriction fragment length polymorphism (RFLP) patterns probed by the insertion element IS6110. Most isolates had patterns indicating the presence of only one or two genomic copies of the IS6110 insertion element. This simple type of pattern was found in all human isolates. In contrast, isolates from M. bovis infections in five herds of farmed deer in Sweden showed a specific RFLP pattern with seven bands, indicating seven copies of the IS6110 sequence. In 1958, Sweden was declared free from M. bovis in cattle. However, in 1987, M. bovis was reintroduced with imported farmed deer, and since 1991, 11 outbreaks in deer herds, but not in other livestock or wildlife, have been diagnosed. Continued RFLP studies of the new Swedish M. bovis isolates can reveal possible transmission of this deer strain to other animals or humans. PMID:8586698
Liu, Ping; He, Xinrong; Guo, Mei
To investigate the correlation effects between single or combined administration of Calculus Bovis or zolpidem and changes of inhibitive neurotransmitter in rat striatum corpora. Sampling from rat striatum corpora was carried out through microdialysis. The content of two inhibitive neurotransmitters in rat corpus striatum- glycine (Gly) and gama aminobutyric acid (GABA), was determined by HPLC, which involved pre-column derivation with orthophthaladehyde, reversed-phase gradient elution and fluorescence detection. GABA content of rat striatum corpora in Calculus Bovis group was significantly increased compared with saline group (P < 0.01). GABA content of zolpidem group and Calculus Boris plus zolpidem group were increased largely compared with saline group as well (P < 0.05). GABA content of Calculus Bovis group was higher than combination group (P < 0.05). GABA content of zolpidem group was not significantly different from combination group. Gly content of Calculus Bovis or zolpidem group was markedly increased compared with saline group or combination group (P < 0.05). Contents of two inhibitive neurotransmitters in rat striatum corpora were all significantly increased in Calculus Bovis group, zolpidem group and combination group. The magnitude of increase was lower in combination group than in Calculus Bovis group and Zolpidem group, suggesting that Calculus Bovis promoted encephalon inhibition is more powerful than zolpidem. The increase in two inhibitive neurotransmitters did not show reinforcing effect in combination group, suggesting that Calculus Bovis and zolpidem may compete the same receptors. Therefore, combination of Calculus Bovis containing drugs and zolpidem has no clinical significance. Calculus Bovis shouldn't as an aperture-opening drugs be used for resuscitation therapy.
Antonio Alvarez, J; Lopez, U; Rojas, C; Borgonio, V M; Sanchez, V; Castañeda, R; Vargas, P; Figueroa, J V
The purpose of this research was to evaluate the recombinant proteins MSA-1, MSA-2c and 12D3 as a combined immunogen for cattle. Fifteen steers were randomly assigned into three groups of five animals each (I, II and III). On day 0, cattle in group I were injected with 50 microg each of rMSA-1, rMSA-2c and r12D3 with the adjuvant Montanide 75; cattle in Group II received adjuvant-PBS, and Group III were untreated controls. On day 14, cattle in Group I received a second injection of the three recombinant proteins in adjuvant and cattle in Group II again received adjuvant alone. On day 28, all groups of cattle were challenged with a field strain of Babesia bovis. After challenge, the experimental cattle were clinically and serologically monitored. Three of the five steers immunized with the combined recombinant B. bovis proteins seroconverted on day 14 post-immunization (P.I.) and the maximum titre was 1 : 1600. All five immunized steers presented strong seropositivity to B. bovis antigens at day 21 P.I. The prepatent periods of vaccinated cattle were delayed until day 10 post-challenge exposure versus 8 and 7 days in Groups II and III, respectively. Cattle in all groups had fever above 41 degrees C; the reduction in packed cell volume was not significantly different (P > 0.05) in vaccinated group I compared with Groups II and III (29% versus 26% and 31%, respectively). Treatment was required for one steer in the control group. During the period of the study, the weight of cattle in Groups I and II increased an average of 9 and 7 kg, whereas the weight of the control cattle was reduced on average 4 kg. Immunization with rMSA-1-rMSA-2c-r12D3 proteins was not sufficient to prevent clinical symptoms against challenge, but the immunologic response was sufficient to protect steers against a mild virulent strain of B. bovis.
Palmero, J; Pusterla, N; Cherry, N A; Kasten, R W; Mapes, S; Boulouis, H J; Breitschwerdt, E B; Chomel, B B
Experimental infection of horses with Bartonella species is not documented. Determine clinical signs, hematologic changes, duration of bacteremia, and pattern of seroconversion in Bartonella henselae or Bartonella bovis-inoculated horses. Twelve (2 groups of 6) randomly selected healthy adult horses seronegative and culture negative for Bartonella spp. Experimental/observational study: Group I: B. henselae or saline control was inoculated intradermally into 4 naïve and 2 sentinel horses, respectively. Group II: same design was followed by means of B. bovis. Daily physical examinations, once weekly CBC, immunofluorescent antibody assay serology, real-time polymerase chain reaction (PCR), and twice weekly blood cultures were performed for 6 weeks and at postinoculation day 80 and 139. Bartonella alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture was performed for horses that seroconverted to B. henselae antigens. Transient clinical signs consistent with bartonellosis occurred in some Bartonella-inoculated horses, but hematological alterations did not occur. Three B. henselae-inoculated horses seroconverted, whereas 1 B. bovis-inoculated horse was weakly seropositive. In Group I, B. henselae was amplified and sequenced from BAPGM blood culture as well as a subculture isolate from 1 horse, blood from a 2nd horse, and BAPGM blood culture from a 3rd horse although a subculture isolate was not obtained. All sentinels remained PCR, culture, and serology negative. Detection of Bartonella sp. in blood after experimental inoculation supports bacteremia and seroconversion. Culture with BAPGM may be required to detect Bartonella sp. Although mild clinical signs followed acute infection, no long-term effects were noted for 2 years postinoculation. Copyright © 2012 by the American College of Veterinary Internal Medicine.
Taubert, Anja; Behrendt, Jan Hillern; Sühwold, Anke; Zahner, Horst; Hermosilla, Carlos
Innate immune reactions conducted by macrophages may affect the outcome of primary infections and are crucial for the transition to adaptive immune responses. In bovine coccidiosis little is known on early monocyte/macrophage-mediated responses. We therefore investigated in vivo, in vitro and ex vivo reactions of monocytes and macrophages against Eimeria bovis, one of the most pathogenic Eimeria species in cattle. Macrophages significantly infiltrate the gut mucosa of E. bovis-infected calves, particularly after challenge infection. Furthermore, peripheral monocytes of infected animals, as precursor cells of macrophages, exhibited enhanced ex vivo phagocytic and oxidative burst activities. Enhanced levels of both activities were found early after infection and towards the end of first merogony. In vitro exposure of macrophages to sporozoites led to phagocytosis of the pathogen, whilst monocytes failed to do so. Phagocytosis occurred independently of the viability of the sporozoites, indicating that active invasion by the parasites was negligible. Phagocytosis occurred in the absence of immune serum, but could clearly be enhanced by addition of immune serum, suggesting macrophage-derived antibody-dependent cytotoxicity. Furthermore, co-culture of macrophages with sporozoites and stimulation with merozoite I antigen induced distinct levels of cytokine and chemokine gene transcription. Thus, the transcription of genes encoding for IFN-gamma, IL-12, TNF-alpha, IL-6, CXCL1, CXCL8, CXCL10 and COX-2 was upregulated after sporozoite encounter. In contrast, soluble merozoite I antigen only induced the gene transcription of IL-6 and IL-12 and failed to upregulate IFN-gamma and TNF-alpha gene transcripts. In monocytes, IFN-gamma and CXCL10 were found upregulated, all other immunoregulatory molecules tested were not affected. In summary, our results strongly suggest that macrophage-mediated, innate immune reactions play an important role in the early immune response to E
Pinto, Rachel; Leotta, Lisa; Shanahan, Erin R; West, Nicholas P; Leyh, Thomas S; Britton, Warwick; Triccas, James A
New therapies to control tuberculosis are urgently required because of the inability of the only available vaccine, BCG, to adequately protect against tuberculosis. Here we demonstrate that proteins of the Mycobacterium tuberculosis sulfate-assimilation pathway (SAP) represent major immunogenic targets of the bacillus, as defined by strong T-cell recognition by both mice and humans infected with M. tuberculosis. SAP proteins displayed increased expression when M. tuberculosis was resident within host cells, which may account in part for their ability to stimulate anti-M. tuberculosis host immunity. Vaccination with the first enzyme in this pathway, adenosine-5'-triphosphate sulfurylase, conferred significant protection against murine tuberculosis and boosted BCG-induced protective immunity in the lung. Therefore, we have identified SAP components as a new family of M. tuberculosis antigens, and we have demonstrated that these components are promising candidate for inclusion in new vaccines to control tuberculosis in humans.
Pinto, Rachel; Leotta, Lisa; Shanahan, Erin R.; West, Nicholas P.; Leyh, Thomas S.; Britton, Warwick; Triccas, James A.
New therapies to control tuberculosis are urgently required because of the inability of the only available vaccine, BCG, to adequately protect against tuberculosis. Here we demonstrate that proteins of the Mycobacterium tuberculosis sulfate-assimilation pathway (SAP) represent major immunogenic targets of the bacillus, as defined by strong T-cell recognition by both mice and humans infected with M. tuberculosis. SAP proteins displayed increased expression when M. tuberculosis was resident within host cells, which may account in part for their ability to stimulate anti-M. tuberculosis host immunity. Vaccination with the first enzyme in this pathway, adenosine-5′-triphosphate sulfurylase, conferred significant protection against murine tuberculosis and boosted BCG-induced protective immunity in the lung. Therefore, we have identified SAP components as a new family of M. tuberculosis antigens, and we have demonstrated that these components are promising candidate for inclusion in new vaccines to control tuberculosis in humans. PMID:23225904
Fine, Amanda E.; O'Brien, Daniel J.; Winterstein, Scott R.; Kaneene, John B.
Deer movements on cattle farms, wildlife feeding, and livestock management practices in Michigan are thought to create opportunities for indirect transmission of Mycobacterium bovis via environmental substrates. To confirm the presence of viable M. bovis in the environment, substrates were collected from 13 farms with culture-confirmed M. bovis in cattle and 5 sites with high prevalence of M. bovis in free-ranging deer. None of the samples processed for mycobacterial culture were positive for M. bovis. Agent, host, and landscape-level factors decrease the probability of detecting M. bovis in the environment using conventional mycobacterial culture. Molecular techniques that increase the probability of M. bovis detection in environmental substrates should be applied to known sites of M. bovis transmission in Michigan. In the interim, epidemiological investigations informed by experimental studies will be most effective in characterizing M. bovis persistence in the environment and its role in the indirect interspecies transmission of M. bovis. PMID:23738108
Poumarat, F; Martel, J L
The in vitro activity of 15 antibiotics was tested with 30-90 Mycoplasma bovis representative strains of bovine lung pathology in France. The distribution of minimal inhibitory concentration (MIC) is homogeneous with low values for spectinomycin, lincomycin, tylosin, gentamicin and baytril, intermediate for chloramphenicol and neomycin, high for nalidixic acid, Flumequine and erythromycin. The MIC distribution is heterogeneous with intermediate values for spiramycin and tetracyclines, and high values for streptomycin. For the later antibiotics, the heterogeneity of the susceptibility suggests a mechanism of acquired resistance.
Cosivi, O.; Grange, J. M.; Daborn, C. J.; Raviglione, M. C.; Fujikura, T.; Cousins, D.; Robinson, R. A.; Huchzermeyer, H. F.; de Kantor, I.; Meslin, F. X.
The World Health Organization (WHO) estimates that human tuberculosis (TB) incidence and deaths for 1990 to 1999 will be 88 million and 30 million, respectively, with most cases in developing countries. Zoonotic TB (caused by Mycobacterium bovis) is present in animals in most developing countries where surveillance and control activities are often inadequate or unavailable; therefore, many epidemiologic and public health aspects of infection remain largely unknown. We review available information on zoonotic TB in developing countries, analyze risk factors that may play a role in the disease, review recent WHO activities, and recommend actions to assess the magnitude of the problem and control the disease in humans and animals. PMID:9452399
Buddle, Bryce M; Shu, Dairu; Parlane, Natalie A; Subharat, Supatsak; Heiser, Axel; Hewinson, R Glyn; Vordermeier, H Martin; Wedlock, D Neil
A study was undertaken to determine whether BCG vaccination of cattle post-challenge could have an effect on a very early Mycobacterium bovis infection. Three groups of calves (n = 12/group) were challenged endobronchially with M. bovis and slaughtered 13 weeks later to examine for tuberculous lesions. One group had been vaccinated prophylactically with BCG Danish vaccine 21 weeks prior to challenge; a second group was vaccinated with a 4-fold higher dose of BCG Danish 3 weeks post-challenge and the third group, remained non-vaccinated. Vaccination prior to challenge induced only minimal protection with just a significant reduction in the lymph node lesion scores. Compared to the non-vaccinated group, BCG vaccination post-challenge produced no reduction in gross pathology and histopathology, but did result in significant increases in mRNA expression of pro-inflammatory mediators (IFN-γ, IL-12p40, IL-17A, IRF-5, CXCL9, CXCL10, iNOs, and TNF-α) in the pulmonary lymph nodes. Although there was no significant differences in the gross pathology and histopathology between the post-challenge BCG and non-vaccinated groups, the enhanced pro-inflammatory immune responses observed in the post-challenge BCG group suggest caution in the use of high doses of BCG where there is a possibility that cattle may be infected with M. bovis prior to vaccination.
Kho, Kai-Ling; Koh, Fui-Xian; Jaafar, Tariq; Nizam, Quaza Nizamuddin Hassan; Tay, Sun-Tee
Bartonellosis is an emerging zoonotic infection responsible for a variety of clinical syndromes in humans and animals. Members of the genus Bartonella exhibit high degrees of genetic diversity and ecologic plasticity. The infection is usually transmitted to animals and humans through blood-feeding arthropod vectors such as fleas, lice, ticks and sandflies. This study was conducted to investigate the prevalence of Bartonella species in 184 beef cattle, 40 dairy cattle, 40 sheep and 40 goats in eight animal farms across Peninsular Malaysia. Bartonella-specific PCR assays and sequence analysis of partial fragments of the citrate synthase gene were used for detection and identification of B. bovis. Isolation of B. bovis was attempted from PCR-positive blood samples. Molecular heterogeneity of the isolates was investigated based on sequence analysis of gltA, ITS, rpoB genes, ERIC-PCR, as well as using an established multilocus sequence typing (MLST) method. The carriage rate of B. bovis in ticks was also determined in this study. B. bovis was detected using Bartonella gltA-PCR assays from ten (4.5 %) of 224 cattle blood samples, of which three (1.3 %) were from beef cattle and seven (3.1 %) were from dairy cattle. None of the blood samples from the sheep and goats understudied were positive for B. bovis. Haemaphysalis bispinosa and Rhipicephalus (Boophilus) microplus were the predominant tick species identified in this study. B. bovis was detected from eight of 200 H. bispinosa ticks and none from the R. microplus ticks. Isolation of B. bovis was successful from all PCR-positive cattle blood samples, except one. Strain differentiation of B. bovis isolates was attempted based on sequence analysis of gltA, ITS, rpoB, and ERIC-PCR assay. B. bovis isolates were differentiated into six genotypes using the approach. The genetic heterogeneity of the isolates was confirmed using MLST method. Of the six MLST sequence types identified, five were designated new sequence types (ST
Berg, Stefan; Garcia-Pelayo, M. Carmen; Müller, Borna; Hailu, Elena; Asiimwe, Benon; Kremer, Kristin; Dale, James; Boniotti, M. Beatrice; Rodriguez, Sabrina; Hilty, Markus; Rigouts, Leen; Firdessa, Rebuma; Machado, Adelina; Mucavele, Custodia; Ngandolo, Bongo Nare Richard; Bruchfeld, Judith; Boschiroli, Laura; Müller, Annélle; Sahraoui, Naima; Pacciarini, Maria; Cadmus, Simeon; Joloba, Moses; van Soolingen, Dick; Michel, Anita L.; Djønne, Berit; Aranaz, Alicia; Zinsstag, Jakob; van Helden, Paul; Portaels, Françoise; Kazwala, Rudovick; Källenius, Gunilla; Hewinson, R. Glyn; Aseffa, Abraham; Gordon, Stephen V.; Smith, Noel H.
We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies. PMID:21097608
CHENG, YAFEN; CHOU, CHUNG-HSI; TSAI, HSIANG-JUNG
The intracellular parasite Mycobacterium bovis (M. bovis) causes tuberculosis in cattle and humans. Understanding the interactions between M. bovis and host cells is essential in developing tools for the prevention, detection, and treatment of M. bovis infection. Gene expression profiles provide a large amount of information regarding the molecular mechanisms underlying these interactions. The present study analyzed changes in gene expression in bovine peripheral blood mononuclear cells (PBMCs) at 0, 4 and 24 h following exposure to M. bovis. Using bovine whole-genome microarrays, a total of 420 genes were identified that exhibited significant alterations in expression (≥2-fold). Significantly enriched genes were identified using the Kyoto Encyclopedia of Genes and Genomes database, of which the highest differentially expressed genes were associated with the immune system, signal transduction, endocytosis, cellular transport, inflammation, and apoptosis. Of the genes associated with the immune system, 84.85% displayed downregulation. These findings support the view that M. bovis inhibits signaling pathways of antimycobacterial host defense in bovine PBMCs. These in vitro data demonstrated that molecular alterations underlying the pathogenesis of tuberculosis begin early, during the initial 24 h following M. bovis infection. PMID:26668602
Woodroffe, Rosie; Donnelly, Christl A; Wei, Gao; Cox, D R; Bourne, F John; Burke, Terry; Butlin, Roger K; Cheeseman, C L; Gettinby, George; Gilks, Peter; Hedges, Simon; Jenkins, Helen E; Johnston, W Thomas; McInerney, John P; Morrison, W Ivan; Pope, Lisa C
1. In most social animals, the prevalence of directly transmitted pathogens increases in larger groups and at higher population densities. Such patterns are predicted by models of Mycobacterium bovis infection in European badgers (Meles meles). 2. We investigated the relationship between badger abundance and M. bovis prevalence, using data on 2696 adult badgers in 10 populations sampled at the start of the Randomized Badger Culling Trial. 3. M. bovis prevalence was consistently higher at low badger densities and in small social groups. M. bovis prevalence was also higher among badgers whose genetic profiles suggested that they had immigrated into their assigned social groups. 4. The association between high M. bovis prevalence and small badger group size appeared not to have been caused by previous small-scale culling in study areas, which had been suspended, on average, 5 years before the start of the current study. 5. The observed pattern of prevalence might occur through badgers in smaller groups interacting more frequently with members of neighbouring groups; detailed behavioural data are needed to test this hypothesis. Likewise, longitudinal data are needed to determine whether the size of infected groups might be suppressed by disease-related mortality. 6. Although M. bovis prevalence was lower at high population densities, the absolute number of infected badgers was higher. However, this does not necessarily mean that the risk of M. bovis transmission to cattle is highest at high badger densities, since transmission risk depends on badger behaviour as well as on badger density.
Olea-Popelka, F J; Flynn, O; Costello, E; McGrath, G; Collins, J D; O'keeffe, J; Kelton, D F; Berke, O; Martin, S W
We investigated whether strains (restriction fragment length polymorphism, RFLP-types) of Mycobacterium bovis isolated from badgers and from cattle clustered among and within four areas in Ireland. The spatial scan test and nearest-neighbor analysis were used as the spatial cluster-detection techniques. In addition, for each of the major strains, associations between the distance to badger setts and the "centroid" of the cattle farm were assessed in a logistic model. Overall, between September 1997 and May 2000, 316 and 287 M. bovis samples, from badgers and cattle, respectively, were strain-typed. The distribution of strains in badgers, and separately in cattle, differed among areas. Within each of the four large areas, badgers and cattle tended to have similar strains; this is consistent with the sharing of M. bovis strains within an area. In more detailed within-area analyses, some spatial clusters of M. bovis strains were detected, separately, in both cattle and badgers. Almost half of the infected badger setts with a specific strain were located outside of the "detected" clusters. There was no association between the number of infected badgers with a specific M. bovis strain within 2 or 5 km distances to cattle herds, and the risk of the same strain in cattle. We speculate about the dynamic nature of badger movements, as an explanation for the absence of more clusters of most of the strains of M. bovis isolated from badgers, and its impact on trying to study transmission of M. bovis between cattle and badger.
Manuel, Christopher; Bagby, Stacey; Reisinger, Julie; Pugazhenthi, Umarani; Pitts, Todd; Keysar, Stephen; Arcaroli, John; Leszczynski, Jori
Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as amodelfor cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retainmany of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strainsused to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At ourinstitution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infectedwith C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfullyharvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our datademonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfullybe used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.
Manuel, Christopher A; Bagby, Stacey M; Reisinger, Julie A; Pugazhenthi, Umarani; Pitts, Todd M; Keysar, Stephen B; Arcaroli, John J; Leszczynski, Jori K
Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as a model for cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retain many of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strains used to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At our institution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infected with C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfully harvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our data demonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfully be used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.
Labidi, A; Thoen, C O
Total DNA from two slowly-growing pathogenic mycobacterial species propagated in vitro was isolated, digested with each of 34 restriction endonucleases and analyzed by agarose gel electrophoresis. The most resolved patterns for M. tuberculosis (ATCC 27294) and for M. bovis (ATCC 19210) were obtained respectively using (BamHI, DraI, ClaI, EcoRI, EcoRV, HindIII, HpaI, SalI, SmaI, XbaI and XmaI). The patterns produced for these strains were reproducible and distinguishable from each other respectively using (HindIII, DraI, EcoRI, MboI, Sau3AI and AvaI). However, with several enzymes (SalI, AsuI, Sau96I, MspI and HpaII) the patterns for M. tuberculosis and M. bovis were similar. Evidence was obtained for the presence of dam and dcmI methylations in the DNA of each mycobacterial species respectively using (MboI, Sau3AI, EcoRII, BstNI, Sau96I and AsuI).
Simuunza, Martin; Bilgic, Huseyin; Karagenc, Tulin; Syakalima, Michelo; Shiels, Brian; Tait, Andy; Weir, William
The tick-borne protozoan parasite, Babesia bovis is one of the causes of bovine babesiosis, an economically important disease of cattle in tropical and sub-tropical countries. Using the recently published genome sequence of the parasite, we developed a panel of eight mini- and micro-satellite markers and used these to investigate the role of genetic exchange in the population structure and diversity of the parasite using isolates from Zambia and Turkey. This population genetic analysis showed that genetic exchange occurs and that there are high levels of genetic diversity, with geographical sub-structuring quantified using Wright's F Index. Linkage disequilibrium was observed when isolates from both countries were treated as one population, but when isolates from Zambia were analysed separately linkage equilibrium was observed. The Turkish isolates were sub-structured, containing two genetically distinct sub-groups, both of which appeared to be in linkage equilibrium. The results of the Zambian study suggest that a sub-set of the parasite population is responsible for the westward spread of babesiosis into the previously disease-free central region of the country. The Zambian isolates had a significantly higher number of genotypes per sample than those from Turkey and age was found to be a significant predictor of the multiplicity of infection. The high levels of diversity seen in the Zambian and Turkish B. bovis populations have implications in the development of subunit vaccines against the disease and the spread of drug resistance.
Walker, Gwen J.
1. The cell-bound α-amylase of Streptococcus bovis has been isolated from other carbohydrases in the cell extract by chromatography on DEAE-cellulose. The enzyme has been compared with the extracellular α-amylase produced by this organism. 2. The two amylases had similar action patterns on amylose, the main product being maltotriose with smaller amounts of maltose and a little glucose. 3. The cell-bound amylase hydrolysed maltopentaose and maltohexaose at a similar rate to the hydrolysis of amylose. Maltotetraose was hydrolysed six times more slowly, and maltotriose 280 times more slowly, than amylose. 4. Studies with end-labelled maltodextrins revealed that the cell-bound α-amylase preferentially hydrolysed the third linkage from the non-reducing end, liberating maltotriose. The linkage at the reducing end of maltotriose was more easily hydrolysed than the other. 5. Egg-white lysozyme and the extracellular enzymes of Streptomyces albus lysed the cell walls of Streptococcus bovis, releasing amylase into the medium. In the presence of 0·6 m-sucrose 10% of the maximal amylase activity was released by lysozyme. Suspension of the spheroplasts in dilute buffer caused the rupture of the cytoplasmic membrane and the liberation of amylase. 6. A sensitive method for determining the ability of amylases to degrade starch granules is described. PMID:14346085
Castillo-Velázquez, Uziel; Aranday-Cortés, Elihú; Gutiérrez-Pabello, José A
The aim of this study was to evaluate the influence of macrophage alternative activation in the intracellular pathogen natural disease resistance phenotype of the host. Macrophage monolayers from resistant (R) (3) or susceptible (S) (3) cattle donors were treated with 10 ng/ml of bovine recombinant IL-4 (rbIL-4), and infected with virulent and avirulent Mycobacterium bovis (MOI 10:1). Bactericidal assays were performed to assess the bacterial phagocytic index and intracellular survival. Total RNA was reverse transcribed and used to analyze the relative changes in gene expression of IL-10, IL-12, IL-18 IL-1β, TNF-α, MCP-1, MCP-2, IL-6, MIP-1, MIP-3, iNOS, ARGII and SLAM by real time PCR. Cell supernatants were collected and nitric oxide and arginase production was assessed. Apoptosis induction was measured by TUNEL. IL-4 treatment increased the phagocytic index in both R and S macrophages; however intracellular survival was augmented mainly in S macrophages. Alternative activation decreased gene expression of pro-inflammatory cytokines, nitric oxide production and DNA fragmentation mainly in R macrophages. On the other hand, arginase production was not different between R and S macrophages. Alternative activation modifies the macrophage response against M. bovis. IL-4 treatment minimized the functional differences that exist between R and S macrophages. Copyright © 2011. Published by Elsevier B.V.
Manuel, Christopher A; Pugazhenthi, Umarani; Leszczynski, Jori K
Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks’ air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection. PMID:26817981
Denis, Michel; Buddle, Bryce M
Bovine and possum macrophages were infected in vitro with a virulent strain of Mycobacterium bovis, and mycobacterial replication was measured in the infected macrophages cultured under a variety of conditions. Virulent M. bovis replicated substantially in alveolar possum macrophages as well as in bovine blood monocyte-derived macrophages. Addition of recombinant bovine interferon-gamma (IFN-gamma) with low concentrations of lipopolysaccharide (LPS) rendered bovine macrophages significantly more resistant to M. bovis replication. Disruption of iron levels in infected macrophages by addition of apotransferrin or bovine lactoferrin blocked replication of M. bovis in both bovine and possum macrophages. On the other hand, addition of exogenous iron, either in the form of iron citrate or iron-saturated transferrin, rendered macrophages of both species much more permissive for the replication of M. bovis. The impact of iron deprivation/loading on the mycobacteriostatic activity of cells was independent of nitric-oxide release, as well as independent of the generation of oxygen radical species in both possum and bovine macrophages. Exogenous iron was shown to reverse the ability of IFN-gamma/LPS pulsed bovine macrophages to restrict M. bovis replication. When autologous possum lymphocytes from animals vaccinated with M. bovis strain BCG were added to infected macrophages, they rendered the macrophages less permissive for virulent M. bovis replication. Loading the cells with iron prior to this macrophage-lymphocyte interaction, reversed this immune effect induced by sensitized cells. We conclude that, in two important animal species, intracellular iron level plays an important role in M. bovis replication in macrophages, irrespective of their activation status.
Manuel, Christopher A; Pugazhenthi, Umarani; Leszczynski, Jori K
Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks' air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection.
Boothby, J T; Schore, C E; Jasper, D E; Osburn, B I; Thomas, C B
This study characterized the immune responses in four vaccinated and four control cows in response to vaccination and experimental intramammary inoculation with Mycoplasma bovis. Specific antibody responses occurred in serum and milk in response to vaccination and experimental infection. Lymphocytes from peripheral blood, but not from the mammary gland of vaccinated cows had increased responsiveness to mitogens. No lymphocytes tested were responsive to M. bovis antigen. Both vaccination and experimental infection resulted in skin test reactivity. These results imply that vaccination results in immune responses which may alter the course of experimental M. bovis mastitis, but may contribute to cellular inflammation. PMID:3167718
Bennett, R H; Jasper, D E
Serologic and cultural observations were made in three herds with and three herds without histories of mycoplasma mastitis. Nasal swabs and sera were collected from dairy animals of various ages over an eight month peiod. The overall prevalence of Myocopalsma bovis in the nares was 34% in diseased herds and 6% in the non-diseased herds without mastitis. Mycoplasma bovis was isolated in the highest prevalence in those young animals fed infected milk. Slight serologic differences were seen in these animals. Nasal prevalence of M. bovis was low but readily detectable in non diseased herds as well as in prepartum heifers in the diseased herds with mycoplasma mastitis.
Kim, Tae-Hyoun; Kim, Dong-Su; Han, Ju-Hee; Chang, Seo-Na; Kim, Kyung-Sul; Seok, Seung-Hyeok; Kim, Dong-Jae; Park, Jong-Hwan; Park, Jae-Hak
Corynebacterium (C.) bovis infection in nude mice causes hyperkeratosis and weight loss and has been reported worldwide but not in Korea. In 2011, nude mice from an animal facility in Korea were found to have white flakes on their dorsal skin. Histopathological testing revealed that the mice had hyperkeratosis and Gram-positive bacteria were found in the skin. We identified isolated bacteria from the skin lesions as C. bovis using PCR and 16S rRNA sequencing. To the best of our knowledge, this is the first report of C. bovis infection in nude mice from Korea.
Forgrave, R; Donaghy, J A; Fisher, A; Rowe, M T
Reports have highlighted the absence of contemporary peer reviewed publications pertaining to Mycobacterium bovis culture from raw milk and cheese. By replicating traditional methods, cheese-making methodology and equipment were devised to produce Cheddar (n = 6) and Caerphilly (n = 3) artificially contaminated with M. bovis (three genotypes) under stringent laboratory-containment guidelines for handling hazardous microbiological material. Middlebrook 7H11, modified for M. bovis isolation, was assessed for capacity to enumerate M. bovis despite changing cheese microflora and prolonged M. bovis exposure to the cheese matrix using maturing cheese test portions (n = 63; up to 16 weeks). Malachite green (MG) containing media isolated M. bovis at significantly (P < 0·05) lower levels than unmodified Middlebrook 7H11 agar despite MG being a common adjunct of Middlebrook 7H11 agar modified for M. bovis growth. Subsequently, a selective MG-free Middlebrook 7H11 agar modified using haemolysed red cells and calf serum was demonstrated as the best performing (P < 0·05) medium for recovery of M. bovis from typical UK cheese types, Cheddar and Caerphilly. Significance and impact of the study: Following increased M. bovis infection of UK cattle, the risk posed to consumers from consumption of unpasteurized milk and dairy products has changed. Furthermore, published methods for the culture and molecular detection of M. bovis in raw milk products are limited. Cheese-making protocols and M. bovis culture media reported here provide tools for further investigation of M. bovis survival during all stages of cheese manufacture and could inform future assessment of the risk to consumers from M. bovis contamination of unpasteurized dairy products.
HIGA, Yumiko; UEMURA, Ryoko; YAMAZAKI, Wataru; GOTO, Shinya; GOTO, Yoshitaka; SUEYOSHI, Masuo
We improved a loop-mediated isothermal amplification (LAMP) assay permitting sensitive and rapid Mycoplasma bovis detection. A total of 55 bacterial strains were examined in this study, including 33 M. bovis strains, 14 non-M. bovis mycoplasmas and eight non-mycoplasma bacterial strains. M. bovis was successfully detected by the LAMP assay within 60 min without cross-reaction to any other bacteria. Furthermore, a total of 135 nasal swab samples were tested directly using our LAMP assays, the previously reported LAMP assay, conventional PCR assay without pre-culture and comparing standard culture methods. The improved LAMP assay showed sensitivity and specificity of 97.2% and 90.9%, respectively (with a kappa coefficient of 0.8231), and the sensitivity of our revised LAMP assay was increased compared to existing methods. PMID:27109067
Pathogenic mycobacteria of the Mycobacterium tuberculosis complex such as Mycobacterium bovis, induce a characteristic lesion known as a granulomas. Granulomas represent a specific host response to chronic antigenic stimuli, such as foreign bodies, certain bacterial components, or persistent pathoge...
Background: Bovine tuberculosis remains one of the most damaging zoonotic diseases. A critical need exists for rapid and inexpensive diagnostics capable of detecting and differentiating M. bovis infection from other pathogenic and environmental mycobacteria at multiple surveillance levels. Method...
