EFFECTS OF OZONE, CHLORINE DIOXIDE, CHLORINE, AND MONOCHLORAMINE ON CRYTOSPORIDIUM PARVUM OOCYST VIABILITY

EPA Science Inventory

Purified Cryptosporiodium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were compareatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlor...

Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts during sporulation.

PubMed

Zhou, Chun-Xue; Zhu, Xing-Quan; Elsheikha, Hany M; He, Shuai; Li, Qian; Zhou, Dong-Hui; Suo, Xun

2016-10-04

Toxoplasma gondii is a medically and economically important protozoan parasite. However, the molecular mechanisms of its sporulation remain largely unknown. Here, we applied iTRAQ coupled with 2D LC-MS/MS proteomic analysis to investigate the proteomic expression profile of T. gondii oocysts during sporulation. Of the 2095 non-redundant proteins identified, 587 were identified as differentially expressed proteins (DEPs). Based on Gene Ontology enrichment and KEGG pathway analyses the majority of these DEPs were found related to the metabolism of amino acids, carbon and energy. Protein interaction network analysis generated by STRING identified ATP-citrate lyase (ACL), GMP synthase, IMP dehydrogenase (IMPDH), poly (ADP-ribose) glycohydrolase (PARG), and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) as the top five hubs. We also identified 25 parasite virulence factors that were expressed at relatively high levels in sporulated oocysts compared to non-sporulated oocysts, which might contribute to the infectivity of mature oocysts. Considering the importance of oocysts in the dissemination of toxoplasmosis these findings may help in the search of protein targets with a key role in infectiousness and ecological success of oocysts, creating new opportunities for the development of better means for disease prevention. The development of new preventative interventions against T. gondii infection relies on an improved understanding of the proteome and chemical pathways of this parasite. To identify proteins required for the development of environmentally resistant and infective T. gondii oocysts, we compared the proteome of non-sporulated (immature) oocysts with the proteome of sporulated (mature, infective) oocysts. iTRAQ 2D-LC-MS/MS analysis revealed proteomic changes that distinguish non-sporulated from sporulated oocysts. Many of the differentially expressed proteins were involved in metabolic pathways and 25 virulence factors were identified upregulated in the sporulated oocysts. This work provides the first quantitative characterization of the proteomic variations that occur in T. gondii oocyst stage during sporulation. Copyright © 2016. Published by Elsevier B.V.

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts.

PubMed Central

Peeters, J E; Mazás, E A; Masschelein, W J; Villacorta Martiez de Maturana, I; Debacker, E

1989-01-01

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable. PMID:2764564

Infectivity to experimental rodents of Cryptosporidium parvum oocysts from Siberian chipmunks (Tamias sibiricus) originated in the People's Republic of China.

PubMed

Matsui, T; Fujino, T; Kajima, J; Tsuji, M

2000-05-01

We isolated Cryptosporidium parvum-type oocysts from naturally infected siberian chipmunks which originated in the People's Republic of China and examined the infectivity to rodents as experimental animals. The naturally infected chipmunks did not show any clinical symptoms. The oocysts were 4.8 x 4.2 microm on average in size. They were ovoid and morphologically similar to the C. parvum oocysts isolated from human and cattle. Experimental rodents were inoculated with 1.6 x 10(6) original oocysts each. SCID mice began to shed oocysts on day 7 and the OPG value was 10(5) from 50 days. The oocysts were found from ICR mice on days 13 and 16 by only sugar flotation method, however, any oocysts were not detected from the rats, guinea pigs and rabbits until 30 days. Two infected SCID mice were necropsied on days 100 and 102 and examined for coccidian organisms. Merozoites and oocysts were found in the low part of jejunum and ileum, however, no parasites were detected in the stomach. Consequently, it was considered that the present species was C. parvum and was probably genotype 2 from result of infectivity to rodents.

Preferential Flow and Transport of Cryptosporidium Parvum Oocysts Through Vadose Zone: Experiments and Modeling

NASA Astrophysics Data System (ADS)

Darnault, C. J.; Darnault, C. J.; Garnier, P.; Kim, Y.; Oveson, K.; Jenkins, M.; Ghiorse, W.; Baveye, P.; Parlange, J.; Steenhuis, T.

2001-12-01

Oocysts of the protozoan Cryptosporidium parvum, when they contaminate drinking water supplies, can cause outbreaks of Cryptosporidiosis, a common waterborne disease. Of the different pathways by which oocysts can wind up in drinking water, one has received very little attention to date; because soils are often considered to be perfect filters, the transport of oocysts through the subsoil to groundwater by preferential flow is generally ignored. To evaluate its significance, three set of laboratory experiments investigated transport of oocysts through vadose zone. Experiment set I was carried out in a vertical 50 cm-long column filled with silica sand, under conditions known to foster fingered flow. Experiment set II investigates the effect of gas-water interfaces by modifying the hydrodynamical conditions in the sand columns with water-repellent sand barriers. Experiment III involved undisturbed soil columns subjected to macropores flow. The sand and soil columns were subjected to artificial rainfall and were allowed to reach steady-state. At that point, feces of contaminated calves were applied at the surface, along with a known amount of KCl to serve as tracer, and rainfall was continued at the same rate. The breakthrough of oocysts and Cl-, monitored in the effluent, demonstrate the importance of preferential flow - fingered flow and macropore flow - on the transport of oocysts through vadose zone. Peak oocyst concentrations were not appreciably delayed, compared to Cl-, and in some cases, occurred even before the Cl- peak. However, the numbers of oocysts present in the effluents were still orders of magnitude higher than the 5 to 10 oocysts per liter that are considerable sufficient to cause cryptosporidiosis in healthy adults. The transport of oocysts was simulated based on a partitioning the soil profile in both a distribution zone and a preferential zone, In particular, the model simulates accurately the markedly asymmetric breakthrough patterns, and the long exponential tailing. The spatial distribution of oocysts suggest a close relationship between oocyst retention and the extent of gas-water interfaces; sharp increases in oocyst numbers are consistently observed in regions of the sand where the water content has steep gradients, and therefore where one expects capillary meniscii to have maximal extent. These observations imply that oocyst transport in the vadose zone is likely to be very limited in the absence of preferential flow. However, experimental results suggest that the transport of oocysts in the subsurface via preferential flow may create a significant risk of groundwater contamination in some situations.

Metam sodium reduces viability and infectivity of Eimeria oocysts.

PubMed

Fetterer, R H; Jenkins, M C; Miska, K B; Cain, G D

2010-06-01

Metam sodium (MS, sodium N-methyldithiocarbamate) is a widely used soil pesticide. Fumigation or chemical sterilization of poultry litter containing infectious oocysts could be an effective strategy to block the transmission of avian coccidia. In the current study, the effect of MS on the viability and infectivity of ocysts was investigated. The development of isolated, unsporulated oocysts of both Eimeria tenella and Eimeria maxima was inhibited, in a dose-related manner (IC(50) 8 to 14 microg/ml), by exposure to aqueous MS. Most treated oocysts failed to develop beyond early stages of sporulation. To determine the effect of MS on infectivity, isolated oocysts of E. tenella , Eimeria acervulina , and E. maxima were exposed for 24 hr to aqueous concentrations of MS ranging from 0 to 1,000 microg/ml. Treated oocysts were inoculated into chickens, and parameters of coccidiosis infection were compared to chickens inoculated with equal numbers of untreated oocysts. In a dose-related manner, MS significantly reduced the infectivity of oocysts with maximum effect observed at a dose of 300 microg/ml. When a mixture of oocysts containing 3 coccidian species was exposed to 300 microg/ml MS, from 0 to 24 hr, infectivity of oocysts was significantly reduced after a minimum of 12 hr of exposure. Treatment of aqueous slurries of litter samples obtained from commercial poultry houses, with 300 microg/ml MS for 24 hr, prevented the sporulation of eimerian oocysts in the litter samples relative to untreated control samples. The results indicate that MS could be used to reduce coccidial contamination of poultry litter.

Evidence for a Structural Role for Acid-Fast Lipids in Oocyst Walls of Cryptosporidium, Toxoplasma, and Eimeria

PubMed Central

Bushkin, G. Guy; Motari, Edwin; Carpentieri, Andrea; Dubey, Jitender P.; Costello, Catherine E.; Robbins, Phillips W.; Samuelson, John

2013-01-01

ABSTRACT Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of β-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin). PMID:24003177

Prevalence and concentration of Cryptosporidium oocysts in beef cattle paddock soils and forage.

PubMed

Boyer, Douglas G; Kuczynska, Ewa

2010-08-01

Cryptosporidium is a parasitic protozoan of great interest because of its widespread occurrence in surface waters, high degree of infectivity, and difficulty of risk management associated with its presence and control. Information about environmental loading and seasonal prevalence of Cryptosporidium oocysts is important for development of watershed management plans to protect public health. Healthy adult beef cattle are known to shed oocysts into the environment, and Cryptosporidium oocysts are often present all year in streams and groundwater in livestock agriculture areas. Surface soil and forage samples from 12 Virginia, United States, paddocks were analyzed bimonthly over 3 years for the presence and concentrations of Cryptosporidium oocysts. Half of the paddocks were grazed by stocker beef from November to September. The other half were managed for hay, but were grazed for a few days by the same animals in late fall and early spring to clean up late fall forage regrowth. Annual mean oocyst prevalences in soil were 57.9% and 48.4% in pasture and hay paddocks, respectively. Mean annual oocyst prevalences on forage were 52.4% and 40.5% in pasture and hay paddocks, respectively. Prevalence and concentration of oocysts on hay forage was highest in summer. Oocyst concentrations increased with increasing prevalences in both management systems. Wild animals appeared to be efficient vectors for oocyst distribution among paddocks. Canopy management, short-cycle rotational grazing, and control of wildlife are potential strategies for reduction of Cryptosporidium oocysts in pasture and lessening risk of contamination of water supplies, but further studies are needed before recommendations can be made.

The kinetics of oocyst shedding and sporulation in two immunologically distinct strains of Eimeria maxima, GS and M6.

PubMed

Al-Badri, Riadh; Barta, John Robert

2012-11-01

The kinetics of oocyst shedding and sporulation of two immunologically distinct strains of Eimeria maxima (GS and M6) were compared. Both strains had a prepatent period of approximately 120 h followed by peak oocyst shedding at 144-150 h post inoculation. Mean total oocyst output determined for each strain demonstrated that the fecundity of the M6 strain (12.8 × 10(3) ± 1.95) of E. maxima was roughly twice that of the GS strain (6.9 × 10(3) ± 3.33) when inoculated at the rate of 1,000 infective oocysts per bird. The process of oocyst sporulation was followed by repetitive sampling of sporulating oocysts at 26 °C with aeration over a 138 hour period. Sporulation was divided into five morphologically distinguishable stages whose abundance peaked at the following times during sporulation: unsporulated oocysts at 0 h; sporoblast anlagen at 18 h; sporoblasts without sporocyst walls at 22 h; and sporocysts without mature sporozoites at 38 h. The time to 50 % sporulation of E. maxima oocysts observed in the present study was approximately 53 h for both strains and all viable oocysts had completed sporulation by 60 h. In the present study, the prepatent periods, duration of oocyst shedding, and the relative kinetics of sporulation of the GS and M6 strains of E. maxima were found to be virtually identical despite the immunological distinctiveness of these two parasite strains.

REMOVAL OF CRYPTOSPORIDIUM BY IN-LINE FILTRATION AS A FUNCTION OF OOCYST AGE AND PRESERVATION METHOD

EPA Science Inventory

This study examined the impacts of oocyst preservation method and age on the removal of seeded Cryptosporidium oocysts by in-line filtration. An existing study has investigated the infectivity of Cryptosporidium parvum as a function of preservation method and oocyst age. Simila...

REMOVAL OF CRYPTOSPORIDIUM BY IN-LINE FILTRATION AS A FUNCTION OF OOCYST AGE AND PRESERVATION METHOD

EPA Science Inventory

This study examined the impacts of oocyst preservation method and age on the removal of seeded Cryptosporidium oocysts by in-line filtration. An existing study has investigated the infectivity of Cryptosporidium Parvum as a function of preservation method and oocyst age. Simila...

Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts

USDA-ARS?s Scientific Manuscript database

The surface properties of aged (stored for 10 years) and fresh (recently excreted) oocysts of Toxoplasma gondii were investigated using monoclonal antibody (mAb) and lectin-binding assays. Fresh oocysts bound a wall-specific mAb labeled with fluorescein isothiocyanate while aged oocysts did not. In ...

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korich, D.G.; Mead, J.R.; Madore, M.S.

1990-05-01

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 timesmore » more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.« less

Sporulation and survival of Toxoplasma gondii oocysts in different types of commercial cat litters

USDA-ARS?s Scientific Manuscript database

Toxoplasma gondii oocysts are environmentally resistant and can survive outdoors for months in the dry and cold climates. In the present study, sporulation and survival of T. gondii oocysts was studied in different types of cat litters commercially available in the US. Oocysts sporulated within 2-...

Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum Oocysts

USDA-ARS?s Scientific Manuscript database

The structure and composition of the oocyst wall are primary factors determining the survival of Cryptosporidium parvum oocysts outside the host. An external polymer matrix (glycocalyx) may mediate interactions with environmental surfaces and, thus, affect the transport of oocysts in water, soil, an...

Determining UV Inactivation of Toxoplasma gondii Oocysts by Using Cell Culture and a Mouse Bioassay

EPA Science Inventory

The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated UV irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro T. gondii oocyst plaque assay (TOP-assay), and a quantitative reve...

Evaluation of USEPA method 1622 for detection of Cryptosporidium oocysts in stream waters

USGS Publications Warehouse

Simmons, O. D.; Sobsey, M.D.; Schaefer, F. W.; Francy, D.S.; Nally, R.A.; Heaney, C.D.

2001-01-01

To improve surveillance for Cryptosporidium oocysts in water, the US Environmental Protection Agency developed method 1622, which consists of filtration, concentration, immunomagnetic separation, fluorescent antibody and 4, 6-diamidino-2-phenylindole (DAPI) counter-staining, and microscopic evaluation. Two filters were compared for analysis of 11 stream water samples collected throughout the United States. Replicate 10-L stream water samples (unspiked and spiked with 100-250 oocysts) were tested to evaluate matrix effects. Oocyst recoveries from the stream water samples averaged 22% (standard deviation [SD] = ??17%) with a membrane disk and 12% (SD = ??6%) with a capsule filter. Oocyst recoveries from reagent water precision and recovery samples averaged 39% (SD = ??13%) with a membrane disk and 47% (SD = ??19%) with a capsule filter. These results demonstrate that Cryptosporidium oocysts can be recovered from stream waters using method 1622, but recoveries are lower than those from reagent-grade water. This research also evaluated concentrations of indicator bacteria in the stream water samples. Because few samples were oocyst-positive, relationships between detections of oocysts and concentrations of indicator organisms could not be determined.

Evaluating the Transport of Bacillus subtilis Spores as a Potential Surrogate for Cryptosporidium parvum Oocysts.

PubMed

Bradford, Scott A; Kim, Hyunjung; Headd, Brendan; Torkzaban, Saeed

2016-02-02

The U.S. Environmental Protection Agency has recommended the use of aerobic spores as an indicator for Cryptosporidium oocysts when determining groundwater under the direct influence of surface water. Surface properties, interaction energies, transport, retention, and release behavior of B. subtilis spores were measured over a range of physicochemical conditions, and compared with reported information for C. parvum oocysts. Interaction energy calculations predicted a much larger energy barrier and a shallower secondary minimum for spores than oocysts when the solution ionic strength (IS) equaled 0.1, 1, and 10 mM, and no energy barrier when the IS = 100 mM. Spores and oocysts exhibited similar trends of increasing retention with IS and decreasing Darcy water velocity (qw), and the predicted setback distance to achieve a six log removal was always larger for spores than oocysts. However, low levels of observed spore and oocyst release significantly influenced the predicted setback distance, especially when the fraction of reversibly retained microbes (Frev) was high. An estimate for Frev was obtained from large release pulses of spore and oocyst when the IS was reduced to deionized water. The value of Frev always increased with qw, whereas an opposition trend for Frev with IS was observed for spores (decreasing) and oocysts (increasing).

Adhesion kinetics of viable Cryptosporidium parvum oocysts to quartz surfaces.

PubMed

Kuznar, Zachary A; Elimelech, Menachem

2004-12-15

The transport and deposition (adhesion) kinetics of viable Cryptosporidium parvum oocysts onto ultrapure quartz surfaces in a radial stagnation point flow system were investigated. Utilizing an optical microscope and an image-capturing device enabled real time observation of oocyst deposition behavior onto the quartz surface in solutions containing either monovalent (KCl) or divalent (CaCl2) salts. Results showed a significantly lower oocyst deposition rate in the presence of a monovalent salt compared to a divalent salt. With a monovalent salt, oocyst deposition rates and corresponding attachment efficiencies were relatively low, even at high KCl concentrations where Derjaguin-Landau-Verwey-Overbeek (DLVO) theory predicts the absence of an electrostatic energy barrier. On the other hand, in the presence of a divalent salt, oocyst deposition rates increased continuously as the salt concentration was increased over the entire range of ionic strengths investigated. The unusually low deposition rate in a monovalent salt solution is attributed to "electrosteric" repulsion between the Cryptosporidium oocyst and the quartz surface, most likely due to proteins on the oocyst surface that extend into the solution. It is further proposed that specific binding of calcium ions to the oocyst surface functional groups results in charge neutralization and conformational changes of surface proteins that significantly reduce electrosteric repulsion.

Fate of Cryptosporidium parvum oocysts within soil, water, and plant environment.

PubMed

McLaughlin, Stephen J; Kalita, Prasanta K; Kuhlenschmidt, Mark S

2013-12-15

Vegetative Filter Strips (VFS) have long been used to control the movement of agricultural nutrients and prevent them from reaching receiving waters. Earlier studies have shown that VFS also dramatically reduce both the kinetics and extent of Cryptosporidium parvum (C. parvum) oocysts overland transport. In this study, we investigated possible mechanisms responsible for the ability of VFS to reduce oocyst overland transport. Measurement of the kinetics of C. parvum adhesion to individual sand, silt, and clay soil particles revealed that oocysts associate over time, albeit relatively slow, with clay but not silt or sand particles. Measurement of oocyst overland transport kinetics, soil infiltration depth, distance of travel, and adhesion to vegetation on bare and vegetated soil surfaces indicate that oocysts move more slowly, and penetrate the soil profile to a greater extent on a vegetated surface than on a bare soil surface. Furthermore, we demonstrate a small fraction of the oocysts become attached to vegetation at the soil-vegetation interface on VFS. These results suggest VFS function to reduce oocyst overland transport by primarily decreasing oocyst surface flow enough to allow penetration within the soil profile followed by subsequent adhesion to or entrapment within clay particle aggregates, and to a lesser extent, adhesion to the surface vegetation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cryptosporidium parvum infection in SCID mice infected with only one oocyst: qPCR assessment of parasite replication in tissues and development of digestive cancer.

PubMed

Benamrouz, Sadia; Guyot, Karine; Gazzola, Sophie; Mouray, Anthony; Chassat, Thierry; Delaire, Baptiste; Chabé, Magali; Gosset, Pierre; Viscogliosi, Eric; Dei-Cas, Eduardo; Creusy, Colette; Conseil, Valerie; Certad, Gabriela

2012-01-01

Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10(5) oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.

Use of a Sentinel System for Field Measurements of Cryptosporidium parvum Oocyst Inactivation in Soil and Animal Waste

PubMed Central

Jenkins, M. B.; Walker, M. J.; Bowman, D. D.; Anthony, L. C.; Ghiorse, W. C.

1999-01-01

A small-volume sentinel chamber was developed to assess the effects of environmental stresses on survival of sucrose-Percoll-purified Cryptosporidium parvum oocysts in soil and animal wastes. Chambers were tested for their ability to equilibrate with external chemical and moisture conditions. Sentinel oocysts were then exposed to stresses of the external environment that affected their viability (potential infectivity), as indicated by results of a dye permeability assay. Preliminary laboratory experiments indicated that temperatures between 35 and 50°C and decreases in soil water potential (−0.003 to −3.20 MPa) increased oocyst inactivation rates. The effects of two common animal waste management practices on oocyst survival were investigated on three dairy farms in Delaware County, N.Y., within the New York City watershed: (i) piling wastes from dairy youngstock (including neonatal calves) and (ii) spreading wastes as a soil amendment on an agricultural field. Sentinel containers filled with air-dried and sieved (2-mm mesh) youngstock waste or field soil were wetted and inoculated with 2 million oocysts in an aqueous suspension and then placed in waste piles on two different farms and in soil within a cropped field on one farm. Controls consisted of purified oocysts in either phosphate-buffered saline or distilled water contained in sealed microcentrifuge tubes. Two microdata loggers recorded the ambient temperature at each field site. Sentinel experiments were conducted during the fall and winter (1996 to 1997) and winter (1998). Sentinel containers and controls were removed at 2- to 4-week intervals, and oocysts were extracted and tested by the dye permeability assay. The proportions of potentially infective oocysts exposed to the soil and waste pile material decreased more rapidly than their counterpart controls exposed to buffer or water, indicating that factors other than temperature affected oocyst inactivation in the waste piles and soil. The effect of soil freeze-thaw cycles was evident in the large proportion of empty sentinel oocysts. The potentially infective sentinel oocysts were reduced to <1% while the proportions in controls did not decrease below 50% potentially infective during the first field experiment. Microscopic observations of empty oocyst fragments indicated that abrasive effects of soil particles were a factor in oocyst inactivation. A similar pattern was observed in a second field experiment at the same site. PMID:10223991

Use of a sentinel system for field measurements of Cryptosporidium parvum oocyst inactivation in soil and animal waste.

PubMed

Jenkins, M B; Walker, M J; Bowman, D D; Anthony, L C; Ghiorse, W C

1999-05-01

A small-volume sentinel chamber was developed to assess the effects of environmental stresses on survival of sucrose-Percoll-purified Cryptosporidium parvum oocysts in soil and animal wastes. Chambers were tested for their ability to equilibrate with external chemical and moisture conditions. Sentinel oocysts were then exposed to stresses of the external environment that affected their viability (potential infectivity), as indicated by results of a dye permeability assay. Preliminary laboratory experiments indicated that temperatures between 35 and 50 degrees C and decreases in soil water potential (-0.003 to -3.20 MPa) increased oocyst inactivation rates. The effects of two common animal waste management practices on oocyst survival were investigated on three dairy farms in Delaware County, N.Y., within the New York City watershed: (i) piling wastes from dairy youngstock (including neonatal calves) and (ii) spreading wastes as a soil amendment on an agricultural field. Sentinel containers filled with air-dried and sieved (2-mm mesh) youngstock waste or field soil were wetted and inoculated with 2 million oocysts in an aqueous suspension and then placed in waste piles on two different farms and in soil within a cropped field on one farm. Controls consisted of purified oocysts in either phosphate-buffered saline or distilled water contained in sealed microcentrifuge tubes. Two microdata loggers recorded the ambient temperature at each field site. Sentinel experiments were conducted during the fall and winter (1996 to 1997) and winter (1998). Sentinel containers and controls were removed at 2- to 4-week intervals, and oocysts were extracted and tested by the dye permeability assay. The proportions of potentially infective oocysts exposed to the soil and waste pile material decreased more rapidly than their counterpart controls exposed to buffer or water, indicating that factors other than temperature affected oocyst inactivation in the waste piles and soil. The effect of soil freeze-thaw cycles was evident in the large proportion of empty sentinel oocysts. The potentially infective sentinel oocysts were reduced to <1% while the proportions in controls did not decrease below 50% potentially infective during the first field experiment. Microscopic observations of empty oocyst fragments indicated that abrasive effects of soil particles were a factor in oocyst inactivation. A similar pattern was observed in a second field experiment at the same site.

Evidence for a structural role for acid-fast lipids in oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria.

PubMed

Bushkin, G Guy; Motari, Edwin; Carpentieri, Andrea; Dubey, Jitender P; Costello, Catherine E; Robbins, Phillips W; Samuelson, John

2013-09-03

Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of β-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin). Oocysts, which are essential for the fecal-oral spread of coccidia, have a wall that is thought responsible for their survival in the environment and for their transit through the stomach and small intestine. While oocyst walls of Toxoplasma and Eimeria are strengthened by a porous scaffold of fibrils of β-1,3-glucan and by proteins cross-linked by dityrosines, both are absent from walls of Cryptosporidium. We show here that all oocyst walls are acid fast, have a rigid bilayer, dissolve in organic solvents, and contain a complex set of triglycerides rich in polyhydroxy and long fatty acyl chains that might be synthesized by an abundant polyketide synthase. These results suggest the possibility that coccidia build a waxy coat of acid-fast lipids in the oocyst wall that makes them resistant to environmental stress.

Method for Detection and Enumeration of Cryptosporidium parvum Oocysts in Feces, Manures, and Soils

PubMed Central

Kuczynska, Ewa; Shelton, Daniel R.

1999-01-01

Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 102, 103, 104, and 105 oocysts g of bovine feces−1. The percentages of recovery ranged from 10.8% (25 oocysts g−1) to 17.0% (104 oocysts g−1) (r2 = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces−1. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (104 oocysts g−1). The percentages of recovery were highest for bovine feces (17.0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO4), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris–0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil−1 depending on the soil type. The percentages of recovery without dispersant (distilled H2O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures. PMID:10388670

Protection of Broiler Chicks Housed with Immunized Cohorts Against Infection with Eimeria maxima and E. acervulina.

PubMed

Fetterer, Raymond H; Barfield, Ruth C; Jenkins, Mark C

2015-03-01

The use of live oocyst vaccines is becoming increasingly important in the control of avian coccidiosis in broilers. Knowledge of the mechanisms employed when chicks uptake oocysts and become immune is important for optimizing delivery of live vaccines. The current study tests the hypothesis that chicks not initially immunized may ingest oocysts by contact with litter containing oocysts shed by immunized cohorts. In Experiment 1, day-old broiler chicks were housed in pens containing clean litter. In Trial 1, 100% of chicks in some pens were immunized with 2.5 X 10(3) Eimeria acervulina oocysts while in other pens only 75% of chicks were immunized and remaining cohorts within the pens were not immunized. Other pens contained chicks that served as nonimmunized nonchallenged controls or nonimmunized challenged controls (NIC). On day 21, birds were given a homologous challenge of 6 X 10(5) oocysts. A second identical trial was conducted, except birds were immunized with 500 Eimeria maxima oocysts and were challenged with 3 X 10(3) E. maxima oocysts. In Experiment 2, 100% of chicks in some pens were immunized with 500 E. acervulina oocysts while in other pens either 75% or 50% of the birds were immunized. On day 14, birds were challenged with 1 X 10(6) oocysts. Trial 2 was identical to Trial 1 except that birds were immunized with 100 E. maxima oocysts and challenged with 1 X 10(6) oocysts. For all experiments weight gain, feed conversion ratio (FCR), plasma carotenoids, and litter oocyst counts were measured. In Experiment 1, the level of protection in groups containing 25% nonimmunized cohorts, as measured by weight gain, carotenoid level, FCR, and oocyst litter counts, was identical to groups containing 100% immunized chicks. In Experiment 2, pens where 50% or 75% of birds were immunized with either E. maxima or E. acervulina were not well protected from decreases in weight gain and plasma carotenoids nor from increases in litter oocyst counts following a challenge infection administered on day 14 relative to NIC. In addition, pens of birds where 100% of chicks were immunized were not well protected compared to NIC, and resistance to coccidiosis infection in immunized chicks was less than resistance in chicks challenged at 21 days. These results in total suggest that, when birds are challenged after 21 days, cohorts are protected from detrimental effects of challenge infection. However, when challenge infection is given at 14 days, cohorts are not well protected. The results support a conclusion that protection to coccidiosis is conveyed to cohorts by contact with oocysts shed into the litter by immunized chicks, but this resistance may take 14 days to develop.

Determining Resistance of Toxoplasma gondii Oocysts to UV Disinfection Using Cell Culture and a Mouse Bioassay

USDA-ARS?s Scientific Manuscript database

The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated irradiated oocysts by three assays: a SCID mouse biassay, an in vitro T. gondii oocyst plaque assay (TOP-assay), and a quantitative reverse-transcriptase real-t...

Cryptosporidium sp. infections in green turtles, Chelonia mydas, as a potential source of marine waterborne oocysts in the Hawaiian Islands

USGS Publications Warehouse

Graczyk, T.K.; Balazs, G.H.; Work, Thierry M.; Aguirre, A.A.; Ellis, D.M.; Murakawa, Shawn K. K.; Morris, Robert

1997-01-01

For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.

Eimeria oocyst concentrations and species composition in litter from commercial broiler farms during anticoccidial drug or live Eimeria oocyst vaccine control programs

USDA-ARS?s Scientific Manuscript database

The purpose of this study was to determine if Eimeria oocyst concentrations and species composition in commercial broiler house litter changed during different cycles of anticoccidial drug (ACD) or live Eimeria oocyst vaccine (VAC) control programs, and if there was a correlation between Eimeria ooc...

Use of aerobic spores as a surrogate for cryptosporidium oocysts in drinking water supplies.

PubMed

Headd, Brendan; Bradford, Scott A

2016-03-01

Waterborne illnesses are a growing concern among health and regulatory agencies worldwide. The United States Environmental Protection Agency has established several rules to combat the contamination of water supplies by cryptosporidium oocysts, however, the detection and study of cryptosporidium oocysts is hampered by methodological and financial constraints. As a result, numerous surrogates for cryptosporidium oocysts have been proposed by the scientific community and efforts are underway to evaluate many of the proposed surrogates. The purpose of this review is to evaluate the suitability of aerobic bacterial spores to serve as a surrogate for cryptosporidium oocysts in identifying contaminated drinking waters. To accomplish this we present a comparison of the biology and life cycles of aerobic spores and oocysts and compare their physical properties. An analysis of their surface properties is presented along with a review of the literature in regards to the transport, survival, and prevalence of aerobic spores and oocysts in the saturated subsurface environment. Aerobic spores and oocysts share many commonalities with regard to biology and survivability, and the environmental prevalence and ease of detection make aerobic spores a promising surrogate for cryptosporidium oocysts in surface and groundwater. However, the long-term transport and release of aerobic spores still needs to be further studied, and compared with available oocyst information. In addition, the surface properties and environmental interactions of spores are known to be highly dependent on the spore taxa and purification procedures, and additional research is needed to address these issues in the context of transport. Published by Elsevier Ltd.

[Eight new species of coccidia (Sporozoa, Coccidia) in fishes from the continental waters of Russia].

PubMed

Belova, L M; Krylov, M V

2001-01-01

Description of coccidian species infecting freshwater fishes is given. Localities: Neman river, Goussia peleci sp. n.--oocyst 37.5-45 x 35-42.5 microns, oocyst residuum (OR) absent, sporocysts 20-22.5 x 15-17.5 microns, Stieda body (SB) absent, sporocyst residuum (SR) present, host (h)--Pelecus cultratus; G. cultrati sp. n.--oocyst 22.5-30 microns, OR absent, sporocysts 12.5 x 15 microns, SR, SB absent, wall 2-2.5 microns, h--P. cultratus; G. arinae sp. n.--oocyst 12.5 x 17.5 microns, OR present, sporocysts 5-7.5 microns, SR, SB absent, h--P. cultratus; G. vimbae sp. n.--oocyst 15-22.5 microns, OR absent, wall 2.5 microns, sporocysts 5-7.5 microns, SR, SB absent, h--Vimba vimba vimba; G. gymnocephali sp. n.--oocyst 25 x 25 microns, OR absent, sporocysts 10 x 12.5 microns, SR, SB absent, h--Gymnocephalus cernuus; G. cernui sp. n.--oocyst 15-22.5 microns, OR present, sporocysts 5-12.5 microns, SR, SB absent, h--Gym. cernuus; G. luciopercae sp. n.--oocyst 30-35 x 30 microns, OR absent, sporocysts 12.5-15 x 10-12.5 microns, SB absent, SR present, h--Stizostedion lucioperca; Eimeria fluviatili sp. n.--oocyst 20-22.5 microns, OR present, sporocysts 7.5 x 12.5 microns, SB present, SR absent, h--Perca fluviatilis.

Depletion of Cryptosporidium parvum Oocysts from Contaminated Sewage by Using Freshwater Benthic Pearl Clams (Hyriopsis schlegeli)

PubMed Central

Yagita, Kenji; Izumiyama, Shinji; Endo, Takuro; Itoh, Yasoo

2012-01-01

The freshwater benthic pearl clam, Hyriopsis schlegeli, was experimentally exposed to Cryptosporidium parvum oocysts, and it was verified that the oocysts were eliminated predominantly via the fecal route, retaining their ability to infect cultured cells (HCT-8). The total fecal oocyst elimination rate was more than 90% within 5 days after exposure to the oocysts. H. schlegeli was able to survive in the final settling pond of a sewage plant for long periods, as confirmed by its pearl production. In the light of these findings, the clam was placed in the final settling pond in a trial to test its long-term efficacy in depleting oocysts contaminating the pond water. The number of clams placed was set to ensure a theoretical oocyst removal rate of around 50%, and the turbidity and the density of feed microbes in the overflow trough water of the pond were about 35% and 40 to 60% lower, respectively, than in the control water throughout the year. It was found that the clam feces containing oocysts were sufficiently heavy for them to settle to the bottom of the pond, despite the upward water flow. From these results, we concluded that efficient depletion of oocysts in the sewage water of small or midscale sewage treatment plants can be achieved by appropriate placement of H. schlegeli clams. PMID:22904053

New filtration system for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts.

PubMed

Al-Sabi, M N S; Gad, J A; Riber, U; Kurtzhals, J A L; Enemark, H L

2015-09-01

To develop a filtration unit for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts ((oo-)cysts) in drinking water. This unit utilizes a metallic filter and an ultrasound transducer for eluting (oo-)cysts, with a fixed retentate backwash volume; approx. 400 μl. Changes in the viability was evaluated by seeding wild type (oo-)cysts (1 × 10(4)) followed by sonication for 5, 10, 20 or 40 s (five replicates for each period). Flow cytometry analysis showed negligible increase in the mortality of (oo-)cysts exposed to 5-10 s of sonication. Recovery rate was assessed by seeding ColorSeed(™) (10 replicates) into the filter unit followed by air backwash to a glass slide and counting of (oo-)cysts by epifluorescent microscopy. High recovery rates (mean ± SD) were found: 84·9% ± 4·8 for Giardia cysts and 70% ± 6·5 for Cryptosporidium oocysts. DNA of seeded wild type (oo-)cysts (1 × 10(2); 10 replicates) was successfully amplified using real-time PCR. The use of a metallic filter, sonication and 'air backwash' were key factors for creating a highly efficient system for recovery of apparently undamaged protozoa. This reagent-less system can be used for monitoring of parasite contamination in drinking water. © 2015 The Society for Applied Microbiology.

[Oocyst structure and problem of coccidian taxonomy].

PubMed

Beĭer, T B; Svezhova, N V; Sidorenko, N V

2001-01-01

A comparative ultrastructural study was made of both thin- and thick-walled oocysts of Cryptosporidium parvum. According to the authors' findings, all the oocysts in C. parvum should be considered as thin-walled, since their walls have been composed of a single membrane or of two, closely apposed membranes without any additional substance in between. Despite the presence of two types of wall-forming bodies (WFB) in the maturing macrogamete or zygote, there is no evidence of their involvement in oocyst wall formation. In this concern, the function and destiny of WFB in C. parvum oocysts still remain obscure. Similar structure of the oocysts wall was reported elsewhere for thin-walled oocysts of fish coccidia of the genera Goussia and Eimeria. In C. parvum, the "thick-walled" oocysts differ from oocysts with thin walls in the availability in the former of a single sporocyst. The sporocyst wall consists of two unequal layers: a thin outer layer and a thicker inner one, in which a characteristic suture line is occasionally seen. By this feature the thick-walled oocysts of C. parvum bear similarities with oocysts of the cyst-forming coccidia (Cystoisospora, Toxoplasma, Sarcocystis) and of the genus Goussia: in all these the valves making up the sporocyst wall are joint just along the suture line. The literary and the authors' own data make it possible to suppose that the suture detected in C. parvum oocysts is located in the sporocyst wall, joining its valves, rather than in the oocyst wall proper, known to be composed of one or two, closely apposed unit membranes. Again, the availability of a suture (or sutures) in the sporocyst hardly provides enough reason to relate C. parvum with either cyst-forming, or fish coccidia, since this structure itself may be of a convergency character, rather than of systematic value. This may be substantiated, at least in part, by the authors' previous findings (Beyer, Sidorenko, 1984) of a similar structure, originally referred to as a "slit channel", in the intraerythrocytic capsule around gamont stage of haemogregarines--the adeleid coccidia of the genus Karyolysus. The suture-like structure could have originated in the evolution independently in different groups of parasitic protozoa to serve eventually as a suitable mechanism for immediate separation of elements involved in protective formation harbouring different developmental stages, including, for example, sporozoites in the eimeriid coccidia, or gamonts in the adeleid coccidia.

Cryptosporidium Attenuation across the Wastewater Treatment Train: Recycled Water Fit for Purpose

PubMed Central

Fanok, Stella; Phillips, Renae; Lau, Melody; van den Akker, Ben; Monis, Paul

2016-01-01

ABSTRACT Compliance with guideline removal targets for Cryptosporidium which do not provide any credit for the inactivation of oocysts through wastewater treatment processes can considerably increase the cost of providing recycled water. Here we present the application of an integrated assay to quantify both oocyst numbers and infectivity levels after various treatment stages at three Victorian and two South Australian (SA) wastewater treatment plants (WWTPs). Oocyst density in the raw sewage was commensurate with community disease burden, with early rounds of sampling capturing a widespread cryptosporidiosis outbreak in Victoria. The level of infectivity of oocysts in sewage was stable throughout the year but was significantly lower at the SA WWTPs. Removals across secondary treatment processes were seasonal, with poorer removals associated with inflow variability; however, no decrease in the oocyst infectivity was identified. For SA WWTPs, those oocysts remaining within the secondary treatment-clarified effluent were proportionally more infectious than those in raw sewage. Lagoon systems demonstrated significant inactivation or removal of oocysts, with attenuation being seasonal. Examination of a UV system emphasized its efficacy as a disinfectant barrier but conversely confirmed the importance of a multibarrier approach with the detection of infectious oocysts postdisinfection. The ability to characterize risk from infectious oocysts revealed that the risk from Cryptosporidium is significantly lower than previously thought and that its inclusion in quantitative risk assessments of reuse systems will more accurately direct the selection of treatment strategies and capital expenditure, influencing the sustainability of such schemes. IMPORTANCE Here we present the application of a recently developed integrated assay not only to quantify the removal of Cryptosporidium oocysts but also to quantify their infectivity across various treatment stages at five wastewater treatment plants (WWTPs), thereby better measuring the “true effect” of the treatment train on oocyst risk reduction. For a number of the WWTPs analyzed in this study the risk, is significantly lower than previously thought. Therefore, the inclusion of oocyst infectivity in guideline values and in quantitative microbial risk assessment (QMRA) has the potential to affect future treatment directions and capital expenditure. PMID:28039137

Eimeria Oocyst Concentrations and Species Composition in Litter from Commercial Broiler Farms During Anticoccidial Drug or Live Eimeria Oocyst Vaccine Control Programs.

PubMed

Jenkins, Mark C; Parker, Carolyn; Ritter, Donald

2017-06-01

The purpose of this study was to determine if Eimeria oocyst concentrations and species composition in commercial broiler house litter changed during different cycles of anticoccidial drug (ACD) or live Eimeria oocyst vaccine (VAC) control programs and if there was a correlation between Eimeria oocyst levels and broiler performance. Litter samples were collected from a total of 15 different broiler farms encompassing a total of 45 individual houses during at least one complete grow-out cycle over a 21-mo period. Of these 15 broiler farms, three were followed for the entire 21-mo period spanning three ACD and four VAC cycles. Samples were collected at 2, 4, and 7-8 wk of grow-out corresponding to starter, grower, and withdraw periods of the ACD cycle. On a number of occasions, litter samples were obtained just prior to chick placement. Eimeria oocysts were isolated from all samples, counted by microscopy, and extracted for DNA to identify Eimeria species by ITS1 PCR. In general, Eimeria oocyst concentration in litter reached peak levels at 2-4 wk of grow-out regardless of coccidiosis control measure being used. However, peak oocyst numbers were sometimes delayed until 7-8 wk, indicating some level of Eimeria spp. drug resistance or incomplete vaccine coverage. Eimeria maxima , Eimeria acervulina , Eimeria praecox, and Eimeria tenella were generally present in all samples, and no difference in the species composition was noted between houses on a particular farm. While Eimeria species composition was similar among houses, Eimeria spp. oocyst levels exhibited sporadic peaks in one house of a given location's houses. Of particular interest was the observed correlation between E. maxima oocyst abundance and chick mortality. However, no correlation was observed in E. maxima oocyst levels, and the performance parameters adjusted feed conversion ratio and average daily weight gain. This study showed that understanding the dynamics of Eimeria spp. oocyst levels and species composition in litter during ACD or VAC programs may provide insight into the effectiveness of coccidiosis control measures in commercial broiler production.

Cryptosporidium Attenuation across the Wastewater Treatment Train: Recycled Water Fit for Purpose.

PubMed

King, Brendon; Fanok, Stella; Phillips, Renae; Lau, Melody; van den Akker, Ben; Monis, Paul

2017-03-01

Compliance with guideline removal targets for Cryptosporidium which do not provide any credit for the inactivation of oocysts through wastewater treatment processes can considerably increase the cost of providing recycled water. Here we present the application of an integrated assay to quantify both oocyst numbers and infectivity levels after various treatment stages at three Victorian and two South Australian (SA) wastewater treatment plants (WWTPs). Oocyst density in the raw sewage was commensurate with community disease burden, with early rounds of sampling capturing a widespread cryptosporidiosis outbreak in Victoria. The level of infectivity of oocysts in sewage was stable throughout the year but was significantly lower at the SA WWTPs. Removals across secondary treatment processes were seasonal, with poorer removals associated with inflow variability; however, no decrease in the oocyst infectivity was identified. For SA WWTPs, those oocysts remaining within the secondary treatment-clarified effluent were proportionally more infectious than those in raw sewage. Lagoon systems demonstrated significant inactivation or removal of oocysts, with attenuation being seasonal. Examination of a UV system emphasized its efficacy as a disinfectant barrier but conversely confirmed the importance of a multibarrier approach with the detection of infectious oocysts postdisinfection. The ability to characterize risk from infectious oocysts revealed that the risk from Cryptosporidium is significantly lower than previously thought and that its inclusion in quantitative risk assessments of reuse systems will more accurately direct the selection of treatment strategies and capital expenditure, influencing the sustainability of such schemes. IMPORTANCE Here we present the application of a recently developed integrated assay not only to quantify the removal of Cryptosporidium oocysts but also to quantify their infectivity across various treatment stages at five wastewater treatment plants (WWTPs), thereby better measuring the "true effect" of the treatment train on oocyst risk reduction. For a number of the WWTPs analyzed in this study the risk, is significantly lower than previously thought. Therefore, the inclusion of oocyst infectivity in guideline values and in quantitative microbial risk assessment (QMRA) has the potential to affect future treatment directions and capital expenditure. Copyright © 2017 American Society for Microbiology.

Quantitative Estimation of the Viability of Toxoplasma gondii Oocysts in Soil

PubMed Central

Villena, Isabelle; Dardé, Marie-Laure; Aubert, Dominique; Geers, Régine; Dupuis, Emilie; Marnef, Francine; Poulle, Marie-Lazarine; Gotteland, Cécile; Dumètre, Aurélien

2012-01-01

Toxoplasma gondii oocysts spread in the environment are an important source of toxoplasmosis for humans and animal species. Although the life expectancy of oocysts has been studied through the infectivity of inoculated soil samples, the survival dynamics of oocysts in the environment are poorly documented. The aim of this study was to quantify oocyst viability in soil over time under two rain conditions. Oocysts were placed in 54 sentinel chambers containing soil and 18 sealed water tubes, all settled in two containers filled with soil. Containers were watered to simulate rain levels of arid and wet climates and kept at stable temperature for 21.5 months. At nine sampling dates during this period, we sampled six chambers and two water tubes. Three methods were used to measure oocyst viability: microscopic counting, quantitative PCR (qPCR), and mouse inoculation. In parallel, oocysts were kept refrigerated during the same period to analyze their detectability over time. Microscopic counting, qPCR, and mouse inoculation all showed decreasing values over time and highly significant differences between the decreases under dry and damp conditions. The proportion of oocysts surviving after 100 days was estimated to be 7.4% (95% confidence interval [95% CI] = 5.1, 10.8) under dry conditions and 43.7% (5% CI = 35.6, 53.5) under damp conditions. The detectability of oocysts by qPCR over time decreased by 0.5 cycle threshold per 100 days. Finally, a strong correlation between qPCR results and the dose infecting 50% of mice was found; thus, qPCR results may be used as an estimate of the infectivity of soil samples. PMID:22582074

Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts

PubMed Central

Quilez, Joaquin; Sanchez-Acedo, Caridad; Avendaño, Catalina; del Cacho, Emilio; Lopez-Bernad, Fernando

2005-01-01

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4′,6′-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection. PMID:15870337

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

PubMed Central

Bushkin, G. Guy; Chatterjee, Aparajita; Robbins, Phillips W.

2013-01-01

Cysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, β-1,3-linked glucose for Toxoplasma, and β-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the β-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (β-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins. PMID:24096907

Adaptation and immunogenicity of Cryptosporidium parvum to immunocompetent mice.

PubMed

Matsuo, Tomohide; Tsuge, Yasuko; Umemiya-Shirafuji, Rika; Fujino, Takashi; Matsui, Toshihiro

2014-03-01

The adaptation and immunogenisity of Cryptosporidium parvum isolated from Siberian chipmunks (SC1 strain) in immunocompetent (ICR) mice were examined. The oocysts were received to the severe combined immunodeficiency (SCID) mice by repeated passage. The oocysts collected from the 18th SCID mice were inoculated to 5 ICR mice. The mice began to shed oocysts from 6 days after inoculation, the patency was 5 days, and the maximum oocysts per gram of feces (OPG) value was 10(4). The maximum of OPG value was gradually increased by successive passage, and finally that in the 22nd mice reached 10(6) (patency: 11 days). It is considered that these results indicate completion of their adaptation to ICR mice. To examine the immunogenicity of C. parvum to ICR mice, 8 groups of 5 mice each were inoculated with 1.3 × 10(6) oocysts of SC1 strain, which were collected after adaptation to SCID mice. All groups shed oocysts from 6th day, and their patency was from 8 to 12 days. On the 21st day after the primary infection, these mice were challenged with 1.3 × 10(6) oocysts. No oocysts shed from any groups, although 2 control groups shed oocysts from the 6th day, and their OPG values were more than 10(6). These results suggest that this strain has strong immunogenicity against ICR mice. Therefore, the immunological healthy mice were considered a useful experimental model to investigate immunological and drug treatments in the strain of C. parvum.

F-actin distribution and function during sexual development in Eimeria maxima.

PubMed

Frölich, Sonja; Wallach, Michael

2015-06-01

To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

Molecules to modeling: Toxoplasma gondii oocysts at the human–animal–environment interface

PubMed Central

VanWormer, Elizabeth; Fritz, Heather; Shapiro, Karen; Mazet, Jonna A.K.; Conrad, Patricia A.

2013-01-01

Environmental transmission of extremely resistant Toxoplasma gondii oocysts has resulted in infection of diverse species around the world, leading to severe disease and deaths in human and animal populations. This review explores T. gondii oocyst shedding, survival, and transmission, emphasizing the importance of linking laboratory and landscape from molecular characterization of oocysts to watershed-level models of oocyst loading and transport in terrestrial and aquatic systems. Building on discipline-specific studies, a One Health approach incorporating tools and perspectives from diverse fields and stakeholders has contributed to an advanced understanding of T. gondii and is addressing transmission at the rapidly changing human–animal–environment interface. PMID:23218130

[Experimental transmission of Cryptosporidium baileyi (Apicomplexa: Cryptosporidiidae) isolated of broiler chicken to Japanese quail (Coturnix japonica)].

PubMed

Cardozo, Sergian V; Teixeira Filho, Walter L; Lopes, Carlos Wilson G

2005-01-01

In this work, oocysts of Cryptosporidium baileyi were isolated and identified in broiler chickens from three different Municipalities of the State of Rio de Janeiro, where they were isolated and identified by using the centrifuge- flotation technique associated to bright-field. Staining techniques, such as: modified Ziehl-Neelsen and safranin-methylene blue, were carried out to confirm natural infection. Oocysts of C. baileyi from broiler chickens were able to infect Coccidia-free Japanese quails, by observation of endogenous stages at histological sections, and the elimination of oocysts in the feces with prepatent period of seven days and patent period of 21 days after infection. Oocysts of C. baileyi from broiler chickens and Japanese quails were similar on bright-field microscopy. With respect to the staining techniques used in this research, all of them left to significant changes in length and width of oocysts, but shape indexes were maintained. Bright-field microscopy was the best technique for oocysts comparison shed by broiler chickens and Japanese quail because of no different among oocysts were observed.

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

PubMed

Everson, William V; Ware, Michael W; Dubey, J P; Lindquist, H D Alan

2002-01-01

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.

Detection of Cryptosporidium sp. Oocyst and Giardia sp. cyst in faucet water samples from cattle and goat farms in Taiwan.

PubMed

Watanabe, Yuko; Kimura, Kenji; Yang, Cheng-Hsiung; Ooi, Hong-Kean

2005-12-01

A survey on the presence of Cryptosporidium oocyst and Giardia cyst in livestock drinking water as well as the urban tap water throughout Taiwan was carried out. Water examination for the presence of the protozoa was conducted by filtering through a PTFE membrane followed by immunomagnetic separation (IMS) and immunostaining the sediment with commercially available monoclonal antibody against Cryptosporidium and Giardia. Of the 55 different water samples from various sources examined, 2 were found to contain both of Cryptosporidium oocyst and Giardia cyst, 1 was found to contain Cryptosporidium oocyst only. These protozoa-positive water samples, originating from underground well and from the mountain spring, were also used as drinking water for livestock. However, no Cryptosporidium oocyst was found in the city tap water. This is the first report of Cryptosporidium oocyst and Giardia cyst being found in the drinking water for livestock.

Motility precedes egress of malaria parasites from oocysts

PubMed Central

Klug, Dennis; Frischknecht, Friedrich

2017-01-01

Malaria is transmitted when an infected Anopheles mosquito deposits Plasmodium sporozoites in the skin during a bite. Sporozoites are formed within oocysts at the mosquito midgut wall and are released into the hemolymph, from where they invade the salivary glands and are subsequently transmitted to the vertebrate host. We found that a thrombospondin-repeat containing sporozoite-specific protein named thrombospondin-releated protein 1 (TRP1) is important for oocyst egress and salivary gland invasion, and hence for the transmission of malaria. We imaged the release of sporozoites from oocysts in situ, which was preceded by active motility. Parasites lacking TRP1 failed to migrate within oocysts and did not egress, suggesting that TRP1 is a vital component of the events that precede intra-oocyst motility and subsequently sporozoite egress and salivary gland invasion. DOI: http://dx.doi.org/10.7554/eLife.19157.001 PMID:28115054

Integrated Cryptosporidium Assay To Determine Oocyst Density, Infectivity, and Genotype for Risk Assessment of Source and Reuse Water

PubMed Central

King, Brendon; Fanok, Stella; Phillips, Renae; Swaffer, Brooke

2015-01-01

Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures. PMID:25769833

Integrated cryptosporidium assay to determine oocyst density, infectivity, and genotype for risk assessment of source and reuse water.

PubMed

King, Brendon; Fanok, Stella; Phillips, Renae; Swaffer, Brooke; Monis, Paul

2015-05-15

Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Association of Cryptosporidium parvum With Suspended Sediments: Implications for Transport in Surface Waters

NASA Astrophysics Data System (ADS)

Searcy, K. E.; Packman, A. I.; Atwill, E. R.; Harter, T.

2003-12-01

Understanding the transport and fate of microorganisms in surface waters is of vital concern in protecting the integrity and safety of municipal water supply systems. The human pathogen Cryptosporidium parvum is a particular public health interest, as it is ubiquitous in the surface waters of the United States, it can persist for long periods in the environment, and it is difficult to disinfect in water treatment plants. Due to its small size (5 um), low specific gravity (1.05 g/cm3), and negative surface charge, C. parvum oocysts are generally considered to move through watersheds from their source to drinking water reservoirs with little attenuation. However, the transport of the oocysts in surface waters may be mediated by interactions with suspended sediments. Batch experiments were conducted to determine the extent of C. parvum oocyst attachment to several inorganic and organic sediments under varying water chemical conditions, and settling column experiments were performed to demonstrate how these associations influence the effective settling velocity of C. parvum oocysts. Results from these experiments showed that C. parvum oocysts do associate with inorganic and organic sediments and often settle at the rate of the suspended sediment. The size and surface charge of the host suspended sediment influenced the extent of oocyst attachment as oocysts preferentially associated with particles greater than 3 um, and fewer oocysts associated with particles having a highly negative surface charge. Background water chemical conditions including ionic strength, ion composition, and pH did not have a significant effect on oocyst attachment to suspended sediments.

Role of Wall Shear Stress in Cryptosporidium parvum Oocyst Attachment to Environmental Biofilms.

PubMed

Luo, Xia; Jedlicka, Sabrina S; Jellison, Kristen L

2017-12-15

This study investigated Cryptosporidium parvum oocyst deposition onto biofilms as a function of shear stress under laminar or turbulent flow. Annular rotating bioreactors were used to grow stabilized stream biofilms at shear stresses ranging from 0.038 to 0.46 Pa. These steady-state biofilms were then used to assess the impact of hydrodynamic conditions on C. parvum oocyst attachment. C. parvum deposition onto biofilms followed a pseudo-second-order model under both laminar (after a lag phase) and turbulent flows. The total number of oocysts attached to the biofilm at steady state decreased as the hydrodynamic wall shear stress increased. The oocyst deposition rate constant increased with shear stress but decreased at high shear, suggesting that increasing wall shear stress results in faster attachment of Cryptosporidium due to higher mass transport until the shear forces exceed a critical limit that prevents oocyst attachment. These data show that oocyst attachment in the short and long term are impacted differently by shear: higher shear (to a certain limit) may be associated with faster initial oocyst attachment, but lower shear is associated with greater numbers of oocysts attached at equilibrium. IMPORTANCE This research provides experimental evidence to demonstrate that shear stress plays a critical role in protozoan-pathogen transport and deposition in environmental waters. The data presented in this work expand scientific understanding of Cryptosporidium attachment and fate, which will further influence the development of timely and accurate sampling strategies, as well as advanced water treatment technologies, to target protozoan pathogens in surface waters that serve as municipal drinking water sources. Copyright © 2017 American Society for Microbiology.

DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN WATER MATRICES

EPA Science Inventory

Since the advent and recognition of waterborne outbreaks of cryptosporidiosis great effort has been expended on development of methods for detecting Cryptosporidium oocysts in water. Oocysts recovery rates using a method originally developed for detecting Giardia cysts ranged fr...

Toxoplasma gondii: A study of oocyst re-shedding in domestic cats.

PubMed

Zulpo, Dauton Luiz; Sammi, Ana Sue; Dos Santos, Joeleni Rosa; Sasse, João Pedro; Martins, Thais Agostinho; Minutti, Ana Flávia; Cardim, Sérgio Tosi; de Barros, Luiz Daniel; Navarro, Italmar Teodorico; Garcia, João Luis

2018-01-15

The aim of the present study was to evaluate the re-shedding of T. gondii oocysts in cats fed tissue cysts of homologous and heterologous strains 12, 24 and 36 months after the first infection. Thirteen cats were used in the present study and were divided into four groups: G1 (n=2), G2 (n=3), G3 (n=5), and G4 (n=3). G1, G3 and G4 cats were infected with brain cysts of ME49 and G2 with TgDoveBr8, both genotype II strains of T. gondii. The G1 and G2 cats were re-infected after twelve months with brain cysts of VEG strain (genotype III), and G3 cats were re-infected with TgDoveBr1 (genotype II). The G3 cats were re-infected a third time after 24 months from the second infection, and the G4 cats were re-infected 36 months after the initial infection with cysts of the VEG strain. The cats' feces were evaluated using fecal flotation and genotyped with PCR-RFLP. The serological responses for IgM, IgA and IgG were determined by ELISA. All cats shed oocysts after the initial infection. Only one G1 cat shed oocysts when re-infected after twelve months with the VEG strain. No G2 cats excreted oocysts after the second infection with VEG. G3 cats, when re-infected after twelve months with the TgDoveBr1 strain, did not shed oocysts. However, when challenged after a third time with the VEG strain, three out of four cats shed oocysts. In the G4 group, when re-infected after thirty-six months with the VEG strain, two out of three cats shed oocysts. All oocyst samples were genotyped and characterized as the same genotype from the inoculum. Protection against oocyst re-excretion occurred in 90%, 25%, and 33.4% of cats after 12, 24, and 36 months from the initial infection, respectively. Therefore, the environmental contamination by oocysts from re-infected adult cats is only 30% lower than from kittens. In conclusion, the excretion of T. gondii oocysts was higher in experimentally re-infected cats throughout the years, especially when a heterologous strain was used. Copyright © 2017 Elsevier B.V. All rights reserved.

Hammondia heydorni oocysts in the faeces of a greyhound in New Zealand.

PubMed

Ellis, J T; Pomroy, W E

2003-02-01

To identify oocysts found in faecal material of a greyhound. Polymerase chain reaction (PCR) and DNA sequencing were used to study genomic DNA isolated from oocysts purified from faeces of a greyhound. Database searches with the DNA sequences obtained showed they were derived from Hammondia heydorni. A species-specific PCR was developed to detect H. heydorni DNA. Light microscopy in conjunction with PCR and DNA sequencing definitively identified the presence of H. heydorni oocysts in faeces of a greyhound. This study confirms the presence of H. heydorni in New Zealand and indicates the need to correctly identify similar oocysts from dogs, rather than assume they are Neospora caninum.

Four new species of coccidia (Apicomplexa: Eimeriidae) from Owen Stanley Skinks, Papuascincus stanleyanus (Sauria: Scincidae), from Papua New Guinea

USGS Publications Warehouse

McAllister, Chris T.; Duszynski, Donald W.; Fisher, Robert N.; Austin, Christopher C.

2014-01-01

Between September and November 1991, 12 Owen Stanley skinks, Papuascincus stanleyanus (Booulenger) were collected from various localities on Papua New Guinea and examined for coccidians. Six (50%) were found to harbour four eimerians that we describe here as new. Oocysts of Eimeria burseyi sp. n. were elongate to ellipsoidal with a bilayered wall and measured (length x width, L x W) 36.0 x 24.0 microm, with a L/W ratio of 1.5. Both micropyle and oocyst residuum were absent, but a polar granule was present. Oocysts of Eimeria goldbergi sp. n. were ellipsoidal, with a bilayered wall, and measured 21.4 x 16.1 microm; L/W ratio was 1.3. Both micropyle and oocyst residuum were absent, but a single or fragmented polar granule was present. Oocysts of Eimeria boulengeri sp. n. were spheroidal to slightly subspheroidal, with a thin, single-layered wall that readily collapses, and measured 16.0 microm, L/W ratio was 1.0. Both micropyle and oocyst residuum were absent, but usually one (sometimes two) polar granule(s) were present. Oocysts of Eimeria niuginiensis sp. n. were oblong to tapered with a bilayered wall, and measured 20.0 x 13.1 microm; L/W ratio was 1.5. A micropyle, oocyst residuum and polar granule were absent. To our knowledge, these represent the only coccidians ever described from P. stanleyanus.