Davidson, Jennifer A; Loutet, Miranda G; O'Connor, Catherine; Kearns, Cathriona; Smith, Robert M M; Lalor, Maeve K; Thomas, H Lucy; Abubakar, Ibrahim; Zenner, Dominik
Despite control efforts, Mycobacterium bovis incidence among cattle remains high in parts of England, Wales, and Northern Ireland, attracting political and public health interest in potential spread from animals to humans. To determine incidence among humans and to identify associated factors, we conducted a retrospective cohort analysis of human M. bovis cases in England, Wales, and Northern Ireland during 2002-2014. We identified 357 cases and observed increased annual case numbers (from 17 to 35) and rates. Most patients were >65 years of age and born in the United Kingdom. The median age of UK-born patients decreased over time. For 74% of patients, exposure to risk factors accounting for M. bovis acquisition, most frequently consumption of unpasteurized milk, was known. Despite the small increase in case numbers and reduction in patient age, M. bovis infection of humans in England, Wales, and Northern Ireland remains rare.
Loutet, Miranda G.; O’Connor, Catherine; Kearns, Cathriona; Smith, Robert M.M.; Lalor, Maeve K.; Thomas, H. Lucy; Abubakar, Ibrahim; Zenner, Dominik
Despite control efforts, Mycobacterium bovis incidence among cattle remains high in parts of England, Wales, and Northern Ireland, attracting political and public health interest in potential spread from animals to humans. To determine incidence among humans and to identify associated factors, we conducted a retrospective cohort analysis of human M. bovis cases in England, Wales, and Northern Ireland during 2002–2014. We identified 357 cases and observed increased annual case numbers (from 17 to 35) and rates. Most patients were >65 years of age and born in the United Kingdom. The median age of UK-born patients decreased over time. For 74% of patients, exposure to risk factors accounting for M. bovis acquisition, most frequently consumption of unpasteurized milk, was known. Despite the small increase in case numbers and reduction in patient age, M. bovis infection of humans in England, Wales, and Northern Ireland remains rare. PMID:28220748
Quan, Zhang; Haiming, Tan; Xiaoyao, Cai; Weifeng, Yuan; Hong, Jia; Hongfei, Zhu
A multiplex PCR (m-PCR) with primers targeting the 16S rRNA, Rv3873 and a 12.7-kb fragment in the genomes of a Mycobacterium tuberculosis complex was designed for the differential diagnosis of M. tuberculosis, M. bovis, M. bovis BCG and non-tuberculosis Mycobacterium (NTM). The specificity of this assay was 100%, and the detection limit was 15 pg of genomic DNA. Of the 206 blinded clinical samples, the detection rate of M. bovis infection by m-PCR was lower than that of the interferon gamma (IFN-γ) release assay; however, the false-positive rate by the tuberculin skin test and false-negative samples in the IFN-γ release assay were reduced. Our findings indicated that our m-PCR method is a useful tool for complementation to differentiate M. bovis from M. tuberculosis and NTM species.
Mycobacterium bovis isolates carry restricted allelic variation yet exhibit a range of disease phenotypes and host preferences. Conventional genotyping methods target small hyper-variable regions of their genome and provide anonymous biallelic information insufficient to develop phylogeny. To resolv...
Chatterjee, D; Bozic, C M; Knisley, C; Cho, S N; Brennan, P J
The glycolipid that characterizes the majority of isolates of Mycobacterium bovis and that has come to be known as M. bovis-identifying lipid is the phenolic glycolipid mycoside B described in the literature by others. However, when mycoside B obtained from M. bovis BCG, field isolates, and infected tissues was examined in detail, it was shown to be different from that described in the literature in some important respects. In particular, the glycosyl substituent is 2-O-methyl-alpha-L-rhamnopyranose rather than 2-O-methyl-beta-D-rhamnopyranose. With this information, a seroreactive neoglycoprotein (neoantigen) containing the 2-O-methyl-alpha-L-rhamnopyranosyl substituent suitable for the serodiagnosis of bovine tuberculosis was synthesized. M. bovis also contains other minor seroreactive phenolic glycolipids, one of which is a deacylated form of mycoside B and another of which contains an alpha-L-rhamnopyranosyl unit rather than 2-O-methyl-alpha-L-rhamnopyranose. Images PMID:2643563
Pérez-Lago, Laura; Navarro, Yurena; García-de-Viedma, Darío
Mycobacterium bovis is both the causative agent of bovine tuberculosis (TB) and a zoonotic pathogen. In humans, considerably fewer cases of TB are caused by M. bovis than M. tuberculosis; nevertheless, diagnostic limitations mean that currently available data on prevalence grossly underestimate the true dimension of the problem. The routes of transmission from animals to humans are well known and include direct exposure to infected animals or consumption of contaminated animal products. Application of fingerprinting tools facilitates analysis of the molecular epidemiology of M. bovis in animal-to-human and human-to-human transmission. Apart from cattle and M. bovis, other animal species and members within the M. tuberculosis complex can contribute to the zoonosis. Improvements in diagnostic techniques, application of more advanced discriminatory genotyping tools, and collaboration between veterinary and human health care researchers are key to our understanding of this zoonosis. Copyright © 2013 Elsevier Ltd. All rights reserved.
Carvalho, Ricardo César Tavares; Vasconcellos, Sidra Ezidio Gonçalves; Issa, Marina de Azevedo; Soares Filho, Paulo Martins; Mota, Pedro Moacyr Pinto Coelho; de Araújo, Flábio Ribeiro; Carvalho, Ana Carolina da Silva; Gomes, Harrison Magdinier; Suffys, Philip Noel; Paschoalin, Vânia Margaret Flosi
Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil. PMID:27631383
Ueti, Massaro W; Olafson, Pia U; Freeman, Jeanne M; Johnson, Wendell C; Scoles, Glen A
Wildlife are an important component in the vector-host-pathogen triangle of livestock diseases, as they maintain biological vectors that transmit pathogens and can serve as reservoirs for such infectious pathogens. Babesia bovis is a tick-borne pathogen, vectored by cattle fever ticks, Rhipicephalus spp., that can cause up to 90% mortality in naive adult cattle. While cattle are the primary host for cattle fever ticks, wild and exotic ungulates, including white-tailed deer (WTD), are known to be viable alternative hosts. The presence of cattle fever tick populations resistant to acaricides raises concerns regarding the possibility of these alternative hosts introducing tick-borne babesial parasites into areas free of infection. Understanding the B. bovis reservoir competence of these alternative hosts is critical to mitigating the risk of introduction. In this study, we tested the hypothesis that WTD are susceptible to infection with a B. bovis strain lethal to cattle. Two groups of deer were inoculated intravenously with either B. bovis blood stabilate or a larval extract supernatant containing sporozoites from infected R. microplus larvae. The collective data demonstrated that WTD are neither a transient host nor reservoir of B. bovis. This conclusion is supported by the failure of B. bovis to establish an infection in deer regardless of inoculum. Although specific antibody was detected for a short period in the WTD, the PCR results were consistently negative at multiple time points throughout the experiment and blood from WTD that had been exposed to parasite, transferred into naïve recipient susceptible calves, failed to establish infection. In contrast, naïve steers inoculated intravenously with either B. bovis blood stabilate or the larval extract supernatant containing sporozoites rapidly succumbed to disease. These findings provide evidence that WTD are not an epidemiological component in the maintenance of B. bovis infectivity to livestock.
Manuel, Christopher A; Pugazhenthi, Umarani; Spiegel, Shannon P; Leszczynski, Jori K
Rodent health-monitoring programs based on sampling an IVC system's exhaust air dust (EAD) has enhanced and even replaced traditional sentinels for some rodent pathogens. EAD testing by qPCR assay is an optimal surveillance method for the rapid detection of Corynebacterium bovis-infected immunodeficient mice. Here we demonstrate that an active EAD surveillance program for C. bovis can be used to maintain nude mice C. bovis-free after the transition from historically enzootically infected colonies. During 3 events over 3 y, rapid detection of infection, elimination of infected mice, aggressive quarantine measures, and local decontamination prevented the spread of C. bovis within 2 barrier rooms. In total, 4 cages of infected nude mice were identified and removed, preventing the spread of infection to 469 other cages of immunodeficient mice. In addition, we present data regarding a refinement to EAD testing which enables row-specific surveillance of an IVC rack. This technique systemically decreases the amount of testing required to locate an individually infected cage. Due to our ability to rapidly detect and localize an infected cage, we were able to investigate the route of C. bovis introduction into our barrier rooms. Our epidemiologic investigation suggested that the transmission of C. bovis occurred through contaminated, cryopreserved, patient-derived xenograft tumor tissue. This previously unknown source of C. bovis can infect mice used to propagate these tumors. Together, these data demonstrate that a remediation program that combines rapid detection, test-and-cull, and local decontamination under quarantine conditions can eliminate C. bovis from a mouse colony. PMID:28315652
Manuel, Christopher A; Pugazhenthi, Umarani; Spiegel, Shannon P; Leszczynski, Jori K
Rodent health-monitoring programs based on sampling an IVC system's exhaust air dust (EAD) has enhanced and even replaced traditional sentinels for some rodent pathogens. EAD testing by qPCR assay is an optimal surveillance method for the rapid detection of Corynebacterium bovis-infected immunodeficient mice. Here we demonstrate that an active EAD surveillance program for C. bovis can be used to maintain nude mice C. bovis-free after the transition from historically enzootically infected colonies. During 3 events over 3 y, rapid detection of infection, elimination of infected mice, aggressive quarantine measures, and local decontamination prevented the spread of C. bovis within 2 barrier rooms. In total, 4 cages of infected nude mice were identified and removed, preventing the spread of infection to 469 other cages of immunodeficient mice. In addition, we present data regarding a refinement to EAD testing which enables row-specific surveillance of an IVC rack. This technique systemically decreases the amount of testing required to locate an individually infected cage. Due to our ability to rapidly detect and localize an infected cage, we were able to investigate the route of C. bovis introduction into our barrier rooms. Our epidemiologic investigation suggested that the transmission of C. bovis occurred through contaminated, cryopreserved, patient-derived xenograft tumor tissue. This previously unknown source of C. bovis can infect mice used to propagate these tumors. Together, these data demonstrate that a remediation program that combines rapid detection, test-and-cull, and local decontamination under quarantine conditions can eliminate C. bovis from a mouse colony.
Chapman, Rosamund; Chege, Gerald; Shephard, Enid; Stutz, Helen; Williamson, Anna-Lise
HIV-1 has resulted in a devastating AIDS pandemic. An effective HIV/AIDS vaccine that can be used to either, prevent HIV infection, control infection or prevent progression of the disease to AIDS is needed. In this review we discuss the use of Mycobacterium bovis BCG, the tuberculosis vaccine, as a vaccine vector for an HIV vaccine. Numerous features make BCG an attractive vehicle to deliver HIV antigens. It has a good safety profile, elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable, a necessary consideration for developing countries. In this review we discuss the numerous factors that influence generation of a genetically stable recombinant BCG vaccine for HIV. PMID:20353397
Long, R; Nobert, E; Chomyc, S; van Embden, J; McNamee, C; Duran, R R; Talbot, J; Fanning, A
Globally, the proportion of all cases of tuberculosis (TB) caused by drug-resistant strains is increasing. We report the case of a Canadian citizen who acquired a highly drug-resistant strain of Mycobacterium bovis while visiting a relative with AIDS-related tuberculosis in Spain. The origin of the strain was traced using spoligotyping, a polymerase chain reaction (PCR)-based fingerprint technology, and the European DNA database. The level of primary drug resistance-all five first-line drugs and 19 of 21 second-line drugs-in this case was unprecedented in Canada. Isolation of this strain from a Canadian citizen represents the first report of its appearance in this hemisphere. The infection was contained and combined medical-surgical treatment delivered.
de Azevedo Issa, Marina; Martins Soares Filho, Paulo; Fonseca Júnior, Antônio Augusto; Arrais Hodon, Mikael; Cristian Dos Santos, Lílian; Karlisson Pimenta Dos Reis, Jenner; Cerqueira Leite, Rômulo
For the definitive diagnosis of bovine tuberculosis, isolation of the etiologic agent is required. However, there is no consensus on the best methodology for isolation of Mycobacterium bovis in Brazil. This study evaluated the most used decontaminants and culture media in the country, in order to identify the best combination for the Brazilian samples. Three decontaminants - 2% sodium hydroxide (w/v), 0.75% hexadecylpiridinium chloride (w/v) and 5% sulphuric acid (v/v) and four culture media - 7H11 Middlebrook with additives and OADC supplement "A" (7H11 A), the same media with another supplement trademark (7H11 B), tuberculosis blood agar (B83) and Stonebrink's medium were compared. Regarding the isolation, there were no significant differences between the decontaminants and media combinations, except 7H11A combined to any decontaminant. However, the mean colonies score was significantly greater when the samples were decontaminated with 5% sulphuric acid and inoculated in 7H11 B or SB, without significant difference between them, although colonies appeared earlier on 7H11B than on SB. The trademark of OADC supplement influenced the isolation rate and the number of isolated colonies in Middlebrook 7H11. An incubation time of four weeks was required to detect all positive samples in 7H11 B after decontamination with 5% sulphuric acid but there was an increase in the number of colonies until the sixth week of incubation. Overall, the best strategy for the primary isolation of M. bovis from Brazilian samples was the decontamination with 5% sulphuric acid (final concentration) and inoculation in Middlebrook 7H11 medium formulated with OADC supplement "B".
Corner, Leigh A L; O'Meara, D; Costello, E; Lesellier, S; Gormley, E
Populations of Eurasian badgers (Meles meles) with tuberculosis (Mycobacterium bovis infection) are a significant reservoir of infection for cattle in Ireland and the United Kingdom. In this study the distribution of infection, histological lesions and gross lesions was determined in a sample of 132 culled badgers from naturally-infected wild populations. Badgers were culled when an epidemiological investigation following a tuberculosis breakdown in a cattle herd implicated badgers as the probable source of infection. The definition of tuberculosis infection was based on the isolation of M. bovis from tissues or clinical samples. An accurate diagnosis of infection was achieved by culturing a wide range of lymph nodes (LN) and organ tissues (mean 32.1) and clinical samples (faeces and urine) from each badger. Infection was detected in 57/132 badgers (43.2%). Histological lesions consistent with tuberculosis were seen in 39/57 (68.4%) culture-positive and 7/75 (9.3%) culture-negative animals. Gross lesions were seen in only 30/57 (52.6%) infected badgers, leaving a high proportion (47.4%) of infected animals with latent infection (no grossly visible lesions). The most frequently infected tissues were the lungs and axillary LN, followed by the deep cervical LN, parotid LN and tracheobronchial LN. The data support the hypotheses that in badgers there are only two significant routes of infection, namely, the lower respiratory tract and bite wounds, and that badgers are very susceptible to infection but resistant to the development and progression of the disease. At all levels of disease severity, infection was found in widely dispersed anatomical locations suggesting that there is early dissemination of infection in the period preceding the development of active immunity.
Fine, Amanda E.; Bolin, Carole A.; Gardiner, Joseph C.; Kaneene, John B.
Reisolation of Mycobacterium bovis from inoculated substrates was used to follow the persistence of viable M. bovis bacteria exposed to natural weather conditions over a 12-month period. Environmental factors were recorded continuously, and factors affecting M. bovis persistence (i.e., temperature, season, and substrate) were studied using survival analysis and Cox's proportional hazards regression. Persistence of M. bovis in the environment was significantly shorter in the spring/summer season, characterized by the highest average daily temperatures over the 12-month period. M. bovis persisted up to 88 days in soil, 58 days in water and hay, and 43 days on corn. These studies demonstrate that M. bovis bacteria persist long enough to represent a risk of exposure for cattle and/or wildlife and strengthen evidence that suggests cattle farm biosecurity and efforts to eliminate supplemental feeding of white-tailed deer will decrease the risk of bovine TB transmission among and between cattle and deer populations. PMID:21547222
Fernández-Ruiz, Mario; Villar-Silva, Julia; Llenas-García, Jara; Caurcel-Díaz, Luis; Vila-Santos, Juan; Sanz-Sanz, Francisca; Chaves, Fernando; Guerra-Vales, Juan Manuel
To characterise the clinical features, associations and outcome in a contemporary series of patients with Streptococcus bovis bacteraemia (SBB). Retrospective analysis of all episodes of SBB at the University Hospital 12 de Octubre (Madrid, Spain) between January 1997 and November 2008 was performed. Patient data were reviewed, focusing on clinical and microbiological associations with the different biotypes of S. bovis. Fifty-nine episodes of SBB were documented in 59 adult patients (30 males; mean age: 70.9 ± 15.0 years). Chronic liver disease was identified in 20 patients (33.9%). Sixteen patients (27.1%) presented infective endocarditis (IE) and 14 (23.7%) had a biliary source of bacteraemia. Thirty-three patients (55.9%) underwent colonic evaluation, adenomatous polyps being the most common finding (21 patients). Malignancy was diagnosed following SBB in 9 cases, including 6 patients with colorectal carcinoma (18.2% of those who underwent colonic evaluation). Of 22 isolates biotyped, 12 were S. bovis biotype I and 10 were S. bovis biotype II. IE was more frequent among patients with S. bovis biotype I (P =0.010), whereas bacteraemia due to biotype II species was more likely to be of biliary origin (P=0.078). S. bovis biotyping identifies some clinically relevant associations. Copyright © 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Heuvelink, Annet; Reugebrink, Constance; Mars, Jet
Control of Mycoplasma bovis infections depends on good husbandry practices and antibiotic treatment. To allow more prudent use of antimicrobial drugs, there is a need for information on the susceptibility profile of this pathogen. The objective of the present study was to analyse the in vitro antimicrobial susceptibility of clinical M. bovis isolates in the Netherlands. The collection comprised 95 bovine isolates, originating from lungs (n=56), mastitis milk (n=27), and synovial fluid (n=12), collected between 2008 and 2014. Minimal inhibitory concentrations (MICs) were assessed by broth microdilution, both by using in-house prepared MIC plates and by using commercially available MIC plates. For each antimicrobial agent, the range of MIC results, the MIC50, and MIC90 values were calculated. M. bovis strains recently isolated in the Netherlands appeared to be characterized by relatively high MIC values for antimicrobial agents that, until now, have been recommended by the Dutch Association of Veterinarians for treating pneumonia caused by Mycoplasma species. Fluoroquinolones appeared to be the most efficacious in inhibiting M. bovis growth, followed by tulathromycin and oxytetracycline. The highest MIC values were obtained for erythromycin, tilmicosin, and tylosin. Future studies should be done on determining M. bovis specific clinical breakpoints, standardization of methods to determine MIC values as well as molecular studies on detection of antimicrobial resistance mechanisms of M. bovis isolates to develop PCR assays for determining resistance.
Lysnyansky, Inna; Freed, Mor; Rosales, Ruben S; Mikula, Inna; Khateb, Nihaya; Gerchman, Irena; van Straten, Michael; Levisohn, Sharon
The prevalence of Mycoplasma bovis in milk samples submitted to the Israeli National Service for Udder Health and Milk Quality was determined during the period 2004-2014 and the genetic pattern of the obtained isolates was assessed by multilocus sequence typing (MLST). Mycoplasma spp. were identified in 66 herds including M. bovis (n = 60), M. cottewii (n = 3), M. bovigenitalium (n = 2), M. alkalescens (n = 2) and M. yeatsii (n = 1). The proportion of M. bovis infected herds was relatively low (0-0.68%) in 2004-2007, increased to 3.77% during the 2008 outbreak, and ranged from 0.77 to 2.77% during the 2009-2014 period. Since 2008, about eight M. bovis positive dairy herds have been identified in Israel annually, with six of which on average being newly infected. MLST of 57 M. bovis isolates revealed that sequence type 10 was the dominant genotype identified in 60% of the herds. In conclusion, these data show that M. bovis is the main mycoplasmal mastitic pathogen in Israel.
Wan, Tien-Chun; Cheng, Fu-Yuan; Liu, Yu-Tse; Lin, Liang-Chuan; Sakata, Ryoichi
The purpose of the study was to investigate bioactive compounds of in vitro cultured Calculus Suis and natural Calculus Bovis obtained as valuable by-products from animals used for meat production. The results showed that the components of natural Calculus Bovis were rich in bilirubin and biliverdin and had higher content of essential amino acids. The major amino acids of in vitro cultured Calculus Suis were identified as glycine, alanine, glutamic acid and aspartic acid, and those for natural Calculus Bovis were found to be glutamic acid, aspartic acid, proline, and arginine. The methionine and cysteine contents of precursors for glutathione in natural Calculus Bovis were significantly higher than those of in vitro cultured Calculus Suis. The mineral contents of zinc, iron and manganese of natural Calculus Bovis were significantly higher than those of in vitro cultured Calculus Suis. The major bile acids in both products were cholic acid and dehydrocholic acid, respectively. The chenodeoxycholic and ursodeoxycholic acid content of in vitro cultured Calculus Suis was significantly higher than that of natural Calculus Bovis.
de Kantor, I N; LoBue, P A; Thoen, C O
Human tuberculosis (TB) caused by Mycobacterium bovis appears to be rare in most of the region of the Americas, although some localities have reported an unusually high prevalence of M. bovis among human TB cases (e.g., San Diego, CA, USA; parts of Mexico). As surveillance data are lacking in many countries, there is substantial uncertainty regarding actual incidence. M. bovis is most often not identified, as the diagnosis of TB is made by smear microscopy alone or using egg-containing culture media lacking pyruvate. Where human M. bovis cases have been studied in the region, they appear to be associated with ingestion of unpasteurized dairy products, or with airborne acquired infection in animal keepers and meat industry workers from countries where bovine TB remains a problem. Human-to-human transmission of M. bovis does occur, but appears to account for a very small proportion of cases. Efforts to eradicate M. bovis in humans in the Americas should therefore be directed at eradicating the disease in cattle, increasing pasteurization of dairy products and providing education about the dangers of consuming unpasteurized dairy products.
Blanco, Federico Carlos; Soria, Marcelo Abel; Klepp, Laura Inés; Bigi, Fabiana
Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that is responsible for significant economic losses worldwide. In spite of its relevance, the limited knowledge about the host immune responses that provide effective protection against the disease has long hampered the development of an effective vaccine. The identification of host proteins with an expression that correlates with protection against bTB would contribute to the understanding of the cattle defence mechanisms against M. bovis infection. In this study, we found that ERAP1 and PDE8A were downregulated in vaccinated cattle that were protected from experimental M. bovis challenge. Remarkably, both genes encode proteins that have been negatively associated with immune protection against bTB. © 2017 S. Karger AG, Basel.
Fecal Volatile Organic Ccompound Profiles from White-Tailed Deer (Odocoileus virginianus) as Indicators of Mycobacterium bovis Exposure or Mycobacterium bovis Bacille Calmette-Guerin (BCG) Vaccination.
Stahl, Randal S; Ellis, Christine K; Nol, Pauline; Waters, W Ray; Palmer, Mitchell; VerCauteren, Kurt C
White-tailed deer (Odocoileus virginianus) serve as a reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and can be a source of infection in cattle. Vaccination with M. bovis Bacille Calmette Guerin (BCG) is being considered for management of bovine tuberculosis in deer. Presently, no method exists to non-invasively monitor the presence of bovine tuberculosis in deer. In this study, volatile organic compound profiles of BCG-vaccinated and non-vaccinated deer, before and after experimental challenge with M. bovis strain 95-1315, were generated using solid phase microextraction fiber head-space sampling over suspended fecal pellets with analysis by gas chromatography/mass spectrometry. Chromatograms were processed using XCMS Online to characterize ion variation among treatment groups. The principal component scores resulting from significant (α = 0.05) ion responses were used to build linear discriminant analysis models. The sensitivity and specificity of these models were used to evaluate the feasibility of using this analytical approach to distinguish within group comparisons between pre- and post-M. bovis challenge: non-vaccinated male or female deer, BCG-vaccinated male deer, and the mixed gender non-vaccinated deer data. Seventeen compounds were identified in this analysis. The peak areas for these compounds were used to build a linear discriminant classification model based on principal component analysis scores to evaluate the feasibility of discriminating between fecal samples from M. bovis challenged deer, irrespective of vaccination status. The model best representing the data had a sensitivity of 78.6% and a specificity of 91.4%. The fecal head-space sampling approach presented in this pilot study provides a non-invasive method to discriminate between M. bovis challenged deer and BCG-vaccinated deer. Additionally, the technique may prove invaluable for BCG efficacy studies with free-ranging deer as well as for use as a non
Fecal Volatile Organic Ccompound Profiles from White-Tailed Deer (Odocoileus virginianus) as Indicators of Mycobacterium bovis Exposure or Mycobacterium bovis Bacille Calmette-Guerin (BCG) Vaccination
Stahl, Randal S.; Ellis, Christine K.; Nol, Pauline; Waters, W. Ray; Palmer, Mitchell; VerCauteren, Kurt C.
White-tailed deer (Odocoileus virginianus) serve as a reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and can be a source of infection in cattle. Vaccination with M. bovis Bacille Calmette Guerin (BCG) is being considered for management of bovine tuberculosis in deer. Presently, no method exists to non-invasively monitor the presence of bovine tuberculosis in deer. In this study, volatile organic compound profiles of BCG-vaccinated and non-vaccinated deer, before and after experimental challenge with M. bovis strain 95–1315, were generated using solid phase microextraction fiber head-space sampling over suspended fecal pellets with analysis by gas chromatography/mass spectrometry. Chromatograms were processed using XCMS Online to characterize ion variation among treatment groups. The principal component scores resulting from significant (α = 0.05) ion responses were used to build linear discriminant analysis models. The sensitivity and specificity of these models were used to evaluate the feasibility of using this analytical approach to distinguish within group comparisons between pre- and post-M. bovis challenge: non-vaccinated male or female deer, BCG-vaccinated male deer, and the mixed gender non-vaccinated deer data. Seventeen compounds were identified in this analysis. The peak areas for these compounds were used to build a linear discriminant classification model based on principal component analysis scores to evaluate the feasibility of discriminating between fecal samples from M. bovis challenged deer, irrespective of vaccination status. The model best representing the data had a sensitivity of 78.6% and a specificity of 91.4%. The fecal head-space sampling approach presented in this pilot study provides a non-invasive method to discriminate between M. bovis challenged deer and BCG-vaccinated deer. Additionally, the technique may prove invaluable for BCG efficacy studies with free-ranging deer as well as for use as a non
Wanzala, Sylvia I; Nakavuma, Jesca; Travis, Dominic A; Kia, Praiscillia; Ogwang, Sam; Sreevatsan, Srinand
Bovine tuberculosis (BTB), a zoonotic infection of cattle caused by Mycobacterium bovis, results in losses of $3 billion to the global agricultural industry and represents the fourth most important livestock disease worldwide. M. bovis as a source of human infection is likely underreported due to the culture medium conditions used to isolate the organism from sputum or other sample sources. We report here the draft genome sequences of M. bovis BZ 31150, isolated from a bronchial washing from a captive chimpanzee, and M. bovis B2 7505, isolated from a human sputum sample in Uganda.
Ramos, Christina M; Cooper, Susan M; Holman, Patricia J
The current study was undertaken to determine if white-tailed deer in south Texas harbor Babesia bovis, a causative agent of bovine babesiosis. Blood samples from free-ranging white-tailed deer (Odocoileus virginianus) on two ranches in LaSalle and Webb Counties were screened for B. bovis and other hemoparasites by the polymerase chain reaction (PCR) to detect the piroplasm 18S rDNA. Serology was conducted on selected samples to detect antibody activity to B. bovis by the immunofluorescent antibody test (IFAT). PCR revealed that 16% of the LaSalle County samples and 4% of the Webb County samples were positive for B. bovis. Five of the LaSalle County and the two Webb County B. bovis 18S rDNA amplicons were cloned and sequenced. The resulting clones shared 99% identity to B. bovis 18S rRNA gene sequences derived from cattle isolates. Weak seroreactivity to B. bovis was shown by the IFAT. The samples were also screened for additional hemoparasites of deer including Theileria cervi, Babesia odocoilei and other Babesia spp. A genotypically unique Theileria sp. was found, along with T. cervi and B. odocoilei. The finding of putative B. bovis in white-tailed deer necessitates further study to determine if deer may act as a transient host or even a reservoir of infection for B. bovis pathogenic to cattle.
Punyapornwithaya, V; Fox, L K; Hancock, D D; Gay, J M; Wenz, J R; Alldredge, J R
The objective was to determine the incidence and transmission of mycoplasma mastitis in the hospital pen in a dairy herd of 650 lactating cows after a hospital pen was established following an outbreak of this disease. Mycoplasma mastitis status was monitored for 3 months through repeated collection of milk samples from cows with clinical mastitis (CM) and from bulk tank milk. During the outbreak 13 cows were diagnosed with Mycoplasma bovis CM, 1 cow with Mycoplasma sp. mastitis and 8 cows showed signs of arthritis, 3 of which were confirmed as having M. bovis arthritis. M. bovis isolates from cows with CM, arthritis and bulk tank milk had indistinguishable chromosomal digest pattern fingerprints. Incidence rates of M. bovis CM cases in the milking and hospital pens were 0.01 and 1.7 cases per 100 cow-days at risk. Approximately 70% of cows with M. bovis CM became infected within 12 days of entering the hospital pen. Transmission of M. bovis in the hospital pen occurred as 3 episodes. Each episode corresponded to the introduction of a cow with M. bovis CM from a milking pen. Evidence indicates that cows with M. bovis CM from milking pens were the source of transmission of the disease in the hospital pen and thus their presence in the hospital pen appeared to be a risk factor for transmission of M. bovis mastitis in this single case study herd.