RESEARCH NOTE: AUTOFLUORESCENCE OF TOXOPLASMA GONDII OOCYSTS

EPA Science Inventory

This is the first report of a blue autofluorescence as a useful characteristic in the microscopic identification of Toxoplasma gondii oocysts. This autofluorescence appears to be of high intensity. Similar to the autofluorescence of related coccidia, the oocysts glow pale blue ...

In vitro activity of natural and chemical products on sporulation of Eimeria species oocysts of chickens.

PubMed

Gadelhaq, Sahar M; Arafa, Waleed M; Abolhadid, Shawky M

2018-02-15

This study was designed to investigate the ability of two herbal extracts and different chemical substances to inhibit or disrupt sporulation of Eimeria species oocysts of the chickens. The two herbal extracts were Allium sativum (garlic) and Moringa olifiera while the chemical substances included commercial disinfectants and diclazuril. Field isolates of Eimeria oocysts were propagated in chickens to obtain a continuous source of oocysts. The collected unsporulated oocysts (10 5 oocysts/5 ml) were dispensed into 5 cm Petri dish. Three replicates were used for each treatment. The treated oocysts were incubated for 48 h at 25-29 °C and 80% relative humidity. The results showed that herbal extracts, the commercial recommended dose of Dettol, TH4, Phenol, Virkon ® S, and Diclazuril 20% have no effect on the sporulation. While Sodium hypochlorite showed a significant degree of sporulation inhibition reached to 49.67%. Moreover, 70% ethanol, and 10% formalin showed 100% sporulation inhibition. It was concluded that 70% ethanol and 10% formalin are the most effective methods to inhibit Eimeria species sporulation. Copyright © 2017 Elsevier B.V. All rights reserved.

Transport of Cryptosporidium oocysts in porous media: Role of straining and physicochemical filtration

USGS Publications Warehouse

Tufenkji, N.; Miller, G.F.; Ryan, J.N.; Harvey, R.W.; Elimelech, M.

2004-01-01

The transport and filtration behavior of Cryptosporidium parvum oocysts in columns packed with quartz sand was systematically examined under repulsive electrostatic conditions. An increase in solution ionic strength resulted in greater oocyst deposition rates despite theoretical predictions of a significant electrostatic energy barrier to deposition. Relatively high deposition rates obtained with both oocysts and polystyrene latex particles of comparable size at low ionic strength (1 mM) suggest that a physical mechanism may play a key role in oocyst removal. Supporting experiments conducted with latex particles of varying sizes, under very low ionic strength conditions where physicochemical filtration is negligible, clearly indicated that physical straining is an important capture mechanism. The results of this study indicate that irregularity of sand grain shape (verified by SEM imaging) contributes considerably to the straining potential of the porous medium. Hence, both straining and physicochemical filtration are expected to control the removal of C. parvum oocysts in settings typical of riverbank filtration, soil infiltration, and slow sand filtration. Because classic colloid filtration theory does not account for removal by straining, these observations have important implications with respect to predictions of oocyst transport.

ISOLATION OF PURIFIED OOCYST WALLS AND SPOROCYSTS FROM TOXOPLASMA GONDII

EPA Science Inventory

Toxaplasma gondii is a parasitic protozoan that infects a wide range of vertebrates, including humans. This report describes methods that have been developed for separation of oocyst components starting with the mechanical fragmentation of oocysts. Use of iodixoanol gradients a...

UTILITY OF SURROGATES FOR MEASURING CRYPTOSPORIDIUM OOCYST INFECTIVITY

EPA Science Inventory

The water industry must assess whether Cryptosporidium oocysts detected in source and finished water are viable and/or infectious. Initial approaches measuring the infectious nature of C. parvum oocysts have focused on in vitro excystation and in vitro vital dye staining. Recen...

UTILITY OF SURROGATES FOR MEASURING CRYPTOSPORIDIUM OOCYSTS INFECTIVITY

EPA Science Inventory

The water industry must assess whether Cryptosporidium oocysts detected in source and finished water are viable and/or infectious. Initial approaches measuring the infectious nature of C. parvum oocysts have focused on in vitro excystation and in vitro vital dye staining. Recen...

Role of predation by zooplankton in transport and fate of protozoan (oo)cysts in granular activated carbon filtration.

PubMed

Bichai, Françoise; Barbeau, Benoit; Dullemont, Yolanda; Hijnen, Wim

2010-02-01

The significance of zooplankton in the transport and fate of pathogenic organisms in drinking water is poorly understood, although many hints of the role of predation in the persistence of microorganisms through water treatment processes can be found in literature. The objective of this study was to assess the impact of predation by natural zooplankton on the transport and fate of protozoan (oo)cysts in granular activated carbon (GAC) filtration process. UV-irradiated unlabelled Cryptosporidium parvum and Giardia lamblia (oo)cysts were seeded into two pilot-scale GAC filtration columns operated under full-scale conditions. In a two-week period after seeding, a reduction of free (oo)cysts retained in the filter bed was observed. Zooplankton was isolated from the filter bed and effluent water on a 30 microm net before and during the two-week period after seeding; it was enumerated and identified. Rotifers, which are potential predators of (oo)cysts, accounted for the major part of the isolated zooplankton. Analytical methods were developed to detect (oo)cysts internalized in natural zooplankton isolated from the filter bed and effluent water. Sample sonication was optimized to disrupt zooplankton organisms and release internalized microorganisms. (Oo)cysts released from zooplankton after sonication were isolated by IMS and stained (EasyStain) for microscopic counting. Both Cryptosporidium and Giardia (oo)cysts were detected in association with zooplankton in the filter bed samples as well as in the effluent of GAC filters. The results of this study suggest that predation by zooplankton can play a role in the remobilization of persistent pathogens such as Cryptosporidium and Giardia (oo)cysts retained in GAC filter beds, and consequently in the transmission of these pathogens in drinking water. Copyright 2009 Elsevier Ltd. All rights reserved.

Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

PubMed Central

Chauret, Christian P.; Radziminski, Chris Z.; Lepuil, Michael; Creason, Robin; Andrews, Robert C.

2001-01-01

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log10 and 2.0 log10 units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log10 units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity. PMID:11425712

GENOTYPING CRYPTOSPORIDIUM OOCYSTS IN WATER SAMPLES AS A TOOL FOR THE IDENTIFICATION OF CONTAMINATION SOURCES

EPA Science Inventory

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of an immunofluorescent assay (IFA). Because IFA detects oocysts from all Cryptosporidium parasites, the species distribution and source of Cryptosporidium parasites in environmental...

EFFECTS OF HIGH PRESSURE PROCESSING ON TOXOPLASMA GONDII OOCYSTS ON RASPBERRIES

USDA-ARS?s Scientific Manuscript database

Oocysts of Toxoplasma gondii are environmentally resistant stages. Humans can become infected by accidentally ingesting the oocysts in water or on contaminated produce. Severe disease can occur in immunocompromised individuals and non-immune pregnant women can infect their offspring. Chronic infect...

CONTROL OF CRYPTOSPORIDIUM OOCYSTS BY STEADY-STATE CONVENTIONAL TREATMENT

EPA Science Inventory

Pilot-scale experiments have been performed to assess the ability of conventional treatment to control Cryptosporidium oocysts under steady-state conditions. The work was performed with a pilot plant that was designed to minimize flow rates and, as a result, the number of oocyst...

Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction.

PubMed

Hill, D E; Liddell, S; Jenkins, M C; Dubey, J P

2001-04-01

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5-specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.

Roles of Tyrosine-Rich Precursor Glycoproteins and Dityrosine- and 3,4-Dihydroxyphenylalanine-Mediated Protein Cross-Linking in Development of the Oocyst Wall in the Coccidian Parasite Eimeria maxima

PubMed Central

Belli, Sabina I.; Wallach, Michael G.; Luxford, Catherine; Davies, Michael J.; Smith, Nicholas C.

2003-01-01

The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host. If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography, together with the detection of a UV autofluorescence in intact oocysts, implicates dityrosine- and possibly DOPA-protein cross-links in oocyst wall hardening. In addition, the identification of peroxidase activity in the wall-forming bodies of macrogametes supports the hypothesis that dityrosine- and DOPA-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes. PMID:12796290

Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

PubMed Central

Di Giovanni, George D.; Rochelle, Paul A.

2012-01-01

This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. PMID:22038611

Effect of Lot Variability on Ultraviolet Radiation Inactivation Kinetics of Cryptosporidium parvum Oocysts

EPA Science Inventory

Numerous studies have demonstrated the efficiency of ultraviolet (UV) radiation for the inactivation of oocysts of Cryptosporidium parvum. In these studies inactivation is measured as reduction in oocysts. A primary goal is to estimate the UV radiation required to achiev...

Toxoplasma gondii oocyst-specific antibodies and sources of infection

USDA-ARS?s Scientific Manuscript database

Sources of post-natal infection with T. gondii include consumption of undercooked meat containing tissue cysts or ingestion of oocysts in contaminated food or environment. This study quantified the risk of acquiring infection from oocysts versus meat in pregnant women from the Valdivia Province, Chi...

IDENTIFICATION OF SPECIES AND SOURCES OF CRYPTOSPORIDIUM OOCYSTS IN STORM WATERS BY A SMALL SUBUNIT RRNA-BASED DIAGNOSTIC AND GENOTYPING TOOL

EPA Science Inventory

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of immunofluorescent assay (IFA). because IFA detects oocysts from all Cryptosporidium parasites, the species distribution and source of Cryptosporidium parasites in environmental sa...

Spinacia oleracea L. leaf stomata harboring Cryptosporidium parvum oocysts: A potential threat for food safety

USDA-ARS?s Scientific Manuscript database

Scientific literature documents the prevalence of Cryptosporidium oocysts in irrigation waters and on fresh produce. In the present study spinach leaves were experimentally exposed to Cryptosporidium oocysts which were subsequently irrigated with clean water daily for 5 days. As determined by confoc...

RNA Extraction Methods for Real-Time PCR and Microarray Analyses of Cryptosporidium and Toxoplasma gondii Oocysts - 2nd Presentation

EPA Science Inventory

The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion pr...

Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalization

USDA-ARS?s Scientific Manuscript database

Toxoplasma gondii is a common parasite of humans and domestic animals, which is transmitted via oocysts in cat faeces or tissue cysts in contaminated meat. The oocyst and sporocyst walls are multilayered polymeric structures that protect the infective sporozoites from deleterious physical and chemic...

RNA Extraction Methods for Reverse Transcriptase Real-Time PCR and Microarray Analysis of Cryptosporidium and Toxoplasma gondii Oocysts

EPA Science Inventory

The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion p...

Immunoproteomic analysis of proteins from unsporulated Oocysts of Eimeria tenella in MALDI TOF/TOF tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

Immunoproteomic approaches were conducted to identify antigenic proteins from the total proteins of unsporulated oocysts of Eimeria tenella (E. tenella). Approximately 101 protein spots were recognized by chicken sera infected experimentally with E. tenella. Fourty-six spots of unsporulated oocysts ...

Effect of Batch-Process Solar Disinfection on Survival of Cryptosporidium parvum Oocysts in Drinking Water

PubMed Central

Méndez-Hermida, F.; Castro-Hermida, J. A.; Ares-Mazás, E.; Kehoe, S. C.; McGuigan, K. G.

2005-01-01

The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m−2) at 40°C. Viability assays (4′,6′-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively. PMID:15746372

Evaluation of Immunomagnetic Separation for Recovery of Infectious Cryptosporidium parvum Oocysts from Environmental Samples

PubMed Central

Rochelle, Paul A.; De Leon, Ricardo; Johnson, Anne; Stewart, Mic H.; Wolfe, Roy L.

1999-01-01

Two commercial immunomagnetic separation (IMS) kits for Cryptosporidium were compared for recovery of oocysts from environmental samples. Oocyst recovery efficiencies with the Dynal and Crypto-Scan kits ranged from 62 to 100% and 34 to 74%, respectively, for seeded environmental water concentrates (turbidity of 210 to 11,480 nephelometric turbidity units). Recovery efficiencies were dependent on the mechanism of agitation during the magnetic capture procedure. An assay combining in vitro cell culture and reverse transcriptase PCR demonstrated that oocysts recovered by IMS retained their infectivity. PMID:9925626

Influence of organic matter on the transport of Cryptosporidium parvum oocysts in a ferric oxyhydroxide-coated quartz sand saturated porous medium

USGS Publications Warehouse

Abudalo, R.A.; Ryan, J.N.; Harvey, R.W.; Metge, D.W.; Landkamer, Lee L.

2010-01-01

To assess the effect of organic matter on the transport of Cryptosporidium parvum oocysts in a geochemically heterogeneous saturated porous medium, we measured the breakthrough and collision efficiencies of oocysts as a function of dissolved organic matter concentration in a flow-through column containing ferric oxyhydroxide-coated sand. We characterized the surface properties of the oocysts and ferric oxyhydroxide-coated sand using microelectrophoresis and streaming potential, respectively, and the amount of organic matter adsorbed on the ferric oxyhydroxide-coated sand as a function of the concentration of dissolved organic matter (a fulvic acid isolated from Florida Everglades water). The dissolved organic matter had no significant effect on the zeta potential of the oocysts. Low concentrations of dissolved organic matter were responsible for reversing the charge of the ferric oxyhydroxide-coated sand surface from positive to negative. The charge reversal and accumulation of negative charge on the ferric oxyhydroxide-coated sand led to increases in oocyst breakthrough and decreases in oocyst collision efficiency with increasing dissolved organic matter concentration. The increase in dissolved organic matter concentration from 0 to 20 mg L-1 resulted in a two-fold decrease in the collision efficiency. ?? 2009 Elsevier Ltd.

Detection of Toxoplasma gondii oocysts in water: proposition of a strategy and evaluation in Champagne-Ardenne Region, France.

PubMed

Aubert, D; Villena, I

2009-03-01

Water is a vehicle for disseminating human and veterinary toxoplasmosis due to oocyst contamination. Several outbreaks of toxoplasmosis throughout the world have been related to contaminated drinking water. We have developed a method for the detection of Toxoplasma gondii oocysts in water and we propose a strategy for the detection of multiple waterborne parasites, including Cryptosporidium spp. and Giardia. Water samples were filtered to recover Toxoplasma oocysts and, after the detection of Cryptosporidium oocysts and Giardia cysts by immunofluorescence, as recommended by French norm procedure NF T 90-455, the samples were purified on a sucrose density gradient. Detection of Toxoplasma was based on PCR amplification and mouse inoculation to determine the presence and infectivity of recovered oocysts. After experimental seeding assays, we determined that the PCR assay was more sensitive than the bioassay. This strategy was then applied to 482 environmental water samples collected since 2001. We detected Toxoplasma DNA in 37 environmental samples (7.7%), including public drinking water; however, none of them were positive by bioassay. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method. Alternative methods can be used in conjunction with this one to determine the infectivity of parasites that were detected by molecular methods.

Prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated sewage sludges.

PubMed

Amorós, Inmaculada; Moreno, Yolanda; Reyes, Mariela; Moreno-Mesonero, Laura; Alonso, Jose L

2016-11-01

Treated sludge from wastewater treatment plants (WWTPs) is commonly used in agriculture as fertilizers and to amend soils. The most significant health hazard for sewage sludge relates to the wide range of pathogenic microorganisms such as protozoa parasites.The objective of this study was to collect quantitative data on Cryptosporidium oocysts and Giardia cysts in the treated sludge in wastewater treatment facilities in Spain. Sludge from five WWTPs with different stabilization processes has been analysed for the presence of Cryptosporidium and Giardia in the raw sludge and after the sludge treatment. A composting plant (CP) has also been assessed. After a sedimentation step, sludge samples were processed and (oo)cysts were isolated by immunomagnetic separation (IMS) and detected by immunofluorescence assay (IFA). Results obtained in this study showed that Cryptosporidium oocysts and Giardia cysts were present in 26 of the 30 samples (86.6%) of raw sludge samples. In treated sludge samples, (oo)cysts have been observed in all WWTP's analysed (25 samples) with different stabilization treatment (83.3%). Only in samples from the CP no (oo)cysts were detected. This study provides evidence that (oo)cysts are present in sewage sludge-end products from wastewater treatment processes with the negative consequences for public health.

Comparative susceptibility of introduced forest-dwelling mosquitoes in Hawai'i to avian malaria, Plasmodium relictum

USGS Publications Warehouse

Lapointe, D.A.; Goff, M.L.; Atkinson, C.T.

2005-01-01

To identify potential vectors of avian malaria in Hawaiian native forests, the innate susceptibility of Aedes albopictus, Wyeomyia mitchellii, and Culex quinquefasciatus from 3 geographical sites along an altitudinal gradient was evaluated using local isolates of Plasmodium relictum. Mosquitoes were dissected 5-8 and 9-13 days postinfective blood meal and microscopically examined for oocysts and salivary-gland sporozoites. Sporogony was completed in all 3 species, but prevalence between species varied significantly. Oocysts were detected in 1-2% and sporozoites in 1-7% of Aedes albopictus that fed on infected ducklings. Wyeomyia mitchellii was slightly more susceptible, with 7-19% and 7% infected with oocysts and sporozoites, respectively. In both species, the median oocyst number was 5 or below. This is only the second Wyeomyia species reported to support development of a malarial parasite. Conversely, Culex quinquefasciatus from all 3 sites proved very susceptible. Prevalence of oocysts and sporozoites consistently exceeded 70%, regardless of gametocytemia or origin of the P. relictum isolate. In trials for which a maximum 200 oocysts were recorded, the median number of oocysts ranged from 144 to 200. It was concluded that Culex quinquefasciatus is the primary vector of avian malaria in Hawai'i. ?? American Society of Parasitologists 2005.

Bioaccumulation and elimination of Cryptosporidium parvum oocysts in experimentally exposed Eastern oysters (Crassostrea virginica) held in static tank aquaria.

PubMed

Willis, Jessica E; McClure, J T; McClure, Carol; Spears, Jonathan; Davidson, Jeff; Greenwood, Spencer J

2014-03-03

A variety of human enteropathogens, including viruses, bacteria, and parasites, have been shown to bioaccumulate in suspension-feeding bivalve shellfish. Cryptosporidium parvum is a zoonotic protozoan parasite that has been detected in many shellfish species within both fecally contaminated and clean oyster growing areas across the globe. For this study, C. parvum oocysts (1000 and 10,000) were spiked into 10 L of water in static tank systems housing Crassostrea virginica. Oysters were either held in the contaminated aquaria for 7 days of exposure or were exposed for 24h and subsequently placed in a clean static tank system for the remainder of the trial. Individual oysters, fecal material, and tank water were analyzed for oocysts up to 7 days post-exposure via direct immunofluorescence. Oysters held under chronic exposure conditions gradually accumulated oocysts (1.5 or 34.4 oocysts/oyster/day for low or high dose exposure groups, respectively) between days 1 and 7, with an exponential uptake in oocysts observed within the first 24h post-exposure (mean uptake of 29.6 or 241.9 oocysts/oyster, respectively). Oysters that were transferred to clean water after 24h were capable of slowly depurating oocysts, following a linear trend. During chronic exposure trials 48-49% of the total spiked inoculum was recovered from oyster tissue, whereas 4.8-5.9% and 38-40% was recovered from tank water and from fecal material at day 7, respectively. In acute exposure trials, 30-31% of the total tank inoculum was found in oysters, suggesting that chronically exposed oysters were likely re-filtering some oocysts. Examinations of oyster fecal material from acute exposures revealed that 72-82% of oocysts recovered were already excreted at the time of oyster transfer (day 1), with only 18-28% being excreted during the static depuration phase. These data support that although most C. parvum oocysts are removed by C. virginica oysters within 24h, elimination after this point occurs slowly. Additionally, chronic exposures demonstrate that wild or cultured oysters in saline environments that are frequently exposed to sources of Cryptosporidium may be unable to eliminate the parasites at a rate that balances initial uptake. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of dissolved organic carbon on the transport and attachment behaviors of Cryptosporidium parvum oocysts and carboxylate-modified microspheres advected through temperate humic and tropical volcanic agricultural soil

USGS Publications Warehouse

Mohanram, Arvind; Ray, Chittaranjan; Metge, David W.; Barber, Larry B.; Ryan, Joseph N.; Harvey, Ronald W.

2012-01-01

Transport of Cryptosporidium parvum oocysts and microspheres in two disparate (a clay- and Fe-rich, volcanic and a temperate, humic) agricultural soils were studied in the presence and absence of 100 mg L–1 of sodium dodecyl benzene sulfonate (SDBS), and Suwannee River Humic Acid (SRHA) at pH 5.0–6.0. Transport of carboxylate-modified, 1.8 μm microspheres in soil columns was highly sensitive to the nature of the dissolved organic carbon (DOC), whereas oocysts transport was more affected by soil mineralogy. SDBS increased transport of microspheres from 48% to 87% through the tropical soil and from 43% to 93% in temperate soil. In contrast, SRHA reduced transport of microspheres from 48% to 28% in tropical soil and from 43% to 16% in temperate soil. SDBS also increased oocysts transport through the temperate soil 5-fold, whereas no oocyst transport was detected in tropical soil. SRHA had only a nominal effect in increasing oocysts transport in tropical soil, but caused a 6-fold increase in transport through the temperate soil. Amendments of only 4 mg L–1 SRHA and SDBS decreased oocyst hydrophobicity from 66% to 20% and from 66% to 5%, respectively. However, SDBS increased microsphere hydrophobicity from 16% to 33%. Soil fines, which includes clays, and SRHA, both caused the oocysts zeta potential (ζ) to become more negative, but caused the highly hydrophilic microspheres to become less negatively charged. The disparate behaviors of the two colloids in the presence of an ionic surfactant and natural organic matter suggest that microspheres may not be suitable surrogates for oocysts in certain types of soils. These results indicate that whether or not DOC inhibits or promotes transport of oocysts and microspheres in agricultural soils and by how much, depends not only on the surface characteristics of the colloid, but the nature of the DOC and the soil mineralogy.

Protecting chickens against coccidiosis in floor pens by administering Eimeria oocysts using gel beads or spray vaccination.

PubMed

Jenkins, Mark C; Parker, Carolyn; O'Brien, Celia; Persyn, Joseph; Barlow, Darren; Miska, Katarzyna; Fetterer, Raymond

2013-09-01

Control of avian coccidiosis is increasingly being achieved by the administration of low doses of Eimeria oocysts to newly hatched chicks. The purpose of this study was to test the efficacy of gel beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine to protect broilers raised in contact with litter. Newly hatched chicks were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel beads. Control, 1-day-old chicks were given an equivalent number of Eimeria oocysts (10(3) total) by oral gavage or received no vaccine (nonimmunized controls). All chicks were raised in floor-pen cages in direct contact with litter. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E. acervulina, E. maxima, and E. tenella challenge infection. Chickens immunized with Eimeria oocysts in gel beads or by spray vaccination displayed significantly (P < 0.05) greater weight gain (WG) compared to nonimmunized controls. Feed conversion ratio (FCR) also showed a significant (P < 0.05) improvement in both groups relative to nonimmunized controls. Moreover, WG and FCR in both groups was not significantly different (P > 0.05) from chickens immunized by oral gavage or from nonimmunized, noninfected controls. Oocyst excretion after Eimeria challenge by all immunized groups was about 10-fold less than in nonimmunized controls. These findings indicate that immunization efficacy of gel beads and spray vaccination is improved by raising immunized chicks in contact with litter.

Human Challenge Pilot Study with Cyclospora cayetanensis

PubMed Central

Eberhard, Mark L.; Seed, John R.; Weber, David J.; Won, Kimberly Y.; Nace, Eva K.; Moe, Christine L.

2004-01-01

We describe a pilot study that attempted to infect human volunteers with Cyclospora cayetanensis. Seven healthy volunteers ingested an inoculum of Cyclospora oocysts (approximately 200–49,000 oocysts). The volunteers did not experience symptoms of gastroenteritis, and no oocysts were detected in any stool samples during the 16 weeks volunteers were monitored. PMID:15200870

Improved Cryptosporidium parvum oocysts propagation using dexamethasone suppressed CF-1 mice

EPA Science Inventory

This study evaluates Cryptosporidium parvum oocyst production in dexamethasone suppressed CF-1 and C57BL/6 mice. Both models can yield 1 x 10⁹ total oocysts over a 20 day production period; however, only 20 CF-1 mice are required to reliably achieve this goal compared...

Increased efficacy of Eimeria oocysts delivery by gel beads or spray vaccination

USDA-ARS?s Scientific Manuscript database

Control of avian coccidiosis is increasingly being achieved by the administration of low doses of Eimeria oocysts to newly hatched chicks. The purpose of this study was to test the efficacy of gel-beads containing a mixture of E. acervulina, E. maxima, and E. tenella oocysts as a vaccine to protect ...

Evaluation of an experimental irradiated oocysts vaccine to protect broiler chicks against avian coccidosis

USDA-ARS?s Scientific Manuscript database

The current study investigates the use of irradiated oocysts to protect broiler chicks, raised on litter, from infection with multiple species of Eimeria. In order to determine the optimum radiation dose for each Eimeria species, day- old chicks were immunized with oocysts of E. maxima, E. acervulin...

Sources and species of cryptosporidium oocysts in the Wachusett Reservoir watershed.

PubMed

Jellison, Kristen L; Hemond, Harold F; Schauer, David B

2002-02-01

Understanding the behavior of Cryptosporidium oocysts in the environment is critical to developing improved watershed management practices for protection of the public from waterborne cryptosporidiosis. Analytical methods of improved specificity and sensitivity are essential to this task. We developed a nested PCR-restriction fragment length polymorphism assay that allows detection of a single oocyst in environmental samples and differentiates the human pathogen Cryptosporidium parvum from other Cryptosporidium species. We tested our method on surface water and animal fecal samples from the Wachusett Reservoir watershed in central Massachusetts. We also directly compared results from our method with those from the immunofluorescence microscopy assay recommended in the Information Collection Rule. Our results suggest that immunofluorescence microscopy may not be a reliable indicator of public health risk for waterborne cryptosporidiosis. Molecular and environmental data identify both wildlife and dairy farms as sources of oocysts in the watershed, implicate times of cold water temperatures as high-risk periods for oocyst contamination of surface waters, and suggest that not all oocysts in the environment pose a threat to public health.

Molecular and biological characterization of first isolates of Hammondia hammondi from cats from Ethiopia.

PubMed

Dubey, J P; Tilahun, G; Boyle, J P; Schares, G; Verma, S K; Ferreira, L R; Oliveira, S; Tiao, N; Darrington, C; Gebreyes, W A

2013-08-01

Toxoplasma gondii oocysts are morphologically and antigenically similar to oocysts of another feline coccidian, Hammondia hammondi. The distinction between H. hammondi and T. gondii is important from an epidemiological perspective because all isolates of T. gondii are potentially pathogenic for humans and animals, whereas H. hammondi is not known to cause clinical disease in any naturally infected intermediate or definitive hosts. In the present report, H. hammondi (designated HhCatEt1 and HhCatEt2) oocysts were found microscopically in the feces of 2 of 36 feral domestic cats (Felis catus) from Addis Ababa, Ethiopia. Oocysts were orally infective to Swiss Webster and gamma interferon gene knockout mice; the inoculated mice developed tissue cysts in their muscles. Laboratory-raised cats fed mouse tissues of infected mice shed H. hammondi oocysts with a prepatent period of 5 days. The DNA extracted from sporulated oocysts reacted with H. hammondi-specific primers, and sequences were deposited in GenBank (accession nos. JX477424, and KC223619). This is the first report of isolation of H. hammondi from cats from the African continent.

Effects of artemisinin in broiler chickens challenged with Eimeria acervulina, E. maxima and E. tenella in battery trials.

PubMed

Pop, Loredana; Györke, Adriana; Tǎbǎran, Alexandru Flaviu; Dumitrache, Mirabela Oana; Kalmár, Zsuzsa; Magdaş, Cristian; Mircean, Viorica; Zagon, Diana; Balea, Anamaria; Cozma, Vasile

2015-12-15

Four experiments were conceived in order to test the efficacy of artemisinin, a sesquiterpene lactone derived from Artemisia annua, in single experimental infection of broiler chickens with Eimeria acervulina (1 × 10(5) oocysts), Eimeria maxima (5 × 10(4) oocysts) or Eimeria tenella (1 × 10(4) oocysts), and mixed infection with all 3 species (3.2 × 10(4) Eimeria spp. oocysts). For each experiment, three different dosages of artemisinin (5, 50 and 500 ppm) were compared with a negative control (uninfected, unmedicated), a positive control (infected, unmedicated) and a classical anticoccidial (monensin). The weight gain (WG), feed conversion ratio (FCR), oocysts shedded per gram of feces (OPG), lesion score, oocysts sporulation rates and mortality rate were recorded in all groups. The dosage of 5 ppm of artemisinin improved the WG and FCR for the chickens infected with E. acervulina. The OPG was significantly decreased in all the groups medicated with artemisinin and challenged with a mixed infection (p ≤ 0.01). The lesion score of the chickens challenged with Eimeria was reduced by different concentrations of artemisinin, depending on the species involved, but this compound did not have a positive effect on the lesions caused by E. acervulina. Histopathological analysis revealed superficial erosions of the intestinal mucosa, mixt. mononuclear and heterophilic inflammatory infiltrate in the lamina propria and intralesional presence of various developmental stages of parasite in groups infected with Eimeria spp.The sporulation rate of E. acervulina and E. maxima oocysts was significantly affected by 500 ppm of artemisinin, whilst the dosage of 5 ppm affected the sporulation of E. tenella oocysts. These data suggest that artemisinin is not effective against single eimerian infections but could be used as an alternative in mixed coccidiosis, especially if its effect on the oocysts sporulation would be fully investigated. Copyright © 2015 Elsevier B.V. All rights reserved.

Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water.

PubMed

McGuigan, K G; Méndez-Hermida, F; Castro-Hermida, J A; Ares-Mazás, E; Kehoe, S C; Boyle, M; Sichel, C; Fernández-Ibáñez, P; Meyer, B P; Ramalingham, S; Meyer, E A

2006-08-01

To determine whether batch solar disinfection (SODIS) can be used to inactivate oocysts of Cryptosporidium parvum and cysts of Giardia muris in experimentally contaminated water. Suspensions of oocysts and cysts were exposed to simulated global solar irradiation of 830 W m(-2) for different exposure times at a constant temperature of 40 degrees C. Infectivity tests were carried out using CD-1 suckling mice in the Cryptosporidium experiments and newly weaned CD-1 mice in the Giardia experiments. Exposure times of > or =10 h (total optical dose c. 30 kJ) rendered C. parvum oocysts noninfective. Giardia muris cysts were rendered completely noninfective within 4 h (total optical dose >12 kJ). Scanning electron microscopy and viability (4',6-diamidino-2-phenylindole/propidium iodide fluorogenic dyes and excystation) studies on oocysts of C. parvum suggest that inactivation is caused by damage to the oocyst wall. Results show that cysts of G. muris and oocysts of C. parvum are rendered completely noninfective after batch SODIS exposures of 4 and 10 h (respectively) and is also likely to be effective against waterborne cysts of Giardia lamblia. These results demonstrate that SODIS is an appropriate household water treatment technology for use as an emergency intervention in aftermath of natural or man-made disasters against not only bacterial but also protozoan pathogens.

Cryptosporidium and Giardia removal by secondary and tertiary wastewater treatment.

PubMed

Taran-Benshoshan, Marina; Ofer, Naomi; Dalit, Vaizel-Ohayon; Aharoni, Avi; Revhun, Menahem; Nitzan, Yeshayahu; Nasser, Abidelfatah M

2015-01-01

Wastewater disposal may be a source of environmental contamination by Cryptosporidium and Giardia. This study was conducted to evaluate the prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated wastewater effluents. A prevalence of 100% was demonstrated for Giardia cysts in raw wastewater, at a concentration range of 10 to 12,225 cysts L(-1), whereas the concentration of Cryptosporidium oocysts in raw wastewater was 4 to 125 oocysts L(-1). The removal of Giardia cysts by secondary and tertiary treatment processes was greater than those observed for Cryptosporidium oocysts and turbidity. Cryptosporidium and Giardia were present in 68.5% and 76% of the tertiary effluent samples, respectively, at an average concentration of 0.93 cysts L(-1) and 9.94 oocysts L(-1). A higher detection limit of Cryptosporidium oocysts in wastewater was observed for nested PCR as compared to immune fluorescent assay (IFA). C. hominis was found to be the dominant genotype in wastewater effluents followed by C. parvum and C. andersoni or C. muris. Giardia was more prevalent than Cryptosporidium in the studied community and treatment processes were more efficient for the removal of Giardia than Cryptosporidium. Zoonotic genotypes of Cryptosporidium were also present in the human community. To assess the public health significance of Cryptosporidium oocysts present in tertiary effluent, viability (infectivity) needs to be assessed.

Characterization of an immunogenic glycocalyx on the surfaces of Cryptosporidium parvum oocysts and sporozoites.

PubMed

Nanduri, J; Williams, S; Aji, T; Flanigan, T P

1999-04-01

Ruthenium red staining of Cryptosporidium parvum oocysts revealed the presence of a carbohydrate matrix on their outer bilayers that is characteristic of a glycocalyx. Surface labeling of intact oocysts identified material of high molecular weight (>10(6)) that reacted positively with sera from cryptosporidium-infected patients and with immunoglobulin A monoclonal antibodies.

Characterization of an Immunogenic Glycocalyx on the Surfaces of Cryptosporidium parvum Oocysts and Sporozoites

PubMed Central

Nanduri, Jayasri; Williams, Selvi; Aji, Toshiki; Flanigan, Timothy P.

1999-01-01

Ruthenium red staining of Cryptosporidium parvum oocysts revealed the presence of a carbohydrate matrix on their outer bilayers that is characteristic of a glycocalyx. Surface labeling of intact oocysts identified material of high molecular weight (>106) that reacted positively with sera from cryptosporidium-infected patients and with immunoglobulin A monoclonal antibodies. PMID:10085053

Development and evaluation of an off-the-slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides

EPA Science Inventory

ABSTRACT Aims This study developed and systematically evaluated performance and limit of detection of an off-the-slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts. Methods and Results Slide standards containing flow sorted (oo)cysts were used to e...

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment

PubMed Central

Staggs, Sarah E.; Beckman, Erin M.; Keely, Scott P.; Mackwan, Reena; Ware, Michael W.; Moyer, Alan P.; Ferretti, James A.; Sayed, Abu; Xiao, Lihua; Villegas, Eric N.

2013-01-01

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources. PMID:23805235

Detection of Toxoplasma gondii oocysts in soils in northwestern China using a new semi-nested PCR assay.

PubMed

Wang, Meng; Meng, Peng; Ye, Qiang; Pu, Yuan-Hua; Yang, Xiao-Yu; Luo, Jian-Xun; Zhang, Nian-Zhang; Zhang, De-Lin

2014-09-28

Toxoplasma gondii is a zoonotic pathogen that can infect a range of animals and humans. Ingestion of T. gondii oocysts in soil is a significant transmission route for humans and animals acquiring toxoplasmosis. In the present study, we developed a new semi-nested PCR method to determine T. gondii oocysts distribution in soils in northwestern China. The one tube semi-nested PCR assay was developed to detect the oocysts of T. gondii in soil, targeting the repetitive 529 bp fragment of T. gondii genomic DNA. Then a total of 268 soil samples, including 148 samples from Gansu Province and 120 samples from Qinghai Province, northwestern China, were examined by the semi-nested PCR method. One third of the positive samples were sequenced. The sensitivity of the semi-nested PCR assay was 10(2)  T. gondii oocysts in 5 g soil sample. Investigation of soil samples from northwestern China showed that 34 out of 268 soil samples (12.69%) were T. gondii positive. Sequences of the partial 529 bp fragments varied from 0-1.2% among the sequenced samples. The prevalence of T. gondii oocysts in soil from cities (24/163) was slightly higher than that in soils from pasturing areas (10/105) (P = 0.21). Among the different regions in cities, the prevalence of T. gondii oocysts in soils from parks was 14.15%, whereas that in soils from schools was 19.05%. The present study firstly reported the prevalence of T. gondii oocysts in soils in northwest China using a novel semi-nested PCR assay, which provided baseline data for the effective prevention and control of toxoplasmosis in this region.

Immunomagnetic Separation of Cryptosporidium parvum from Source Water Samples of Various Turbidities

PubMed Central

Bukhari, Z.; McCuin, R. M.; Fricker, C. R.; Clancy, J. L.

1998-01-01

Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts. PMID:9797313

Development and evaluation of an off-the-slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides.

PubMed

Ware, M W; Keely, S P; Villegas, E N

2013-07-01

This study developed and systematically evaluated performance and limit of detection of an off-the-slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts. Slide standards containing flow-sorted (oo)cysts were used to evaluate the off-the-slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide. This study successfully developed and evaluated recovery rates and limits of detection of an off-the-slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide. This off-the-slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions. Journal of Applied Microbiology Published [2013]. This article is a U.S. Government work and is in the public domain in the USA.

Thermal Contribution to the Inactivation of Cryptosporidium in Plastic Bottles during Solar Water Disinfection Procedures

PubMed Central

Gómez-Couso, Hipólito; Fontán-Sainz, María; Ares-Mazás, Elvira

2010-01-01

To determine the thermal contribution, independent of ultraviolet radiation, on the inactivation of Cryptosporidium parvum during solar water disinfection procedures (SODIS), oocysts were exposed for 4, 8, and 12 hours to temperatures recorded in polyethylene terephthalate bottles in previous SODIS studies carried out under field conditions. Inclusion/exclusion of the fluorogenic vital dye propidium iodide, spontaneous excystation, and infectivity studies were used to determine the inactivation of oocysts. There was a significant increase in the percentage of oocysts that took up propidium iodide and in the number of oocysts that excysted spontaneously. There was also a significant decrease in the intensity of infection elicited in suckling mice at the end of all exposure times. The results of the study demonstrate the importance of temperature in the inactivation of C. parvum oocysts during application of SODIS under natural conditions. PMID:20064992

Electrical cream separator coupled with vacuum filtration for the purification of eimerian oocysts and trichostronglyid eggs

NASA Astrophysics Data System (ADS)

El-Ashram, Saeed; Suo, Xun

2017-02-01

Several methods have been proposed for separation of eimerian oocysts and trichostronglyid eggs from extraneous debris; however, these methods have been considered to be still inconvenient in terms of time and wide-ranging applications. We describe herein an alternative way using the combination of electrical cream separator and vacuum filtration for harvesting and purifying eimerian oocysts and haemonchine eggs on large-scale applications with approximately 81% and 92% recovery rates for oocysts and nematode eggs obtained from avian and ovine faeces, correspondingly. The sporulation percentages as a measure of viability in the harvested oocysts and eggs from dry faecal materials are nearly 68% and 74%, respectively, and 12 liters of faecal suspension can be processed in approximately 7.5 min. The mode of separation in terms of costs (i.e. simple laboratory equipments and comparably cheap reagents) and benefits renders the reported procedure an appropriate pursuit to harvest and purify parasite oocysts and eggs on a large scale in the shortest duration from diverse volumes of environmental samples compared to the modified traditional sucrose gradient, which can be employed on a small scale.

Interaction of low-pathogenicity reoviruses and low levels of infection with several coccidial species.

PubMed

Ruff, M D; Rosenberger, J K

1985-01-01

Various combinations of reoviruses and coccidia were studied to see if interactions would occur. Two reoviruses were used: virus 2035, a moderate to low pathogen, and virus 2177, a nonpathogen. Coccidia used were Eimeria acervulina, E. mitis, and E. maxima at dosages of 10(3) or 10(4) sporulated oocysts/chick and E. brunetti at 10(4) sporulated oocysts/chick. In Hubbard-Hubbard cockerels, a combination of virus 2035 and E. acervulina (10(4) oocysts/chick) or E. maxima (10(3) oocysts/chick) significantly (P less than or equal to 0.05) increased the frequency of stunting (% of chicks with body weight less than 80% of controls) and further depressed weight gain over that seen with either virus or coccidia alone. Conversely, virus 2177 ameliorated the same effects in Shaver-Arbor Acre cockerels given 10(4) oocysts/chick of E. mitis or E. maxima. The interaction could not be attributed to changes in the degree of coccidial infection based on oocyst production. Reovirus did not generally change the effect of coccidia on levels of plasma pigment and plasma protein. In Hubbard-Hubbard cockerels, coccidia-induced effects were not ameliorated by virus 2177, suggesting that breed difference in interaction can be expected.

Electrical cream separator coupled with vacuum filtration for the purification of eimerian oocysts and trichostronglyid eggs

PubMed Central

El-Ashram, Saeed; Suo, Xun

2017-01-01

Several methods have been proposed for separation of eimerian oocysts and trichostronglyid eggs from extraneous debris; however, these methods have been considered to be still inconvenient in terms of time and wide-ranging applications. We describe herein an alternative way using the combination of electrical cream separator and vacuum filtration for harvesting and purifying eimerian oocysts and haemonchine eggs on large-scale applications with approximately 81% and 92% recovery rates for oocysts and nematode eggs obtained from avian and ovine faeces, correspondingly. The sporulation percentages as a measure of viability in the harvested oocysts and eggs from dry faecal materials are nearly 68% and 74%, respectively, and 12 liters of faecal suspension can be processed in approximately 7.5 min. The mode of separation in terms of costs (i.e. simple laboratory equipments and comparably cheap reagents) and benefits renders the reported procedure an appropriate pursuit to harvest and purify parasite oocysts and eggs on a large scale in the shortest duration from diverse volumes of environmental samples compared to the modified traditional sucrose gradient, which can be employed on a small scale. PMID:28233853

Effect of ferric oxyhydroxide grain coatings on the transport of bacteriophage PRD1 and Cryptosporidium parvum oocysts in saturated porous media

USGS Publications Warehouse

Abudalo, R.A.; Bogatsu, Y.G.; Ryan, J.N.; Harvey, R.W.; Metge, D.W.; Elimelech, M.

2005-01-01

To test the effect of geochemical heterogeneity on microorganism transport in saturated porous media, we measured the removal of two microorganisms, the bacteriophage PRD1 and oocysts of the protozoan parasite Cryptosporidium parvum, in flow-through columns of quartz sand coated by different amounts of a ferric oxyhydroxide. The experiments were conducted over ranges of ferric oxyhydroxide coating fraction of ?? = 0-0.12 for PRD1 and from ?? = 0-0.32 for the oocysts at pH 5.6-5.8 and 10-4 M ionic strength. To determine the effect of pH on the transport of the oocysts, experiments were also conducted over a pH range of 5.7-10.0 at a coating fraction of ?? = 0.04. Collision (attachment) efficiencies increased as the fraction of ferric oxyhydroxide coated quartz sand increased, from ?? = 0.0071 to 0.13 over ?? = 0-0.12 for PRD1 and from ?? = 0.059 to 0.75 over ?? = 0-0.32 for the oocysts. Increasing the pH from 5.7 to 10.0 resulted in a decrease in the oocyst collision efficiency as the pH exceeded the expected point of zero charge of the ferric oxyhydroxide coatings. The collision efficiencies correlated very well with the fraction of quartz sand coated by the ferric oxyhydroxide for PRD1 but not as well for the oocysts. ?? 2005 American Chemical Society.

A comparison of fecal percent dry matter and number of Cryptosporidium parvum oocysts shed to observational fecal consistency scoring in dairy calves.

PubMed

Bellosa, Mary L; Nydam, Daryl V; Liotta, Janice L; Zambriski, Jennifer A; Linden, Thomas C; Bowman, Dwight D

2011-04-01

Evaluation of dairy calf feces is often used in research and for clinical decision making to assess severity of diarrhea. However, this has not been validated for agreement between dry matter content and observed fecal consistency. Therefore, a comparison of observed fecal consistency score to fecal percent dry matter and Cryptosporidium parvum oocyst shedding was performed to assess the accuracy of observational scoring as a measure of diarrhea and its association with number of oocysts shed. Fecal samples from 20 dairy calves experimentally infected with C. parvum oocysts were collected daily post-infection and scored on a scale from 1 to 4, with 1 being normal feces to 4 being severe diarrhea. An aliquot of each sample was analyzed for percent dry matter and Cryptosporidium oocyst counts by using immunofluorescent microscopy. Fecal consistency scores of 1, 2, 3, and 4 had median percent dry matter of 20.9, 16.3, 9.6, and 5.8, respectively. Using percent dry matter assessed by fecal consistency scoring were significantly different from each other (P < 0.001). A higher fecal consistency score also was associated with a greater number of Cryptosporidium oocysts shed (P < 0 .0001). Scores of 1, 2, 3, and 4 had median oocyst counts of 0, 0, 1.3 × 10⁶, and 2.8 × 10⁶, respectively. These results suggest that observational scoring is a useful proxy to assess diarrhea in dairy calves.

Infectious Cryptosporidium parvum oocysts in final reclaimed effluent

USGS Publications Warehouse

Gennaccaro, A.L.; McLaughlin, M.R.; Quintero-Betancourt, W.; Huffman, D.E.; Rose, J.B.

2003-01-01

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.

Characterization of Cryptosporidium parvum by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Magnuson, Matthew L.; Owens, James H.; Kelty, Catherine A.

2000-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used to investigate whole and freeze-thawed Cryptosporidium parvum oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtained after the oocysts were lysed with a freeze-thaw procedure. Spectral-marker patterns for C. parvum were distinguishable from those obtained for Cryptosporidium muris. One spectral marker appears specific for the genus, while others appear specific at the species level. Three different C. parvum lots were investigated, and similar spectral markers were observed in each. Disinfection of the oocysts reduced and/or eliminated the patterns of spectral markers. PMID:11055915

Detection of Infectious Cryptosporidium parvum Oocysts in Mussels (Mytilus galloprovincialis) and Cockles (Cerastoderma edule)

PubMed Central

Gomez-Bautista, M.; Ortega-Mora, L. M.; Tabares, E.; Lopez-Rodas, V.; Costas, E.

2000-01-01

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 103 oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination. PMID:10788352

Cryptosporidium and Giardia in Humans, Domestic Animals, and Village Water Sources in Rural India

PubMed Central

Daniels, Miles E.; Shrivastava, Arpit; Smith, Woutrina A.; Sahu, Priyadarshi; Odagiri, Mitsunori; Misra, Pravas R.; Panigrahi, Pinaki; Suar, Mrutyunjay; Clasen, Thomas; Jenkins, Marion W.

2015-01-01

Cryptosporidium parvum and Giardia lamblia are zoonotic enteric protozoa of significant health concern where sanitation, hygiene, and water supplies are inadequate. We examined 85 stool samples from diarrhea patients, 111 pooled fecal samples by species across seven domestic animal types, and water from tube wells (N = 207) and ponds (N = 94) across 60 villages in coastal Odisha, India, for Cryptosporidium oocysts and Giardia cysts to measure occurrence, concentration/shedding, and environmental loading rates. Oocysts/cysts were detected in 12% of diarrhea patients. Detection ranged from 0% to 35% for Cryptosporidium and 0% to 67% for Giardia across animal hosts. Animal loading estimates indicate the greatest contributors of environmental oocysts/cysts in the study region are cattle. Ponds were contaminated with both protozoa (oocysts: 37%, cysts: 74%), as were tube wells (oocysts: 10%, cysts: 14%). Future research should address the public health concern highlighted from these findings and investigate the role of domestic animals in diarrheal disease transmission in this and similar settings. PMID:26123963

Shedding of Toxoplasma gondii oocysts by Felidae in zoos in the Czech Republic.

PubMed

Lukesová, D; Literák, I

1998-01-15

In 1995 and 1996, the shedding of Toxoplasma gondii oocysts was monitored in the faeces of Felidae in six zoos in the Czech Republic. In all, 2287 samples of faeces from 19 species of Felidae were examined. In Ostrava Zoo, four episodes of shedding of Toxoplasma-like oocysts were identified, using a flotation examination, in a pair of wild cats (Felis silvestris), six episodes in a wild cat held separately, and three episodes in a pair of Amur leopard cats (F. euptilurus). After the passage of sporulated oocysts through laboratory mice, T. gondii was confirmed in the pair of wild cats (three episodes), in the wild cat held separately (three episodes) and in the pair of Amur leopard cats (one episode). In Jihlava Zoo, one episode of shedding of T. gondii oocyst was identified in Geoffroy's cat (Oncifelis geoffroyi) using flotation and isolation examination. The possible sources of toxoplasmosis of the Felidae in zoos are discussed.

Effects of pH and Magnetic Material on Immunomagnetic Separation of Cryptosporidium Oocysts from Concentrated Water Samples

PubMed Central

Kuhn, Ryan C.; Rock, Channah M.; Oshima, Kevin H.

2002-01-01

In this study, we examined the effect that magnetic materials and pH have on the recoveries of Cryptosporidium oocysts by immunomagnetic separation (IMS). We determined that particles that were concentrated on a magnet during bead separation have no influence on oocyst recovery; however, removal of these particles did influence pH values. The optimal pH of the IMS was determined to be 7.0. The numbers of oocysts recovered from deionized water at pH 7.0 were 26.3% higher than those recovered from samples that were not at optimal pH. The results indicate that the buffers in the IMS kit did not adequately maintain an optimum pH in some water samples. By adjusting the pH of concentrated environmental water samples to 7.0, recoveries of oocysts increased by 26.4% compared to recoveries from samples where the pH was not adjusted. PMID:11916735

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

USGS Publications Warehouse

Simmons, O. D.; Sobsey, M.D.; Heaney, C.D.; Schaefer, F. W.; Francy, D.S.

2001-01-01

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4???,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.

Contamination of River Water by Cryptosporidium parvum Oocysts in Western Japan

PubMed Central

Ono, Kazuo; Tsuji, Hidetaka; Rai, Shiba Kumar; Yamamoto, Akio; Masuda, Kuniyoshi; Endo, Takuro; Hotta, Hak; Kawamura, Takashi; Uga, Shoji

2001-01-01

In Japan, only a few rivers have been inspected for Cryptosporidium parvum contamination, and the methods used had low sensitivity. In 1998 and 1999, we used a method with higher sensitivity to examine all large rivers used as sources of water supply in one prefecture (which we divided into four areas) in western Japan for Cryptosporidium oocysts. One sample was collected at each of 156 sites along 18 rivers, and samples were tested for Cryptosporidium oocysts by immunomagnetic separation. Samples were classified as being obtained on an island with livestock and fishing industries, a densely populated urban area, a western region including farming villages, or a still more rural northern area with agriculture and fishing. Restriction fragment length polymorphism analysis was used for identification of the C. parvum found as the bovine or human type. C. parvum was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the samples from each area contained C. parvum oocysts. The number of C. parvum oocysts per 20 liters of river water varied in the same pattern as the number of cattle kept in the four kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the C. parvum detected in rivers probably came from cattle kept in that valley. As we had expected, when tested with a more sensitive method, river water in western Japan was found to be greatly contaminated with C. parvum oocysts, as reported in other countries. PMID:11525974

Effects of kefir on coccidial oocysts excretion and performance of dairy goat kids following weaning.

PubMed

Daş, Gürbüz; Ataşoğlu, Cengiz; Akbağ, Hande Işıl; Tölü, Cemil; Yurtman, Ismail Yaman; Savaş, Türker

2012-06-01

The aim of this study was to investigate effects of kefir, a traditional source of probiotic, on coccidial oocysts excretion and on the performance of dairy goat kids following weaning. Twin kids were randomly allocated to one of two groups at weaning. Kids of the first group received 20 ml of kefir daily for 6 weeks (KEF), while kids in the control group were given a placebo (CON). Individual faecal samples were regularly (n = 18 per kid) taken to quantify the number of coccidial oocysts per gram of faeces (OpG). There were no differences between the groups in terms of body weight development (P > 0.05) and feed consumption. Kids of both groups were not able to consume enough feed to meet their nutrient requirements during the first 3 weeks following weaning. KEF had a lower frequency of OpG positive samples than CON (P = 0.043). Kefir did not affect the maximum oocyst excretion and age of the kids at the highest oocyst excretion (P > 0.05). KEF shed numerically 35% lower coccidial oocysts than the controls, which corresponded to a statistical tendency (P = 0.074) in lowering Log-OpG in comparison to CON. While KEF had a lower frequency of OpG positive samples and tended to shed lower OPG by around one-third, the frequency of diarrhea, level of highest oocyst excretion, and performance of the kids remained unaffected. Therefore, it is concluded that overall effects of kefir do not have a significant impact on sub-clinical infection and performance in weaned kids under relatively high-hygienic farming conditions.

10-year parasitological examination results (2003 to 2012) of faecal samples from horses, ruminants, pigs, dogs, cats, rabbits and hedgehogs.

PubMed

Raue, Katharina; Heuer, Lea; Böhm, Claudia; Wolken, Sonja; Epe, Christian; Strube, Christina

2017-12-01

The results of coproscopical examinations in domestic animals and hedgehogs carried out as routine diagnostics in the years 2003 to 2012 at the Institute for Parasitology, University of Veterinary Medicine Hannover, Germany, are presented. Of 3475 horse faecal samples, 30.1% contained stages of strongyles and 1.3% eggs of Strongyloides westeri and Parascaris equorum, respectively. The most frequently observed parasite stages in 1416 cattle faecal samples were Eimeria oocysts (21.3%) and strongyle eggs or larvae (15.9%). Dictyocaulus viviparus larvae and Fasciola hepatica eggs were identified in 0.9 and 1.3% of samples. Of 574 bovine faecal samples analysed by carbol-fuchsin staining, 39.9% were positive for Cryptosporidium oocysts. Stages of strongyles were found in 52.4% of sheep (n = 374) and 44.9% of goat faeces (n = 98) and Eimeria oocysts in 41.4 and 32.7% of their faeces, respectively. Of 1848 pig faecal samples, 3.0% contained stages of strongyles, 1.6% eggs of Ascaris suum and 3.3% coccidian (Eimeria or Cystoisospora spp.) oocysts. The most frequently detected helminth eggs in faecal samples of dogs (n = 2731) and cats (n = 903) were Toxocara spp. (2.8 and 3.9%, respectively). Cystoisospora oocysts were identified in 5.6% of dog and 2.4% of cat faeces. Furthermore, 0.7% of the cat samples were positive for small Toxoplasma gondii-like oocysts. The faecal samples of rabbits (n = 434) contained eggs of Passalurus ambiguus (3.0%), strongyles (1.8%) and Trichuris leporis (0.2%) as well as Eimeria oocysts (21.2%). The most abundant nematodes in the samples of hedgehogs (n = 205) were Capillaria spp. (39.5%) and Crenosoma striatum (26.8%); coccidian oocysts were found in 14.2% of the samples.

Eimeria peltocephali n. sp., (Apicomplexa:Eimeriidae) from the freshwater turtle Peltocephalus dumerilianus (Chelonia:Pelomusidae) and Eimeria molossi n. sp., from the bat, Molossus ater (Mammalia:Chiroptera).

PubMed

Lainson, R; Naiff, R D

1998-01-01

The oocyst is described of Eimeria peltocephali n.sp. from faeces of the freshwater turtle Peltocephalus dumerilianus from Barcelos, State of Amazonas, Brazil. Sporulation is exogenous and fully developed oocysts are elongate, ellipsoidal or cylindrical, frequently curved to a banana-shape, 54.4 x 19.1 (37.5-68.7 x 18.7-20.0 microns), shape-index 2.8 (1.8-3.9). The oocyst wall is a single thin, colourless layer about 1 micron thick, with no micropyle. There is a bulky oocyst residuum, at first spherical to ellipsoidal, 19 x 16 (16.2-26.2 x 16-21.5 microns), but becoming dispersed on maturation. There are no polar bodies. The sporocysts, 19.1 x 6.8 (17.5-21.2 x 6.2-7.5 microns), shape-index 2.8 (2.3-3.2), are usually disposed in pairs at each end of the oocyst, and bear an inconspicuous Stieda body in the form of a flat cap. The sporozoites are elongate and slightly curved around the residuum. No refractile bodies were seen. Eimeria molossi n.sp., is described from the molossid bat Molossus ater. Sporulation is exogenous and the mature oocysts are predominantly broadly ellipsoidal, 23.4 x 17.5 (18-30 x 15-22.5 microns), shape-index 1.3 (1-1.6). The oocyst wall is about 2 microns thick, and of three layers: an inner thin, colourless one and two outer layers which are thicker, yellowish-brown, prominently striated and in close apposition. There is no micropyle or oocyst residuum, but one and occasionally two polar bodies are usually present. Sporocysts are ellipsoidal, 10.2 x 7.5 (10-12.5 x 7.5 microns), shape-index 1.4 (1.3-1.7) with an inconspicuous Stieda body. Endogenous stages are described in the epithelial cells of the small intestine.

Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites.

PubMed

Orfano, Alessandra S; Nacif-Pimenta, Rafael; Duarte, Ana P M; Villegas, Luis M; Rodrigues, Nilton B; Pinto, Luciana C; Campos, Keillen M M; Pinilla, Yudi T; Chaves, Bárbara; Barbosa Guerra, Maria G V; Monteiro, Wuelton M; Smith, Ryan C; Molina-Cruz, Alvaro; Lacerda, Marcus V G; Secundino, Nágila F C; Jacobs-Lorena, Marcelo; Barillas-Mury, Carolina; Pimenta, Paulo F P

2016-08-02

Malaria is transmitted when an infected mosquito delivers Plasmodium sporozoites into a vertebrate host. There are many species of Plasmodium and, in general, the infection is host-specific. For example, Plasmodium gallinaceum is an avian parasite, while Plasmodium berghei infects mice. These two parasites have been extensively used as experimental models of malaria transmission. Plasmodium falciparum and Plasmodium vivax are the most important agents of human malaria, a life-threatening disease of global importance. To complete their life cycle, Plasmodium parasites must traverse the mosquito midgut and form an oocyst that will divide continuously. Mature oocysts release thousands of sporozoites into the mosquito haemolymph that must reach the salivary gland to infect a new vertebrate host. The current understanding of the biology of oocyst formation and sporozoite release is mostly based on experimental infections with P. berghei, and the conclusions are generalized to other Plasmodium species that infect humans without further morphological analyses. Here, it is described the microanatomy of sporozoite escape from oocysts of four Plasmodium species: the two laboratory models, P. gallinaceum and P. berghei, and the two main species that cause malaria in humans, P. vivax and P. falciparum. It was found that sporozoites have species-specific mechanisms of escape from the oocyst. The two model species of Plasmodium had a common mechanism, in which the oocyst wall breaks down before sporozoites emerge. In contrast, P. vivax and P. falciparum sporozoites show a dynamic escape mechanism from the oocyst via polarized propulsion. This study demonstrated that Plasmodium species do not share a common mechanism of sporozoite escape, as previously thought, but show complex and species-specific mechanisms. In addition, the knowledge of this phenomenon in human Plasmodium can facilitate transmission-blocking studies and not those ones only based on the murine and avian models.

Effect of High-Rate Algal Ponds on Viability of Cryptosporidium parvum Oocysts

PubMed Central

Araki, S.; Martín-Gomez, S.; Bécares, E.; De Luis-Calabuig, E.; Rojo-Vazquez, F.

2001-01-01

The physicochemical conditions of high-rate algal ponds were responsible for a more than 97% reduction in the infectivity of Cryptosporidium parvum oocysts in neonatal mice. The use of semipermeable bags of cellulose showed that pH, ammonia, and/or light seems to be a major factor for the inactivation of oocysts in wastewater, supporting the importance of alga-based systems for safer reuse of treated wastewater. PMID:11425762

Detection and molecular status of Isospora sp. from the domestic pigeon (Columba livia domestica).

PubMed

Matsubara, Ryuma; Fukuda, Yasuhiro; Murakoshi, Fumi; Nomura, Osamu; Suzuki, Toru; Tada, Chika; Nakai, Yutaka

2017-10-01

The domestic pigeon, Columba livia domestica, is reared for meat production, as a pet, or for racing. Few reports have characterized the parasitic protists from the genus Isospora isolated from Columbiformes. We detected Isospora-like oocysts from C. livia reared for racing. The oocyst contained two sporocysts, and each sporocyst included four sporozoites. The sporulated oocysts (n=4) were spherical; their mean diameters were 25.6 (24.0-27.2)×24.7 (23.4-26.0) μm. Micropyles, polar granules, and oocyst residuum were absent. The mean length and width of the sporocysts (n=8) were 19.5 (18.5-20.5) and 11.2 (10.2-12.1) μm, respectively. Stieda and sub-Stieda bodies were observed. Single-oocyst PCR revealed two different 18S rRNA gene sequences and one 28S rRNA gene sequence in a single oocyst of Isospora sp. Based on a phylogenetic analysis of the 18S rRNA gene, the two sequences made a group which fell within a cluster of known avian Isospora species. A tree based on the 28S rRNA gene sequence indicated that sequences from the pigeon Isospora sp. fell within a cluster of avian Isospora species. Both trees failed to clarify the phylogenetic relationships among the avian Isospora species due to limited resolution. Because the morphological description of Isospora sp. is based on only four oocysts, Isospora sp. is not proposed as a novel species here. This is the first description of Isospora sp. isolated from the domestic pigeon C. livia. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of organic carbon loading, sediment associated metal oxide content and sediment grain size distributions upon Cryptosporidium parvum removal during riverbank filtration operations, Sonoma County, CA.

PubMed

Metge, D W; Harvey, R W; Aiken, G R; Anders, R; Lincoln, G; Jasperse, J

2010-02-01

This study assessed the efficacy for removing Cryptosporidium parvum oocysts of poorly sorted, Fe- and Al-rich, subsurface sediments collected from 0.9 to 4.9 and 1.7-13.9 m below land surface at an operating riverbank filtration (RBF) site (Russian River, Sonoma County, CA). Both formaldehyde-killed oocysts and oocyst-sized (3 microm) microspheres were employed in sediment-packed flow-through and static columns. The degree of surface coverage of metal oxides on sediment grain surfaces correlated strongly with the degrees of oocyst and microsphere removals. In contrast, average grain size (D(50)) was not a good indicator of either microsphere or oocyst removal, suggesting that the primary mechanism of immobilization within these sediments is sorptive filtration rather than physical straining. A low specific UV absorbance (SUVA) for organic matter isolated from the Russian River, suggested that the modest concentration of the SUVA component (0.8 mg L(-1)) of the 2.2 mg L(-1) dissolved organic carbon (DOC) is relatively unreactive. Nevertheless, an amendment of 2.2 mg L(-1) of isolated river DOC to column sediments resulted in up to a 35.7% decrease in sorption of oocysts and (or) oocyst-sized microspheres. Amendments (3.2 microM) of the anionic surfactant, sodium dodecyl benzene sulfonate (SDBS) also caused substantive decreases (up to 31.9 times) in colloid filtration. Although the grain-surface metal oxides were found to have a high colloid-removal capacity, our study suggested that any major changes within the watershed that would result in long-term alterations in either the quantity and (or) the character of the river's DOC could alter the effectiveness of pathogen removal during RBF operations. Published by Elsevier Ltd.

Recovery and Enumeration of Cryptosporidium parvum from Animal Fecal Matrices

PubMed Central

Davies, Cheryl M.; Kaucner, Christine; Deere, Daniel; Ashbolt, Nicholas J.

2003-01-01

Accurate quantification of Cryptosporidium parvum oocysts in animal fecal deposits on land is an essential starting point for estimating watershed C. parvum loads. Due to the general poor performance and variable recovery efficiency of existing enumeration methods, protocols were devised based on initial dispersion of oocysts from feces by vortexing in 2 mM tetrasodium pyrophosphate, followed by immunomagnetic separation. The protocols were validated by using an internal control seed preparation to determine the levels of oocyst recovery for a range of fecal types. The levels of recovery of 102 oocysts from cattle feces (0.5 g of processed feces) ranged from 31 to 46%, and the levels of recovery from sheep feces (0.25 g of processed feces) ranged from 21% to 35%. The within-sample coefficients of variation for the percentages of recovery from five replicates ranged from 10 to 50%. The ranges for levels of recovery of oocysts from cattle, kangaroo, pig, and sheep feces (juveniles and adults) collected in a subsequent watershed animal fecal survey were far wider than the ranges predicted by the validation data. Based on the use of an internal control added to each fecal sample, the levels of recovery ranged from 0 to 83% for cattle, from 4 to 62% for sheep, from 1 to 42% for pigs, and from 40 to 73% for kangaroos. Given the variation in the levels of recovery of oocysts from different fecal matrices, it is recommended that an internal control be added to at least one replicate of every fecal sample analyzed to determine the percentage of recovery. Depending on the animal type and based on the lowest approximate percentages of recovery, between 10 and 100 oocysts g of feces−1 must be present to be detected. PMID:12732556

Fecal antibodies to Cryptosporidium parvum in healthy volunteers.

PubMed

Dann, S M; Okhuysen, P C; Salameh, B M; DuPont, H L; Chappell, C L

2000-09-01

This study examined the intestinal antibody response in 26 healthy volunteers challenged with Cryptosporidium parvum oocysts. Fecal extracts were assayed for total secretory immunoglobulin A (IgA) and C. parvum-specific IgA reactivity. Specific IgA reactivity was standardized to IgA concentration and expressed as a reactivity index (RI). Anti-C. parvum fecal IgA (fIgA) increased significantly in 17 of 26 (65.4%) following oocyst ingestion. Of those with detectable responses, 59, 76.5, and 94.1% were positive by days 7, 14, and 30, respectively. Volunteers receiving high challenge doses (>1,000 and 300 to 500 oocysts) had higher RIs (RI = 5.57 [P = 0. 027] and RI = 1.68 [P = 0.039], respectively) than those ingesting low doses (30 to 100 oocysts; RI = 0.146). Subjects shedding oocysts and experiencing a diarrheal illness had the highest fIgA reactivity. When evaluated separately, oocyst excretion was associated with an increased fIgA response compared to nonshedders (RI = 1.679 versus 0. 024, respectively; P = 0.003). However, in subjects experiencing diarrhea with or without oocyst shedding, a trend toward a higher RI (P = 0.065) was seen. Extracts positive for fecal IgA were further examined for IgA subclass. The majority of stools contained both IgA1 and IgA2, and the relative proportions did not change following challenge. Also, no C. parvum-specific IgM or IgG was detected in fecal extracts. Thus, fecal IgA to C. parvum antigens was highly associated with infection in subjects who had no evidence of previous exposure and may provide a useful tool in detecting recent infections.

Survival of Cryptosporidium parvum oocysts in calf housing facilities in the New York City watersheds.

PubMed

Collick, A S; Fogarty, E A; Ziegler, P E; Walter, M T; Bowman, D D; Steenhuis, T S

2006-01-01

Pathogen contamination of the public drinking water supply in the New York City watersheds is a serious concern. New York City's Watershed Agriculture Program is working with dairy farms in the watersheds to implement management practices that will reduce the risk of pathogens contaminating the water supply. Solar calf housing (SCH) was suggested as a best management practice (BMP) to control Cryptosporidium parvum, a common protozoan parasite that causes disease in humans. This BMP targets young calves because they are the primary source of C. parvum in dairy herds. The objective of this project was to assess and compare the survivability of C. parvum in SCH and in conventional calf housing (CCH), usually located in the main barn. C. parvum oocysts were secured in sentinel chambers and placed in SCH and CCH bedding on four farms. The chambers were in thermal, chemical, and moisture equilibrium with their microenvironments. An oocyst-filled control chamber, sealed from its surroundings, was placed near each chamber. Chambers and controls were sampled after 4, 6, and 8 wk. Oocyst viability in the chambers decreased to less than 10% in warm months and between 15 and 30% in the winter months. The viability of the control oocysts was similar to the chambers during warm months and generally higher during winter months. There was no significant (P > 0.05) difference in the viability decrease between SCH and CCH. Although oocyst viability was similar in both types of calf housing, SCH allow contaminated calf manure to be isolated from the main barn manure and potentially managed differently and in a way to decrease the number of viable oocysts entering the environment during field spreading.

Dispersion and Transport of Cryptosporidium Oocysts from Fecal Pats under Simulated Rainfall Events

PubMed Central

Davies, Cheryl M.; Ferguson, Christobel M.; Kaucner, Christine; Krogh, Martin; Altavilla, Nanda; Deere, Daniel A.; Ashbolt, Nicholas J.

2004-01-01

The dispersion and initial transport of Cryptosporidium oocysts from fecal pats were investigated during artificial rainfall events on intact soil blocks (1,500 by 900 by 300 mm). Rainfall events of 55 mm h−1 for 30 min and 25 mm h−1 for 180 min were applied to soil plots with artificial fecal pats seeded with approximately 107 oocysts. The soil plots were divided in two, with one side devoid of vegetation and the other left with natural vegetation cover. Each combination of event intensity and duration, vegetation status, and degree of slope (5° and 10°) was evaluated twice. Generally, a fivefold increase (P < 0.05) in runoff volume was generated on bare soil compared to vegetated soil, and significantly more infiltration, although highly variable, occurred through the vegetated soil blocks (P < 0.05). Runoff volume, event conditions (intensity and duration), vegetation status, degree of slope, and their interactions significantly affected the load of oocysts in the runoff. Surface runoff transported from 100.2 oocysts from vegetated loam soil (25-mm h−1, 180-min event on 10° slope) to up to 104.5 oocysts from unvegetated soil (55-mm h−1, 30-min event on 10° slope) over a 1-m distance. Surface soil samples downhill of the fecal pat contained significantly higher concentrations of oocysts on devegetated blocks than on vegetated blocks. Based on these results, there is a need to account for surface soil vegetation coverage as well as slope and rainfall runoff in future assessments of Cryptosporidium transport and when managing pathogen loads from stock grazing near streams within drinking water watersheds. PMID:14766600

Effects of Surfactants on Cryptosporidium parvum Mobility in Agricultural Soils from Illinois and Utah

NASA Astrophysics Data System (ADS)

Darnault, C. J.; Koken, E.; Jacobson, A. R.; Powelson, D.

2011-12-01

The occurence of the parasitic protozoan Cryptosporidium parvum in rural and agricultural watersheds due to agricultural activities and wildlife is inevitable. Understanding the behavior of C. parvum oocysts in the environment is critical for the protection of public health and the environment. To better understand the mechanisms by which the pathogen moves through soils and contaminates water resources, we study their mobility under conditions representative of real-world scenarios, where both C. parvum and chemicals that affect their fate are present in soils. Surfactants occur widely in soils due to agricultural practices such as wastewater irrigation and the application of pesticides or soil wetting agents. They affect water tension and, consequently, soil infiltration processes and the air-water interfaces in soil pores where C. parvum may be retained. We investigate the effects of surfactants on the mobility of C. parvum oocysts in agricultural soils from Illinois and Utah under unsaturated flow conditions. As it is critical to examine C. parvum in natural settings, we also developed a quantification method using RT-PCR for monitoring C. parvum oocysts in environmental soil and water samples. We optimized physico-chemical parameters to disrupt C. parvum oocysts and extract their DNA, and developed isolation methods to separate C. parvum oocysts from colloids in natural soil samples. The results of this research will lead to the development of an accurate and sensitive molecular method for the monitoring of C. parvum oocysts in environmental soil and water samples, and will further our understanding of the mechanisms controlling the behavior of C. parvum oocysts in soils, in particular the role of vadose zone processes, sorption to soil and surfactants.

Occurrence of Giardia and Cryptosporidium (oo)cysts in the river water of two recreational areas in Selangor, Malaysia.

PubMed

Azman, J; Init, I; Wan Yusoff, W S

2009-12-01

This study is the first report on the occurrence of Giardia and Cryptosporidium (oo)cysts in recreational rivers water from Malaysia. It was carried out in water samples at two rivers, 'Sungai Congkak' and 'Sungai Batu', located in Selangor State. The occurrence of both Giardia lamblia and Cryptosporidium parvum (oo)cysts was higher in Sungai Congkak (50% or 15/30 and 10% or 3/30 respectively) than Sungai Batu (16% or 5/30 and 3.3% or 1/30 respectively). The mean density of cysts/L was 0.72 in Sungai Congkak and 0.023 in Sungai Batu, and that of oocysts/L was 0.023 in Sungai Congkak and 0.0033 in Sungai Batu, showing that the occurrence of Giardia was higher and more frequent than Cryptosporidium in both rivers. Sungai Congkak also showed higher faecal coliforms count (ranging from 0.48x10³ to 73x10³ CFU/100 mL) than Sungai Batu (0.41x10³ to 16x10³ CFU/100 mL). On the other hand, the Giardia and Cryptosporidium (oo)cysts and faecal coliforms were more concentrated at the downstream station, followed by midstream and upstream stations which might be due to human factors where settlements and recreation areas were located around and between midstream and downstream stations. The (oo)cysts and faecal coliforms also increased during public holidays due to the significantly higher number of visitors (bathers) compared with the week days. All the parameters (physical, faecal coliforms and rainfall) did not show consistent significant correlation (based on r values of Pearson correlation analysis) with both protozoa, therefore these parameters are not suitable as indicator for the presence of Giardia and Cryptosporidium (oo)cysts in both rivers.

Concentrating Toxoplasma gondii and Cyclospora cayetanensis from surface water and drinking water by continuous separation channel centrifugation.

PubMed

Borchardt, M A; Spencer, S K; Bertz, P D; Ware, M W; Dubey, J P; Alan Lindquist, H D

2009-10-01

To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters. Ready-to-seed vials with known quantities of T. gondii and C. cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l(-1) into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were > or =84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64% to 100%, with the lowest recoveries in the most turbid waters. Method precision was between 10% and 20% coefficient of variation. Toxoplasma gondii and C. cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation. Waterborne transmission of T. gondii and C. cayetanensis presents another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from ambient water, otherwise false negatives may result. Validation data specific to T. gondii and C. cayetanensis concentration methods are limited. Continuous separation channel centrifugation recovers oocysts with high efficiency and precision, the method attributes required to accurately assess the risk of waterborne transmission.

Detection of Toxoplasma gondii DNA by qPCR in the feces of a cat that recently ingested infected prey does not necessarily imply oocyst shedding.

PubMed

Poulle, Marie-Lazarine; Forin-Wiart, Marie-Amélie; Josse-Dupuis, Émilie; Villena, Isabelle; Aubert, Dominique

2016-01-01

Detection of Toxoplasma gondii DNA in cat feces is considered indicative of the presence of T. gondii oocysts. This study aims to demonstrate that the high sensitivity of qPCR can lead to T. gondii DNA detection in cat feces in the absence of oocysts. A cat immune to toxoplasmosis was fed with a mouse experimentally infected with T. gondii. Detection of DNA of this parasite was performed by qPCR on feces passed: (i) on the day the cat ingested the infected prey; (ii) during the three previous days; and (iii) during the three following days. The kinetics of qPCR results are clearly not linked to oocyst shedding and this result demonstrates that qPCR can detect T. gondii DNA related to bradyzoites from an infected prey, in the absence of oocysts. Caution is thus recommended when interpreting T. gondii qPCR results for samples of cat feces. © M.-L. Poulle et al., published by EDP Sciences, 2016.

Deformability Assessment of Waterborne Protozoa Using a Microfluidic-Enabled Force Microscopy Probe

PubMed Central

Seddon, James R. T.; Lai, Stanley C. S.; Lemay, Serge G.; Bridle, Helen L.

2016-01-01

Many modern filtration technologies are incapable of the complete removal of Cryptosporidium oocysts from drinking-water. Consequently, Cryptosporidium-contaminated drinking-water supplies can severely implicate both water utilities and consumers. Existing methods for the detection of Cryptosporidium in drinking-water do not discern between non-pathogenic and pathogenic species, nor between viable and non-viable oocysts. Using FluidFM, a novel force spectroscopy method employing microchannelled cantilevers for single-cell level manipulation, we assessed the size and deformability properties of two species of Cryptosporidium that pose varying levels of risk to human health. A comparison of such characteristics demonstrated the ability of FluidFM to discern between Cryptosporidium muris and Cryptosporidium parvum with 86% efficiency, whilst using a measurement throughput which exceeded 50 discrete oocysts per hour. In addition, we measured the deformability properties for untreated and temperature-inactivated oocysts of the highly infective, human pathogenic C. parvum to assess whether deformability may be a marker of viability. Our results indicate that untreated and temperature-inactivated C. parvum oocysts had overlapping but significantly different deformability distributions. PMID:26938220

Cryptosporidium and Giardia in Humans, Domestic Animals, and Village Water Sources in Rural India.

PubMed

Daniels, Miles E; Shrivastava, Arpit; Smith, Woutrina A; Sahu, Priyadarshi; Odagiri, Mitsunori; Misra, Pravas R; Panigrahi, Pinaki; Suar, Mrutyunjay; Clasen, Thomas; Jenkins, Marion W

2015-09-01

Cryptosporidium parvum and Giardia lamblia are zoonotic enteric protozoa of significant health concern where sanitation, hygiene, and water supplies are inadequate. We examined 85 stool samples from diarrhea patients, 111 pooled fecal samples by species across seven domestic animal types, and water from tube wells (N = 207) and ponds (N = 94) across 60 villages in coastal Odisha, India, for Cryptosporidium oocysts and Giardia cysts to measure occurrence, concentration/shedding, and environmental loading rates. Oocysts/cysts were detected in 12% of diarrhea patients. Detection ranged from 0% to 35% for Cryptosporidium and 0% to 67% for Giardia across animal hosts. Animal loading estimates indicate the greatest contributors of environmental oocysts/cysts in the study region are cattle. Ponds were contaminated with both protozoa (oocysts: 37%, cysts: 74%), as were tube wells (oocysts: 10%, cysts: 14%). Future research should address the public health concern highlighted from these findings and investigate the role of domestic animals in diarrheal disease transmission in this and similar settings. © The American Society of Tropical Medicine and Hygiene.

An epidemiological study of Cryptosporidium parvum in two herds of adult beef cattle.

PubMed

Scott, C A; Smith, H V; Mtambo, M M; Gibbs, H A

1995-04-01

Prevalences of Cryptosporidium parvum oocysts in faeces and of isotype-specific anti-C. parvum antibodies in serum of apparently healthy adult cattle on two farms were determined. On Farm 1 cryptosporidial diarrhoea had been recorded in more than 80% of calves born over the previous 5 years, whereas on Farm 2 cryptosporidiosis had never been reported. No differences were demonstrated in oocyst excretion or presence of antibodies between the two farms. C. parvum oocysts were detected in 62.4% of faecal smears collected from a total of 553 apparently healthy adult cattle. Sucrose flotation was performed on a proportion of the faecal samples. This proved a more sensitive technique, detecting oocysts in 92% of the samples tested, and highlighting the insensitivity of direct smears for detecting oocysts. More than 90% of the cattle had specific anti-C. parvum IgG, IgG1, IgG2 and IgM antibodies and 58% specific anti-C. parvum IgA antibodies. Results suggest that asymptomatic adults may play an important role in the epidemiology of cryptosporidiosis in calves.

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

PubMed

Wang, Yunzhou; Tao, Geru; Cui, Yujuan; Lv, Qiyao; Xie, Li; Li, Yuan; Suo, Xun; Qin, Yinghe; Xiao, Lihua; Liu, Xianyong

2014-09-01

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists. Copyright © 2014 Elsevier Inc. All rights reserved.

Coccidia in passerines from the Nevado de Toluca National Park, Mexico.

PubMed

Medina, Juan P; Salgado-Miranda, Celene; García-Conejo, Michele; Galindo-Sánchez, Karla P; Mejía-García, Cristian J; Janczur, Mariusz K; Gomes Lopes, Carlos W; Berto, Bruno P; Soriano-Vargas, Edgardo

2014-03-01

In this study, we found unsporulated coccidia oocysts in passerines from the Nevado de Toluca National Park, Mexico. We captured birds and took samples of their droppings during three field visits. We examined a total of 72 fecal samples and found unsporulated coccidia oocysts in 10 samples from five passerine species: Atlapetes pileatus (3), Cardelina ruber (1), Mniotilta varia (1), Oreothlypis celata (2) and Regulus calendula (3). This appears to be the first recorded study of unsporulated coccidia oocysts in passerine species from Mexico.

Molecular-Based Detection Systems for Cryptosporidium Oocysts

EPA Science Inventory

The presentation describes on-going studies in collaboration with US EPA Region 2, 3, and the CDC on identifying sources of Cryptosporidium oocyst contamination in source waters using conventional and real-time PCR approaches.

Giardia spp. and Cryptosporidium spp. In the Ivaí Indigenous Land, Brazil.

PubMed

Nishi, Letícia; Bergamasco, Rosângela; Toledo, Max Jean de Ornelas; Falavigna, Dina Lúcia Morais; Gomes, Mônica Lúcia; Mota, Lúcio Tadeu; Falavigna-Guilherme, Ana Lúcia

2009-10-01

The objective of this study was to investigate the occurrence of cysts of Giardia spp. and oocysts of Cryptosporidium spp. in waters of the Ivaí Indigenous Land, Brazil. Samples of river and spring water and of treated water were filtered and analyzed by direct immunofluorescence (Merifluor kit, Meridian Bioscience, Cincinnati, Ohio). Of 21 samples, 7 from each locality, 3 (3/7, 42.8%) from a river were positive for Giardia (mean concentration 2.57 cysts/L), and 1 (1/7, 14.3%) was positive for Cryptosporidium (6 oocysts/L). From springs, 1 sample (1/7, 14.3%) was positive for Cryptosporidium (6 oocysts/L). One sample (1/7, 14.3%) from treated water was positive for both, with 4 oocysts/L and 2 cysts/L. Giardia was the more frequent protozoan present.

A simplified protocol for molecular identification of Eimeria species in field samples.

PubMed

Haug, Anita; Thebo, Per; Mattsson, Jens G

2007-05-15

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats.

PubMed

Zulpo, Dauton Luiz; Igarashi, Michelle; Sammi, Ana Sue; Santos, Joeleni Rosa Dos; Sasse, João Pedro; Cunha, Ivo Alexandre Leme da; Taroda, Alessandra; Barros, Luiz Daniel de; Almeida, Jonatas Campos de; Jenkins, Mark Christopher; Navarro, Italmar Teodorico; Garcia, João Luis

2017-01-01

The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

β-1,3-Glucan, Which Can Be Targeted by Drugs, Forms a Trabecular Scaffold in the Oocyst Walls of Toxoplasma and Eimeria

PubMed Central

Bushkin, G. Guy; Motari, Edwin; Magnelli, Paula; Gubbels, Marc-Jan; Dubey, Jitender P.; Miska, Katarzyna B.; Bullitt, Esther; Costello, Catherine E.; Robbins, Phillips W.; Samuelson, John

2012-01-01

ABSTRACT The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., β-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, β-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is β-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) β-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of β-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, β-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls. PMID:23015739

Comparison of transport and attachment behaviors of Cryptosporidium parvum oocysts and oocyst-sized microspheres being advected through three minerologically different granular porous media.

PubMed

Mohanram, Arvind; Ray, Chittaranjan; Harvey, Ronald W; Metge, David W; Ryan, Joseph N; Chorover, Jon; Eberl, D D

2010-10-01

In order to gain more information about the fate of Cryptosporidium parvum oocysts in tropical volcanic soils, the transport and attachment behaviors of oocysts and oocyst-sized polystyrene microspheres were studied in the presence of two soils. These soils were chosen because of their differing chemical and physical properties, i.e., an organic-rich (43-46% by mass) volcanic ash-derived soil from the island of Hawaii, and a red, iron (22-29% by mass), aluminum (29-45% by mass), and clay-rich (68-76% by mass) volcanic soil from the island of Oahu. A third agricultural soil, an organic- (13% by mass) and quartz-rich (40% by mass) soil from Illinois, was included for reference. In 10-cm long flow-through columns, oocysts and microspheres advecting through the red volcanic soil were almost completely (98% and 99%) immobilized. The modest breakthrough resulted from preferential flow-path structure inadvertently created by soil-particle aggregation during the re-wetting process. Although a high (99%) removal of oocysts and microsphere within the volcanic ash soil occurred initially, further examination revealed that transport was merely retarded because of highly reversible interactions with grain surfaces. Judging from the slope of the substantive and protracted tail of the breakthrough curve for the 1.8-μm microspheres, almost all (>99%) predictably would be recovered within ∼4000 pore volumes. This suggests that once contaminated, the volcanic ash soil could serve as a reservoir for subsequent contamination of groundwater, at least for pathogens of similar size or smaller. Because of the highly reversible nature of organic colloid immobilization in this soil type, C. parvum could contaminate surface water should overland flow during heavy precipitation events pick up near-surface grains to which they are attached. Surprisingly, oocyst and microsphere attachment to the reference soil from Illinois appeared to be at least as sensitive to changes in pH as was observed for the red, metal-oxide rich soil from Oahu. In contrast, colloidal attachment in the organic-rich, volcanic ash soil was relatively insensitive to changes in pH in spite of the high iron content. Given the fundamental differences in transport behavior of oocyst-sized colloids within the two volcanic soils of similar origin, agricultural practices modified to lessen C. parvum contamination of ground or surface water would necessitate taking the individual soil properties into account. Copyright © 2010 Elsevier Ltd. All rights reserved.

Comparison of transport and attachment behaviors of Cryptosporidium parvum oocysts and oocyst-sized microspheres being advected through three minerologically different granular porous media

USGS Publications Warehouse

Mohanram, Arvind; Ray, Chittaranjan; Harvey, Ronald W.; Metge, David W.; Ryan, Joseph N.; Chorover, Jon; Eberl, D.D.

2010-01-01

In order to gain more information about the fate of Cryptosporidium parvum oocysts in tropical volcanic soils, the transport and attachment behaviors of oocysts and oocyst-sized polystyrene microspheres were studied in the presence of two soils. These soils were chosen because of their differing chemical and physical properties, i.e., an organic-rich (43–46% by mass) volcanic ash-derived soil from the island of Hawaii, and a red, iron (22–29% by mass), aluminum (29–45% by mass), and clay-rich (68–76% by mass) volcanic soil from the island of Oahu. A third agricultural soil, an organic- (13% by mass) and quartz-rich (40% by mass) soil from Illinois, was included for reference. In 10-cm long flow-through columns, oocysts and microspheres advecting through the red volcanic soil were almost completely (98% and 99%) immobilized. The modest breakthrough resulted from preferential flow-path structure inadvertently created by soil-particle aggregation during the re-wetting process. Although a high (99%) removal of oocysts and microsphere within the volcanic ash soil occurred initially, further examination revealed that transport was merely retarded because of highly reversible interactions with grain surfaces. Judging from the slope of the substantive and protracted tail of the breakthrough curve for the 1.8-μm microspheres, almost all (>99%) predictably would be recovered within ∼4000 pore volumes. This suggests that once contaminated, the volcanic ash soil could serve as a reservoir for subsequent contamination of groundwater, at least for pathogens of similar size or smaller. Because of the highly reversible nature of organic colloid immobilization in this soil type, C. parvum could contaminate surface water should overland flow during heavy precipitation events pick up near-surface grains to which they are attached. Surprisingly, oocyst and microsphere attachment to the reference soil from Illinois appeared to be at least as sensitive to changes in pH as was observed for the red, metal-oxide rich soil from Oahu. In contrast, colloidal attachment in the organic-rich, volcanic ash soil was relatively insensitive to changes in pH in spite of the high iron content. Given the fundamental differences in transport behavior of oocyst-sized colloids within the two volcanic soils of similar origin, agricultural practices modified to lessen C. parvum contamination of ground or surface water would necessitate taking the individual soil properties into account.

Comparison of transport and attachment behaviors of Cryptosporidium parvum oocysts and oocyst-sized microspheres being advected through three minerologically different granular porous media

USGS Publications Warehouse

Mohanram, A.; Ray, C.; Harvey, R.W.; Metge, D.W.; Ryan, J.N.; Chorover, J.; Eberl, D.D.

2010-01-01

In order to gain more information about the fate of Cryptosporidium parvum oocysts in tropical volcanic soils, the transport and attachment behaviors of oocysts and oocyst-sized polystyrene microspheres were studied in the presence of two soils. These soils were chosen because of their differing chemical and physical properties, i.e., an organic-rich (43-46% by mass) volcanic ash-derived soil from the island of Hawaii, and a red, iron (22-29% by mass), aluminum (29-45% by mass), and clay-rich (68-76% by mass) volcanic soil from the island of Oahu. A third agricultural soil, an organic- (13% by mass) and quartz-rich (40% by mass) soil from Illinois, was included for reference. In 10-cm long flow-through columns, oocysts and microspheres advecting through the red volcanic soil were almost completely (98% and 99%) immobilized. The modest breakthrough resulted from preferential flow-path structure inadvertently created by soil-particle aggregation during the re-wetting process. Although a high (99%) removal of oocysts and microsphere within the volcanic ash soil occurred initially, further examination revealed that transport was merely retarded because of highly reversible interactions with grain surfaces. Judging from the slope of the substantive and protracted tail of the breakthrough curve for the 1.8-??m microspheres, almost all (>99%) predictably would be recovered within ~4000 pore volumes. This suggests that once contaminated, the volcanic ash soil could serve as a reservoir for subsequent contamination of groundwater, at least for pathogens of similar size or smaller. Because of the highly reversible nature of organic colloid immobilization in this soil type, C. parvum could contaminate surface water should overland flow during heavy precipitation events pick up near-surface grains to which they are attached. Surprisingly, oocyst and microsphere attachment to the reference soil from Illinois appeared to be at least as sensitive to changes in pH as was observed for the red, metal-oxide rich soil from Oahu. In contrast, colloidal attachment in the organic-rich, volcanic ash soil was relatively insensitive to changes in pH in spite of the high iron content. Given the fundamental differences in transport behavior of oocyst-sized colloids within the two volcanic soils of similar origin, agricultural practices modified to lessen C. parvum contamination of ground or surface water would necessitate taking the individual soil properties into account. ?? 2010.

Differences in fecundity of Eimeria maxima strains exhibiting different levels of pathogenicity in its avian host.

PubMed

Jenkins, Mark C; Dubey, J P; Miska, Katarzyna; Fetterer, Raymond

2017-03-15

Eimeria maxima is one of the most pathogenic species of avian coccidia, yet it is unknown why different E. maxima strains differ in the pathogenic effects they cause in chickens. The purpose of this study was to determine if a more pathogenic E. maxima strain (APU1) was also more fecund than a less pathogenic E. maxima strain (APU2). At identical doses, E. maxima APU1 always produces greater intestinal lesions and lower weight gain compared to E. maxima APU2. Using a dose response study, median and mean intestinal lesion scores in E. maxima APU1-infected chickens were greater by a score of 1-1.5 compared to chickens infected with E. maxima APU2. Likewise, weight gain depression in E. maxima APU1-infected chickens was 20-25% greater (equivalent to 110-130g body weight) than in E. maxima APU2-infected chickens. In order to understand the underlying cause of these observed clinical effects, 120 broiler chicks (5 oocyst levels, 6 replicates/level) were inoculated with various doses of E. maxima APU1 or APU2 oocysts. The dynamics of oocyst shedding was investigated by collecting fecal material every 12h from 114 to 210h post-inoculation (p.i.) and every 24h thereafter from 210 to 306h, and then processed for measuring E. maxima oocyst output. Oocysts were first observed at 138h p.i., and time of peak oocyst production was nearly identical for both E. maxima APU1 and APU2 around 150-162h. Total oocyst production was 1.1-2.6 fold higher at all dose levels for E. maxima APU1 compared to E. maxima APU2, being significantly higher (P<0.05) at the log 1.5 dose level. Other groups of chickens were infected with higher doses of E. maxima APU1 or APU2 oocysts, and intestinal lesions were assessed by histology at 72, 96, 120, and 144h p.i. Although schizonts, gamonts, and oocysts were observed at expected time-points, no obvious differences were noted in lesions induced by the two E. maxima strains. This study showed that the greater fecundity of E. maxima APU1 compared to E. maxima APU2 explains in part the observed differences in pathogenicity of the two E. maxima strains, but that other factors may contribute to differences in observed clinical effects. Published by Elsevier B.V.

Morphological and molecular characterization of Eimeria paludosa coccidian parasite (Apicomplexa:Eimeriidae) in a dusky moorhen (Gallinula tenebrosa, Gould, 1846) in Australia.

PubMed

Yang, Rongchang; Brice, Belinda; Elloit, Aileen; Lee, Elvina; Ryan, Una

2014-12-01

An Eimeria species is described from a dusky moorhen (Gallinula tenebrosa). Sporulated oocysts (n = 40) are ovoid, with a pitted single-layered oocyst wall in young oocysts and a relatively smooth wall in the mature oocysts. Oocyst wall was 1.0 µm thick, oocysts measured 17.3 × 13.3 (16.3-17.9 × 12.7-13.9) µm, oocyst length/width (L/W) ratio, 1.3. Oocyst residuum was absent. A large polar granule was always observed in the centre of the micropyle and many small polar granules were observed when the focus was on the wall. Sporocysts are elongate-ovoid, 8.4 × 5.1 (8.0-8.9 × 4.9-5.5) µm, sporocyst L/W ratio, 1.6 (1.5-1.8), sporocyst residuum was present, composed of numerous granules in a spherical or ovoid mass. Each sporocyst contained 2 elongate sporozoites, 7.7 × 2.6 (7-10 × 2.2-3) µm. A spherical-ellipsoid posterior refractile body was found in the sporozoites. A nucleus is located immediately anterior to the posterior refractile body. When the oocyst measurements and features were compared with valid Eimeria species from hosts in the Rallidae family, this Eimeria species was identified as E. paludosa. This is the first report of E. paludosa in Australia and the dusky moorhen (Gallinula tenebrosa) in a new host for this species. Molecular analysis was conducted at three loci; the 18S and 28S ribosomal RNA genes and the mitochondrial cytochrome oxidase gene (COI). At the 18 S locus, E. paludosa shared 97.3% genetic similarity with Eimeria gruis (GenBank accession number: AB544336). It also shared 99.2% genetic similarity with Eimeria crecis (GenBank accession numbers: HE653904 and HE653905) and 98.5% similarity with Eimeria nenei (GenBank accession numbers: HE653906), both of which were identified from a corncrake (Crex crex) in the United Kingdom. At the 28S locus, E. paludosa shared 91.4% similarity with E. papillata from a chicken (Gallus gallus) in the USA. At COI locus, E. paludosa was in a clade by itself and shared 87.2% similarity with E. irresidua, from a European rabbit (Oryctolagus cuniculus) from the Czech Republic. This is the first molecular characterization of E. paludosa. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Regulation of Plasmodium yoelii oocyst development by strain- and stage-specific small-subunit rRNA.

PubMed

Qi, Yanwei; Zhu, Feng; Eastman, Richard T; Fu, Young; Zilversmit, Martine; Pattaradilokrat, Sittiporn; Hong, Lingxian; Liu, Shengfa; McCutchan, Thomas F; Pan, Weiqing; Xu, Wenyue; Li, Jian; Huang, Fusheng; Su, Xin-zhuan

2015-03-10

One unique feature of malaria parasites is the differential transcription of structurally distinct rRNA (rRNA) genes at different developmental stages: the A-type genes are transcribed mainly in asexual stages, whereas the S-type genes are expressed mostly in sexual or mosquito stages. Conclusive functional evidence of different rRNAs in regulating stage-specific parasite development, however, is still absent. Here we performed genetic crosses of Plasmodium yoelii parasites with one parent having an oocyst development defect (ODD) phenotype and another producing normal oocysts to identify the gene(s) contributing to the ODD. The parent with ODD--characterized as having small oocysts and lacking infective sporozoites--was obtained after introduction of a plasmid with a green fluorescent protein gene into the parasite genome and subsequent passages in mice. Quantitative trait locus analysis of genome-wide microsatellite genotypes of 48 progeny from the crosses linked an ~200-kb segment on chromosome 6 containing one of the S-type genes (D-type small subunit rRNA gene [D-ssu]) to the ODD. Fine mapping of the plasmid integration site, gene expression pattern, and gene knockout experiments demonstrated that disruption of the D-ssu gene caused the ODD phenotype. Interestingly, introduction of the D-ssu gene into the same parasite strain (self), but not into a different subspecies, significantly affected or completely ablated oocyst development, suggesting a stage- and subspecies (strain)-specific regulation of oocyst development by D-ssu. This study demonstrates that P. yoelii D-ssu is essential for normal oocyst and sporozoite development and that variation in the D-ssu sequence can have dramatic effects on parasite development. Malaria parasites are the only known organisms that express structurally distinct rRNA genes at different developmental stages. The differential expression of these genes suggests that they play unique roles during the complex life cycle of the parasites. Conclusive functional proof of different rRNAs in regulating parasite development, however, is still absent or controversial. Here we functionally demonstrate for the first time that a stage-specifically expressed D-type small-subunit rRNA gene (D-ssu) is essential for oocyst development of the malaria parasite Plasmodium yoelii in the mosquito. This study also shows that variations in D-ssu sequence and/or the timing of transcription may have profound effects on parasite oocyst development. The results show that in addition to protein translation, rRNAs of malaria parasites also regulate parasite development and differentiation in a strain-specific manner, which can be explored for controlling parasite transmission. Copyright © 2015 Qi et al.

Hollow-fiber ultrafiltration of Cryptosporidium parvum oocysts from a wide variety of 10-L surface water samples.

PubMed

Kuhn, Ryan C; Oshima, Kevin H

2002-06-01

An optimized hollow-fiber ultrafiltration system (50 000 MWCO) was developed to concentrate Cryptosporidium oocysts from 10-L samples of environmental water. Seeded experiments were conducted using a number of surface-water samples from the southwestern U.S.A. and source water from four water districts with histories of poor oocyst recovery. Ultrafiltration produced a mean recovery of 47.9% from 19 water samples (55.3% from 39 individual tests). We also compared oocyst recoveries using the hollow-fiber ultrafiltration system with those using the Envirochek filter. In limited comparison tests, the hollow-fiber ultrafiltration system produced recoveries similar to those of the Envirochek filter (hollow fiber, 74.1% (SD = 2.8); Envirochek, 71.9% (SD = 5.2)) in low-turbidity (3.9 NTU) samples and performed better than the Envirochek filter in high-turbidity (159.0 NTU) samples (hollow fiber, 27.5%; Envirochek, 0.4%). These results indicate that hollow-fiber ultrafiltration can efficiently recover oocysts from a wide variety of surface waters and may be a cost-effective alternative for concentrating Cryptosporidium from water, given the reusable nature of the filter.

Toxoplasmosis Update and Public Health Implications

PubMed Central

Fayer, R.

1981-01-01

Toxoplasma gondii has a coccidian life cycle in the intestine of domestic and wild felids that includes a series of asexual and sexual stages and an oocyst stage that is shed in the feces. Oocysts complete their development outside the body, eventually becoming infective for about 350 species of vertebrates including cats and man. The effects of climate on oocyst survival and the physical and biological means of oocyst dispersal are discussed. Infectivity and pathogenicity for livestock species vary. Acute disease results from rapidly multiplying tachyzoites that may be transmitted by carnivorism, transfusion, vertical transmission and other routes. Patent infections may persist for the life of a host as bradyzoites within tissue cysts. Bradyzoites initiate acute infection in other hosts after carnivorism or organ transplantation or in the same host after immunosuppression. Also discussed are: (a) prevalence of T. gondii in livestock as determined by digestion and serological techniques, (b) identification in humans as accomplished by isolation, serological and skin test techniques and (c) identification in cats as accomplished primarily by fecal examinations for oocysts infective for mice. Source of human infections, major outbreaks, treatment, effects on mental health and methods for preventing toxoplasmosis in man and livestock are listed. ImagesFigure 1. PMID:7337909

Comparison of different diagnostic techniques for the detection of cryptosporidiosis in bovines

PubMed Central

Rekha, K. M. H.; Puttalakshmamma, G. C.; D’Souza, Placid E.

2016-01-01

Aim: Aim of the present study was to compare different methods, viz., Sheather's sugar flotation (SSF), Ziehl-Neelsen (ZN), Kinyoun's acid-fast method (KAF), safranin-methylene blue staining (SMB), and negative staining techniques such as nigrosin staining, light green staining, and malachite green staining for the detection of Cryptosporidium spp. oocysts in bovines. Materials and Methods: A total of 455 fecal samples from bovines were collected from private, government farms and from the clinical cases presented to Department of Medicine, Veterinary College, Bengaluru. They were subjected for SSF, ZN, KAF, SMB and negative staining methods. Results: Out of 455 animal fecal samples screened 5.71% were found positive for Cryptosporidium spp. oocysts. The species were identified as Cryptosporidium parvum in calves and Cryptosporidium andersoni in adults based on the morphological characterization and micrometry of the oocysts. Conclusions: Of all the techniques, fecal flotation with sheather's was found to be more specific and sensitive method for the detection of Cryptosporidium spp. oocysts. Among the conventional staining methods, the SMB gives better differentiation between oocysts and yeast. Among the three negative staining methods, malachite green was found sensitive over the other methods. PMID:27051211

Global Cryptosporidium Loads from Livestock Manure.

PubMed

Vermeulen, Lucie C; Benders, Jorien; Medema, Gertjan; Hofstra, Nynke

2017-08-01

Understanding the environmental pathways of Cryptosporidium is essential for effective management of human and animal cryptosporidiosis. In this paper we aim to quantify livestock Cryptosporidium spp. loads to land on a global scale using spatially explicit process-based modeling, and to explore the effect of manure storage and treatment on oocyst loads using scenario analysis. Our model GloWPa-Crypto L1 calculates a total global Cryptosporidium spp. load from livestock manure of 3.2 × 10 23 oocysts per year. Cattle, especially calves, are the largest contributors, followed by chickens and pigs. Spatial differences are linked to animal spatial distributions. North America, Europe, and Oceania together account for nearly a quarter of the total oocyst load, meaning that the developing world accounts for the largest share. GloWPa-Crypto L1 is most sensitive to oocyst excretion rates, due to large variation reported in literature. We compared the current situation to four alternative management scenarios. We find that although manure storage halves oocyst loads, manure treatment, especially of cattle manure and particularly at elevated temperatures, has a larger load reduction potential than manure storage (up to 4.6 log units). Regions with high reduction potential include India, Bangladesh, western Europe, China, several countries in Africa, and New Zealand.

Attenuation of a drug-sensitive strain of a turkey protozoan parasite Eimeria meleagrimitis by selection for precocious development.

PubMed

Rathinam, T; Gadde, U; Chapman, H D

2016-01-30

An attenuated line of Eimeria meleagrimitis was established by repeated propagation of the parasite in 9-day old turkey poults and subsequent selection for precocious development. Following 20 passages, the prepatent period decreased from 120 to 104h. A series of experiments were conducted to evaluate the pathogenicity, immunogenicity and fecundity of the newly selected line. Judged by body weight gain, feed consumption and feed efficiency following infection, the attenuated line had appreciably reduced pathogenicity. Immunogenicity of the attenuated line was examined by infecting poults successively with incremental doses of 10(2), 10(3) and 10(4) oocysts at 0, 7, and 14 days of age respectively. No oocysts were detected following challenge with 5×10(2) oocysts, indicating that the attenuated line had retained immunogenicity. Fecundity was assessed by infecting two-week old birds with 5×10(2) oocysts of either parent or attenuated line. Oocyst production from 96 to 240h post-infection showed that the patent period of the attenuated line commenced earlier and was of shorter duration than the parent line. Copyright © 2015 Elsevier B.V. All rights reserved.

Influence of organic carbon loading, sediment associated metal oxide content and sediment grain size distributions upon Cryptosporidium parvum removal during riverbank filtration operations, Sonoma County, CA

USGS Publications Warehouse

Metge, D.W.; Harvey, R.W.; Aiken, G.R.; Anders, R.; Lincoln, G.; Jasperse, James

2010-01-01

This study assessed the efficacy for removing Cryptosporidium parvum oocysts of poorly sorted, Fe- and Al-rich, subsurface sediments collected from 0.9 to 4.9 and 1.7–13.9 m below land surface at an operating riverbank filtration (RBF) site (Russian River, Sonoma County, CA). Both formaldehyde-killed oocysts and oocyst-sized (3 μm) microspheres were employed in sediment-packed flow-through and static columns. The degree of surface coverage of metal oxides on sediment grain surfaces correlated strongly with the degrees of oocyst and microsphere removals. In contrast, average grain size (D50) was not a good indicator of either microsphere or oocyst removal, suggesting that the primary mechanism of immobilization within these sediments is sorptive filtration rather than physical straining. A low specific UV absorbance (SUVA) for organic matter isolated from the Russian River, suggested that the modest concentration of the SUVA component (0.8 mg L−1) of the 2.2 mg L−1dissolved organic carbon (DOC) is relatively unreactive. Nevertheless, an amendment of 2.2 mg L−1 of isolated river DOC to column sediments resulted in up to a 35.7% decrease in sorption of oocysts and (or) oocyst-sized microspheres. Amendments (3.2 μM) of the anionic surfactant, sodium dodecyl benzene sulfonate (SDBS) also caused substantive decreases (up to 31.9 times) in colloid filtration. Although the grain-surface metal oxides were found to have a high colloid-removal capacity, our study suggested that any major changes within the watershed that would result in long-term alterations in either the quantity and (or) the character of the river's DOC could alter the effectiveness of pathogen removal during RBF operations.

Effects of Artemisia annua extracts on sporulation of Eimeria oocysts.

PubMed

Fatemi, Ahmadreza; Razavi, Seyyed Mostafa; Asasi, Keramat; Goudarzi, Majid Torabi

2015-03-01

The present study aimed to compare the effect of different Artemisia annua extracts on sporulation rate of mixed oocysts of Eimeria acervulina, Eimeria necatrix, and Eimeria tenella. Three types of A. annua extracts including petroleum ether (PE), ethanol 96° (E), and water (W) extracts were prepared. Artemisinin, a sesquiterpene lactone endoperoxide derived from the A. annua analysis of each extract was done by high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Fresh fecal samples containing three Eimeria species were floated and counted, and the oocysts were transferred into 50 tubes, each containing 10(5) oocysts per milliliter. Five tubes were control. Each of the other 45 tubes contained one of three doses (1 part per thousand (ppt), 2 ppt, and 5 ppt) and one of three extracts (PE, E, and W extracts) with five replications. The tubes were incubated for 48 h at 25-29 °C and aerated. Sporulation inhibition assay was used to evaluate the activity of extracts. The results showed that the E and PE extracts inhibit sporulation in 2 and 5 ppt concentrations, but the W extract stimulates it in all concentrations. The proportions of oocyst inhibition relative to control were 31 % (5 ppt) and 29 % (2 ppt) for PE and 34 % (5 ppt) and 46 % (2 ppt) for E extract. Furthermore, many oocysts in PE and E groups were wrinkled and contained abnormal sporocysts. The proportions of sporulation stimulation relative to control were 22 % (5 ppt), 24 % (2 ppt), and 27 % (1 ppt) in W extract. Our study is the first to demonstrate that all types of A. annua extracts do not necessarily have a similar activity, and the interaction of all contents and their relative concentrations is an important factor for sporulation stimulation or inhibition. It seems, some parts of unmetabolized excreted PE and E extracts could inhibit oocyst sporulation and eventually affect infection transmission.

A New Pathogen Transmission Mechanism in the Ocean: The Case of Sea Otter Exposure to the Land-Parasite Toxoplasma gondii

PubMed Central

Mazzillo, Fernanda F. M.; Shapiro, Karen; Silver, Mary W.

2013-01-01

Toxoplasma gondii is a land-derived parasite that infects humans and marine mammals. Infections are a significant cause of mortality for endangered southern sea otters (Enhydra lutris nereis), but the transmission mechanism is poorly understood. Otter exposure to T. gondii has been linked to the consumption of marine turban snails in kelp (Macrocystis pyrifera) forests. It is unknown how turban snails acquire oocysts, as snails scrape food particles attached to surfaces, whereas T. gondii oocysts enter kelp beds as suspended particles via runoff. We hypothesized that waterborne T. gondii oocysts attach to kelp surfaces when encountering exopolymer substances (EPS) forming the sticky matrix of biofilms on kelp, and thus become available to snails. Results of a dietary composition analysis of field-collected snails and of kelp biofilm indicate that snails graze the dense kelp-biofilm assemblage composed of pennate diatoms and bacteria inserted within the EPS gel-like matrix. To test whether oocysts attach to kelp blades via EPS, we designed a laboratory experiment simulating the kelp forest canopy in tanks spiked with T. gondii surrogate microspheres and controlled for EPS and transparent exopolymer particles (TEP - the particulate form of EPS). On average, 19% and 31% of surrogates were detected attached to kelp surfaces covered with EPS in unfiltered and filtered seawater treatments, respectively. The presence of TEP in the seawater did not increase surrogate attachment. These findings support a novel transport mechanism of T. gondii oocysts: as oocysts enter the kelp forest canopy, a portion adheres to the sticky kelp biofilms. Snails grazing this biofilm encounter oocysts as ‘bycatch’ and thereby deliver the parasite to sea otters that prey upon snails. This novel mechanism can have health implications beyond T. gondii and otters, as a similar route of pathogen transmission may be implicated with other waterborne pathogens to marine wildlife and humans consuming biofilm-feeding invertebrates. PMID:24386100

The pathological changes caused by Eimeria falciformis var pragensis in mice.

PubMed Central

Mesfin, G M; Bellamy, J E; Stockdale, P H

1978-01-01

Groups of Swiss white mice weighing 25-28 grams were infected orally with 500, 2,000, 5,000 or 20,000 oocysts of Eimeria falciformis var pragensis. Depression, anorexia, weight loss, diarrhea or dysentery, and dehydration were most pronounced at eight to ten days postinfection. The highest mortality, 31%, occurred in mice infected with 20,000 oocysts. None of the mice infected with 500 oocysts died. The pathological findings were equally severe in mice infected with 5,000 and 20,000 oocysts. The enteric lesions, most pronounced at eight to ten days postinfection, were restricted mainly to the large intestine and consisted initially of both cryptal and absorptive epithelial cell destruction and submucosal edema. These changes were followed in 12 to 24 hours by a transient influx of neutrophils into the lamina propria followed by mononuclear cell infiltration which lasted for five to ten days. As the infective dose decreased, the inflammatory response occurred later and was less extensive. When seen, hemorrhage occurred seven to 11 days postinfection. In 50% of the mice infected with 5,000 and 20,000 oocysts, varying degrees of a nonselective mucosal necrosis were seen at eight to 12 days postinfection. In mice infected with 500 oocysts, mucosal destruction was restricted to the epithelium. Neutrophils predominated when necrosis was extensive, otherwise, mononuclear cells were the main inflammatory cells. Two to three days following necrosis, crypt hyperplasia was marked and mucosal integrity was restored. Ulcers, some of which extended into the submucosa, healed by days 14 to 20. Localized granulomatous colitis, induced by trapped oocysts within the lamina propria, was seen until the experiment was terminated at 25 days postinfection. Infection was followed by lymphoid hyperplasia in the lymph nodes and the spleen. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10. PMID:743602

RESEARCH IN FILTRATION FOR CRYPTOSPORIDIUM REMOVAL

EPA Science Inventory

The USEPA has conducted pilot plant studies for the removal of Cryptosporidium oocysts from drinking water. Fourteen pilot-scale tests were performed to assess the ability of conventional treatment to control Cryptosporidium oocysts and three surrogates; turbidity, total particle...

Sporulation dynamics of poultry Eimeria oocysts in Chennai.

PubMed

Venkateswara Rao, P; Raman, M; Gomathinayagam, S

2015-12-01

The infective form of Eimeria is the highly resistant oocyst, which is shed in the faeces of infected animals. Present study was carried out to understand the sporulation dynamics of six Eimeria oocysts viz. E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix and E. tenella in Chennai. Faecal samples of poultry were collected from various poultry farms located in and around Tamil Nadu. Oocysts of various Eimeria species were examined microscopically for sporulation on a 6 h interval basis till complete sporulation is acheived. The sporulation time recorded was 168, 120, 216, 192, 96 and 96 h for E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix and E. tenella respectively. It can be concluded on comparison with previous studies that humid weather conditions delay the sporulation time and dry weather and wet litter is the ideal condition for rapid sporulation.

Hyperimmune bovine colostrum treatment of moribund Leopard geckos (Eublepharis macularius) infected with Cryptosporidium sp.

PubMed

Graczyk, T K; Cranfield, M R; Bostwick, E F

1999-01-01

Therapy based on the protective passive immunity of hyperimmune bovine colostrum (HBC) was applied to 12 moribund Leopard geckos (Eublepharis macularius) infected with Cryptosporidium sp. The geckos were lethargic and moderately to severely emaciated, weighing on average 36% of the baseline body weight value. Seven gastric HBC treatments at 1-week intervals each decreased the relative output of Cryptosporidium sp. oocysts and the prevalence of oocyst-positive fecal specimens. Histologically, after 8 weeks of therapy, seven out of 12 geckos had only single developmental stages of Cryptosporidium sp. in the intestinal epithelium, and three, one and one geckos had low, moderate and high numbers, respectively, of the pathogen developmental stages. The HBC therapy was efficacious in decreasing the parasite load in moribund geckos. Morphometric and immunologic analysis of Cryptosporidium sp. oocyst isolates originating from Leopard geckos (E. macularius) demonstrated differences between gecko-derived oocyst isolates and isolates of C. serpentis recovered from snakes.

Transport and Retention of Toxoplasma gondii Oocysts in Loamy Sand and Sandy Loam Soils

NASA Astrophysics Data System (ADS)

Kinsey, E. N.; Korte, C.; L'Ollivier, C.; Dubey, J. P.; Aurélien, D.; Darnault, C. J. G.

2016-12-01

Toxoplasma gondii is one of the most prevalent parasites affecting warm-blooded animals and humans. It has a complex life cycle that involves a wide variety of intermediate hosts with felids as a definitive host. Humans may contract it through consumption of infected, undercooked meat or by water or food sources contaminated with the oocyst form of the parasite. Infection of pregnant women can cause stillbirth, neurological effects or blindness. Because of the prevalence of cats, including on farms where oocyst-contaminated cat feces, animal feed, soil and water have been found, T. gondii is spread almost throughout the entire globe. It has been implicated or suspected in waterborne infections since the 1990s. This study aims to characterize the transport and retention of T. gondii oocysts in field soils. The four soils used were collected from fallow and cultivated fields in Illinois and Utah, USA. They are classified as loamy sands and sandy loams. Soil columns were subjected to continuous artificial rainfall until they reached steady state at which point pulses that included 2.5 million T. gondii oocysts (Me49 strain) and KBr as a tracer were added. After the pulse infiltrated, continuous rainfall was resumed. Rain applied all columns was a 1 mM KCl solution. Leachate samples were collected, analyzed using qPCR for T. gondii and bromide ions and breakthrough curves were produced. Soil was sliced into 1 to 2 cm sections, for which water content and T. gondii concentration were measured to access degree of saturation and oocyst retention.

INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH OZONE

EPA Science Inventory

Ozone inactivation rates for Cryptosporidium parvum (C. parvum) oocysts were determined with an in-vitro excystation method based on excysted sporozoite counts. Results were consistent with published animal infectivity data for the same C. parvum strain. The inactivation kinetics...

Efficacy of halofuginone lactate against experimental cryptosporidiosis in goat neonates.

PubMed

Petermann, Julie; Paraud, Carine; Pors, Isabelle; Chartier, Christophe

2014-05-28

Preliminary results obtained in calves, lambs and goat kids infected by Cryptosporidium sp. have indicated a partial prophylactic efficacy of halofuginone lactate when administered at 100 μg/kg body weight (BW). In this study, the efficacy of halofuginone lactate was evaluated in goat neonates experimentally inoculated with Cryptosporidium parvum oocysts per oral route. The trial consisted in 2 replicated experiments carried out successively at 2 months of interval. Twenty-two 2- to 4-day-old kids were experimentally inoculated once, 2-3 days after the arrival in premises, with 10(6)C. parvum oocysts per oral route and were allocated into 2 groups. Animals of group 1 acted as untreated control whereas animals of group 2 received halofuginone lactate for 10 days from the infection day to day 9 post-infection (DPI) at a daily oral dose rate of 100 μg/kg BW. Individual oocyst shedding was monitored by daily examination of faecal smears stained by carbol fuchsin and scored semi-quantitatively (0-5) until 19 DPI. Daily diarrhoea scores, weight gain and mortality were recorded. In the first experiment, oocyst excretion started 1 DPI in the control group, was highest on 4 DPI (mean score 3.6) and became undetectable from 16-19 DPI. In the treated group, oocyst shedding started 1 day later, showed lower scores compared to control on 4, 5, 6, 7 and 10 DPI and vanished from 16 to 19 DPI. No significant difference was seen for weight gains between groups. Five kids died in the control group compared to 1 kid in the treated group. In the second (replicated) experiment, oocyst excretion started 2 DPI in the control group, was highest on 4 DPI (mean score 4.5) and became undetectable 18 and 19 DPI. In the treated group, oocyst shedding started 2 days later, peaked on 13 DPI (mean score 2.3) and persisted until the end of the experiment. No significant difference was seen for weight gains between groups. Ten kids died in the control group compared to 3 kids in the treated group. The results demonstrated the efficacy of halofuginone lactate when given as a prophylactic treatment at 100 μg/kg BW during 10 days in reducing oocyst shedding, diarrhoea and mortality in goat kid cryptosporidiosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Aquatic biomonitoring of Giardia cysts and Cryptosporidium oocysts in peninsular Malaysia.

PubMed

Lee, Soo Ching; Ngui, Romano; Tan, Tiong Kai; Roslan, Muhammad Aidil; Ithoi, Init; Lim, Yvonne A L

2014-01-01

An aquatic biomonitoring of Giardia cysts and Cryptosporidium oocysts in river water corresponding to five villages situated in three states in peninsular Malaysia was determined. There were 51.3% (20/39) and 23.1% (9/39) samples positive for Giardia and Cryptosporidium (oo)cysts, respectively. Overall mean concentration between villages for Giardia cysts ranged from 0.10 to 25.80 cysts/l whilst Cryptosporidium oocysts ranged from 0.10 to 0.90 oocysts/l. Detailed results of the river samples from five villages indicated that Kuala Pangsun 100% (9/9), Kemensah 77.8% (7/9), Pos Piah 33.3% (3/9) and Paya Lebar 33.3% (1/3) were contaminated with Giardia cysts whilst Cryptosporidium (oo)cysts were only detected in Kemensah (100 %; 9/9) and Kuala Pangsun (66.6%; 6/9). However, the water samples from Bentong were all negative for these waterborne parasites. Samples were collected from lower point, midpoint and upper point. Midpoint refers to the section of the river where the studied communities are highly populated. Meanwhile, the position of the lower point is at least 2 km southward of the midpoint and upper point is at least 2 km northward of the midpoint. The highest mean concentration for (oo)cysts was found at the lower points [3.15 ± 6.09 (oo)cysts/l], followed by midpoints [0.66 ± 1.10 (oo)cysts/l] and upper points [0.66 ± 0.92 (oo)cysts/l]. The mean concentration of Giardia cysts was highest at Kuala Pangsun (i.e. 5.97 ± 7.0 cysts/l), followed by Kemensah (0.83 ± 0.81 cysts/l), Pos Piah (0.20 ± 0.35 cysts/l) and Paya Lebar (0.10 ± 0.19 cysts/l). On the other hand, the mean concentration of Cryptosporidium oocysts was higher at Kemensah (0.31 ± 0.19 cysts/l) compared to Kuala Pangsun (0.03 ± 0.03cysts/l). All the physical and chemical parameters did not show significant correlation with both protozoa. In future, viability status and molecular characterisation of Giardia and Cryptosporidium should be applied to identify species and genotypes/subgenotypes for better understanding of the epidemiology of these waterborne parasites.