Smith, Steven G; Zelmer, Andrea; Blitz, Rose; Fletcher, Helen A; Dockrell, Hazel M
Vaccination with Bacillus Calmette Guerin (BCG) protects infants against childhood tuberculosis however the immune mechanisms involved are not well understood. Further elucidation of the infant immune response to BCG will aid with the identification of immune correlates of protection against tuberculosis and with the design of new improved vaccines. The purpose of this study was to investigate BCG-induced CD4+ T-cell responses in blood samples from infants for cytokine secretion profiles thought to be important for protection against tuberculosis and compare these to PBMC-mediated in vitro mycobacterial growth inhibition. Blood from BCG-vaccinated or unvaccinated infants was stimulated overnight with Mycobacterium tuberculosis (M. tb) purified protein derivative (PPD) or controls and intracellular cytokine staining and flow cytometry used to measure CD4+T-cell responses. PBMC cryopreserved at the time of sample collection were thawed and incubated with live BCG for four days following which inhibition of BCG growth was determined. PPD-specific IFNγ+TNFα+IL-2+CD4+T-cells represented the dominant T-cell response at 4monthsand1yearafter infant BCG. These responses were undetectable in age-matched unvaccinated infants. IL-17+CD4+T-cells were significantly more frequent in vaccinated infants at 4monthsbut not at 1-year post-BCG. PBMC-mediated inhibition of mycobacterial growth was significantly enhanced at 4monthspost-BCG as compared to unvaccinated controls. In an analysis of all samples with both datasets available, mycobacterial growth inhibition correlated significantly with the frequency of polyfunctional (IFNγ+TNFα+IL-2+) CD4+T-cells. These data suggest that BCG vaccination of infants induces specific polyfunctional T-helper-1 and T-helper-17 responses and the ability, in the PBMC compartment, to inhibit the growth of mycobacteria in vitro. We also demonstrate that polyfunctional T-helper-1 cells may play a role in growth inhibition as evidenced by a
Perez-Llaneza, Agustina; Caballero, Marina; Baravalle, Eugenia; Mesplet, Maria; Mosqueda, Juan; Suarez, Carlos E; Echaide, Ignacio; Katzer, Frank; Pacheco, Gabriela M; Florin-Christensen, Monica; Schnittger, Leonhard
Mini- and microsatellite sequences have proven to be excellent tools for the differentiation of strains and populations in several protozoan parasites due to their high variability. In the present work we have searched the genome of the tick-transmitted bovine hemoprotozoon Babesia bovis for tandem repeats (TRs) that could be useful for a multilocus typing system. Hundred and nineteen sequences were shortlisted and tested in five common B. bovis reference isolates originating from distinct geographic locations of North and South America: Texas, USA (T2Bo), Mexico (RAD and Mo7), and Santa Fe and Salta, Argentina (R1A and S2P, respectively). Satellite sequences were PCR-amplified using specific primers, separated by polyacrylamide gel electrophoresis, visualized by silver staining and sized. Fourteen TR sequences could be reliably amplified in all isolates and displayed length polymorphism. All primers used were specific for B. bovis and did not amplify genomic DNA from the bovine host or from Babesia bigemina, the principal co-infecting bovine parasite in the Americas, allowing their future use in field surveys. The 14 satellite markers identified are distributed throughout the four chromosomes of B. bovis as follows: chromosome 1 (n=3), chromosome 2 (n=2), chromosome 3 (n=5), and chromosome 4 (n=4). Within the five B. bovis isolates we identified nine satellite marker loci with two alleles, three with three alleles, one with four and another with five alleles. In comparison to Theileria parva, a bovine hemoprotozoan that pertains to the same piroplasmida order and own a genome of similar size, the number of polymorphic TRs and the average number of alleles per TR locus seem to be significantly reduced in the B. bovis genome. Furthermore, the ratio of micro- to minisatellites in both B. bovis and T. parva is considerably lower than in other eukaryotes, as confirmed by bioinformatic analysis. The multilocus genotype of the five B. bovis isolates was assessed and the
Alzan, Heba F; Silva, Marta G; Davis, William C; Herndon, David R; Schneider, David A; Suarez, Carlos E
Babesia bovis is an intra-erythrocytic tick-transmitted apicomplexan protozoan parasite. It has a complex lifestyle including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed that certain B. bovis genes, including members of 6-Cys gene family, are differentially expressed during tick and mammalian stages of the parasite's life cycle. Moreover, the 6-Cys E gene is differentially expressed in the T3Bo strain of B. bovis tick stages, and anti 6-Cys E antibodies were shown to be able to inhibit in vitro growth of the phenotypically distinct B. bovis Mo7clonal line. In this study, the 6-Cys E gene of B. bovis T3Bo strain was disrupted by transfection using a plasmid containing 6-Cys gene E 5' and 3' regions to guide homologous recombination, and the egfp-bsd fusion gene under control of a ef-1α promoter, yielding a B. bovis clonal line designated 6-Cys EKO-cln. Full genome sequencing of 6-Cys EKO-cln parasites was performed and in vitro inhibition assays using anti 6-Cys E antibodies. Full genome sequencing of 6-Cys EKO-cln B. bovis demonstrated single insertion of egfp-bsd gene that disrupts the integrity of 6-Cys gene E. Undistinguishable growth rate of 6-Cys EKO-cln line compared to wild-type 6-Cys E intact T3Bo B. bovis strain in in vitro cultures indicates that expression of gene 6-Cys E is not essential for blood stage replication in this strain. In vitro inhibition assays confirmed the ability of anti-6 Cys E antibodies to inhibit the growth of the wild-type Mo7 and T3Bo B. bovis parasites, but no significant inhibition was found for 6-Cys EKO-cln line parasites. Overall, the data suggest that the anti-6 Cys E antibody neutralising effect on the wild type strains is likely due to mechanical hindrance, or cross-reactivity, rather than due to functional requirements of 6-Cys gene E product for survival and development of the erythrocyte stages
Alcaraz-López, Omar A; García-Gil, Cindy; Morales-Martínez, Claudia; López-Rincón, Gonzalo; Estrada-Chávez, Ciro; Gutiérrez-Pabello, José A; Esquivel-Solís, Hugo
Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), is a successful pathogen that remains an important global threat to livestock. Cattle naturally exposed to M. bovis normally become reactive to the M. bovis-purified protein derivative (tuberculin) skin test; however, some individuals remain negative, suggesting that they may be resistant to infection. To better understand host innate resistance to infection, 26 cattle from herds with a long history of high TB prevalence were included in this study. We investigated the bactericidal activity, the production of reactive oxygen and nitrogen species and the TB-related gene expression profile after in vitro M. bovis challenge of monocyte-derived macrophages from cattle with TB (n=17) and from non-infected, exposed cattle (in-contacts, n=9). The disease status was established based on the tuberculin skin test and blood interferon-gamma test responses, the presence of visible lesions at inspection on abattoirs and the histopathology and culture of M. bovis. Although macrophages from TB-infected cattle enabled M. bovis replication, macrophages from healthy, exposed cattle had twofold lower bacterial loads, overproduced nitric oxide and had lower interleukin (IL)-10 gene expression (P⩽0.05). Higher mRNA expression levels of inducible nitric oxide synthase, C-C motif chemokine ligand 2 and IL-12 were observed in macrophages from all in-contact cattle than in macrophages from their TB-infected counterparts, which expressed more tumour necrosis factor-α; however, the differences were not statistically significant owing to individual variation. These results confirm that macrophage bactericidal responses have a crucial role in innate resistance to M. bovis infection in cattle.
ter Laak, E A; Noordergraaf, J H; Verschure, M H
The purpose of this study was to determine the susceptibility of various strains of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum, which are prevalent causes of pneumonia in calves, to 16 antimicrobial agents in vitro. The MICs of the antimicrobial agents were determined by a serial broth dilution method for 16 field strains and the type strain of M. bovis, for 19 field strains and the type strain of M. dispar, and for 17 field strains of U. diversum. Final MICs for M. bovis and M. dispar were read after 7 days and final MICs for U. diversum after 1 to 2 days. All strains tested were susceptible to tylosin, kitasamycin, and tiamulin but were resistant to nifuroquine and streptomycin. Most strains of U. diversum were intermediately susceptible to oxytetracycline but fully susceptible to chlortetracycline; most strains of M. bovis and M. dispar, however, were resistant to both agents. Strains of M. dispar and U. diversum were susceptible to doxycycline and minocycline, but strains of M. bovis were only intermediately susceptible. Susceptibility or resistance to chloramphenicol, spiramycin, spectinomycin, lincomycin, or enrofloxacin depended on the species but was not equal for the three species. The type strains of M. bovis and M. dispar were more susceptible to various antimicrobial agents, including tetracyclines, than the field strains. This finding might indicate that M. bovis and M. dispar strains are becoming resistant to these agents. Antimicrobial agents that are effective in vitro against all three mycoplasma species can be considered for treating mycoplasma infections in pneumonic calves. Therefore, tylosin, kitasamycin, and tiamulin may be preferred over oxytetracycline and chlortetracycline.
ter Laak, E A; Noordergraaf, J H; Verschure, M H
The purpose of this study was to determine the susceptibility of various strains of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum, which are prevalent causes of pneumonia in calves, to 16 antimicrobial agents in vitro. The MICs of the antimicrobial agents were determined by a serial broth dilution method for 16 field strains and the type strain of M. bovis, for 19 field strains and the type strain of M. dispar, and for 17 field strains of U. diversum. Final MICs for M. bovis and M. dispar were read after 7 days and final MICs for U. diversum after 1 to 2 days. All strains tested were susceptible to tylosin, kitasamycin, and tiamulin but were resistant to nifuroquine and streptomycin. Most strains of U. diversum were intermediately susceptible to oxytetracycline but fully susceptible to chlortetracycline; most strains of M. bovis and M. dispar, however, were resistant to both agents. Strains of M. dispar and U. diversum were susceptible to doxycycline and minocycline, but strains of M. bovis were only intermediately susceptible. Susceptibility or resistance to chloramphenicol, spiramycin, spectinomycin, lincomycin, or enrofloxacin depended on the species but was not equal for the three species. The type strains of M. bovis and M. dispar were more susceptible to various antimicrobial agents, including tetracyclines, than the field strains. This finding might indicate that M. bovis and M. dispar strains are becoming resistant to these agents. Antimicrobial agents that are effective in vitro against all three mycoplasma species can be considered for treating mycoplasma infections in pneumonic calves. Therefore, tylosin, kitasamycin, and tiamulin may be preferred over oxytetracycline and chlortetracycline. PMID:8452363
Zbinden, Christina; Pilo, Paola; Frey, Joachim; Bruckmaier, Rupert M; Wellnitz, Olga
Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1β, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites.
Malama, Sydney; Johansen, Tone Bjordal; Muma, John Bwalya; Mwanza, Sydney; Djønne, Berit; Godfroid, Jacques
Bovine tuberculosis (BTB) is a chronic bacterial disease caused by Mycobacterium bovis. Infections due to M. bovis, which serves as a stable reservoir, can pose serious challenge to control and eradicate in both wildlife and livestock at the interface. This study aimed at isolating and characterizing M. bovis from Kafue lechwe (Kobus leche kafuensis) and black lechwe (Kobus leche smithemani) at the animal/human interface in Zambia. The samples with lesions compatible with BTB collected during the hunting seasons of 2009 and 2010 were cultured for isolation of mycobacteria using Stonebrink with pyruvate (BD Diagnostics, MD, USA) and Middlebrook 7H10 (BD Diagnostics) slants. Isolated mycobacteria were identified using IS6110 polymerase chain reaction and deletion analysis. Molecular characterization of the isolates was performed using spoligotyping and mycobacteria interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) with nine loci. Data was analyzed using BioNumerics software 6.1. Out of the 39 samples, acid fast bacilli were detected in 27 (69.2 %) based on smear microscopy. Seven isolates were found to belong to Mycobacterium tuberculosis complex, and all were identified as M. bovis based on deletion analysis. All seven isolates were identical on spoligotyping as belonging to the SB0120 (SIT 482). MIRU-VNTR differentiated the isolates into five different patterns. This study has confirmed that M. bovis circulates in the Kafue lechwe, and non-tuberculous mycobacteria were detected in the black lechwe in Zambia which represents a wildlife reservoir, with a potential to spillover to cattle and humans. Isolates of M. bovis from lechwe antelopes are much conserved as only one spoligotype was detected. The study has shown that three loci differentiated fairly well. This option is cheap and less laborious, and hence a better option in resource-strained country like Zambia. The study further showed that some of the loci recommended by the European
Li, Pei; Wang, Rui; Dong, Wenqi; Hu, Linlin; Zong, Bingbing; Zhang, Yanyan; Wang, Xiangru; Guo, Aizhen; Zhang, Anding; Xiang, Yaozu; Chen, Huanchun; Tan, Chen
Mycobacterium bovis (M. bovis), the most common pathogens of tuberculosis (TB), is virulent to human and cattle, and transmission between cattle and humans warrants reconsideration concerning food safety and public health. Recently, efforts have begun to analyze cellular proteomic responses induced by Mycobacterium tuberculosis (M. tb). However, the underlying mechanisms by which virulent M. bovis affects human hosts are not fully understood. For the present study, we utilized a global and comparative labeling strategy of isobaric tag for relative and absolute quantitation (iTRAQ) to assess proteomic changes in the human monocyte cell line (THP-1) using a vaccine strain and two virulent strains H37Rv and M. bovis. We measured 2,032 proteins, of which 61 were significantly differentially regulated. Ingenuity Pathway Analysis was employed to investigate the canonical pathways and functional networks involved in the infection. Several pathways, most notably the phagosome maturation pathway and TNF signaling pathway, were differentially affected by virulent strain treatment, including the key proteins CCL20 and ICAM1. Our qRT-PCR results were in accordance with those obtained from iTRAQ. The key enzyme MTHFD2, which is mainly involved in metabolism pathways, as well as LAMTOR2 might be effective upon M. bovis infection. String analysis also suggested that the vacuolar protein VPS26A interacted with TBC1D9B uniquely induced by M. bovis. In this study, we have first demonstrated the application of iTRAQ to compare human protein alterations induced by virulent M. bovis infections, thus providing a conceptual understanding of mycobacteria pathogenesis within the host as well as insight into preventing and controlling TB in human and animal hosts' transmission. PMID:28337427
Herrera-Rodríguez, Sara E; Gordiano-Hidalgo, María Alejandra; López-Rincón, Gonzálo; Bojorquez-Narváez, Luis; Padilla-Ramírez, Francisco Javier; Pereira-Suárez, Ana Laura; Flores-Valdez, Mario Alberto; Estrada-Chávez, Ciro
Bovine tuberculosis (bTB) remains a problem on many dairy farms in Mexico, as well as a public health risk. We previously found a high frequency of Mycobacterium bovis DNA in colostrum from dairy cows using a nested PCR to detect mpb70. Since there are no reliable in vivo tests to determine the effectiveness of booster Mycobacterium bovis BCG vaccination against bTB, in this work we monitored M. bovis DNA in colostrum by using this nested PCR. In order to decrease the risk of adverse reactions in animals likely containing viable M. bovis, a single application of BCG and a subunit vaccine (EEP-1) formulated with M. bovis culture filtrate proteins (CFP) and a copolymer as the adjuvant was performed in tuberculin skin test-negative cattle (TST(-)), while TST reactor animals (TST(+)) received EEP-1 only. Booster immunization using EEP-1 was applied to both groups, 2 months after primary vaccination to whole herds and 12 months later to lactating cows. Colostrum samples were collected from 6 farms where the cows were vaccinated over a 12-month period postvaccination and, for comparison, from one control farm where the cows were not vaccinated with comparable bTB prevalence. We observed an inverse relationship between the frequency of M. bovis DNA detection and time postvaccination at the first (P < 0.001) and second (P < 0.0001) 6-month periods. Additionally, the concentration of gamma interferon (IFN-γ) was higher in mpb70 PCR-positive colostrum samples (P = 0.0003). These results suggest that M. bovis DNA frequency in colostrum could be a potentially useful biomarker for bTB vaccine efficacy on commercial dairy farms.
Herrera-Rodríguez, Sara E.; Gordiano-Hidalgo, María Alejandra; López-Rincón, Gonzálo; Bojorquez-Narváez, Luis; Padilla-Ramírez, Francisco Javier; Pereira-Suárez, Ana Laura; Flores-Valdez, Mario Alberto
Bovine tuberculosis (bTB) remains a problem on many dairy farms in Mexico, as well as a public health risk. We previously found a high frequency of Mycobacterium bovis DNA in colostrum from dairy cows using a nested PCR to detect mpb70. Since there are no reliable in vivo tests to determine the effectiveness of booster Mycobacterium bovis BCG vaccination against bTB, in this work we monitored M. bovis DNA in colostrum by using this nested PCR. In order to decrease the risk of adverse reactions in animals likely containing viable M. bovis, a single application of BCG and a subunit vaccine (EEP-1) formulated with M. bovis culture filtrate proteins (CFP) and a copolymer as the adjuvant was performed in tuberculin skin test-negative cattle (TST−), while TST reactor animals (TST+) received EEP-1 only. Booster immunization using EEP-1 was applied to both groups, 2 months after primary vaccination to whole herds and 12 months later to lactating cows. Colostrum samples were collected from 6 farms where the cows were vaccinated over a 12-month period postvaccination and, for comparison, from one control farm where the cows were not vaccinated with comparable bTB prevalence. We observed an inverse relationship between the frequency of M. bovis DNA detection and time postvaccination at the first (P < 0.001) and second (P < 0.0001) 6-month periods. Additionally, the concentration of gamma interferon (IFN-γ) was higher in mpb70 PCR-positive colostrum samples (P = 0.0003). These results suggest that M. bovis DNA frequency in colostrum could be a potentially useful biomarker for bTB vaccine efficacy on commercial dairy farms. PMID:23425597
Matos, A C; Figueira, L; Martins, M H; Pinto, M L; Matos, M; Coelho, A C
A survey to determine the prevalence of Mycobacterium bovis in wild mammals in Portugal was conducted by testing samples from hunted animals and those found dead between 2009 and 2013. In this study, we investigated 2116 wild mammals. Post-mortem examinations were performed, and tissues were collected from wild mammals representing 8 families and 11 different species, with a total of 393 animals analysed. Cultures were performed, and acid-fast isolates were identified by PCR. Tissues were also screened for Mycobacterium bovis by directly extracting DNA and testing for the Mycobacterium bovis-specific sequences. Mycobacterium bovis prevalence was 26.9% (95% CI: 22.8-31.5%). Mycobacterium bovis was recorded in 106 of the 393 studied species: prevalence by species were 26.9% (95% CI: 16.8-40.2%) in red foxes, 20.0% (95% CI: 7.0-45.2%) in Egyptian mongooses, 21.4% (95% CI: 16.2-27.7%) in wild boar and 38.3% (95% CI: 29.9-47.4%) in red deer. Mycobacterium bovis infection was detected in six of eight taxonomic families. For some species, the small sample sizes obtained were a reflection of their restricted range and low abundance, making estimates of infection prevalence very difficult (1 beech marten of 4; 1 Eurasian otter of 3; 2 common genet of 3). Infection was not detected in European badgers, hedgehog, wild rabbits and hare. The results of this study confirm the presence of Mycobacterium bovis infection in wild carnivores in Portugal. © 2014 Blackwell Verlag GmbH.
Cherry, Natalie A; Maggi, Ricardo G; Cannedy, Allen L; Breitschwerdt, Edward B
Although an organism primarily associated with non-clinical bacteremia in domestic cattle and wild ruminants, Bartonella bovis was recently defined as a cause of bovine endocarditis. The purpose of this study was to develop a B. bovis species-specific PCR assay that could be used to confirm the molecular prevalence of Bartonella spp. infection. Blood samples from 142 cattle were tested by conventional PCR targeting the Bartonella 16S-23S intergenic spacer (ITS) region. Overall, Bartonella DNA was detected in 82.4% (117/142) of the cattle using either Bartonella genus primers or B. bovis species-specific primers. Based upon size, 115 of the 117 Bartonella genus ITS PCR amplicons were consistent with B. bovis infection, which was confirmed by PCR using B. bovis species-specific primers and by sequencing three randomly selected, appropriately sized Bartonella genus PCR amplicons. By DNA sequencing, Bartonella henselae was confirmed as the two remaining amplicons, showing sequence similarity to B. henselae URBHLIE 9 (AF312496) and B. henselae Houston 1 (NC_005956), respectively. Following pre-enrichment blood culture of 12 samples in Bartonella alpha Proteobacteria growth medium (BAPGM) B. henselae infection was found in another three cows. Four of the five cows infected with B. henselae were co-infected with B. bovis. To our knowledge this study describes the first detection of B. henselae in any large ruminant species in the world and supports the need for further investigation of prevalence and pathogenic potential of B. henselae and B. bovis in cattle.
Asada, Masahito; Goto, Yasuyuki; Yahata, Kazuhide; Yokoyama, Naoaki; Kawai, Satoru; Inoue, Noboru; Kaneko, Osamu; Kawazu, Shin-ichiro
Background Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite. Methodology/Principal Findings Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion. Conclusions/Significance This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding. PMID:22506073
Monitoring serum antibody production kinetics to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and efficacy of intervention strategies in several species. In the presen...
Monitoring serum antibody production kinetics to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and efficacy of intervention strategies in several species. Humoral immun...
Understanding immune responses of white-tailed deer (WTD) to infection with Mycobacterium bovis provides insight into mechanisms of pathogen control and may provide clues to development of effective vaccine strategies. WTD were vaccinated with either BCG strain Pasteur or BCG Danish. Both vaccinates...
Efficacy of vaccination of cattle with the Leptospira interrogans serovar hardjo type hardjoprajitno component of a pentavalent Leptospira bacterin against experimental challenge with Leptospira borgpetersenii serovar hardjo type hardjo-bovis.
Rinehart, Carol L; Zimmerman, Alicia D; Buterbaugh, Robin E; Jolie, Rika A; Chase, Christopher C L
To evaluate the efficacy of vaccination with the Leptospira interrogans serovar hardjo type hardjoprajitno component of a pentavalent Leptospira bacterin against a virulent experimental challenge with Leptospira borgpetersenii serovar hardjo type hardjo-bovis strain 203 in cattle. Fifty-five 6-month-old Holstein heifers. Heifers that were negative for persistent infection with bovine viral diarrhea virus determined via immunohistochemical testing and negative for Leptospira interrogans serovar pomona, Leptospira interrogans serovar hardjo, Leptospira interrogans serovar grippotyphosa, Leptospira interrogans serovar bratislava, Leptospira interrogans serovar canicola, and Leptospira interrogans serovar icterohaemorrhagiae determined via microscopic agglutination assay were enrolled in the study. Two heifers were separated and used for the challenge passage. The remaining heifers were vaccinated twice with a commercial pentavalent bacterin or a sham vaccine 21 days apart and subsequently challenged with L borgpetersenii serovar hardjo type hardjo-bovis strain 203. Urinary shedding, antibody titers, and clinical signs of leptospirosis infection were recorded for 8 weeks after challenge. Heifers that received the pentavalent bacterin did not shed the organism in urine after challenge and did not have renal colonization at necropsy. Heifers that were sham vaccinated shed the organism in urine and had renal colonization. Results provided evidence that a pentavalent Leptospira vaccine containing L interrogans serovar hardjo type hardjoprajitno can provide protection against challenge with L borgpetersenii serovar hardjo type hardjo-bovis strain 203. It is important to demonstrate cross-protection that is vaccine specific against disease-causing strains of organisms that are prevalent under field conditions.
Salguero, F J; Gibson, S; Garcia-Jimenez, W; Gough, J; Strickland, T S; Vordermeier, H M; Villarreal-Ramos, B
Cattle vaccination against bovine tuberculosis (bTB) has been proposed as a supplementary method to help control the incidences of this disease. Bacillus Calmette-Guérin (BCG) is currently the only viable candidate vaccine for immunization of cattle against bTB, caused by Mycobacterium bovis (M. bovis). In an attempt to characterize the differences in the immune response following M. bovis infection between BCG-vaccinated and non-vaccinated animals, a combination of gross pathology, histopathology and immunohistochemical (IHC) analyses was used. BCG vaccination was found to significantly reduce the number of gross and microscopic lesions present within the lungs and lymph nodes. Additionally, the microscopically visible bacterial load of stages III and IV granulomas was reduced. IHC using cell surface markers revealed the number of CD68+ (macrophages), CD3+ (T lymphocytes) and WC1+ cells (γδ T cells) to be significantly reduced in lymph node granulomas of BCG-vaccinated animals, when compared to non-vaccinated animals. B lymphocytes (CD79a+) were significantly increased in BCG-vaccinated cattle for granulomas at stages II, III and IV. IHC staining for iNOS showed a higher expression in granulomas from BCG-vaccinated animals compared to non-vaccinated animals for all stages, being statistically significant in stages I and IV. TGFβ expression decreased alongside the granuloma development in non-vaccinated animals, whereas BCG-vaccinated animals showed a slight increase alongside lesion progression. IHC analysis of the cytokines IFN-γ and TNF-α demonstrated significantly increased expression within the lymph node granulomas of BCG-vaccinated cattle. This is suggestive of a protective role for IFN-γ and TNF-α in response to M. bovis infection. Findings shown in this study suggest that the use of BCG vaccine can reduce the number and severity of lesions, induce a different phenotypic response and increase the local expression of key cytokines related to
de los Monteros, Luz Elena Espinosa; Galán, Juan Carlos; Gutiérrez, Montserrat; Samper, Sofía; García Marín, Juan F.; Martín, Carlos; Domínguez, Lucas; de Rafael, Luis; Baquero, Fernando; Gómez-Mampaso, Enrique; Blázquez, Jesús
An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform. PMID:9431955
Cavanaugh, Joseph S.; Silk, Benjamin J.; Ershova, Julia; Mazurek, Gerald H.; LoBue, Philip A.; Moonan, Patrick K.
Current US guidelines recommend longer treatment for tuberculosis (TB) caused by pyrazinamide-resistant organisms (e.g., Mycobacterium bovis) than for M. tuberculosis TB. We compared treatment response times for patients with M. bovis TB and M. tuberculosis TB reported in the United States during 2006–2013. We included culture-positive, pulmonary TB patients with genotyping results who received standard 4-drug treatment at the time of diagnosis. Time to sputum-culture conversion was defined as time between treatment start date and date of first consistently culture-negative sputum. We analyzed 297 case-patients with M. bovis TB and 30,848 case-patients with M. tuberculosis TB. After 2 months of treatment, 71% of M. bovis and 65% of M. tuberculosis TB patients showed conversion of sputum cultures to negative. Likelihood of culture conversion was higher for M. bovis than for M. tuberculosis, even after controlling for treatment administration type, sex, and a composite indicator of bacillary burden. PMID:28221125
Hang'ombe, Mudenda B; Munyeme, Musso; Nakajima, Chie; Fukushima, Yukari; Suzuki, Haruka; Matandiko, Wigganson; Ishii, Akihiro; Mweene, Aaron S; Suzuki, Yasuhiko
In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important. A PCR identified 15 and 37 M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia. These results provide strong evidence of M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host.
Mon, María Laura; Moyano, Roberto Damián; Viale, Mariana Noelia; Colombatti Olivieri, María Alejandra; Gamietea, Ignacio José; Montenegro, Valeria Noely; Alonso, Bernardo; Santangelo, María de la Paz; Singh, Mahavir; Duran, Rosario; Romano, María Isabel
The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria. PMID:25110654
Michel, A L; Coetzee, M L; Keet, D F; Maré, L; Warren, R; Cooper, D; Bengis, R G; Kremer, K; van Helden, P
Bovine tuberculosis is endemic in African buffalo and a number of other wildlife species in the Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP) in South Africa. It was thought that the infection had been introduced into the KNP ecosystem through direct contact between cattle and buffalo, a hypothesis which was confirmed in this study by IS6110 and PGRS restriction fragment length polymorphism (RFLP) typing. The molecular characterisation of 189 Mycobacterium bovis isolates from nine wildlife species in the HiP, including three smaller associated parks, and the Kruger National Park with adjacent areas showed that the respective epidemics were each caused by an infiltration of a single M. bovis genotype. The two M. bovis strains had different genetic profiles, as demonstrated by hybridisation with the IS6110 and PGRS RFLP probes, as well as with regard to evidence of evolutionary changes to the IS profile. While the M. bovis type in HiP was transmitted between buffaloes and to at least baboon, bushpig and lion without obvious genetic changes in the RFLP patterns, in the KNP a dominant strain was represented in 73% of the M. bovis isolates, whilst the remaining 27% were variants of this strain. No species-specific variants were observed, except for one IS6110 type which was found only in a group of five epidemiologically related greater kudu. This finding was attributed to species-specific behaviour patterns rather than an advanced host-pathogen interaction.
Genis, Alma D; Mosqueda, Juan J; Borgonio, Verónica M; Falcón, Alfonso; Alvarez, Antonio; Camacho, Minerva; de Lourdes Muñoz, Maria; Figueroa, Julio V
Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2), and msa-2b). Small subunit ribosomal (ssr)RNA gene is subject to evolutive pressure and has been used in phylogenetic studies. To determine the phylogenetic relationship among B. bovis Mexican isolates using different genetic markers, PCR amplicons, corresponding to msa-1, msa-2c, msa-2b, and ssrRNA genes, were cloned and plasmids carrying the corresponding inserts were sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 12 geographically different B. bovis isolates and a reference strain. Overall sequence identities of 47.7%, 72.3%, 87.7%, and 94% were determined for msa-1, msa-2b, msa-2c, and ssrRNA, respectively. A robust phylogenetic tree was obtained with msa-2b sequences. The phylogenetic analysis suggests that Mexican B. bovis isolates group in clades not concordant with the Mexican geography. However, the Mexican isolates group together in an American clade separated from the Australian clade. Sequence heterogeneity in msa-1, msa-2b, and msa-2c coding regions of Mexican B. bovis isolates present in different geographical regions can be a result of either differential evolutive pressure or cattle movement from commercial trade.
Francisco, Tolani I; Orloski, Kathleen A; Roberts, Nancy J
To describe an epidemiological investigation of a bovine tuberculosis outbreak on a Colorado dairy operation. A cull dairy cow infected with Mycobacterium bovis (index cow) was detected at a Texas abattoir during routine slaughter surveillance and subsequent diagnostic testing. This initiated an epidemiological investigation that was performed in accordance with USDA regulations. The index cow was traced back to a Colorado dairy (index herd). Of the 908 cattle in the index herd, 101 (11.1%; 86 adult cattle > 2 years old and 15 immature cattle ≤ 2 years old) were infected with M bovis. Fourteen M bovis-infected cattle ≤ 2 years old were identified on 5 additional premises that had purchased cattle from the index herd directly or indirectly. All 115 affected cattle were infected with the same genetic type (spoligotype) of M bovis. A substantial proportion of cattle that left the index herd during the 5 years previous to the identification of the index cow were untraceable because of a lack of unique animal identification and inadequate records. Results indicated that neonatal calves can have an important role in the transmission of M bovis. Also, this report highlights the exigent need for unique individual identification of livestock, including neonatal animals, so that thorough epidemiological investigations of reportable (zoonotic or foreign animal) diseases can be conducted when necessary.
Scott, Colleen; Cavanaugh, Joseph S; Silk, Benjamin J; Ershova, Julia; Mazurek, Gerald H; LoBue, Philip A; Moonan, Patrick K
Current US guidelines recommend longer treatment for tuberculosis (TB) caused by pyrazinamide-resistant organisms (e.g., Mycobacterium bovis) than for M. tuberculosis TB. We compared treatment response times for patients with M. bovis TB and M. tuberculosis TB reported in the United States during 2006-2013. We included culture-positive, pulmonary TB patients with genotyping results who received standard 4-drug treatment at the time of diagnosis. Time to sputum-culture conversion was defined as time between treatment start date and date of first consistently culture-negative sputum. We analyzed 297 case-patients with M. bovis TB and 30,848 case-patients with M. tuberculosis TB. After 2 months of treatment, 71% of M. bovis and 65% of M. tuberculosis TB patients showed conversion of sputum cultures to negative. Likelihood of culture conversion was higher for M. bovis than for M. tuberculosis, even after controlling for treatment administration type, sex, and a composite indicator of bacillary burden.
Abdala, Alejandro A; Garbaccio, Sergio; Zumárraga, Martín; Tarabla, Héctor D
Control eradication campaigns of bovine tuberculosis based on the «test and slaughter» approach were successful in many countries and regions; however, in some areas the infection persists and one of the main reasons is Mycobacterium bovis infection in wild life species. Argentina has applied the same approach since 1999, achieving progress in dairy cattle herds. Nonetheless, the wildlife role has never been investigated. The objective of this study was to determine if wildlife from the Santa Fe dairy area is infected with M. bovis. Wildlife species having a positive tuberculin skin test were captured in five dairy farms. Ninety five wildlife mammals were captured; M. bovis was recovered from 7 possums (Didelphys albiventris), from one fox (Lycolapex gimnocercus) and from one rat (Rattus norvegicus). None of the animals exhibited macroscopic lesions. The most frequently isolated M. bovis spoligotypes were types 34 (4 isolates) and 12 (3 isolates). Spoligotype 34 is the most frequently isolated type in Argentine cattle. The role of D. albiventris as spillover host of M. bovis is discussed in this study. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.
Jang, Yunho; Ryoo, Soyoon; Lee, Hyunkyoung; Kim, Narae; Lee, Hang; Park, So-Young; Song, Woong-Seog; Kim, Jong-Taek; Lee, Hee Soo; Myung Kim, Jae
We demonstrate Mycobacterium bovis infection in wild boar ( Sus scrofa ) in South Korea. During 2012-15, we attempted to isolate M. bovis from 847 wild animals, mainly Korean water deer ( Hydropotes inermis argyropus), raccoon dogs ( Nyctereutes procyonoides ), and wild boar, from 11 regions in South Korea. We isolated M. bovis from three of 118 wild boar (2.5%) captured in Gyeonggi Province, where bovine tuberculosis (bTB) outbreaks have also occurred in livestock. Spoligotypes and mycobacterial interspersed repetitive units-variable number tandem repeats types of these M. bovis isolates (SB0140 and SB1040, 4-2-3-3-7-5-5-4-4-3-4-3 and 5-2-3-3-7-5-5-4-3-10-5-2; MIRU4, MIRU16, MIRU27, MIRU31, ETR-A, ETR-B, ETR-C, QUB11b, QUB26, QUB3336, VNTR2401, and VNTR3171) have also been identified from farmed livestock such as cattle ( Bos taurus coreanae), Formosan sika deer ( Cervus nippon taiouanus), and American elk ( Cervus canadensis ) in the country. In South Korea, bTB appears to be endemic in livestock, and there are numerous opportunities for contact between wild boar and livestock due to high population densities and broad activity ranges. Our results support the hypothesis that M. bovis is transmitted between domestic and wild animals.