Experimentally induced clinical Cystoisospora canis coccidiosis in dogs with prior natural patent Cystoisospora ohioensis-like or C. canis infections.

PubMed

Houk, Alice E; O'Connor, Thomas; Pena, Hilda F J; Gennari, Solange Maria; Zajac, Anne M; Lindsay, David S

2013-10-01

Diarrhea caused by intestinal coccidia (Cystoisospora species) is a common problem in pet dogs and in dogs in animal shelters. Cystoisospora canis has the largest oocysts of the 4 named species of coccidia infecting dogs. The present study examined an isolate of C. canis obtained from a dog from São Paulo, SP, Brazil. Oocysts sporulated within 2 days at room temperature, and 20 sporulated oocysts were measured at 37.6 by 28.6 μm (range 35-42 by 26-31 μm). Most sporulated oocysts contained 2 sporocysts, each with 4 sporozoites, although a few (<1%) were Caryospora-like and contained 1 sporocyst with 8 sporozoites. Two experiments using a total of 11 female 6-wk-old beagles were conducted to determine the pathogenicity of oral infection with 5 × 10(4) sporulated oocysts of this isolate of C. canis. Five of the 11 dogs had natural infections with Cystoisospora ohioensis-like (n = 4) or C. canis (n = 1) species prior to the predicted patent period of 9-10 days. Ten of the dogs developed diarrhea with occasional blood, and 3 dogs were affected to the extent that clinical treatment for coccidiosis using sulfadimethoxine was recommended. Dog CRU had a natural C. canis infection and did not develop clinical disease after oral infection with C. canis oocysts. This dog had a prepatent period of 9 days and a patent period of 3 days, corresponding to experimental infection with the new isolate of C. canis. It excreted fewer C. canis oocysts than did the other dogs. The 4 dogs with natural C. ohioensis-like infection all developed clinical disease, and 1 required treatment. The prepatent period was 9-10 days, and the patent period was 10-11 days in these dogs. All 6 dogs not naturally infected with Cystoisospora developed clinical disease, and 2 required treatment. The prepatent period was 9-10 days, and the patent period was 8-12 days. The present study confirms that C. canis is a primary pathogen for young dogs. It demonstrates that prior infection with C. canis but not C. ohioensis-like coccidia confers some resistance to clinical disases and a decrease in oocyst production in dogs challenged with C. canis.

EVALUATION OF CRYPTOSPORIDIUM OOCYSTS AND GIARDIA CYSTS IN A WATERSHED RESERVOIR

EPA Science Inventory

This investigation evaluated the occurrence of Cryptosporidium oocysts and Giardia cysts at 17 sampling locations in Lake Texoma reservoir using method 1623 with standard Envirocheck" capsule filters. The watershed serves rural agricultural communities active in cattle ranching,...

EVALUATION OF CRYPTOSPORIDIUM OOCYSTS AND GIARDIA CYSTS IN A WATERSHED RESERVOIR

EPA Science Inventory

This investigation evaluated the occurrence of Cryptosporidium oocysts and Giardia cysts at 17 sampling locations in Lake Texoma reservoir using method 1623 with standard Envirocheck™ capsule filters. The watershed serves rural agricultural communities active in cattle ranching, ...

Mechanics of the Toxoplasma gondii oocyst wall

USDA-ARS?s Scientific Manuscript database

The ability of microorganisms to survive under extreme conditions is closely related to the physicochemical properties of their wall. In the ubiquitous protozoan parasite Toxoplasma gondii, the oocyst stage possesses a bilayered wall that protects the dormant but potentially infective parasites from...

THE INFECTIVITY OF CRYPTOSPORIDIUM PARVUM IN HEALTHY VOLUNTEERS

EPA Science Inventory

Background. Small numbers of Cryptosporidium parvum oocysts can contaminate even treated drinking water, and ingestion of oocysts can cause diarrheal disease in normal as well as immunocompromised hosts. Since the number of organisms necessary to cause infection in humans is unkn...

Identification of algae which interfere with the detection of Giardia cysts and Cryptosporidium oocysts and a method for alleviating this interference.

PubMed Central

Rodgers, M R; Flanigan, D J; Jakubowski, W

1995-01-01

Fifty-four algal species were tested for cross-reaction in the American Society for Testing and Materials Giardia/Cryptosporidium indirect immunofluorescence assay, and 24 showed some degree of fluorescence. Two species, Navicula minima and Synechococcus elongatus, exhibited a bright apple green fluorescence. The addition of goat serum to the assay mixture blocked the fluorescence of most nontarget organisms tested and also decreased the background fluorescence. Goat serum did not interfere with the fluorescence of Giardia cysts or Cryptosporidium oocysts or the identification of cyst and oocyst internal structures. PMID:7487013

Global Cryptosporidium Loads from Livestock Manure

PubMed Central

2017-01-01

Understanding the environmental pathways of Cryptosporidium is essential for effective management of human and animal cryptosporidiosis. In this paper we aim to quantify livestock Cryptosporidium spp. loads to land on a global scale using spatially explicit process-based modeling, and to explore the effect of manure storage and treatment on oocyst loads using scenario analysis. Our model GloWPa-Crypto L1 calculates a total global Cryptosporidium spp. load from livestock manure of 3.2 × 1023 oocysts per year. Cattle, especially calves, are the largest contributors, followed by chickens and pigs. Spatial differences are linked to animal spatial distributions. North America, Europe, and Oceania together account for nearly a quarter of the total oocyst load, meaning that the developing world accounts for the largest share. GloWPa-Crypto L1 is most sensitive to oocyst excretion rates, due to large variation reported in literature. We compared the current situation to four alternative management scenarios. We find that although manure storage halves oocyst loads, manure treatment, especially of cattle manure and particularly at elevated temperatures, has a larger load reduction potential than manure storage (up to 4.6 log units). Regions with high reduction potential include India, Bangladesh, western Europe, China, several countries in Africa, and New Zealand. PMID:28654242

A Waterborne Outbreak and Detection of Cryptosporidium Oocysts in Drinking Water of an Older High-Rise Apartment Complex in Seoul

PubMed Central

Yang, Jin-Young; Lee, Eun-Sook; Kim, Se-Chul; Cha, So-Yang; Kim, Sung-Tek; Lee, Man-Ho; Han, Sun-Hee; Park, Young-Sang

2013-01-01

From May to June 2012, a waterborne outbreak of 124 cases of cryptosporidiosis occurred in the plumbing systems of an older high-rise apartment complex in Seoul, Republic of Korea. The residents of this apartment complex had symptoms of watery diarrhea and vomiting. Tap water samples in the apartment complex and its adjacent buildings were collected and tested for 57 parameters under the Korean Drinking Water Standards and for additional 11 microbiological parameters. The microbiological parameters included total colony counts, Clostridium perfringens, Enterococcus, fecal streptococcus, Salmonella, Shigella, Pseudomonas aeruginosa, Cryptosporidium oocysts, Giardia cysts, total culturable viruses, and Norovirus. While the tap water samples of the adjacent buildings complied with the Korean Drinking Water Standards for all parameters, fecal bacteria and Cryptosporidium oocysts were detected in the tap water samples of the outbreak apartment complex. It turned out that the agent of the disease was Cryptosporidium parvum. The drinking water was polluted with sewage from a septic tank in the apartment complex. To remove C. parvum oocysts, we conducted physical processes of cleaning the water storage tanks, flushing the indoor pipes, and replacing old pipes with new ones. Finally we restored the clean drinking water to the apartment complex after identification of no oocysts. PMID:24039290

A new Eimeria species (Protozoa: Eimeriidae) from caribou in Ameralik, West Greenland.

PubMed

Skirnisson, K; Cuyler, C

2016-04-01

Fecal samples of 11 calves shot in the Ameralik area, West Greenland, in August-September 2014 were examined for coccidian parasites. The calves belonged to a population of interbreeding indigenous caribou Rangifer tarandus groenlandicus and feral semi-domestic Norwegian reindeer Rangifer tarandus tarandus. Two coccidian species were found: Eimeria rangiferis and a coccidium that was identified and described as a new species. The latter's sporulated oocyst is spherical or slightly subspherical. Average size is 25.6 × 24.8 μm. The oocyst has two distinct walls. Wall thickness is ∼1.4 μm. The unicolored outer wall is brown, the inner wall is dark gray. The oocysts contain a small polar granule but are devoid of a microphyle. The oocysts enclose four ovoid-shaped sporocysts with a rounded end opposite to the Stieda body. The average size of sporocysts is 15.2 × 7.8 μm. Sporocysts contain a granular sporocyst residuum that forms a spherical cluster between the sporocysts, one large refractile body is present in each sporozoite. The spherical form easily distinguishes oocysts of the new species from the seven previously described eimerid species in R. tarandus. This is the first eimerid described as a new species to the sciences from caribou in the Nearctic.

The epizootiology of Eimeria infections in commercial broiler chickens where anticoccidial drug programs were employed in six successive flocks to control coccidiosis.

PubMed

Chapman, H D; Barta, J R; Hafeez, M A; Matsler, P; Rathinam, T; Raccoursier, M

2016-08-01

The course of natural Eimeria infections in 6 successive broiler flocks at a commercial farm comprising 4 houses, where different anticoccidial drug programs were employed, was studied by counting the number of oocysts in the litter at weekly intervals. The course of infection in all flocks followed a bell shaped curve in which oocyst numbers, initially low, increased to a peak ranging from 36 × 10(3) to 74 × 10(3) oocysts/g (OPG) of litter around 3 to 4 wk of age. Numbers subsequently declined to 3 × 10(3) to 15 × 10(3) OPG. Oocysts could be detected between flocks when birds were not present. Species of Eimeria identified included E. acervulina, E. maxima, and E. tenella Despite the presence of large numbers of oocysts in the litter, coccidial lesions were not observed in the intestines of the birds. The performance of broilers at the study site was comparable to that of other farms in the area where birds from the same settlement were reared to a similar age using the same drug programs. The results indicate the ubiquitous nature of Eimeria spp. infections in commercial broilers despite prophylactic medication. © 2016 Poultry Science Association Inc.

A waterborne outbreak and detection of cryptosporidium oocysts in drinking water of an older high-rise apartment complex in seoul.

PubMed

Cho, Eun-Joo; Yang, Jin-Young; Lee, Eun-Sook; Kim, Se-Chul; Cha, So-Yang; Kim, Sung-Tek; Lee, Man-Ho; Han, Sun-Hee; Park, Young-Sang

2013-08-01

From May to June 2012, a waterborne outbreak of 124 cases of cryptosporidiosis occurred in the plumbing systems of an older high-rise apartment complex in Seoul, Republic of Korea. The residents of this apartment complex had symptoms of watery diarrhea and vomiting. Tap water samples in the apartment complex and its adjacent buildings were collected and tested for 57 parameters under the Korean Drinking Water Standards and for additional 11 microbiological parameters. The microbiological parameters included total colony counts, Clostridium perfringens, Enterococcus, fecal streptococcus, Salmonella, Shigella, Pseudomonas aeruginosa, Cryptosporidium oocysts, Giardia cysts, total culturable viruses, and Norovirus. While the tap water samples of the adjacent buildings complied with the Korean Drinking Water Standards for all parameters, fecal bacteria and Cryptosporidium oocysts were detected in the tap water samples of the outbreak apartment complex. It turned out that the agent of the disease was Cryptosporidium parvum. The drinking water was polluted with sewage from a septic tank in the apartment complex. To remove C. parvum oocysts, we conducted physical processes of cleaning the water storage tanks, flushing the indoor pipes, and replacing old pipes with new ones. Finally we restored the clean drinking water to the apartment complex after identification of no oocysts.

First isolation of Hammondia hammondi from a cat from Ethiopia

USDA-ARS?s Scientific Manuscript database

Toxoplasma gondii oocysts are morphologically and antigenically similar to oocysts of another feline coccidian, Hammondia hammondi. The distinction between H. hammondi and T. gondii is important from an epidemiological perspective because all isolates of T. gondii are potentially pathogenic for hum...

CHARACTERIZATION OF CRYPTOSPORIDIUM PARVUM BY MATRIX-ASSISTED LASER DESORPTION -- IONIZATION TIME OF FLIGHT MASS SPECTROMETRY

EPA Science Inventory

Matrix assisted laser desorption/ionization (MALDI) mass spectrometry was used to investigate whole and freeze thawed Cryptosporidium parvum oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtai...

ENHANCED CONCENTRATION AND ISOLATION OF CYCLOSPORA CAYETANENSIS OOCYSTS FROM HUMAN FECAL SAMPLES

EPA Science Inventory

Cyclospora cayetanensis is the causative agent of cyclosporiasis, an emerging infections disease. A new method for the purification of Cycloposra cayetanensis oocysts from fecal matter has been developed, using a modified detachment solution and a Renocal-sucrose gradient sedimen...

A COMPARISON OF ENUMERATION TECHNIQUES FOR CRYPTOSPORIDIUM PARVUM OOCYSTS

EPA Science Inventory

A variety of methods have been used to enumerate Cryptosporidium parvum oocysts from source or drinking waters. The reliability of these counting methods varies, in part, with suspension density, sample purity, and other factors. Frequently, the method of determination of suspens...

Three new species of Coccidia (Apicomplexa: Eimeriidae) from Skinks, Lipinia spp. (Sauria: Scincidae), from Oceania

USGS Publications Warehouse

McAllister, Chris T.; Duszynski, Donald W.; Austin, Christopher C.; Fisher, Robert N.

2013-01-01

Between September 1991 and March 1993, 25 moth skinks (Lipinia noctua) were collected from various localities on the Cook Islands, Fiji, Papua New Guinea (PNG), and Vanuatu and examined for coccidians. In addition, a single Roux's lipinia skink (Lipinia rouxi) was collected from PNG and examined for coccidia. Sixteen (64%) L. noctua were found to harbor 2 new eimerians, and L. rouxi harbored another new Eimeria sp. Oocysts of Eimeria lipinia n. sp. from 9 (36%) L. noctua from the Cook Islands, Fiji, and PNG were subspherical with a bilayered wall and measured (L × W) 18.6 × 16.9 μm, with a L/W ratio of 1.1. Both micropyle and oocyst residuum were absent, but a polar granule was present. Oocysts of Eimeria melanesia n. sp. from 6 (24%) L. noctua from Fiji and Vanuatu and a single L. rouxi from PNG were subspherical to ellipsoidal with a bilayered wall and measured 19.8 × 17.5 μm, and L/W was 1.1. Both micropyle and oocyst residuum were absent, but a single or fragmented polar granule was present. Oocysts of Eimeria lessoni n. sp. from 1 (4%) L. noctua from PNG were cylindroidal with a bilayered wall and measured 28.1 × 15.7 μm, and L/W was 1.8. Both micropyle and oocyst residuum were absent, but a single polar granule was present. These represent the third report of Eimeria spp. reported from any host on PNG and the only coccidians, to our knowledge, ever described from L. noctua and L. rouxi and from the Cook Islands and Vanuatu.

Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

PubMed

Gonzalez-Ceron, L; Rodriguez, M H; Wirtz, R A; Sina, B J; Palomeque, O L; Nettel, J A; Tsutsumi, V

1998-11-01

The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur. Copyright 1998 Academic Press.

Disease surveillance of Atlantic herring: molecular characterization of hepatic coccidiosis and a morphological report of a novel intestinal coccidian

USGS Publications Warehouse

Friend, Sarah E; Lovey, J; Hershberger, Paul

2016-01-01

Surveillance for pathogens of Atlantic herring, including viral hemorrhagic septicemia virus (VHSV),Ichthyophonus hoferi, and hepatic and intestinal coccidians, was conducted from 2012 to 2016 in the NW Atlantic Ocean, New Jersey, USA. Neither VHSV nor I. hoferi was detected in any sample. Goussia clupearum was found in the livers of 40 to 78% of adult herring in varying parasite loads; however, associated pathological changes were negligible. Phylogenetic analysis based on small subunit 18S rRNA gene sequences placed G. clupearum most closely with other extraintestinal liver coccidia from the genus Calyptospora, though the G. clupearum isolates had a unique nucleotide insertion between 604 and 729 bp that did not occur in any other coccidian species. G. clupearum oocysts from Atlantic and Pacific herring were morphologically similar, though differences occurred in oocyst dimensions. Comparison of G. clupearum genetic sequences from Atlantic and Pacific herring revealed 4 nucleotide substitutions and 2 gaps in a 1749 bp region, indicating some divergence in the geographically separate populations. Pacific G. clupearum oocysts were not directly infective, suggesting that a heteroxenous life cycle is likely. Intestinal coccidiosis was described for the first time from juvenile and adult Atlantic herring. A novel intestinal coccidian species was detected based on morphological characteristics of exogenously sporulated oocysts. A unique feature in these oocysts was the presence of 3 long (15.1 ± 5.1 µm, mean ±SD) spiny projections on both ends of the oocyst. The novel morphology of this coccidian led us to tentatively name this parasite G. echinata n. sp.

Occurrence of Cryptosporidium and Giardia in raw and finished drinking water in north-eastern Spain.

PubMed

Ramo, Ana; Del Cacho, Emilio; Sánchez-Acedo, Caridad; Quílez, Joaquín

2017-02-15

This paper collects the first large-sample-size study on the presence of Cryptosporidium oocysts and Giardia cysts in drinking water plants at the 20 most populated towns in Aragón (north-eastern Spain). Samples of influent raw water and effluent finished water were collected from each plant during different seasons and processed according to USEPA Method 1623. Cryptosporidium oocysts and Giardia cysts were detected in samples collected from 55% and 70% plants, respectively, with nine plants being positive for both protozoa and only four plants being negative over the study period. Both parasites were identified in the raw water throughout the year, with a lower frequency in autumn and a peak in winter, at a mean concentration of 67±38 oocysts per 100l and 125±241 cysts per 100l. The turbidity of raw water was not related to the presence or concentration of (oo)cysts, and the (oo)cyst removal efficiency was not related to the type of water treatment. One or both pathogens were identified in the finished water in 7 out of 11 plants with a conventional treatment process (coagulation, flocculation, sedimentation, filtration, and disinfection processes) compared to 4 out of 9 plants that did not apply one of the pre-chlorination treatment steps. Protozoa were detected in the finished water of positive plants at a mean concentration of 88±55 oocysts per 100l and 37±41 cysts per 100l, and most of them excluded propidium iodide so were considered potentially viable. The ubiquity of these parasites in the drinking water sources and the inefficiency of conventional water treatment in reducing/inactivating them may present a serious public health issue in this geographical area. Copyright © 2016 Elsevier B.V. All rights reserved.

Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona.

PubMed

Kitajima, Masaaki; Haramoto, Eiji; Iker, Brandon C; Gerba, Charles P

2014-06-15

We investigated the occurrence of Cryptosporidium, Giardia, and Cyclospora at two wastewater treatment plants (WWTPs) in Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent wastewater samples were collected monthly, and protozoan (oo)cysts were concentrated using an electronegative filter, followed by the detection of protozoa using fluorescent microscopy (Cryptosporidium oocysts and Giardia cysts) and PCR-based methods (Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis). The concentration of Giardia cysts in the influent was always higher than that of Cryptosporidium oocysts (mean concentration of 4.8-6.4×10(3) versus 7.4×10(1)-1.0×10(2)(oo)cysts/l) with no clear seasonality, and log10 reduction of Giardia cysts was significantly higher than that of Cryptosporidium oocysts for both WWTPs (P<0.05). Log10 reduction of Giardia cysts at the WWTP utilizing activated sludge was significantly higher than the other WWTP using trickling filter (P=0.014), while no statistically significant difference between the two WWTPs was observed for the log10 reduction of Cryptosporidium oocysts (P=0.207). Phylogenetic analysis revealed that G. intestinalis strains identified in wastewater belonged to two assemblages, AII and B, which are potentially infectious to humans. C. cayetanensis was also detected from both influent and effluent using a newly developed quantitative PCR, with the highest influent concentration of 1.2×10(4)copies/l. Our results demonstrated that these protozoan pathogens are prevalent in the study area and that efficacy of the conventional wastewater treatment processes at physically removing (oo)cysts is limited. Copyright © 2014 Elsevier B.V. All rights reserved.

In vivo and in vitro efficacy of sainfoin (Onobrychis viciifolia) against Eimeria spp in lambs.

PubMed

Saratsis, Anastasios; Regos, Ionela; Tzanidakis, Nikolaos; Voutzourakis, Nikolaos; Stefanakis, Alexandros; Treuter, Dieter; Joachim, Anja; Sotiraki, Smaragda

2012-08-13

The effect of sainfoin (Onobrychis viciifolia) against ovine coccidia was evaluated in vivo and in vitro. In 3 in vivo trials weaned lambs were allocated into two treatment groups receiving diets with either lucerne (Medicago sativa) or sainfoin. During the trials, which lasted for 7 (trial 1) or 8 weeks (trials 2 and 3), oocysts per gram of faeces (OPGs), faecal scores and weight gain were recorded. In two of the experiments (trials 1 and 3) a reduction in the mean oocyst excretion rates was observed, starting three to four weeks after sainfoin hay feeding. This reduction ranged between 21.3% (trial 1) and 61.7% (trial 3) compared to the control values. As a result, a decrease in the total number of oocysts excreted (expressed as the mean area under the curve of the OPG) was observed from week 4 to the end of the two trials, respectively (trial 1: 42.6% reduction, p=0.05; trial 3: 52.4% reduction, p=0.06). The results did not show any significant diet effect on lamb growth rates and faecal scores. In the in vitro experiments the effect of 39 sainfoin extracts were tested in an oocyst sporulation inhibition assay. The Eimeria oocysts sporulation inhibition throughout the experiments did not exceed 10.7%, showing that extracts of this forages do not have a significant inhibitory effect on Eimeria oocyst sporulation. This was an initial attempt to investigate a possible anticoccidial effect of sainfoin and further studies are needed in order to better understand its mode of action against Eimeria. Copyright © 2012 Elsevier B.V. All rights reserved.

Two COWP-like cysteine rich proteins from Eimeria nieschulzi (coccidia, apicomplexa) are expressed during sporulation and involved in the sporocyst wall formation.

PubMed

Jonscher, Ernst; Erdbeer, Alexander; Günther, Marie; Kurth, Michael

2015-07-25

The family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can also be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well. Genome sequence analysis of Eimeria suggests that these proteins may also exist in this genus and led us to the assumption that these proteins may also play a role in oocyst wall formation. In this study, COWP-like encoding sequences had been identified in Eimeria nieschulzi. The predicted gene sequences were subsequently utilized in reporter gene assays to observe time of expression and localisation of the reporter protein in vivo. Both investigated proteins, EnOWP2 and EnOWP6, were expressed during sporulation. The EnOWP2-promoter driven mCherry was found in the cytoplasm and the EnOWP2, respectively EnOWP6, fused to mCherry was initially observed in the extracytoplasmatic space between sporoblast and oocyst wall. This, so far unnamed compartment was designated as circumplasm. Later, the mCherry reporter co-localised with the sporocyst wall of the sporulated oocysts. This observation had been confirmed by confocal microscopy, excystation experiments and IFA. Transcript analysis revealed the intron-exon structure of these genes and confirmed the expression of EnOWP2 and EnOWP6 during sporogony. Our results allow us to assume a role, of both investigated EnOWP proteins, in the sporocyst wall formation of E. nieschulzi. Data mining and sequence comparisons to T. gondii and other Eimeria species allow us to hypothesise a conserved process within the coccidia. A role in oocyst wall formation had not been observed in E. nieschulzi.

Pathogen and chemical transport in the karst limestone of the Biscayne aquifer: 3. Use of microspheres to estimate the transport potential of Cryptosporidium parvum oocysts

USGS Publications Warehouse

Harvey, Ronald W.; Metge, David W.; Shapiro, Allen M.; Renken, Robert A.; Osborn, Christina L.; Ryan, Joseph N.; Cunningham, Kevin J.; Landkamer, Lee L.

2008-01-01

The vulnerability of a municipal well in the Northwest well field in southeastern Florida to potential contamination by Cryptosporidium parvum oocysts was assessed in a large‐scale, forced‐gradient (convergent) injection and recovery test. The field study involved a simultaneous pulse introduction of a nonreactive tracer (SF6, an inert gas) and oocyst‐sized (1.6, 2.9, and 4.9 μm diameter) carboxylated polystyrene microspheres into karst limestone of the Biscayne aquifer characterized by a complex triple (matrix, touching‐vug, and conduit) porosity. Fractional recoveries 97 m down gradient were inversely related to diameter and ranged from 2.9% for the 4.9 μm microspheres to 5.8% for 1.6 μm microspheres. Their centers of mass arrived at the pumping well approximately threefold earlier than that of the nonreactive tracer SF6 (gas), underscoring the need for use of colloid tracers and field‐scale tracer tests for these kinds of evaluations. In a modified triaxial cell using near in situ chemical conditions, 2.9 and 4.9 μm microspheres underestimated by fourfold to sixfold the attachment potential of the less electronegative 2.9–4.1 μm oocysts in the matrix porosity of limestone core samples. The field and laboratory results collectively suggested that it may take 200–300 m of transport to ensure even a 1‐log unit removal of oocysts, even though the limestone surfaces exhibited a substantive capability for their sorptive removal. The study further demonstrated the utility of microspheres as oocyst surrogates in field‐scale assessments of well vulnerability in limestone, provided that differences in attachment behaviors between oocysts and microspheres are taken into account.

Unrecognized Ingestion of Toxoplasma gondii Oocysts Leads to Congenital Toxoplasmosis and Causes Epidemics in North America

PubMed Central

Boyer, Kenneth; Hill, Dolores; Mui, Ernest; Wroblewski, Kristen; Karrison, Theodore; Dubey, J. P.; Sautter, Mari; Noble, A. Gwendolyn; Withers, Shawn; Swisher, Charles; Heydemann, Peter; Hosten, Tiffany; Babiarz, Jane; Lee, Daniel

2011-01-01

(See the Editorial Commentary by Linn, on pages 1090–1.) Background. Congenital toxoplasmosis presents as severe, life-altering disease in North America. If mothers of infants with congenital toxoplasmosis could be identified by risks, it would provide strong support for educating pregnant women about risks, to eliminate this disease. Conversely, if not all risks are identifiable, undetectable risks are suggested. A new test detecting antibodies to sporozoites demonstrated that oocysts were the predominant source of Toxoplasma gondii infection in 4 North American epidemics and in mothers of children in the National Collaborative Chicago-based Congenital Toxoplasmosis Study (NCCCTS). This novel test offered the opportunity to determine whether risk factors or demographic characteristics could identify mothers infected with oocysts. Methods. Acutely infected mothers and their congenitally infected infants were evaluated, including in-person interviews concerning risks and evaluation of perinatal maternal serum samples. Results. Fifty-nine (78%) of 76 mothers of congenitally infected infants in NCCCTS had primary infection with oocysts. Only 49% of these mothers identified significant risk factors for sporozoite acquisition. Socioeconomic status, hometown size, maternal clinical presentations, and ethnicity were not reliable predictors. Conclusions. Undetected contamination of food and water by oocysts frequently causes human infections in North America. Risks are often unrecognized by those infected. Demographic characteristics did not identify oocyst infections. Thus, although education programs describing hygienic measures may be beneficial, they will not suffice to prevent the suffering and economic consequences associated with congenital toxoplasmosis. Only a vaccine or implementation of systematic serologic testing of pregnant women and newborns, followed by treatment, will prevent most congenital toxoplasmosis in North America. PMID:22021924

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA ▿

PubMed Central

Liang, Zhanbei; Keeley, Ann

2011-01-01

Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for Cryptosporidium detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of Cryptosporidium with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella enterica serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102 oocysts g−1 of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104 C. parvum oocysts g−1 soil for sandy, loamy, and clay samples, respectively. PMID:21803904

SEROLOGICAL RESPONSES TO CRYPTOSPORIDIUM ANTIGENS AMONG USERS OF SURFACE VERSUS UNDERGROUND DRINKING WATER SOURCES

EPA Science Inventory

Cryptosporidium oocysts have been detected in source and treated drinking waters in the United States and elsewhere. Enhanced enteric disease surveillance, initiated following detection of oocysts, has not often detected elevated rates of infection or of symptoms compatible with...

TOXOPLASMA GONDII : UPTAKE AND SURVIVAL OF OOCYSTS IN FREE-LIVING AMOEBAE

USDA-ARS?s Scientific Manuscript database

Waterborne transmission of the oocyst stage of Toxoplasma gondii can cause outbreaks of clinical toxoplasmosis in humans and infection of marine mammals. In water-related environments and soil, free-living amoebae are considered potential carriers of various pathogens, but knowledge on interactions ...

MOLECULAR DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN WATER: THE CHALLENGE AND PROMISE

EPA Science Inventory

Because of the presence of host-adapted Cryptosporidium species and genotypes, molecular tools can help assess the source and hazardous potential of Cryptosporidium oocysts in water. The development and use of molecular tools in the analysis of environmental samples have gone tho...

CRYPTOSPORIDIUM OOCYST RECOVERY IN WATER BY EPA METHOD 1623: EVALUATION OF A MODIFIED IMS DISSOCIATION

EPA Science Inventory

EPA Methods 1622 and 1623 are the benchmarks for detection of Cryptosporidium spp. oocysts in water. These methods consist of filtration, elution, purification by immunomagnetic separation (IMS), and microscopic analysis after staining with a fluorescein isothiocyanate conjugate...

DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN SOURCE AND FINISHED WATERS

EPA Science Inventory

Numerous waterborne outbreaks of cryptosporidiosis have occurred with the most notable being the 1993 episode in Milwaukee. As a result, the past decade has seen a massive effort expended on the development of methods to detect Cryptosporidium parvum oocysts in source and finish...

Modeling Cryptosporidium spp. Oocyst Inactivation in Bubble-Diffuser Ozone Contactors

DTIC Science & Technology

1998-07-01

requirements for Giardia lamblia (G. lamblia) and viruses under the Surface Water Treatment Rule (SWTR). Minimum CT requirements include relatively...parvum and C. muris ) oocysts in ozone bubble-diffuser contactors. The model is calibrated with semi-batch kinetic data, verified with pilot-scale

Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating.

PubMed Central

Visvesvara, G S; Moura, H; Kovacs-Nace, E; Wallace, S; Eberhard, M L

1997-01-01

Cyclospora, a coccidian protist, is increasingly being identified as an important, newly emerging parasite that causes diarrhea, flatulence, fatigue, and abdominal pain leading to weight loss in immunocompetent persons with or without a recent travel history as well as in patients with AIDS. Modified Kinyoun's acid-fast stain is the most commonly used stain to identify the oocyst of this parasite in fecal smears. Oocysts of Cyclospora stain variably by the modified acid-fast procedure, resulting in the possible misidentification of this parasite. We examined fecal smears stained by six different procedures that included Giemsa, trichrome, chromotrope, Gram-chromotrope, acid-fast, and safranin stains. We report on safranin-based stain that uniformly stains oocysts of Cyclospora a brilliant reddish orange, provided that the fecal smears are heated in a microwave oven prior to staining. This staining procedure, besides being superior to acid-fast staining, is fast, reliable, and easy to perform in most clinical laboratories. PMID:9041421

Limiting swimming pool outbreaks of cryptosporidiosis - the roles of regulations, staff, patrons and research.

PubMed

Ryan, Una; Lawler, Sheleigh; Reid, Simon

2017-02-01

Cryptosporidium is the leading cause of swimming pool outbreaks of gastroenteritis. Transmission occurs through the ingestion of oocysts that are passed in the faeces of an infected person or animal when an accidental faecal release event occurs. Cryptosporidium parasites present specific challenges for infection control as oocysts are highly resistant to chlorine levels used for pool disinfection, infected individuals can shed large numbers of oocysts, there is a long incubation period and shedding of oocysts occurs even after symptom resolution. The purposes of this review are to identify key barriers to limiting swimming pool-associated outbreaks of cryptosporidiosis and to outline needs for research and collaboration to advance co-ordinated management practices. We reviewed swimming pool-associated cryptosporidiosis outbreaks, disinfection teachniques, current regulations and the role of staff and patrons. Key barriers to limiting swimming pool-associated outbreaks of cryptosporidiosis are a lack of uniform national and international standards, poor adherence and understanding of regulations governing staff and patron behaviour, and low levels of public knowledge and awareness.

A new coccidian parasite, Isospora samoaensis, from the Wattled Honeyeater (Foulehaio carunculata) from American Samoa

USGS Publications Warehouse

Adamczyk, Kelly J.; McQuistion, Thomas E.; LaPointe, Dennis

2004-01-01

A new species of Isospora is described from the feces of the wattled honeyeater, Foulehaio carunculata from American Samoa. Numerous oocysts of similar morphology were found in a single adult wattled honeyeater. Sporulated oocysts are ovoid, 28.9 × 26.1 (25-32 × 23-30) µm, with a smooth, colorless, bilayered wall; the inner wall is slightly thicker and darker than the outer wall. The average shape index is 1.1. No micropyle or oocyst residuum are present but the oocyst contains one or two ovoid polar granules. Sporocysts are ovoid, 17.1 × 10.9 (16-18 × 10-11) µm with a smooth single layered wall and an average shape index of 1.6. The Stieda body is broad, dome-like with a rather rectangular-shaped substieda body. Within the sporocyst is a large amorphous residuum composed of coarse granules and 4 randomly arranged, sausage-shaped sporozoites with a subspherical, posterior refractile body and a centrally located nucleus.

Evaluation of solar photocatalysis using TiO2 slurry in the inactivation of Cryptosporidium parvum oocysts in water.

PubMed

Abeledo-Lameiro, María Jesús; Ares-Mazás, Elvira; Gómez-Couso, Hipólito

2016-10-01

Cryptosporidium is a genus of enteric protozoan parasites of medical and veterinary importance, whose oocysts have been reported to occur in different types of water worldwide, offering a great resistant to the water treatment processes. Heterogeneous solar photocatalysis using titanium dioxide (TiO2) slurry was evaluated on inactivation of Cryptosporidium parvum oocysts in water. Suspensions of TiO2 (0, 63, 100 and 200mg/L) in distilled water (DW) or simulated municipal wastewater treatment plant (MWTP) effluent spiked with C. parvum oocysts were exposed to simulated solar radiation. The use of TiO2 slurry at concentrations of 100 and 200mg/L in DW yielded a high level of oocyst inactivation after 5h of exposure (4.16±2.35% and 15.03±4.54%, respectively, vs 99.33±0.58%, initial value), representing a good improvement relative to the results obtained in the samples exposed without TiO2 (51.06±9.35%). However, in the assays carried out using simulated MWTP effluent, addition of the photocatalyst did not offer better results. Examination of the samples under bright field and epifluorescence microscopy revealed the existence of aggregates comprising TiO2 particles and parasitic forms, which size increased as the concentration of catalyst and the exposure time increased, while the intensity of fluorescence of the oocyst walls decreased. After photocatalytic disinfection process, the recovery of TiO2 slurry by sedimentation provided a substantial reduction in the parasitic load in treated water samples (57.81±1.10% and 82.10±2.64% for 200mg/L of TiO2 in DW and in simulated MWTP effluent, respectively). Although further studies are need to optimize TiO2 photocatalytic disinfection against Cryptosporidium, the results obtained in the present study show the effectiveness of solar photocatalysis using TiO2 slurry in the inactivation of C. parvum oocysts in distilled water. Copyright © 2016 Elsevier B.V. All rights reserved.

Metam sodium reduces viability and infectivity of Eimeria oocysts

USDA-ARS?s Scientific Manuscript database

Metam sodium (MS, sodium N-methyldithiocarbamate) is a widely used soil pesticide. Fumigation or chemical sterilization of poultry litter containing infectious oocysts could be an effective strategy to block the transmission of avian coccidia. In the current study the effect of MS on the viability ...

BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

EPA Science Inventory

An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts

EPA Science Inventory

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels o...

MODELING CRYPTOSPORIDIUM PARVUM OOCYST INACTIVATION AND BROMATE IN A FLOW-THROUGH OZONE CONTACTOR TREATING NATURAL WATER

EPA Science Inventory

A reactive transport model was developed to simultaneously predict Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of natural water. A mechanistic model previously established to predict bromate formation in organic-free synthetic waters w...

EVALUATING IN VITRO INFECTIVITY FOR MEASURING UV DISINFECTION OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN FINISHED WATER

EPA Science Inventory

UV technology to inactivate Cryptosporidium parvum oocysts has become well established in the US. The challenge now is to effectively demonstrate UV reactor performance and disinfection capacity with various finished water matrices and under different operational conditions. In s...

COMPARISON OF TWO METHODS FOR DETECTION OF GIARDIA CYSTS AND CRYTOSPORIDIUM OOCYSTS IN WATER

EPA Science Inventory

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts a...

SEROLOGICAL EVALUATION OF CRYPTOSPORIDIUM OOCYSTS FINDINGS IN THE WATER SUPPLY OF SYDNEY, AUSTRALIA

EPA Science Inventory

From July to September, 1998, high levels of Cryptospordium oocysts and Giardia cysts were detected in Sydney, Australia drinking water. To evaluate whether Sydney residents had an elevated risk of infection, serological responses to two Cryptospordium antigen groups (15/17 - an...

Prevalence and molecular characterisation of Eimeria species in Ethiopian village chickens.

PubMed

Luu, Lisa; Bettridge, Judy; Christley, Robert M; Melese, Kasech; Blake, Damer; Dessie, Tadelle; Wigley, Paul; Desta, Takele T; Hanotte, Olivier; Kaiser, Pete; Terfa, Zelalem G; Collins, Marisol; Lynch, Stacey E

2013-10-15

Coccidiosis, caused by species of the apicomplexan parasite Eimeria, is a major disease of chickens. Eimeria species are present world-wide, and are ubiquitous under intensive farming methods. However, prevalence of Eimeria species is not uniform across production systems. In developing countries such as Ethiopia, a high proportion of chicken production occurs on rural smallholdings (i.e. 'village chicken production') where infectious diseases constrain productivity and surveillance is low. Coccidiosis is reported to be prevalent in these areas. However, a reliance on oocyst morphology to determine the infecting species may impede accurate diagnosis. Here, we used cross-sectional and longitudinal studies to investigate the prevalence of Eimeria oocyst shedding at two rural sites in the Ethiopian highlands. Faecal samples were collected from 767 randomly selected chickens in May or October 2011. In addition, 110 chickens were sampled in both May and October. Eimeria oocysts were detected microscopically in 427 (56%, 95% confidence interval (95% CI) 52-59%) of the 767 faecal samples tested. Moderate clustering of positive birds was detected within households, perhaps suggesting common risk factors or exposure pathways. Seven species of Eimeria were detected by real time PCR in a subset of samples further analysed, with the prevalence of some species varying by region. Co-infections were common; 64% (23/36, 95% CI 46-79%) of positive samples contained more than one Eimeria spp. Despite frequent infection and co-infection overt clinical disease was not reported. Eimeria oocysts were detected significantly more frequently in October (248/384, 65%, 95% CI 60-69%), following the main rainy season, compared to May (179/383, 47%, 95% CI 42-52%, p < 0.001). Eimeria oocyst positivity in May did not significantly affect the likelihood of detecting Eimeria oocyst five months later perhaps suggesting infection with different species or immunologically distinct strains. Eimeria spp oocysts may be frequently detected in faecal samples from village chickens in Ethiopia. Co-infection with multiple Eimeria spp was common and almost half of Eimeria positive birds had at least one highly pathogenic species detected. Despite this, all sampled birds were free of overt disease. Although there was no evidence of a difference in the prevalence of oocysts in faecal samples between study regions, there was evidence of variation in the prevalence of some species, perhaps suggesting regional differences in exposure to risk factors associated with the birds, their management and/or location-specific environmental and ecological factors.

Prevalence and molecular characterisation of Eimeria species in Ethiopian village chickens

PubMed Central

2013-01-01

Background Coccidiosis, caused by species of the apicomplexan parasite Eimeria, is a major disease of chickens. Eimeria species are present world-wide, and are ubiquitous under intensive farming methods. However, prevalence of Eimeria species is not uniform across production systems. In developing countries such as Ethiopia, a high proportion of chicken production occurs on rural smallholdings (i.e. 'village chicken production’) where infectious diseases constrain productivity and surveillance is low. Coccidiosis is reported to be prevalent in these areas. However, a reliance on oocyst morphology to determine the infecting species may impede accurate diagnosis. Here, we used cross-sectional and longitudinal studies to investigate the prevalence of Eimeria oocyst shedding at two rural sites in the Ethiopian highlands. Results Faecal samples were collected from 767 randomly selected chickens in May or October 2011. In addition, 110 chickens were sampled in both May and October. Eimeria oocysts were detected microscopically in 427 (56%, 95% confidence interval (95% CI) 52-59%) of the 767 faecal samples tested. Moderate clustering of positive birds was detected within households, perhaps suggesting common risk factors or exposure pathways. Seven species of Eimeria were detected by real time PCR in a subset of samples further analysed, with the prevalence of some species varying by region. Co-infections were common; 64% (23/36, 95% CI 46-79%) of positive samples contained more than one Eimeria spp. Despite frequent infection and co-infection overt clinical disease was not reported. Eimeria oocysts were detected significantly more frequently in October (248/384, 65%, 95% CI 60-69%), following the main rainy season, compared to May (179/383, 47%, 95% CI 42-52%, p < 0.001). Eimeria oocyst positivity in May did not significantly affect the likelihood of detecting Eimeria oocyst five months later perhaps suggesting infection with different species or immunologically distinct strains. Conclusions Eimeria spp oocysts may be frequently detected in faecal samples from village chickens in Ethiopia. Co-infection with multiple Eimeria spp was common and almost half of Eimeria positive birds had at least one highly pathogenic species detected. Despite this, all sampled birds were free of overt disease. Although there was no evidence of a difference in the prevalence of oocysts in faecal samples between study regions, there was evidence of variation in the prevalence of some species, perhaps suggesting regional differences in exposure to risk factors associated with the birds, their management and/or location-specific environmental and ecological factors. PMID:24125076

Four New Species of Eimeria (Apicomplexa: Eimeriidae) from Emoia spp. Skinks (Sauria: Scincidae), from Papua New Guinea and the Insular Pacific.

PubMed

McAllister, Chris T; Duszynski, Donald W; Austin, Christopher C; Fisher, Robert N

2017-02-01

Between September and November 1991, 54 adult skinks from 15 species were collected by hand or blowpipe from several localities on Rarotonga, Cook Islands, Ovalau Island, Fiji, and Papua New Guinea (PNG), and their feces were examined for coccidians. Species included 5 seaside skinks (Emoia atrocostata), 1 Pacific blue-tailed skink (Emoia caeroleocauda), 2 Fiji slender treeskinks (Emoia concolor), 15 white-bellied copper-striped skinks (Emoia cyanura), 1 Bulolo River forest skink (Emoia guttata), 6 dark-bellied copper-striped skinks (Emoia impar), 5 Papua five-striped skinks (Emoia jakati), 2 Papua slender treeskinks (Emoia kordoana), 3 Papua robust treeskinks (Emoia longicauda), 1 brown-backed forest skink (Emoia loveridgei), 3 Papua black-sided skinks (Emoia pallidiceps), 2 Papua white-spotted skinks (Emoia physicae), 2 Papua yellow-head skinks (Emoia popei), 1 Papua brown forest skink (Emoia submetallica), and 5 Fiji barred treeskinks (Emoia trossula) Species of Eimeria (Ei.) were detected from these Emoia (Em.) spp. and are described here as new. Oocysts of Eimeria iovai n. sp. from Em. pallidiceps from PNG were ellipsoidal with a bilayered wall (L × W) 26.5 × 18.1 μm, with a length/width ratio (L/W) of 1.1. Both micropyle and oocyst residuum were absent, but a fragmented polar granule was present. This eimerian also was found in Em. atrocostata from PNG. Oocysts of Eimeria kirkpatricki n. sp. from Em. atrocostata from PNG were ellipsoidal with a bilayered wall, 18.6 × 13.5 μm, L/W 1.4. A micropyle and oocyst residuum were absent, but a fragmented polar granule was present. This eimerian was also shared by Em. cyanura from the Cook Islands and Fiji, Em. impar from the Cook Islands, Em. loveridgei from PNG, Em. pallidiceps from PNG, Em. popei from PNG, and Em. submetallica from PNG. Oocysts of Eimeria stevejayuptoni n. sp. from Em. longicauda were subspheroidal to ellipsoidal with a bilayered wall, 18.7 × 16.6 μm, L/W 1.1. A micropyle and oocyst residuum were absent, but a fragmented polar granule was present. Oocysts of Eimeria emoia n. sp. from Em. longicauda from PNG were cylindroidal with a bilayered wall, 29.2 × 15.7 μm, L/W 1.9. A micropyle and oocyst residuum were absent, but a polar granule was present. These are the first eimerians reported from Emoia spp. and they add to our growing knowledge of the coccidian fauna of scincid lizards of the South Pacific.

Waterborne toxoplasmosis investigated and analysed under hydrogeological assessment: new data and perspectives for further research

PubMed Central

Vieira, Flávia Pereira; Alves, Maria da Glória; Martins, Livia Mattos; Rangel, Alba Lucínia Peixoto; Dubey, Jitender Prakash; Hill, Dolores; Bahia-Oliveira/, Lilian Maria Garcia

2015-01-01

We present a set of data on human and chicken Toxoplasma gondii seroprevalence that was investigated and analysed in light of groundwater vulnerability information in an area endemic for waterborne toxoplasmosis in Brazil. Hydrogeological assessment was undertaken to select sites for water collection from wells for T. gondii oocyst testing and for collecting blood from free-range chickens and humans for anti-T. gondii serologic testing. Serologic testing of human specimens was done using conventional commercial tests and a sporozoite-specific embryogenesis-related protein (TgERP), which is able to differentiate whether infection resulted from tissue cysts or oocysts. Water specimens were negative for the presence of viable T. gondii oocysts. However, seroprevalence in free-range chickens was significantly associated with vulnerability of groundwater to surface contamination (p < 0.0001; odds ratio: 4.73, 95% confidence interval: 2.18-10.2). Surprisingly, a high prevalence of antibodies against TgERP was detected in human specimens, suggesting the possibility of a continuous contamination of drinking water with T. gondii oocysts in this endemic setting. These findings and the new proposed approach to investigate and analyse endemic toxoplasmosis in light of groundwater vulnerability information associated with prevalence in humans estimated by oocyst antigens recognition have implications for the potential role of hydrogeological assessment in researching waterborne toxoplasmosis at a global scale. PMID:26560984

Simultaneous detection of the protozoan parasites Toxoplasma, Cryptosporidium and Giardia in food matrices and their persistence on basil leaves.

PubMed

Hohweyer, Jeanne; Cazeaux, Catherine; Travaillé, Emmanuelle; Languet, Emilie; Dumètre, Aurélien; Aubert, Dominique; Terryn, Christine; Dubey, Jitender P; Azas, Nadine; Houssin, Maryline; Loïc, Favennec; Villena, Isabelle; La Carbona, Stéphanie

2016-08-01

Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis are emerging pathogen parasites in the food domain. However, without standardized methods for their detection in food matrices, parasitic foodborne outbreaks remain neglected. In this study, a new immunomagnetic separation assay (IMS Toxo) targeting the oocyst's wall of T. gondii was developed using a specific purified monoclonal antibody. Performance of this IMS Toxo coupled to microscopic and qPCR analyses was evaluated in terms of limit of detection (LOD) and recovery rate (RR) on: i) simple matrix (LOD = 5 oocysts; RR between 5 and 56%); ii) raspberries and basil (LOD = 33 oocysts/g; RR between 0.2 and 35%). Finally, to simultaneously extract the three protozoa from these food matrices, T. gondii oocysts were directly concentrated (without IMS Toxo) from the supernatant of the IMS of Cryptosporidium and Giardia (oo)cysts. This strategy associated to qPCR detection led to LOD <1 to 3 (oo)cysts/g and RR between 2 and 35%. This procedure was coupled to RT-qPCR analyses and showed that the three protozoa persisted on the leaves of basil and remained viable following storage at 4 °C for 8 days. These data strengthen the need to consider these protozoa in food safety. Copyright © 2016 Elsevier Ltd. All rights reserved.

Predation and transport of persistent pathogens in GAC and slow sand filters: a threat to drinking water safety?

PubMed

Bichai, Françoise; Dullemont, Yolanda; Hijnen, Wim; Barbeau, Benoit

2014-11-01

Zooplankton has been shown to transport internalized pathogens throughout engineered drinking water systems. In this study, experimental measurements from GAC and SSF filtration tests using high influent concentrations of Cryptosporidium (1.3 × 10(6) and 3.3 × 10(4) oocysts L(-1)) and Giardia (4.8 × 10(4) cysts L(-1)) are presented and compared. A predation and transport conceptual model was developed to extrapolate these results to environmental conditions of typical (oo)cyst concentrations in surface water in order to predict concentrations of internalized (oo)cysts in filtered water. Pilot test results were used to estimate transport and survival ratios of internalized (oo)cysts following predation by rotifers in the filter beds. Preliminary indications of lower transport and survival ratios in SSF were found as compared with GAC filters. A probability of infection due to internalized (oo)cysts in filtered water was calculated under likeliest environmental conditions and under a worst-case scenario. Estimated risks under the likeliest environmental scenario were found to fall below the tolerable risk target of 10(-4) infections per person per year. A discussion is presented on the health significance of persistent pathogens that are internalized by zooplankton during granular filtration processes and released into treated water. Copyright © 2014 Elsevier Ltd. All rights reserved.

Detection of Cryptosporidium oocysts in green mussels (Perna viridis) from shell-fish markets of Thailand.

PubMed

Srisuphanunt, M; Wiwanitkit, Viroj; Saksirisampant, W; Karanis, P

2009-09-01

Mussels filter large volumes of water and can concentrate pathogenic organisms, which may act as potential vehicles of transmission to the consumer. A survey study was carried out to investigate the presence of Cryptosporidium protozoan parasites in green mussels (Perna viridis), the smussles pecies most destined for consumption in Thailand. In total, 56 samples were examined from Bangkok (n = 24) and Samut Prakan (n = 32) a wholesale shell-fish markets located at the mouth of the Chao Phraya River. The market for green mussels was closed to the mussel culture placed along the coastal line and this localization may have significant economical impact if the mussels' cultures are found contaminated. Cryptosporidium spp. oocysts were detected by the immunofluorescence antibody method (IFA) in 12.5% of the samples examined. The detection of Cryptosporidium oocysts in green mussels' population of Samut Prakan was higher (15.6%) than in Bangkok market (8.3%). These differences in positive samples from the two locations may be caused by physical, ecological and anthropogenic conditions. This could relay to different contamination levels of marine water by Cryptosporidium oocysts and consequently to contamination of harvested shellfish populations. The results demonstrate that the Cryptosporidium spp. oocysts were found indigenous in mussels from the coastal line of Thailand, indicating that mussels may act as a reservoir of Cryptosporidium foodborne infections for humans.

Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa)

PubMed Central

Wiedmer, Stefanie; Erdbeer, Alexander; Volke, Beate; Randel, Stephanie; Kapplusch, Franz; Hanig, Sacha; Kurth, Michael

2017-01-01

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain. PMID:29210668

Waterborne toxoplasmosis investigated and analysed under hydrogeological assessment: new data and perspectives for further research.

PubMed

Vieira, Flávia Pereira; Alves, Maria da Glória; Martins, Livia Mattos; Rangel, Alba Lucínia Peixoto; Dubey, Jitender Prakash; Hill, Dolores; Bahia-Oliveira, Lilian Maria Garcia

2015-11-01

We present a set of data on human and chicken Toxoplasma gondii seroprevalence that was investigated and analysed in light of groundwater vulnerability information in an area endemic for waterborne toxoplasmosis in Brazil. Hydrogeological assessment was undertaken to select sites for water collection from wells for T. gondii oocyst testing and for collecting blood from free-range chickens and humans for anti-T. gondii serologic testing. Serologic testing of human specimens was done using conventional commercial tests and a sporozoite-specific embryogenesis-related protein (TgERP), which is able to differentiate whether infection resulted from tissue cysts or oocysts. Water specimens were negative for the presence of viable T. gondii oocysts. However, seroprevalence in free-range chickens was significantly associated with vulnerability of groundwater to surface contamination (p < 0.0001; odds ratio: 4.73, 95% confidence interval: 2.18-10.2). Surprisingly, a high prevalence of antibodies against TgERP was detected in human specimens, suggesting the possibility of a continuous contamination of drinking water with T. gondii oocysts in this endemic setting. These findings and the new proposed approach to investigate and analyse endemic toxoplasmosis in light of groundwater vulnerability information associated with prevalence in humans estimated by oocyst antigens recognition have implications for the potential role of hydrogeological assessment in researching waterborne toxoplasmosis at a global scale.

Farm factors associated with reducing Cryptosporidium loading in storm runoff from dairies.

PubMed

Miller, W A; Lewis, D J; Pereira, M D G; Lennox, M; Conrad, P A; Tate, K W; Atwill, E R

2008-01-01

A systems approach was used to evaluate environmental loading of Cryptosporidium oocysts on five coastal dairies in California. One aspect of the study was to determine Cryptosporidium oocyst concentrations and loads for 350 storm runoff samples from dairy high use areas collected over two storm seasons. Selected farm factors and beneficial management practices (BMPs) associated with reducing the Cryptosporidium load in storm runoff were assessed. Using immunomagnetic separation (IMS) with direct fluorescent antibody (DFA) analysis, Cryptosporidium oocysts were detected on four of the five farms and in 21% of storm runoff samples overall. Oocysts were detected in 59% of runoff samples collected near cattle less than 2 mo old, while 10% of runoff samples collected near cattle over 6 mo old were positive. Factors associated with environmental loading of Cryptosporidium oocysts included cattle age class, 24 h precipitation, and cumulative seasonal precipitation, but not percent slope, lot acreage, cattle stocking number, or cattle density. Vegetated buffer strips and straw mulch application significantly reduced the protozoal concentrations and loads in storm runoff, while cattle exclusion and removal of manure did not. The study findings suggest that BMPs such as vegetated buffer strips and straw mulch application, especially when placed near calf areas, will reduce environmental loading of fecal protozoa and improve stormwater quality. These findings are assisting working dairies in their efforts to improve farm and ecosystem health along the California coast.

IDENTIFICATION OF CRYPTOSPORIDIUM SPECIES AND SOURCES IN RAW WASTEWATER USING A SMALL SUBUNIT RRNA-BASED PCR-RFLP TOOL

EPA Science Inventory

The species composition and source of Cryptosporidium oocysts in wastewater have never been determined, even though it is widely assumed that these oocysts are from human sewage. Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate hum...

IDENTIFICATION OF CRYPTOSPORIDIUM SPECIES AND THE SOURCES IN RAW WASTEWATER USING A SMALL SUBUNIT RRNA-BASED PCR-RFLP TOOL

EPA Science Inventory

The species composition and source of Cryptosporidium oocysts in wastewater have never been determined, even though it is widely assumed that these oocysts are from human sewage. Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate hum...

ENHANCED PRODUCTION OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN IMMUNOSUPPRESSED MICE

EPA Science Inventory

Recently there has been an increase in the need for fresh C. parvum oocysts for engineering and biomedical research applications. In our laboratory the emphsis has shifted from the use of dairy calves to inbred C57BL/67n mice, primarily for reasons of ease of collection and proce...

Using Propidium Monoazide as an Enrichment Step to Remove Extraneous DMA and Non-Viable Organisms for Cryptosporidium Genotyping

EPA Science Inventory

Cryptosporidium is an important protozoan parasite that continues to cause waterborne disease outbreaks worldwide. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels of oocysts in s...

Evaluating the transport of bacillus subtilis spores as a potential surrogate for Cryptosporidium parvum Oocysts

USDA-ARS?s Scientific Manuscript database

The USEPA has recommended the use of aerobic spores as an indicator for Cryptosporidium oocysts when determining groundwater under the direct influence of surface water. Surface properties, interaction energies, transport, retention, and release behavior of B. subtilis spores were measured over a r...

A sensitive method for detecting and genotyping Cryptosporidium parvum oocysts

USDA-ARS?s Scientific Manuscript database

Cryptosporidium parvum oocysts represent a considerable health risk to humans and animals because the parasite has a low infectious dose and usually exists in low numbers in environmental samples, which makes detection problematic. The purpose of this study was to evaluate Cryspovirus as a target f...

The use of an atmospheric cold plasma jet to inactivate Cryptosporidium parvum oocysts on cilantro

USDA-ARS?s Scientific Manuscript database

Introduction: In 2015, the CDC reported a rise in outbreaks linked to parasites like Cryptosporidium. Outbreaks of Cryptosporidium parvum have been associated with contaminated drinking or recreational water; however, there is growing concern that oocysts may become a more common contaminant in food...

EVALUATION OF CRYPTOSPORIDIUM OOCYST RECOVERY IN WATER BY EPA METHOD 1623 WITH A MODIFIED IMS DISSOCIATION PROCEDURE

EPA Science Inventory

U.S.EPA Methods 1622 and 1623 are used for the detection of waterborne Cryptosporium. These methods consist of filtration, elution, purificaiton by immunomagnetic separation (IMS), and microscopic analysis for oocysts stained by a fluorescent monoclonal antibody and counter stai...

EVALUATION OF CRYPTOSPORIDIUM OOCYST RECOVERY IN WATER BY EPA METHOD 1623 WITH A MODIFIED IMS DISSOCIATION PROCEDURE.

EPA Science Inventory

EPA Methods 1622 and 1623 are the benchmarks for detection of Cryptosporidium spp. oocysts in water. 5-7 These methods consist of filtration, elution, purification by immunomagnetic separation (IMS), and microscopic analysis after staining with a fluorescein isothiocyanate conju...

Implications of biofilm-associated waterborne Cryptosporidium oocysts for the water industry.

PubMed

Angles, Mark L; Chandy, Joseph P; Cox, Peter T; Fisher, Ian H; Warnecke, Malcolm R

2007-08-01

Waterborne Cryptosporidium has been responsible for drinking water-associated disease outbreaks in a number of developed countries. As a result of the resistance of Cryptosporidium to chlorine, which is typically applied as a final barrier to protect the quality of distributed drinking water, current management practices are focused on source-water management and water treatment as ways of preventing Cryptosporidium from entering drinking-water supplies. In the event that treatment barriers fail, surprisingly little is known of the fate of oocysts once they enter a distribution system. To assess properly the risks of waterborne Cryptosporidium, a more thorough understanding of the fate of oocysts in water distribution systems, with emphasis on Cryptosporidium-biofilm interactions, is required.

Coccidia of whooping cranes

USGS Publications Warehouse

Forrester, Donald J.; Carpenter, J.W.; Blankinship, D.R.

1978-01-01

Coccidial oocysts were observed in 6 of 19 fecal samples from free-ranging whooping cranes (Grus americana) and 4 of 16 samples from captive whooping cranes. Eimeria gruis occurred in four free-ranging whooping cranes and E. reichenowi in two free-ranging and two captive whooping cranes. Fecal samples from two captive cranes contained oocysts of Isospora lacazei which was considered a spurious parasite. Oocysts of both species of Eimeria were prevalent in fecal samples collected from three free-ranging Canadian sandhill cranes (G. canadensis rowani) from whooping crane wintering grounds in Texas. These coccidia were prevalent also in fecal samples from 14 sandhill cranes (of 4 subspecies) maintained in captivity at the Patuxent Wildlife Research Center in Maryland.

INACTIVATION OF CRYPTOSPORIDIUM OOCYSTS IN A PILOT-SCALE OZONE BUBBLE-DIFFUSER CONTACTOR - II: MODEL VALIDATION AND APPLICATION

EPA Science Inventory

The ADR model developed in Part I of this study was successfully validated with experimenta data obtained for the inactivation of C. parvum and C. muris oocysts with a pilot-scale ozone-bubble diffuser contactor operated with treated Ohio River water. Kinetic parameters, required...

EFFECTS OF SEEDING PROCEDURES AND WATER QUALITY ON RECOVERY OF CRYPTOSPORIDIUM OOCYSTS FROM STREAM WATER BY USING U.S. ENVIRONMENTAL PROTECTION AGENCY METHOD 1623

EPA Science Inventory

U.S.EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluor...

The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment

EPA Science Inventory

Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...

Two new Eimeria species (Apicomplexa: Eimeriidae) from the yellow-crowned Amazon Amazona ochrocephala (Aves: Psittacidae) in Brazil.

PubMed

Hofstatter, P G; Kawazoe, U

2011-06-01

In this study, we describe 2 new species of Eimeria associated with the yellow-crowned Amazon Amazona ochrocephala. Eimeria amazonae n. sp. has bilayered, ellipsoidal, and smooth oocysts that measure 48.9 × 36.2 µm; the length/width ratio is 1.35. The micropyle and oocyst residuum are both absent, but the polar granule is present. Ovoidal sporocysts are 22.2 × 11.9 µm. Stieda and sub-Stieda bodies and sporocyst residuum are present. The 2 elongate sporozoites are curved and measure 18.1 × 3.4 µm; both have 2 refractile bodies. Eimeria ochrocephalae n. sp. has bilayered, ellipsoidal, and smooth oocysts that measure 43.8 × 27.7 µm; the length/width ratio is 1.58. The micropyle and oocyst residuum are absent, but the polar granule is present; ovoidal sporocysts are 20.6 × 10.1 µm. Stieda and sub-Stieda bodies and sporocyst residuum are present; 2 elongate and curved sporozoites are 15.8 × 3.4 µm, each of which has 2 refractile bodies.

Prevalence of Cryptosporidium sp. infection in diarrheic and non-diarrheic humans in Iran

PubMed Central

2007-01-01

For evaluation of the prevalence of Cryptosporidium sp. infection in diarrheic and non-diarrheic humans in Iran, fecal specimens from diarrheic (n = 129) and non-diarrheic humans (n = 271) were collected and examined for the presence of Cryptosporidium sp. oocysts. The presence of Cryptosporidium sp. oocysts was determined by Ziehl-Neelsen acid-fast staining. Humans were grouped according to their age as follows: younger than 15, 16-25, 26-35, 36-50, and over 51 years. The results showed that the overall prevalence of infection in all 400 samples was 10.8%, but the prevalence (25.6%) in diarrheic humans was higher than that (3.7%) in non-diarrheic humans. Oocysts of Cryptosporidium sp. were detected in the feces of 21.4%, 9.3%, 8.8%, 6.7% and 5.7% of different age groups, respectively. The intensity of oocysts was significantly higher in diarrheic humans than in non-diarrheic ones. There was a significant association between Cryptosporidium sp. infection and occurrence of diarrhea (P < 0.05). The results indicate that Cryptosporidium sp. infection is prevalent in diarrheic humans in Iran. PMID:17570977

High pathogenicity and strong immunogenicity of a Chinese isolate of Eimeria magna Pérard, 1925.

PubMed

Tao, Geru; Wang, Yunzhou; Li, Chao; Gu, Xiaolong; Cui, Ping; Fang, Sufang; Suo, Xun; Liu, Xianyong

2017-06-01

Coccidia infection of rabbits with one or several species of parasites of the genus Eimeria causes coccidiosis, a disease leading to huge economic losses in the rabbit industry. Eimeria magna, one of the causal agents of rabbit coccidiosis, was characterized as mildly pathogenic and moderately immunogenic in previous studies. In this study, we identified a Chinese isolate of E. magna by testing its biological features (oocyst morphology and size, prepatent time) and sequencing its internal transcribed spacer 1 (ITS-1) DNA fragment. This isolate is highly pathogenic; infection of rabbits with only 1×10 2 oocysts caused a 55% reduction in weight gain in 14days. In addition, immunization with 1×10 2 oocysts prevented body weight loss against re-infection with 5×10 4 oocysts, indicating the high immunogenicity of this isolate. Our study described the distinctive phenotype of the Chinese isolate of E. magna and contributed to the research of geographic variation of rabbit coccidia. Copyright © 2017 Elsevier B.V. All rights reserved.

A new avian Cryptosporidium genotype in a 1-month-old caged brown wood owl (Strix leptogrammica) with severe dehydration and diarrhea.

PubMed

Makino, Ikuko; Inumaru, Mizue; Abe, Niichiro; Sato, Yukita

2018-06-06

A 1-month-old brown wood owlet (Strix leptogrammica) purchased from a wholesaler and housed as a companion bird by an individual owner in Japan showed severe dehydration and anorexia following a week of vomiting and severe diarrhea. A great number of approximately 5 × 4-μm-sized Cryptosporidium oocysts were found in the feces by microscopy. The owlet was administered subcutaneous fluid and intragastric tube feeding for 2 weeks, resulting in improvement of the condition with a decreased number of oocysts in the feces. At days 51 and 119, no oocysts were found in the feces by microscope and PCR detection. These results suggested that this parasite was a possible agent of severe diarrhea in the affected bird. Molecular analysis of DNA extracted from oocysts based on the 18SrRNA loci identified C. avium; however, analysis of actin and hsp (heat shock protein) genes identified a novel genotype indicating a mixed infection with C. avium and a novel genotype.

Renal coccidiosis in interior Canada geese, Branta canadensis interior Todd, of the Mississippi Valley population

USGS Publications Warehouse

Tuggle, Benjamin N.; Crites, John L.

1984-01-01

Kidneys from 309 Interior Canada geese from three locations in the Mississippi Flyway were examined for renal coccidia. Oocysts and/or young zygotes of Eimeria sp. were found in 6.8% of goose kidneys sampled. Only one type of renal coccidian oocyst was observed. Significantly more immature geese were infected than adults; however, there was no significant difference observed between the prevalences of infection in male and female birds. A host cellular response to zygotes and oocysts was noted in the majority of infected adult geese. Heavily infected kidneys were hypertrophic with minute foci on the surface of the organ. Histological examinations showed large numbers of unsporulated oocysts accumulated in distended collecting tubules, resulting in pressure necrosis to adjacent tissue and urate retention. Zygotes were observed in the cytoplasm of tubule cells and extracellularly in interstitial tissue. Infected tubule cells were characterized by the peripheral location of the nuclei, cytoplasmic basophilia, and cellular hypertrophy. This is the first report of an Eimeria sp. in the kidneys of Canada geese of the Mississippi Valley population.

Recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip for equipment-free detection of Cryptosporidium spp. oocysts in dairy cattle feces.

PubMed

Wu, Yao-Dong; Zhou, Dong-Hui; Zhang, Long-Xian; Zheng, Wen-Bin; Ma, Jian-Gang; Wang, Meng; Zhu, Xing-Quan; Xu, Min-Jun

2016-09-01

Cryptosporidium is a widespread protozoan parasite that infects a large number of vertebrate animals, resulting in varying degrees of diarrhea or even death. As dairy cattle feces is an important source of Cryptosporidium spp. infection, development of a handy and accurate detection method via its oocysts in dairy cattle feces would be interesting and necessary. We herein developed a quick detecting method using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip to detect DNA of Cryptosporidium oocysts in dairy cattle feces. The DNA was released by boiled water with 0.1 % N-lauroylsarcosine sodium salt (LSS). The established method was proven to be of higher sensitivity than normal polymerase chain reaction (PCR) amplification with the lowest detection of 0.5 oocyst per reaction, and specificity with no cross reactivity to other common protozoan species in the intestine of dairy cattle. The diagnostic method established herein is simple, rapid, and cost-effective, and has potential for further development as a diagnostic kit for the diagnosis of cryptosporidiosis of dairy cattle.

Co-infection of chickens with Eimeria praecox and Eimeria maxima does not prevent development of immunity to Eimeria maxima.

PubMed

Jenkins, M; Fetterer, R; Miska, K

2009-05-12

Previous studies revealed an ameliorating effect of Eimeria praecox on concurrent E. maxima infection, such that weight gain, feed conversion ratio, and intestinal lesions were nearly identical to uninfected or E. praecox-infected controls. The purpose of the present study was to determine if protective immunity against E. maxima challenge infection developed in chickens infected with both E. praecox and E. maxima. Day-old chickens were infected with 10(3)E. praecox, 10(3)E. maxima, or a mixture of 10(3)E. praecox and 10(3)E. maxima oocysts. Chickens were then challenged at 4 weeks of age with 5x10(4)E. praecox or 5x10(3)E. maxima oocysts and clinical signs of coccidiosis were assessed 7 days post-challenge. Relative to non-challenged controls, naïve chickens or chickens immunized with E. praecox displayed a 32-34% weight gain depression after challenge with 5x10(3)E. maxima oocysts. In contrast, chickens immunized with either E. maxima oocysts alone or a combination of E. praecox and E. maxima oocysts displayed complete protection against lower weight gain associated with E. maxima challenge. Also, protection against decreased feed conversion ratio and intestinal lesions was observed in single E. maxima- or dual E. maxima+E. praecox-immunized chickens. These findings indicate that co-infection of chickens with E. maxima and E. praecox does not prevent development of immunity against E. maxima or E. praecox challenge.

Transport of Cryptosporidium parvum Oocysts in Charge Heterogeneous Porous Media: Microfluidics Experiment and Numerical Simulation

NASA Astrophysics Data System (ADS)

Liu, Y.; Meng, X.; Guo, Z.; Zhang, C.; Nguyen, T. H.; Hu, D.; Ji, J.; Yang, X.

2017-12-01

Colloidal attachment on charge heterogeneous grains has significant environmental implications for transport of hazardous colloids, such as pathogens, in the aquifer, where iron, manganese, and aluminium oxide minerals are the major source of surface charge heterogeneity of the aquifer grains. A patchwise surface charge model is often used to describe the surface charge heterogeneity of the grains. In the patchwise model, the colloidal attachment efficiency is linearly correlated with the fraction of the favorable patches (θ=λ(θf - θu)+θu). However, our previous microfluidic study showed that the attachment efficiency of oocysts of Cryptosporidium parvum, a waterborne protozoan parasite, was not linear correlated with the fraction of the favorable patches (λ). In this study, we developed a pore scale model to simulate colloidal transport and attachment on charge heterogeneous grains. The flow field was simulated using the LBM method and colloidal transport and attachment were simulated using the Lagrange particle tracking method. The pore scale model was calibrated with experimental results of colloidal and oocyst transport in microfluidic devices and was then used to simulate oocyst transport in charge heterogeneous porous media under a variety of environmental relative conditions, i.e. the fraction of favorable patchwise, ionic strength, and pH. The results of the pore scale simulations were used to evaluate the effect of surface charge heterogeneity on upscaling of oocyst transport from pore to continuum scale and to develop an applicable correlation between colloidal attachment efficiency and the fraction of the favorable patches.

Functional genomics of gam56: characterisation of the role of a 56 kilodalton sexual stage antigen in oocyst wall formation in Eimeria maxima.

PubMed

Belli, Sabina I; Witcombe, David; Wallach, Michael G; Smith, Nicholas C

2002-12-19

Gam56 (M(r) 56,000) is an antigen found in the sexual (macrogametocyte) stage of the intestinal parasite Eimeria maxima that is implicated in protective immunity. The gene (gam56) encoding this protein was cloned and sequenced. It is a single-copy, intronless gene, that localises to a 1,754 bp transcript, and is first detected at 120 h p.i. The gene predicts two distinct protein domains; a tyrosine-serine rich region, composed of amino acids implicated in oocyst wall formation in Eimeria spp., and a proline-methionine rich region often detected in extensins, protein components of plant cell walls. The tyrosine-serine rich region predicts a secondary structure commonly seen in the structural protein fibroin, a component of the cocoon of the caterpillar Bombyx mori. The inference that gam56 is a structural component of the oocyst wall was confirmed when a specific antibody to gam56 recognised the wall forming bodies in macrogametocytes, and the walls of oocysts and sporocysts. Together, these data identify a developmentally regulated, sexual stage gene in E. maxima that shares primary and secondary structure features in common with intrinsic structural proteins in other parasites such as Schistosoma mansoni and Fasciola hepatica, and other organisms across different phyla, including the caterpillar Bombyx mori. In addition, these findings provide evidence for the molecular mechanisms underlying oocyst wall formation in Eimeria and the role of gametocyte antigens in this process.

Effects of sediment-associated extractable metals, degree of sediment grain sorting, and dissolved organic carbon upon Cryptosporidium parvum removal and transport within riverbank filtration sediments, Sonoma County, California.

PubMed

Metge, David W; Harvey, Ronald W; Aiken, George R; Anders, Robert; Lincoln, George; Jasperse, Jay; Hill, Mary C

2011-07-01

Oocysts of the protozoan pathogen Cryptosporidium parvum are of particular concern for riverbank filtration (RBF) operations because of their persistence, ubiquity, and resistance to chlorine disinfection. At the Russian River RBF site (Sonoma County, CA), transport of C. parvum oocysts and oocyst-sized (3 μm) carboxylate-modified microspheres through poorly sorted (sorting indices, σ(1), up to 3.0) and geochemically heterogeneous sediments collected between 2 and 25 m below land surface (bls) were assessed. Removal was highly sensitive to variations in both the quantity of extractable metals (mainly Fe and Al) and degree of grain sorting. In flow-through columns, there was a log-linear relationship (r(2) = 0.82 at p < 0.002) between collision efficiency (α, the probability that colloidal collisions with grain surfaces would result in attachment) and extractable metals, and a linear relationship (r(2) = 0.99 at p < 0.002) between α and σ(1). Collectively, variability in extractable metals and grain sorting accounted for ∼83% of the variability in α (at p < 0.0002) along the depth profiles. Amendments of 2.2 mg L(-1) of Russian River dissolved organic carbon (DOC) reduced α for oocysts by 4-5 fold. The highly reactive hydrophobic organic acid (HPOA) fraction was particularly effective in re-entraining sediment-attached microspheres. However, the transport-enhancing effects of the riverine DOC did not appear to penetrate very deeply into the underlying sediments, judging from high α values (∼1.0) observed for oocysts being advected through unamended sediments collected at ∼2 m bls. This study suggests that in evaluating the efficacy of RBF operations to remove oocysts, it may be necessary to consider not only the geochemical nature and size distribution of the sediment grains, but also the degrees of sediment sorting and the concentration, reactivity, and penetration of the source water DOC.

A study of the level and dynamics of Eimeria populations in naturally infected, grazing beef cattle at various stages of production in the Mid-Atlantic USA.

PubMed

Lucas, Aaron S; Swecker, William S; Lindsay, David S; Scaglia, Guillermo; Neel, James P S; Elvinger, Francois C; Zajac, Anne M

2014-05-28

There is little information available on the species dynamics of eimerian parasites in grazing cattle in the central Appalachian region of the United States. Therefore, the objective of this study was to describe the level of infection and species dynamics of Eimeria spp. in grazing beef cattle of various age groups over the course of a year in the central Appalachian region. Rectal fecal samples were collected from male and female calves (n=72) monthly from May through October 2005, heifers only (n=36) monthly from November 2005 to April 2006, and cows (n=72) in May, July, and September, 2005. Eimeria spp. oocysts were seen in 399 of 414 (96%) fecal samples collected from the calves from May through October. Fecal oocysts counts (FOC) in the calves were lower (P<0.05) in May than all other months and no significant differences were detected from June through September. Eimeria spp. oocysts were detected in 198 of 213 (92%) of fecal samples collected from the 36 replacement heifers monthly from November to April and monthly mean FOC did not differ during this time period. The prevalence of oocyst shedding increased to 100% in calves in September and remained near 100% in the replacement heifers during the sampling period. Eimeria spp. oocysts were also detected in 150 of 200 (75%) samples collected in May, July, and September from the cows and mean FOC did not differ significantly over the sampling period. Eimeria spp. composition was dominated by Eimeria bovis in fecal samples collected from calves, replacement heifers and cows. Mixed Eimeria spp. infections were, however, common in all groups and 13 Eimeria spp. oocysts were identified throughout the sampling period. Copyright © 2014 Elsevier B.V. All rights reserved.

Four new species of Eimeria (Apicomplexa: Eimeriidae) from Emoia spp. Skinks (Sauria: Scincidae), from Papua New Guinea and the Insular Pacific

USGS Publications Warehouse

McAllister, Chris T.; Duszynski, Donald W.; Austin, Christopher C.; Fisher, Robert N.

2017-01-01

Between September and November 1991, 54 adult skinks from 15 species were collected by hand or blowpipe from several localities on Rarotonga, Cook Islands, Ovalau Island, Fiji, and Papua New Guinea (PNG), and their feces were examined for coccidians. Species included 5 seaside skinks (Emoia atrocostata), 1 Pacific blue-tailed skink (Emoia caeroleocauda), 2 Fiji slender treeskinks (Emoia concolor), 15 white-bellied copper-striped skinks (Emoia cyanura), 1 Bulolo River forest skink (Emoia guttata), 6 dark-bellied copper-striped skinks (Emoia impar), 5 Papua five-striped skinks (Emoia jakati), 2 Papua slender treeskinks (Emoia kordoana), 3 Papua robust treeskinks (Emoia longicauda), 1 brown-backed forest skink (Emoia loveridgei), 3 Papua black-sided skinks (Emoia pallidiceps), 2 Papua white-spotted skinks (Emoia physicae), 2 Papua yellow-head skinks (Emoia popei), 1 Papua brown forest skink (Emoia submetallica), and 5 Fiji barred treeskinks (Emoia trossula) Species of Eimeria (Ei.) were detected from these Emoia (Em.) spp. and are described here as new. Oocysts of Eimeria iovai n. sp. from Em. pallidiceps from PNG were ellipsoidal with a bilayered wall (L × W) 26.5 × 18.1 μm, with a length/width ratio (L/W) of 1.1. Both micropyle and oocyst residuum were absent, but a fragmented polar granule was present. This eimerian also was found in Em. atrocostata from PNG. Oocysts of Eimeria kirkpatricki n. sp. from Em. atrocostata from PNG were ellipsoidal with a bilayered wall, 18.6 × 13.5 μm, L/W 1.4. A micropyle and oocyst residuum were absent, but a fragmented polar granule was present. This eimerian was also shared by Em. cyanura from the Cook Islands and Fiji, Em. imparfrom the Cook Islands, Em. loveridgei from PNG, Em. pallidicepsfrom PNG, Em. popei from PNG, and Em. submetallica from PNG. Oocysts of Eimeria stevejayuptoni n. sp. from Em. longicaudawere subspheroidal to ellipsoidal with a bilayered wall, 18.7 × 16.6 μm, L/W 1.1. A micropyle and oocyst residuum were absent, but a fragmented polar granule was present. Oocysts of Eimeria emoia n. sp. from Em. longicauda from PNG were cylindroidal with a bilayered wall, 29.2 × 15.7 μm, L/W 1.9. A micropyle and oocyst residuum were absent, but a polar granule was present. These are the first eimerians reported from Emoia spp. and they add to our growing knowledge of the coccidian fauna of scincid lizards of the South Pacific.

DEVELOPMENT OF A CT EQUATION TAKING INTO CONSIDERATION THE EFFECT OF LOT VARIABILITY ON THE INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH OZONE

EPA Science Inventory

Cryptosporidium parvum oocysts are prevalent in surface water and ground water under the influence of surface water, and are difficult to inactivate using free chlorine, the most common disinfectant currently used for treating drinking water. In contrast, it has been shown...

A BAYESIAN METHOD OF ESTIMATING KINETIC PARAMETERS FOR THE INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH CHLORINE DIOXIDE AND OZONE

EPA Science Inventory

The main objective of this paper is to use Bayesian methods to estimate the kinetic parameters for the inactivation kinetics of Cryptosporidium parvum oocysts with chlorine dioxide or ozone which are characterized by the delayed Chick-Watson model, i.e., a lag phase or shoulder f...

Protection of non-immunized broiler chicks housed with immunized cohorts against infection with Eimeria maxima and E. acervulina

USDA-ARS?s Scientific Manuscript database

The use of live oocyst vaccines is becoming increasingly important in the control of avian coccidosis in broiler chicks. Knowledge of the mechanisms of how chicks uptake oocysts and become immune is important for optimizing delivery of live vaccines. The current study tests the hypothesis that chick...

Lectin-Magnetic Separation (LMS) for isolation of Toxoplasma gondii oocysts from concentrated water samples prior to detection by microscopy or qPCR

USDA-ARS?s Scientific Manuscript database

Although standard methods for analyzing water samples for the protozoan parasites Cryptosporidium spp. and Giardia duodenalis are available and widely used, equivalent methods for analyzing water samples for Toxoplasma oocysts are lacking. This is partly due to the lack of a readily available, relia...

Detection of viable Cyptosporidium parvum in soil by reverse transcription real time PCR targeting hsp70 mRNA

EPA Science Inventory

Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive proce...

Natural infection of Cryptosporidium muris (Apicomplexa: Cryptosporiidae) in Siberian chipmunks.

PubMed

Hůrková, Lada; Hajdusek, Ondrej; Modrý, David

2003-04-01

Coprologic examination of nine Siberian chipmunks (Eutamias sibiricus) imported from Southeast Asia revealed infection with Cryptosporidium sp. Experimental inoculation of BALB/c mice proved their susceptibility to the infection. Infected mice shed oocysts 14-35 days postinfection. Oocyst morphology was similar to that reported for C. muris in previous studies, oocysts were 8.1 (7.0-9.0) x 5.9 (5.0-6.5) microns. Clinical signs were absent in naturally infected chipmunks and experimental mice. Histologic examinations of mice revealed numerous developmental stages of C. muris in the glandular stomach. Analysis of partial small subunit rRNA gene sequences confirmed identity of these isolates as C. muris. Our results represent the first report of C. muris in members of the family Sciuridae.

Identification of Species and Sources of Cryptosporidium Oocysts in Storm Waters with a Small-Subunit rRNA-Based Diagnostic and Genotyping Tool

PubMed Central

Xiao, Lihua; Alderisio, Kerri; Limor, Josef; Royer, Michael; Lal, Altaf A.

2000-01-01

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of an immunofluorescent assay. In this study, we have used a small-subunit rRNA-based PCR-restriction fragment length polymorphism technique to identify species and sources of Cryptosporidium oocysts present in 29 storm water samples collected from a stream in New York. A total of 12 genotypes were found in 27 positive samples; for 4 the species and probable origins were identified by sequence analysis, whereas the rest represent new genotypes from wildlife. Thus, this technique provides an alternative method for the detection and differentiation of Cryptosporidium parasites in environmental samples. PMID:11097935

Morphological and molecular characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa: Eimeriidae, 1989, Paperna and Landsberg) in a western bearded dragon (Pogona minor minor).

PubMed

Yang, Rongchang; Brice, Belinda; Ryan, Una

2016-01-01

A new species, Choleoeimeria pogonae n. sp. is described from a Western bearded dragon (Pogona minor minor) in Western Australia. Sporulated oocysts (n = 48) were cylindroidal in shape. Oocyst length, 27.0 (26.0-28.3) μm, oocyst width, 15.2 (14.0-16.5) μm, oocyst length/width ratio (L/W) 1.8 (1.6-1.9), each with 4 sporocysts (Eimeria-like) and a polar granule, but lacking a micropyle and oocyst residuum. Sporocysts are ovoidal in shape, sporocyst length, 10.0 (9.0-11.0) μm, sporocyst width 8.5 (7.0-9.5) μm, sporocyst L/W ratio, 1.2 (1.1-1.3). Stieda, substieda and parasubstieda bodies were all absent. Molecular analysis was conducted at the 18S rRNA and cytochrome c oxidase I (COI) loci. Phylogenetic analysis of 18S sequences revealed that C. pogonae n. sp. grouped together with another four Choleoeimeria spp. and exhibited 99.1%-99.4% genetic similarity. At the COI locus, C. pogonae n. sp. was in an independent clade and had the highest similarity (80.4%) to Eimeria cf. mivati from a chicken (Gallus gallus domesticus). According to the morphological and molecular data, this isolate is a new species of coccidian parasite. This study further supports the taxonomy of Choleoeimeria spp. as a new genus based on molecular phylogenetic analysis. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

PubMed Central

Kaucner, Christine; Stinear, Timothy

1998-01-01

We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvum oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-μl packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable Giardia cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable C. parvum oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four samples by IF microscopy (14%), suggesting that Cryptosporidium species other than C. parvum were present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology. PMID:9572946

Oral oocyst-induced mouse model of toxoplasmosis: Effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality

PubMed Central

DUBEY, J.P.; FERREIRA, L. R.; MARTINS, J.; MCLEOD, RIMA

2013-01-01

SUMMARY Humans and other hosts acquire Toxoplasma gondii infection by ingesting tissue cysts in undercooked meat, or by food or drink contaminated with oocysts. Currently, there is no vaccine to prevent clinical disease due this parasite in humans, although, various T. gondii vaccine candidates are being developed. Mice are generally used to test the protective efficacy of vaccines because they are susceptible, reagents are available to measure immune parameters, in mice, and they are easily managed in the laboratory. In the present study, pathogenesis of toxoplasmosis was studied in mice of different strains, including Human leukocyte antigen(HLA) transgenic mice infected with different doses of T. gondii strains of different genotypes derived from several countries. Based on many experiments, the decreasing order of infectivity and pathogenicity of oocysts was: interferon gamma gene knock out (KO), HLA 3.11, HLA 2.1, HLA B7, Swiss Webster, C57/black, and BALB/c. Mice fed as few as 1 oocyst of Type I and several atypical strains died of acute toxoplasmosis within 21 days p.i. Type II, and III strains were less virulent. The model developed herein should prove to be extremely useful for testing vaccines because it is possible to accurately quantitate a challenge inoculum, test response to different strains of T. gondii using the same preparations of oocysts which are stable for up to a year, and to have highly reproducible responses to the infection. PMID:22078010

Effects of Monovalent and Divalent Salt Solutions on the Transport of Toxoplasma gondii in Saturated Porous Media

NASA Astrophysics Data System (ADS)

Darnault, C. J. G.; Pullano, C. P.; Mutty, T.; L'Ollivier, C.; Dubey, J. P.; Dumetre, A.

2017-12-01

The pathogenic microorganism Toxoplasma gondii is a current public health threat. Knowledge of the fate and transport of T. gondii in the environment, especially the subsurface, is critical to evaluate the risk of soil and groundwater contaminations. The physico-chemcial properties of groundwater systems, i.e. solution chemistry and aquifer materials, play a key role in the interaction of biocolloids with surfaces and therefore their mobility. This research examines how different salt solutions alter the mobility of T. gondii through saturated porous media. Salt solutions containing varying ionic strengths and concentrations of sodium chloride, calcium chloride, and magnesium chloride were used to test the transport of the T. gondii oocysts. These tests were performed using quartz silica sand columns fed by a peristaltic pump in order to generate flow and transport of the biocolloids. The salt solution was pumped though the column followed by a pulse of the T. gondii oocysts, then a pulse of salt solution without oocysts, and then lastly a pulse of distilled water. Sampling of the solution exiting the columns was tested for T. gondii oocysts using qPCR in order to quantify the oocysts present. The breakthough curve results were then compared to a conservative bromide tracer test in order to determine the factors associated with the movement of these biocolloids through the sand columns. A model of the flow of the toxoplasma colloids through the sand matrix was made in order to characterize the parameters affecting the transport and retention of T. gondii occysts though saturated porous media.

Parental development of eimerian coccidia in sandhill and whooping cranes

USGS Publications Warehouse

Novilla, M.N.; Carpenter, J.W.; Spraker, T.R.; Jeffers, T.K.

1981-01-01

In contrast with isosporoid species of coccidia that have established extraintestinal phases of development, the eimeriids, except for a few species, generally have been considered inhabitants of the intestinal tract. Eimeria infection in sandhill cranes (Grus canadensis) and whooping cranes (G. americana) may result in disseminated visceral coccidiosis. Nodules were observed in the oral cavity of 33% (n = 95) of the G. canadensis at the Patuxent Wildlife Research Center (PWRC) in Laurel, MD. Necropsy of six of the afflicted cranes revealed granulomatous nodules in many tissues and organs. Histologic studies disclosed protozoan organisms morphologically resembling schizonts in the granulomas, and endogenous stages of coccidia were present in the intestines of four birds. Fecalysis of three of four sandhill cranes yielded oocysts of E. reichenowi and E. gruis. Only E. reichenowi-type oocysts were recovered from a dead whooping crane sample. Domestic broiler chicks each intubated with about 1 times 106 pooled sporulated oocysts of E. reichenowi and E. gruis were not infected. Exposure of six incubator-hatched and hand-reared sandhill crane chicks to oocysts artificially (two chicks) and naturally (four chicks) resulted in typical infection of intestinal epithelium with invasion of subepithelial tissues extending to the muscular layer and widespread extraintestinal development. Asexual and sexual stages occurred primarily in macrophages in the liver, spleen, heart, and lung. In the lung, oocysts were found in bronchial exudate and epithelial lining cells. Six of ten G. canadensis chicks, one adult G. americana, and three of five G. americana chicks that died naturally at PWRC had disseminated visceral coccidiosis.

Cryptosporidium Oocyst Detection in Water Samples: Floatation Technique Enhanced with Immunofluorescence Is as Effective as Immunomagnetic Separation Method

PubMed Central

Koompapong, Khuanchai; Sutthikornchai, Chantira

2009-01-01

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 101, 102, and 103 per 10 µl were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 102 per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting. PMID:19967082

Cryptosporidium oocyst detection in water samples: floatation technique enhanced with immunofluorescence is as effective as immunomagnetic separation method.

PubMed

Koompapong, Khuanchai; Sutthikornchai, Chantira; Sukthana, Yowalark

2009-12-01

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 10(1), 10(2), and 10(3) per 10 microl were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 10(2) per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.

Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

EPA Science Inventory

Purified oocysts of Cryptosporidium parvum were used to evaluate applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-...

Inactivation of Toxoplasma gondii on blueberries using low dose irradiation without affecting quality

USDA-ARS?s Scientific Manuscript database

Blueberries (10 g) inoculated with T. gondii (5 log oocysts/g) were exposed to an absorbed dose of 0 (control), 0.2, 0.4 or 0.6 kGy gamma radiation at 4°C. After treatment, oocysts were recovered from berries by washing, and excysted sporozoites were enumerated using a plaque assay. Vero cells wer...

Loop-Mediated Isothermal Amplification-Lateral-Flow Dipstick (LAMP-LFD) to detect Toxoplasma gondii oocyst in ready-to-eat salad.

PubMed

Lalle, Marco; Possenti, Alessia; Dubey, Jitender P; Pozio, Edoardo

2018-04-01

The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a foodborne zoonosis with a global distribution and estimated to cause up to 20% of the total foodborne disease burden in Europe. Association between T. gondii infection and the consumption of unwashed raw fruits and vegetables contaminated with oocysts has been reported and the increasing habit to eat pre-washed ready-to-eat salads poses a new potential risk for consumers. It is therefore important to trace the occurrence of potential contamination with this parasite to guarantee the safety of ready-to-eat vegetables. Detection of T. gondii in vegetables by molecular techniques has been achieved but low sensitivity (PCR) or expensive equipments (qPCR) limit routine applicability. Here, we describe the development and validation of a sensitive and robust method relying on a LAMP assay, targeting the 529 bp locus, to detect T. gondii oocysts down to 25 oocysts/50 g in ready-to-eat baby lettuce. The LAMP has been also adapted for a faster visualization of the result by a lateral flow dipstick chromatographic detection method. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation and biological and molecular characterization of Neospora caninum (NC-SP1) from a naturally infected adult asymptomatic cattle (Bos taurus) in the state of São Paulo, Brazil.

PubMed

Oliveira, Solange; Soares, Rodrigo Martins; Aizawa, Juliana; Soares, Herbert Sousa; Chiebao, Daniela Pontes; Ortega-Mora, Luis Miguel; Regidor-Cerrillo, Javier; Silva, Natália Quadros Bressa; Gennari, Solange Maria; Pena, Hilda Fátima Jesus

2017-05-01

The biological and genetic diversity of Neospora caninum is very limited because of availability of only a few viable isolates worldwide. This study describes the isolation and biological and molecular characterization of a new viable isolate of N. caninum (NC-SP1), from a cattle in Brazil. Approximately 400 g of brain from a naturally infected adult male cattle from an abattoir was fed to a 2-month-old dog. Neospora-like oocysts were observed on day 7 post-inoculation (PI) and the duration of oocyst shedding was 14 days. The DNA obtained from oocysts was characterized molecularly and the final sequence was 99% identical to homologous sequences of N. caninum available in GenBank®. For bioassay, gerbils (Meriones unguiculatus) were orally inoculated with 10 100 and 1000 oocysts; all gerbils remained clinically normal but developed N. caninum antibodies 14 days PI. Cell culture isolation was successful using the brain homogenate from one of the gerbils and tachyzoites were observed 24 days PI. Microsatellite genotyping revealed a unique genetic profile for this new reference isolate.

[Coccidies genus Eimeria as a bioindicator of radioactive pollution of the biocenose].

PubMed

Pel'gunov, A N

2006-01-01

The data on coccidies of rodents were collected in Chernobil (1989-1991) and in the regions of radioactive pollution in the Bryansk region of Russia (1992-1999). The surface pollution of experimental plots was different and come from 0.11 to 11.8 MBq/m2. 2185 rodent were examined in all. Thirteen types of coccidies p. Eimeria were found out in 525 small animals. The analysis of changes in morphological characters and oocysts sporulation in dependence of the level of radioactive pollution of biocenose was carried out. It was found out that parametric signs (length, width and form index of oocysts) were independent from radioactive pollution. At the some time the radioactive pollution renders a significant influence on the nonparametric signs (different types of capsule deformation and internal texture of oocysts) and the process of sporulation. With the increase of radioactive pollution the part of nonsporulated oocycts increased and the quantity of oocysts, corresponding to the description of given type (normal), decreased. This dependence is well described by the equation of logarithmic regression, that allows to use this indexes in the bioindication of the radioactive pollution of the biocenose.

Point prevalence of Cryptosporidium oocyst in calves grazing along River Rima bank in Sokoto, Nigeria.

PubMed

Faleke, O O; Yabo, Y A; Olaleye, A O; Dabai, Y U; Ibitoye, E B

2014-02-01

The present study was conducted to investigate the point prevalence of Cryptosporidium oocysts infection in calves grazing along the bank of Rima River Sokoto in October 2011. The river bank is a converging zone for domestic animals reared in different quarters of the town and the surrounding settlements. A total number of 2,959 cattle were enumerated out of which 147 (4.97%) were calves. Faecal samples were collected from 100 (68.02%) calves by convenient sampling technique. Formol-Ether sedimentation and modified Ziehl-Neelsen staining techniques were used to identify the Cryptosporidium oocysts in the faecal samples. Faecal consistency was also used to identify diarrhoeic and non-diarrhoeic calves. Cryptosporidium oocysts were identified in 33 (33.0%) of the calves examined. The detection rate was higher among the male calves (38.46%) than females while the Rahaji breed had the highest prevalence of 62.5%. A total of 6 (18.18%) among the positive cases were diarrhoeic. The differences in prevalence based on sex, breeds and presence of diarrhoea were not statistically significant. Calves may become sources of Cryptosporidia infection to man and other animals in the study area through unrestricted movements and interactions with the environment.

Monitoring of Noxious Protozoa for Management of Natural Water Resources.

PubMed

Bahk, Young Yil; Cho, Pyo Yun; Ahn, Sung Kyu; Park, Sangjung; Jheong, Won Hwa; Park, Yun-Kyu; Shin, Ho-Joon; Lee, Sang-Seob; Rhee, Okjae; Kim, Tong-Soo

2018-04-01

Waterborne parasitic protozoa, particularly Giardia lamblia and Cryptosporidium spp., are common causes of diarrhea and gastroenteritis worldwide. The most frequently identified source of infestation is water, and exposure involves either drinking water or recreation in swimming pools or natural bodies of water. In practice, studies on Cryptosporidium oocysts and Giardia cysts in surface water are challenging owing to the low concentrations of these microorganisms because of dilution. In this study, a 3-year monitoring of Cryptosporidium parvum, Giardia lamblia , and Naegleria fowleri was conducted from August 2014 to June 2016 at 5 surface water sites including 2 lakes, 1 river, and 2 water intake plants. A total of 50 water samples of 40 L were examined. Cryptosporidium oocysts were detected in 22% of samples and Giardia cysts in 32%. Water at the 5 sampling sites was all contaminated with Cryptosporidium oocysts (0-36/L), Giardia cysts (0-39/L), or both. The geometric mean concentrations of Cryptosporidium and Giardia were 1.14 oocysts/L and 4.62 cysts/L, respectively. Thus, effective monitoring plans must take into account the spatial and temporal parameters of contamination because they affect the prevalence and distribution of these protozoan cysts in local water resources.

Occurrence of Cryptosporidium and Giardia and the Relationship between Protozoa and Water Quality Indicators in Swimming Pools.

PubMed

Xiao, Shumin; Yin, Pengna; Zhang, Yan; Hu, Sike

2017-04-01

A total of 60 samples were collected from 35 swimming pools in Beijing, China, and the presence of Cryptosporidium and Giardia were investigated. The results showed that 16.7% and 15.0% of samples were positive for Cryptosporidium oocyst and Giardia cysts, respectively, with a mean concentration of 0.30 oocysts/10 L and 0.27 cysts/10 L. The oocysts and cysts were found to have higher rates of occurrence in August than in May. Genotyping confirmed the presence of Cryptosporidium hominis, C. parvum , and Giardia assemblages A and B, all of which were associated with human infections. The predominant species/assemblages were C. hominis and Giardia assemblage A. Analyses of the relationships between parasite oocysts/cysts, indicator bacteria, and physical-chemical parameters revealed that there was no correlation between 2 parasites and fecal bacterial indicators, whilst there was a significant correlation between protozoa and urea concentration, which indicates that urea concentration rather than fecal bacterial indicators might be an appropriate index for chlorine-resistant protozoa in swimming pools. This study provides useful information to improve the safety of swimming pool water and deduce the risk of protozoan infections.

Detection of Toxoplasma oocysts from soil by modified sucrose flotation and PCR methods.

PubMed

Matsuo, Junji; Kimura, Daisuke; Rai, Shiba Kumar; Uga, Shoji

2004-06-01

A detection method of Toxoplasma gondii oocysts from soil was evaluated using the sucrose flotation technique with modification involving addition of 0.1% gelatin into washing and floating solutions. PCR was performed on untreated samples and after treatment with polyvinylpyrrolidone (PVP), heating and cooling, and NaCl. The addition of gelatin in the sucrose solution yielded a higher number of oocysts. A very thin band was observed when DNA extract was diluted to 1:1024, indicating the presence of PCR inhibitor in the soil. PCR performed on untreated DNA, on PVP-treated, and on PVP-treated with heating and cooling without added bovine serum albumin (BSA) showed a band only at higher dilutions (1:1024 and 1:512) but at a much lower dilution (1:8) with BSA. In contrast, DNA treated with all three agents showed a band at a much lower dilution (1:64), even without added BSA, and no dilution was required when BSA was added. The PCR inhibitors present in the soil were removed by employing various treatment procedures during DNA extraction, and BSA in PCR. Furthermore, the detection limit with the method was 1 oocyst/g of soil, indicating that this method is useful in epidemiological studies.

IDENTIFICATION OF TWO NOVEL COCCIDIAN SPECIES SHED BY CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS)

PubMed Central

Carlson-Bremer, Daphne; Johnson, Christine K.; Miller, Robin H.; Gulland, Frances M. D.; Conrad, Patricia A.; Wasmuth, James D.; Colegrove, Kathleen M.

2016-01-01

Routine fecal examination revealed novel coccidian oocysts in asymptomatic California sea lions (Zalophus californianus) in a rehabilitation facility. Coccidian oocysts were observed in fecal samples collected from 15 of 410 California sea lions admitted to The Marine Mammal Center between April 2007 and October 2009. Phylogenetic analysis using the full ITS-1 region, partial small subunit 18S rDNA sequence, and the Apicomplexa rpoB region identified 2 distinct sequence clades, referred to as Coccidia A and Coccidia B, and placed them in the Sarcocystidae, grouped with the tissue-cyst–forming coccidia. Both sequence clades resolved as individual taxa at ITS-1 and rpoB and were most closely related to Neospora caninum. Coccidia A was identified in 11 and Coccidia B in 4 of 12 sea lion oocyst samples successfully sequenced (3 of those sea lions were co-infected with both parasites). Shedding of Coccidia A oocysts was not associated with age class, sex, or stranding location, but yearlings represented the majority of shedders (8/15). This is the first study to use molecular phylogenetics to identify and describe coccidian parasites shed by a marine mammal. PMID:22091999

Identification of two novel coccidian species shed by California sea lions (Zalophus californianus).

PubMed

Carlson-Bremer, Daphne; Johnson, Christine K; Miller, Robin H; Gulland, Frances M D; Conrad, Patricia A; Wasmuth, James D; Colegrove, Kathleen M; Grigg, Michael E

2012-04-01

Routine fecal examination revealed novel coccidian oocysts in asymptomatic California sea lions (Zalophus californianus) in a rehabilitation facility. Coccidian oocysts were observed in fecal samples collected from 15 of 410 California sea lions admitted to The Marine Mammal Center between April 2007 and October 2009. Phylogenetic analysis using the full ITS-1 region, partial small subunit 18S rDNA sequence, and the Apicomplexa rpoB region identified 2 distinct sequence clades, referred to as Coccidia A and Coccidia B, and placed them in the Sarcocystidae, grouped with the tissue-cyst-forming coccidia. Both sequence clades resolved as individual taxa at ITS-1 and rpoB and were most closely related to Neospora caninum. Coccidia A was identified in 11 and Coccidia B in 4 of 12 sea lion oocyst samples successfully sequenced (3 of those sea lions were co-infected with both parasites). Shedding of Coccidia A oocysts was not associated with age class, sex, or stranding location, but yearlings represented the majority of shedders (8/15). This is the first study to use molecular phylogenetics to identify and describe coccidian parasites shed by a marine mammal.

Comparative susceptibility of Culex tarsalis, a Nopheles franciscanus, and Culiseta inornata (Diptera: Culicidae) to Plasmodium relictum (Haemosporidia: Plasmodiidae)

USGS Publications Warehouse

Work, Thierry M.; Washino, Robert K.; van Riper, Charles

1990-01-01

Repeated laboratory attempts failed to infect Culiseta inornata (Williston) and Anopheles franciscanus McCracken with Plasmodium relictum as efficiently as Culex tarsalis Coquillett controls. Of 210 An. franciscanus that imbibed a replete meal from a parasitemic canary, two were found with oocysts and none with sporozoites. Of 112 Cs. inornata similarly fed, seven contained oocysts and one contained sporozoites. In contrast, of 94 Cx. tarsalis tested, 80 (85%) were found with oocysts, thus confirming that this mosquito is a suitable host of P. relictum . Lack of An. franciscanus and Cs. inornata with sporozoites precluded transmission studies and indicated that these species do not play a major role in the maintenance of P. relictum during fall and winter in the Sacramento Valley, Calif.

Internal parasites of reptiles.

PubMed

Raś-Noryńska, Małgorzata; Sokół, Rajmund

2015-01-01

Nowadays a growing number of exotic reptiles are kept as pets. The aim of this study was to determine the species of parasites found in reptile patients of veterinary practices in Poland. Fecal samples obtained from 76 lizards, 15 turtles and 10 snakes were examined by flotation method and direct smear stained with Lugol's iodine. In 63 samples (62.4%) the presence of parasite eggs and oocysts was revealed. Oocysts of Isospora spp. (from 33% to 100% of the samples, depending on the reptilian species) and Oxyurids eggs (10% to 75%) were predominant. In addition, isolated Eimeria spp. oocysts and Giardia intestinalis cysts were found, as well as Strongylus spp. and Hymenolepis spp. eggs. Pet reptiles are often infected with parasites, some of which are potentially dangerous to humans. A routine parasitological examination should be done in such animals.

Eimeria collieie n. sp. (Apicomplexa:Eimeriidae) from the western long-necked turtle (Chelodina colliei).

PubMed

Yang, Rongchang; Brice, Belinda; Elloit, Aileen; Lee, Elvina; Ryan, Una

2015-07-01

A new species, Eimeria collieie n. sp., is described from the western long-necked turtle (Chelodina colliei). Sporulated oocysts (n = 35) are spherical to subspherical, with colourless single layer oocyst wall, 0.6 ± 0.2 (0.4-0.7) µm thick. Oocyst with elongated ellipsoid sporocysts. Oocyst length, 29.8 ± 0.4 (28.2-31.0) µm; oocyst width, 29.4 ± 0.3 (28.0-30.8) µm; oocyst length/width (L/W) ratio, 1.0 ± 0.03 (1.0-1.05). Micropyle, oocyst residuum and polar granule were absent. Sporocysts with sporocyst residuum and 2 sporozoites. Sporocyst length, 21.6 ± 0.4 (21.2-22.0) µm; sporocyst width, 6.0 ± 0.3 (5.7-6.3) µm; sporocyst L/W ratio, 3.6 ± 0.2 (3.4-3.8). Stieda, parastieda and substieda bodies were absent. Sporozoite length, 14.0 ± 0.2 (13.8-14.2) µm; sporozoite width, 2.6 ± 0.2 (2.4-2.8) µm; sporozoite L/W ratio, 5.46 ± 0.10 (5.4-5.6). Molecular analysis was conducted at three loci: the 18S and 28S ribosomal RNA (rRNA), and the mitochondrial cytochrome oxidase gene (COI). At the 18S rRNA locus, E. collieie n. sp. shared 96.4% and 98.3% genetic similarity to E. ranae (GenBank accession number: EU717219) and E. arnyi (AY613853) respectively. At the 28S rRNA locus, E. collieie n. sp. shared 91.6% genetic similarity to E. papillata (GenBank accession number: GU593706) and phylogenetic analysis at this locus placed E. collieie n. sp. in aseparateclade. At the COI locus, E. collieie n. sp. shared 92.7% genetic similarity to Eimeria setonicis (GenBankaccession number: KF225638) from a quokka (Setonix brachyurus) in Western Australia. Reptile-derived sequences were not available for the 28S rRNA and the COI loci. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that, to date, has only been found in western long-necked turtles. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

Association of Cryptosporidium with bovine faecal particles and implications for risk reduction by settling within water supply reservoirs.

PubMed

Brookes, Justin D; Davies, Cheryl M; Hipsey, Matthew R; Antenucci, Jason P

2006-03-01

Artificial cow pats were seeded with Cryptosporidium oocysts and subjected to a simulated rainfall event. The runoff from the faecal pat was collected and different particle size fractions were collected within settling columns by exploiting the size-dependent settling velocities. Particle size and Cryptosporidium concentration distribution at 10 cm below the surface was measured at regular intervals over 24 h. Initially a large proportion of the total volume of particles belonged to the larger size classes (> 17 microm). However, throughout the course of the experiment, there was a sequential loss of the larger size classes from the sampling depth and a predominance of smaller particles (< 17 microm). The Cryptosporidium concentration at 10 cm depth did not change throughout the experiment. In the second experiment samples were taken from different depths within the settling column. Initially 26% of particles were in the size range 124-492 microm. However, as these large particles settled there was an enrichment at 30 cm after one hour (36.5-49.3%). There was a concomitant enrichment of smaller particles near the surface after 1 h and 24 h. For Pat 1 there was no difference in Cryptosporidium concentration with depth after 1 h and 24 h. In Pat 2 there was a difference in concentration between the surface and 30 cm after 24 h. However, this could be explained by the settling velocity of a single oocyst. The results suggested that oocysts are not associated with large particles, but exist in faecal runoff as single oocysts and hence have a low (0.1 m(d-1)) settling velocity. The implications of this low settling velocity on Cryptosporidium risk reduction within water supply reservoirs was investigated through the application of a three-dimensional model of oocyst fate and transport to a moderately sized reservoir (26 GL). The model indicated that the role of settling on oocyst concentration reduction within the water column is between one and three orders of magnitude less than that caused by advection and dilution, depending on the strength of hydrodynamic forcing.

Cryptosporidium parvum infection and associated risk factors in dairy calves in western France.

PubMed

Delafosse, A; Chartier, C; Dupuy, M C; Dumoulin, M; Pors, I; Paraud, C

2015-03-01

This study was conducted to determine the prevalence and risk factors for Cryptosporidium infection in calf neonates on dairy farms in Normandy. Fecal samples were randomly collected between July 2010 and September 2011 from 968 calves (7-21 days old) on 97 farms. Up to 10 calves were selected and sampled per farm, and feces examined for oocysts by microscopy. C. parvum oocyst shedding was scored semi-quantitatively (0-5). A questionnaire about calf-level care and management was completed, and mortality rates were obtained from the French national registration database (BDNI). Bivariable and multivariable analyses of potential risk factors for C. parvum oocyst shedding were conducted using generalized estimating equation (GEE) models (family=Binomial).Overall, 402 out of 968 calves (41.5%) were positive for oocysts, and 25.1% of animals had a shedding score >2. Seven of the 97 farms (7%) were negative for oocysts in all fecal samples. At the time of collection, 375 calves (39%) had diarrhea, and its prevalence strongly correlated with the score for C. parvum oocyst shedding (p<0.0001). The mortality rate at 90 days was significantly greater for calves with high combined scores of diarrhea and shedding. Factors associated with the shedding of C. parvum were the Normande breed (odds ratio=1.49; 95% confidence interval (CI): 0.93-2.37), dispensing of colostrum using a bucket (odds ratio=1.37; 95% CI: 1.00-1.89), treatment with halofuginone (odds ratio=0.46; 95% CI: 0.19-1.15) and feeding with fermented milk (odds ratio=0.32; 95% CI: 0.17-0.63). C. parvum is widespread among calves under 21 days old in dairy herds of western France. Shedding of C. parvum is associated with a high incidence of diarrhea and increased risk of mortality in young calves. This study identified some associated calf-level factors, although further investigations are necessary to determine appropriate measures that farmers and veterinary practitioners should take to reduce the prevalence of C. parvum. Copyright © 2015 Elsevier B.V. All rights reserved.

Assessment of the efficacy of the first water system for emergency hospital use.

PubMed

Long, Sharon C; Olstadt, Jeremy

2011-03-01

The First Water Responder B package water treatment device was evaluated for its ability to reduce the levels of spiked indicators and pathogens (Escherichia coli, MS2 coliphage, murine adenovirus, and Cryptosporidium oocysts) in a surface water to partially evaluate its appropriateness to be used to provide safe drinking water to hospitals during emergency situations. Lake water was collected in 50-L carboys and spiked with selected indicators and pathogens (E coli, MS2 coliphage, murine adenovirus, and Cryptosporidium oocysts) at 2 different spike levels (low and high). This water was treated using the First Water Responder B, and the microorganisms were enumerated before and after treatment using US Environmental Protection Agency and Standard Methods. Microbial removal efficiencies were compared with Environmental Protection Agency guidelines. E coli spikes ranged from 2.9 to 1059 colony-forming units (CFU)/100 mL with removals to below detection limits (1 CFU/100 mL) to 2.8 CFU/100 mL or 0.98 to 3.5 log(10) reductions. MS2 coliphage spikes ranged from 3 plaque-forming units (PFU) to 837 PFU/100 mL with removals to below detection limits (1 PFU/100 mL) to 11.7 PFU/100 mL or 0.65 to 1.9 log(10) reductions. Murine adenovirus spikes ranged from 203 to 8410 most probable number (MPN) of infectious units/100 mL with removals to below detection limits (23 MPN infectious units/100 mL) to 1370 MPN infectious units/100 mL or 0.79 to >1.2 log(10) reductions. Cryptosporidium parvum oocyst spikes ranged from 52 to 853 oocysts per liter with removals to below detection limits (<1 oocyst per liter) to 0.3 oocysts per liter or >2.2 to 3.4 log(10) reductions. Although the First Water system could remove a significant portion of the spiked organisms, it is recommended that this point-of-use system be coupled with chemical disinfection in a multiple-barrier approach to provide water of the highest reasonably achievable quality for hospital use in emergency situations. ©2011 American Medical Association. All rights reserved.

Effect of whole yeast cell product supplementation (CitriStim®) on immune responses and cecal microflora species in pullet and layer chickens during an experimental coccidial challenge.

PubMed

Markazi, Ashley D; Perez, Victor; Sifri, Mamduh; Shanmugasundaram, Revathi; Selvaraj, Ramesh K

2017-07-01

Three separate experiments were conducted to study the effects of whole yeast cell product supplementation in pullets and layer hens. Body weight gain, fecal and intestinal coccidial oocyst counts, cecal microflora species, cytokine mRNA amounts, and CD4+ and CD8+ T-cell populations in the cecal tonsils were analyzed following an experimental coccidial infection. In Experiment I, day-old Leghorn layer chicks were fed 3 experimental diets with 0, 0.1, or 0.2% whole yeast cell product (CitriStim®, ADM, Decatur, IL). At 21 d of age, birds were challenged with 1 × 105 live coccidial oocysts. Supplementation with whole yeast cell product decreased the fecal coccidial oocyst count at 7 (P = 0.05) and 8 (P < 0.01) d post-challenge. In Experiment II, 27-week old Leghorn layer hens were fed 3 experimental diets with 0, 0.05 or 0.1% whole yeast cell product and challenged with 1 × 105 live coccidial oocysts on d 25 of whole yeast cell product feeding. Supplementation with whole yeast cell product decreased the coccidial oocyst count in the intestinal content (P < 0.01) at 5, 13, and 38 d post-coccidial challenge. Supplementation with whole yeast cell product increased relative proportion of Lactobacillus (P < 0.01) in the cecal tonsils 13 d post-coccidial challenge. Supplementation with whole yeast cell product decreased CD8+ T cell percentages (P < 0.05) in the cecal tonsils at 5 d post-coccidial challenge. In Experiment III, 32-week-old Leghorn layer hens were fed 3 experimental diets with 0, 0.1, or 0.2% whole yeast cell product and challenged with 1 × 105 live coccidial oocysts on d 66 of whole yeast cell product feeding. At 5 d post-coccidial challenge, whole yeast cell product supplementation down-regulated (P = 0.01) IL-10 mRNA amount. It could be concluded that supplementing whole yeast cell product can help minimize coccidial infection in both growing pullets and layer chickens. © 2017 Poultry Science Association Inc.

Comparative efficacy of curcumin and paromomycin against Cryptosporidium parvum infection in a BALB/c model.

PubMed

Asadpour, Mohammad; Namazi, Fatemeh; Razavi, Seyed Mostafa; Nazifi, Saeed

2018-01-30

Cryptosporidium is a ubiquitous protozoan parasite causing gastrointestinal disorder in various hosts worldwide. The disease is self-limiting in the immunocompetent but life-threatening in immunodeficient individuals. Investigations to find an effective drug for the complete elimination of the Cryptosporidium infection are ongoing and urgently needed. The current study was undertaken to examine the anti-cryptosporidial efficacy of curcumin in experimentally infected mice compared with that of paromomycin. Oocysts were isolated from a pre-weaned dairy calf and identified as Cryptosporidium parvum using a nested- polymerase chain reaction (PCR) on Small subunit ribosomal ribonucleic acid (SSU rRNA) gene and sequencing analysis. One hundred and ten female BALB/c mice were divided into five groups. Group 1 was infected and treated with curcumin; Group 2 infected and treated with paromomycin; Group 3 infected without treatment; Group 4 included uninfected mice treated with curcumin, and Group 5 included uninfected mice treated with distilled water for 11 successive days, starting on the first day of oocyst shedding. The oocyst shedding was recorded daily. At days 0, 3, 7, and 11 of post treatments, five mice from each group were killed humanly; jejunum and ileum tissue samples were processed for histopathological evaluation and counting of oocyst on villi, simultaneously. Furthermore, total antioxidant capacity (TAC) and malondialdehyde (MDA) concentrations in affected tissues were also measured in different groups. By treatments, tissue lesions and the number of oocyst on villi of both jejunum and ileum were decreased with a time-dependent manner. In comparison with Group 3, oocyst shedding was stopped at the end of treatment period in both groups 1 and 2 without recurrence at 10days after drug withdrawal. Also, TAC was increased and the MDA concentrations were decreased in Group 1. Moreover, paromomycin showed acceptable treatment outcomes during experiment and its anti-cryptosporidial activity was faster than curcumin. The results confirmed the anti-cryptosporidial and antioxidant activity of curcumin against C. parvum and further evaluation of immunosuppressed animal models needs to be carried out. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR.

PubMed

Murphy, Helen R; Lee, Seulgi; da Silva, Alexandre J

2017-07-01

Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for detection of C. cayetanensis.

Hammondia sp. oocysts shed by a Brazilian fox (Lycalopex vetulus) differ from Hammondia heydorni and Hammondia triffittae.

PubMed

Gondim, Luís F P; Soares, Rodrigo M; Osaki, Silvia C; Snak, Alessandra; Grillo, Laura R; Fernandes, Nelson L M; de Carvalho, Anderson L

2018-05-21

A Brazilian fox (Lycalopex vetulus) was rescued from a highway, and 16 days after maintained in captivity, the fox shed oocysts with sizes compatible with Hammondia sp. and Neospora caninum. DNA extracted from oocysts were initially tested in two PCRs targeting the internal transcribed spacer 1 (ITS-1) of the rDNA of Hammondia heydorni and the Nc-5 gene of N. caninum. A 270-bp product was visualized in the PCR for H. heydorni. No amplification was observed for N. caninum PCR. Since ITS-1-based PCR is not sufficient to differentiate Hammondia species derived from canids, oocyst DNA was examined using multilocus sequence analysis of five genetic fragments [intron 1 of the alpha tubulin gene (intron 1), internal transcribed spaces 1 and 2 (ITS-1 and ITS-2) of the rDNA, 28S rRNA gene (D2/D3 domain), and heat shock protein 70 (Hsp70)]. The Hammondia sp. oocyst from the Brazilian fox, referred here as H-FOXBR isolate, is closely related to H. heydorni and Hammondia triffittae, but differs from these parasites in three genetic markers (alpha tubulin gene, ITS-2, and 28S rRNA). As reported by other research groups, Hammondia spp. excreted by canids are genetically diverse and may encompass additional species besides H. heydorni and H. triffittae. In this study, we confirmed that H-FOXBR has significant genetic differences in comparison to H. heydorni and H. triffittae and may represent a separate species. Further studies are needed to identify the life cycle of this parasite and to characterize the parasite stages in the intermediate and definitive hosts.

Significance of Timing on Effect of Metaphylactic Toltrazuril Treatment against Eimeriosis in Calves.

PubMed

Enemark, Heidi Larsen; Dahl, Jan; Enemark, Jörg Matthias Dehn

2015-08-01

In this multicentric, randomised, blinded and placebo-controlled field study, the effect of treatment with toltrazuril (Baycox(®) Bovis, Bayer) on oocyst excretion, diarrhoea score and weight gain was studied in Danish dairy herds with confirmed history of eimeriosis (coccidiosis) and prevalence of Eimeria bovis and Eimeria zuernii. Three commercial herds and a total of 71 calves, aged 48 - 135 days, were included. Treatment with a single oral dose of toltrazuril (15 mg/kg) was given after relocation to common pens and one week before expected outbreak of eimeriosis. The effect of treatment was followed by weekly faecal sampling and weighing initially and at the end of a study period of 8 weeks. In Herd 2 and 3 toltrazuril treated calves gained on average 7.95 kg more than placebo treated calves (p = 0.007), and both oocyst excretion and prevalence of Eimeria spp. were significantly reduced the first weeks post treatment. In Herd 1, by contrast, the farmer made some unforeseen changes in the management which entailed relocation to large deep-litter pens 3 - 6 weeks post treatment. In addition, many calves were not treated metaphylactically while few calves excreted oocysts when the trial was initiated. Thus, no significant difference in weight gain was found between toltrazuril and placebo treated calves (p = 0.523), and the oocyst excretion of toltrazuril treated calves was significantly higher during week 7 and 8. Significant differences in faecal scores were observed between the herds (p<0.002) but not between treatment groups in any of the herds. In conclusion, timing of treatment is crucial for optimal effect of metaphylactic toltrazuril treatment on weight gain and oocyst excretion.