Background In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important. Results A PCR identified 15 and 37 M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units–Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia. Conclusions These results provide strong evidence of M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host. PMID:23151267
Liu, Chunfa; Yue, Ruichao; Yang, Yang; Cui, Yongyong; Yang, Lifeng; Zhao, Deming; Zhou, Xiangmei
Mycobacteria can trigger the AIM2 inflammasome, autophagy activation and type-I interferon release, which are both activated by cytosolic DNA. We have recently demonstrated that activation of the AIM2 inflammasome during M. bovis infection is the result of mycobacterial translocation into the cytosol. To elucidate the effects of inflammasome activation on autophagy, we investigated the role of the AIM2 inflammasome from macrophages infected with a virulent strain of M. bovis. The results showed that the M. bovis-induced AIM2 inflammasome activation decreases autophagy in immortalized and primary murine macrophages. This relied on the inflammasome sensor AIM2 which conjugates with cytosolic DNA to inhibit the STING-dependent pathway involved in selective autophagy and interferon-β release in Mycobacterium-infected macrophages. These results suggest that the AIM2 cytosolic DNA sensor may conjugate competitively with cytosolic M. bovis DNA to restrict M. bovis induced STING-TBK1-dependent autophagy activation and IFN-β secretion. PMID:27409673
Kelly, Pamela; Jahns, Hanne; Power, Eugene; Bainbridge, John; Kenny, Kevin; Corpa, Juan M; Cassidy, Joseph P; Callanan, John J
Avian tuberculosis rarely affects ratites compared to other bird species and is typically caused by Mycobacterium avium species. This study describes the pathological and microbiological findings in three adult ostriches with mycobacteriosis, in one of which Mycobacterium bovis was isolated from the lesions. Post mortem examinations on ostriches from two different zoological collections in Ireland revealed multifocal caseous granulomas affecting the spleen and liver in all cases, with additional involvement of intestines in two cases. In one case, granulomas were present within the pharynx, at the thoracic inlet and multifocally on the pleural surface. Acid-fast bacilli were observed in all lesions. Mycobacterium sp. of the M. avium complex was isolated from the intestinal lesions in the two cases with intestinal involvement, and M. bovis sp. oligotype SB0140 was cultured from the liver of the third ostrich. This represents the first reported case of M. bovis infection in an ostrich. Avian tuberculosis due to M. bovis is rare and to date has been reported in only parrots and experimentally inoculated birds. Mycobacterium bovis needs to be considered as a possible cause of tuberculosis in ostriches because the lesions are similar to those observed with M. avium complex infection.
Behrendt, Jan Hillern; Ruiz, Antonio; Zahner, Horst; Taubert, Anja; Hermosilla, Carlos
Eimeria bovis infections are under immunological control and recent studies have emphasized the role of early PMN-mediated innate immune responses in infected calves. Neutrophil extracellular traps (NETs) have recently been demonstrated to act as a killing mechanism of PMN against several pathogens. In the present study, the interactions of bovine PMN with sporozoites of E. bovis were investigated in this respect in vitro. For demonstration and quantification of NET formation, extracellular DNA was stained by Sytox Orange. Fluorescence images after Sytox Orange staining as well as scanning electron microscopy (SEM) showed NET formation to occur upon contact with E. bovis sporozoites. Exposure of PMN to viable sporozoites induced stronger NET formation than to dead or homogenized parasites. NET formation was abolished by treatment with DNase and could be reduced by diphenylene iodonium, which is described as a potent inhibitor of NADPH oxidase. After sporozoite and PMN co-culture, extracellular fibres were found attached to sporozoites and seemed to trap them, strongly suggesting that NETs immobilize E. bovis sporozoites and thereby prevent them from infecting host cells. Thus, transfer of sporozoites, previously being confronted with PMN, to adequate host cells resulted in clearly reduced infection rates when compared to PMN-free controls. NET formation by PMN may therefore represent an effector mechanism in early innate immune reactions against E. bovis. This is the first report indicating Eimeria-induced NET formation.
Bangoura, Berit; Mundt, Hans-Christian; Schmäschke, Ronald; Westphal, Bernhard; Daugschies, Arwid
The present study was designed to investigate the prevalence of the pathogenic coccidia species Eimeria bovis and Eimeria zuernii in shed-reared animals in German dairy and fattening facilities. Samples were obtained from 65 cattle farms distributed randomly across all the regions of Germany regardless of the occurrence of clinical problems. The samples were obtained rectally. Faecal consistency and the total number of oocysts per gram of faeces (OPG) were determined, along with the OPG values for E. bovis and E. zuernii. A questionnaire was completed for each farm to record information about herd size and management, along with individual animal data. Eimeria oocysts were detected in 62 of these farms, which give a prevalence of 95.4%. The farm prevalence of the pathogenic species was 76.9% for E. bovis and 83.1% for E. zuernii. The number of oocysts excreted could not be correlated significantly with farm type or farm management but depended on the floor type, the age of the calves and the time after rehousing. Furthermore, there was a positive correlation between OPG and the observation of diarrhoea. E. zuernii had a greater influence on the occurrence of diarrhoea than E. bovis. This study confirms that herd management frequently does not meet the requirements of effective coccidia control despite the fact that the pathogenic coccidia E. bovis and E. zuernii are ubiquitous in German cattle populations.
Meije, Y; Martínez-Montauti, J; Caylà, J A; Loureiro, J; Ortega, L; Clemente, M; Sanz, X; Ricart, M; Santomà, M J; Coll, P; Sierra, M; Calsina, M; Vaqué, M; Ruiz-Camps, I; López-Sánchez, C; Montes, M; Ayestarán, A; Carratalà, J; Orcau, A
Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is widely used as adjunctive therapy for superficial bladder cancer. Intravesical administration of BCG has been associated with systemic infection. Disseminated infection due to M. bovis is otherwise uncommon. After identification of three patients with health-care associated BCG infection (HCBCGI) who had never received intravesical BCG administration, an epidemiologic study was performed. All patients with HCBCGI in the Barcelona tuberculosis (TB) program were reviewed from January 1, 2005 to December 31, 2015 searching for infections caused by M. bovis-BCG. Patients with HCBCGI who had not received intravesical BCG instillation were selected and the source of infection was investigated. Nine oncology patients with infection caused by M. bovis-BCG were studied. All had permanent central venous catheters. Catheter maintenance was performed at four different outpatient clinics in the same room in which other patients underwent BCG instillations for bladder cancer without required biological precautions. All patients developed pulmonary TB, either alone or with extrapulmonary disease. Catheter-related infection was considered the mechanism of acquisition based on the epidemiologic association and positive catheter cultures for BCG in patients in whom mycobacterial cultures were performed. Physicians should be alerted to the possibility of TB due to nosocomially acquired, catheter-related infections with M. bovis-BCG in patients with indwelling catheters. This problem may be more common than expected in centers providing BCG therapy for bladder cancer without adequate precautions.
Mackintosh, C G; Qureshi, T; Waldrup, K; Labes, R E; Dodds, K G; Griffin, J F
Tuberculosis (Tb) caused by Mycobacterium bovis is a worldwide threat to livestock and humans. One control strategy is to breed livestock that are more resistant to Mycobacterium bovis. In a 3-year heritability study 6 farmed red deer stags were selected from 39 on the basis of their differing responses to experimental challenge via the tonsillar sac with approximately 500 CFU of M. bovis. Two stags remained uninfected, two were moderately affected, and two developed serious spreading Tb. Seventy offspring, bred from these six stags by artificial insemination using stored semen, were similarly challenged with M. bovis. The offspring showed patterns of response to M. bovis challenge similar to those of their sires, providing evidence for a strong genetic basis to resistance to Tb, with an estimated heritability of 0.48 (standard error, 0.096; P < 0. 01). This is the first time the heritability of Tb resistance in domestic livestock has been measured. The breeding of selection lines of resistant and susceptible deer will provide an ideal model to study the mechanisms of Tb resistance in a ruminant and could provide an additional strategy for reducing the number and severity of outbreaks of Tb in farmed deer herds. Laboratory studies to identify genetic and immunological markers for resistance to Tb are under way. Preliminary studies showed no associations between NRAMP or DRB genes and resistance to Tb in deer. Patterns of immune responses seen in resistant animals suggest that both innate and acquired pathways of immunity are necessary to produce the resistant phenotype.
Shah, Neha; Flood, Jennifer
We conducted a retrospective review of California tuberculosis (TB) registry and genotyping data to evaluate trends, analyze epidemiologic differences between adult and child case-patients with Mycobacterium bovis disease, and identify risk factors for M. bovis disease. The percentage of TB cases attributable to M. bovis increased from 3.4% (80/2,384) in 2003 to 5.4% (98/1,808) in 2011 (p = 0.002). All (6/6) child case-patients with M. bovis disease during 2010–2011 had >1 parent/guardian who was born in Mexico, compared with 38% (22/58) of child case-patients with M. tuberculosis disease (p = 0.005). Multivariate analysis of TB case-patients showed Hispanic ethnicity, extrapulmonary disease, diabetes, and immunosuppressive conditions, excluding HIV co-infection, were independently associated with M. bovis disease. Prevention efforts should focus on Hispanic binational families and adults with immunosuppressive conditions. Collection of additional risk factors in the national TB surveillance system and expansion of whole-genome sequencing should be considered. PMID:25693687
Kieser, T; Moss, M T; Dale, J W; Hopwood, D A
The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.
Suleman, Muhammad; Prysliak, Tracy; Windeyer, Claire; Perez-Casal, Jose
Mycoplasma bovis is a pathogen globally affecting cattle and bison herds, causing pneumonia, arthritis, mastitis, abortions, and other symptoms, leading to huge economic losses. Many studies have been done regarding the antimicrobial susceptibility of M. bovis isolated from cattle, but no such study is available for isolates recovered from bison. For the first time, in vitro susceptibilities of 40 M. bovis clinical isolates collected from bison herds in Canada are reported here. Minimal inhibitory concentration (MIC) values were determined using Sensititre® plates. The most effective MIC50 and MIC90 were for spectinomycin (1 and >64 μg/mL), tiamulin (1 and >32 μg/mL), and tulathromycin (16 and 64 μg/mL), whereas tetracyclines, fluoroquinolones, and florfenicol failed to inhibit growth of M. bovis bison isolates. Isolates were nonsusceptible to tetracyclines (100%), fluoroquinolones (97.5%), and tilmicosin (100%), whereas the highest susceptibility of bison clinical isolates was seen with spectinomycin (95%) and tulathromycin (67.5%). Two lung isolates (Mb283 and 348) were found resistant to both spectinomycin and tulathromycin. These results show a marked difference in antimicrobial susceptibility of bison isolates as compared with previously reported and laboratory reference cattle isolates, emphasizing the necessity of testing antimicrobial susceptibility of M. bovis bison isolates and to generate better therapeutic regime for improved recovery chances for infected bison herds across North America.
Register, Karen B; Thole, Luke; Rosenbush, Ricardo F; Minion, F Chris
Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and the bacterium most frequently isolated from the polymicrobial syndrome known as bovine respiratory disease complex. Recently, M. bovis has emerged as a significant health problem in bison, causing necrotic pharyngitis, pneumonia, dystocia and abortion. Whether isolates from cattle and bison comprise genetically distinct populations is unknown. This study describes the development of a highly discriminatory multilocus sequencing typing (MLST) method for M. bovis and its use to investigate the population structure of the bacterium. Genome sequences from six M. bovis isolates were used for selection of gene targets. Seven of 44 housekeeping genes initially evaluated were selected as targets on the basis of sequence variability and distribution within the genome. For each gene target sequence, four to seven alleles could be distinguished that collectively define 32 sequence types (STs) from a collection of 94 cattle isolates and 42 bison isolates. A phylogeny based on concatenated target gene sequences of each isolate revealed that bison isolates are genetically distinct from strains that infect cattle, suggesting recent disease outbreaks in bison may be due to the emergence of unique genetic variants. No correlation was found between ST and disease presentation or geographic origin. MLST data reported here were used to populate a newly created and publicly available, curated database to which researchers can contribute. The MLST scheme and database provide novel tools for exploring the population structure of M. bovis and tracking the evolution and spread of strains.
Dyer, Neil; Register, Karen B; Miskimins, Dale; Newell, Teresa
Mycoplasma bovis has emerged as a significant and costly infectious disease problem in bison, generally presenting as severe, caseonecrotic pneumonia. Three diagnostic cases in which M. bovis is associated with necrotic pharyngitis in bison are described in the current study. The bacterium was isolated from lesions of the pharynx or lung of 3 American bison (Bison bison), at 2 different locations in the upper Midwestern United States, with severe, necrotic pharyngeal abscesses. Chronic caseonecrotic inflammation typical of M. bovis infection in bovines was observed microscopically in the pharynxes of affected bison. A mixed population of bacteria was recovered from the pharyngeal lesions, and Trueperella pyogenes, a frequent secondary pathogen in ruminant respiratory disease, was consistently isolated from the affected animals. Distinctive histopathological features of the pharyngeal lesions favor causation by M. bovis, although a role for T. pyogenes in the clinical presentation cannot be excluded. Veterinarians and producers working with bison should be aware that M. bovis may be associated with pharyngitis in bison.
Malama, Sydney; Johansen, Tone Bjordal; Muma, John Bwalya; Munyeme, Musso; Mbulo, Grace; Muwonge, Adrian; Djønne, Berit; Godfroid, Jacques
Tuberculosis remains a major public health problem in Zambia. While human to human transmission of Mycobacterium tuberculosis is of major importance in driving the tuberculosis epidemic, the impact of Mycobacterium bovis transmission from infected cattle is largely unknown. This cross-sectional study aimed at molecular characterization of M. bovis in humans and cattle. A total of 100 human sputum samples and 67 bovine tissues were collected and analyzed for the presence of mycobacteria. Of 65 human samples that harbored acid fast bacteria (AFB), 55 isolates were obtained of which 34 were identified as M. tuberculosis and 2 as M. bovis. AFB-positive bovine samples (n = 67) yielded 47 mycobacterial isolates among which 25 were identified as M. bovis and no M. tuberculosis was found. Among the M. bovis isolates, spoligotyping revealed a high homogeneity in genotypes circulating in Namwala district. Human and cattle isolates shared identical MIRU-VNTR genotypes, suggesting that transmission between the two hosts may occur. Therefore, this study has documented zoonotic TB in human patients in Namwala district of Zambia. However, further molecular epidemiological studies in the study area are recommended.
Ruhnke, H L; Thawley, D; Nelson, F C
Bovine mastitis caused by Mycoplasma agalactiae subsp. bovis was first diagnosed in 16 of 55 cows in an Ontario herd in Feburary 1972. A total of 182 of 598 (30.4%) cows from 33 of 64 (51.5%) farms in widely separated areas of the province were culturally positive. Herd incidence varied from 15 to 40% with one closed herd having an incidence of 61%. Four herds were investigated culturally and serologically by the growth inhibition test for 15 months. In the acute phase the organism was present in the milk in extremely high numbers and could still be isolated from a few cows after eight to 12 months. The sera from 89.5% of the animals with clinical mycoplasma mastitis produced a zone of surface "film" and/or colony inhibition and some cows remained positive for six to 12 months. The disease was experimentally reproduced with a pure culture of the organism isolated from the milk of a cow from one of the herds. PMID:1000385
Perumaalla, V S; Adams, L G; Payeur, J B; Jarnagin, J L; Baca, D R; Suárez Güemes, F; Ficht, T A
Seventy-nine Mycobacterium bovis isolates recovered from Mexican and Texas cattle were categorized into 16 and 25 distinct types on the basis of IS6110 and direct-repeat fingerprint patterns, respectively. By using a combination of both fingerprint patterns, 30 distinct restriction fragment length polymorphism types were defined. Fifty-eight of 79 isolates (73%) were distributed among nine clusters. Clustered isolates were identified within herds, as well as in geographically disperse herds in Texas and Mexico. This observation is consistent with active transmission within herds and among herds, presumably as a result of active or historical cattle movements. The majority of bovine isolates (64 of 79) exhibited a single copy of IS6110. Interestingly, in contrast to previous studies, a high percentage of bovine isolates (15 of 79) exhibited multiple IS6110 copies (two to five) distributed among 11 different restriction fragment length polymorphism types. It is speculated that transmission from noncattle sources may be responsible. Continued fingerprinting of isolates originating from nonbovine sources and herd surveys is expected to provide useful information regarding the epidemiology of tuberculosis in this region. PMID:8862559
Milian-Suazo, Feliciano; Harris, Beth; Arriaga Díaz, Camila; Romero Torres, Cecilia; Stuber, Tod; Alvarez Ojeda, Genoveva; Morales Loredo, Alberto; Perez Soria, Martina; Payeur, Janet B
Tuberculosis (TB) represents a barrier for free trade of livestock between Mexico and the United States of America (US). In spite of efforts from Mexico to export TB-free animals, some of those found with TB lesions in slaughterhouses in the US are traced back to that country. Therefore, the purpose of this study was to determine, through molecular epidemiology, the most probable source of infection for cattle found with TB lesions in the US. Ninety M. bovis isolates, 50 from Mexico obtained from cattle in 8 different states, and 40 from the US from cattle, deer, elk and feral pigs from 7 different states were included in the study. All samples were analyzed in both laboratories, Mexico and the US, following the same protocol for molecular analysis by spoligotyping. Twenty-seven clusters, ranging from 1 to 18 genetically similar strains were found. Some clustering by country was observed, strains from cattle and deer in Michigan in the US fell into the same cluster, suggesting transmission between species. These results, combined with epidemiological information suggest that despite of the possibility that some animals with lesions in the US come from Mexico as false negatives, the US has its own source of infection, must probably in dairy cattle and wildlife. Genetic diversity of isolates from Mexico was larger than that in the US, which could be a consequence of the endemic status of the disease and the indiscriminate movement of animals between regions.
Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. This report demonstrates that ELISAs for detection of M. bovis-specific antibody in cattle are not optimal for identification of seropositive bison. An ELISA optimized for use with bison sera is ...
Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis from livestock, particularly cattle. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to other deer and cattle. One approach in dealing with this wildlife reservoir is to vaccina...
Numerous species of wild mammals are susceptible to Mycobacterium bovis, a cause of bovine tuberculosis (TB). Eurasian badgers, white-tailed deer, brushtail possums, and wild boar are implicated in the maintenance of wildlife reservoirs of M. bovis infection in different countries, fueling bovine TB...
Mycoplasma bovis causes mastitis, pneumonia and arthritis in cattle and is a major contributor to bovine respiratory disease complex. Recently, it has emerged as a significant respiratory and reproductive health problem in bison. Understanding why M. bovis, known to cause disease in cattle for ove...
Rodwell, Timothy C.; Kapasi, Anokhi J.; Moore, Marisa; Milian-Suazo, Feliciano; Harris, Beth; Guerrero, L.P.; Moser, Kathleen; Strathdee, Steffanie A.; Garfein, Richard S.
Objectives To compare genotypes of Mycobacterium bovis strains from humans in Southern California with genotypes of M. bovis strains in cattle in Mexico and the USA to explore the possible origins of human infections. Methods We conducted a descriptive analysis of M. bovis genotypes from a binational population of humans and cattle using spacer oligonucleotide typing (spoligotyping). Results One hundred six human M. bovis spoligotypes were compared to spoligotypes from 496 Mexican cattle and 219 US cattle. Twelve spoligotype patterns were identified among human cases and 126 spoligotype patterns were detected in cattle. Over 91% (97/106) of the human M. bovis isolates had spoligotypes that were identical to those found in Mexican cattle. Four human cases had spoligotypes that matched both cattle born in Mexico and in the USA. Nine human cases had spoligotypes that did not match cattle born in Mexico or the USA. Conclusions Our data indicate that the population of M. bovis strains causing human TB disease in Southern California is closely related to the M. bovis strain population found in Mexican cattle and supports existing epidemiological evidence that human M. bovis disease in San Diego likely originated from Mexican cattle. PMID:20399697
Completion of the Babesia bovis (T2Bo strain) genome provides detailed data concerning the predicted proteome of this parasite, and allows for a bioinformatics approach to gene discovery. Comparative genomics of the hemoprotozoan parasites B. bovis and Theileria parva revealed a highly conserved syn...
Bovicin HC5, a broad spectrum lantibiotic produced by Streptococcus bovis HC5, catalyzed the efflux of intracellular potassium from Streptococcus bovis JB1, a sensitive strain. ATP also decreased, but this decline appeared to be caused by the activity of the F1FO ATPase rather than efflux per se....
Brooks, B W; Barnum, D A
Twenty bovine udder quarters colonized with Corynebacterium bovis SR6 and 20 uncolonized quarters were challenged by inoculation of Staphylococcus aureus Newbould 305 (ATCC 29740) into the teat cistern. The percentage of infection in quarters colonized with C. bovis (50%) was significantly lower than that in controls (100%). By similar challenge no significant difference was observed between the percentage of infection with Streptococcus agalactiae ATCC 27956 in 33 quarters colonized with C. bovis (70%) compared to 33 controls (87.9%). A total of 37 quarters colonized with C. bovis and 37 control quarters were challenged with Staph. aureus Newbould 305 (ATCC 29740) and Maxi (ATCC 27543) and Strep. agalactiae (ATCC 27956) by exposure of the teat orifice. The percentage of teat ducts colonized with C. bovis which became infected with either pathogen was not different from that for controls. PMID:6372969
Coenzyme Q1 is herein proposed as the best catalyst among coenzymes Q and vitamins K for quinone-catalyzed luminol chemiluminescent assays applied to rapid determination of viability or rapid antimicrobial susceptibility tests of Mycobacterium bovis. Luminol chemiluminescence intensity (LCI) was determined 10 min after the incubation of M. bovis with coenzyme Q1, and was proportional to CFU (colony-forming unit)/ml in the range of 9,000 to 2,250,000. LCI depended on the the production of the superoxide anion (O2-) rather than H2O2 during a 10-min incubation of M. bovis with coenzyme Q1, as superoxide dismutase reduced LCI more effectively than catalase. The minimal inhibitory concentrations (MICs) of 10 kinds of antituberculous agents estimated on the basis of decrease in LCI after one or two days' cultivation were in good agreement with MICs determined by turbidity analysis, which requires upwards of 1 week to complete.
Stable luminol chemiluminescence was observed 10 min after the addition of menadione to a suspension of Mycobacterium bovis homogenized in Middlebrook 7H9 broth base including OADC enrichment. The chemiluminescence intensity was proportional to the absorbance of the bacterial suspension at 600 nm in a range of 0.005 to 0.15. Luminol chemiluminescence disappeared after 10 min incubation of M. bovis at over 60% of ethanol or 4 days of cultivation of M. bovis in the presence of 40 microg/ml of streptomycin. The bacterium showing the disappearance of chemiluminescence could not grow after being washed, suggesting that the inhibition concentration of the antimicrobials can be estimated on the basis of the disappearance of chemiluminescence. Menadione-catalyzed luminol chemiluminescent assay was rapid and sensitive in comparison to turbidimetry, tetrazolium (WST-8) reduction assay, and the assay using the Mycobacteria growth indicator tube (MGIT).
Kakuda, Tsutomu; Sarataphan, Nopporn; Tanaka, Tetsuya; Takai, Shinji
The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.
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
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.
Nakamura, Mutsumi; Nagamine, Takafumi; Harada, Chisato; Tajima, Kiyoshi; Matsui, Hiroki; Benno, Yoshimi
The objective of this study was to ligate the xylanase gene A ( xynA) isolated from Ruminococcus albus 7 into the promoter and signal-peptide region of the lichenase [beta-(1,3-1,4)-glucanase] gene of Streptococcus bovis JB1. This fusion gene was inserted into the pSBE11 vector, and the resulting recombinant, plasmid pXA, was used to transform S. bovis 12-U-1 cells. The transformant, S. bovis 12UXA, secreted the xylanase, which was stable against freeze-thaw treatment and long-time incubation at 37 degrees C. The introduction of pXA and production of xylanase did not affect cell growth, and the xylanase produced degraded xylan from oat-spelt and birchwood.
Aljada, Ibrahim S.; Crane, John K.; Corriere, Nancy; Wagle, Datta G.; Amsterdam, Daniel
We report a case of Mycobacterium bovis BCG vertebral osteomyelitis in a 79-year-old man 2.5 years after intravesical BCG therapy for bladder cancer. The recovered isolate resembled M. tuberculosis biochemically, but resistance to pyrazinamide (PZA) rendered that diagnosis suspect. High-pressure liquid chromatographic studies confirmed the diagnosis of M. bovis BCG infection. The patient was originally started on a four-drug antituberculous regimen of isoniazid, rifampin, ethambutol, and PZA. When susceptibility studies were reported, the regimen was changed to isoniazid and rifampin for 12 months. Subsequently, the patient was transferred to a skilled nursing facility for 3 months, where he underwent intensive physical therapy. Although extravesical adverse reactions are rare, clinicians and clinical microbiologists need to be aware of the possibility of disseminated infection by M. bovis BCG in the appropriate setting of clinical history, physical examination, and laboratory investigation. PMID:10325395
Parra, A; García, A; Inglis, N F; Tato, A; Alonso, J M; Hermoso de Mendoza, M; Hermoso de Mendoza, J; Larrasa, J
Recreational hunting of indigenous wild artiodactyls has been one of the most lucrative and rapidly growing industries in Western Spain over the last five years. In the absence of careful ecological management, one consequence of the commercial exploitation of this natural resource has been the appearance of outbreaks of infectious disease; most notably bovine tuberculosis. From the outset of the study in 1997, we have observed a steady increase in prevalence of Mycobacterium bovis (M. bovis) in both species reaching 1.74 (+/-0.17) in deer in 2002 and 2.32 (+/-0.24) in wild boar. The latter species seems to be most severely affected with pulmonary lesions appearing more chronic than those observed in deer. In this study, we describe the epidemiology of M. bovis in European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus) in Extremadura (W. Spain); a region where there are large areas of natural habitat for these species.
Hermosilla, Carlos; Schröpfer, Elmar; Stowasser, Michael; Eckstein-Ludwig, Ursula; Behrendt, Jan Hillern; Zahner, Horst
The first merogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macromeronts up to 250 microm. In this study, we investigated the host cell cytoskeleton (actin filaments, microtubules, spectrin, vimentin intermediate filaments) associated with parasitic development in vitro by confocal laser scanning microscopy (CLSM) using primary bovine umbilical vein endothelial cells (BUVEC) and bovine spleen lymphatic endothelial cells (BSLEC) as host cells. No prominent changes in the host cell cytoskeleton were detected 1-3 days after E. bovis sporozoite invasion. With ongoing meront maturation a significant increase in microtubules and actin filaments close to the parasitophorous vacuole (PV) was found. Mature macromeronts within the PV were completely enclosed by these cytoskeletal elements. Our findings suggest, that in order to guarantee the survival of the host cell on the enlargement of macromeronts, E. bovis needs not only to augment but also to rearrange its cytoskeletal system.
Behrendt, J H; Clauss, W; Zahner, H; Hermosilla, C
In vitro Eimeria bovis sporozoites invade a wide range of cell types, and in the case of bovine cells, they may develop to first-generation schizonts. Often, however, they subsequently leave their host cell to invade a new one, which seems contrary to the classical way of infecting a cell by forming a parasitophorous vacuole. Using a standard, "cell wound assay," we show that E. bovis can invade bovine endothelial cells by breaching the plasma membrane and may again leave the surviving cell. Eimeria bovis sporozoites also infected VERO and HT29 cells but obviously without damaging the plasma membrane. The same held true when bovine endothelial cells were exposed to tachyzoites of Toxoplasma gondii and Neospora caninum. According to a literature report dealing with Plasmodium yoelii sporozoites, breaching the membrane of certain host cells may be a common phenomenon for coccidian sporozoites but may not be for merozoites.
Skuce, R A; Brittain, D; Hughes, M S; Beck, L A; Neill, S D
Two insertion sequences, IS6110 and IS1081, specific to the tuberculosis complex mycobacteria and a highly reiterated DNA element (pTBN12) cloned from Mycobacterium tuberculosis were systematically used to identify restriction fragment length polymorphism (RFLP) types among bovine isolates of Mycobacterium bovis in Northern Ireland. In a sample of 109 isolates, probes IS6110, IS1081, and pTBN12 identified 10, 2, and 12 distinct patterns, respectively. By combining the patterns generated by the three probes it was possible to identify 28 distinct RFLP types. The standard protocol advocated for RFLP analysis of M. tuberculosis was used and would facilitate computer-based gel documentation and image analysis to establish a database of M. bovis types for large-scale epidemiological studies. These procedures will facilitate interlaboratory comparisons of M. bovis isolates and will help to elucidate the precise epidemiology of bovine tuberculosis in different countries. Images PMID:7814471
MOSSAAD, Ehab; ASADA, Masahito; NAKATANI, Daichi; INOUE, Noboru; YOKOYAMA, Naoaki; KANEKO, Osamu; KAWAZU, Shin-ichiro
Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca2+ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca2+ concentration in the cytosol of the parasite cells. The increased intracellular Ca2+ concentration following these treatments was confirmed using live cell Ca2+ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca2+ signalling pathway in the egress of B. bovis merozoites. PMID:25298241
Gross, W M; Hawkins, J E
In the context of a busy reference laboratory, radiometric selective inhibition tests were evaluated for rapid differentiation of Mycobacterium tuberculosis and Mycobacterium bovis and of the M. tuberculosis complex from other mycobacteria. p-Nitro-alpha-acetylamino-beta-hydroxypropiophenone at 5 micrograms and hydroxylamine hydrochloride at 62.5 and 125 micrograms per ml of 7H12 medium were used to separate the M. tuberculosis complex from other mycobacteria (MOTT bacilli). Since it is important epidemiologically to distinguish M. tuberculosis from M. bovis, susceptibility to 1 microgram of thiophene-2-carboxylic acid per ml was also determined radiometrically. By using these three agents as selective inhibitors, M. tuberculosis, M. bovis, and MOTT bacilli were differentiated with a high degree of specificity by a BACTEC radiometric procedure. Results of tests performed on clinical isolates submitted on solid medium to our reference laboratory were available within 5 days. PMID:3921561
Gómez-Camarasa, Cristina; Gutiérrez Soto, Blanca; Jiménez-Guerra, Gemma; Sorlózano Puerto, Antonio; Navarro-Marí, José María; Gutiérrez-Fernández, José
Given the relevance of proper clinical validation of Streptococcus bovis, we here consider revising its presence in urine samples in order to determine its relative frequency and the pattern of antibiotic susceptibility. The susceptibility to antibiotics of 91 isolates of S. bovis from urine samples was retrospectively reviewed over a period of 4 years (2012-2015). The mean age of patients was 55 years, 81% of whom were women and 37.4% were hospitalized patients suffering from urological diseases (61%). Susceptibility to penicillin, vancomycin and teicoplanin was 97.8%. Due to the fact that S. bovis can be infrequent in urine isolates and given its presence in patients suffering from urological diseases, further pathogenic studies, showing the true ability of this group of bacteria to produce disease, are required. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.
Hart, Bryan E.; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D.; Lukose, Regy; Souther, Sommer J. R.; Rayasam, Swati D. G.; Saelens, Joseph W.; Chen, Ching-ju; Seay, Sarah A.; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E.; Ng, Tony W.; Tobin, David M.; Porcelli, Steven A.; Larsen, Michelle H.; Schmitz, Joern E.; Haynes, Barton F.; Jacobs, William R.; Lee, Sunhee
The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766
McMurray, D N; Mintzer, C L; Bartow, R A; Parr, R L
Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated. They were maintained for 6 weeks on defined, isocaloric diets containing either 30% (control animals) or 10% (animals receiving low protein) ovalbumin as the sole protein source. Animals were challenged by the respiratory route with a low dose of virulent M. tuberculosis H37Rv and killed 4 weeks later. Protein-malnourished animals were not protected by previous vaccination with BCG. Lymphocytes isolated from various tissues were tested in vitro for proliferative responses to mitogen (concanavalin A) and antigen (purified protein derivative [PPD]), production of interleukin-2 (IL-2), and response to exogenous recombinant IL-2 (rIL-2). Protein-malnourished guinea pigs responded only weakly to PPD skin tests, and their blood and lymph node lymphocytes exhibited impaired proliferation when cultured with PPD in vitro. IL-2 levels were consistently low in cultures of stimulated blood and spleen lymphocytes from protein-deprived animals. BCG vaccination of nutritionally normal guinea pigs, on the other hand, induced significantly more IL-2 production by PPD- and concanavalin A-stimulated lymphocytes. The addition of exogenous mouse rIL-2 (40 and 80 U/ml) in vitro to PPD-stimulated blood and lymph node cells from nonvaccinated, protein-deprived guinea pigs resulted in no improvement of the proliferative response. Previous vaccination of malnourished guinea pigs did not consistently enhance the response of PPD-stimulated lymphocytes to added rIL-2. Dietary protein deficiency and BCG vaccination appear to modulate antigen-driven cellular immunity in animals with tuberculosis by altering the production of, and the response to, IL-2 by PPD-stimulated lymphocytes. PMID:2788135
Corredoira, J; Alonso, M P; Coira, A; Casariego, E; Arias, C; Alonso, D; Pita, J; Rodriguez, A; López, M J; Varela, J
The purpose of this study was to evaluate the characteristics of infective endocarditis (IE) caused by S. bovis and compare them to those caused by streptococci of the viridans group (SVG). A prospective study was undertaken considering 55 consecutive cases of IE due to S. bovis and 41 to SVG over 18 years. The study was divided into two periods (1988-1996 and 1997-2005). S. bovis caused 24% of the IE in our centre and constituted the main aetiology for this disease, showing an increase of 358% during the second period studied. Biotype I was responsible for 94.5% of cases and there was a high degree of association with colon tumours (53%). Over the period of the study, 107 patients admitted to our hospital had bacteraemia caused by S. bovis and 310 patients had bacteraemia caused by SVG. In the first group, 55 (51%) were endocarditis cases, but only 41 (13%) of the patients with SVG bacteraemia had endocarditis (p < 0.0001). The distinguishing features of endocarditis caused by S. bovis in comparison with those caused by SGV were: a greater increase in cases during the 2nd period studied (from 12 to 43 vs. from 19 to 22, p < 0.01), a higher percentage of males (93% vs. 71%, p < 0.004), patients significantly older (median age 66 vs. 58.5, p < 0.004), less predisposing cardiopathy (42% vs. 76%, p < 0.0009), more bivalvular involvement (42% vs. 22%, p < 0.04), more spondylitis (9% vs. 0%, p < 0.04), a higher association with colonic tumours (53% vs. 5%, p < 0.0001), and a higher percentage of antibiotic resistance: erythromycin 66% vs. 19%, p < 0.0001; clindamycin 67% vs. 11%, p < 0.0001; cotrimoxazole 77% vs. 30.5%, p < 0.0001, respectively. IE due to S. bovis is an emergent disease in our environment, presenting different characteristics to those produced by SVG.