Tomaculocystis corpulenta n. gen., n. sp. (Apicomplexa: Eugregarinorida) parasitizing the little yellow cockroach, Cariblatta lutea (Blattodea: Ectobiidae), in Alabama and Florida with recognition of Tomaculocystis cylindrosa n. comb. and Tomaculocystis mukundai n. comb. parasitizing ectobiid cockroaches in India.

PubMed

Clopton, Richard E

2015-02-01

Tomaculocystis corpulenta n. gen., n. sp. (Apicomplexa: Eugregarinorida: Septatorina: Gregarinidae) is described from populations of the little yellow cockroach, Cariblatta lutea (Blattodea: Ectobiidae), established in laboratory culture from samples collected in Alabama and Florida. Tomaculocystis n. gen. are differentiated from other members of Gregarina by a markedly elliptoid gametocyst inside a persistent, lomentiform hyaline epicyst; developmental organization and growth of the spore tubes from gametocyst surface tumidi; and dehiscence by extrusion of non-chain forming oocysts through spore tubes that barely extend beyond the epicyst wall. Gregarina cylindrosa, Gregarina discocephala, and Gregarina mukundai are recognized as members of Tomaculocystis, and G. cylindrosa is recognized as the senior synonym of G. discocephala. Thus, Tomaculocystis cylindrosa n. comb. and Tomaculocystis mukundai n. comb. are formed. Species of Tomaculocystis are distinguished based on gamont deutomerite and oocyst shape and size. The oocysts of T. corpulenta are broadly dolioform, lack 4 polar knobs, and possess distinct, unique polar plates. Oocysts of all other known species in the genus are more oblong in shape, possess 4 polar knobs, and lack the distinct polar plates observed in the oocysts of T. corpulenta. Host utilization and geographic distribution among gregarine genera parasitizing the cockroach family Ectobiidae reveal a pattern of host-parasite specificity linking gregarine genera with ectobiidid subfamilies. Overall patterns suggest a hypothesis of European endemicy for Gamocystis, but hypotheses for the origin and radiation of Tomaculocystis or species of Gregarina infecting cockroaches are confounded by the cosmopolitan spread of pest cockroach species among humans.

Recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip for detection of Toxoplasma gondii in the environment.

PubMed

Wu, Y D; Xu, M J; Wang, Q Q; Zhou, C X; Wang, M; Zhu, X Q; Zhou, D H

2017-08-30

Toxoplasma gondii infects all warm-blooded vertebrates, resulting in a great threat to human health and significant economic loss to the livestock industry. Ingestion of infectious oocysts of T. gondii from the environment is the major source of transmission. Detection of T. gondii oocysts by existing methods is laborious, time-consuming and expensive. The objective of the present study was to develop a recombinase polymerase amplification (RPA) method combined with a lateral flow (LF) strip for detection of T. gondii oocysts in the soil and water. The DNA of T. gondii oocysts was amplified by a pair of specific primers based on the T. gondii B1 gene over 15min at a constant temperature ranging from 30°C to 45°C using RPA. The amplification product was visualized by the lateral flow (LF) strip within 5min using the specific probe added to the RPA reaction system. The sensitivity of the established assay was 10 times higher than that of nested PCR with a lower detection limit of 0.1 oocyst per reaction, and there was no cross-reactivity with other closely related protozoan species. Fifty environmental samples were further assessed for the detection validity of the LF-RPA assay (B1-LF-RPA) and compared with nested PCR based on the B1 gene sequence. The B1-LF-RPA and nested PCR both showed that 5 out of the 50 environmental samples were positive. The B1-LF-RPA method was also proven to be sufficiently tolerant of existing inhibitors in the environment. In addition, the advantages of simple operation, speediness and cost-effectiveness make B1-LF-RPA a promising molecular detection tool for T. gondii. Copyright © 2017 Elsevier B.V. All rights reserved.

[Achatina fulica Bowdich (1822) a new host of Cryptosporidium (Apicomplexa, Cryptosporidiidae) species].

PubMed

Schiffler, Cinthia L; Gomes, Francimar F; Ederli, Nicole B; De Oliveira, Francisco Carlos R

2008-09-01

With the objective of isolate Cryptosporidium spp. in Achatina fulica s feces, 50 mollusks were collected in nine neighborhoods of the municipal of Campos dos Goytacazes, RJ to the observation of oocysts in feces. The snails were put in individuals containers and fed with water and green vegetables ad libitum until be collected a gram of feces per animal. The samples were conditioned in tubes with formalin 10% and later smear of feces were made and dyed by Ziechl-Neelsen modified technique. Of the 50 samples examined, 26 (52%) were positive for the presence of oocysts of Cryptosporidium spp. The morphology and morphometry of the oocysts showed that are a great morphologic variability. Considering the obtained results, the mollusk Achatina fulica is a host of Cryptosporidium species and can participate in the epidemic chain of the cryptosporidiosis.

[Development of a technique for recovering Giardia cysts and Cryptosporidium oocysts from fresh vegetables].

PubMed

Di Benedetto, M A; Di Piazza, F; Oliveri, R; Cerame, G; Valenti, R; Firenze, A

2006-01-01

Techniques described for recovering Giardia and Cryptosporidium (oo)cysts from fruit and vegetables are generally inadequate and present variable recovery efficience and elevated costs. The aim of our study was to evaluate the recovery efficiency of a simple and economic technique to apply either to berry vegetables, like tomatoes and peppers, or to large leave vegetables, like lettuce and chicory. The method include contamination and further elution of the vegetables. Then sedimentation of (oo)cysts by centrifugation of the eluate of vegetables and their visualization by means of direct immunofluorescence. The higher recovery values for both protozoa were obtained in large leave vegetables with mean data above 70% for Giardia and 76% for Cryptosporidium, whereas the values observed in the berry vegetables were above 43% for Giardia and above 37% for Cryptosporidium on average.

Toxoplasmosis in sentinel chickens (Gallus domesticus) in New England farms: seroconversion, distribution of tissue cysts in brain, heart, and skeletal muscle by bioassay in mice and cats

USDA-ARS?s Scientific Manuscript database

Free-range chickens are a good indicator of soil contamination with oocysts because they feed from the ground and they are also an important source of infection for cats that in turn shed oocysts after eating tissues of intermediate hosts. Little is known of the epidemiology of toxoplasmosis in chic...

A fast method for detecting Cryptosporidium parvum oocysts in real world samples

NASA Astrophysics Data System (ADS)

Stewart, Shona; McClelland, Lindy; Maier, John

2005-04-01

Contamination of drinking water with pathogenic microorganisms such as Cryptosporidium has become an increasing concern in recent years. Cryptosporidium oocysts are particularly problematic, as infections caused by this organism can be life threatening in immunocompromised patients. Current methods for monitoring and analyzing water are often laborious and require experts to conduct. In addition, many of the techniques require very specific reagents to be employed. These factors add considerable cost and time to the analytical process. Raman spectroscopy provides specific molecular information on samples, and offers advantages of speed, sensitivity and low cost over current methods of water monitoring. Raman spectroscopy is an optical method that has demonstrated the capability to identify and differentiate microorganisms at the species and strain levels. In addition, this technique has exhibited sensitivities down to the single organism detection limit. We have employed Raman spectroscopy and Raman Chemical Imaging, in conjunction with chemometric techniques, to detect small numbers of oocysts in the presence of interferents derived from real-world water samples. Our investigations have also indicated that Raman Chemical Imaging may provide chemical and physiological information about an oocyst sample which complements information provided by the traditional methods. This work provides evidence that Raman imaging is a useful technique for consideration in the water quality industry.

[Investigation on contamination of Cryptosporidium and Giardia in drinking water and environmental water in Shanghai].

PubMed

Zhang, Xiao-Ping; He, Yan-Yan; Zhu, Qian; Ma, Xiao-Jiang; Cai, Li

2010-12-30

To understand the contamination status of Cryptosporidium sp. and Giardia lamblia in drinking water, source water and environmental water in Shanghai. All water samples collected from drinking water, source water and environmental water were detected by a procedure of micromembrane filtration, immune magnetic separation (IMS), and immunofluorescent assay (IFA). Cryptosporidium oocysts and Giardia cysts were not found in 156 samples of the drinking water including finished water, tap water, or pipe water for directly drinking in communities. Among 70 samples either source water of water plants (15 samples), environmental water from Huangpu River(25), canal water around animal sheds(15), exit water from waste-water treatment plants(9), or waste water due to daily life(6), Cryptosporidium oocysts were detected in 1(6.7%), 2(8.0%), 7(46.7%), 1(11.1%), and 1(16.7%) samples, respectively; and Giardia cysts were detected in 1(6.7%), 3(12.0%), 6 (40.0%), 2(22.2%), and 2(33.3%), respectively. The positive rate of Cryptosporidium oocysts and Giardia cysts was 17.1% (12/70) and 20.0% (14/70), respectively. No Cryptosporidium oocysts and Giardia cysts have been detected in drinking water, but found in source water and environmental water samples in Shanghai.

Occurrence and removal of Giardia spp. cysts and Cryptosporidium spp. oocysts from a municipal wastewater treatment plant in Brazil.

PubMed

Santos, Priscila Ribeiro Dos; Daniel, Luiz Antonio

2017-05-01

Sewage and sewage sludge have been recognized as potential sources of two important waterborne pathogenic protozoa: Giardia spp. and Cryptosporidium spp. Due to the lack of studies about the occurrence of these pathogens in sewage and sludge in Brazil, an investigation was conducted at various stages of a municipal wastewater treatment plant (WWTP) aiming to assess the occurrence of Giardia spp. cysts and Cryptosporidium spp. oocysts, their removal by the treatment processes, which are upflow anaerobic sludge blanket (UASB) reactor and dissolved air flotation process, and also the correlations between protozoa and indicator microorganisms. Significant quantities of cysts were detected in 100% of the analyzed wastewater samples, while oocysts were detected only in 39.0% of all wastewater samples. The overall removal of Giardia spp. cysts from the WWTP was on average 2.03 log, and the UASB reactor was more efficient than flotation. The sludge samples presented high quantities of (oo)cysts, implying the risks of contamination in the case of sludge reuse or inadequate disposal. Giardiasis prevalence was estimated between 2.21% and 6.7% for the population served by the WWTP, while cryptosporidiosis prevalence was much lower. Significant positive correlation was obtained only between cysts and Clostridium spores in anaerobic effluent.

Coagulation, flocculation, dissolved air flotation and filtration in the removal of Giardia spp. and Cryptosporidium spp. from water supply.

PubMed

Andreoli, Fernando César; Sabogal-Paz, Lyda Patricia

2017-11-15

Removing protozoa from a water supply using coagulation, flocculation, dissolved air flotation (DAF) and filtration on a bench scale was evaluated. Calcium carbonate flocculation with and without immunomagnetic separation (IMS) was chosen to detect Giardia spp. cysts and Cryptosporidium spp. oocysts in the studied samples. The results indicated that DAF removed between 1.31 log and 1.79 log of cysts and between 1.08 log and 1.42 log of oocysts. The performance was lower in filtration, with the removal of 1.07 log-1.44 log for cysts and 0.82 log-0.98 log for oocysts. The coagulation, flocculation, DAF and filtration steps removed more than 2.2 log of cysts and oocysts from the water studied. However, protozoa were detected in the filtered water, even with turbidity values of 0.2 NTU. The recovery of the detection method met the international criteria and was higher when there was no IMS. Including the third acid dissociation in the IMS was critical to improve the performance of the protocol tested. However, there was an increase in the technical and analytical complexity and costs. It was also observed that the efficiency of the treatment was linked to the performance of the selected method of detecting protozoa.

Oocysts of Hepatozoon canis in Rhipicephalus (Boophilus) microplus collected from a naturally infected dog.

PubMed

de Miranda, Renata Lima; de Castro, Jacqueline Ribeiro; Olegário, Maria Marlene Martins; Beletti, Marcelo Emílio; Mundim, Antonio Vicente; O'Dwyer, Lucia Helena; Eyal, Osnat; Talmi-Frank, Dalit; Cury, Márcia Cristina; Baneth, Gad

2011-05-11

Canine hepatozoonosis is a tick-borne disease caused by protozoans of the genus Hepatozoon. Several tick species have been implicated as potential vectors. Therefore, extensive studies are needed to determine the 'natural' endemic cycle of this parasite. This paper presents the first report of the presence of Hepatozoon canis oocysts in Rhipicephalus (Boophilus) microplus collected from an infected dog. Copyright © 2011 Elsevier B.V. All rights reserved.

Fatal toxoplasmosis and enteroepithelial stages of Toxoplasma gondii in a Pallas cat (Felis manul).

PubMed

Dubey, J P; Gendron-Fitzpatrick, A P; Lenhard, A L; Bowman, D

1988-11-01

Toxoplasma gondii was found in tissues of a six-year-old female Pallas cat (Felis manul) from the Milwaukee County Zoo. Toxoplasma gondii meronts (types D and E), gamonts, and oocysts were present in the epithelium of the small intestine. Numerous unsporulated oocysts were present in the intestinal lumen. The cat died of acute, overwhelming toxoplasmosis. Necrotic enteritis, multifocal necrotizing granulomatous hepatitis, and pneumonia were the prominent lesions.

Reverse Osmosis Water Purification Unit: Efficacy of Cartridge Filters for Removal of Bacteria and Protozoan Cysts when Ro Elements are Bypassed

DTIC Science & Technology

1993-04-01

were Klebsiella terrigena, Cryptosporidium parvum oocysts, Rhodotorula rubra, and 3.7 pm latex beads. Challenge waters were dechlorinated tap water and...The morphological and size characteristics of Rhodotorula rubra (ATCC 36053) made the yeast suitable as a protozoan cyst simulant. The yeast cells...representative enteric bacterium), Cryptosporidium parvum (an enteric protozoan pathogen) oocysts, Rhodotorula rubra (a yeast, used to test prefilters only

In vitro evaluation of the disinfection efficacy on Eimeria tenella unsporulated oocysts isolated from broilers.

PubMed

Guimarães, José S; Bogado, Alexey L Gomel; da Cunha, Thiago Cezar B; Garcia, João Luis

2007-01-01

The objective of this study was to evaluate in vitro the action of eight chemical principles by disinfection efficacy (DE) of Eimeria tenella oocysts. Disinfection efficacy was evaluated by either destruction or sporulation inhibition of the oocysts. Eight treatments were performed: T1 (Glutaraldehyde 42.5 g + Benzalkonium Chloride 7.5 g); T2 (Benzalkonium chloride + quaternary ammonium salt); T3 (formol 37% + Sodium Dodecylbenzene Sulfonate 12%); T4 (sodium hypochlorite 2%); T5 (Orthodichlorobenzene 60% + Xylene 30%); T6 (Polyoctyl polyamino ethyl glycine + Polyoxyethylene alkylphenol ether + Sodium Chloride); T7 (Chloramine T) and finally T8 (free iodine 2.25% + Phosphoric acid 15 g). The control test was carried out with distilled water (T9). The best DE were observed, respectively, in T3 (79.49%), T5 (75.60%) and T4 (65.56%) treatments.

Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina

PubMed Central

Belli, Sabina I.; Ferguson, David J.P.; Katrib, Marilyn; Slapetova, Iveta; Mai, Kelly; Slapeta, Jan; Flowers, Sarah A.; Miska, Kate B.; Tomley, Fiona M.; Shirley, Martin W.; Wallach, Michael G.; Smith, Nicholas C.

2009-01-01

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins – GAM56 and GAM82 – were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity. PMID:19477178

Goussia girellae n. sp. (Apicomplexa: Eimeriorina) in the opaleye, Girella nigricans.

PubMed

Kent, M L; Fournie, J W; Snodgrass, R E; Elston, R A

1988-05-01

Goussia girellae n. sp. is described from the opaleye fish, Girella nigricans. Merogonic stages were observed in the apices of intestinal epithelial cells, in the lamina propria, and in extra-intestinal sites including liver, gills, and spleen. Gamonts were observed in the intestinal epithelial cells. Only unsporulated oocysts were detected in the intestine, and sporulation occurred when feces containing oocysts were incubated for 48 h in seawater at 21 degrees C. Oocysts are elongated (24.8 x 14.7 micron) with a wall about 200 nm thick and have no residuum, micropyle, or polar granule. Sporocysts are ellipsoid (8.5 x 4.5 micron), have a thin two-layered wall approximately 30 nm thick, and consist of two valves joined by a suture. Although moribund opaleye were also infected with Gyrodactylus sp., Cryptobia sp., Cardicola sp., and epitheliocystis organisms (chlamydia), all fish were heavily infected with G. girellae and morbidity was thus attributed to the coccidium.

Water used to moisten vegetables is a source of Escherichia coli and protozoan parasite contamination at markets in Hanoi, Vietnam.

PubMed

Tram, Nguyen Thuy; Dalsgaard, Anders

2014-12-01

The study was done to assess the level of fecal (Escherichia coli) and protozoan parasite (Cryptosporidium spp. and Giardia spp.) contamination in water used by traders to moisten vegetables at markets in Hanoi, Vietnam. A total of 200 splashing water samples from markets located within eight districts were analyzed for E. coli and Cryptosporidium spp. and Giardia spp. (oo)cysts. Giardia cysts were found in 17 splashing water samples and Cryptosporidium oocysts in nine samples, with median values of 20 cysts ml(-1) and 10 oocysts ml(-1), respectively. E. coli was found with a median concentration of 636 cfu ml(-1) and its occurrence was negatively correlated with the numbers of protozoan parasites. The splashing water was kept in buckets that were rarely cleaned and often used for handwashing. The finding of these pathogens in splashing water is likely to represent real food safety hazards.

Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum.

PubMed

Dubey, J P; Jenkins, M C; Rajendran, C; Miska, K; Ferreira, L R; Martins, J; Kwok, O C H; Choudhary, S

2011-09-27

The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. Neospora-like oocysts were found microscopically in the feces of three of 73 wolves from Minnesota examined at necropsy. N. caninum-specific DNA was amplified from the oocysts of all three wolves. Oocysts from one wolf were infective for the gamma interferon gene knock out (KO) mice. Viable N. caninum (designated NcWolfUS1) was isolated in cell cultures seeded with tissue homogenate from the infected mouse. Typical thick walled tissue cysts were found in outbred mice inoculated with the parasite from the KO mouse. Tissue stages in mice stained positively with N. caninum-specific polyclonal antibodies. Our observation suggests that wolves may be an important link in the sylvatic cycle of N. caninum. Copyright © 2011 Elsevier B.V. All rights reserved.

Seasonality in the proportions of domestic cats shedding Toxoplasma gondii or Hammondia hammondi oocysts is associated with climatic factors.

PubMed

Schares, G; Ziller, M; Herrmann, D C; Globokar, M V; Pantchev, N; Conraths, F J

2016-04-01

A previous study on domestic cats in Germany and neighbouring countries suggested seasonality in shedding Toxoplasma gondii oocysts. The aim of the present study was to elucidate whether this seasonality in shedding could be explained by climatic effects and whether differences between years in the proportions of cats shedding oocysts could also be explained by climatic factors. To this end, a long-term study over a period of 55 months on domestic cats for T. gondii and Hammondia hammondi oocysts was performed and the results compared with climatic data. Using species-specific PCR, T. gondii oocysts were identified in 0.14% (84/61,224) and H. hammondi in 0.10% (61/61,224) of the samples. Toxoplasma gondii oocysts were predominantly observed from summer to autumn, while H. hammondi oocysts were mainly found during autumn and winter. In statistical analyses using climatic data, even differences in parasitological findings between years could be partially modelled using monthly temperature, North Atlantic Oscillation indices and precipitation. Of the three climatic variables analysed, precipitation as an explanatory variable had the lowest impact in the statistical models while those taking only temperature and North Atlantic Oscillation indices into account were sufficiently predictive. Interestingly, time lags between the climatic event and the parasitological findings had to be implemented in all models. For T. gondii, North Atlantic Oscillation indices with a time lag of 7 months and temperature with a time lag of 2 months had the best predictive value. In contrast, temperature (with a time lag of 6 months) and the interaction of precipitation (with a time lag of 5 months) and North Atlantic Oscillation indices (with a time lag of 11 months) were optimal for predicting the seasonality of H. hammondi. These results suggest prominent differences in the life cycles of the two closely related parasites. Previous findings showed that H. hammondi lack avian hosts, in contrast to T. gondii, and the coincidence in the periods of high abundance of birds and high proportions of cats shedding T. gondii suggest that birds may play an important role in the epidemiology of this infection. The result that North Atlantic Oscillation index is an important variable in modelling variations in the proportion of cats shedding T. gondii and H. hammondi over the year is an indication that global warming may also influence the infection risk of animals and humans with T. gondii and H. hammondi. The findings have important implications for planning epidemiological studies and for estimating the risk of human infection. Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Incidence of single and mixed infections with Eimeria kofoidi, E. caucasica and E. legionensis on the health of experimentally infected red-legged partridges (Alectoris rufa).

PubMed

Naciri, M; Fort, G; Briant, J; Duperray, J; Benzoni, G

2014-09-15

Little is known about Eimeria-induced coccidiosis in partridges. After a coccidiosis outbreak in a farm rearing red-legged partridges (Alectoris rufa) in Brittany (France), three Eimeria species were identified as Eimeria kofoidi, Eimeria caucasica and Eimeria legionensis. This study aimed to reproduce the effects of the disease occurring in field conditions, in the absence of preventive treatments, to further build a coccidiosis model, helpful for coccidiostatic development. The pathogenic effects of a single infection with Eimeria kofoidi, E. caucasica and E. legionensis were evaluated, as well as the effects of multiple infections associating two or three of these species in red-legged partridges. Thirty-one-day-old birds were individually inoculated with Eimeria spp. and clinically followed up until 49 days of age. Mortality, lesion scores, daily oocyst production and growth were used as assessment criteria. Single infections with 250,000 E. kofoidi, 30,000 E. caucasica or 100,000 E. legionensis oocysts did not increase mortality rate compared to uninfected birds, whereas the combination of 3 species caused significant 28% mortality (P<0.05). Five days post inoculation, lesions scores in the proximal intestine (duodenum/jejunum) decreased in dual-infected birds and tended to decrease in triple-infected birds compared to lesions in mono-infected birds. The highest caecal lesion score was recorded in birds co-infected with E. kofoidi and E. legionensis. In multi-infected birds, the total number of oocysts excreted per gram of faeces was lower than the sum of oocysts excreted by mono-infected birds. Each single infection caused significant growth depression and even weight loss in E. legionensis infected birds (P<0.05). Dual or triple infections worsened this effect. Eighteen days post inoculation, only mono-infected birds had recovered. Their weight gains were not different from that of uninfected birds, whereas growth was significantly depressed in dual-infected birds, notably in co-infected E. kofoidi and E. legionensis birds and worsened in triple-infected birds (-34% and -78% growth depression, respectively). Our results indicate that the oocyst doses used were suitable in single infections or should be slightly reduced for future studies requiring significant growth retardation (about 25%). In mixed-infections, the effects on growth are considerably increased; therefore, oocyst doses must be reduced, approximately 125,000 E. kofoidi+15,000 E. caucasica+50,000 E. legionensis oocysts can be recommended. The study model presented here is valuable to conduct further research such as for assessing the ability of these birds to mount immune response against Eimeria spp. or for selecting efficient molecules to struggle coccidiosis of red-legged partridges. Copyright © 2014 Elsevier B.V. All rights reserved.

Systemic acute phase proteins response in calves experimentally infected with Eimeria zuernii.

PubMed

Lassen, Brian; Bangoura, Berit; Lepik, Triin; Orro, Toomas

2015-09-15

Acute phase proteins (APPs) have been demonstrated to be useful in evaluating general health stress and diseases in cattle. Serum amyloid A (SAA) and haptoglobin (Hp) are APPs that are produced during inflammation, and likely play a role in host immunological defence against Eimeria infection and the associated intestinal tissue damage. We investigated the involvement of SAA and HP in an experimental study, including three groups of calves: a control group (group 0, n=11), and two groups infected with either 150,000 or 250,000 Eimeria zuernii oocysts (group 1 (n=11) and group 2 (n=12), respectively). The calves were monitored for 28 days and data was collected on oocyst excretion, faecal score, animal weight, and SAA and Hp serum concentrations. Generalized linear mixed models showed that the clinical symptoms, indicated by an increase in the number of oocysts in the faeces and severe diarrhoea, manifested at patency for group 1 and 2. Serum Hp and SAA levels also increased during this period. Hp appeared to be a more sensitive marker than SAA, and differences between groups 1 and 2 were observed only for Hp. Linear regression models showed a negative association between weight gain and Hp concentrations, calculated as the area under the curve (AUC) during the overall experimental period and the patency period. A similar result was seen for SAA only during the patency period. This result supports the assumption that reduced weight gain due to E. zuernii infection is an immunologically driven process that involves an increase in APPs. A random intercept regression model of oocyst shedding groups showed that calves shedding 1-500 oocysts had reduced concentrations of Hp, indicating that a different immunological reaction occurs during mild shedding of E. zuernii oocysts than during more intensive shedding. A similar model was used to examine associations between faecal scores and Hp concentrations for each group. Group 2 calves with haemorrhagic diarrhoea displayed higher Hp levels than calves in that group with lower faecal scores, which may be in response to an increased demand for Hp in the repair process as a result of haemolysis. APPs seem to play an important role in determining the course of E. zuernii infection in calves, which may enhance our understanding of the immunological reaction and development of this disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Large-scale investigation of the parameters in response to Eimeria maxima challenge in broilers.

PubMed

Hamzic, E; Bed'Hom, B; Juin, H; Hawken, R; Abrahamsen, M S; Elsen, J M; Servin, B; Pinard-van der Laan, M H; Demeure, O

2015-04-01

Coccidiosis, a parasitic disease of the intestinal tract caused by members of the genera Eimeria and Isospora, is one of the most common and costly diseases in chicken. The aims of this study were to assess the effect of the challenge and level of variability of measured parameters in chickens during the challenge with Eimeria maxima. Furthermore, this study aimed to investigate which parameters are the most relevant indicators of the health status. Finally, the study also aimed to estimate accuracy of prediction for traits that cannot be measured on large scale (such as intestinal lesion score and fecal oocyst count) using parameters that can easily be measured on all animals. The study was performed in 2 parts: a pilot challenge on 240 animals followed by a large-scale challenge on 2,024 animals. In both experiments, animals were challenged with 50,000 Eimeria maxima oocysts at 16 d of age. In the pilot challenge, all animals were measured for BW gain, plasma coloration, hematocrit, and rectal temperature and, in addition, a subset of 48 animals was measured for oocyst count and the intestinal lesion score. All animals from the second challenge were measured for BW gain, plasma coloration, and hematocrit whereas a subset of 184 animals was measured for intestinal lesion score, fecal oocyst count, blood parameters, and plasma protein content and composition. Most of the parameters measured were significantly affected by the challenge. Lesion scores for duodenum and jejunum (P < 0.001), oocyst count (P < 0.05), plasma coloration for the optical density values between 450 and 490 nm (P < 0.001), albumin (P < 0.001), α1-globulin (P < 0.01), α2-globulin (P < 0.001), α3-globulin (P < 0.01), and β2-globulin (P < 0.001) were the most strongly affected parameters and expressed the greatest levels of variation. Plasma protein profiles proved to be a new, reliable parameter for measuring response to Eimeria maxima. Prediction of intestinal lesion score and fecal oocyst count using the other parameters measured was not very precise (R2 < 0.7). The study was successfully performed in real raising conditions on a large scale. Finally, we observed a high variability in response to the challenge, suggesting that broilers' response to Eimeria maxima has a strong genetic determinism, which may be improved by genetic selection.

Use of Artemisia annua as a natural coccidiostat in free-range broilers and its effects on infection dynamics and performance.

PubMed

de Almeida, Gustavo F; Horsted, Klaus; Thamsborg, Stig M; Kyvsgaard, Niels C; Ferreira, Jorge F S; Hermansen, John E

2012-05-25

This work investigated the preventive effect of Artemisia annua L. dried leaves supplied as a botanical coccidiostat to two broiler genotypes reared in a Danish free-range system in a factorial experiment (two genotypes and ± supplement of dried A. annua leaves). The genotypes White Bresse L40, a pure slow-growing line, and Kosmos 8 Ross, a hybrid genotype with medium growing characteristics, were used. Broilers were raised indoor until 29-days-old and kept free of parasites. Twelve groups of 30 randomly selected broilers were placed in the range forming three replicates for each treatment combination. The paddocks were cultivated with a mix of grass and clover. A separate group of broilers was naturally infected with Eimeria spp. oocysts and five animals nominated as "seeders" were introduced to the above mentioned 12 groups, 10 days after its formation, with each group consisting of 35 animals per plot. This infection strategy was meant to imitate the transmission pathway observed at farm level. Ten individual birds from each of the 12 groups, in total 120 animals of mixed sex, were monitored twice weekly for 30 days for oocysts excretion. PCR of pooled faecal samples, oocyst morphology and localization upon necropsy were used to identify the Eimeria species involved in the infection. In general, broilers from both genotypes in the range coped well with a coccidia infection caused by Eimeria acervulina and Eimeria maxima as no clinical symptoms, or deaths, were reported during the experiment. In general, broilers supplemented with A. annua dried leaves showed a significantly (p<0.05) reduced number of excreted oocysts during the infection with no interaction to genotype. Females generally had a significantly higher shedding of oocysts than males (p<0.05). The overall body weight gain and the daily weight gain when infection was subdued showed a three-way interaction among genotype, sex and treatment - accounted mainly for the fact that Kosmos females responded positively to the Artemisia treatment while Kosmos males responded negatively, and only minor differences were found between sexes for the White Bresse genotype. In conclusion, supply of A. annua dried leaves as a botanical coccidiostat significantly reduced oocyst output in free ranged broilers and thus may form part of a strategy to prevent commercial losses. Copyright © 2011 Elsevier B.V. All rights reserved.

Detection of Toxoplasma gondii oocysts in different water resources by Loop Mediated Isothermal Amplification (LAMP).

PubMed

Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Mahmoodi, Mohammad Reza; Karanis, Panagiotis

2013-02-01

Human toxoplasmosis is potentially contracted due to consumption of contaminated drinking water and represents an increasing public health risk worldwide. Toxoplasma gondii oocysts can be resistant to standard disinfection processes, including UV radiation. Increased awareness of the risk of waterborne toxoplasmosis outbreaks has led to an increase in research interest in the detection of oocysts in environmental water systems. Ninety-five environmental water samples from the Lower Rhine area in Germany have been included in the study and examined for the presence of Toxoplasma. Water samples were filtered or flocculated by aluminum sulfate and purified by sucrose density gradient. DNA was then extracted, and the DNA samples were then examined by LAMP analysis. T. gondii DNA was detected in eight out of 83 (9.6%) influent and effluent samples obtained from wastewater treatment plants. All samples (n=12) from the surface, ground, raw and tap waters tested negative. The purpose of this work was to investigate the occurrence and distribution of Toxoplasma oocysts on the Lower Rhine in Germany. Our study provides evidence that the assay is a sensitive, specific, rapid and cost effective method for the detection of T. gondii and is useful for both the investigations of cases of waterborne outbreaks and for identifying the source of contamination. Copyright © 2012 Elsevier B.V. All rights reserved.

Modified flotation method with the use of Percoll for the detection of Isospora suis oocysts in suckling piglet faeces.

PubMed

Karamon, Jacek; Ziomko, Irena; Cencek, Tomasz; Sroka, Jacek

2008-10-01

The modification of flotation method for the examination of diarrhoeic piglet faeces for the detection of Isospora suis oocysts was elaborated. The method was based on removing fractions of fat from the sample of faeces by centrifugation with a 25% Percoll solution. The investigations were carried out in comparison to the McMaster method. From five variants of the Percoll flotation method, the best results were obtained when 2ml of flotation liquid per 1g of faeces were used. The limit of detection in the Percoll flotation method was 160 oocysts per 1g, and was better than with the McMaster method. The efficacy of the modified method was confirmed by results obtained in the examination of the I. suis infected piglets. From all faecal samples, positive samples in the Percoll flotation method were double the results than that of the routine method. Oocysts were first detected by the Percoll flotation method on day 4 post-invasion, i.e. one-day earlier than with the McMaster method. During the experiment (except for 3 days), the extensity of I. suis invasion in the litter examined by the Percoll flotation method was higher than that with the McMaster method. The obtained results show that the modified flotation method with the use of Percoll could be applied in the diagnostics of suckling piglet isosporosis.

Guar meal ameliorates Eimeria tenella infection in broiler chicks.

PubMed

Hassan, Sherif M; El-Gayar, Amal K; Cadwell, David J; Bailey, Christopher A; Cartwright, Aubrey L

2008-10-20

Guar meal contains relatively high levels of saponins, which are known to have antiprotozoal activity and may be effective against coccidiosis. A 2x2 factorial experiment investigated the impact of guar meal (0 or 5%) corn-soy-based starter broiler diets on chicks unchallenged or challenged with Eimeria tenella. At 1 day of age, 120 unsexed RossxRoss broiler chicks were randomly distributed among four treatment groups. Chicks were challenged with 5x10(3) sporulated oocysts of E. tenella in 0.5 ml at 10 days of age by oral gavage. Weekly body weight, body weight gains, feed conversion ratio and mortality rate were recorded for chicks fed from 0 to 21 days of age. Oocysts shed per gram feces were recorded from 6 to 10 days post-challenge. Results showed that challenged chicks fed 0% guar meal had significantly higher oocysts per gram shed in feces than the other groups. No significant differences among treatment groups in mortality rate were observed. Body weights of unchallenged and challenged chicks fed 0% guar meal were significantly higher than those fed 5% guar meal at 2 weeks of age. Results indicated that including 5% guar meal in the diet of chicks challenged with E. tenella decreased oocysts shed per gram feces and prevented bloody diarrhea, but without affects on body weight and feed conversion ratio at 11 days post-challenge.

[Investigation on contamination of Giardia and Cryptosporidium in drinking water in Jiangsu Province].

PubMed

Bi-Xian, N I; Ming-Xue, S; Xiang-Zhen, X U; Xiao-Ting, W; Yang, D; Xiao-Lin, J

2017-05-17

Objective To know the contamination status of Giardia lamblia and Cryptosporidium in drinking water of Jiangsu Province, so as to provide the evidence for producing hygiene and safety drinking water. Methods A total of 28 water plants of 13 cities in Jiangsu Province were selected, and the source water (10 L), chlorinated water (100 L) and tap water (100 L) were collected separately in each site. The water samples were then treated by filtration, washing, centrifuging concentration, immune magnetic separation, and immunofluorescent assay, to detect the numbers of Giardia cysts and Cryptosporidium oocysts. Results Totally 84 samples from 13 cities were collected, including 28 source water, 28 chlorinated water and 28 tap water samples. Among the chlorinated water and tap water samples, no Giardia cysts and Cryptosporidium oocysts were found. However, Giardia cysts were detected in 3 (10.71%, 3/28) source water samples (Yancheng, Lianyungang, Changzhou cities), with the density of 1 cyst/10 L of all. Cryptosporidium oocysts were also detected in 3 (10.71%, 3/28) source water samples (Nanjing, Zhenjiang, Yangzhou cities), with the density of 1 oocyst/10 L of all. Conclusions The source water in partial areas of Jiangsu Province has been contaminated by Giardia and Cryptosporidium . To ensure the safety of drinking, the regulation of source water and surveillance of drinking water should be strengthened.

Genotypic Characterization of Cryptosporidium hominis from Water Samples in São Paulo, Brazil

PubMed Central

Araújo, Ronalda S.; Dropa, Milena; Fernandes, Licia N.; Carvalho, Terezinha T.; Sato, Maria Inês Z.; Soares, Rodrigo M.; Matté, Glavur R.; Matté, Maria Helena

2011-01-01

The protozoan parasite Cryptosporidium has emerged as one of the most important water contaminants, causing waterborne outbreaks of diarrheal diseases worldwide. The small size of oocysts under the microscope and the possibility of changes in characteristics of oocysts, mainly in environmental samples, make the taxonomy of the genus difficult if morphologic characteristics are considered. This limitation encouraged the application of molecular methods to identify this microorganism. The aim of this study was to detect and identify by nested-polymerase chain reaction oocysts of Cryptosporidium present in water samples in the state of São Paulo, Brazil. Water samples were concentrated through a membrane filter, DNA was extracted by using a standard technique, and both amplification reactions used forward and reverse oligonucleotides that were complementary to Cryptosporidium 18S ribosomal RNA gene sequences. Thirty water samples from different sites of collection in the state of São Paulo were evaluated. Cryptosporidium oocysts were detected in 30% of the samples. By genoptyping, C. hominis and Cryptosporidium sp. were identified in recreational water and C. meleagridis was identified in surface water samples. This is the first report of C. hominis in environmental samples in Brazil. Although identification of Cryptosporidium is still a difficult task, molecular methods are essential for specific identification and are a helpful tool to aid to understand the epidemiology of this parasite in Brazil. PMID:22049036

Impact of confinement housing on study end-points in the calf model of cryptosporidiosis.

PubMed

Graef, Geneva; Hurst, Natalie J; Kidder, Lance; Sy, Tracy L; Goodman, Laura B; Preston, Whitney D; Arnold, Samuel L M; Zambriski, Jennifer A

2018-04-01

Diarrhea is the second leading cause of death in children < 5 years globally and the parasite genus Cryptosporidium is a leading cause of that diarrhea. The global disease burden attributable to cryptosporidiosis is substantial and the only approved chemotherapeutic, nitazoxanide, has poor efficacy in HIV positive children. Chemotherapeutic development is dependent on the calf model of cryptosporidiosis, which is the best approximation of human disease. However, the model is not consistently applied across research studies. Data collection commonly occurs using two different methods: Complete Fecal Collection (CFC), which requires use of confinement housing, and Interval Collection (IC), which permits use of box stalls. CFC mimics human challenge model methodology but it is unknown if confinement housing impacts study end-points and if data gathered via this method is suitable for generalization to human populations. Using a modified crossover study design we compared CFC and IC and evaluated the impact of housing on study end-points. At birth, calves were randomly assigned to confinement (n = 14) or box stall housing (n = 9), or were challenged with 5 x 107 C. parvum oocysts, and followed for 10 days. Study end-points included fecal oocyst shedding, severity of diarrhea, degree of dehydration, and plasma cortisol. Calves in confinement had no significant differences in mean log oocysts enumerated per gram of fecal dry matter between CFC and IC samples (P = 0.6), nor were there diurnal variations in oocyst shedding (P = 0.1). Confinement housed calves shed significantly more oocysts (P = 0.05), had higher plasma cortisol (P = 0.001), and required more supportive care (P = 0.0009) than calves in box stalls. Housing method confounds study end-points in the calf model of cryptosporidiosis. Due to increased stress data collected from calves in confinement housing may not accurately estimate the efficacy of chemotherapeutics targeting C. parvum.

Sequence-based genotyping clarifies conflicting historical morphometric and biological data for 5 Eimeria species infecting turkeys.

PubMed

El-Sherry, S; Ogedengbe, M E; Hafeez, M A; Sayf-Al-Din, M; Gad, N; Barta, J R

2015-02-01

Unlike with Eimeria species infecting chickens, specific identification and nomenclature of Eimeria species infecting turkeys is complicated, and in the absence of molecular data, imprecise. In an attempt to reconcile contradictory data reported on oocyst morphometrics and biological descriptions of various Eimeria species infecting turkey, we established single oocyst derived lines of 5 important Eimeria species infecting turkeys, Eimeria meleagrimitis (USMN08-01 strain), Eimeria adenoeides (Guelph strain), Eimeria gallopavonis (Weybridge strain), Eimeria meleagridis (USAR97-01 strain), and Eimeria dispersa (Briston strain). Short portions (514 bp) of mitochondrial cytochrome c oxidase subunit I gene (mt COI) from each were amplified and sequenced. Comparison of these sequences showed sufficient species-specific sequence variation to recommend these short mt COI sequences as species-specific markers. Uniformity of oocyst features (dimensions and oocyst structure) of each pure line was observed. Additional morphological features of the oocysts of these species are described as useful for the microscopic differentiation of these Eimeria species. Combined molecular and morphometric data on these single species lines compared with the original species descriptions and more recent data have helped to clarify some confusing, and sometimes conflicting, features associated with these Eimeria spp. For example, these new data suggest that the KCH and KR strains of E. adenoeides reported previously represent 2 distinct species, E. adenoeides and E. meleagridis, respectively. Likewise, analysis of the Weybridge strain of E. adenoeides, which has long been used as a reference strain in various studies conducted on the pathogenicity of E. adenoeides, indicates that this coccidium is actually a strain of E. gallopavonis. We highly recommend mt COI sequence-based genotyping be incorporated into all studies using Eimeria spp. of turkeys to confirm species identifications and so that any resulting data can be associated correctly with a single named Eimeria species. © 2015 Poultry Science Association Inc.

Toxoplasma gondii: infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts.

PubMed

Costa, Gustavo Henrique Nogueira; da Costa, Alvimar José; Lopes, Welber Daniel Zanetti; Bresciani, Katia Denise Saraiva; dos Santos, Thais Rabelo; Esper, César Roberto; Santana, Aureo Evangelista

2011-01-01

Two studies, of a natural infection and an experimental infection, were performed in order to study congenital transmission of Toxoplasma gondii in cattle. In the first study, 50 fetuses were harvested from gestating cows that were eutanasied at a municipal slaughterhouse in Jaboticabal, São Paulo state, Brazil. In the second study, 11 gestating cows were divided into four groups for inoculation with T. gondii: GI consisted of three cows inoculated with 1.0 × 10(5) oocysts during their first trimester of gestation; GII consisted of three cows inoculated with 1.0 × 10(5) oocysts during their second trimester of gestation; GIII consisted of three cows inoculated with 1.0 × 10(5) oocysts during their last trimester of gestation; and GIV consisted of two control cows, one during its first and the other during its second trimester of gestation. In both studies, the presence of T. gondii was confirmed both indirectly by immunofluorescence assay (IFAT). In the natural infection experiment, 18% (9/50) of the gestating cows were confirmed to have specific antibodies (IFAT--1:64) against T. gondii. The bioassay was able to diagnose the presence of T. gondii in the tissue samples from three calves. In the second experiment, the nine cows from groups I, II and III presented with specific antibodies (IFAT) against T. gondii. In contrast, T. gondii could not be detected by IFAT, histopathological examination or the bioassay in any of the nine calves born to cows experimentally infected with T. gondii oocysts. Based on the results from both studies, we conclude that congenital infection of T. gondii in cattle, while infrequent, does occur naturally. The pathogenicity of the strain of T. gondii may influence the likelihood of this route of transmission. Copyright © 2010 Elsevier Inc. All rights reserved.

[Clinical epidemiology of cryptosporidiosis in calves].

PubMed

Weber, S E; Lippuner, C; Corti, S; Deplazes, P; Hässig, M

2016-05-01

The aim of this study was to determine whether there is an association between Cryptosporidium infections in calves and immunological factors, as well as farm-related factors or the application of the anti-cryptosporidiosis drug Halofuginone. From January to June 2010, 63 cow-calf-pairs from 20 different farms near Zürich, Switzerland have been investigated. Each cowcalf- pair was visited three times within the first 6 weeks of life to collect data of the farm and animals, as well as blood, faecal, colostral and milk samples. An ELISA using sporozoite antigen was developed for the specific detection of anti-Cryptosporidium-IgG in blood- and colostral serum. The IgG concentration in the bloodand colostral serum was determined using radial immuno diffusion test (RID). White blood cell isolation and differential blood cell counts and California Mastitis Test were performed. Bacteriological studies on quarter-milk-samples were carried out. Cryptosporidium oocysts were diagnosed with the modified Ziehl-Neelsen staining, other protozoa with the SAFC method and Eimeria oocysts and helminth eggs were diagnosed with the combined sedimentation/floatation test. ELISAs were performed for the detection of rota- and coronavirus, E. coli F5 and Cryptosporidium spp. in bovine feces (bio-X Diagnostics®, Belgium). The highest prevalence of Cryptosporidium oocysts was 54.0% and found 7 to 20 days post natum, whereas 47.1% were suffering from diarrhea. The transfer of total IgG with the colostrum and the humoral immunity of the calf could not prevent any infection with Cryptosporidium, but the severity of the diarrhea symptoms decreased with increasing total IgG concentrations. Calves housed in open sheds showed significantly more often diarrhea, i. e. they shed more Cryptosporidium oocysts during the first 4 days and 7 to 20 days post natum, respectively. Halofuginone (Halocur®) is approved for prophylaxis against cryptosporidiosis, but it showed no effect on the excretion of Cryptosporidium oocysts in the present study.

[Measuring resistant forms of two pathogenic protozoa (Giardia spp and Cryptosporidium spp) in two aquatic biotopes in Yaoundé (Cameroon)].

PubMed

Gideon, Aghaindum Ajeagah; Njiné, Thomas; Nola, Moïse; Menbohan, Samuel Foto; Ndayo, Marguerite Wouafo

2007-01-01

Organisms of the genera Cryptosporidium and Giardia are enteric parasites that are responsible for cryptosporidiosis and giardiasis respectively. They are characterised by profuse diarrhoea, abdominal pain and cramping. The symptoms can be fatal for immunodepressed subjects and children because these are opportunistic pathogens. These pathogenic infections are rampant in developing countries due to the unhygienic and improper disposal of wastewater and the use of surface water as the major sources of potable water. Little information is available about the resistant forms of these microorganisms in the hydrosystems of Cameroon in particular and other sub-Saharan ecosystems in general. The aim of this research was to isolate, identify, and measure these forms of these emerging protozoa in the waste and surface waters of Yaoundé, Cameroon. Giardia spp cysts were identified by Lugol iodine staining, and the Cryptosporidium spp oocysts by the Ziehl-Neelsen technique. The cysts were oval in structure and varied in length from 7 um to 12.0 um. Axostyles were visible along the median line and nuclei were present. The oocysts were round in structure and varied in diameter from 4 um to 6 um. A double cell wall was seen in the apicomplexa. The highest concentrations of Giardia spp cysts were 560 cysts/L in the surface water and 650 cysts/L in the residual effluent, while the corresponding figures for Cryptosporidium spp were 1,110 oocysts/L in the stream and 1,500 oocysts/L in the effluent. The cyst and oocyst counts were higher in the direct effluent because they are released directly into the wastewater by feces, while they are diluted in the surface water, reducing their abundance. Turbidity has a significant effect (p<0.05) on the population dynamics of these microorganisms, thereby indicating the importance of suspended or colloidal particles in the dissemination of these pathogens in aquatic media. The suspended particles in the wastewater adsorb the oocysts and cysts, probably because of the interactions between the electric charges carried by suspended particles and those present at the surface of the parasites. The positive correlation observed between these parasites and ammonia may account for some of the die-off of these resistant forms, because ammonium ions in water are known to inactivate trophozoites and sporozoites present in cysts and oocysts. This may be due to the permeability of the protozoan membrane to the ammonium ion and other alkaline earth metals present in aqueous form in the biotope. These parasite forms in these media represent a considerable health risk to the inhabitants of the study zone who depend on this surface water for drinking. The small size of these parasitic forms, the nonfunctional water purification stations and ineffective water treatment systems expose the community to outbreaks of giardiasis and cryptosporidiosis.

A Comparative Analysis of Coprologic Diagnostic Methods for Detection of Toxoplama gondii in Cats

PubMed Central

Salant, Harold; Spira, Dan T.; Hamburger, Joseph

2010-01-01

The relative role of transmission of Toxoplasma gondii infection from cats to humans appears to have recently increased in certain areas. Large-scale screening of oocyst shedding in cats cannot rely on microscopy because oocyst identification lacks sensitivity and specificity, or on bioassays, which require test animals and weeks before examination. We compared a sensitive and species-specific coprologic–polymerase chain reaction (copro-PCR) for detection of T. gondii infected cats with microscopy and a bioassay. In experimentally infected cats followed over time, microscopy was positive occasionally, and positive copro-PCR and bioassay results were obtained continuously from days 2 to 24 post-infection. The copro-PCR is at least as sensitive and specific as the bioassay and is capable of detecting infective oocysts during cat infection. Therefore, this procedure can be used as the new gold standard for determining potential cat infectivity. Its technologic advantages over the bioassay make it superior for large-scale screening of cats. PMID:20439968

Temperature alters Plasmodium blocking by Wolbachia

NASA Astrophysics Data System (ADS)

Murdock, Courtney C.; Blanford, Simon; Hughes, Grant L.; Rasgon, Jason L.; Thomas, Matthew B.

2014-02-01

Very recently, the Asian malaria vector (Anopheles stephensi) was stably transinfected with the wAlbB strain of Wolbachia, inducing refractoriness to the human malaria parasite Plasmodium falciparum. However, conditions in the field can differ substantially from those in the laboratory. We use the rodent malaria P. yoelii, and somatically transinfected An. stephensi as a model system to investigate whether the transmission blocking potential of wAlbB is likely to be robust across different thermal environments. wAlbB reduced malaria parasite prevalence and oocyst intensity at 28°C. At 24°C there was no effect on prevalence but a marked increase in oocyst intensity. At 20°C, wAlbB had no effect on prevalence or intensity. Additionally, we identified a novel effect of wAlbB that resulted in reduced sporozoite development across temperatures, counterbalancing the oocyst enhancement at 24°C. Our results demonstrate complex effects of temperature on the Wolbachia-malaria interaction, and suggest the impacts of transinfection might vary across diverse environments.

Removal of Giardia and Cryptosporidium in drinking water treatment: a pilot-scale study.

PubMed

Hsu, Bing Mu; Yeh, Hsuan Hsien

2003-03-01

Giardia and Cryptosporidium have emerged as waterborne pathogens of concern for public health. The aim of this study is to examine both parasites in the water samples taken from three pilot-scale plant processes located in southern Taiwan, to upgrade the current facilities. Three processes include: conventional process without prechlorination (Process 1), conventional process plus ozonation and pellet softening (Process 2), and integrated membrane process (MF plus NF) followed conventional process (Process 3). The detection methods of both parasites are modified from USEPA Methods 1622 and 1623. Results indicated that coagulation, sedimentation and filtration removed the most percentage of both protozoan parasites. The pre-ozonation step can destruct both parasites, especially for Giardia cysts. The microfiltration systems can intercept Giardia cysts and Cryptosporidium oocysts completely. A significant correlation between water turbidity and Cryptosporidium oocysts was found in this study. The similar results were also found between three kinds of particles (phi=3-5,5-8 and 8-10 microm) and Cryptosporidium oocysts.

[The effect of betain on biology and morphology of developmental stages of Eimeria acervulina in broiler chicks experimentally infected].

PubMed

Teixeira, Marcel; Niang, Tania Marcia S; Gomes, Augusto V da C; Lopes, Carlos Wilson G

2006-01-01

Purposing to investigate the betaine effect on biology and morphology of developmental stages of Eimeria acervulina, 420 broiler chicks Cobb were experimentally inoculated with 2 x 10(5) sporulated oocysts and housed in battery cages in a block design with five treatments and six replicates each, including a positive control, a group treated with salinomycin and growth promoter plus three levels of betaine as additive in the feed at 0.05, 0.10 and 0.15%. Measurements of oocysts, sporocysts and endogenous stages were performed as morphological parameters, while pre patent and patent periods and sporulation time were taken as biological parameters. Morphology was also associated with the mathematical constant Phi (1.618) to evaluate possible relationship. Betaine was able to cause modifications in both biology and morphology of oocysts and sporocysts, whereas it was weakly able to affect developmental stages based on trophozoites and macrogamonts measurements. According to the measures of sporocysts E. acervulina development was closely related to Phi.

Effect of irradiation on the viability of Toxoplasma gondii cysts in tissues of mice and pigs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dubey, J.P.; Brake, R.J.; Murrell, K.D.

1986-03-01

Muscles from tongue, heart, and limbs of 14 pigs inoculated orally with Toxoplasma gondii oocysts were irradiated with 10, 20, 25, and 30 krad of gamma (cesium-137 and cobalt-60) irradiation. Viability of T gondii cysts was assayed by feeding porcine muscles to T gondii-free cats and/or by inoculation of sediment from acid-pepsin digested porcine muscle into mice. Cats fed 500-g samples of muscles irradiated with up to 20 krad shed T gondii oocysts. Cats fed muscles irradiated with 25 or 30 krad did not shed oocysts. Mice were inoculated with 8 isolates of T gondii, and tissue cysts in theirmore » brains irradiated with up to 40 krad were infective to mice; however, there was a 10,000-fold reduction in the viability of organisms in tissue cysts irradiated with 40 krad, compared with that in nonirradiated cysts. At 50 krad of gamma irradiation, there were no detectable infective organisms in infected mouse brains.« less

Studies on the resistance/reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation.

PubMed

Belosevic, M; Craik, S A; Stafford, J L; Neumann, N F; Kruithof, J; Smith, D W

2001-10-16

The ex vivo and in vivo reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts after exposure to different doses of ultraviolet (UV) radiation was determined using animal infectivity. The infectivity of UV-treated parasites stored for 1-4 days (G. muris) or 1-17 days (C. parvum) at room temperature in the dark was similar to that of organisms administered immediately after UV treatment, indicating that the parasites did not reactivate ex vivo. In contrast, we observed in vivo reactivation of G. muris in three of seven independent animal infectivity experiments, when parasites were treated with relatively low doses of medium-pressure UV (<25 mJ/cm(2)). Our observations indicate that G. muris cysts and C. parvum oocysts exposed to medium-pressure UV doses of 60 mJ/cm(2) or higher did not exhibit resistance to and/or reactivation following treatment. This suggests that when appropriate doses of UV are used, significant and permanent inactivation of these parasites may be achieved.

Investigating seagrass in Toxoplasma gondii transmission in Florida (Trichechus manatus latirostris) and Antillean (T. m. manatus) manatees.

PubMed

Wyrosdick, Heidi M; Gerhold, Richard; Su, Chunlei; Mignucci-Giannoni, Antonio A; Bonde, Robert K; Chapman, Alycia; Rivera-Pérez, Carla I; Martinez, Jessica; Miller, Debra L

2017-12-19

Toxoplasma gondii is a feline protozoan reported to cause morbidity and mortality in manatees and other marine mammals. Given the herbivorous nature of manatees, ingestion of oocysts from contaminated water or seagrass is presumed to be their primary mode of infection. The objectives of this study were to investigate oocyst contamination of seagrass beds in Puerto Rico and determine the seroprevalence of T. gondii in Antillean (Trichechus manatus manatus) and Florida (T. m. latirostris) manatees. Sera or plasma from Antillean (n = 5) and Florida (n = 351) manatees were tested for T. gondii antibodies using the modified agglutination test. No T. gondii DNA was detected via PCR in seagrass samples (n = 33) collected from Puerto Rico. Seroprevalence was 0%, suggesting a lower prevalence of T. gondii in these manatee populations than previously reported. This was the first study to investigate the potential oocyst contamination of the manatee diet, and similar studies are important for understanding the epidemiology of T. gondii in herbivorous marine mammals.

Prevalence and burden of gastrointestinal parasites in cattle and buffaloes in Jabalpur, India.

PubMed

Marskole, Priyanka; Verma, Yamini; Dixit, Alok Kumar; Swamy, Madhu

2016-11-01

The study was conducted to determine the prevalence and burden of gastrointestinal (GI) parasites in cattle and buffaloes of Jabalpur, Madhya Pradesh. The presence of helminths eggs and coccidial oocysts in fecal samples were detected using standard qualitative and quantitative methods. Identification of eggs or oocysts was done on the basis of morphology and size of the eggs or oocysts. Out of 120 cattle and buffaloes examined, 73.33% were found positive for eggs of one or more species of GI parasite. The prevalence of parasitic infection was higher in cattle (75%) as compared to that of buffaloes (70.45%), but the difference was nonsignificant (p>0.05). Sex wise prevalence of GI parasites was higher in males as compared to that of females, but the difference was nonsignificant (p>0.05). The animals above 2 years of age were more affected by GI parasites as compared to animals of 6 months - 2 years of age, but the age wise differences were nonsignificant (p>0.05). Single parasitic infections were more common than mixed infections. The monthly prevalence of GI parasites in cattle and buffaloes were highest in the month of September (81.81%) and least in December (61.11%). The eggs/oocysts per gram in most of the animals, was in the range of 201-300. GI parasites are problem in cattle and buffaloes of Jabalpur, Madhya Pradesh. The prevalence rate of GI parasites varied with month. The burden of parasitic infection was moderate in most animals warranting treatment.

Concentration and retention of Toxoplasma gondii oocysts by marine snails demonstrate a novel mechanism for transmission of terrestrial zoonotic pathogens in coastal ecosystems

USGS Publications Warehouse

Krusor, Colin; Smith, Woutrina A.; Tinker, M. Tim; Silver, Mary; Conrad, Patricia A.; Shapiro, Karen

2015-01-01

The parasite Toxoplasma gondii is an environmentally persistent pathogen that can cause fatal disease in humans, terrestrial warm-blooded animals and aquatic mammals. Although an association between T. gondii exposure and prey specialization on marine snails was identified in threatened California sea otters, the ability of kelp-dwelling snails to transmit terrestrially derived pathogens has not been previously investigated. The objective of this study was to measure concentration and retention of T. gondii by marine snails in laboratory aquaria, and to test for natural T. gondii contamination in field-collected snails. Following exposure to T. gondii-containing seawater, oocysts were detected by microscopy in snail faeces and tissues for 10 and 3 days respectively. Nested polymerase chain reaction was also applied as a method for confirming putative T. gondii oocysts detected in snail faeces and tissues by microscopy. Toxoplasma gondiiwas not detected in field-collected snails. Results suggest that turban snails are competent transport hosts for T. gondii. By concentrating oocysts in faecal pellets, snails may facilitate entry of T. gondii into the nearshore marine food web. This novel mechanism also represents a general pathway by which marine transmission of terrestrially derived microorganisms can be mediated via pathogen concentration and retention by benthic invertebrates.

Excystation of Eimeria tenella Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56▿

PubMed Central

Krücken, Jürgen; Hosse, Ralf J.; Mouafo, Aimdip N.; Entzeroth, Rolf; Bierbaum, Stefan; Marinovski, Predrag; Hain, Karolina; Greif, Gisela; Wunderlich, Frank

2008-01-01

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins. PMID:18083827

Excystation of Eimeria tenella sporozoites impaired by antibody recognizing gametocyte/oocyst antigens GAM22 and GAM56.

PubMed

Krücken, Jürgen; Hosse, Ralf J; Mouafo, Aimdip N; Entzeroth, Rolf; Bierbaum, Stefan; Marinovski, Predrag; Hain, Karolina; Greif, Gisela; Wunderlich, Frank

2008-02-01

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.

Cryptosporidium and Giardia safety margin increase in leafy green vegetables irrigated with treated wastewater.

PubMed

Domenech, Eva; Amorós, Inmaculada; Moreno, Yolanda; Alonso, José L

2018-01-01

The presence of Cryptosporidium and Giardia in waste water is a main concern because water reuse for irrigation can jeopardize human health. Spanish Legislation for water reuse does not oblige to analyze the presence of both pathogens Cryptosporidium and Giardia in reused water for irrigation. Therefore, the objective of this paper is to determine the influence of wastewater treatment in the increase of the consumer safety margin in relation to the presence of Cryptosporidium and Giardia in leafy green vegetables. With this aim in mind, a total of 108 samples from raw (influent) and treated wastewater (effluent) from three wastewater treatment plants in Spain were analysed according to USEPA Method 1623. Effluent results show that Cryptosporidium oocysts average counts ranged from 1.38 to 2.6/L oocysts and Giardia cysts ranged from 0.6 to 1.7/L cysts, which means a removal values of 2.7 log, 2.5 log and 1.8 log for Cryptosporidium oocysts and 1 log, 2 log and 2.2 log for Giardia cysts in the three wastewater treatment plants analysed. In relation to safety margin the highest probability that exposure exceed the dose response was observed for Giardia. In addition, the sensitivity analysis showed that (oo)cysts concentration present in the leafy green vegetables and the human dose-response were the most influential inputs in the safety margin obtained. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

[Detection of Cryptosporidium infection among HIV/AIDS patients with chronic diarrhea in Beijing, Henan and Xinjiang of China].

PubMed

Wang, Hui-zhu; Jiao, Bing-xin; Tian, Jing-hua; Li, Min; Guo, Jie; Liu, Ying; Li, Xing-wang; Wang, Yu-guang

2011-09-01

To investigate the Cryptosporidium infection and its epidemiological characteristics in HIV/AIDS patients with chronic diarrhea. Stool samples collected from HIV/AIDS confirmed patients with chronic diarrhea who lived in Beijing, Henan and Xinjiang. Samples were concentrated by Formalin-Ethyl Acetate Sedimentation technique and stained by modified acid-fast stain (AFS) for the identification of oocysts by microscopy. CD4(+)T cells count was performed by Flow Cytometry. The overall infection rate of Cryptosporidium in AIDS patients was 12.6% (32/253). The infection rates of oocysts in the area of Beijing, Henan and Xinjiang were 5.97% (4/67), 16.1% (24/149) and 10.8% (4/37) respectively. The infection rate of oocysts in the urban areas was 6.5% (7/104) while in the countryside it was 16.8% (25/149) and the difference was significantly different. However, there were no any differences discovered between the infection rates on patient's gender or on infection occurred in different seasons. The infectious rates of oocyst in patients on different stages of the disease were also significantly different (P < 0.01). AIDS patients infected by Cryptosporidium were not rarely seen in northern China. The rate of infection was not associated with patient's gender but was associated with patient's living environments. Patients living in the countryside, with lower lever of CD4(+)T cells counts and at the middle/late stage of the disease, Cryptosporidium infection appeared to be high.

Clinical, serological, and parasitological analysis of snakes naturally infected with Cryptosporidium serpentis.

PubMed

Paiva, Philipp Ricardo S O; Grego, Kathleen F; Lima, Valéria M F; Nakamura, Alex A; da Silva, Deuvânia C; Meireles, Marcelo V

2013-11-15

Infection by Cryptosporidium serpentis is one of the most important diseases in reptiles and is characterized by chronic clinical or subclinical infection and the presence of hypertrophic gastritis, food regurgitation, progressive weight loss, mortality, and intermittent or continuous shedding of oocysts in the feces. The objectives of this study were to standardize an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against C. serpentis and to evaluate the clinical, parasitological, and humoral immune response in snakes naturally infected with C. serpentis. Twenty-one snakes naturally infected with C. serpentis and housed at the Butantan Institute, São Paulo, Brazil, underwent clinical and parasitological analyses for C. serpentis infection through daily records of clinical signs and a monthly survey of fecal shedding of oocysts using the Kinyoun's acid-fast staining. The serological evaluation was performed monthly by indirect ELISA using crude total antigen from oocysts of C. serpentis to detect anti-C. serpentis antibodies. Clinical symptoms consisted of food regurgitation, inappetence, and progressive weight loss. The parasitological analysis revealed intermittent fecal shedding of a variable number of oocysts in all snakes, with positivity in 85.32% (157/184) of the samples. The indirect ELISA was positive in 68.25% (86/126) of the samples. A humoral immune response was observed in most animals; however, fluctuating antibodies levels, leading to alternating positive and negative results, were observed in most snakes. Copyright © 2013 Elsevier B.V. All rights reserved.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

PubMed Central

Rhodes, Eric R.; Villegas, Leah Fohl; Shaw, Nancy J.; Miller, Carrie; Villegas, Eric N.

2012-01-01

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy. Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocystsand Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 . This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms. PMID:22805201

Comparison of field-based xenodiagnosis and direct membrane feeding assays for evaluating host infectiousness to malaria vector Anopheles gambiae.

PubMed

Gouagna, Louis Clément; Yao, Frank; Yameogo, Bienvenue; Dabiré, Roch K; Ouédraogo, Jean-Bosco

2014-02-01

Several techniques are currently being used to study host infectiousness to mosquitoes, including the experimental possibility of laboratory reared mosquitoes acquiring infections through membrane feeders or directly on host skin. Here, the relative performance of the laboratory-based membrane feeding method (DMFA) and the field-based xenodiagnosis (XD) of malaria infectious hosts using wild Anopheles mosquitoes were compared. A cross-sectional survey involving a sample of 70 children (aged 3-12 years) living in a malaria endemic area in Western Burkina Faso, was carried out to measure their infectiousness to Anopheles mosquitoes using two approaches. The first approach used the xenodiagnostic procedure in which children were exposed to mosquito bites overnight, being sleeping individually in different sentinel huts from 6 pm to 6 am (4 nights per child). Anopheles sp that had acquired blood-meal on each child were subsequently collected early in the morning, and examined for Plasmodium falciparum oocyst infection on day 7 post-feeding. In the second approach, the infectiousness of the same children was estimated by whole-blood membrane feeding procedure using F0 An. gambiae s.l. that emerged from field-collected larvae cohorts. In the DMFA, 41.4% of the children successfully infected at least one mosquito with the mean oocyst prevalence of only 4.6±1.1% in the 2171 mosquitoes that were examined (mean oocyst intensity: 2.0±(std error of mean) 0.3 oocysts per infected midgut). Comparatively 78.6% of children yielded oocysts infection in mosquitoes during the XD approach (Chi square=20.11, df=1; p<0.001), with a mean rate of 19.6±2.0 in the 3752 wild caught mosquitoes (mean intensity: 3.93±0.2 oocysts per infected mosquito). The DMFA failed to reveal a portion (n=26) of infectious individuals that were sharply evidenced by the XD, particularly at low gametocyte densities or at levels that could not be detected by the classical microscopic examination of blood smears. As opposed to the resource consuming DMFA, which is often mined by technical constraints, using the XD method could be an advantage in experimental investigations of host infectiousness in areas where anopheline species cannot be conveniently reared for the experimental studies. Ethical aspects of this approach, mainly related to exposure of the human subjects to potentially infectious mosquito bites are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of sediment-associated extractable metals, degree of sediment grain sorting, and dissolved organic carbon upon Cryptosporidium parvum removal and transport within riverbank filtration sediments, Sonoma County, California

USGS Publications Warehouse

Metge, D.W.; Harvey, R.W.; Aiken, G.R.; Anders, R.; Lincoln, G.; Jasperse, James; Hill, M.C.

2011-01-01

Oocysts of the protozoan pathogen Cryptosporidium parvum are of particular concern for riverbank filtration (RBF) operations because of their persistence, ubiquity, and resistance to chlorine disinfection. At the Russian River RBF site (Sonoma County, CA), transport of C. parvumoocysts and oocyst-sized (3 μm) carboxylate-modified microspheres through poorly sorted (sorting indices, σ1, up to 3.0) and geochemically heterogeneous sediments collected between 2 and 25 m below land surface (bls) were assessed. Removal was highly sensitive to variations in both the quantity of extractable metals (mainly Fe and Al) and degree of grain sorting. In flow-through columns, there was a log–linear relationship (r2 = 0.82 at p < 0.002) between collision efficiency (α, the probability that colloidal collisions with grain surfaces would result in attachment) and extractable metals, and a linear relationship (r2 = 0.99 at p < 0.002) between α and σ1. Collectively, variability in extractable metals and grain sorting accounted for ∼83% of the variability in α (at p < 0.0002) along the depth profiles. Amendments of 2.2 mg L–1 of Russian River dissolved organic carbon (DOC) reduced α for oocysts by 4–5 fold. The highly reactive hydrophobic organic acid (HPOA) fraction was particularly effective in re-entraining sediment-attached microspheres. However, the transport-enhancing effects of the riverine DOC did not appear to penetrate very deeply into the underlying sediments, judging from high α values (∼1.0) observed for oocysts being advected through unamended sediments collected at ∼2 m bls. This study suggests that in evaluating the efficacy of RBF operations to remove oocysts, it may be necessary to consider not only the geochemical nature and size distribution of the sediment grains, but also the degrees of sediment sorting and the concentration, reactivity, and penetration of the source water DOC.

Effect of nitazoxanide on cryptosporidiosis in experimentally infected neonatal dairy calves.

PubMed

Ollivett, T L; Nydam, D V; Bowman, D D; Zambriski, J A; Bellosa, M L; Linden, T C; Divers, T J

2009-04-01

Cryptosporidium is a zoonotic protozoan that is most often diagnosed in association with diarrhea in 1- to 3-wk-old dairy calves. There are neither consistently effective nor approved antimicrobial drugs for treatment in animals. The objective of this study was to test nitazoxanide (NTZ) as a treatment for cryptosporidiosis in experimentally infected dairy calves. A randomized, controlled, and blinded trial was performed using Holstein bull calves obtained from a large commercial dairy. All births were attended by study personnel and calves were fed 4 L of heat-treated colostrum within 1 h of birth. Calves were randomly assigned to treatment or placebo group and maintained for a 32-feeding (16 d) study period. Twenty-three calves were enrolled with 3 lost to follow up. Thirteen calves were assigned to the treatment group and 7 calves to the placebo group. All calves were inoculated with 1 x 10(6) viable Cryptosporidium parvum oocysts at feeding 3. Treatment was a commercially available NTZ product and the placebo was the carrier of the same product. Nitazoxanide was administered at 1.5 g twice per day for 5 d. Nitazoxanide or placebo treatment began after feeding 10 and when the fecal score was greater than 1 out of 3. Outcome measurements included twice-daily fecal and health scores and a once-daily oocyst count by an immunofluorescent antibody assay. Data were analyzed by nonparametric and time-to-event methods. Measures of passive transfer of antibodies, initial body weight, and onset of oocyst shedding were not different between treatment and control calves. Eighty-five percent of the NTZ-treated calves stopped shedding oocysts by the end of the observation period whereas only 15% of the placebo group stopped shedding. The median number of feedings with a fecal score equal to 3 was 2 in the NTZ group while it was 6 in the placebo group. Calves receiving NTZ were 0.13 times as likely to have severe and sustained diarrhea than control calves (95% confidence interval, 0.02-0.98). Treating calves with NTZ reduced the duration of oocyst shedding and improved fecal consistency.

Keeping it cool: Survival of Giardia cysts and Cryptosporidium oocysts on lettuce leaves.

PubMed

Utaaker, Kjersti Selstad; Skjerve, Eystein; Robertson, Lucy J

2017-08-16

Fresh produce has been recognized as a vehicle for transmission of protozoan parasites for many years, and there are numerous publications regarding their occurrence on such foodstuffs, indicating their potential importance as foodborne parasites. Nevertheless, few studies have been published regarding the effectiveness of this transmission route, and whether contamination is likely to result in transmission. The purpose of this study was to assess the viability of Cryptosporidium oocysts and Giardia cysts, two protozoa associated with both waterborne and foodborne transmission, by spiking fresh produce (lettuce leaves) with viable transmission stages and determining changes in viability. These investigations were performed under different conditions and over time spans that may be used in a regular household; a fridge at 4°C, under ambient temperatures exposed to natural cycles of light during night and day, and inside a cupboard to ensure no light exposure, for a duration of up to two weeks, or as long as the produce remained visually palatable. The major finding from this study is that whereas both Cryptosporidium oocysts and Giardia cysts survive well when kept moist and refrigerated, survival of Giardia cysts was abrogated on lettuce at room temperature. Indeed, almost 50% die-off of Giardia cysts was recorded within the first 24h. Cryptosporidium oocysts had a stable viability throughout the experiment under all the conditions investigated, indicating that fresh produce is a suitable transmission vehicle for Cryptosporidium, even if contamination occurs on-farm and the parasites are exposed to non-favourable storage conditions, as may be common in developing countries. Giardia cysts were not as robust as Cryptosporidium oocysts, and would be probably unlikely to survive under ambient storage conditions on-farm, during sale, or at home. However, if kept refrigerated, then some contaminating Giardia cysts may remain viable and therefore may pose a threat to the consumer. Thus, as the cold chain for transport and storage of fresh produce improves, it is important that similar improvements are implemented to reduce the contamination of fresh produce with parasite transmission stages. Copyright © 2017 Elsevier B.V. All rights reserved.

Cyclospore cayetanensis in Anhui, China

PubMed Central

Wang, Ke-Xia; Li, Chao-Pin; Wang, Jian; Tian, Ye

2002-01-01

AIM: To investigate the infection of Cyclospore cayetanensis in Anhui Province. METHODS: Identification of Cyclospore cayetanensis was made microscopically by finding the oocysts of Cyclospore cayetanensis in fecal smears taken from the infants, pupils and adults with obstinate diarrhea, and immunocompromised individuals by using a auramine-phenol stain and modified acid-fast stain. Cellular immune function was detected with biotin-streptavidin (BSA), and the specific antibody against Cyclospore cayetanensis was detected with method of ELISA. RESULTS: (1) The positive rates of Cyclospore cayetanensis infection in infants, pupils, infants and adults with obstinate diarrhea and with immunocompromised individuals were significantly different (P < 0.01), with the rates of 0%, 0.50% (1/200), 5.62% (10/178), and 9.38% (3/32) respectively. (2) The infection rates of males and females were 2.61% (10/383) and 1.44% (4/227) respectively, with no significant difference (P < 0.05). (3) The positive rates of population with oocysts in urban and rural areas were 0.92% (3/325) and 3.86% (11/285) respectively. (4) The positive rates of CD3+, CD4+, CD8+ and the ratio of CD4+/CD8+ of individuals with and without oocysts were significantly different (P < 0.05, P < 0.01), and their values were (64.28% ± 6.55%), (43.55% ± 5.80%), (28.23% ± 4.32%), 1.52 ± 0.32 and (58.97% ± 5.23%), (39.26% ± 4.93%), (30.54% ± 5.17%), 1.26 ± 0.21, respectively. (5) Specific IgG, IgM and IgG+IgM in serum of the patients with oocyst were significantly different (P < 0.01) with the positive rates of 63.41% (9/14), 17.07% (1/14) and 19.51% (4/14) respectively. CONCLUSION: Cyclospore cayetanensis infection is present in Anhui, China and it was confirmed to be a new pathogen associated with children diarrhea, adults obstinate diarrhea and diarrhea in immunocompromised individuals. Among all the infected individuals, adult obstinate diarrhea patients and immunocompromised individuals are common. Feces examination of oocysts and serological examination of the specific antibody will be of much help in the diagnosis of Cyclospore cayetanensis infection. PMID:12439942

Removals of cryptosporidium parvum oocysts and cryptosporidium-sized polystyrene microspheres from swimming pool water by diatomaceous earth filtration and perlite-sand filtration.

PubMed

Lu, Ping; Amburgey, James E; Hill, Vincent R; Murphy, Jennifer L; Schneeberger, Chandra L; Arrowood, Michael J; Yuan, Tao

2017-06-01

Removal of Cryptosporidium-sized microspheres and Cryptosporidium parvum oocysts from swimming pools was investigated using diatomaceous earth (DE) precoat filtration and perlite-sand filtration. In pilot-scale experiments, microsphere removals of up to 2 log were obtained with 0.7 kg·DE/m 2 at a filtration rate of 5 m/h. A slightly higher microsphere removal (2.3 log) was obtained for these DE-precoated filters when the filtration rate was 3.6 m/h. Additionally, pilot-scale perlite-sand filters achieved greater than 2 log removal when at least 0.37 kg/m 2 of perlite was used compared to 0.1-0.4 log removal without perlite both at a surface loading rate of 37 m/h. Full-scale testing achieved 2.7 log of microspheres and oocysts removal when 0.7 kg·DE/m 2 was used at 3.6 m/h. Removals were significantly decreased by a 15-minute interruption of the flow (without any mechanical agitation) to the DE filter in pilot-scale studies, which was not observed in full-scale filters. Microsphere removals were 2.7 log by perlite-sand filtration in a full-scale swimming pool filter operated at 34 m/h with 0.5 kg/m 2 of perlite. The results demonstrate that either a DE precoat filter or a perlite-sand filter can improve the efficiency of removal of microspheres and oocysts from swimming pools over a standard sand filter under the conditions studied.

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

PubMed Central

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

2016-01-01

The morphological, biological, and molecular characteristics of Cryptosporidium avian genotype V are described, and the species name Cryptosporidium avium is proposed to reflect its specificity for birds under natural and experimental conditions. Oocysts of C. avium measured 5.30–6.90 μm (mean = 6.26 μm) × 4.30–5.50 μm (mean = 4.86 μm) with a length to width ratio of 1.29 (1.14–1.47). Oocysts of C. avium obtained from four naturally infected red-crowned parakeets (Cyanoramphus novaezealandiae) were infectious for 6-month-old budgerigars (Melopsittacus undulatus) and hens (Gallus gallus f. domestica). The prepatent periods in both susceptible bird species was 11 days post infection (DPI). The infection intensity of C. avium in budgerigars and hens was low, with a maximum intensity of 5,000 oocysts per gram of faeces. Oocysts of C. avium were microscopically detected at only 12–16 DPI in hens and 12 DPI in budgerigars, while PCR analyses revealed the presence of specific DNA in faecal samples from 11 to 30 DPI (the conclusion of the experiment). Cryptosporidium avium was not infectious for 8-week-old SCID and BALB/c mice (Mus musculus). Naturally or experimentally infected birds showed no clinical signs of cryptosporidiosis and no pathology was detected. Developmental stages of C. avium were detected in the ileum and caecum using scanning electron microscopy. Phylogenetic analyses based on small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. avium is genetically distinct from previously described Cryptosporidium species. PMID:26905074

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PubMed Central

2014-01-01

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis. PMID:25031466

DNA extraction from protozoan oocysts/cysts in feces for diagnostic PCR.

PubMed

Hawash, Yousry

2014-06-01

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

Intestinal coccidia (Apicomplexa: Eimeriidae) of Brazilian lizards. Eimeria carmelinoi n.sp., from Kentropyx calcarata and Acroeimeria paraensis n.sp. from Cnemidophorus lemniscatus lemniscatus (Lacertilia: Teiidae).

PubMed

Lainson, Ralph

2002-03-01

Eimeria carmelinoi n.sp., is described in the teiid lizard Kentropyx calcarata Spix, 1825 from north Brazil. Oocysts subspherical to spherical, averaging 21.25 x 20.15 micro m. Oocyst wall smooth, colourless and devoid of striae or micropyle. No polar body or conspicuous oocystic residuum, but frequently a small number of fine granules in Brownian movement. Sporocysts, averaging 10.1 x 9 microm, are without a Stieda body. Endogenous stages characteristic of the genus: intra-cytoplasmic, within the epithelial cells of the ileum and above the host cell nucleus. A re-description is given of a parasite previously described as Eimeria cnemidophori, in the teiid lizard Cnemidophorus lemniscatus lemniscatus. A study of the endogenous stages in the ileum necessitates renaming this coccidian as Acroeimeria cnemidophori (Carini, 1941) nov.comb., and suggests that Acroeimeria pintoi Lainson & Paperna, 1999 in the teiid Ameiva ameiva is a synonym of A. cnemidophori. A further intestinal coccidian, Acroeimeria paraensis n.sp. is described in C. l. lemniscatus, frequently as a mixed infection with A. cnemidophori. Mature oocysts, averaging 24.4 x 21.8 microm, have a single-layered, smooth, colourless wall with no micropyle or striae. No polar body, but the frequent presence of a small number of fine granules exhibiting Brownian movements. Sporocysts 9 x 8, without a Stieda body. Endogenous stages epicytoplasmic, characteristic of the genus, in the upper ileum. The importance of a study of the endogenous stages of eimeriid coccidia is discussed.

IDENTIFICATION OF A SPOROZOITE-SPECIFIC ANTIGEN FROM TOXOPLASMA GONDII

PubMed Central

Hill, Dolores; Coss, Cathleen; Dubey, J. P.; Wroblewski, Kristen; Sautter, Mari; Hosten, Tiffany; Muñoz-Zanzi, Claudia; Mui, Ernest; Withers, Shawn; Boyer, Kenneth; Hermes, Gretchen; Coyne, Jessica; Jagdis, Frank; Burnett, Andrew; McLeod, Patrick; Morton, Holmes; Robinson, Donna; McLeod, Rima

2013-01-01

Reduction of risk of human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst versus tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis in combination with tandem mass spectroscopy and Western blot to identify a sporozoite-specific protein (Toxoplasma gondii embryogenesis-related protein, TgERP) which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites, and this protein was used in Western blots and probed with sera from T. gondii infected humans. Serum antibody to TgERP was detected in humans within 6–8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti-Toxoplasma IgM detected in sera, or <30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti-Toxoplasma IgM detected in sera, or >30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early Toxoplasma infection and implicates oocysts as the agent of infection. PMID:21506817

Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes

PubMed Central

Ferguson, David J. P.; Kaindama, Mbinda L.; Brusini, Lorenzo; Joshi, Nimitray; Rchiad, Zineb; Brady, Declan; Guttery, David S.; Wheatley, Sally P.; Yamano, Hiroyuki; Holder, Anthony A.; Pain, Arnab; Wickstead, Bill; Tewari, Rita

2015-01-01

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei. PMID:26565797

Leaching of Cryptosporidium parvum Oocysts, Escherichia coli, and a Salmonella enterica Serovar Typhimurium Bacteriophage through Intact Soil Cores following Surface Application and Injection of Slurry▿

PubMed Central

Forslund, Anita; Markussen, Bo; Toenner-Klank, Lise; Bech, Tina B.; Jacobsen, Ole Stig; Dalsgaard, Anders

2011-01-01

Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions. PMID:21948848

THE EFFICACY OF THREE MEDICINAL PLANTS; GARLIC, GINGER AND MIRAZID AND A CHEMICAL DRUG METRONIDAZOLE AGAINST CRYPTOSPORIDIUM PARVUM: II-HISTOLOGICAL CHANGES.

PubMed

Abouel-Nour, Mohamed F; El-Shewehy, Dina Magdy M; Hamada, Shadia F; Morsy, Tosson A

2016-04-01

Cryptosporidiosis parvum is a zoonotic protozoan parasite infects intestinal epithelial cells of man and animals causing a major health problem. This study was oriented to evaluate the protective and curative capacity of garlic, ginger and mirazid in comparison with metronidazole drug (commercially known) against Cryptosporidium in experimental mice. Male Swiss Albino mice experimentally infected with C. parvum were treated with medicinal plants extracts (Ginger, Mirazid, and Garlic) as compared to chemical drug Metronidazole. Importantly, C. parvum-infected mice treated with ginger, Mirazid, garlic and metronidazole showed a complete elimination in shedding oocysts by 9th day PI. The reduction and elimination of shedding oocysts in response to the treatments might be attributable to a direct effect on parasite growth in intestines, sexual phases production and/or the formation of oocysts. The results were evaluated histopathological examination of ideum section of control mice (uninfected, untreated) displayed normal architecture of the villi. Examiination of infected mice ileum section (infected, untreated) displayed histopathological alterations from uninfected groups. Examination of ileum section prepared from mice treated with garlic, ginger, mirazid, and metronidazole displayed histopathological alterations from that of the control groups, and showed marked histologic correction in the pattern with the four regimes used in comparison to control mice. Garlic successfully eradicated oocysts of infected mice from stool and intestine. Supplementation of ginger to infected mice markedly corrected elevation in the inflammatory risk factors and implied its potential antioxidant, anti-inflammatory and immunomodulatory capabilities. Infected mice treated with ginger, mirazid, garlic and metronidazole showed significant symptomatic improvements during treatment.

Investigating seagrass in Toxoplasma gondii transmission in Florida (Trichechus manatus latirostris) and Antillean (T. m. manatus) manatees

USGS Publications Warehouse

Wyrosdick, Heidi M; Gerhold, Richard; Su, Chunlei; Mignucci-Giannoni, Antonio A.; Bonde, Robert K.; Chapman, Alycia; Riviera-Perez, Carla; Martinez, Jessica; Miller, Debra L.

2017-01-01

Toxoplasma gondii is a feline protozoan reported to cause morbidity and mortality in manatees and other marine mammals. Given the herbivorous nature of manatees, ingestion of oocysts from contaminated water or seagrass is presumed to be their primary mode of infection. The objectives of this study were to investigate oocyst contamination of seagrass beds in Puerto Rico and determine the seroprevalence of T. gondii in Antillean (Trichechus manatus manatus) and Florida (T. m. latirostris) manatees. Sera or plasma from Antillean (n = 5) and Florida (n = 351) manatees were tested for T. gondii antibodies using the modified agglutination test. No T. gondii DNA was detected via PCR in seagrass samples (n = 33) collected from Puerto Rico. Seroprevalence was 0%, suggesting a lower prevalence of T. gondii in these manatee populations than previously reported. This was the first study to investigate the potential oocyst contamination of the manatee diet, and similar studies are important for understanding the epidemiology of T. gondii in herbivorous marine mammals.

Development of electrochemical based sandwich enzyme linked immunosensor for Cryptosporidium parvum detection in drinking water.

PubMed

Thiruppathiraja, Chinnasamy; Saroja, Veerappan; Kamatchiammal, Senthilkumar; Adaikkappan, Periyakaruppan; Alagar, Muthukaruppan

2011-10-01

Cryptosporidium parvum is one of the most important biological contaminants in drinking water and generates significant risks to public health. Due to low infectious dose of C. parvum, remarkably sensitive detection methods are required for water and food industry analysis. This present study describes a simple, sensitive, enzyme amplified sandwich form of an electrochemical immunosensor using dual labeled gold nanoparticles (alkaline phosphatase and anti-oocysts monoclonal antibody) in indium tin oxide (ITO) as an electrode to detect C. parvum. The biosensor was fabricated by immobilizing the anti-oocysts McAb on a gold nanoparticle functionalized ITO electrode, followed by the corresponding capture of analytes and dual labeled gold nanoparticle probe to detect the C. parvum target. The outcome shows the sensitivity of electrochemical immune sensor enhanced by gold nanoparticles with a limit of detection of 3 oocysts/mL in a minimal processing period. Our results demonstrated the sensitivity of the new approach compared to the customary method and the immunosensors showed acceptable precision, reproducibility, stability, and could be readily applied to multi analyte determination for environmental monitoring.

Effect of colostrum quantity and quality on neonatal calf diarrhoea due to Cryptosporidium spp. infection.

PubMed

Arsenopoulos, K; Theodoridis, A; Papadopoulos, E

2017-08-01

This study was conducted to assess the effect of colostrum quality and quantity on Cryptosporidium spp. calf diarrhoea in an intensive dairy cattle farm in Greece. Faecal samples were collected from 100 dairy calves randomly selected and born during all 4 seasons (March 2015 to May 2016) of the year. In total, 71% of the selected calves were positive for Cryptosporidium spp. oocysts. The statistical analysis revealed influence of colostrum quality on faecal score. Linear regression showed that the colostrum quantity during the first day of life was negatively associated with the number of Cryptosporidium spp. oocysts in faeces. During multivariable analysis, the variables representing the quality of colostrum and the season of the calf's birth were identified as confounders. Cryptosporidium spp. is a common pathogen participating in neonatal calf diarrhoea. Colostrum management and season influence the number of Cryprosporidium spp. oocysts and faecal consistency. The above findings demonstrate novel risk factors that should be included in the strategic approaches to control cryptosporidiosis in newborn calves. Copyright © 2017 Elsevier Ltd. All rights reserved.

Experimental Toxoplasmosis in Chimpanzees

PubMed Central

Draper, C. C.; Killick-Kendrick, R.; Hutchison, W. M.; Siim, J. Chr.; Garnham, P. C. C.

1971-01-01

Two chimpanzees were given by mouth large numbers of viable oocysts of Toxoplasma gondii obtained from the faeces of experimentally infected cats. Before the experiment the first chimpanzee had a positive dye test reaction (1:250), an indication that it had undergone an earlier infection of toxoplasmosis; the serum antibody titres remained unchanged, no evidence of illness was found, and oocysts did not appear in its faeces during the subsequent six weeks. The second chimpanzee showed a negative dye test reaction before infection, and this converted to positive on the 7th day, rose to a peak on the 35th day, and remained high for six months. This animal appeared unwell during the first week, and on the 7th day its blood proved infective to mice; on the 40th day the lymph nodes became enlarged and biopsy specimens of a node and muscle in the 11th week were also infective to mice. No oocysts were passed in the faeces. The presumed cycle in the chimpanzee and in man and the relationships between Toxoplasma and Isospora are discussed. PMID:5575975

First report of predation of Giardia sp. cysts by ciliated protozoa and confirmation of predation of Cryptosporidium spp. oocysts by ciliate species.

PubMed

Siqueira-Castro, Isabel Cristina Vidal; Greinert-Goulart, Juliane Araújo; Bonatti, Tais Rondello; Yamashiro, Sandra; Franco, Regina Maura Bueno

2016-06-01

Ciliated protozoa are important components of the microbial food web in various habitats, especially aquatic environments. These organisms are useful bioindicators for both environmental quality assessment and the wastewater purification process. The pathogenic parasitic protozoan species Giardia and Cryptosporidium represent a significant concern for human health, being responsible for numerous disease outbreaks worldwide. The predation of cysts and oocysts in 15 ciliate species from water and sewage samples collected in Campinas, São Paulo, Brazil were verified under laboratory conditions. The ciliated protozoan species were selected based on their mode of nutrition, and only bacterivorous and suspension-feeders were considered for the experiments. The species Blepharisma sinuosum, Euplotes aediculatus, Sterkiella cavicola, Oxytricha granulifera, Vorticella infusionum, Spirostomum minus, and Stentor coeruleus ingested cysts and oocysts, the resistance forms of Giardia spp. and Cryptosporidium spp., respectively. This is the first time that the ingestion of Giardia cysts by ciliated protozoa has been reported. These findings may contribute to a better understanding of the biological removal of these pathogens from aquatic environments.

Elevation and vegetation determine Cryptosporidium oocyst shedding by yellow-bellied marmots (Marmota flaviventris) in the Sierra Nevada Mountains.

PubMed

Montecino-Latorre, Diego; Li, Xunde; Xiao, Chengling; Atwill, Edward R

2015-08-01

Wildlife are increasingly recognized as important biological reservoirs of zoonotic species of Cryptosporidium that might contaminate water and cause human exposure to this protozoal parasite. The habitat range of the yellow-bellied marmot (Marmota flaviventris) overlaps extensively with the watershed boundaries of municipal water supplies for California communities along the foothills of the Sierra Nevada. We conducted a cross-sectional epidemiological study to estimate the fecal shedding of Cryptosporidium oocysts by yellow-bellied marmots and to quantify the environmental loading rate and determine risk factors for Cryptosporidium fecal shedding in this montane wildlife species. The observed proportion of Cryptosporidium positive fecal samples was 14.7% (33/224, positive number relative to total number samples) and the environmental loading rate was estimated to be 10,693 oocysts animal(-1) day(-1). Fecal shedding was associated with the elevation and vegetation status of their habitat. Based on a portion of the 18s rRNA gene sequence of 2 isolates, the Cryptosporidium found in Marmota flaviventris were 99.88%-100% match to multiple isolates of C. parvum in the GenBank.

Evaluation of the Solar Water Disinfection Process (SODIS) Against Cryptosporidium parvum Using a 25-L Static Solar Reactor Fitted with a Compound Parabolic Collector (CPC)

PubMed Central

Fontán-Sainz, María; Gómez-Couso, Hipólito; Fernández-Ibáñez, Pilar; Ares-Mazás, Elvira

2012-01-01

Water samples of 0, 5, and 30 nephelometric turbidity units (NTU) spiked with Cryptosporidium parvum oocysts were exposed to natural sunlight using a 25-L static solar reactor fitted with a compound parabolic collector (CPC). The global oocyst viability was calculated by the evaluation of the inclusion/exclusion of the fluorogenic vital dye propidium iodide and the spontaneous excystation. After an exposure time of 8 hours, the global oocyst viabilities were 21.8 ± 3.1%, 31.3 ± 12.9%, and 45.0 ± 10.0% for turbidity levels of 0, 5, and 30 NTU, respectively, and these values were significantly lower (P < 0.05) that the initial global viability of the isolate (92.1 ± 0.9%). The 25-L static solar reactor that was evaluated can be an alternative system to the conventional solar water disinfection process for improving the microbiological quality of drinking water on a household level, and moreover, it enables treatment of larger volumes of water (> 10 times). PMID:22302852

Evaluation of the solar water disinfection process (SODIS) against Cryptosporidium parvum using a 25-L static solar reactor fitted with a compound parabolic collector (CPC).

PubMed

Fontán-Sainz, María; Gómez-Couso, Hipólito; Fernández-Ibáñez, Pilar; Ares-Mazás, Elvira

2012-02-01

Water samples of 0, 5, and 30 nephelometric turbidity units (NTU) spiked with Cryptosporidium parvum oocysts were exposed to natural sunlight using a 25-L static solar reactor fitted with a compound parabolic collector (CPC). The global oocyst viability was calculated by the evaluation of the inclusion/exclusion of the fluorogenic vital dye propidium iodide and the spontaneous excystation. After an exposure time of 8 hours, the global oocyst viabilities were 21.8 ± 3.1%, 31.3 ± 12.9%, and 45.0 ± 10.0% for turbidity levels of 0, 5, and 30 NTU, respectively, and these values were significantly lower (P < 0.05) that the initial global viability of the isolate (92.1 ± 0.9%). The 25-L static solar reactor that was evaluated can be an alternative system to the conventional solar water disinfection process for improving the microbiological quality of drinking water on a household level, and moreover, it enables treatment of larger volumes of water (> 10 times).

Immunomodulatory and antiparasitic effects of garlic extract on Eimeria vermiformis-infected mice.

PubMed

Khalil, Atef Mohammed; Yasuda, Masahiro; Farid, Ayman Samir; Desouky, Mohamed Ibrahim; Mohi-Eldin, Mouchira Mohammed; Haridy, Mohie; Horii, Yoichiro

2015-07-01

We investigated the immunomodulatory and parasiticidal effects of garlic extract on coccidiosis caused by Eimeria vermiformis infection in male ICR mice. One group received garlic extract daily until the end of the experiment by the oral route from 10 days prior to oral infection with 300 sporulated E. vermiformis oocysts (infected-garlic(+)). The other group served as a control positive with E. vermiformis infection alone (infected-garlic(-)). In the infected-garlic(+) group, garlic extract treatment induced a significant reduction in fecal oocyst output when compared with the infected-garlic(-) group. Histopathological, immunohistochemical, and gene expression analysis for inflammatory cytokines in ileal tissues showed that the garlic extract treatment impaired intracellular development of E. vermiformis during the early stages by increasing the number of intraepithelial CD8(+) T cells and decreasing IL-10 expression. This induced cell cytotoxicity which was reflected by a decrease in oocyst numbers in the intestinal villi and the feces, indicating anticoccidial effects of the garlic extract. However, further studies to explore the precise mechanism of the observed effects of garlic treatment during Eimeria infection are needed to verify our results.

Oligonucleotide-Gold Nanoparticle Networks for Detection of Cryptosporidium parvum Heat Shock Protein 70 mRNA ▿

PubMed Central

Javier, David J.; Castellanos-Gonzalez, Alejandro; Weigum, Shannon E.; White, A. Clinton; Richards-Kortum, Rebecca

2009-01-01

We report on a novel strategy for the detection of mRNA targets derived from Cryptosporidium parvum oocysts by the use of oligonucleotide-gold nanoparticles. Gold nanoparticles are functionalized with oligonucleotides which are complementary to unique sequences present on the heat shock protein 70 (HSP70) DNA/RNA target. The results indicate that the presence of HPS70 targets of increasing complexity causes the formation of oligonucleotide-gold nanoparticle networks which can be visually monitored via a simple colorimetric readout measured by a total internal reflection imaging setup. Furthermore, the induced expression of HSP70 mRNA in Cryptosporidium parvum oocysts via a simple heat shock process provides nonenzymatic amplification such that the HSP70 mRNA derived from as few as 5 × 103 purified C. parvum oocysts was successfully detected. Taken together, these results support the use of oligonucleotide-gold nanoparticles for the molecular diagnosis of cryptosporidiosis, offering new opportunities for the further development of point-of-care diagnostic assays with low-cost, robust reagents and simple colorimetric detection. PMID:19828740

Monitoring of Cryptosporidium and Giardia in Czech drinking water sources.

PubMed

Dolejs, P; Ditrich, O; Machula, T; Kalousková, N; Puzová, G

2000-01-01

In Czech raw water sources for drinking water supply, Cryptosporidium was found in numbers from 0 to 7400 per 100 liters and Giardia from 0 to 485 per 100 liters. The summer floods of 1997 probably brought the highest numbers of Cryptosporidium oocysts into one of the reservoirs sampled; since then these numbers decreased steadily. A relatively high number of Cryptosporidium oocysts was found in one sample of treated water. Repeated sampling demonstrated that this was a sporadic event. The reason for the presence of Cryptosporidium in a sample of treated drinking-water is unclear and requires further study.

[Detection of Cryptospordium spp. in environmental water samples by FTA-PCR].

PubMed

Zhang, Xiao-Ping; Zhu, Qian; He, Yan-Yan; Jiang, Li; Jiang, Shou-Fu

2011-02-01

To establish a FTA-polymeras chain reaction (FTA-PCR) method in detection of Cryptospordium spp. in different sources of water. The semi automated immunomagnetic separation (IMS) of Cryptospordium oocysts in environmental water samples was performed firstly, and then genomic DNA of Cryptospordium oocysts was extracted by FTA filters disk. Oligonucleotide primers were designed based on the DNA fragment of the 18 S rRNA gene from C. parvum. Plate DNA was amplified with primers in PCR. The control DNA samples from Toxoplasma gondii,Sarcocystis suihominis, Echinococcus granulosus, and Clonorchis sinensis were amplified simultaneously. All PCR products were detected by agar electrophoresis dyed with ethidium bromide. The 446 bp fragment of DNA was detected in all samples of C. parvum, C. andersoni, and C. baileyi, while it was not detected in control groups in laboratory. No positive samples were found from 10 samples collected from tape water in 5 districts of Shanghai City by FTA-PCR. Nine positive samples were detected totally from 70 different environmental water samples, there were 0 out of 15 samples from the source of tape water, 2 out of 25 from the Huangpu River, 5 out of 15 from rivers around the animal farmers, 1 out of 9 from output water of contaminating water treatment factory, 1 out of 6 from the out gate of living contaminating water. The 446 bp fragment was detected from all the amplified positive water samples. FTA-PCR is an efficient method for gene detection of Cryptospordium oocysts, which could be used in detection of environmental water samples. The contamination degree of Cryptospordium oocysts in the river water around animal farms is high.

Movement of Toxoplasma gondii Oocysts in Unsaturated Natural Soils

NASA Astrophysics Data System (ADS)

Kinsey, Erin; Korte, Caroline; L'Ollivier, Coralie; Dubey, Jitender; Dumetre, Aurélien; Darnault, Christophe

2017-04-01

Toxoplasma gondii has a complex lifecycle that involves a wide variety of intermediate hosts with felids as the definitive host. Because of its numerous hosts and the prevalence of cats, T.gondii has spread throughout nearly the entire globe. Oocysts have been found not only in the feces of cats, but also in soils, animal feeds and water. Exposure through consumption of infected meat or following contact with cat feces can cause damage to the eyes, brain and other organs of immunocompromised populations as well as fetuses if they are exposed in utero. The prevalence of T.gondii and potential health risks necessitate a better understanding of the transport of T.gondii through soils, which to this point has not been well studied. This work aims to characterize the transport and retention of T.gondii oocysts in a number of unsaturated natural soils where fast transport and preferential flow paths have been prevented. The soils used are classified as loamy sands and sandy loams. They were placed in soil columns at a known bulk density and were then subjected to an artificial rain of 1 mM KCl solution. Flow in the columns was vertical and gravity driven. After steady state was reached, a pulse containing 2.5 million T.gondii oocysts and KBr as a conservative tracer was applied to the top of the column, after which steady rainfall was resumed. Leachate samples were collected throughout the experiment. qPCR for T.gondii was performed and KBr ions were measured to create breakthrough curves for both. After the completion of the rainfall portion of the experiment, soil columns were cut into 1 to 2 cm sections and analyzed for T.gondii with qPCR to characterize retention within the column and for soil water content.

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites.

PubMed

Gonzalez-Ceron, L; Rodriguez, M H; Santillan, F; Chavez, B; Nettel, J A; Hernandez-Avila, J E; Kain, K C

2001-07-01

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites. Copyright 2001 Academic Press.

Identification of Cryptosporidium Species and Genotypes in Scottish Raw and Drinking Waters during a One-Year Monitoring Period▿

PubMed Central

Nichols, R. A. B.; Connelly, L.; Sullivan, C. B.; Smith, H. V.

2010-01-01

We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. Cryptosporidium andersoni, Cryptosporidium parvum, and the Cryptosporidium cervine genotype (now Cryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%). PMID:20639357

Cryptosporidiosis and other intestinal parasitic infections in patients with chronic diarrhea.

PubMed

Mahdi, Nadham K; Ali, Naeel H

2004-09-01

To consider the relationship of the parasitic infections including cryptosporidium with chronic diarrhea. Also the effect of chronic disease as pulmonary tuberculosis (TB) and nosocomial infection on the occurrence rate of parasites in cases of chronic diarrhea. Stool samples were collected from 205 patients in teaching, general, child and maternity hospitals in Basrah, Iraq, suffering from chronic diarrhea during 2000. Out of these patients, there were 40 patients with pulmonary TB and 50 inpatients with nosocomial infection. Also 175 apparently healthy individuals who have no episodes of diarrhea for at least 2-months were served as a control group. Direct smear method and then formalin ether sedimentation method were carried out for stool samples to detect intestinal parasites. Fecal smears were prepared from the sediment and stained by the modified Ziehl Neelsen stain for the recovery of red pink oocysts of cryptosporidium. Out of the 205 examined patients, cryptosporidium oocysts were found to be excreted in 20 (9.7%) patients in comparing to 1.1% of the control group. The difference is statistically significant. There were 109 (53.2%) patients found to be positive for intestinal parasitic infections compared to 26 (14.8%) of the control group. The difference is also statistically significant. Out of the 40 TB patients, 2 (5%) were found to excrete cryptosporidium oocysts and also 27 (67.3%) were positive for intestinal parasites. In addition, there were 4 (8%) excreting cryptosporidium oocysts and 23 (46%) infecting by intestinal parasites among the in patients with nosocomial infection. Both acid and non-acid fast parasites should be considered in the differential diagnosis of undiagnosed chronic diarrhea especially among patients with pulmonary TB or nosocomial infection.

Efficacy of toltrazuril 5 % suspension against Eimeria bovis and Eimeria zuernii in calves and observations on the associated immunopathology.

PubMed

Jonsson, Nicholas N; Piper, Emily K; Gray, Christian P; Deniz, Abdulkerim; Constantinoiu, Constantin C

2011-08-01

16 Calves were each infected with suspensions containing a mixture of approximately 230,000 Eimeria bovis and 70,000 E. zuernii oocysts, which resulted in detection of oocysts in faeces of 12 of 16 calves by day +14 after infection. On day +14 after infection calves were either treated (n = 8) with toltrazuril at 15 mg/kg body weight or with a placebo. Observations were made on the clinical condition, faecal score and liveweight of calves daily from one day post infection (pi) until 24 days pi when all calves were euthanised and examined post mortem. Samples were collected from ileum and colon for histological, immunohistochemical and gene expression studies. The study demonstrated an efficacy of toltrazuril for the treatment of E. bovis and E. zuernii infections in calves reaching 99 % (based on arithmetic mean oocyst counts in faeces) within three days of treatment and remaining at or above this level for six days. Toltrazuril did not have a significant effect on the pattern and extent of immune cellular infiltration in the mucosa of ileum and colon, but the expression of the genes coding IL-2, IL-10 and TNF-α in the ileum and TNF-α in the colon were elevated in calves treated with toltrazuril. Higher levels of oocyst shedding were significantly associated with lower expression of genes coding for IL-2, IL-10 and higher IP-10. It is concluded that toltrazuril is effective for the treatment of coccidiosis due to E. bovis and E. zuernii in calves and enables the development of a normal or enhanced immune response to infection.

Detection of Cryptosporidium and Giardia in clinical laboratories in Europe--a comparative study.

PubMed

Manser, M; Granlund, M; Edwards, H; Saez, A; Petersen, E; Evengard, B; Chiodini, P

2014-01-01

To determine the routine diagnostic methods used and compare the performance in detection of oocysts of Cryptosporidium species and cysts of Giardia intestinalis in faecal samples by European specialist parasitology laboratories and European clinical laboratories. Two sets of seven formalin-preserved faecal samples, one containing cysts of Giardia intestinalis and the other, containing oocysts of Cryptosporidium, were sent to 18 laboratories. Participants were asked to examine the specimens using their routine protocol for detecting these parasites and state the method(s) used. Eighteen laboratories answered the questionnaire. For detection of Giardia, 16 of them used sedimentation/concentration followed by light microscopy. Using this technique the lower limit of detection of Giardia was 17.2 cysts/mL of faeces in the best performing laboratories. Only three of 16 laboratories used fluorescent-conjugated antibody-based microscopy. For detection of Cryptosporidium acid-fast staining was used by 14 of the 17 laboratories that examined the samples. With this technique the lower limit of detection was 976 oocysts/mL of faeces. Fluorescent-conjugated antibody-based microscopy was used by only five of the 17 laboratories. There was variation in the lower limit of detection of cysts of Giardia and oocysts of Cryptosporidium between laboratories using the same basic microscopic methods. Fluorescent-conjugated antibody-based microscopy was not superior to light microscopy under the conditions of this study. There is a need for a larger-scale multi-site comparison of the methods used for the diagnosis of these parasites and the development of a Europe-wide laboratory protocol based upon its findings. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

PubMed Central

Payment, P; Franco, E

1993-01-01

To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8368831

Two new species of Eimeria (Apicomplexa, Eimeriidae) from tree skinks, Prasinohaema spp. (Sauria: Scincidae), from Papua New Guinea

USGS Publications Warehouse

McAllister, Chris T.; Duszynski, Donald W.; Fisher, Robert N.; Austin, Christopher C.

2014-01-01

Between September 1991 and June 1992, feces from 4 species of tree skinks, Prasinohaema spp. from Papua New Guinea, were collected and examined for coccidia. Two species, P. flavipes and P. prehensicauda were found to harbor eimerians which are described as new. Oocysts of Eimeria krausi sp. nov. from P. flavipes were ellipsoidal to subspheroidal with a smooth bilayered wall and measured (L × W) 19.2 × 16.9 μm, with a length/width (L/W) ratio of 1.1. Micropyle and oocyst residuum were absent but a fragmented polar granule was present. Sporocysts were ellipsoidal, 9.7 × 6.7 μm, L/W of 1.5. Stieda, subStieda and paraStieda bodies were absent. The sporocyst residuum was composed of many small granules in a compact mass between sporozoites. The sporozoites were sausage-shaped, 11.7 × 2.7 μm, in situ, with an ellipsoidal posterior refractile body and a spheroidal anterior refractile body. Oocysts of Eimeria greeri sp. nov. from P. prehensicauda were ellipsoidal with a smooth bilayered wall, (L × W) 23.0 × 18.3 μm, with a L/W of 1.3. Micropyle and oocyst residuum were absent but a fragmented polar granule was present. Sporocysts were ellipsoidal, 9.7 × 8.4 μm, with a L/W of 1.2. Stieda, subStieda and paraStieda bodies were absent. The sporocyst residuum was composed of many large granules in a compact mass between sporozoites. The sporozoites were sausage-shaped, with an ellipsoidal posterior refractile body and a spheroidal anterior refractile body. We document here the first report of coccidia from skinks of the genus Prasinohaema.

Re-description of a genetically typed, single oocyst line of the turkey coccidium, Eimeria dispersa Tyzzer, 1929.

PubMed

El-Sherry, S; Ogedengbe, M E; Hafeez, M A; Sayf-Al-Din, M; Gad, N; Barta, J R

2017-10-01

The Briston strain of Eimeria dispersa Tyzzer, 1929 was isolated originally from a commercial turkey flock from Briston, Norfolk, UK. A single oocyst-derived line of E. dispersa was propagated and used to re-describe biological and morphological features of E. dispersa in the turkey. Oocysts of the Briston strain measured 26 ± 1.1 μm (24-28) by 21 ± 1 μm (19-23); these were larger than oocysts described originally by Tyzzer in 1929 (22.75 by 18.84 μm) but within dimensions (26.07 by 21.04 μm) reported by Hawkins (1952) in his description of E. dispersa isolated from turkeys. In the present study, endogenous development started mainly in duodenum and upper jejunum and then spread down toward the lower jejunum. A few parasites were detected in the ileum beginning 96 h post-infection; only few gamonts were observed in the cecal neck area at 120 h, and no parasites were detected in cecal pouches or rectum. Four asexual generations were observed before the start of gametogony, and only one large type of first generation meront was detected in duodenum and upper jejunum at 32 h. This strain has a prepatent period of 120 h. The Briston strain of E. dispersa is a mildly pathogenic coccidium. Duodenum and jejunum of infected birds were slightly dilated and paler in color than of uninfected controls. There was whitish green mucoid material in the lumen of the duodenum and jejunum. The mucosa looked slightly congested and edematous with a few scattered petechial hemorrhages.

Validation of a quantitative Eimeria spp. PCR for fresh droppings of broiler chickens.

PubMed

Peek, H W; Ter Veen, C; Dijkman, R; Landman, W J M

2017-12-01

A quantitative Polymerase Chain Reaction (qPCR) for the seven chicken Eimeria spp. was modified and validated for direct use on fresh droppings. The analytical specificity of the qPCR on droppings was 100%. Its analytical sensitivity (non-sporulated oocysts/g droppings) was 41 for E. acervulina, ≤2900 for E. brunetti, 710 for E. praecox, 1500 for E. necatrix, 190 for E. tenella, 640 for E. maxima, and 1100 for E. mitis. Field validation of the qPCR was done using droppings with non-sporulated oocysts from 19 broiler flocks. To reduce the number of qPCR tests five grams of each pooled sample (consisting of ten fresh droppings) per time point were blended into one mixed sample. Comparison of the oocysts per gram (OPG)-counting method with the qPCR using pooled samples (n = 1180) yielded a Pearson's correlation coefficient of 0.78 (95% CI: 0.76-0.80) and a Pearson's correlation coefficient of 0.76 (95% CI: 0.70-0.81) using mixed samples (n = 236). Comparison of the average of the OPG-counts of the five pooled samples with the mixed sample per time point (n = 236) showed a Pearson's correlation coefficient (R) of 0.94 (95% CI: 0.92-0.95) for the OPG-counting method and 0.87 (95% CI: 0.84-0.90) for the qPCR. This indicates that mixed samples are practically equivalent to the mean of five pooled samples. The good correlation between the OPG-counting method and the qPCR was further confirmed by the visual agreement between the total oocyst/g shedding patterns measured with both techniques in the 19 broiler flocks using the mixed samples.

Prevalence of cryptosporidium infection and characteristics of oocyst shedding in a breeding colony of leopard geckos (Eublepharis macularius).

PubMed

Deming, Clare; Greiner, Ellis; Uhl, Elizabeth W

2008-12-01

Cryptosporidiosis is an emerging problem in reptile medicine and has been associated with a wasting syndrome in leopard geckos (Eublepharis macularius). This study determined the prevalence of infection in a breeding colony of leopard geckos to be 9.8%. Two groups of 20 geckos, one that was fecal positive for oocysts of Cryptosporidium sp., and one, whose individuals were fecal negative at the inception of the study, were followed for 2 mo. Fecal samples were tested for oocysts every 2 wk, body weights were measured, and a body condition score was assigned for each gecko. Selected geckos from these two groups were euthanized and necropsied. There were statistically significant differences (P < 0.05) between the two groups for mean body weight, mean body condition score, and prevalence of infection. Cryptosporidium sp. infection is endemic in this breeding colony, and there were a large number of geckos with a subclinical or carrier state of infection. These animals continued to be infected with Cryptosporidium sp. but gained weight and remained in good body condition. Only one gecko in the entire group of 40 was confirmed to be negative for oocysts or developmental stages by repeated fecal exams and histopathology. An additional 37 severely emaciated geckos from the breeding colony were euthanized, and all were positive for Cryptosporidium sp. on histopathologic examination of the gastrointestinal tract. The results of this study indicate that although some animals can recover from a clinical infection, if a gecko is severely wasted, it should be euthanized because of the poor prognosis and possible source of infection to other geckos.

Vector Competence of Anopheles kleini and Anopheles sinensis (Diptera: Culicidae) From the Republic of Korea to Vivax Malaria-Infected Blood From Patients From Thailand.

PubMed

Ubalee, Ratawan; Kim, Heung-Chul; Schuster, Anthony L; McCardle, Patrick W; Phasomkusolsil, Siriporn; Takhampunya, Ratree; Davidson, Silas A; Lee, Won-Ja; Klein, Terry A

2016-11-01

In total, 1,300 each of Anopheles kleini Rueda and Anopheles sinensis Wiedemann sensu stricto (s.s.) females (colonized from the Republic of Korea) and Anopheles dirus Peyton & Harrison (Thai strain) were allowed to feed on blood from Thai malaria patients naturally infected with Plasmodium vivax The overall oocyst infection rates for An. dirus, An. kleini, and An. sinensis s.s. were 77.4, 46.1, and 45.9%, respectively. The mean number of oocysts was significantly higher for An. dirus (82.7) compared with An. kleini (6.1) and An. sinensis s.s. (8.6), whereas the mean number of oocysts for An. kleini and An. sinensis s.s. was similar. The overall sporozoite infection rates for An. dirus, An. kleini, and An. sinensis s.s. dissected on days 14-15, 21, and 28 days post-feed were significantly higher for An. dirus (90.0%) than An. kleini (5.4%), whereas An. kleini sporozoite rates were significantly higher than An. sinensis s.s. (<0.1%). The overall sporozoite indices for positive females with +3 (100-1,000 sporozoites) and +4 (>1,000 sporozoites) salivary gland indices were significantly higher for An. dirus (85.7%), compared with An. kleini (47.1%). Only one An. sinensis s.s. had sporozoites (+2; >10-100 sporozoites). These results indicate that An. kleini is a competent vector of vivax malaria. Although An. sinensis s.s. develops relatively high numbers of oocysts, it is considered a very poor vector of vivax malaria due to a salivary gland barrier. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

Transstadial transmission of Hepatozoon canis from larvae to nymphs of Rhipicephalus sanguineus.

PubMed

Giannelli, Alessio; Ramos, Rafael Antonio Nascimento; Di Paola, Giancarlo; Mencke, Norbert; Dantas-Torres, Filipe; Baneth, Gad; Otranto, Domenico

2013-09-01

Hepatozoon canis is an apicomplexan parasite of dogs, which is known to become infected by ingesting Rhipicephalus sanguineus adult ticks. To investigate the possibility of H. canis transovarial and transstadial transmission from larvae to nymphs, engorged adult female ticks were collected from a private animal shelter in southern Italy, where H. canis infection is highly prevalent. Female ticks (n=35) and egg batches were tested by PCR for H. canis. All eggs examined were PCR-negative whereas 88.6% of females from the environment tested positive. Additionally, fed larvae (n=120) from a dog naturally infected by H. canis were dissected at different time points post collection (i.e. 0, 10, 20 and 30 days). Molted nymphs dissected at 20 days post collection revealed immature oocysts displaying an amorphous central structure in 50% of the specimens, and oocysts containing sporocysts with sporozoites were found in 53.3% of the nymphs dissected at 30 days post collection. This study demonstrates that H. canis is not transmitted transovarially, but it is transmitted transstadially from larvae to nymphs of R. sanguineus and develops sporozoites in oocysts that may infect dogs. Copyright © 2013 Elsevier B.V. All rights reserved.

A description of Isospora amphiboluri (Apicomplexa: Eimeriidae) from the inland bearded dragon, Pogona vitticeps (Sauria: Agamidae).

PubMed

McAllister, C T; Upton, S J; Jacobson, E R; Kopit, W

1995-04-01

Fecal samples from 50 captive inland bearded dragons, Pogona vitticeps (Ahl, 1926), bred in California, were examined for coccidian parasites. Sixteen (32%) of the lizards were found to be passing oocysts of Isospora amphiboluri Cannon, 1967, previously described from bearded dragons Pogona barbata (Cuvier, 1829) from Australia. Sporulated oocytes were spherical to subspherical, 25.3 x 25.1 (23-26 x 23-26) microns, with a shape index (length/width) of 1.0 (1.0-1.1). A micropyle, oocyst residuum, and polar granule were absent. Sporocyts were ovoidal, 17.0 x 11.4 (16-18 x 11-12) microns, with a shape index of 1.5 (1.4-1.7). A sporocyst residuum, Stieda, and substieda bodies were present, but parastieda bodies were absent. Sporozoites were elongated, 13.9 x 3.5 (12-15 x 3-4) microns in situ, containing spherical anterior and posterior refractile bodies. The occurrence of I. amphiboluri in P. vitticeps is a new host and geographic record for the parasite. Photomicrographs of the oocysts and endogenous life cycle stages of I. amphiboluri are presented for the first time.

Risk factors and infection due to Cryptosporidium spp. in dogs and cats in southern Rio Grande do Sul.

PubMed

Moreira, Andrios da Silva; Baptista, Cristiane Telles; Brasil, Carolina Litchina; Valente, Júlia de Souza Silveira; Bruhn, Fábio Raphael Pascoti; Pereira, Daniela Isabel Brayer

2018-01-01

This study investigated the frequency of oocysts of Cryptosporidium spp. in feces from dogs and cats in five municipalities in the southern region of the state of Rio Grande do Sul. The risk factors associated with infection were also investigated. Feces samples from 110 dogs and 18 cats were stained using the auramine method. At the time of feces sampling, a questionnaire with semi-open-ended questions was applied to the animal guardians and all data obtained underwent statistical analysis. The real frequency of oocysts of Cryptosporidium spp. was 24.63% (27 dogs and two cats). Only four samples of dog feces were diarrheic and no presence of oocysts was observed in any of them. Variables that represented risk factors for infection were: homemade food, untreated water, circulation of animals on grassy terrain and living in the same environment as other animals (cattle). The results made it possible to inferring that within the population studied, the frequency of parasitism due to Cryptosporidium spp. in dogs was relevant and emphasize the asymptomatic nature of this infection. The adopting control measures are highlighted, particularly in relation to variables that represent risk factors for this infection.

Detection of Cryptosporidium oocysts in fresh and frozen cattle faeces: comparison of three methods.