Esquivel-Solís, Hugo; Vallecillo, Antonio J.; Benítez-Guzmán, Alejandro; Adams, L. Garry; López-Vidal, Yolanda; Gutiérrez-Pabello, José A.
To identify the resistance phenotype against Mycobacterium bovis in cattle, we used a bactericidal assay that has been considered a marker of this trait. Three of 24 cows (12.5%) were phenotyped as resistant and 21 as susceptible. Resistance of bovine macrophages (MΦ) to BCG challenge was evaluated for its association with SLC11A1 GT microsatellite polymorphisms within 3′UTR region. Twenty-three cows (95.8%) had a GT13 genotype, reported as resistant, consequently the SLC11A1polymorphism was not in agreement with our bactericidal assay results. MΦ of cows with resistant or susceptible phenotype were challenged in vitro with virulent M. bovis field strain or BCG, and nitric oxide production, bacterial killing and apoptosis induction were measured in resting and LPS-primed states. M. bovis field strain induced more apoptosis than BCG, although the difference was not significant. Resistant MΦ controlled better the replication of M. bovis (P<0.01), produced more nitric oxide (P<0.05) and were slightly more prone to undergo apoptosis than susceptible cells. LPS pretreatment of MΦ enhanced all the functional parameters analyzed. Inhibition of nitric oxide production with nG-monomethyl-L-arginine monoacetate enhanced replication of M. bovis but did not modify apoptosis rates in both resistant and susceptible MΦ. We conclude that nitric oxide production not apoptosis is a major determinant of macrophage resistance to M. bovis infection in cattle and that the influence of SLC11A1 gene 3′UTR polymorphism is not associated with this event. PMID:23691050
Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki
Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand.
Teng, Lee-Jene; Hsueh, Po-Ren; Ho, Shen-Wu; Luh, Kwen-Tay
Susceptibilities to 13 antimicrobial agents were determined by measurement of MICs for 60 isolates of Streptococcus bovis from blood cultures. Thirty-eight isolates (63.3%) had high-level resistance to erythromycin (MICs, ≥128 μg/ml). Among the 38 erythromycin-resistant strains, 21 isolates (55%) had inducible resistance to macrolides-lincosamides-streptogramin B (iMLS isolates) and 17 (45%) had constitutive resistance to macrolides-lincosamides-streptogramin B (cMLS isolates). Tetracycline resistance was also found among all of the erythromycin-resistant strains. None of the strains displayed resistance to penicillin, chloramphenicol, or vancomycin. Detection of erythromycin resistance genes by PCR and sequencing indicated that all 17 cMLS isolates were positive for the ermB gene and that 7 of 21 iMLS isolates carried the ermB gene and the remaining 14 iMLS isolates carried the ermT gene. Sequence analysis of amplified partial ermB fragments (594 bp) from S. bovis isolates revealed a 99.8% nucleotide identity and a 100% amino acid homology compared with the sequences from gene banks. The sequences of amplified fragments with primers targeted for ermC were shown to be very similar to that of ermGT (ermT) from Lactobacillus reuteri (98.5% nucleotide identity). This is the first report to describe the detection of the ermT class of erythromycin resistance determinants in S. bovis. The high rate of inducible erythromycin resistance among S. bovis isolates in Taiwan was not reported before. The iMLS S. bovis isolates were shown to be heterogeneous by randomly amplified polymorphic DNA analysis. These results indicate that the prevalence of inducible erythromycin resistance in S. bovis in Taiwan is very high and that most of the resistant strains carry the ermT or the ermB gene. PMID:11709309
Munkhjargal, Tserendorj; Ishizaki, Takahiro; Guswanto, Azirwan; Takemae, Hitoshi; Yokoyama, Naoaki; Igarashi, Ikuo
Aminopeptidases are increasingly being investigated as therapeutic targets in various diseases. In this study, we cloned, expressed, and biochemically characterized a member of the methionine aminopeptidase (MAP) family from Babesia bovis (B. bovis) to develop a potential molecular drug target. Recombinant B. bovis MAP (rBvMAP) was expressed in Escherichia coli (E. coli) as a glutathione S-transferase (GST)-fusion protein, and we found that it was antigenic. An antiserum against the rBvMAP protein was generated in mice, and then a native B. bovis MAP was identified in B. bovis by Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of the B. bovis merozoite. Analysis of the biochemical properties of rBvMAP revealed that it was enzymatically active, with optimum activity at pH 7.5. Enhanced enzymatic activity was observed in the presence of divalent manganese cations and was effectively inhibited by a metal chelator, ethylenediaminetetraacetic acid (EDTA). Moreover, the enzymatic activity of BvMAP was inhibited by amastatin and bestatin as inhibitors of MAP (MAPi) in a dose-dependent manner. Importantly, MAPi was also found to significantly inhibit the growth of Babesia parasites both in vitro and in vivo; additionally, they induced high levels of cytokines and immunoglobulin (IgG) titers in the host. Therefore, our results suggest that BvMAP is a molecular target of amastatin and bestatin, and those inhibitors may be drug candidates for the treatment of babesiosis, though more studies are required to confirm this.
Cao, Shinuo; Aboge, Gabriel Oluga; Terkawi, Mohamad Alaa; Yu, Longzheng; Kamyingkird, Ketsarin; Luo, Yuzi; Li, Yan; Goo, Youn-Kyoung; Yamagishi, Junya; Nishikawa, Yoshifumi; Yokoyama, Naoaki; Suzuki, Hiroshi; Igarashi, Ikuo; Maeda, Ryuichiro; Inpankaew, Tawin; Jittapalapong, Sathaporn; Xuan, Xuenan
Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.
Hamid, Penny H; Hirzmann, Joerg; Kerner, Katharina; Gimpl, Gerald; Lochnit, Guenter; Hermosilla, Carlos R; Taubert, Anja
During first merogony Eimeria bovis forms large macromeronts in endothelial host cells containing >120 000 merozoites I. During multiplication, large amounts of cholesterol are indispensable for the enormous offspring membrane production. Cholesterol auxotrophy was proven for other apicomplexan parasites. Consequently they scavenge cholesterol from their host cell apparently in a parasite-specific manner. We here analyzed the influence of E. bovis infection on endothelial host cell cholesterol metabolism and found considerable differences to other coccidian parasites. Overall, free cholesterol significantly accumulated in E. bovis infected host cells. Furthermore, a striking increase of lipid droplet formation was observed within immature macromeronts. Artificial host cell lipid droplet enrichment significantly improved E. bovis merozoite I production confirming the key role of lipid droplet contents for optimal parasite proliferation. The transcription of several genes being involved in both, cholesterol de novo biosynthesis and low density lipoprotein-(LDL) mediated uptake, was significantly up-regulated at a time in infected cells suggesting a simultaneous exploitation of these two cholesterol acquisition pathways. E. bovis scavenges LDL-derived cholesterol apparently through significantly increased levels of surface LDL receptor abundance and LDL binding to infected cells. Consequently, LDL supplementation significantly improved parasite replication. The up-regulation of the oxidized LDL receptor 1 furthermore identified this scavenger receptor as a key molecule in parasite-triggered LDL uptake. Moreover, cellular cholesterol processing was altered in infected cells as indicated by up-regulation of cholesterol-25-hydroxylase and sterol O-acyltransferase. Overall, these results show that E. bovis considerably exploits the host cell cholesterol metabolism to guarantee its massive intracellular growth and replication.
Esquivel-Solís, Hugo; Vallecillo, Antonio J; Benítez-Guzmán, Alejandro; Adams, L Garry; López-Vidal, Yolanda; Gutiérrez-Pabello, José A
To identify the resistance phenotype against Mycobacterium bovis in cattle, we used a bactericidal assay that has been considered a marker of this trait. Three of 24 cows (12.5%) were phenotyped as resistant and 21 as susceptible. Resistance of bovine macrophages (MΦ) to BCG challenge was evaluated for its association with SLC11A1 GT microsatellite polymorphisms within 3'UTR region. Twenty-three cows (95.8%) had a GT13 genotype, reported as resistant, consequently the SLC11A1 polymorphism was not in agreement with our bactericidal assay results. MΦ of cows with resistant or susceptible phenotype were challenged in vitro with virulent M. bovis field strain or BCG, and nitric oxide production, bacterial killing and apoptosis induction were measured in resting and LPS-primed states. M. bovis field strain induced more apoptosis than BCG, although the difference was not significant. Resistant MΦ controlled better the replication of M. bovis (P<0.01), produced more nitric oxide (P<0.05) and were slightly more prone to undergo apoptosis than susceptible cells. LPS pretreatment of MΦ enhanced all the functional parameters analyzed. Inhibition of nitric oxide production with n (G)-monomethyl-L-arginine monoacetate enhanced replication of M. bovis but did not modify apoptosis rates in both resistant and susceptible MΦ. We conclude that nitric oxide production not apoptosis is a major determinant of macrophage resistance to M. bovis infection in cattle and that the influence of SLC11A1 gene 3'UTR polymorphism is not associated with this event.
Zumárraga, Martín J.; Martin, Carlos; Samper, Sofia; Alito, Alicia; Latini, Omar; Bigi, Fabiana; Roxo, Eliana; Cicuta, María Elena; Errico, Francisco; Ramos, Miguel Castro; Cataldi, Angel; van Soolingen, Dick; Romano, María Isabel
Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while AluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovis isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes. PMID:9889207
Gutiérrez-Pabello, José A.; McMurray, David N.; Adams, L. Garry
Bovine macrophages underwent apoptosis as a result of infection with a Mycobacterium bovis field strain. Macrophages infected with a multiplicity of infection (MOI) of 25:1 developed chromatin condensation and DNA fragmentation at 4 h and 8 h, respectively, whereas changes in chromatin condensation induced by MOIs of 10:1 and 1:1 required more time and had a reduced number of apoptotic cells. Not only infected macrophages underwent apoptosis, but also uninfected bystander macrophages became apoptotic. Increased differential expression of thymosin β-10 was identified in M. bovis-infected bovine macrophages by differential display reverse transcriptase PCR. Phagocytosis of latex beads had no effect on the expression of thymosin β-10, whereas bacterial suspensions upregulated thymosin β-10 expression, suggesting that M. bovis or mycobacterial products are essential in the process. Heat-inactivated M. bovis induced a slight increase in thymosin β-10 mRNA, whereas live virulent and attenuated M. bovis organisms increased the gene expression almost twofold. A mouse macrophage cell line (RAW 264.7) overexpressing the bovine thymosin β-10 transgene had spontaneous apoptosis at a higher rate (66.5%) than parental cells (4.7%) or RAW cells harboring the empty vector (22.8%). The apoptotic rates of the overexpressing cells were significantly higher when compared with both the empty vector transfected (P < 0.01) and parental cells (P < 0.001). Our evidence suggests that upregulation of thymosin β-10 in M. bovis-infected macrophages is linked with increased cell death due to apoptosis. PMID:11895978
Wilkinson, S; Bishop, S C; Allen, A R; McBride, S H; Skuce, R A; Bermingham, M; Woolliams, J A; Glass, E J
Susceptibility to Mycobacterium bovis infection in cattle is governed in part by host genetics. However, cattle diagnosed as infected with M. bovis display varying signs of pathology. The variation in host response to infection could represent a continuum since time of exposure or distinct outcomes due to differing pathogen handling. The relationships between host genetics and variation in host response and pathological sequelae following M. bovis infection were explored by genotyping 1966 Holstein-Friesian dairy cows at 538,231 SNPs with three distinct phenotypes. These were: single intradermal cervical comparative tuberculin (SICCT) test positives with visible lesions (VLs), SICCT-positives with undetected visible lesions (NVLs) and matched controls SICCT-negative on multiple occasions. Regional heritability mapping identified three loci associated with the NVL phenotype on chromosomes 17, 22 and 23, distinct to the region on chromosome 13 associated with the VL phenotype. The region on chromosome 23 was at genome-wide significance and candidate genes overlapping the mapped window included members of the bovine leukocyte antigen class IIb region, a complex known for its role in immunity and disease resistance. Chromosome heritability analysis attributed variance to six and thirteen chromosomes for the VL and NVL phenotypes, respectively, and four of these chromosomes were found to explain a proportion of the phenotypic variation for both the VL and NVL phenotype. By grouping the M. bovis outcomes (VLs and NVLs) variance was attributed to nine chromosomes. When contrasting the two M. bovis infection outcomes (VLs vs NVLs) nine chromosomes were found to harbour heritable variation. Regardless of the case phenotype under investigation, chromosome heritability did not exceed 8% indicating that the genetic control of bTB resistance consists of variants of small to moderate effect situated across many chromosomes of the bovine genome. These findings suggest the host
de la Torre-Escudero, Eduardo; Pérez-Sánchez, Ricardo; Manzano-Román, Raúl; Oleaga, Ana
Schistosoma bovis is a blood-dwelling fluke of ruminants that lives for years inside the vasculature of their hosts. The parasite tegument covers the surface of the worms and plays a key role in the host-parasite relationship. The parasite molecules expressed at the tegument surface are potential targets for immune or drug intervention. The purpose of this work was the identification of the proteins expressed in vivo on the surface of the tegument of S. bovis adult worms. To accomplish this we used a method based on in vivo vascular perfusion of mice infected with S. bovis which allowed the labelling of the surface of the worms inside the blood vasculature. The biotinylation of parasite inside blood vessels prevents the handling of worms in vitro and hence possible damage to the tegument that could produce results that would be difficult to interpret. Trypsin digestion of biotinylated proteins and subsequent liquid chromatography and tandem mass spectrometry analysis (LC-MS/MS) resulted in the identification on the S. bovis tegument of 80 parasite proteins and 28 host proteins. The proteins identified were compared with the findings from other proteomic studies of the schistosome surface. The experimental approach used in this work is a reliable method for selective investigation of the surface of the worms and provides valuable information about the exposed protein repertoire of the tegument of S. bovis in the environmental conditions that the parasite faces inside the blood vessels. To identify the proteins expressed on the surface of the tegument of S. bovis adult worms we used a method based on in vivo vascular perfusion, with biotin, of mice infected with S. bovis which allowed the labelling of the surface of the worms inside the blood vasculature. This methodology prevents the handling of worms in vitro and hence possible damage to the tegument that could produce results that would be difficult to interpret. This work is the first in which vascular perfusion
Lavin, S; Lastras, M E; Marco, I; Cabañes, F X
A case of fibrinopurulent bronchopneumonia associated with Moraxella bovis infection in a chamois (Rupicapra pyrenaica) is described. The animal, a 4-month-old female, was referred by the staff warden of the National Game Reserve of Freser-Setcases (Catalonia, north-eastern Spain). The animal was in good general condition and was found 4 h before death. On necropsy the lungs were congested and oedematous, with haemorrhagic areas in the cranial and middle lobes. The microscopic lesions were those of a fibrinopurulent bronchopneumonia. Microbiological study of the samples obtained showed numerous small beta-haemolytic colonies in pure culture, identified as Moraxella (Moraxella) bovis.
Murakami, Patricia Sayuri; Monego, Fernanda; Ho, John L; Gibson, Andrea; Vilani, Ricardo Guilherme D'Otaviano de Castro; Soresini, Grazielle Cristina Garcia; Brockelt, Sonia Regina; Biesdorf, Sonia Maria; Fuverki, Renata Benicio Neves; Nakatani, Sueli Massumi; Riediger, Irina Nastassja; Grazziotin, Ana Laura; do Santos, Andrea Pires; de Barros Filho, Ivan Roque; Biondo, Alexander Welker
Mycobacterium tuberculosis complex, which includes Mycobacterium bovis, infrequently causes severe or lethal disease in captive wildlife populations. A dead coati from a wildlife triage center showing pulmonary lesions compatible with tuberculosis had raised suspicion of a potential disease caused by mycobacteria species and was further investigated. Four native coatis (Nasua nasua) with suspected mycobacterial infection were sedated, and bronchoalveolar lavages and tuberculin skin tests (TSTs) were performed. All animals tested positive upon TST. Mycobacterial culturing, Ziehl-Neelsen staining, and genetic testing were performed on postmortem samples and the etiologic agent was identified as M. bovis. Molecular genetic identification using a polymerase chain reaction panel was crucial to achieving a definitive diagnosis.
Çavuşoğlu, Cengiz; Edeer Karaca, Neslihan; Azarsız, Elif; Ulusoy, Ezgi; Kütükçüler, Necil
It is well known that disseminated Mycobacterium bovis BCG infection is developed after BCG vaccination in infants with congenital cellular immune deficiencies such as mutations in genes along the interleukin (IL)-12/interferon (IFN)-γ pathway and mutations in nuclear factor-kB essential modulator (NEMO). In this report, a rifampicin-resistant M.bovis BCG strain isolated from an infant with NEMO defect was presented. An 8-month-old male infant with NEMO defect admitted to the pediatric outpatient clinic of our hospital with fever, generalized lymphadenopathy and hepatosplenomegaly. Microscopic examination of the smears prepared from lymph node and liver biopsy specimens revealed abundant amount (3+) of acid-fast bacilli (AFB). Rifampicin-susceptible Mycobacterium tuberculosis complex (MTC) was detected by real-time PCR (GeneXpert MTB/RIF; Cepheid, USA) in the samples. The growth of mycobacteria was determined on the 20th day of culture performed in MGIT960 system (Becton Dickinson, USA). The isolate was identified as M.bovis BCG by GenoType MTBC kit (Hain Lifescience, Germany) and defined as M.bovis BCG [SIT 482 (BOV_1)] by spoligotyping. In the primary anti-tuberculosis drug susceptibility test performed by MGIT960 system, the isolate was found susceptible to rifampicin (RIF), isoniazid (INH), streptomycin (STM) and ethambutol (EMB). Then anti-tuberculosis treatment was started to the patient. However, the patient at the age of 2 years, re-admitted to the hospital with the complaint of hepatosplenomegaly. Smear of spontaneously draining abscess material obtained from subcutaneous nodules revealed intensive AFB positivity (3+) once again. In the present instance RIF-resistant MTC was detected with GeneXpert system in the specimen. The growth of mycobacteria was determined on the 13th day of culture and isolate was identified as M.bovis BCG. The present isolate was found susceptible to INH, STM and EMB but resistant to RIF. A mutation in the rpoB gene (codon 531, S
In view of the considerable and continuing public health significance of Mycobacterium bovis infection in humans and animals, WHO convened a meeting on zoonotic tuberculosis in Geneva in November 1993. The participants at the meeting reviewed the human and animal tuberculosis situation worldwide, discussed the zoonotic aspects of M. bovis infection in United Republic of Tanzania and Zambia, exchanged views on methodologies in epidemiology, immunology and molecular biology, and identified areas for further research and intersectoral collaboration. A project protocol to investigate the zoonotic aspects of bovine tuberculosis was elaborated by the group and included in their report. This Memorandum is a summary of the full report of the meeting. PMID:7867130
Hantsis-Zacharov, Elionora; Senderovich, Yigal; Halpern, Malka
Three Gram-negative, rod-shaped, oxidase-positive, aerobic, non-motile bacterial strains, designated H9T, H10 and H15, were isolated during a study on the diversity of culturable psychrotolerant bacteria in raw cow's milk. Comparisons of 16S rRNA gene sequences showed that the three strains were very closely related to each other (sequence similarities of 99.6-99.8 %). A polyphasic taxonomic study of the isolates resulted in their identification as members of the genus Chryseobacterium (family Flavobacteriaceae, phylum Bacteroidetes). The three strains showed < or =96.9 % sequence similarity with respect to the type strains of described Chryseobacterium species, indicating that H9T, H10 and H15 represent a novel species of the genus Chryseobacterium. The three strains grew at 7-37 degrees C (strain H10 grew at up to 41 degrees C), with 0-2.5 % NaCl and at pH 5.0-9.8. The dominant cellular fatty acids of strain H9T were 15 : 0 iso (38.9 %), 15 : 0 anteiso (15.6 %) and 17 : 0 iso 3-OH (12.7 %). Strain H10 also possessed 17 : 1 iso omega 9c (14.8 %) as a major fatty acid. On the basis of phenotypic properties and phylogenetic distinctiveness, the three milk isolates represent a novel species in the genus Chryseobacterium, for which the name Chryseobacterium bovis sp. nov. is proposed. The type strain is H9T (=LMG 24227T =DSM 19482T).
Doherty, Rachael; Whiston, Ronan; Cormican, Paul; Finlay, Emma K.; Couldrey, Christine; Brady, Colm; O’Farrelly, Cliona; Meade, Kieran G.
We hypothesised that epigenetic regulation of CD4+ T lymphocytes contributes to a shift toward a dysfunctional T cell phenotype which may impact on their ability to clear mycobacterial infection. Combined RNA-seq transcriptomic profiling and Reduced Representation Bisulfite Sequencing identified 193 significantly differentially expressed genes and 760 differentially methylated regions (DMRs), between CD4+ T cells from M. bovis infected and healthy cattle. 196 DMRs were located within 10 kb of annotated genes, including GATA3 and RORC, both of which encode transcription factors that promote TH2 and TH17 T helper cell subsets respectively. Gene-specific DNA methylation and gene expression levels for the TNFRSF4 and Interferon-γ genes were significantly negatively correlated suggesting a regulatory relationship. Pathway analysis of DMRs identified enrichment of genes involved in the anti-proliferative TGF-β signaling pathway and TGFB1 expression was significantly increased in peripheral blood leukocytes from TB-infected cattle. This first analysis of the bovine CD4+ T cell methylome suggests that DNA methylation directly contributes to a distinct gene expression signature in CD4+ T cells from cattle infected with M. bovis. Specific methylation changes proximal to key inflammatory gene loci may be critical to the emergence of a non-protective CD4+ T cell response during mycobacterial infection in cattle. PMID:27507428
Activity of rifapentine and its metabolite 25-O-desacetylrifapentine compared with rifampicin and rifabutin against Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis and M. bovis BCG.
Rastogi, N; Goh, K S; Berchel, M; Bryskier, A
The in vitro activity of rifapentine and its metabolite, 25-O:-desacetylrifapentine, as compared with that of rifampicin and rifabutin, was determined against Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis and M. bovis BCG. MICs were determined radiometrically and by the 1% proportional method using Middlebrook 7H11 agar. The bactericidal effect of the drugs was determined in parallel at selected concentrations. For drugsusceptible isolates of M. tuberculosis, the Bactec MICs of rifapentine and 25-O:-desacetylrifapentine were 0.03-0.06 mg/L and 0. 125-0.25 mg/L, respectively. Similar MICs were obtained for M. africanum (0.03-0.125 and 0.125-0.50 mg/L, respectively), and M. bovis (0.063-0.25 and 0.125-1.0 mg/L, respectively), but MICs were considerably lower for M. bovis BCG (0.008-0.063 mg/L for rifapentine and 0.016-0.125 mg/L for its metabolite). In general, MICs determined using 7H11 agar medium were usually one or two dilutions higher than those obtained using Bactec broth. When compared with rifampicin and rifabutin, the inhibitory activity of rifapentine for drug-susceptible isolates was roughly equal to that of rifabutin, and the inhibitory activity of 25-O:-desacetylrifapentine was comparable to that of rifampicin; however, rifapentine was somewhat more bactericidal than rifabutin at equal concentrations. Clinical isolates of M. tuberculosis with a high degree of resistance to rifampicin (MIC >/= 32 mg/L) were also highly resistant to rifabutin, rifapentine and 25-O:-desacetylrifapentine, although the MICs of rifabutin in this case were somewhat lower than the MICs of rifapentine.
Sacco, Randy E.; Olsen, Steven C.
Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. A method for the detection of M. bovis-specific serum antibodies is needed in order to establish prevalence and transmission patterns. Enzyme-linked immunosorbent assays (ELISAs) validated for the detection of M. bovis-specific serum IgG in cattle are commercially available, but their suitability for bison sera has not been determined. A collection of bison sera, most from animals with a known history of infection or vaccination with M. bovis, was tested for M. bovis-specific IgG using commercially available kits as well as an in-house ELISA in which either cattle or bison M. bovis isolates were used as a source of antigen. Comparison of the results demonstrates that ELISAs optimized for cattle sera may not be optimal for the identification of bison seropositive for M. bovis, particularly those with low to moderate antibody levels. The reagent used for the detection of bison IgG and the source of the antigen affect the sensitivity of the assay. Optimal performance was obtained when the capture antigen was derived from bison isolates rather than cattle isolates and when a protein G conjugate rather than an anti-bovine IgG conjugate was used for the detection of bison IgG. PMID:23843427
Tomlinson, A J; Chambers, M A; Wilson, G J; McDonald, R A; Delahay, R J
Heterogeneity in the progression of disease amongst individual wild animals may impact on both pathogen and host dynamics at the population level, through differential effects on transmission, mortality and reproductive output. The role of the European badger (Meles meles) as a reservoir host for Mycobacterium bovis infection in the UK and Ireland has been the focus of intense research for many years. Here, we investigate life-history correlates of infection in a high-density undisturbed badger population naturally infected with M. bovis. We found no evidence of a significant impact of M. bovis infection on female reproductive activity or success, with evidence of reproduction continuing successfully for several years in the face of M. bovis excretion. We also found evidence to support the hypothesis that female badgers are more resilient to established M. bovis infection than male badgers, with longer survival times following the detection of bacterial excretion. We discuss the importance of infectious breeding females in the persistence of M. bovis in badger populations, and how our findings in male badgers are consistent with testosterone-induced immunosuppression. In addition, we found significant weight loss in badgers with evidence of disseminated infection, based on the culture of M. bovis from body systems other than the respiratory tract. For females, there was a gradual loss of weight as infection progressed, whereas males only experienced substantial weight loss when infection had progressed to the point of dissemination. We discuss how these differences may be explained in terms of resource allocation and physiological trade-offs.
Boothby, J T; Jasper, D E; Thomas, C B
Four cows were vaccinated with Mycoplasma bovis five times at two week intervals: three times subcutaneously in Freund's complete adjuvant, and two times with M. bovis alone in two of four quarters by intramammary infusion. The effect of vaccination on the immune response was evaluated in the serum and whey of the four vaccinated and control (placebo) cows experimentally challenged in two of four quarters with live M. bovis. Vaccination resulted in markedly increased M. bovis-specific, serum IgM, IgG and IgG2, but not IgA, reactivity. Challenge exposure with live M. bovis by intramammary infusion resulted in high specific serum IgM, IgG1 and IgG2 reactivity and a noticeable IgA response in both vaccinated and control cows. Whey from quarters on vaccinated cows had elevated, specific IgG1 reactivity at the time of challenge but no other differences were observed. Challenge exposure with live M. Bovis resulted in high antibody levels of all isotypes in quarters which were challenged, but highly elevated reactivities in unchallenged quarters occurred only with IgG1 and IgG2. These results indicate that vaccination elevated M. bovis-specific IgG1 but not other immunoglobulin reactivity in quarters on vaccinated cows, and that live organisms are necessary to elicit a local, specific IgA response. PMID:3567746
Ferreira, Daniele J; Zanine, Anderson M; Lana, Rogério P; Ribeiro, Marinaldo D; Alves, Guilherme R; Mantovani, Hilário C
The objective of the present study was to assess the chemical and bromatological composition and in situ degradability of elephant grass silages inoculated with Streptococcus bovis isolated from cattle rumen. A complete randomized design was used with four treatments and six replications: elephant grass silage, elephant grass silage inoculated with 10(6) CFU/g Streptococcus bovis JB1 strains; elephant grass silage inoculated with 106 CFU/g Streptococcus bovis HC5 strains; elephant grass silage inoculated with 106 CFU/g Enterococcus faecium with six replications each. The pH and ammoniacal nitrogen values were lower (P<0.05) for the silages inoculated with Streptococcus bovis JB1 and HC5, respectively. The silage inoculated with Streptococcus bovis had a higher crude protein content (P<0.05) and there were no differences for the fiber contents in the silage. The (a)soluble fraction degradability, especially in the silages inoculated with Streptococcus bovis JB1 and HC5, had higher values, 30.77 and 29.97%, for dry matter and 31.01 and 36.66% for crude protein, respectively. Inoculation with Streptococcus bovis improved the fermentation profile, protein value and rumen degradability of the nutrients.
Boothby, J T; Jasper, D E; Thomas, C B
Four cows were vaccinated with Mycoplasma bovis five times at two week intervals: three times subcutaneously in Freund's complete adjuvant, and two times with M. bovis alone in two of four quarters by intramammary infusion. The effect of vaccination on the immune response was evaluated in the serum and whey of the four vaccinated and control (placebo) cows experimentally challenged in two of four quarters with live M. bovis. Vaccination resulted in markedly increased M. bovis-specific, serum IgM, IgG and IgG2, but not IgA, reactivity. Challenge exposure with live M. bovis by intramammary infusion resulted in high specific serum IgM, IgG1 and IgG2 reactivity and a noticeable IgA response in both vaccinated and control cows. Whey from quarters on vaccinated cows had elevated, specific IgG1 reactivity at the time of challenge but no other differences were observed. Challenge exposure with live M. Bovis resulted in high antibody levels of all isotypes in quarters which were challenged, but highly elevated reactivities in unchallenged quarters occurred only with IgG1 and IgG2. These results indicate that vaccination elevated M. bovis-specific IgG1 but not other immunoglobulin reactivity in quarters on vaccinated cows, and that live organisms are necessary to elicit a local, specific IgA response.
Lombardi, G; Botti, I; Pacciarini, M L; Boniotti, M B; Roncarati, G; Dal Monte, P
Human tuberculosis (TB) caused by Mycobacterium bovis surveillance is affected by a lack of data. The aims of the present study were: (i) to estimate the proportion of human TB caused by M. bovis over a period of 5 years in Bologna, Northern Italy, which, like most Western European countries, has been declared bovine TB-free; (ii) to compare the genetic profiles of M. bovis strains identified in humans with those circulating in cattle in the last 15 years in Italy. Among 511 TB patients, the proportion of human TB caused by M. bovis was 1·76%, significantly associated to extra-pulmonary localization (P = 0·004) and to being elderly (P < 0·001) and Italy-born (P = 0·036). The molecular epidemiology analysis by spoligotyping and Multilocus Variable Tandem Repeat Analysis confirmed that most M. bovis strains from Italy-born patients matched those circulating in cattle herds in Italy between 2001 and 2016. Two cases of Mycobacterium bovis BCG infection were also characterized. In conclusion, the rate of human TB caused by M. bovis was not negligible, highlighting the relevance of molecular typing in evaluating the effectiveness of programmes designed to eradicate TB in cattle in Italy.
Streptococcus bovis (S. bovis) bacteria are associated with colorectal cancer and adenoma. S. bovis is currently named S. gallolyticus. 25 to 80% of patients with S. bovis/gallolyticus bacteremia have concomitant colorectal tumors. Colonic neoplasia may arise years after the presentation of bacteremia or infectious endocarditis of S. bovis/gallolyticus. The presence of S. bovis/gallolyticus bacteremia and/or endocarditis is also related to the presence of villous or tubular-villous adenomas in the large intestine. In addition, serological relationship of S. gallolyticus with colorectal tumors and direct colonization of S. gallolyticus in tissues of colorectal tumors were found. However, this association is still under controversy and has long been underestimated. Moreover, the etiological versus non-etiological nature of this associationis not settled yet. Therefore, by covering the most of up to date studies, this review attempts to clarify the nature and the core of S. bovis/gallolyicus association with colorectal tumors and analyze the possible underlying mechanisms. PMID:21247505
Register, Karen B; Sacco, Randy E; Olsen, Steven C
Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. A method for the detection of M. bovis-specific serum antibodies is needed in order to establish prevalence and transmission patterns. Enzyme-linked immunosorbent assays (ELISAs) validated for the detection of M. bovis-specific serum IgG in cattle are commercially available, but their suitability for bison sera has not been determined. A collection of bison sera, most from animals with a known history of infection or vaccination with M. bovis, was tested for M. bovis-specific IgG using commercially available kits as well as an in-house ELISA in which either cattle or bison M. bovis isolates were used as a source of antigen. Comparison of the results demonstrates that ELISAs optimized for cattle sera may not be optimal for the identification of bison seropositive for M. bovis, particularly those with low to moderate antibody levels. The reagent used for the detection of bison IgG and the source of the antigen affect the sensitivity of the assay. Optimal performance was obtained when the capture antigen was derived from bison isolates rather than cattle isolates and when a protein G conjugate rather than an anti-bovine IgG conjugate was used for the detection of bison IgG.
Minden, P; Kelleher, P J; Freed, J H; Nielsen, L D; Brennan, P J; McPheron, L; McClatchy, J K
A component of Mycobacterium bovis BCG referred to as BCG-a was isolated through the combined use of monoclonal antibody directed to BCG and affinity chromatography. Analysis of BCG-a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single prominent band with a molecular weight of ca. 10,000. Structural characterization of BCG-a consisting of amino acid composition and amino-terminal sequence determination was carried out. The intact BCG-a antigen was bound by neither the lectin from common lentils nor concanavalin A, implying that BCG-a does not carry any asparagine-linked oligosaccharides. Immunoprecipitation of 125I-labeled BCG-a with polyclonal and monoclonal antibodies directed against BCG resulted in bands having the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as did free 125I-BCG-a. In radioimmunoassays 125I-BCG-a was bound by the monoclonal antibody and by polyclonal antibodies from rabbits that had been immunized to BCG and to Mycobacterium tuberculosis H37Rv. Antibodies to nontuberculous and to nonacid-fast bacteria bound BCG-a poorly or not at all. The binding of 125I-BCG-a by the monoclonal antibody was readily inhibited by extracts of BCG and H37Rv, but it was not as readily inhibited by extracts of nontuberculous mycobacteria and was not at all inhibited by extracts of nonacid-fast bacteria. Considerable inhibition was similarly observed by surface antigens of nonviable, intact BCG organisms. Delayed cutaneous hypersensitivity reactions to small concentrations of BCG-a were elicited in guinea pigs that had been immunized with BCG or H37Rv antigens, but such reactions were not elicited in unimmunized animals. Images PMID:6389346
Stewart, Linda D.; McNair, James; McCallan, Lyanne; Thompson, Suzan; Kulakov, Leonid A.
This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 105 CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis. PMID:22322353
Corredoira, J C; Alonso, M P; García, J F; Casariego, E; Coira, A; Rodriguez, A; Pita, J; Louzao, C; Pombo, B; López, M J; Varela, J
The aim of this study was to determine the clinical significance of Streptococcus salivarius isolates recovered from blood cultures and compare them with isolates of Streptococcus bovis biotypes I and II. Seventeen of the 52 (32%) S. salivarius isolates recovered were considered clinically significant, compared with 62 of the 64 (97%) S. bovis isolates (p<0.0001). Bacteremia caused by S. salivarius occurred mostly in patients who showed relevant disruption of the mucous membranes and/or serious underlying diseases. Patients with S. salivarius bacteremia were younger than those with S. bovis bacteremia (57 vs. 67 years; p<0.01). Patients with S. salivarius bacteremia and patients with S. bovis II bacteremia had similar rates of endocarditis, colon tumors, and non-colon cancer. On the other hand, when compared with S. bovis I bacteremia, S. salivarius bacteremia was associated with lower rates of endocarditis (18% vs. 74%, respectively) (p<0.01) and colon tumors (0% vs. 57%, respectively) (p<0.005) and higher rates of non-colon cancer (53% vs. 9.5%, respectively) (p<0.01). Bacteremia caused by S. bovis II had a hepatobiliary origin in 50% of the patients, while, in contrast, that due to S. salivarius or S. bovis I was less frequently associated with a hepatobiliary origin (12% and 5%, respectively) (p<0.00001). The rate of penicillin resistance was 31% among S. salivarius isolates and 0% among S. bovis isolates (p<0.0001). In conclusion, the clinical characteristics of S. salivarius bacteremia and S. bovis II bacteremia are similar, and the isolation of S. salivarius in blood should not be systematically regarded as contamination.
Calder, J A; Reddy, G R; Chieves, L; Courtney, C H; Littell, R; Livengood, J R; Norval, R A; Smith, C; Dame, J B
The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%. PMID:8897177
We recently reported that on 14 dairy farms from Vermont to Florida ~85% of pre-weaned dairy calves were infected with zoonotic Cryptosporidium parvum whereas only 1-2% of post-weaned calves and 1-2 year-old heifers were infected with this species. Cryptosporidium bovis and the deer-like genotype w...
Background Over the past two decades, highly virulent strains of Mycobacterium tuberculosis have emerged and spread rapidly in humans, suggesting a selective advantage based upon virulence. A similar scenario has not been described for Mycobacterium bovis infection in cattle (i.e., Bovine Tuberculos...
Mini and microsatellite sequences have proven to be excellent tools for the differentiation of strains and populations in several protozoan parasites due to their high variability. In the present work we have searched the genome of the tick-transmitted bovine hemoprotozoon Babesia bovis for tandem r...
Amram, E.; Mikula, I.; Schnee, C.; Ayling, R. D.; Nicholas, R. A. J.; Rosales, R. S.; Harrus, S.
Mycoplasma bovis isolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3 and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16 μg/ml. Mutations at positions A965T, A967T/C (Escherichia coli numbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetracycline (MICs, ≥2 μg/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and G1058) of the two rrs alleles. No tet(M), tet(O), or tet(L) determinants were found in the genome of any of the 70 M. bovis isolates. The data presented correlate (P < 0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with decreased susceptibility to tetracycline. PMID:25403668
Wildlife are an important component in the vector-host-pathogen triangle of livestock diseases, as they maintain biological vectors that transmit pathogens and can serve as reservoirs for such infectious pathogens. Babesia bovis is a tick-borne pathogen, vectored by cattle fever ticks, Rhipicephalus...
Wibberg, Daniel; Winkler, Anika; Straube, Eberhard; Karrasch, Matthias; Keller, Peter M.
Here, we present the draft genome sequence of Mycobacterium bovis BCG S4-Jena, a tuberculosis vaccine strain. The genome of S4-Jena is represented by 48 scaffolds, consisting of 132 scaffolded contigs and amounting to a size of about 4.2 Mb. New genes potentially encoding a phage fragment were identified in the genome. PMID:27103721
Gutiérrez, M; Samper, S; Gavigan, J A; García Marín, J F; Martín, C
Forty Mycobacterium bovis isolates from cattle and goats were analyzed by using different repetitive genetic markers. The 23 M. bovis strains from goats were found to carry six to eight copies of the insertion sequence IS6110. In contrast, most of the bovine isolates contained only a single copy of this element. The standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP), described for Mycobacterium tuberculosis strains, allowed the differentiation of caprine strains. Although this method was not useful for typing bovine isolates, the repetitive elements pTBN12 and DR proved to be suitable for this purpose. A procedure using PCR which amplifies IS6110 in the outward direction was found to be as sensitive as RFLP for typing M. bovis strains from goats. The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis. The results are consistent with different strains of M. bovis being implicated in bovine and caprine tuberculosis. PMID:8576352
Romero, R E; Garzón, D L; Mejía, G A; Monroy, W; Patarroyo, M E; Murillo, L A
Tuberculosis, caused by Mycobacterium bovis is emerging as the most important disease affecting cattle. Furthermore, it results in a major public health problem when transmitted to humans. Due to its difficult and non-specific diagnosis, M. bovis has been declared to be one of the etiologic agents causing significant economic loss in the cattle industry. Our group evaluated a more rapid and specific method, based on a new polymerase chain reaction species-specific primers, which amplifies a 470-base pair fragment of the M. bovis genome. A total of 275 milk-producing cows were studied by intradermal tuberculin test (ITT) which gave 184 positive and 91 negative cases. From them, 50 animals were taken from a cattle ranch free of tuberculosis. Three different samples were collected from each animal (blood, nasal mucus, and milk). Positive results were obtained from 26 animals by PCR (11.4%), 1 by bacteriological culturing (0.4%) and 1 by bacilloscopy (0.4%). This finding suggests, as in previous reports, that ITT, normally used for bovine tuberculosis detection, has the inconvenience of having a broad range of specificity and sensitivity, and the PCR technique is a more specific and sensitive test to detect infection associated with M. bovis. Therefore, we propose this PCR assay as a useful tool in the epidemiological characterization of infected animals in areas considered to be at high risk of transmission. Images Figure 1. PMID:10369566
We report the draft genome sequences of Streptococcus bovis type strain ATTC 33317 (CVM42251) isolated from cow dung and strain JB1 (CVM42252) isolated from a cow rumen in 1977. Strains were subjected to Next Generation sequencing and the genome sizes are approximately 2 MB and 2.2 MB, respectively....
Fitzgerald, Scott D; Zwick, Laura S; Diegel, Kelly L; Berry, Dale E; Church, Steven V; Sikarskie, James G; Kaneene, John B; Reed, Willie M
The goal of this study was to evaluate the susceptibility of North American opossums (Didelphis virginiana) to aerosol inoculation of Mycobacterium bovis at two dose levels in order to gain information on disease pathogenesis, fecal shedding of the organism, and the potential role that opossums play in the spread of this disease in nature. Six opossums received high dose (1 x 10(7) colony forming units (cfu) by aerosol inoculation, six opossums received low dose (1 x 10(3) cfu inoculation, and six opossums were sham-inoculated with sterile water and served as controls. Lungs were the most frequently infected tissues, with nine of 12 inoculated opossums positive for M. bovis on culture. Gross lesions consisted of multifocal pneumonia and enlarged lymph nodes. Microscopically, granulomatous pneumonia and granulomatous lymphadenitis associated with acid-fast bacilli were present in eight of 12 inoculated opossums. Fecal shedding of M. bovis was uncommon at both inoculation doses. While opossums were highly susceptible to aerosol inoculation of M. bovis, they did not become emaciated or develop widely disseminated lesions. From this study, opossums may transmit tuberculosis by aerosol infection to other opossums in close contact and serve as a source of infection to carnivores that feed upon them, however, transmission of the disease to large herbivores by fecal shedding or direct contact may be less likely.
Balseiro, Ana; González-Quirós, Pablo; Rodríguez, Óscar; Francisca Copano, M; Merediz, Isabel; de Juan, Lucía; Chambers, Mark A; Delahay, Richard J; Marreros, Nelson; Royo, Luis J; Bezos, Javier; Prieto, José M; Gortázar, Christian
Recent studies suggest that badgers may be a potential reservoir of Mycobacterium bovis infection for cattle in Northern Spain. The objective of this study was to investigate potential epidemiological links between cattle and badgers. Culture and molecular typing data were available for cattle culled during the national tuberculosis (TB) eradication campaigns between 2008 and 2012, as well as from 171 necropsied badgers and 60 live animals trapped and examined over the same time period. Mycobacterium tuberculosis complex strains were isolated from pooled tissues of 14 (8.2%) necropsied badgers, of which 11 were identified as M. bovis: six different spoligotypes of M. bovis were subsequently identified. In two geographical locations where these isolates were shared between cattle and badgers, infected cattle herds and badgers lived in close contact. Although it remains unclear if badgers are a maintenance or spill-over host of M. bovis in this setting, it would appear prudent to have precautionary measures in place to reduce contact between cattle and badgers.
Lesellier, Sandrine; Corner, Leigh; Costello, Eamon; Sleeman, Paddy; Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; Glyn Hewinson, R; Chambers, Mark; Gormley, Eamonn
The Eurasian badger (Meles meles) is a wildlife reservoir for Mycobacterium bovis infection in Ireland and Great Britain and has been implicated in the transmission of tuberculosis to cattle. Vaccination of badgers is an option that could be used as part of a strategy to control the disease. In this study we used an endobronchial infection procedure to inoculate groups of badgers with three different doses (3x10(3), 2x10(2) and <10 Colony Forming Units (CFUs)) of M. bovis. After 17 weeks the disease status of each animal was determined by post-mortem pathology and culture for M. bovis. Each of the inoculum doses resulted in establishment of infection in the badgers. The cell-mediated immune (CMI) responses were measured by lymphocyte transformation assay (LTA) of peripheral blood mononuclear cells (PBMCs) cultured with bovine tuberculin (PPD-B). In each infected group the CMI responses increased with a kinetic profile corresponding to the delivered dose and the post-mortem pathology. The serological responses were measured by ELISA and a multi-antigen print immunoassay (MAPIA) in order to investigate any changes in the antigenic repertoire associated with different infective doses. In contrast to the CMI responses, the ELISA and MAPIA showed that the recognition of antigens by the badgers was intermittent and not strongly influenced by the dose of M. bovis.
Chen, Chang-Hung; Lee, Chao-Tai; Chang, Tsung-Chain
We describe an immunocompromised patient with Tsukamurella tyrosinosolvens bacteremia and coinfection of Mycobacterium bovis pneumonia. A 75-year-old male was admitted to our hospital complaining of persistent fever with general malaise. His medical history showed that he had diabetes mellitus (HbA1C 9.2%). A chest computed tomography (CT) showed left upper lung consolidation . Two sets of blood culture at admission finally showed Tsukamurella tyrosinosolvens. Moreover, three transbronchoscopy washing specimen cultures revealed Mycobacterium bovis. The organism Tsukamurella tyrosinosolvens was identified using conventional biochemical identification methods, PCR-restriction DNA fragment analysis, and 16S rRNA gene sequencing. The clinical mycobacterial isolates were identified to the species level by combining Polymerase Chain Reaction (PCR) with an oligonucleotide microarray to detect the M. bovis amplicons. According to our literature review, our patient's case was the first of a coinfection with Tsukamurella tyrosinosolvens and Mycobacterium bovis. Prolonged antibiotic treatment and underlying disease control are necessary for this type of patient.
Boophilus ticks are vectors of Babesia bovis, the protozoan causative agent of cattle fever, a disease which is responsible for significant production losses to cattle producers in much of Africa, Central and South America and Australia. We utilized subtractive cDNA library synthesis techniques to o...
Buss, Peter E.; van Helden, Paul D.; Parsons, Sven D.C.
In 2016, an emaciated black rhinoceros (Diceros bicornis) was found in Kruger National Park, South Africa. An interferon-γ response was detected against mycobacterial antigens, and lung tissue was positive for Mycobacterium bovis. This case highlights the risk that tuberculosis presents to rhinoceros in M. bovis–endemic areas. PMID:28221132
Complex and dynamic interactions involving domestic animals, wildlife and humans create environments favorable to the emergence of new diseases, or re-emergence of diseases in new host species. Today, reservoirs of Mycobacterium bovis, the causative agent of tuberculosis in animals and a serious zoo...
Babesia bovis is an intra-erythrocytic tick transmitted apicomplexan protozoan parasite. It has a complex life style including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed th...
Giovannini, S; Zanoni, M G; Salogni, C; Cinotti, S; Alborali, G L
Mycoplasma bovis is an important cause of bovine respiratory disease, especially in young calves where it can also cause arthritis, tenosynovitis and otitis. During 2009 and 2010 a survey was carried out on carcasses of calves less than one month old sent to the Diagnostic Laboratory of the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna in Brescia, regardless of the presence of lung lesions, to detect this pathogen. PCR tests for Mycoplasma spp. and M. bovis were applied. 83 out of 224 (37%) lung tissue samples examined were positive at PCR test for Mycoplasma spp.; in 64 cases of these we observed typical respiratory lesions (P<0.001). M. bovis was identified in 26 out of 83 (31%) lung tissue samples positive at PCR test for Mycoplasma spp.; in 24 cases of these we observed typical respiratory lesions (P=0.039). Our data demonstrate that presence of Mycoplasma spp. and M. bovis positively correlates with pneumonic lung lesions in young dairy calves.
The objective of this study was to identify microRNAs associated with a serum antibody response to Mycoplasma bovis in beef cattle. Serum from sixteen beef calves was collected at three points: in summer after calves were born, in fall at weaning, and in the following spring. All sera collected in t...
A gene coding for a protein with sequence similarity to the Toxoplasma gondii micronemal 1 (MIC1) protein that contains a copy of a domain described as a sialic acid-binding micronemal adhesive repeat was identified in the Babesia bovis genome. The single copy gene, located in chromosome 3, contains...
The causative agent of tuberculosis (TB) in cattle is Mycobacterium bovis. The characteristic lesions of bovine TB are well-organized pulmonary granulomas. Gamma delta T cells are a unique subset of nonconventional T cells that play major roles in both the innate and adaptive arms of the immune sys...
Byrne, W; Markey, B; McCormack, R; Egan, J; Ball, H; Sachse, K
To study the course of clinical mycoplasma mastitis and investigate its potential for persistence, 10(8) colony-forming units (cfu) of an Irish isolate of Mycoplasma bovis was inoculated aseptically into the right fore teat canal of three lactating cows. M bovis rapidly colonised the infected quarters and grew exponentially to more than 10(10) cfu/ml within the first three days, and spread to other quarters of each of the three cows within five to 10 days. After periods of between 24 and 72 hours the infected quarters became distended and sensitive to touch, and their secretions changed from containing visible particles, to a seropurulent exudate, to an aqueous suspension of fine particles which formed a sediment after a sample was collected. M bovis-specific antibody levels increased to varying degrees in all three cows. Subsequently, the concentrations of mycoplasma decreased to less than 10(7) cfu/ml in two of the cows, but remained at more than 10(8) cfu/ml to the end of the lactation of the other cow. Apparently normal milk was secreted by one of the cows within a month of the challenge, and by the other two cows at the start of their next lactation. However, in two of the cows subclinical M bovis infection persisted through the dry periods and into their next lactations.
King, Hayley C; Murphy, Andrew; James, Phillip; Travis, Emma; Porter, David; Sawyer, Jason; Cork, Jennifer; Delahay, Richard J; Gaze, William; Courtenay, Orin; Wellington, Elizabeth M
The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, in cattle herds in the United Kingdom is increasing, resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir and is the subject of control measures aimed at reducing the incidence of infection in cattle populations. Understanding the epidemiology of M. bovis in badger populations is essential for directing control interventions and understanding disease spread; however, accurate diagnosis in live animals is challenging and currently uses invasive methods. Here we present a noninvasive diagnostic procedure and sampling regimen using field sampling of latrines and detection of M. bovis with quantitative PCR tests, the results of which strongly correlate with the results of immunoassays in the field at the social group level. This method allows M. bovis infections in badger populations to be monitored without trapping and provides additional information on the quantities of bacterial DNA shed. Therefore, our approach may provide valuable insights into the epidemiology of bovine tuberculosis in badger populations and inform disease control interventions.
Lantos, Akos; Niemann, Stefan; Mezõsi, Lásló; Sós, Endre; Erdélyi, Károly; Dávid, Sándor; Parsons, Linda M; Kubica, Tanja; Rüsch-Gerdes, Sabine; Somoskövi, Akos
We report the first case of pulmonary tuberculosis caused by Mycobacterium bovis subsp. caprae in a captive Siberian tiger, an endangered feline. The pathogen was isolated from a tracheal aspirate obtained by bronchoscopy. This procedure provided a reliable in vivo diagnostic method in conjunction with conventional and molecular tests for the detection of mycobacteria.
Garbaccio, Sergio; Barandiaran, Soledad; Fernandez, Analía; Macias, Analía; Magnano, Gabriel; Martinez Vivot, Marcela; Peyrú, Maite; Cataldi, Angel
Mycobacterium bovis is the causative agent of bovine tuberculosis. The diagnostic laboratory confirmation is made through bacterial isolation. The aim of interlaboratory tests is to assess the performance of each participant in comparison with other of similar capacities. The test objective was to determine the efficiency of isolation of M. bovis. Four laboratories were part of the test and processed 25 blind tissue samples from granulomatous lesions and with previous M. bovis isolation. The laboratory that had the highest proportion of isolates was A (68%), followed by C (60%) and then B and D (both with 52%). The greatest concordance was observed between B-D and B-C laboratories (68%). The differences could be due to specific factors in each laboratory procedures. This type of interlaboratory tests highlights errors in the bacteriology and identifies critical points in the process to detect M. bovis accurately. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.
Gille, L; Pilo, P; Valgaeren, B R; Van Driessche, L; Van Loo, H; Bodmer, M; Bürki, S; Boyen, F; Haesebrouck, F; Deprez, P; Pardon, B
Mycoplasma bovis is a highly contagious bacterium, which predominantly causes chronic pneumonia, otitis and arthritis in calves and mastitis in adult cattle. In humans, Mycoplasma species have been associated with post-surgical infections. The present study aimed to identify the bacteria associated with three outbreaks of infected seromas after caesarian section in Belgian Blue beef cattle. A total of 10 cases occurred in three herds which were in close proximity of each other and shared the same veterinary practice. M. bovis could be cultured from seroma fluid in five of the six referred animals, mostly in pure culture and was isolated from multiple chronic sites of infection (arthritis and mastitis) as well. DNA fingerprinting of the isolates targeting two insertion sequence elements suggested spread of M. bovis from chronic sites of infection (udder and joints) to the postsurgical seromas. Identical genetic profiles were demonstrated in two animals from two separate farms, suggesting spread between farms. Mortality rate in the referred animals positive for M. bovis in a seroma was 80% (4/5), despite intensive treatment. A massive increase in antimicrobial use was observed in every affected farm. These observations demonstrate involvement of mycoplasmas in outbreaks of postsurgical seromas in cattle. Copyright © 2016 Elsevier B.V. All rights reserved.
Glycosylphosphatidyl-anchored proteins are particularly abundant on the surface of pathogenic protozoans and might play an important role for parasite survival. In the present work the relevance of GPI-anchored proteins for erythrocyte invasion of Babesia bovis, one of the tick-transmitted causative...
The presence of tuberculosis due to Mycobacterium bovis in captive and free-ranging wildlife remains one of the greatest challenges to eradication of tuberculosis in the United States. A possible addition to current control measures could be vaccination of deer to prevent infection, disease, or tran...
Viljoen, Ignatius M; van Helden, Paul D; Millar, Robert P
Mycobacterium bovis has global public-health and socio-economic significance and can infect a wide range of species including the lion (Panthera leo) resulting in tuberculosis. Lions are classified as vulnerable under the IUCN Red List of Threatened Species and have experienced a 30% population decline in the past two decades. However, no attempt has been made to collate and critically evaluate the available knowledge of M. bovis infections in lions and potential effects on population. In this review we set out to redress this. Arguments suggesting that ingestion of infected prey animals are the main route of infection for lions have not been scientifically proven and research is needed into other possible sources and routes of infection. The paucity of knowledge on host susceptibility, transmission directions and therefore host status, manifestation of pathology, and epidemiology of the disease in lions also needs to be addressed. Advances have been made in diagnosing the presence of M. bovis in lions. However, these diagnostic tests are unable to differentiate between exposure, presence of infection, or stage of disease. Furthermore, there are contradictory reports on the effects of M. bovis on lion populations with more data needed on disease dynamics versus the lion population's reproductive dynamics. Knowledge on disease effects on the lion reproduction and how additional stressors such as drought or co-morbidities may interact with tuberculosis is also lacking. Filling these knowledge gaps will contribute to the understanding of mycobacterial infections and disease in captive and wild lions and assist in lion conservation endeavours.
The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan pro...
Background. Mycoplasma bovis is a cause of respiratory disease in cattle and the bacterium most frequently isolated from bovine respiratory disease complex. It has recently emerged as a major health problem in bison, causing pharyngitis, pneumonia, arthritis, dystocia and abortion. In cattle, M. b...
Fructans from pasture can be fermented by Gram-positive bacteria (e.g., Streptococcus bovis) in the equine hindgut, increasing production of lactic acid and decreasing pH. The degree of polymerization (DP) of fructans has been suggested to influence fermentation rates. The objective of the current ...
In 1994, white-tailed deer in northeast Michigan were found to be harboring Mycobacterium bovis, the causative agent of tuberculosis in most animals including humans. Although deer likely contracted tuberculosis from cattle in the early 20th century, when the disease was present in Michigan cattle, ...
Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related...
Hermosilla, Carlos; Zahner, Horst; Taubert, Anja
Eimeria bovis is an important coccidian parasite of cattle causing severe diarrhea in young animals. Its first schizogony takes place in endothelial cells of the ileum resulting in the formation of macroschizonts 14-18 days p.i. This longlasting development suggests a particular immune evasion strategy of the parasite. Here, we analyse early innate immune reactions to E. bovis by determining the adhesion of polymorphonuclear neutrophils (PMN) to infected endothelial cell layers under flow conditions and the transcription of adhesion molecule genes in infected host cells. Bovine umbilical vein endothelial cells (BUVEC) were infected with E. bovis sporozoites. Sporozoites invaded BUVEC within 1h and the first mature macroschizonts occurred 14 days p.i. PMN adhesion was enhanced in E. bovis-infected BUVEC layers as early as 8h p.i.; maximum adhesion occurred 48 h p.i. Increased adhesion rates persisted until the end of the observation period at 14 days p.i. PMN adhered to both infected and uninfected cells within monolayers, suggesting paracrine cell activation. E. bovis infection upregulated the transcription of genes encoding for P-selectin, E-selectin, vascular cellular adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Most marked effects concerned E-selectin followed by P-selectin, VCAM-1 and ICAM-1. Increased transcript levels were found beginning 30 min p.i. and maximum values occurred 1-2h p.i. (P-selectin) and 2-4h p.i. (E-selectin, VCAM-1, ICAM-1). By 12-24h p.i. levels had decreased to those of uninfected controls. Tumor necrosis factor alpha (TNFalpha)-induced PMN adhesion was significantly reduced in infected vs. uninfected BUVEC. Eimeria bovis also had suppressive effects on TNFalpha-mediated upregulation of adhesion molecule gene transcription. The data presented here suggest that infection of BUVEC with E. bovis on one hand induces proinflammatory reactions resulting in enhanced PMN adhesion mediated by upregulated adhesion
Guo, Yongpeng; Zhu, Hongmei; Wang, Jiayao; Huang, Jing; Khan, Farhan Anwar; Zhang, Jingjing; Guo, Aizhen; Chen, Xi
Mycoplasma bovis is an important pathogenic mycoplasma, causing the cattle industry serious economic losses. Adhesion is a crucial step in the mycoplasmas’ infection and colonization process; fibronectin (Fn), an extracellular matrix glycoprotein, is a molecular bridge between the bacterial adhesins and host cell receptors. The present study was designed to characterize the Fn-binding ability of methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) and its role in M. bovis cytoadherence. The trmFO (MBOV_RS00785) gene was cloned and expressed in E. coli BL21, and polyclonal antibodies against the recombinant TrmFO (rTrmFO) were raised in rabbits. Immunoblotting demonstrated that TrmFO was an immunogenic component, and the TrmFO expression was conserved in different M. bovis isolates. The mycoplasmacidal assay further showed that in the presence of complement, rabbit anti-recombinant TrmFO serum exhibited remarkable mycoplasmacidal efficacy. TrmFO was detected in both the M. bovis membrane and cytoplasm. By ligand dot blot and enzyme-linked immunosorbent assay (ELISA) binding assay, we found that rTrmFO bound Fn in a dose-dependent manner. Immunostaining visualized by confocal laser scanning microscopy showed that rTrmFO had capacity to adhere to the embryonic bovine lung (EBL) cells. In addition, the adhesion of M. bovis and rTrmFO to EBL cells could be inhibited by anti-rTrmFO antibodies. To the best of our knowledge, this is the first report to characterize the Fn-binding ability of TrmFO and its role in the bacterial adhesion to host cells. PMID:28792486
Mackintosh, Colin G.; Qureshi, Tariq; Waldrup, Ken; Labes, Robert E.; Dodds, Ken G.; Griffin, J. Frank T.
Tuberculosis (Tb) caused by Mycobacterium bovis is a worldwide threat to livestock and humans. One control strategy is to breed livestock that are more resistant to Mycobacterium bovis. In a 3-year heritability study 6 farmed red deer stags were selected from 39 on the basis of their differing responses to experimental challenge via the tonsillar sac with approximately 500 CFU of M. bovis. Two stags remained uninfected, two were moderately affected, and two developed serious spreading Tb. Seventy offspring, bred from these six stags by artificial insemination using stored semen, were similarly challenged with M. bovis. The offspring showed patterns of response to M. bovis challenge similar to those of their sires, providing evidence for a strong genetic basis to resistance to Tb, with an estimated heritability of 0.48 (standard error, 0.096; P < 0.01). This is the first time the heritability of Tb resistance in domestic livestock has been measured. The breeding of selection lines of resistant and susceptible deer will provide an ideal model to study the mechanisms of Tb resistance in a ruminant and could provide an additional strategy for reducing the number and severity of outbreaks of Tb in farmed deer herds. Laboratory studies to identify genetic and immunological markers for resistance to Tb are under way. Preliminary studies showed no associations between NRAMP or DRB genes and resistance to Tb in deer. Patterns of immune responses seen in resistant animals suggest that both innate and acquired pathways of immunity are necessary to produce the resistant phenotype. PMID:10678981
Lerner, Uri; Amram, Eytan; Ayling, Roger D; Mikula, Inna; Gerchman, Irena; Harrus, Shimon; Teff, Dina; Yogev, David; Lysnyansky, Inna
The molecular mechanism of acquired resistance to the 16-membered macrolides tylosin (Ty) and tilmicosin (Tm) was investigated in Mycoplasma bovis field isolates. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 54 M. bovis isolates showing different minimal inhibitory concentrations (MIC). The presence of any one of the point mutations G748A, C752T, A2058G, A2059G or A2059C (Escherichia coli numbering) in one or both alleles of the 23S rRNAs was correlated with decreased susceptibility to Ty (8-1024 μg/ml) and to Tm (32 to >256 μg/ml) in 27/27 and 27/31 M. bovis isolates, respectively. Although a single mutation in domain II or V could be sufficient to cause decreased susceptibility to Ty, our data imply that a combination of mutations in two domains is necessary to achieve higher MICs (≥ 128 μg/ml). The influence of a combination of mutations in two domains II and V on enhancement of resistance to Tm was less clear. In addition, the amino acid (aa) substitution L22-Q90H was found in 24/32 representative M. bovis isolates with different MICs, but no correlation with decreased susceptibility to Ty or Tm was identified. Multiple aa substitutions were also identified in the L4 protein, including at positions 185-186 (positions 64 and 65 in E. coli) which are adjacent to the macrolide-binding site. This is the first description of the molecular mechanism of acquired resistance to the 16-membered macrolides in M. bovis. Copyright © 2013 Elsevier B.V. All rights reserved.
Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Ybanez, Adrian Patalinghug; Ybanez, Rochelle Haidee Daclan; Perez, Zandro Obligado; Guswanto, Azirwan; Igarashi, Ikuo; Yokoyama, Naoaki
Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite. © 2013.
Keet, D F; Michel, A L; Bengis, R G; Becker, P; van Dyk, D S; van Vuuren, M; Rutten, V P M G; Penzhorn, B L
African lions in the southern half of Kruger National Park (KNP) are infected with Mycobacterium bovis. Historically, reliable detection of mycobacteriosis in lions was limited to necropsy and microbiological analysis of lesion material collected from emaciated and ailing or repeat-offender lions. We report on a method of cervical intradermal tuberculin testing of lions and its interpretation capable of identifying natural exposure to M. bovis. Infected lions (n=52/95) were identified by detailed necropsy and mycobacterial culture. A large proportion of these confirmed infected lions (45/52) showed distinct responses to bovine tuberculin purified protein derivative (PPD) while responses to avian tuberculin PPD were variable and smaller. Confirmed uninfected lions from non-infected areas (n=11) responded variably to avian tuberculin PPD only. Various non-tuberculous mycobacteria (NTM) were cultured from 45/95 lions examined, of which 21/45 were co-infected with M. bovis. Co-infection with M. bovis and NTM did not influence skin reactions to bovine tuberculin PPD. Avian tuberculin PPD skin reactions were larger in M. bovis-infected lions compared to uninfected ones. Since NTM co-infections are likely to influence the outcome of skin testing, stricter test interpretation criteria were applied. When test data of bovine tuberculin PPD tests were considered on their own, as for a single skin test, sensitivity increased (80.8-86.5%) but false positive rate for true negatives (18.75%) remained unchanged. Finally, the adapted skin test procedure was shown not to be impeded by persistent Feline Immunodeficiency Virus(Ple) co-infection. Copyright 2010 Elsevier B.V. All rights reserved.
Genis, Alma D; Perez, Jocelin; Mosqueda, Juan J; Alvarez, Antonio; Camacho, Minerva; Muñoz, Maria de Lourdes; Rojas, Carmen; Figueroa, Julio V
Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2) and msa-2b). To determine the sequence variation among B. bovis Mexican isolates using msa-2b as a genetic marker, PCR amplicons corresponding to msa-2b were cloned and plasmids carrying the corresponding inserts were purified and sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 16 geographically different Mexican B. bovis isolates and a reference strain. Clustal-W multiple alignments of the MSA-2b deduced amino acid sequences performed with the 17 B. bovis Mexican isolates, revealed the identification of three genotypes with a distinct set each of amino acid residues present at the variable region: Genotype I represented by the MO7 strain (in vitro culture-derived from the Mexico isolate) as well as RAD, Chiapas-1, Tabasco and Veracruz-3 isolates; Genotype II, represented by the Jalisco, Mexico and Veracruz-2 isolates; and Genotype III comprising the sequences from most of the isolates studied, Tamaulipas-1, Chiapas-2, Guerrero-1, Nayarit, Quintana Roo, Nuevo Leon, Tamaulipas-2, Yucatan and Guerrero-2. Moreover, these three genotypes could be discriminated against each other by using a PCR-RFLP approach. The results suggest that occurrence of indels within the variable region of msa-2b sequences can be useful markers for identifying a particular genotype present in field populations of B. bovis isolated from infected cattle in Mexico.
McCluskey, B; Lombard, J; Strunk, S; Nelson, D; Robbe-Austerman, S; Naugle, A; Edmondson, A
From 2002 to 2013, bovine tuberculosis (bTB) caused by Mycobacterium bovis (M. bovis) has been detected on numerous dairies in California. In total, twelve herds had bTB detected and are included in the case series which describes these recent outbreaks and discusses potential pathways of introduction. Epidemiological investigations to determine the initial source of bTB in each herd included obtaining data on likely pathways of pathogen introduction. Pathways included purchasing cattle, use of heifer-raising operations, commingling of cattle at greater risk of exposure to infected cattle with cattle destined for California dairies, contact with infected wildlife, exposure to humans with bTB infections, community and neighboring herds and others. Epidemiologic and molecular typing data confirmed the source of infection in 3 herds and probable sources of infection in 2 herds. In the 7 remaining herds described in this case series an epidemiologic link to a source could not be determined and molecular typing results did not associate M. bovis isolates acquired from these herds with another specific U.S. herd or U.S.-born animal. Preventing new introductions of M. bovis onto California dairies will require rigorous epidemiologic investigation of all the potential pathways of introduction discussed here. The root cause(s) of bTB on California dairies is certainly multifactorial with complex interactions of herd management practices, importation of cattle at greater risk of exposure to infected cattle, and the potential of human M. bovis exposure. The extensive use of molecular typing has improved epidemiologists' ability to narrow the scope of potential sources.
Guo, Mengyao; Wang, Guoqing; Lv, Tingting; Song, Xiaojing; Wang, Tiancheng; Xie, Guanghong; Cao, Yongguo; Zhang, Naisheng; Cao, Rongfeng
Mycoplasma bovis infection can cause endometrial inflammation leading to infertility and involuntary culling in dairy cows. Because extracellular matrix (ECM) proteins affect the adherence of mycoplasma to eukaryotic cell surface, they may play a role in the pathogenesis of the bacteria. The objective of the present study was to evaluate the endometrial inflammatory response and ECM protein expression induced by M bovis. Endometrial concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and mRNA and protein expression of collagen IV (CL-IV), fibronectin (FN), and laminin (LN) were evaluated 10, 20, and 30 days after M bovis intrauterine infusion in breed cows 18 days postpartum. The presence of the bacteria in the uterus was detected by nested polymerase chain reaction and denaturing gradient gel electrophoresis. Endometrial TNF-α, IL-1β, and IL-6 concentrations in the treatment group were greater (P < 0.05) than in the positive and negative control groups 20 and 30 days after infusion. Endometrial CL-IV, FN, and LN mRNA and protein expression increased (P < 0.01) 20 days after infusion in all groups. However, the increase was more pronounced in the treatment group and reactive expressions were greater (P < 0.05) than in the positive and negative control groups 10, 20, and 30 days after infusion. In conclusion, M bovis triggered endometrial inflammatory response and increased CL-IV, FN, and LN mRNA and protein expression. The abnormal expression of ECM these proteins may promote the pathogenic effects of M bovis that lead to endometrial tissue damage and infertility.
Guo, Yongpeng; Zhu, Hongmei; Wang, Jiayao; Huang, Jing; Khan, Farhan Anwar; Zhang, Jingjing; Guo, Aizhen; Chen, Xi
Mycoplasma bovis is an important pathogenic mycoplasma, causing the cattle industry serious economic losses. Adhesion is a crucial step in the mycoplasmas' infection and colonization process; fibronectin (Fn), an extracellular matrix glycoprotein, is a molecular bridge between the bacterial adhesins and host cell receptors. The present study was designed to characterize the Fn-binding ability of methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) and its role in M. bovis cytoadherence. The trmFO (MBOV_RS00785) gene was cloned and expressed in E. coli BL21, and polyclonal antibodies against the recombinant TrmFO (rTrmFO) were raised in rabbits. Immunoblotting demonstrated that TrmFO was an immunogenic component, and the TrmFO expression was conserved in different M. bovis isolates. The mycoplasmacidal assay further showed that in the presence of complement, rabbit anti-recombinant TrmFO serum exhibited remarkable mycoplasmacidal efficacy. TrmFO was detected in both the M. bovis membrane and cytoplasm. By ligand dot blot and enzyme-linked immunosorbent assay (ELISA) binding assay, we found that rTrmFO bound Fn in a dose-dependent manner. Immunostaining visualized by confocal laser scanning microscopy showed that rTrmFO had capacity to adhere to the embryonic bovine lung (EBL) cells. In addition, the adhesion of M. bovis and rTrmFO to EBL cells could be inhibited by anti-rTrmFO antibodies. To the best of our knowledge, this is the first report to characterize the Fn-binding ability of TrmFO and its role in the bacterial adhesion to host cells.
Wilson, D J; Justice-Allen, A; Goodell, G; Baldwin, T J; Skirpstunas, R T; Cavender, K B
The objective of this study was to evaluate the possible transmission of Mycoplasma bovis from positive sand bedding to naïve dairy calves. Twelve preweaned Holstein bull calves were blocked in pairs and randomly assigned as unexposed controls (n=6) bedded with control sand, or exposed calves (n=6) bedded with sand previously positive for M. bovis at a dairy farm. Bedding sand was cultured weekly. Nasal and ear swabs and sera were collected weekly, tracheal swabs were collected monthly, and by the end of the 105-d study, all calves were euthanized (n=10) or died (n=2). Sera were tested for M. bovis-specific antibody. Mycoplasma spp. culture was performed on nasal and ear swabs; culture and a PCR differentiating multiple Mycoplasma spp. were performed on postmortem samples of lung, retropharyngeal lymph node, and trachea from each calf. A complete necropsy also was performed. During 6 wk, mycoplasma concentration in exposed group sand was between 200 and 32,000 cfu/g. All 166 tracheal swabs, nasal and ear swabs, and postmortem tests from all calves were negative for mycoplasma. All 94 sera were negative for M. bovis-specific antibody. No gross pathology suggestive of mycoplasma disease was detected. The probability of mycoplasma detection, if an exposed calf had become infected 4 wk after exposure, ranged between 97 and 99% depending on time of exposure for individual calves. There was no evidence that sand bedding contaminated with M. bovis might serve as a source of transmission to naïve dairy calves.
Lutz, Kathleen; Schmitt, Sigrid; Linder, Monica; Hermosilla, Carlos; Zahner, Horst; Taubert, Anja
The proteome of Eimeria bovis meront I-carrying host cells was analyzed by two-dimensional gel electrophoresis (2DE) at 14 days p.i. and compared to non-infected control cells. A total of 221 protein spots were modulated in their abundance in E. bovis-infected host cells and were subsequently analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectometry (MALDI-TOF-MS). These analyses identified 104 proteins in total with 25 host cell proteins being up-regulated and 79 proteins being down-regulated in E. bovis-infected host cells. Moreover, 20 newly expressed proteins were identified exclusively in E. bovis-infected host cells and were most likely of parasite origin. Parasite-induced differences in protein abundance concerned distinct functional categories, with most proteins being involved in host cell metabolism, cell structure, protein fate and gene transcription. Some of the modulated molecules also indicated regulatory processes on the level of host cell stress response (HSP70, HSP90), host cell apoptosis (caspase 8) and actin elongation/depolymerization (α-actinin-1, gelsonin, tropomodulin-3, transgelin). Since merozoites I were already released shortly after cell sampling, the current data reflect the situation at the end of first merogony. This is the first proteomic approach on E. bovis-infected host cells that was undertaken to gain a rather broad insight into Eimeria-induced host cell modulation. The data processed in this investigation should provide a useful basis for more detailed analyses concerning Eimeria-host cell interactions.
Rojas-Martínez, C; Rodríguez-Vivas, R I; Figueroa Millán, J V; Acosta Viana, K Y; Gutiérrez Ruiz, E J; Álvarez Martínez, J A
This study reports the effect of putrescine addition, either alone or in combination with insulin, transferrin and selenite (ITS), to serum-free Advanced DMEM/F12 (A-DMEM/F12) medium, on the in vitro culture of Babesia bovis and using a perfusion bioreactor to improve efficiency of the process. A B. bovis strain previously adapted to proliferate in serum-free medium (Bbovis-SF) was evaluated using eight increasing concentrations of putrescine. The percentage of parasitized erythrocytes (PPE) obtained from cultures supplemented with 0.101 mg/L was 6.23% compared with 2.3% for control cultures with M199 with Earle's salts (M199) and 40% serum. The combination of putrescine (0.101 mg/L) and a mixture of ITS (2000, 1100, and 1.34 mg/L, respectively) (Pu-ITS), in A-DMEM/F12 culture medium without serum yielded a maximum PPE of 17.26% compared to 2.58% in the control medium. This new formulation of culture medium, together with the use of a hollow-fiber perfusion bioreactor system (HFPBS), caused a substantial increase in the proliferation of B. bovis, yielding a maximum cumulative PPE of 118.8% after five days, compared to 58.6% in cultures treated with control medium M199 and 40% serum. We concluded that the addition of the ITS mixture and putrescine to the culture medium stimulated the proliferation of B. bovis in vitro. This new medium formulation, used in a HFPBS culture system, can be an effective, automated-prone system that can induce massive proliferation of B. bovis for use as a source of parasite antigens and immunogens.
Martín-Hernando, M P; Torres, M J; Aznar, J; Negro, J J; Gandía, A; Gortázar, C
Wild deer have an important role in the epidemiology of bovine tuberculosis (bTB). The aims of this study were (1) to compare the pattern of lesions present in wild red (Cervus elaphus) and fallow (Dama dama) deer that were naturally infected with Mycobacterium bovis, and (2) to use this information to develop a sampling strategy for the isolation of M. bovis from the lymphoid tissues of the head of these animals. Culture of head lymphoid tissues demonstrated that 28 of 95 red deer and 22 of 100 fallow deer sampled were infected with M. bovis. Approximately 30% of each deer population had no gross lesions. Fallow deer were significantly more likely to have thoracic lesions than red deer. Lesions were observed in the retropharyngeal lymph nodes of 64% of the culture-positive red deer and 43% of the culture positive fallow deer. One third of the red deer, but none of the fallow deer, had well-encapsulated abscess lesions. There were no microscopical differences in the lesions in the lymph nodes of the red and fallow deer. Bacteriological culture from both the tonsil and retropharyngeal lymph nodes increased the rate of isolation of M. bovis by 22% over culture of the retropharyngeal lymph nodes alone in both species. These findings indicate that investigation of wild deer for bTB-compatible lesions should include examination of the medial retropharyngeal, left tracheobronchial, mediastinal, mesenteric and ileocaecal lymph nodes. Sampling for bacteriological culture from head lymphoid tissues should be from the tonsil and the medial retropharyngeal lymph nodes. These protocols may prove useful in bTB surveillance and control in regions where wild deer contribute to the circulation of M. bovis.
Müller, Borna; Steiner, Benjamin; Bonfoh, Bassirou; Fané, Adama; Smith, Noel H; Zinsstag, Jakob
Background Mali is one of the most important livestock producers of the Sahel region of Africa. A high frequency of bovine tuberculosis (BTB) has been reported but surveillance and control schemes are restricted to abattoir inspections only. The objective of this study was to conduct, for the first time, molecular characterisation of Mycobacterium bovis strains isolated from cattle slaughtered at the Bamako abattoir. Of 3330 animals screened only 60 exhibited gross visible lesions. From these animals, twenty strains of M. bovis were isolated and characterised by spoligotyping. Results Organ lesions typical of BTB were most often detected in the liver, followed by the lung and the peritoneum. M. bovis was isolated from 20 animals and 7 different spoligotypes were observed among these 20 strains; three of the patterns had not been previously reported. Spoligotype patterns from thirteen of the strains lacked spacer 30, a characteristic common in strains of M. bovis found in Chad, Cameroon and Nigeria. However, unlike the other three Central African countries, the majority of spoligotype patterns observed in Mali also lacked spacer 6. Of the remaining seven strains, six had spoligotype patterns identical to strains commonly isolated in France and Spain. Conclusion Two groups of M. bovis were detected in cattle slaughtered at the Bamako abattoir. The spoligotype pattern of the first group has similarities to strains previously observed in Chad, Cameroon and Nigeria. The additional absence of spacer 6 in the majority of these strains suggests a Mali specific clone. The spoligotype patterns of the remaining strains suggest that they may have been of European origin. PMID:18637160
Macuamule, C L S; Wiid, I J; van Helden, P D; Tanner, M; Witthuhn, R C
Mycobacterium bovis that causes Bovine tuberculosis (BTB) can be transmitted to humans thought consumption of raw and raw fermented milk products from diseased animals. Lactic acid bacteria (LAB) used in popular traditional milk products in Africa produce anti-microbial compounds that inhibit some pathogenic and spoilage bacteria. M. bovis BCG is an attenuated non-pathogenic vaccine strain of M. bovis and the aim of the study was to determine the effect of the fermentation process on the survival of M. bovis BCG in milk. M. bovis BCG at concentrations of 6 log CFU/ml was added to products of kefir fermentation. The survival of M. bovis BCG was monitored at 12-h intervals for 72 h by enumerating viable cells on Middlebrook 7H10 agar plates enriched with 2% BD BACTEC PANTA™. M. bovis BCG was increasingly reduced in sterile kefir that was fermented for a period of 24h and longer. In the milk fermented with kefir grains, Lactobacillus paracasei subsp. paracasei or Lactobacillus casei, the viability of M. bovis BCG was reduced by 0.4 logs after 24h and by 2 logs after 48 h of fermentation. No viable M. bovis BCG was detected after 60 h of fermentation. Results from this study show that long term fermentation under certain conditions may have the potential to inactivate M. bovis BCG present in the milk. However, to ensure safety of fermented milk in Africa, fermentation should be combined with other hurdle technologies such as boiling and milk pasteurisation.
Pardo, J; Carranza, C; Turrientes, M C; Pérez Arellano, J L; López Vélez, R; Ramajo, V; Muro, A
Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive.
Pardo, J.; Carranza, C.; Turrientes, M. C.; Arellano, J. L. Pérez; Vélez, R. López; Ramajo, V.; Muro, A.
Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive. PMID:15539523
Background Mycoplasma bovis (M. bovis) is a major, but often overlooked, pathogen documented to cause respiratory disease, mastitis, and arthritis in cattle throughout China since 2008. Here, we report the development of a direct competitive enzyme-linked immunosorbent assay (Dc-ELISA) to detect M. bovis antibody. Results We used a recombinant P48 protein and monoclonal antibody (mAb) 10E. MAb 10E, prepared against the recombinant P48 protein of M. bovis, identified all M. bovis strains with no cross-reactivity with other related pathogens. Coating micro plates with P48 protein instead of whole M. bovis cells as well as the use of mAb 10E produced a specific and sensitive Dc-ELISA for M. bovis antibody detection with a cut-off percent inhibition (PI) value of 32%. Compared with two commercial indirect ELISA (i-ELISA) kits, our Dc-ELISA offered a higher positive detection rate in 165 clinical bovine serum samples. Conclusions A rapid, sensitive, and reliable serological diagnosis method was developed for M. bovis, which can facilitate M. bovis surveillance, assisting researchers in understanding the ecology and epidemiology of M. bovis. PMID:24533468
Ozeki, Yuriko; Hirayama, Yukio; Takii, Takemasa; Yamamoto, Saburo; Kobayashi, Kazuo; Matsumoto, Sohkichi
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the most often used vaccine worldwide and sole vaccine against tuberculosis. BCG is protective against severe form of childhood tuberculosis but less or not protective to adult pulmonary tuberculosis. Therefore, improved vaccination strategies and development of new tuberculosis vaccines are urgent demands. For those purposes, appropriate animal models that reflect human are critically useful. However, in animal models, BCG vaccination protects well against subsequent challenge of Mycobacterium tuberculosis. In this study we evaluated the duration of protective efficacy of the BCG vaccination in mice over time and found that efficacy was diminished 40 weeks after vaccination. The aged mice older than 45 weeks are protected sufficiently after the vaccination with BCG, suggesting that loss of its efficacy is not dependent on the age of mice but rather depends on the period from vaccination. The loss of protection occurred in TH1 polarized STAT6 deficient mice despite the maintenance of interferon (IFN)-gamma production activity of lymph node cells and splenic CD4(+) T cells against M. tuberculosis antigens. Our data suggest that the duration from vaccination may explain the variation in BCG efficacy against adult pulmonary tuberculosis. Copyright © 2011 Elsevier Ltd. All rights reserved.
Hlokwe, Tiny; Raseleka, Keneilwe; Getz, Wayne M.; Marcotty, Tanguy
Background Unpasteurised fresh and souring dairy products form an essential component of household diets throughout many rural communities in southern Africa. The presence of milk-borne zoonotic pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis and zoonotic tuberculosis in humans, constitute a public health threat, especially in remote areas with poor disease surveillance in livestock and highly compromised human health due to HIV/AIDS. Methods In this study we used culture to determine the longevity of M. bovis in experimentally inoculated fresh and naturally souring milk obtained from communal cattle in the KwaZulu-Natal province of South Africa. The effect of bacterial load and storage temperature on the survival of M. bovis was evaluated by spiking mixtures of fresh milk and starter soured milk (aMasi) culture with three concentrations of bacteria (102, 104, 107 colony forming units/ml), followed by incubation under controlled laboratory conditions that mimicked ambient indoor (20°C) and outdoor (33°C) temperatures and periodic sampling and testing over time (0-56 days). Results M. bovis cultured from samples of the fresh and souring milk was identified by PCR analysis. At the highest spiking concentration (107cfu/ml), M. bovis survived for at least 2 weeks at 20°C; but, at all concentrations in the 33°C treatment, M. bovis was absent by three days after inoculation. Logistic regression analysis was used to assess the effects of bacterial concentration and time since inoculation, as well as determine the potential half-life of M. bovis in raw souring milk. Given the most favourable tested conditions for bacterial survival (20°C), approximately 25% of mycobacteria were alive after one day of storage (95% CI: 9-53%), giving an estimated half-life of M. bovis in raw souring milk of approximately 12 hours (95% CI: 7-27 hours). Conclusions This study demonstrates that M. bovis may survive in fresh and souring milk for
Kupferwasser, I; Darius, H; Muller, A; Mohr-Kahaly, S; Westermeier, T; Oelert, H; Erbel, R; Meyer, J
Aim—To compare the clinical and morphological characteristics of patients with Streptococcus bovis endocarditis with those of patients with endocarditis caused by other microorganisms. Methods—177 consecutive patients (Streptococcus bovis, 22; other streptococci, 94; staphylococci, 44; other, 17) with definite infective endocarditis according to the Duke criteria were included. All patients underwent transthoracic and transoesophageal echocardiography. In 88 patients, findings from surgery/necropsy were obtained. Results—S bovis endocarditis was associated with older patients, with a higher mortality (p = 0.04), and with a higher rate of cardiac surgery (p < 0.001) than other microorganisms, although embolic events were observed less often (p = 0.02). Pathological gastrointestinal lesions were detected in 45% of the patients. Multiple valves were affected in 68% of the patients with S bovis endocarditis and in 20% of those with other organisms (p < 0.001). Moderate or severe regurgitation occurred more often in S bovis endocarditis than with other microorganisms (p = 0.05). When surgery or necropsy was performed, infectious myocardial infiltration of the left ventricle was confirmed histopathologically in 36% of the patients with S bovis endocarditis and in 10% of those with other organisms (p = 0.002). Conclusions—S bovis endocarditis is a severe illness because of the more common involvement of multiple valves, and of the frequent occurrence of haemodynamically relevant valvar regurgitation and infectious myocardial infiltration. Keywords: infective endocarditis; Streptococcus bovis; transoesophageal echocardiography; valvar disease PMID:9875088
Becker, Claire A M; Thibault, François M; Arcangioli, Marie-Anne; Tardy, Florence
Mycoplasma (M.) bovis has recently emerged as a major, worldwide etiological agent of bovine respiratory diseases leading to huge economic losses mainly due to high morbidity and mortality as well as poor growth rates. The spread of M. bovis infections between different animals, herds, regions or countries has been often reported to be connected to the movement of animals. However, despite recent considerable efforts, no universal subtyping method is yet available to trace M. bovis isolates circulation at an international scale. Moreover in France, the overall population diversity of M. bovis isolates has not been assessed since the early 1990s. This study was conducted to fill in these gaps. The genotypic diversity between sixty isolates collected in France over the last 35 years was assessed using two molecular subtyping methods that addressed either the long-term epidemiological relationships (Multi Locus Sequence Typing, MLST) or the genetic microvariations (Multiple Locus VNTR Analysis, MLVA) between isolates. Phenotypic diversity was also analyzed by using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) to compare the main protein patterns of isolates. All proposed subtyping approaches were optimized and led to the same pattern in the French M. bovis population that consisted of two clusters, the first one comprising isolates collected before 2000 and the second, those collected after 2000. Recent strains were further shown to be more homogeneous than older ones, which is consistent with the spread of a single clone throughout the country. Because this spread was concomitant with the emergence of multiresistant M. bovis isolates, several hypotheses are discussed to explain the homogeneity of M. bovis isolates in France, even though the M. bovis species is fully equipped to generate diversity.
Vega-Manriquez, X; López-Vidal, Y; Moran, J; Adams, L G; Gutiérrez-Pabello, J A
Mycobacterium tuberculosis complex species survive and replicate in phagosomes of the host cell. Cell death (CD) has been highlighted as one of the probable outcomes in this host-pathogen interaction. Previously, our group demonstrated macrophage apoptosis as a consequence of Mycobacterium bovis infection. In this study, we aimed to identify the contribution of apoptotic effector elements in M. bovis-induced CD. Bovine macrophages were either infected with M. bovis (multiplicity of infection, 10:1) or treated with an M. bovis cell extract (CFE). Structural changes compatible with CD were evaluated. Chromatin condensation was increased three times by the CFE. On the other hand, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay demonstrated that levels of DNA fragmentation induced by M. bovis and CFE were 53.7% +/- 24% and 38.9% +/- 14%, respectively, whereas control cells had a basal proportion of 8.9% +/- 4.1%. Rates of DNA fragmentation were unaffected by the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (z-VAD). Cells treated with 100 mug of CFE for 12 h had a fivefold decrease in the level of mitochondrial outer membrane permeabilization compared to that of untreated cells. Neither M. bovis infection nor CFE treatment induced activation of caspase 3, 8, or 9. Translocation of apoptosis-inducing factor (AIF) to the nucleus was identified in 32% +/- 3.5% and 26.3% +/- 4.9% of M. bovis-infected and CFE-treated cells, respectively. Incubation of macrophages with z-VAD prior to infection did not alter the percentage of cells showing AIF translocation. Our data suggest that M. bovis-induced CD in bovine macrophages is caspase independent with AIF participation.
Barbier, E; Boschiroli, M L; Gueneau, E; Rochelet, M; Payne, A; de Cruz, K; Blieux, A L; Fossot, C; Hartmann, A
The aim of the study was to determine the prevalence of Mycobacterium bovis (the causative agent of bovine tuberculosis, bTB) in environmental matrices within a French region (Côte d'Or) affected by this zoonotic disease. We report here the development and the use of molecular detection assays based on qPCR (double fluorescent dye-labelled probe) to monitor the occurrence of Mycobacterium tuberculosis complex (MTBC) or Myco. bovis in environmental samples collected in pastures where infected cattle and wildlife had been reported. Three qPCR assays targeting members of the MTBC (IS1561' and Rv3866 loci) or Myco. bovis (RD4 locus) were developed or refined from existing assays. These tools were validated using Myco. bovis spiked soil, water and faeces samples. Environmental samples were detected positive for the presence of MTBC strains and Myco. bovis in the environment of bTB-infected farms in the Côte d'Or region. The development of molecular assays permitted testing of several types of environmental samples including spring water, sediment samples and soils from badger setts entrance located in the vicinity of these farms, which were repeatedly contaminated with Myco. bovis (up to 8·7 × 10(3) gene copies per gram of badger sett soil). For the first time, direct spoligotyping of soil DNA enabled identification of Myco. bovis genotypes from environmental matrices. All together, these results suggest that Myco. bovis occurs at low levels in environmental matrices in Côte d'Or within the bTB-infected area. Drinking contaminated water or inhaling contaminated bioaerosols might explain cattle infection in some cases. © 2016 The Society for Applied Microbiology.
Rodgers, J D; Connery, N L; McNair, J; Welsh, M D; Skuce, R A; Bryson, D G; McMurray, D N; Pollock, J M
Non-aerosol models of bovine tuberculosis are limited in reproducibility and relevance to natural cases seen in farmed animals. Therefore, there is a need for aerosol models of infection in cattle that can reproduce bovine tuberculosis as seen in natural cases of the disease. This manuscript describes a cattle tuberculosis model based on the inhalation of a precisely defined dose of Mycobacterium bovis in aerosol form, and defines those sites of M. bovis deposition following aerosol inhalation. The dissemination of bacilli and the resultant pathological change following infection is also described. Cattle aged 4-5 months, were infected with approximately 10(4) colony forming units (CFU), using a Madison chamber that had been modified to deliver aerosols to calves. In Experiment 1, calves were examined for gross pathology at post mortem (PM) examination at 93 and 132 days post-infection (PI), respectively. In Experiment 2, pairs of calves were examined for gross pathology at PM examination at 1 day PI and 7 days PI, respectively. At PM examination, samples were taken for bacteriology. Retrospective counts showed that the calves inhaled between 3 x 10(4) and 8 x 10(4)CFU of M. bovis. In Experiment 1, pathology indicative of tuberculosis and detection of M. bovis by qualitative bacteriology was found throughout the lower respiratory tract (LRT). In Experiment 2, pathology was only observed in a single site of one calf at day 7 PI. Samples positive for M. bovis by bacteriology were predominantly in the LRT. The numbers of M. bovis CFU recovered and the distributions of positive sites were greater at day 7 PI than day 1 PI. This study describes an aerosol exposure method that can deliver a defined dose of M. bovis almost exclusively to the LRT. The distribution of M. bovis and lesions indicative of tuberculosis suggests this aerosol method replicates the primary mode of tuberculosis transmission in cattle.
Roug, Annette; Geoghegan, Claire; Wellington, Elizabeth; Miller, Woutrina A; Travis, Emma; Porter, David; Cooper, David; Clifford, Deana L; Mazet, Jonna A K; Parsons, Sven
A real-time PCR protocol for detecting Mycobacterium bovis in feces was evaluated in bovine tuberculosis-infected African buffalo (Syncerus caffer). Fecal samples spiked with 1.42 × 10(3) cells of M. bovis culture/g and Bacille Calmette-Guérin standards with 1.58 × 10(1) genome copies/well were positive by real-time PCR but all field samples were negative.
Vega-Manriquez, X.; López-Vidal, Y.; Moran, J.; Adams, L. G.; Gutiérrez-Pabello, J. A.
Mycobacterium tuberculosis complex species survive and replicate in phagosomes of the host cell. Cell death (CD) has been highlighted as one of the probable outcomes in this host-pathogen interaction. Previously, our group demonstrated macrophage apoptosis as a consequence of Mycobacterium bovis infection. In this study, we aimed to identify the contribution of apoptotic effector elements in M. bovis-induced CD. Bovine macrophages were either infected with M. bovis (multiplicity of infection, 10:1) or treated with an M. bovis cell extract (CFE). Structural changes compatible with CD were evaluated. Chromatin condensation was increased three times by the CFE. On the other hand, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay demonstrated that levels of DNA fragmentation induced by M. bovis and CFE were 53.7% ± 24% and 38.9% ± 14%, respectively, whereas control cells had a basal proportion of 8.9% ± 4.1%. Rates of DNA fragmentation were unaffected by the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (z-VAD). Cells treated with 100 μg of CFE for 12 h had a fivefold decrease in the level of mitochondrial outer membrane permeabilization compared to that of untreated cells. Neither M. bovis infection nor CFE treatment induced activation of caspase 3, 8, or 9. Translocation of apoptosis-inducing factor (AIF) to the nucleus was identified in 32% ± 3.5% and 26.3% ± 4.9% of M. bovis-infected and CFE-treated cells, respectively. Incubation of macrophages with z-VAD prior to infection did not alter the percentage of cells showing AIF translocation. Our data suggest that M. bovis-induced CD in bovine macrophages is caspase independent with AIF participation. PMID:17158896
Cui, Yongyong; Zhao, Deming; Sreevatsan, Srinand; Liu, Chunfa; Yang, Wei; Song, Zhiqi; Yang, Lifeng; Barrow, Paul; Zhou, Xiangmei
Mycobacterium bovis (M. bovis) is highly adapted to macrophages and has developed multiple mechanisms to resist intracellular assaults. However, the host cells in turn deploy a multipronged defense mechanism to control bacterial infection. Endoplasmic reticulum (ER) stress-mediated apoptosis is one such primary defense mechanism. However, the role of interferon regulatory factor 3 (IRF3) between ER stress and apoptosis during M. bovis infection is unknown. Here, we demonstrate that M. bovis effectively induced apoptosis in murine macrophages. Caspase-12, caspase-9, and caspase-3 were activated over a 48 h infection period. The splicing of XBP-1 mRNA and the level of phosphorylation of eIF2α, indicators of ER stress, significantly increased at early time points after M. bovis infection. The expansion of the ER compartment, a morphological hallmark of ER stress, was observed at 6 h. Pre-treatment of Raw 264.7 cells with 4-PBA (an ER stress-inhibitor) reduced the activation of the ER stress indicators, caspase activation and its downstream poly (ADP-ribose) polymerase (PARP) cleavage, phosphorylation of TBK1 and IRF3 and cytoplasmic co-localization of STING and TBK1. M. bovis infection led to the interaction of activated IRF3 and cytoplasmic Bax leading to mitochondrial damage. Role of IRF3 in apoptosis was further confirmed by blocking this molecule with BX-795 that showed significant reduction expression of caspase-8 and caspase-3. Intracellular survival of M. bovis increased in response to 4-PBA and BX-795. These findings indicate that STING-TBK1-IRF3 pathway mediates a crosstalk between ER stress and apoptosis during M. bovis infection, which can effectively control intracellular bacteria. PMID:28018864
Jaramillo Ortiz, José Manuel; Del Médico Zajac, María Paula; Zanetti, Flavia Adriana; Molinari, María Paula; Gravisaco, María José; Calamante, Gabriela; Wilkowsky, Silvina Elizabeth
In this study, a recombinant modified vaccinia virus Ankara vector expressing a chimeric multi-antigen was obtained and evaluated as a candidate vaccine in homologous and heterologous prime-boost immunizations with a recombinant protein cocktail. The chimeric multi-antigen comprises immunodominant B and T cell regions of three Babesia bovis proteins. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity induced by different immunization schemes. The best vaccination scheme was achieved with a prime of protein cocktail and a boost with the recombinant virus. This scheme induced high level of specific IgG antibodies and secreted IFN and a high degree of activation of IFNγ(+) CD4(+) and CD8(+) specific T cells. This is the first report in which a novel vaccine candidate was constructed based on a rationally designed multi-antigen and evaluated in a prime-boost regime, optimizing the immune response necessary for protection against bovine babesiosis.
Abadie, Valérie; Badell, Edgar; Douillard, Patrice; Ensergueix, Danielle; Leenen, Pieter J M; Tanguy, Myriam; Fiette, Laurence; Saeland, Sem; Gicquel, Brigitte; Winter, Nathalie
The early innate response after Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination is poorly characterized but probably decisive for subsequent protective immunity against tuberculosis. Therefore, we vaccinated mice with fluorescent BCG strains in the ear dorsum, as a surrogate of intradermal vaccination in humans. During the first 3 days, we tracked BCG host cells migrating out of the dermis to the auricular draining lymph nodes (ADLNs). Resident skin dendritic cells (DCs) or macrophages did not play a predominant role in early BCG capture and transport to ADLNs. The main BCG host cells rapidly recruited both in the dermis and ADLNs were neutrophils. Fluorescent green or red BCG strains injected into nonoverlapping sites were essentially sheltered by distinct neutrophils in the ADLN capsule, indicating that neutrophils had captured bacilli in peripheral tissue and transported them to the lymphoid organ. Strikingly, we observed BCG-infected neutrophils in the lumen of lymphatic vessels by confocal microscopy on ear dermis. Fluorescence-labeled neutrophils injected into the ears accumulated exclusively into the ipsilateral ADLN capsule after BCG vaccination. Thus, we provide in vivo evidence that neutrophils, like DCs or inflammatory monocytes, migrate via afferent lymphatics to lymphoid tissue and can shuttle live microorganisms.
Bartow, R A; McMurray, D N
Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated and challenged 6 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. By using a double rosette assay with isotype-specific antibody-coated ox and uncoated rabbit erythrocytes, the proportions of T lymphocytes bearing Fc receptors for immunoglobulin G (IgG) (T gamma cells) or IgM (T mu cells) were quantified in tissues taken from animals that were killed within 4 weeks postchallenge. Tuberculin reactivity in vivo and in vitro and antimycobacterial resistance were also measured. BCG vaccination protected the guinea pigs and resulted in significantly enhanced proportions of T mu cells in the blood during the first 3 weeks and in the spleen during weeks 2 and 3 postchallenge. Levels of T gamma cells declined in all tissues during the first 3 weeks of infection and were unaffected by prior vaccination with BCG. Increased proportions of T mu cells in the blood were accompanied by dramatic tuberculin skin reactions and purified protein derivative-induced lymphoproliferation in BCG-vaccinated guinea pigs during the first 2 weeks following virulent pulmonary challenge. Peak levels of T mu cells in the spleens of vaccinated animals at 2 weeks coincided with the first appearance of virulent mycobacteria in that organ. BCG vaccination appears to influence immunoregulatory events in pulmonary tuberculosis through effects on the distribution of IgM Fc receptor-bearing (T mu cell) T lymphocytes. PMID:2523350
Background Currently used mouse models fail to fully reflect human immunity to tuberculosis (TB), which hampers progress in research and vaccine development. Bone marrow-liver-thymus (BLT) mice, generated by engrafting human fetal liver, thymus, and hematopoietic stem cells in severely immunodeficient NOD/SCID/IL-2Rγ-/- (NSG) mice, have shown potential to model human immunity to infection. We engrafted HLA-A2-positive fetal tissues into NSG mice transgenically expressing human leukocyte antigen (HLA)-A2.1 (NSG-A2) to generate NSG-A2-BLT mice and characterized their human immune response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection to assess the utility of this model for investigating human TB. Results NSG-A2-BLT mice were infected intravenously with BCG and the immune response of engrafted human immune cells was characterized. After ex vivo antigenic stimulation of splenocytes, interferon (IFN)-γ-producing cells were detected by ELISPOT from infected, but not uninfected NSG-A2-BLT mice. However, the levels of secreted IFN-γ, determined by ELISA, were not significantly elevated by antigenic stimulation. NSG-A2-BLT mice were susceptible to BCG infection as determined by higher lung bacillary load than the non-engrafted control NSG-A2 mice. BCG-infected NSG-A2-BLT mice developed lung lesions composed mostly of human macrophages and few human CD4+ or CD8+ T cells. The lesions did not resemble granulomas typical of human TB. Conclusions Engrafted human immune cells in NSG-A2-BLT mice showed partial function of innate and adaptive immune systems culminating in antigen-specific T cell responses to mycobacterial infection. The lack of protection was associated with low IFN-γ levels and limited numbers of T cells recruited to the lesions. The NSG-A2-BLT mouse is capable of mounting a human immune response to M. tuberculosis in vivo but a quantitatively and possibly qualitatively enhanced effector response will be needed to improve the utility of this
Su, Feng; Wang, Yongsheng; Liu, Guanghui; Ru, Kun; Liu, Xin; Yu, Yuan; Liu, Jun; Wu, Yongyan; Quan, Fusheng; Guo, Zekun; Zhang, Yong
Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection. © 2016 Federation of European Biochemical Societies.
Vázquez, Cristina Lourdes; Bianco, María Verónica; Blanco, Federico Carlos; Forrellad, Marina Andrea; Gutierrez, Maximiliano Gabriel
ABSTRACT Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis. Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host. PMID:28031264
Palmer, Mitchell V; Thacker, Tyler C; Waters, W Ray; Robbe-Austerman, Suelee
Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis from livestock, particularly cattle. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to other deer and cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer, thus interfering with the intraspecies and interspecies transmission cycles. Thirty-three white-tailed deer were assigned to one of two groups; oral vaccination with 1 × 10(8) colony-forming units of M. bovis BCG Danish (n = 17); and non-vaccinated (n = 16). One hundred eleven days after vaccination deer were infected intratonsilarly with 300 colony-forming units of virulent M. bovis. At examination, 150 days after challenge, BCG vaccinated deer had fewer gross and microscopic lesions, fewer tissues from which M. bovis could be isolated, and fewer late stage granulomas with extensive liquefactive necrosis. Fewer lesions, especially those of a highly necrotic nature should decrease the potential for dissemination of M. bovis within the host and transmission to other susceptible hosts.
Palmer, Mitchell V.; Thacker, Tyler C.; Waters, W. Ray; Robbe-Austerman, Suelee
Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis from livestock, particularly cattle. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to other deer and cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer, thus interfering with the intraspecies and interspecies transmission cycles. Thirty-three white-tailed deer were assigned to one of two groups; oral vaccination with 1×108 colony-forming units of M. bovis BCG Danish (n = 17); and non-vaccinated (n = 16). One hundred eleven days after vaccination deer were infected intratonsilarly with 300 colony-forming units of virulent M. bovis. At examination, 150 days after challenge, BCG vaccinated deer had fewer gross and microscopic lesions, fewer tissues from which M. bovis could be isolated, and fewer late stage granulomas with extensive liquefactive necrosis. Fewer lesions, especially those of a highly necrotic nature should decrease the potential for dissemination of M. bovis within the host and transmission to other susceptible hosts. PMID:24804678
Zanine, Anderson de Moura; Bonelli, Emerson Alencar; de Souza, Alexandre Lima; Ferreira, Daniele de Jesus; Santos, Edson Mauro; Ribeiro, Marinaldo Divino; Geron, Luiz Juliano Valério; Pinho, Ricardo Martins Araujo
This study aimed to evaluate the effects of Streptococcus bovis on the fermentation characteristics and nutritive value of Tanzania grass silage. Tanzania grass was chopped and left untreated (U) or treated with Streptococcus bovis JB1 at 1 × 106 colony-forming units per gram (cfu/g) of fresh forage or Streptococcus bovis HC5 at 1 × 106 cfu/g of fresh forage and packed into sixtuplicate laboratory silos. The largest number of enterobacteria, molds and yeast (M&Y) occurred in untreated silages and the smallest populations of enterobacteria and M&Y and the largest numbers of lactic acid bacteria (LAB), at 9.81 and 9.87 log cfu/g, were observed in Streptococcus bovis JB1 and HC5, respectively (P < 0.05). Silages treated with JB1 and HC5 had lower (P < 0.05) silage pHs and concentrations of ammoniacal nitrogen (NH3-N) than untreated silages. The application of Streptococcus bovis JB1 and HC5 resulted in fewer losses through gases and effluents (P < 0.05), which resulted in greater dry matter recovery (DMR) and crude protein recovery (CPR) (P < 0.05). Streptococcus bovis JB1 and HC5 improved the fermentative profile and increased the concentration of crude protein and DMR and CPR in Tanzania grass silage. PMID:27073806
Fenton, Karla A; Fitzgerald, Scott D; Bolin, Steve; Kaneene, John; Sikarskie, James; Greenwald, Rena; Lyashchenko, Konstantin
An endemic focus of Mycobacterium bovis (M. bovis) infection in the state of Michigan has contributed to a regional persistence in the animal population. The objective of this study was to determine if Virginia opossums (Didelphis virginiana) contribute to disease persistence by experimentally assessing intraspecies lateral transmission. One wild caught pregnant female opossum bearing 11 joeys (young opossum) and one age-matched joey were obtained for the study. Four joeys were aerosol inoculated with M. bovis (inoculated), four joeys were noninoculated (exposed), and four joeys plus the dam were controls. Four replicate groups of one inoculated and one exposed joey were housed together for 45 days commencing 7 days after experimental inoculation. At day 84 opossums were sacrificed. All four inoculated opossums had a positive test band via rapid test, culture positive, and gross/histologic lesions consistent with caseogranulomatous pneumonia. The exposed and control groups were unremarkable on gross, histology, rapid test, and culture. In conclusion, M. bovis infection within the inoculated opossums was confirmed by gross pathology, histopathology, bacterial culture, and antibody tests. However, M. bovis was not detected in the control and exposed opossums. There was no appreciable lateral transmission of M. bovis after aerosol inoculation and 45 days of cohabitation between infected and uninfected opossums.
Fenton, Karla A.; Fitzgerald, Scott D.; Bolin, Steve; Kaneene, John; Sikarskie, James; Greenwald, Rena; Lyashchenko, Konstantin
An endemic focus of Mycobacterium bovis (M. bovis) infection in the state of Michigan has contributed to a regional persistence in the animal population. The objective of this study was to determine if Virginia opossums (Didelphis virginiana) contribute to disease persistence by experimentally assessing intraspecies lateral transmission. One wild caught pregnant female opossum bearing 11 joeys (young opossum) and one age-matched joey were obtained for the study. Four joeys were aerosol inoculated with M. bovis (inoculated), four joeys were noninoculated (exposed), and four joeys plus the dam were controls. Four replicate groups of one inoculated and one exposed joey were housed together for 45 days commencing 7 days after experimental inoculation. At day 84 opossums were sacrificed. All four inoculated opossums had a positive test band via rapid test, culture positive, and gross/histologic lesions consistent with caseogranulomatous pneumonia. The exposed and control groups were unremarkable on gross, histology, rapid test, and culture. In conclusion, M. bovis infection within the inoculated opossums was confirmed by gross pathology, histopathology, bacterial culture, and antibody tests. However, M. bovis was not detected in the control and exposed opossums. There was no appreciable lateral transmission of M. bovis after aerosol inoculation and 45 days of cohabitation between infected and uninfected opossums. PMID:22701815
Sylvester, Tashnica Taime; Martin, Laura Elizabeth Rosen; Buss, Peter; Loxton, Andre Gareth; Hausler, Guy Anton; Rossouw, Leana; van Helden, Paul; Parsons, Sven David Charles; Olea-Popelka, Francisco; Miller, Michele Ann
Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), is endemic in the Kruger National Park (KNP), South Africa. African lions ( Panthera leo ) are susceptible to BTB, but the impact of the disease on lion populations is unknown. In this study, we used a novel gene expression assay for chemokine (C-X-C motif) ligand 9 (CXCL9) to measure the prevalence of M. bovis infection in 70 free-ranging lions that were opportunistically sampled in the southern and central regions of the KNP. In the southern region of the KNP, the apparent prevalence of M. bovis infection was 54% (95% confidence interval [CI]=36.9-70.5%), compared with 33% (95% CI=18.0-51.8%) in the central region, an important difference (P=0.08). Prevalence of M. bovis infection in lions showed similar patterns to estimated BTB prevalence in African buffaloes ( Syncerus caffer ) in the same areas. Investigation of other risk factors showed a trend for older lions, males, or lions with concurrent feline immunodeficiency virus infection to have a higher M. bovis prevalence. Our findings demonstrate that the CXCL9 gene expression assay is a useful tool for the determination of M. bovis status in free-ranging lions and identifies important epidemiologic trends for future studies.
Bürki, Sibylle; Spergser, Joachim; Bodmer, Michèle; Pilo, Paola
Mycoplasma bovis is the most frequent etiologic agent of bovine mycoplasmosis. It causes various diseases in bovines and considerable economic loss due to the lack of effective treatment or preventive measures such as vaccination. In contrast to the US, where M. bovis-mastitis has been reported for a long time, M. bovis infections in Switzerland and Austria were predominantly associated with pneumonia and subclinical mastitis. However, since 2007 the situation has changed with the emergence of severe M. bovis-associated mastitis cases in both countries. In order to evaluate the molecular epidemiology of the bacteria isolated from these infections, recent and old Swiss, along with recent Austrian M. bovis isolates were analyzed by a typing method displaying intermediate resolution of evolutionary relationships among isolates called Multi-Locus Sequence Typing (MLST). The analysis of Swiss and Austrian M. bovis isolates revealed two major lineages. Isolates collected since 2007 in both countries cluster in the lineage I including ST5, ST33, ST34, 36, and ST38-40 (clonal complex 1), while all Swiss isolates recovered before 2007 cluster in the lineage II comprising ST17 and ST35 (clonal complex 5). Further investigations are necessary to understand if lineage I has a higher predilection or virulence toward mammary gland cells than the old lineage or if other factors are involved in the increased number of severe mastitis cases. Copyright © 2016 Elsevier B.V. All rights reserved.
Olivier, T T; Viljoen, I M; Hofmeyr, J; Hausler, G A; Goosen, W J; Tordiffe, A S W; Buss, P; Loxton, A G; Warren, R M; Miller, M A; van Helden, P D; Parsons, S D C
Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON(®) -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions. © 2015 Blackwell Verlag GmbH.
Bangoura, Berit; Mundt, Hans-Christian; Schmäschke, Ronald; Westphal, Bernhard; Daugschies, Arwid
The present study was designed to investigate the prevalence of the pathogenic coccidia species E. bovis and E. zuernii in shed-reared animals in German dairy and fattening facilities.Samples were obtained from 65 cattle farms distributed randomly across all the regions of Germany, regardless of the occurrence of clinical problems. The samples were obtained rectally. Faecal consistency and the total number of oocysts per gram of faeces (OPG) were determined for Eimeria spp., along with the separate OPG values for Eimeria (E.) bovis and E. zuernii. A questionnaire was completed for each farm to record information about herd size and management together with individual animal data. Eimeria oocysts, regardless of the kind of Eimeria spp., were detected in 62 of these farms, which gives a prevalence of 95.4 %. The farm prevalence of the pathogenic species was 76.9 % for E. bovis and 83.1 % for E. zuernii. The average oocyst excretion level was 2,950 OPG in terms of total Eimeria spp. oocyst excretion, 700 OPG for E. bovis and 1,500 OPG for E. zuernii.The number of oocysts excreted could not be correlated significantly with farm type or farm management but depended on the floor type which influences the infection pressure, on the age of the calves and the time after rehousing. In general, higher oocyst excretion rates were found in calves kept on litter compared to rearing on slatted floor. Younger calves and calves sampled early after housing shed higher amounts of oocysts than older calves and calves stabled a longer period before sampling, respectively. Furthermore, there was a positive correlation between OPG and the observation of diarrhoea, defined as observation of a loose to liquid faecal consistency. Excretion of E. zuernii oocysts was more closely linked to the occurrence of diarrhoea than E. bovis oocyst excretion. This study confirms that the pathogenic coccidia E. bovis and E. zuernii are ubiquitous in German cattle populations and a significant cause of
Lefèvre, Philippe; Denis, Olivier; De Wit, Lucas; Tanghe, Audrey; Vandenbussche, Paul; Content, Jean; Huygen, Kris
Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test. PMID:10678905
Liu, Yonggang; Tan, Peng; Liu, Shanshan; Shi, Hang; Feng, Xin; Ma, Qun
Calculus bovis have been widely used in Chinese herbology for the treatment of hyperpyrexia, convulsions, and epilepsy. Nowadays, due to the limited source and high market price, the substitutes, artificial and in vitro cultured Calculus bovis, are getting more and more commonly used. The adulteration phenomenon is serious. Therefore, it is crucial to establish a fast and simple method in discriminating the natural, artificial and in vitro cultured Calculus bovis. Bile acids, one of the main active constituents, are taken as an important indicator for evaluating the quality of Calculus bovis and the substitutes. Several techniques have been built to analyze bile acids in Calculus bovis. Whereas, as bile acids are with poor ultraviolet absorbance and high structural similarity, effective technology for identification and quality control is still lacking. In this study, high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) was applied in the analysis of bile acids, which effectively identified natural, artificial and in vitro cultured Calculus bovis and provide a new method for their quality control. Natural, artificial and in vitro cultured Calculus bovis were differentiated by bile acids analysis. A new compound with protonated molecule at m/z 405 was found, which we called 3α, 12α-dihydroxy-7-oxo-5α-cholanic acid. This compound was discovered in in vitro cultured Calculus bovis, but almost not detected in natural and artificial Calculus bovis. A total of 13 constituents was identified. Among them, three bio-markers, including glycocholic acid, glycodeoxycholic acid and taurocholic acid (TCA) were detected in both natural and artificial Calculus bovis, but the density of TCA was different in two kinds of Calculus bovis. In addition, the characteristics of bile acids were illustrated. The HPLC coupled with tandem MS (LC/MS/MS) method was feasible, easy, rapid and accurate in identifying natural, artificial and in vitro
Liu, Yonggang; Tan, Peng; Liu, Shanshan; Shi, Hang; Feng, Xin; Ma, Qun
Objective: Calculus bovis have been widely used in Chinese herbology for the treatment of hyperpyrexia, convulsions, and epilepsy. Nowadays, due to the limited source and high market price, the substitutes, artificial and in vitro cultured Calculus bovis, are getting more and more commonly used. The adulteration phenomenon is serious. Therefore, it is crucial to establish a fast and simple method in discriminating the natural, artificial and in vitro cultured Calculus bovis. Bile acids, one of the main active constituents, are taken as an important indicator for evaluating the quality of Calculus bovis and the substitutes. Several techniques have been built to analyze bile acids in Calculus bovis. Whereas, as bile acids are with poor ultraviolet absorbance and high structural similarity, effective technology for identification and quality control is still lacking. Methods: In this study, high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) was applied in the analysis of bile acids, which effectively identified natural, artificial and in vitro cultured Calculus bovis and provide a new method for their quality control. Results: Natural, artificial and in vitro cultured Calculus bovis were differentiated by bile acids analysis. A new compound with protonated molecule at m/z 405 was found, which we called 3α, 12α-dihydroxy-7-oxo-5α-cholanic acid. This compound was discovered in in vitro cultured Calculus bovis, but almost not detected in natural and artificial Calculus bovis. A total of 13 constituents was identified. Among them, three bio-markers, including glycocholic acid, glycodeoxycholic acid and taurocholic acid (TCA) were detected in both natural and artificial Calculus bovis, but the density of TCA was different in two kinds of Calculus bovis. In addition, the characteristics of bile acids were illustrated. Conclusions: The HPLC coupled with tandem MS (LC/MS/MS) method was feasible, easy, rapid and accurate in
Devriese, Luc A.; Vandamme, Peter; Pot, Bruno; Vanrobaeys, Mia; Kersters, Karel; Haesebrouck, Freddy
Strains formerly identified as Streptococcus bovis were allotted to two groups by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell proteins. Strains from humans with infections, mostly patients with endocarditis, and strains from pigeons with septicemia clustered with the recently described species Streptococcus gallolyticus. The original S. bovis type strain and strains exclusively from ruminants formed the second cluster. The findings indicate that S. gallolyticus is more likely to be involved in human and animal infections than S. bovis. Growth characteristics and several biochemical reactions were found to be useful in the differentiation of S. gallolyticus from S. bovis. PMID:9817865
Lopez, A; Maxie, M G; Savan, M; Ruhnke, H L; Thomson, R G; Barnum, D A; Geissinger, H D
Based on current literature which commonly associates bovine virus diarrhea virus and Mycoplasma bovis with "pneumonic pasteurellosis," an investigation was conducted into the effect of these two pathogens on the capacity of bovine lung to clear inhaled Pasteurella haemolytica. There was no significant effect (p less than 0.05) of either bovine virus diarrhea virus or M. bovis on the mean clearance rate of P. haemolytica, nor did the time interval of three, five or seven days between the first inoculation and exposure to P. haemolytica and adversely affect the lung clearance rates. However, it was found that the left lungs and a higher bacterial retention (p less than 0.05) than the right lungs. PMID:7127194
Martín-Atance, P; León-Vizcaíno, L; Palomares, F; Revilla, E; González-Candela, M; Calzada, J; Cubero-Pablo, M J; Delibes, M
We conducted a retrospective serologic survey for antibodies against the MPB70 protein of Mycobacterium bovis in wild carnivores from Doñana National Park (southwestern Spain). Serum samples from 118 red foxes (Vulpes vulpes), 39 Iberian lynx (Lynx pardinus), 31 Eurasian badgers (Meles meles), five Egyptian mongoose (Herpestes ichneumon), four European genet (Genetta genetta), and one Eurasian otter (Lutra lutra) were analyzed using an indirect competitive enzyme-linked immunoassay. Antibodies against the MPB70 protein of M. bovis were detected in seven badgers, five foxes, and one lynx. The frequency of positive animals was significantly higher in badger (23%) than in lynx (3%) and fox (4%). Antibodies were not detected in other species. Annual antibody frequency peaked at 38% in badgers and 11% for red fox. These species may contribute to persistence of bovine tuberculosis in Doñana.
Tomlinson, A J; Chambers, M A; Carter, S P; Wilson, G J; Smith, G C; McDonald, R A; Delahay, R J
The behaviour of certain infected individuals within socially structured populations can have a disproportionately large effect on the spatio-temporal distribution of infection. Endemic infection with Mycobacterium bovis in European badgers (Meles meles) in Great Britain and Ireland is an important source of bovine tuberculosis in cattle. Here we quantify the risk of infection in badger cubs in a high-density wild badger population, in relation to the infection status of resident adults. Over a 24-year period, we observed variation in the risk of cub infection, with those born into groups with resident infectious breeding females being over four times as likely to be detected excreting M. bovis than cubs from groups where there was no evidence of infection in adults. We discuss how our findings relate to the persistence of infection at both social group and population level, and the potential implications for disease control strategies.
Dekker, John P.
The Streptococcus bovis group has undergone significant taxonomic changes over the past 2 decades with the advent of new identification methods with higher discriminatory power. Although the current classification system is not yet embraced by all researchers in the field and debate remains over the performance of molecular techniques for identification to the species level within the group, important disease associations for several members of the group have been clarified. Here, we provide a brief overview of the history of the S. bovis group, an outline of the currently accepted classification scheme, a review of associated clinical syndromes, and a summary of the performance and diagnostic accuracy of currently available identification methods. PMID:26912760
Maragou, Chrysoula; Theologie-Lygidakis, Nadia; Ioannidis, Panayotis; Stenou, Antonia; Kanavaki, Sophia; Iatrou, Ioannis; Tsolia, Maria N
Bovine tuberculosis is a zoonotic disease, and although its incidence has dramatically decreased in developed countries where effective control measures are applied, it still remains a potential health hazard in the developing world. Tuberculosis of the oral cavity is extremely rare and is usually secondary to pulmonary involvement. We present the unusual case of an immunocompetent 6-year-old child residing in an urban area with primary oral tuberculosis due to Mycobacterium bovis, which was confirmed by the application of a molecular genetic approach. M. bovis belongs to Mycobacterium tuberculosis complex which comprises species with close genetic relationship, and for this reason, the use of new molecular techniques is a useful tool for the differentiation at species level of the closely related members of this complex.
De Castro, Cristina; Grice, I Darren; Daal, Terese-Marie; Peak, Ian R; Molinaro, Antonio; Wilson, Jennifer C
Moraxella bovis is a Gram-negative microorganism that causes Infectious Bovine Keratoconjunctivitis (IBK), colloquially known as 'Pink eye' in cattle worldwide. Lipopolysaccharides/lipooligosaccharides are the predominant glycans on the surface of Gram-negative microorganisms. Structural elucidation of the oligosaccharide structure of the rough phenotype of Moraxella bovis strain Epp63 was determined using GC-MS, methylation analysis, and NMR spectroscopy. The oligosaccharide is a branched structure that comprises 10 sugars in addition to KDO. The unusual features of this oligosaccharide include the fact that the oligosaccharide is devoid of heptose. The KDO residue is directly attached to a (→4,6)-branched glucose and additionally contains a terminal open chain acetal-linked N-acetylgalactosamine, (1S)-GalaNAc residue →4,6-linked to a sub-terminal galactose residue.
Mukherjee, Sanjay; Subramaniam, Renuka; Chen, Han; Smith, Anthony; Keshava, Shiva
Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia. PMID:28686604
Background Cattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world’s tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, Babesia bovis, and transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus, with the most prevalent species being Rhipicephalus (Boophilus) microplus. We studied the reaction of the R. microplus larval transcriptome in response to infection by B. bovis. Methods Total RNA was isolated for both uninfected and Babesia bovis-infected larval samples. Subtracted libraries were prepared by subtracting the B. bovis-infected material with the uninfected material, thus enriching for expressed genes in the B. bovis-infected sample. Expressed sequence tags from the subtracted library were generated, assembled, and sequenced. To complement the subtracted library method, differential transcript expression between samples was also measured using custom high-density microarrays. The microarray probes were fabricated using oligonucleotides derived from the Bmi Gene Index database (Version 2). Array results were verified for three target genes by real-time PCR. Results Ticks were allowed to feed on a B. bovis-infected splenectomized calf and on an uninfected control calf. RNA was purified in duplicate from whole larvae and subtracted cDNA libraries were synthesized from Babesia-infected larval RNA, subtracting with the corresponding uninfected larval RNA. One thousand ESTs were sequenced from the larval library and the transcripts were annotated. We used a R. microplus microarray designed from a R. microplus gene index, BmiGI Version 2, to look for changes in gene expression that were associated with infection of R. microplus larvae. We found 24 transcripts were expressed at a statistically significant higher level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf
Mandal, Sema; Bradshaw, Louise; Anderson, Laura F.; Brown, Tim; Evans, Jason T.; Drobniewski, Francis; Smith, Grace; Magee, John G.; Barrett, Anne; Blatchford, Oliver; Laurenson, Ian F.; Seagar, Amie-Louise; Ruddy, Michael; White, P. Lewis; Myers, Richard; Hawkey, Peter; Abubakar, Ibrahim
Due to an increase in bovine tuberculosis in cattle in the United Kingdom, we investigated the characteristics of Mycobacterium bovis infection in humans and assessed whether extensive transmission of M. bovis between humans has occurred. A cross-sectional study linking demographic, clinical, and DNA fingerprinting (using 15-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat [MIRU-VNTR] typing) data on cases reported between 2005 and 2008 was undertaken. A total of 129 cases of M. bovis infection in humans were reported over the period, with a decrease in annual incidence from 0.065 to 0.047 cases per 100,000 persons. Most patients were born pre-1960, before widespread pasteurization was introduced (73%), were of white ethnicity (83%), and were born in the United Kingdom (76%). A total of 102 patients (79%) had MIRU-VNTR typing data. A total of 31 of 69 complete MIRU-VNTR profiles formed eight distinct clusters. The overall clustering proportion determined using the n − 1 method was 33%. The largest cluster, comprising 12 cases, was indistinguishable from a previously reported West Midlands outbreak strain cluster and included those cases. This cluster was heterogeneous, having characteristics supporting recent zoonotic and human-to-human transmission as well as reactivation of latent disease. Seven other, smaller clusters identified had demographics supporting recrudescence rather than recent infection. A total of 33 patients had incomplete MIRU-VNTR profiles, of which 11 may have yielded 2 to 6 further small clusters if typed to completion. The incidence of M. bovis in humans in the United Kingdom remains low, and the epidemiology is predominantly that of reactivated disease. PMID:21430093
Aboge, Gabriel Oluga; Cao, Shinuo; Terkawi, Mohamad Alaa; Masatani, Tatsunori; Goo, Younkyoung; AbouLaila, Mahmoud; Nishikawa, Yoshifumi; Igarashi, Ikuo; Suzuki, Hiroshi; Xuan, Xuenan
The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 μM, respectively, while the Ki was 2210 ± 358 μM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 μM] than B. gibsoni [IC50 = 460.8 ± 114.45 μM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.
Farrell, Damien; Stuber, Tod P.; Schubert, Olga T.; Aebersold, Ruedi; Robbe-Austerman, Suelee
ABSTRACT We report here an update to the reference genome sequence of the bovine tuberculosis bacillus Mycobacterium bovis AF2122/97, generated using an integrative multiomics approach. The update includes 42 new coding sequences (CDSs), 14 modified annotations, 26 single-nucleotide polymorphism (SNP) corrections, and disclosure that the RD900 locus, previously described as absent from the genome, is in fact present. PMID:28385856
Leclercq, Roland; Huet, Corinne; Picherot, Mélanie; Trieu-Cuot, Patrick; Poyart, Claire
Among 128 Streptococcus gallolyticus (Streptococcus bovis) isolates, 77.7% were resistant to tetracyclines and contained tet(M) and/or tet(L) and/or tet(O). A total of 59.4% had macrolide resistance and contained erm(B) and, rarely, mef(A). Among the one-third of isolates highly resistant to kanamycin and streptomycin, most harbored aphA3 and aad-6 genes. PMID:15793162
Taubert, Anja; Wimmers, Klaus; Ponsuksili, Siriluck; Jimenez, Cristina Arce; Zahner, Horst; Hermosilla, Carlos
Within its life cycle Eimeria bovis undergoes a long lasting intracellular development into large macromeronts in endothelial cells. Since little is known about the molecular basis of E. bovis-triggered host cell regulation we applied a microarray-based approach to define transcript variation in bovine endothelial cells early after sporozoite invasion (4 h post inoculation (p.i.)), during trophozoite establishment (4 days p.i.), during early parasite proliferation (8 days p.i.) and towards macromeront maturation (14 days p.i.). E. bovis infection led to significant changes in the abundance of many host cell gene transcripts. As infection progressed, the number of regulated genes increased such that 12, 45, 175 and 1184 sequences were modulated at 4 h, 4, 8 and 14 days p.i., respectively. These genes significantly interfered with several host cell functions, networks and canonical pathways, especially those involved in cellular development, cell cycle, cell death, immune response and metabolism. The correlation between stage of infection and the number of regulated genes involved in different aspects of metabolism suggest parasite-derived exploitation of host cell nutrients. The modulation of genes involved in cell cycle arrest and host cell apoptosis corresponds to morphological in vitro findings and underline the importance of these aspects for parasite survival. Nevertheless, the increasing numbers of modulated transcripts associated with immune responses also demonstrate the defensive capacity of the endothelial host cell. Overall, this work reveals a panel of novel candidate genes involved in E. bovis-triggered host cell modulation, providing a valuable tool for future work on this topic.
Bigi, F; Alito, A; Fisanotti, J C; Romano, M I; Cataldi, A
Serum from naturally infected cattle was used to identify a novel Mycobacterium bovis antigen from an expression library. The first recombinant product identified was a fusion protein with lacZ (55 kDa). A clone containing the whole gene was also obtained. This clone expressed a 38-kDa protein. A rabbit serum against the recombinant antigen reacts in M. bovis supernatants with two proteins of 36 and 34 kDa. The new protein was called P36/P34. The gene cloned has a deduced amino acid sequence with a predicted molecular mass of 28 kDa, showing a characteristic signal sequence for exportation. The protein bears partial homology to a 28-kDa protein from M. leprae. An interesting feature of the P36/P34 sequence is that it contains several PGLTS repeats, which are not present in the M. leprae protein. Antigenic determinants seem also to be conserved between the two proteins because sera from leprosy patients recognized the recombinant M. bovis protein. The discrepancy among the molecular mass deduced from the sequence (28 kDa), that of the recombinant protein in Escherichia coli (38 kDa), and that of the native protein in M. bovis (36 and 34 kDa) could be attributed to posttranslational modifications or to the high proline content that may alter the migration properties of the protein. This antigen seems to be immunodominant during bovine tuberculosis, because 8 of 9 serum specimens from diseased cattle are reactive. The homology among the M. leprae 28-kDa protein, the protein described in this article, and a recently described M. tuberculosis protein suggests the existence of a new protein family in mycobacteria.