PubMed

Brook, E J; Christley, R M; French, N P; Hart, C A

2008-01-01

The aim of this study was to compare the performance of three commonly used screening tests for Cryptosporidium oocysts in fresh and frozen cattle faeces. Twenty-nine freshly voided faecal samples were collected from calves from three farms in the northwest of England. Three diagnostic tests for Cryptosporidium were carried out on each sample both before and after freezing - the modified Ziehl-Neelsen (MZN) and auramine phenol (APh) stains and a commercial enzyme immunoassay (EIA) kit, the ProSpecT Cryptosporidium Microplate assay (Remel, Lenexa, KS). Twelve samples were deemed positive by the reference standard (polymerase chain reaction, PCR). There were some discrepancies between the results of the screening tests and the levels of agreement were quantified. The sensitivity and specificity of each method was determined, with PCR as the gold standard. Sensitivity and specificity of the MZN stain was optimized when samples with fewer than two oocyst-like bodies were classified as negative. All three screening methods used were effective in detecting Cryptosporidium infection in both fresh and frozen calf faeces. This study has highlighted the value of determining characteristics of tests used for diagnosis and epidemiological studies.

Cryptosporidiosis in Haiti: surprisingly low level of species diversity revealed by molecular characterization of Cryptosporidium oocysts from surface water and groundwater

PubMed Central

Damiani, Céline; Balthazard-Accou, Ketty; Clervil, Elmyre; Diallo, Aïssata; Da Costa, Cécilia; Emmanuel, Evens; Totet, Anne; Agnamey, Patrice

2013-01-01

The protozoan parasite Cryptosporidium sp. has emerged as one of the most important water contaminants, causing waterborne outbreaks of diarrhoeal diseases worldwide. In Haiti, cryptosporidiosis is a frequent cause of diarrhoea in children under the age of five years, HIV-infected individuals, and people living in low socioeconomic conditions, mainly due to the consumption of water or food polluted by Cryptosporidium oocysts. The aim of this study was to detect and identify Cryptosporidium oocysts present in 12 water samples collected in Port-au-Prince and 4 water samples collected in Cap Haïtien. Initial detection consisted of immunomagnetic separation – immunofluorescence assay (IMS-IFA), which was confirmed by nested PCR, targeting the most polymorphic region of the 18S rRNA gene in 15/16 samples. Genotyping was performed by PCR-restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Under our working conditions, neither nested PCR-RFLP nor direct DNA sequencing revealed the expected species diversity, as only Cryptosporidium parvum was identified in the water samples studied. This study highlights the difficulty of detecting mixed populations of Cryptosporidium species in environmental samples. PMID:24252814

Elevation and vegetation determine Cryptosporidium oocyst shedding by yellow-bellied marmots (Marmota flaviventris) in the Sierra Nevada Mountains

PubMed Central

Montecino-Latorre, Diego; Li, Xunde; Xiao, Chengling; Atwill, Edward R.

2015-01-01

Wildlife are increasingly recognized as important biological reservoirs of zoonotic species of Cryptosporidium that might contaminate water and cause human exposure to this protozoal parasite. The habitat range of the yellow-bellied marmot (Marmota flaviventris) overlaps extensively with the watershed boundaries of municipal water supplies for California communities along the foothills of the Sierra Nevada. We conducted a cross-sectional epidemiological study to estimate the fecal shedding of Cryptosporidium oocysts by yellow-bellied marmots and to quantify the environmental loading rate and determine risk factors for Cryptosporidium fecal shedding in this montane wildlife species. The observed proportion of Cryptosporidium positive fecal samples was 14.7% (33/224, positive number relative to total number samples) and the environmental loading rate was estimated to be 10,693 oocysts animal-1 day-1. Fecal shedding was associated with the elevation and vegetation status of their habitat. Based on a portion of the 18s rRNA gene sequence of 2 isolates, the Cryptosporidium found in Marmota flaviventris were 99.88%–100% match to multiple isolates of C. parvum in the GenBank. PMID:25834788

Evaluation of garlic plant and indinavir drug efficacy in the treatment of cryptosporidiosis in experimentally immumosuppressed rats.

PubMed

Toulah, Fawzia H; El-Shafei, Amal A; Al-Rashidi, Hyat S

2012-08-01

The present study evaluated the efficacy of garlic plant and Indinavir on cryptosporidiosis in experimentally immunosuppressed infected rats. One hundred forty five Wister rats aging 3 weeks were divided into five groups: GI: normal control, GII: Indinavir treated control, GIII: immunosuppressed infected, GIV: immunosuppressed infected and treated with garlic and GV: immunosuppressed infected and treated with Indinavir. All were subjected to clinical, parasitological and histopathological examination at different days post infection (P.I.). The results showed that in GIII, all rats had diarrhea, loss of appetite, weakness and limited movement, with 51.4% death rate. In both treated groups, some rats regained activities, with death rate of 33.3% (GV) and non GIV. There was significant decrease in the number of excreted oocysts at 5th and 10th day post treatment (P.T.) in treated groups. One week P.T., in GIV, the number of excreted oocysts had continued in decreasing while in GV, it was insignificantly increased. No cure rate was detected in both treated groups as oocysts still excreted till the end of experiment. The histopathological changes improved in treated groups in spite of the presence of some parasites on the epithelial surfaces of ileum.

A NEW SPECIES OF EIMERIA (APICOMPLEXA: EIMERIIDAE) FROM THE EASTERN PIPISTRELLE, PERIMYOTIS SUBFLAVUS (CHIROPTERA: VESPERTILIONIDAE), IN ARKANSAS

PubMed Central

McAllister, Chris T.; Burt, Scott; Seville, R. Scott; Robison, Henry W.

2011-01-01

During November 2009 and March 2010, 20 adult eastern pipistrelles, Perimyotis (=Pipistrellus) subflavus were collected from Polk County, Arkansas, and their feces examined for coccidian parasites. Two (10%) of the bats were found to be passing oocysts of an undescribed species of Eimeria. Oocysts of Eimeria heidti n. sp. were ovoidal to ellipsoidal, 26.1 × 20.5 (23-31 × 18-23) μm, with a bilayered wall, externally rough, internally smooth, and with a shape index of 1.3. Micropyle and oocyst residuum were absent, but a subspherical polar granule was often present. Sporocysts were ovoidal, 13.0 × 8.8 (11-15 × 7-13) μm, the shape index was 1.6, a Stieda body was present and sub-Stieda and para-Stieda bodies were absent. A sporocyst residuum consisting of multiple globules dispersed along the perimeter of the sporocyst and between the sporozoites were present, sporozoites were elongate, with a subspherical anterior refractile body and elongate posterior refractile body; a nucleus not discernable. This is the second coccidian reported from this host and the fourth instance of a coccidian species reported from an Arkansas bat. PMID:21506799

First amplification of Eimeria hessei DNA from the lesser horseshoe bat (Rhinolophus hipposideros) and its phylogenetic relationships with Eimeria species from other bats and rodents.

PubMed

Afonso, Eve; Baurand, Pierre-Emmanuel; Tournant, Pierline; Capelli, Nicolas

2014-04-01

Although coccidian parasites of the genus Eimeria are among the best-documented parasites in bats, few Eimeria species found in bats have been characterised using molecular tools, and none of the characterised species are found in European countries. Phylogenetic relationships of Eimeria species that parasitise bats and rodents can be related to the morphology of oocysts, independently from host range, suggesting that these species are derived from common ancestors. In the present study, we isolated a partial sequence of the Eimeria hessei 18S rRNA gene from the lesser horseshoe bat (Rhinolophus hipposideros), a European bat species. Droppings from lesser horseshoe bats were collected from 11 maternity roosts located in France that were positive for the presence of the parasite. Through morphological characterisation, the oocysts detected in the lesser horseshoe bat droppings were confirmed to be E. hessei. The unique E. hessei sequence obtained through molecular analysis belonged to a clade that includes both rodent and bat Eimeria species. However, the E. hessei oocysts isolated from the bat droppings did not show morphological similarities to rodent Eimeria species. Copyright © 2014 Elsevier Inc. All rights reserved.

Cryptosporidium erinacei n. sp. (Apicomplexa: Cryptosporidiidae) in hedgehogs.

PubMed

Kváč, Martin; Hofmannová, Lada; Hlásková, Lenka; Květoňová, Dana; Vítovec, Jiří; McEvoy, John; Sak, Bohumil

2014-03-17

The morphological, biological, and molecular characteristics of Cryptosporidium hedgehog genotype are described, and the species name Cryptosporidium erinacei n. sp. is proposed to reflect its specificity for hedgehogs under natural and experimental conditions. Oocysts of C. erinacei are morphologically indistinguishable from Cryptosporidium parvum, measuring 4.5-5.8 μm (mean=4.9 μm) × 4.0-4.8 μm (mean=4.4 μm) with a length to width ratio of 1.13 (1.02-1.35) (n=100). Oocysts of C. erinacei obtained from a naturally infected European hedgehog (Erinaceus europaeus) were infectious for naïve 8-week-old four-toed hedgehogs (Atelerix albiventris); the prepatent period was 4-5 days post infection (DPI) and the patent period was longer than 20 days. C. erinacei was not infectious for 8-week-old SCID and BALB/c mice (Mus musculus), Mongolian gerbils (Meriones unguiculatus), or golden hamsters (Mesocricetus auratus). Phylogenetic analyses based on small subunit rRNA, 60 kDa glycoprotein, actin, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, and heat shock protein 70 gene sequences revealed that C. erinacei is genetically distinct from previously described Cryptosporidium species. Copyright © 2014 Elsevier B.V. All rights reserved.

Morphological and molecular characterization of Eimeria labbeana-like (Apicomplexa:Eimeriidae) in a domestic pigeon (Columba livia domestica, Gmelin, 1789) in Australia.

PubMed

Yang, Rongchang; Brice, Belinda; Elloit, Aileen; Ryan, Una

2016-07-01

An Eimeria species is described from a domestic pigeon (Columba livia domestica). Sporulated oocysts (n = 35) were subspherical, with a smooth bi-layered oocyst wall (1.0 μm thick). Oocysts measured 20.2 × 16.1 (22.0-18.9 × 15.7-18.9) μm, oocyst length/width (L/W) ratio, 1.38. Oocyst residuum and a polar granule were present. The micropyle was absent. Sporocysts are elongate-ovoid, 13.0 × 6.1 (14.5-12.5 × 5.5-7.0) μm, sporocyst L/W ratio, 2.13 (2.0-2.2), sporocyst residuum was present, composed of numerous granules in a spherical or ovoid mass. Each sporocyst contained 2 banana-shaped sporozoites, 12.3 × 3.5 (11.8-13.0 × 3.3-3.6) μm. A spherical-ellipsoid posterior refractile body was found in the sporozoites. A nucleus was located immediately anterior to the posterior refractile body. Molecular analysis was conducted at three loci; the 18S and 28S ribosomal RNA genes and the mitochondrial cytochrome oxidase gene (COI). At the 18S locus, the new isolate shared 98.0% genetic similarity with three Isospora isolates from Japan from the domestic pigeon (Columba livia domestica). At the 28S locus, it grouped separately and shared 92.4% and 92.5% genetic similarity with Isospora anthochaerae (KF766053) from a red wattlebird (Anthochaera carunculata) from Australia and an Isospora sp. (MS-2003 - AY283845) from a Himalayan grey-headed bullfinch (Pyrrhula erythaca) respectively. At COI locus, this new isolate was in a separate clade and shared 95.6% and 90.0% similarity respectively with Eimeria tiliquae n. sp. from a shingleback skink in Australia and an Eimeria sp. from a common pheasant (Phasianus colchicus) from America. Based on the morphological data, this isolate is most similar to Eimeria labbeana. As no molecular data for E. labbeana is available and previous morphological data is incomplete, we refer to the current isolate as E. labbeana-like. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Presence of Cryptosporidium parvum and Giardia lamblia in water samples from Southeast Asia: towards an integrated water detection system.

PubMed

Kumar, Thulasi; Abd Majid, Mohamad Azlan; Onichandran, Subashini; Jaturas, Narong; Andiappan, Hemah; Salibay, Cristina C; Tabo, Hazel A L; Tabo, Norbel; Dungca, Julieta Z; Tangpong, Jitbanjong; Phiriyasamith, Sucheep; Yuttayong, Boonyaorn; Polseela, Raxsina; Do, Binh Nhu; Sawangjaroen, Nongyao; Tan, Tian-Chye; Lim, Yvonne A L; Nissapatorn, Veeranoot

2016-01-13

Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans, is still an issue in Southeast Asia (SEA). This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays. A total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia (53), Thailand (120), the Philippines (33), and Vietnam (15). A physicochemical analysis was conducted. The water samples were processed in accordance with the US Environmental Protection Agency's methods 1622/1623.1, microscopically observed and subsequently screened using qPCR assays. Cryptosporidium oocysts were detected in treated water samples from the Philippines (1/10), with a concentration of 0.06 ± 0.19 oocyst/L, and untreated water samples from Thailand (25/93), Malaysia (17/44), and the Philippines (11/23), with concentrations ranging from 0.13 ± 0.18 to 0.57 ± 1.41 oocyst/L. Giardia cysts were found in treated water samples from the Philippines (1/10), with a concentration of 0.02 ± 0.06 cyst/L, and in untreated water samples from Thailand (20/93), Vietnam (5/10), Malaysia (22/44), and the Philippines (16/23), with concentrations ranging from 0.12 ± 0.3 to 8.90 ± 19.65 cyst/L. The pathogens C. parvum and G. lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene, respectively. C. parvum was detected in untreated water samples from the Philippines (1/23) and Malaysia (2/44), whilst, G. lamblia detected was detected in treated water samples from the Philippines (1/10) and in untreated water samples from Thailand (21/93), Malaysia (12/44), and the Philippines (17/23). Nitrate concentration was found to have a high positive correlation with (oo)cyst (0.993). The presence of (oo)cysts in the water samples means that there is potential risk for zoonotic disease transmission in the studied countries. Detection using qPCR is feasible for quantifying both pathogenic C. parvum and G. lamblia in large water samples.

Investigating Attachment Behaviors of Cryptosporidium Parvum Oocysts Using Collision Efficiency in Laboratory Column Experiments

NASA Astrophysics Data System (ADS)

Park, Y.; Hou, L.; Atwill, R.; Packman, A. I.; Harter, T.

2009-12-01

Cryptosporidium is one of the most common enteric parasites of humans and domestic animals, and a number of outbreaks of Cryprosporidiosis, a diarrheal disease caused by Cryptosporidium have been reported worldwide. Natural porous media has been demonstrated to be an effective filter for removing Cryptosporidium parvum from contaminated water and the amount of Cryptosporidium filtered is known to be highly dependent on physical and chemical conditions of the porous media and the water. Cryptosporidium deposition in saturated porous media involves two main steps: approach and attachment. In contrast to the approach mechanisms, attachment processes have not been systematically described to predict a priori because theories that represent attachment behavior (colloid stability) such as DLVO are insufficient to explain experimental data. For this reason, attachment efficiency is calculated based on empirical data, typically experimental breakthrough curves in laboratory columns or field experiments. In this study, collision (attachment) efficiencies (α) of C. parvum oocyst were calculated to test the effect of chemical property changes on the association of oocysts with sand grains. The breakthrough curve data obtained from twelve column experiments and three models were employed to calculate single collector efficiency (η) and α. The first ten experiments were conducted by changing ionic strength and pH, and mixing with natural sediments under the same physical properties (same η). Our experiment results show that iron coating or clay/suspended solids mixture drastically enhanced oocyst deposition. The experiments also showed that increase in ionic strength and decrease in pH enhanced the attachment efficiency. However, the experiment with 100mM NaCl resulted in low attachment efficiency and the experiment with pH 8.5 showed similar attachment efficiency to the one at pH 7. Based on the results from two additional experiments with different flow velocities, it appears that attachment efficiency changes when the flow velocity changes, which contradicts CFT. The results prove that predicting attachment efficiency of C. parvum oocyst using ionic strength or pH is inappropriate when non-DLVO interactions are involved. A review of our results and comparison to existing data shows that it is challenging to accurately predict the attachment efficiency using single peak value of breakthrough curve data from geochemical information of porous media.

Cryptosporidium and Giardia in Danish organic pig farms: Seasonal and age-related variation in prevalence, infection intensity and species/genotypes.

PubMed

Petersen, Heidi H; Jianmin, Wang; Katakam, Kiran K; Mejer, Helena; Thamsborg, Stig M; Dalsgaard, Anders; Olsen, Annette; Enemark, Heidi L

2015-11-30

Although pigs are commonly infected with Cryptosporidium spp. and Giardia duodenalis, including potentially zoonotic species or genotypes, little is known about age-related infection levels, seasonal differences and genetic variation in naturally infected pigs raised in organic management systems. Therefore, the current study was conducted to assess seasonal and age-related variations in prevalence and infection intensity of Cryptosporidium and Giardia, evaluate zoonotic potential and uncover correlations between species/genotypes, infection intensity and faecal consistency. Shedding of oocysts and cysts ((oo-)cysts) was monitored at quarterly intervals (September 2011-June 2012) in piglets (n = 152), starter pigs (n = 234), fatteners (n = 230) and sows (n = 240) from three organic farms in Denmark. (oo-)Cysts were quantified by immunofluorescence microscopy; and 56/75 subsamples from Cryptosporidium infected pigs were successfully analysed by PCR amplification and partial sequencing of the small subunit (SSU) 18S rRNA and hsp70genes, while 13/67 Giardia subsamples were successfully analysed by amplification and partial sequencing of the 18S rRNA and the gdh genes. Altogether, Cryptosporidium or Giardia infections were observed in 40.9% (350/856) and 14.0% (120/856) of the pigs, respectively, including 8.2% (70/856) infected with both parasites. Prevalence, intensity of infections and presence of Cryptosporidium species varied significantly between age-groups; 53.3% piglets, 72.2% starter pigs, 40.4% fatteners and 2.9% sows were infected with Cryptosporidium, whereas 2.0% piglets, 27.4% starter pigs, 17.8% fatteners and 5.0% sows were infected with Giardia. The overall prevalence was stable throughout the year, except for dual-infections that were more prevalent in September and December (p < 0.05). The infection intensity was age-related for both parasites, and dual-infected pigs tended to excrete lower levels of oocysts compared to pigs harbouring only Cryptosporidium. Likewise, pigs infected with Cryptosporidium scrofarum excreted fewer oocysts (mean CPG: 54,848 ± 194,508CI: 9085-118,781) compared to pigs infected with Cryptosporidium suis (mean OPG: 351,035 ± 351,035CI: 67,953-634,117). No correlation between faecal consistency and (oo-)cyst excretion levels was observed. Of the successfully genotyped isolates, 38/56 (67.9%) were C. scrofarum and 18/56 (32.1%) were C. suis, while the livestock specific G. duodenalis Assemblage E was detected in 11/13 (84.6%) isolates and the potentially zoonotic Assemblage A was identified in 2/13 (15.4%) isolates. Piglets exclusively hosted C. suis, with one exception, while starter pigs and fatteners predominantly hosted C. scrofarum. As organic pigs are partly reared outdoors, environmental contamination with Cryptosporidium and Giardia is inevitable. Nevertheless, the present data indicate that the potential public health risk associated with both of these parasites in Danish organic pig production seems to be negligible. Copyright © 2015 Elsevier B.V. All rights reserved.

New host and distributional records for Cryptosporidium sp. (Apicomplexa: Cryptosporidiidae) from lizards (Sauria: Gekkonidae, Scincidae) from the Cook Islands and Vanuatu, South Pacific

USGS Publications Warehouse

McAllister, Chris T.; Duszynski, Donald W.; Fisher, Robert N.

2013-01-01

Between 1991 and 1993, 295 lizards, comprising 21 species in 2 families (Gekkonidae, Scincidae) from the Cook Islands, Fiji, Palau, Takapoto, and Vanuatu in the South Pacific, were examined for Cryptosporidium oocysts. Only 6 lizards (2%) were found to be passing Cryptosporidium oocysts in their feces, including 2 of 30 (7%) Oceania geckos, Gehyra oceanica, from Rarotonga, Cook Islands, and 4 of 26 (15%) Pacific blue-tailed skinks, Emoia caeruleocauda, from Efate Island, Vanuatu. This represents the largest survey for Cryptosporidium in Pacific island lizards, and we document 2 new host and 2 new locality records for this parasite genus.

Caryospora neofalconis and other enteroparasites in raptors from Mexico

PubMed Central

Santana-Sánchez, G.; Flores-Valle, I.T.; González-Gómez, M.; Vega-Sánchez, V.; Salgado-Miranda, C.; Soriano-Vargas, E.

2015-01-01

A coprological survey of enteroparasites in raptors (60 Falconiformes) from Central Mexico is reported. Three samples contained coccidian unsporulated oocysts, one contained Eimeria sp., one contained trematode eggs and one contained capillarid and trematode eggs and Eimeria sp. After sporulation at the laboratory, oocysts from a Falco peregrinus were identified as Caryospora neofalconis. The phylogenetic analysis of the C. neofalconis (GenBank accession number KT037081) showed a close relationship to the Australian strain RY 2014 isolate 16710 (GenBank accession number KJ634019) of Caryosporadaceloe, with 99.2% similarity. As far as we are aware, this is the first report of C. neofalconis in raptors from Mexico and the Americas. PMID:26543806

Caryospora neofalconis and other enteroparasites in raptors from Mexico.

PubMed

Santana-Sánchez, G; Flores-Valle, I T; González-Gómez, M; Vega-Sánchez, V; Salgado-Miranda, C; Soriano-Vargas, E

2015-12-01

A coprological survey of enteroparasites in raptors (60 Falconiformes) from Central Mexico is reported. Three samples contained coccidian unsporulated oocysts, one contained Eimeria sp., one contained trematode eggs and one contained capillarid and trematode eggs and Eimeria sp. After sporulation at the laboratory, oocysts from a Falco peregrinus were identified as Caryospora neofalconis. The phylogenetic analysis of the C. neofalconis (GenBank accession number KT037081) showed a close relationship to the Australian strain RY 2014 isolate 16710 (GenBank accession number KJ634019) of Caryospora daceloe, with 99.2% similarity. As far as we are aware, this is the first report of C. neofalconis in raptors from Mexico and the Americas.

Diagnostic stages of the parasites of the Florida manatee, Trichechus manatus latirostris.

PubMed

Bando, Monica; Larkin, Iskande V; Wright, Scott D; Greiner, Ellis C

2014-02-01

Limited information is available on diagnostic stages of parasites in Florida manatees (Trichechus manatus latirostris). We examined 67 fecal samples from captive and wild manatees to define the diagnostic stages of the parasite fauna known to occur in Florida manatees. Parasite eggs were freshly extracted ex utero from identified mature helminths and subsequently characterized, illustrated, and matched to those isolated from fecal samples. In addition, coccidian oocysts in the fecal samples were identified. These diagnostic stages included eggs from 5 species of trematodes (Chiorchis fabaceus, Chiorchis groschafti, Pulmonicola cochleotrema, Moniligerum blairi, and Nudacotyle undicola), 1 nematode (Heterocheilus tunicatus), and oocysts of 2 coccidians (Eimeria manatus and Eimeria nodulosa).

Contribution of treated wastewater to the microbiological quality of Seine River in Paris.

PubMed

Moulin, Laurent; Richard, Fanny; Stefania, Sabrina; Goulet, Marion; Gosselin, Sylvie; Gonçalves, Alexandre; Rocher, Vincent; Paffoni, Catherine; Dumètre, Aurélien

2010-10-01

Urban part of Seine River serving as drinking water supply in Paris can be heavily contaminated by Cryptosporidium spp. and Giardia duodenalis. In the absence of agricultural practice in this highly urbanized area, we investigated herein the contribution of treated wastewater to the microbiological quality of this river focusing on these two parasites. Other microorganisms such as faecal bacterial indicators, enteroviruses and oocysts of Toxoplasma gondii were assessed concurrently. Raw wastewaters were heavily contaminated by Cryptosporidium and Giardia (oo)cysts, whereas concentrations of both protozoa in treated wastewater were lower. Treated wastewater, flowed into Seine River, had a parasite concentration closed to the one found along the river, in particular at the entry of a drinking water plant (DWP). Even if faecal bacteria were reliable indicators of a reduction in parasite concentrations during the wastewater treatment, they were not correlated to protozoal contamination of wastewater and river water. Oocysts of T. gondii were not found in both raw and treated wastewater, or in Seine River. Parasitic contamination was shown to be constant in the Seine River up to 40 km upstream Paris. Altogether, these results strongly suggest that treated wastewater does not contribute to the main parasitic contamination of the Seine River usually observed in this urbanized area. Copyright © 2010 Elsevier Ltd. All rights reserved.

Validation of a new technique to detect Cryptosporidium spp. oocysts in bovine feces.

PubMed

Inácio, Sandra Valéria; Gomes, Jancarlo Ferreira; Oliveira, Bruno César Miranda; Falcão, Alexandre Xavier; Suzuki, Celso Tetsuo Nagase; Dos Santos, Bianca Martins; de Aquino, Monally Conceição Costa; de Paula Ribeiro, Rafaela Silva; de Assunção, Danilla Mendes; Casemiro, Pamella Almeida Freire; Meireles, Marcelo Vasconcelos; Bresciani, Katia Denise Saraiva

2016-11-01

Due to its important zoonotic potential, cryptosporidiosis arouses strong interest in the scientific community, because, it was initially considered a rare and opportunistic disease. The parasitological diagnosis of the causative agent of this disease, the protozoan Cryptosporidium spp., requires the use of specific techniques of concentration and permanent staining, which are laborious and costly, and are difficult to use in routine laboratory tests. In view of the above, we conducted the feasibility, development, evaluation and intralaboratory validation of a new parasitological technique for analysis in optical microscopy of Cryptosporidium spp. oocysts, called TF-Test Coccidia, using fecal samples from calves from the city of Araçatuba, São Paulo. To confirm the aforementioned parasite and prove the diagnostic efficiency of the new technique, we used two established methodologies in the scientific literature: parasite concentration by centrifugal sedimentation and negative staining with malachite green (CSN-Malachite) and Nested-PCR. We observed good effectiveness of the TF-Test Coccidia technique, being statistically equivalent to CSN-Malachite. Thus, we verified the effectiveness of the TF-Test Coccidia parasitological technique for the detection of Cryptosporidium spp. oocysts and observed good concentration and morphology of the parasite, with a low amount of debris in the fecal smear. Copyright © 2016 Elsevier B.V. All rights reserved.

Cryptosporidium oocysts and giardia cysts on salad products irrigated with contaminated water.

PubMed

Amorós, Inmaculada; Alonso, José L; Cuesta, Gonzalo

2010-06-01

A field study in Valencia, Spain, was done to determine the occurrence of Giardia and Cryptosporidium on salad products that are frequently eaten raw, such as lettuces and Chinese cabbage, and in irrigation waters. Four water samples were taken weekly 1 month before harvesting the vegetables. All water samples were analyzed using techniques included in the U.S. Environmental Protection Agency Method 1623. Standard methods for detecting protozoan parasites on salad vegetables are not available. Published techniques for the isolation of parasites from vegetables generally have low and variable recovery efficiencies. In this study, vegetables were analyzed using a recently reported method for detection of Cryptosporidium oocysts and Giardia cysts on salad products. The waters tested were positive for both Cryptosporidium and Giardia. Of 19 salad products studied, we observed Cryptosporidium in 12 samples and Giardia in 10 samples. Recoveries of the Texas Red-stained Cryptosporidium and Giardia, which were used as internal controls, were 24.5% +/- 3.5% for Cryptosporidium and 16.7% +/- 8.1% for Giardia (n = 8). This study provides data on the occurrence of Cryptosporidium and Giardia in salad products in Spain. The method was useful in the detection of Cryptosporidium oocysts and Giardia cysts on the vegetables tested, and it provides a useful analytical tool for occurrence monitoring.

Experimental Infection with Sporulated Oocysts of Eimeria maxima (Apicomplexa: Eimeriidae) in Broiler

PubMed Central

Brito, Luciana da S.; Pereira, Elder N.; da Silva, Augusta A.; Bentivóglio Costa Silva, Vinícius; Freitas, Fagner L. da C.

2014-01-01

Through this study we assessed the metabolic and pathological changes in broilers experimentally infected with oocysts of Eimeria maxima. To perform the experiment, we used 150 broiler strain cooB males, with ten days of age, were randomized according to weight and randomly assigned to two experimental groups: the control group was inoculated with 0.5 mL of distilled water; the infected group inoculated with 0.5 mL of solution containing 5 × 104 sporulated oocysts of Eimeria maxima. The live performance was evaluated on day 0 (day of inoculation), 5°, 10°, 15°, 25°, and 35° dpi, being slaughtered by cervical dislocation, fifteen birds/group. Although the sum in meat production was higher in the control group, the weight of the heart and gizzard of the experimental animals showed no significant difference, while the liver had difference on day 5°, 15°, and 35° dpi. The pathologic evaluation showed congested mucosa and presence of large amounts of mucus at 6 dpi. Therefore, it is concluded that the dose of 5 × 104  E. maxima inoculated in the experimental group was enough to cause harm to the animal organism. PMID:26464925

Mechanisms for parasites removal in a waste stabilisation pond.

PubMed

Reinoso, Roberto; Blanco, Saúl; Torres-Villamizar, Linda A; Bécares, Eloy

2011-04-01

A waste stabilisation pond (WSP) system formed by two anaerobic ponds, a facultative pond and a maturation pond was studied from December 2003 to September 2004 in north-western Spain in order to evaluate its efficiency in the removal of faecal indicator bacteria (total coliforms, Escherichia coli, faecal streptococci), coliphages, helminth eggs and protozoan (oo)cysts (Cryptosporidium and Giardia). Furthermore, sediment samples were collected from the bottom of the ponds to assess the settling rates and thus determine the main pathogen removal mechanisms in the WSPs system. The overall removal ranged from 1.4 log units for coliphages in the cold period to 5.0 log units for E. coli in the hot period. Cryptosporidium oocysts were reduced by an average of 96%, Giardia cysts by 98% and helminth eggs by 100%. The anaerobic ponds showed significantly higher surface removal rates (4.6, 5.2 and 3.7 log (oo)cysts/eggs removed m(-2) day(-1), respectively) than facultative and maturation ponds. Sunlight and water physicochemical conditions were the main factors influencing C. parvum oocysts removal both in the anaerobic and maturation ponds, whereas other factors like predation or natural mortality were more important in the facultative pond. Sedimentation, the most commonly proposed mechanism for cyst removal had, therefore, a negligible influence in the studied ponds.

Serum Metabolic Profiling of Oocyst-Induced Toxoplasma gondii Acute and Chronic Infections in Mice Using Mass-Spectrometry

PubMed Central

Zhou, Chun-Xue; Cong, Wei; Chen, Xiao-Qing; He, Shen-Yi; Elsheikha, Hany M.; Zhu, Xing-Quan

2018-01-01

Toxoplasma gondii is an obligate intracellular parasite causing severe diseases in immunocompromised individuals and congenitally infected neonates, such as encephalitis and chorioretinitis. This study aimed to determine whether serum metabolic profiling can (i) identify metabolites associated with oocyst-induced T. gondii infection and (ii) detect systemic metabolic differences between T. gondii-infected mice and controls. We performed the first global metabolomics analysis of mice serum challenged with 100 sporulated T. gondii Pru oocysts (Genotype II). Sera from acutely infected mice (11 days post-infection, dpi), chronically infected mice (33 dpi) and control mice were collected and analyzed using LC-MS/MS platform. Following False Discovery Rate filtering, we identified 3871 and 2825 ions in ESI+ or ESI− mode, respectively. Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) identified metabolomic profiles that clearly differentiated T. gondii-infected and -uninfected serum samples. Acute infection significantly influenced the serum metabolome. Our results identified common and uniquely perturbed metabolites and pathways. Acutely infected mice showed perturbations in metabolites associated with glycerophospholipid metabolism, biosynthesis of amino acid, and tyrosine metabolism. These findings demonstrated that acute T. gondii infection induces a global perturbation of mice serum metabolome, providing new insights into the mechanisms underlying systemic metabolic changes during early stage of T. gondii infection. PMID:29354104

Experimental cryptosporidiosis in kids.

PubMed

Koudela, B; Jirí, V

1997-08-01

The clinical, pathological and parasitological features of cryptosporidiosis resulting from experimental inoculation with 6 x 10(6) Cryptosporidium parvum oocysts were studied in kids. Decreased appetite and depression became apparent 72 h post inoculation. Subsequently watery feces with clumps of mucus and color changes from brown to yellow were observed. The mean duration of diarrhea was 4.2 days. Oocyst shedding started 4 days post inoculation (DPI), started to decrease at 7 DPI, and lasted until 12 DPI. The evidence of high infectivity and fast transmission of C. parvum oocysts was observed under standard zoohygienic conditions. The characteristics of intestinal lesions were similar to those found in other neonatal ruminants infected with C. parvum. The most severe lesions were seen in the posterior jejunum and ileum from 3 to 7 DPI, characterized by villus atrophy, villus blunting, fusion of atrophic villi, crypt hyperplasia, inflammatory infiltration in the lamina propria, and metaplasia of mucosal epithelium. Scanning electron microscopy of ileal epithelium revealed ultrastructural changes on the surface of intestinal mucosa. No cryptosporidia or associated pathological lesions were found in the large intestine or other tissues. The distribution of cryptosporidia in the intestine and number of cryptosporidia per ileal villus on different DPI were also estimated for detailed characterization of the infection in kids as a model for experimental cryptosporidiosis.

Removal of model viruses, E. coli and Cryptosporidium oocysts from surface water by zirconium and chitosan coagulants.

PubMed

Christensen, Ekaterina; Nilsen, Vegard; Håkonsen, Tor; Heistad, Arve; Gantzer, Christophe; Robertson, Lucy J; Myrmel, Mette

2017-10-01

The present work evaluates the effect of contact filtration, preceded by coagulation with zirconium (Zr) and chitosan coagulants, on model microorganisms and waterborne pathogens. River water intended for potable water production was spiked with MS2 and Salmonella Typhimurium 28B bacteriophages, Escherichia coli, and Cryptosporidium parvum oocysts prior to coagulation. The hygienic performance demonstrated by Zr comprised 3.0-4.0 log 10 removal of viruses and 5.0-6.0 log 10 removal of E. coli and C. parvum oocysts. Treatment with chitosan resulted in a removal of 2.5-3.0 log 10 of viruses and parasites, and 4.5-5.0 log 10 of bacteria. A reference coagulant, polyaluminium chloride (PACl), gave a 2.5-3.0 log 10 removal of viruses and 4.5 log 10 of E. coli. These results indicate that both Zr and chitosan enable adequate removal of microorganisms from surface water. The present study also attempts to assess removal rates of the selected microorganisms with regard to their size and surface properties. The isoelectric point of the Salmonella Typhimurium 28B bacteriophage is reported for the first time. The retention of the selected microorganisms in the filter bed appeared to have some correlation with their size, but the effect of the charge remained unclear.

Microscopic and Molecular Detection of Cryptosporidium andersoni and Cryptosporidium xiaoi in Wastewater Samples of Tehran Province, Iran

PubMed Central

HATAM-NAHAVANDI, Kareem; MOHEBALI, Mehdi; MAHVI, Amir-Hossein; KESHAVARZ, Hossein; NAJAFIAN, Hamid-Reza; MIRJALALI, Hamed; REZAEI, Sasan; REZAEIAN, Mostafa

2016-01-01

Background: As a waterborne pathogen, Cryptosporidium is one of the most common causes of gastroenteritis in human and hoofed livestock animals. This study aimed to investigate the distribution of Cryptosporidium spp. in human and livestock wastewaters in Iran, by the 18S rRNA sequence analysis. Methods: A total of 54 raw wastewater samples collected from three urban treatment plants and two slaughterhouses during 2014–2015 in Tehran, Iran. The presence of the Cryptosporidium oocysts was assessed by immunofluorescence with monoclonal antibodies. To characterize the oocysts at the molecular level, the 18S rRNA gene of Cryptosporidium was PCR amplified and sequenced. Results: Of the 54 wastewater samples examined, 34 (62.9%) were positive for Cryptosporidium oocysts using the IFA. Of these, 70.5% (24/34) were positive by PCR, that 91.6% (22/24) were successfully sequenced. The species of C. andersoni (95.4%) and C. xiaoi (4.6%) were detected in livestock wastewater samples. Conclusion: C. andersoni was the major Cryptosporidium sp. found in the aquatic environmental wastewater samples. The high rate of detection of C. andersoni in domestic wastewater was probably the result of the predominancy of this species in cattle herds in Iran. The current study is the first report of C. xiaoi in Iran. PMID:28127361

Coastal development and precipitation drive pathogen flow from land to sea: evidence from a Toxoplasma gondii and felid host system

PubMed Central

VanWormer, Elizabeth; Carpenter, Tim E; Singh, Purnendu; Shapiro, Karen; Wallender, Wesley W.; Conrad, Patricia A.; Largier, John L.; Maneta, Marco P.; Mazet, Jonna A. K.

2016-01-01

Rapidly developing coastal regions face consequences of land use and climate change including flooding and increased sediment, nutrient, and chemical runoff, but these forces may also enhance pathogen runoff, which threatens human, animal, and ecosystem health. Using the zoonotic parasite Toxoplasma gondii in California, USA as a model for coastal pathogen pollution, we examine the spatial distribution of parasite runoff and the impacts of precipitation and development on projected pathogen delivery to the ocean. Oocysts, the extremely hardy free-living environmental stage of T. gondii shed in faeces of domestic and wild felids, are carried to the ocean by freshwater runoff. Linking spatial pathogen loading and transport models, we show that watersheds with the highest levels of oocyst runoff align closely with regions of increased sentinel marine mammal T. gondii infection. These watersheds are characterized by higher levels of coastal development and larger domestic cat populations. Increases in coastal development and precipitation independently raised oocyst delivery to the ocean (average increases of 44% and 79%, respectively), but dramatically increased parasite runoff when combined (175% average increase). Anthropogenic changes in landscapes and climate can accelerate runoff of diverse pathogens from terrestrial to aquatic environments, influencing transmission to people, domestic animals, and wildlife. PMID:27456911

Toxoplasma gondii, source to sea: higher contribution of domestic felids to terrestrial parasite loading despite lower infection prevalence.

PubMed

Vanwormer, Elizabeth; Conrad, Patricia A; Miller, Melissa A; Melli, Ann C; Carpenter, Tim E; Mazet, Jonna A K

2013-09-01

Environmental transmission of Toxoplasma gondii, a global zoonotic parasite, adversely impacts human and animal health. Toxoplasma is a significant cause of mortality in threatened Southern sea otters, which serve as sentinels for disease threats to people and animals in coastal environments. As wild and domestic felids are the only recognized hosts capable of shedding Toxoplasma oocysts into the environment, otter infection suggests land-to-sea pathogen transmission. To assess relative contributions to terrestrial parasite loading, we evaluated infection and shedding among managed and unmanaged feral domestic cats, mountain lions, and bobcats in coastal California, USA. Infection prevalence differed among sympatric felids, with a significantly lower prevalence for managed feral cats (17%) than mountain lions, bobcats, or unmanaged feral cats subsisting on wild prey (73-81%). A geographic hotspot of infection in felids was identified near Monterey Bay, bordering a high-risk site for otter infection. Increased odds of oocyst shedding were detected in bobcats and unmanaged feral cats. Due to their large populations, pet and feral domestic cats likely contribute more oocysts to lands bordering the sea otter range than native wild felids. Continued coastal development may influence felid numbers and distribution, increase terrestrial pathogens in freshwater runoff, and alter disease dynamics at the human-animal-environment interface.

A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China.

PubMed

Liu, Xuehan; He, Tingmei; Zhong, Zhijun; Zhang, Hemin; Wang, Rongjun; Dong, Haiju; Wang, Chengdong; Li, Desheng; Deng, Jiabo; Peng, Guangneng; Zhang, Longxian

2013-10-01

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60×3.99 μm (n=50). The isolate was genetically analyzed using the partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype. © 2013 Elsevier Ireland Ltd. All rights reserved.

Reduction of Cryptosporidium, Giardia, and Fecal Indicators by Bardenpho Wastewater Treatment.

PubMed

Schmitz, Bradley W; Moriyama, Hitoha; Haramoto, Eiji; Kitajima, Masaaki; Sherchan, Samendra; Gerba, Charles P; Pepper, Ian L

2018-06-19

Increased demand for water reuse and reclamation accentuates the importance for optimal wastewater treatment to limit protozoa in effluents. Two wastewater treatment plants utilizing advanced Bardenpho were investigated over a 12-month period to determine the incidence and reduction of Cryptosporidium, Giardia, Cyclospora, and fecal indicators. Results were compared to facilities that previously operated in the same geographical area. Protozoa (oo)cysts were concentrated using an electronegative filter and subsequently detected by fluorescent microscopy and/or PCR methods. Cryptosporidium and Giardia were frequently detected in raw sewage, but Cyclospora was not detected in any wastewater samples. Facilities with Bardenpho treatment exhibited higher removals of (oo)cysts than facilities utilizing activated sludge or trickling filters. This was likely due to Bardenpho systems having increased solid wasting rates; however, this mechanism cannot be confirmed as sludge samples were not analyzed. Use of dissolved-air-flotation instead of sedimentation tanks did not result in more efficient removal of (oo)cysts. Concentrations of protozoa were compared with each other, Escherichia coli, somatic coliphage, and viruses (pepper mild mottle virus, Aichi virus 1, adenovirus, and polyomaviruses JC and BK). Although significant correlations were rare, somatic coliphage showed the highest potential as an indicator for the abundance of protozoa in wastewaters.

Use of fluorescent nanoparticles to investigate nutrient acquisition by developing Eimeria maxima macrogametocytes.

PubMed

Frölich, Sonja; Wallach, Michael

2016-06-29

The enteric disease coccidiosis, caused by the unicellular parasite Eimeria, is a major and reoccurring problem for the poultry industry. While the molecular machinery driving host cell invasion and oocyst wall formation has been well documented in Eimeria, relatively little is known about the host cell modifications which lead to acquisition of nutrients and parasite growth. In order to understand the mechanism(s) by which nutrients are acquired by developing intracellular gametocytes and oocysts, we have performed uptake experiments using polystyrene nanoparticles (NPs) of 40 nm and 100 nm in size, as model NPs typical of organic macromolecules. Cytochalasin D and nocodazole were used to inhibit, respectively, the polymerization of the actin and microtubules. The results indicated that NPs entered the parasite at all stages of macrogametocyte development and early oocyst maturation via an active energy dependent process. Interestingly, the smaller NPs were found throughout the parasite cytoplasm, while the larger NPs were mainly localised to the lumen of large type 1 wall forming body organelles. NP uptake was reduced after microfilament disruption and treatment with nocodazole. These observations suggest that E. maxima parasites utilize at least 2 or more uptake pathways to internalize exogenous material during the sexual stages of development.

A NEW SPECIES OF EIMERIA (APICOMPLEXA: EIMERIIDAE) FROM THE NORTHERN MYOTIS, MYOTIS SEPTENTRIONALIS (CHIROPTERA: VESPERTILIONIDAE), IN OKLAHOMA

PubMed Central

McAllister, Chris T.; Seville, R. Scott; Roehrs, Zachary P.

2012-01-01

During September 2004, 4 adult northern myotis, Myotis septentrionalis, were collected from LeFlore County, Oklahoma (n = 2), and Logan (n = 1) and Yell (n = 1) counties, Arkansas, and their feces examined for coccidian parasites. Three of 4 bats (75%) were passing oocysts of Eimeria spp. Oocysts of Eimeria tumlisoni n. sp. were ovoidal, 17.6 × 16.8 (16–19 × 14–18) μm with a shape index of 1.0 (1.0–1.1). A micropyle and oocyst residuum were absent, although 1–2 bilobed polar granules were often present. Sporocysts were ovoidal, 10.5 × 5.9 (9–12 × 5–7) μm with a shape index of 1.8 (1.6–2.0). A Stieda body was present, but sub–Stieda and para–Stieda bodies were absent. A sporocyst residuum was present consisting of compact to dispersed granules between the sporozoites. The sporozoites were elongate, with subspherical anterior refractile body and spherical posterior refractile body; a nucleus was not discernable. This is the second coccidian reported from this host and the first instance of a bat coccidian reported from Oklahoma. We also document a new geographic record for Eimeria catronensis in Oklahoma, and provide an emended description. PMID:22509940

Morphological and molecular characterization of Eimeria haematodi, coccidian parasite (Apicomplexa: Eimeriidae) in a rainbow lorikeet (Trichoglossus haematodus).

PubMed

Yang, Rongchang; Brice, Belinda; Ryan, Una

2015-06-01

Eimeria haematodi was first described in 1977 from the rainbow lorikeet (Trichoglossus haematodus) in Papua New Guinea. In the present study, we re-describe this coccidian species morphologically and molecularly from a rainbow lorikeet bird in Western Australia (WA). The oocysts were ovoid to slightly piriform and measured 28.5-37.8 by 25.8-33.0 µm (33.3 by 28.1 µm). Oocyst wall was approximately 1.5 µm thick and bilayered. Micropyle (5-7 µm) and oocyst residuum (8.0-10.0 µm) present; polar granule was absent. Sporocysts ellipsoidal, 11.8-13.6 by 8.0-9.6 µm (12.2 by 8.3 µm), with thin convex Stieda body and granular sporocyst residuum (4.0-5.0 µm). Molecular characterization of E. haematodi was conducted at 18S ribosomal RNA and the mitochondrial cytochrome oxidase gene (COI) loci. At the 18S ribosomal RNA locus, E. haematodi shared 98.1% genetic similarity to E. alabamensis from cattle in New South Wales, Australia. At COI locus, E. haematodi was closest (92.3% similarity) to E. praecox from domestic chickens (Gallus gallus domesticus) from Canada and China. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

Detection of Cryptosporidium and Cyclospora Oocysts from Environmental Water for Drinking and Recreational Activities in Sarawak, Malaysia.

PubMed

Bilung, Lesley Maurice; Tahar, Ahmad Syatir; Yunos, Nur Emyliana; Apun, Kasing; Lim, Yvonne Ai-Lian; Nillian, Elexson; Hashim, Hashimatul Fatma

2017-01-01

Cryptosporidiosis and cyclosporiasis are caused by waterborne coccidian protozoan parasites of the genera Cryptosporidium and Cyclospora, respectively. This study was conducted to detect Cryptosporidium and Cyclospora oocysts from environmental water abstracted by drinking water treatment plants and recreational activities in Sarawak, Malaysia. Water samples (12 each) were collected from Sungai Sarawak Kanan in Bau and Sungai Sarawak Kiri in Batu Kitang, respectively. In addition, 6 water samples each were collected from Ranchan Recreational Park and UNIMAS Lake at Universiti Malaysia Sarawak, Kota Samarahan, respectively. Water physicochemical parameters were also recorded. All samples were concentrated by the iron sulfate flocculation method followed by the sucrose floatation technique. Cryptosporidium and Cyclospora were detected by modified Ziehl-Neelsen technique. Correlation of the parasites distribution with water physicochemical parameters was analysed using bivariate Pearson correlation. Based on the 24 total samples of environmental water abstracted by drinking water treatment plants, all the samples (24/24; 100%) were positive with Cryptosporidium , and only 2 samples (2/24; 8.33%) were positive with Cyclospora . Based on the 12 total samples of water for recreational activities, 4 samples (4/12; 33%) were positive with Cryptosporidium , while 2 samples (2/12; 17%) were positive with Cyclospora . Cryptosporidium oocysts were negatively correlated with dissolved oxygen (DO).

Detection of Cryptosporidium and Cyclospora Oocysts from Environmental Water for Drinking and Recreational Activities in Sarawak, Malaysia

PubMed Central

Tahar, Ahmad Syatir; Yunos, Nur Emyliana; Apun, Kasing; Nillian, Elexson; Hashim, Hashimatul Fatma

2017-01-01

Cryptosporidiosis and cyclosporiasis are caused by waterborne coccidian protozoan parasites of the genera Cryptosporidium and Cyclospora, respectively. This study was conducted to detect Cryptosporidium and Cyclospora oocysts from environmental water abstracted by drinking water treatment plants and recreational activities in Sarawak, Malaysia. Water samples (12 each) were collected from Sungai Sarawak Kanan in Bau and Sungai Sarawak Kiri in Batu Kitang, respectively. In addition, 6 water samples each were collected from Ranchan Recreational Park and UNIMAS Lake at Universiti Malaysia Sarawak, Kota Samarahan, respectively. Water physicochemical parameters were also recorded. All samples were concentrated by the iron sulfate flocculation method followed by the sucrose floatation technique. Cryptosporidium and Cyclospora were detected by modified Ziehl-Neelsen technique. Correlation of the parasites distribution with water physicochemical parameters was analysed using bivariate Pearson correlation. Based on the 24 total samples of environmental water abstracted by drinking water treatment plants, all the samples (24/24; 100%) were positive with Cryptosporidium, and only 2 samples (2/24; 8.33%) were positive with Cyclospora. Based on the 12 total samples of water for recreational activities, 4 samples (4/12; 33%) were positive with Cryptosporidium, while 2 samples (2/12; 17%) were positive with Cyclospora. Cryptosporidium oocysts were negatively correlated with dissolved oxygen (DO). PMID:29234679

Does the use of tubular digesters to treat livestock waste lower the risk of infection from Cryptosporidium parvum and Giardia lamblia?

PubMed

Kinyua, Maureen N; Wald, Ileana; Camacho-Céspedes, Fabricio; Izurieta, Ricardo; Haas, Charles N; Ergas, Sarina J

2016-10-01

Worldwide, high incidences of cryptosporidiosis and giardiasis are attributed to livestock waste. Quantitative microbial risk assessment can be used to estimate the risk of livestock related infections from Cryptosporidium parvum and Giardia lamblia. The objective of this paper was to assess the occupational and public health risks associated with management of raw and anaerobically digested livestock waste in two rural communities in Costa Rica based on fomite, soil and crop contamination and livestock waste management exposure pathways. Risks related to cattle waste were greater than swine waste due to cattle shedding more (oo)cysts. Cryptosporidium parvum also posed a greater risk than Giardia lamblia in all exposure pathways due to livestock shedding high loads of Cryptosporidium parvum oocysts and oocysts' lower inactivation rates during anaerobic digestion compared with Giardia lamblia cysts. The risk of infection from exposure to contaminated soil and crops was significantly lower for a community using tubular anaerobic digesters to treat livestock waste compared to a community where the untreated waste was applied to soil. The results indicate that treatment of livestock waste in small-scale tubular anaerobic digesters has the potential to significantly decrease the risk of infection below the World Health Organization's acceptable individual annual risk of infection (10 -4 ).

A NEW SPECIES OF CARYOSPORA (APICOMPLEXA: EIMERIIDAE) FROM THE FLATHEAD SNAKE, TANTILLA GRACILIS (OPHIDIA: COLUBRIDAE), IN SOUTHEASTERN OKLAHOMA

PubMed Central

McAllister, Chris T.; Roehrs, Zachary P.; Seville, R. Scott

2012-01-01

A single flathead snake, Tantilla gracilis, collected in early October 2010 from Choctaw County, Oklahoma, was found to harbor an undescribed species of Caryospora. Oocysts of Caryospora choctawensis n. sp. were spherical to subspherical, 15.8 × 15.0 (14–18 × 14–16) μm with a thick bilayered wall and a shape index (length/width) of 1.1. A micropyle and an oocyst residuum are absent but prominent Stieda and bubble-like sub-Stieda bodies were present as well as a bilobed polar granule near the oocyst wall. Sporocysts were ovoidal, 10.8 × 9.0 (10–12 × 8–9) μm with a shape index of 1.3. The sporocyst residuum was spherical and composed of a cluster of granules often membrane-bound. This is the second time a caryosporan species has been reported from T. gracilis, but the first coccidian ever described from a reptilian host in Oklahoma. Additional T. gracilis from Arkansas (n = 6), Oklahoma (n = 1), and Texas (n = 7) were examined and a single specimen from Newton County, Arkansas, harbored Caryospora gracilis Upton, McAllister, Trauth, and Bibb, 1992, previously reported from T. gracilis collected in Arkansas and Texas. PMID:22191621

A New Host for Caryospora lampropeltis (Apicomplexa: Eimeriidae) from the Eastern Hognose Snake, Heterodon platirhinos (Ophidia: Colubroidea: Dipsadinae), from Arkansas, U.S.A., with a Summary of Hosts of This Coccidian

PubMed Central

Seville, R. Scott; Connior, Matthew B.

2016-01-01

Between May 2012 and July 2013, four eastern hognose snakes (Heterodon platirhinos) were collected from Arkansas (n = 2) and Oklahoma (n = 2), U.S.A., and examined for coccidians. A single H. platirhinos from Arkansas was found to be passing oocysts of Caryospora lampropeltis Anderson, Duszynski, and Marquardt. Oocysts of C. lampropeltis were spheroidal to slightly subspheroidal with a rough, colourless, bi-layered wall, measure 23.5 × 22.8 µm, and have a length/width (L/W) ratio of 1.0; both micropyle and oocyst residuum were absent, but a prominent polar granule was present. Sporocysts are ovoidal, 16.8 × 12.8 µm, L/W 1.3; a prominent Stieda and subStieda body was present; a sporocyst residuum was present and composed of numerous spheroidal granules dispersed into small and large granules. Sporozoites lie lengthwise and parallel in a semi-spiral in sporocyst; a spheroidal anterior refractile and posterior refractile body is present; a single nucleus is located between the 2 refractile bodies. This represents the first report of a caryosporan reported from H. platirhinos as well as the only known coccidian from this host. A summary of hosts of C. lampropeltis is provided. PMID:27917072

Coastal development and precipitation drive pathogen flow from land to sea: evidence from a Toxoplasma gondii and felid host system.

PubMed

VanWormer, Elizabeth; Carpenter, Tim E; Singh, Purnendu; Shapiro, Karen; Wallender, Wesley W; Conrad, Patricia A; Largier, John L; Maneta, Marco P; Mazet, Jonna A K

2016-07-26

Rapidly developing coastal regions face consequences of land use and climate change including flooding and increased sediment, nutrient, and chemical runoff, but these forces may also enhance pathogen runoff, which threatens human, animal, and ecosystem health. Using the zoonotic parasite Toxoplasma gondii in California, USA as a model for coastal pathogen pollution, we examine the spatial distribution of parasite runoff and the impacts of precipitation and development on projected pathogen delivery to the ocean. Oocysts, the extremely hardy free-living environmental stage of T. gondii shed in faeces of domestic and wild felids, are carried to the ocean by freshwater runoff. Linking spatial pathogen loading and transport models, we show that watersheds with the highest levels of oocyst runoff align closely with regions of increased sentinel marine mammal T. gondii infection. These watersheds are characterized by higher levels of coastal development and larger domestic cat populations. Increases in coastal development and precipitation independently raised oocyst delivery to the ocean (average increases of 44% and 79%, respectively), but dramatically increased parasite runoff when combined (175% average increase). Anthropogenic changes in landscapes and climate can accelerate runoff of diverse pathogens from terrestrial to aquatic environments, influencing transmission to people, domestic animals, and wildlife.

Removal of Giardia spp. and Cryptosporidium spp. from water supply with high turbidity: analytical challenges and perspectives.

PubMed

Maciel, P M F; Sabogal-Paz, L P

2016-06-01

Giardia and Cryptosporidium species are a serious problem if present in water supplies. The removal of these protozoans and the adaptation of existing protocols are essential for supplying drinking water to developing countries. Considering this, the aim of this study is to evaluate, on a bench level, the removal of Giardia spp. cysts and of Cryptosporidium spp. oocysts from water with high turbidity, using polyaluminium chloride as a coagulant. Filtration using mixed cellulose ester membranes, followed, or not, by purification through immunomagnetic separation (IMS) was used for detecting protozoans. By evaluating the adopted protocol, without using IMS, retrievals of 80% of cysts and 5% of oocysts were obtained, whereas by using IMS, recoveries of 31.5% of cysts and 5.75% of oocysts were reached. When analyzing the coagulant performance, a dosage of 65 mg L(-1) showed contamination from protozoans in all the samples of filtered water. A dosage of 25 mg L(-1) presented protozoans in 50% of the filtered water samples. The results showed an improved performance for the 25 mg L(-1) dosage; therefore, the control of coagulation and adaptation of detection protocols must be evaluated according to the features of raw water and availability of local resources.

A multi-event capture-recapture analysis of Toxoplasma gondii seroconversion dynamics in farm cats.

PubMed

Simon, Julie Alice; Pradel, Roger; Aubert, Dominique; Geers, Régine; Villena, Isabelle; Poulle, Marie-Lazarine

2018-06-08

Domestic cats play a key role in the epidemiology of the parasite Toxoplasma gondii by excreting environmentally-resistant oocysts that may infect humans and other warm-blooded animals. The dynamics of Toxoplasma gondii seroconversion, used as a proxy for primo-infection dynamics, was investigated in five cat populations living on farms. Serological tests on blood samples from cats were performed every three months over a period of two years, for a total of 400 serological tests performed on 130 cats. Variations in seroconversion rates and associated factors were investigated using a multi-event capture-recapture modelling approach that explicitly accounted for uncertainties in cat age and serological status. Seroprevalence varied between farms, from 15 to 73%, suggesting differential exposure of cats to T. gondii. In farms with high exposure, cats could become infected before reaching the age of six months. Seroconversion rates varied from 0.42 to 0.96 seroconversions per cat per year and were higher in autumn and winter than in spring and summer. Our results suggest inter-farm and seasonal variations in the risks of exposure to T. gondii oocysts for humans and livestock living on farms. The paper also discusses the role of young cats in the maintenance of environmental contamination by T. gondii oocysts on farms.

Post-challenge hematological evaluation with virulent strain of Eimeria tenella in broilers immunized with attenuated strain or sporozoite proteins from homologous strain.

PubMed

Bogado, Alexey Leon Gomel; Garcia, João Luis; Silva, Patrícia Fernandes Nunes da; Balarin, Mara Regina Stipp; Junior, José da Silva Guimarães

2010-01-01

Hematological parameters were evaluated in broilers immunized and challenged with Eimeria tenella. Broiler chickens of Hubbard strain, females, coccidian-free, were kept in wire cages and inoculated on the third day. The experiment was designed to include five sorts of treatment with three replicates each. T1 was the negative control group, T2 received 500 attenuated sporulated oocysts by gavage, T3 was the positive control, T4 received 50 μg of sporozoite protein + Quil A vaccine, and T5 received Quil A without sporozoite protein + PBS, the last two through nasal route on days 0, 7, and 21. On the 31st day, all treatments were challenged with homologous virulent strain of E. tenella in the dose of 8.0 x 10⁴ oocysts, with the exception of T1. One week later, blood sampling, lesion scores, and cecal oocyst count were carried out. The parasitological parameters showed statistical significance (p < 0.05) and there was no damage to the hematological parameters of birds (p > 0.05) by ANOVA test. The correlations suggest that the blood parameters were impaired by effects of the parasite on tissue, showing levels of hemorrhage and/or hydration.

Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis.

PubMed

Castro-Hermida, José Antonio; García-Presedo, Ignacio; Almeida, André; González-Warleta, Marta; Correia Da Costa, José Manuel; Mezo, Mercedes

2008-07-01

Samples of the influent and final effluent from 12 wastewater treatment plants from Galicia (NW, Spain) were analyzed for the presence of Cryptosporidium spp. oocysts and Giardia duodenalis cysts. All of the plants discharge effluent to a hydrographic basin in which there are numerous recreational areas and fluvial beaches. The samples (25-50 liters) were collected in spring, summer, autumn and winter of 2007. A total of 96 samples were analyzed using techniques included in the US Environmental Protection Agency Method 1623. To identify the genotypes present, the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and beta-giardina (G. duodenalis). Both parasites were detected in influent and effluent samples from all treatment plants (100%) throughout the year, and G. duodenalis always outnumbered Cryptosporidium spp. The mean concentration of G. duodenalis per liter of influent was significantly higher (P<0.05) than the mean concentration of Cryptosporidium spp. per liter of influent. The mean concentrations of parasites in influent samples ranged from 6 to 350 Cryptosporidium spp. oocysts per liter and from 89 to 8305 G. duodenalis cysts per liter. In final treated effluent, the mean concentration of parasites ranged from 2 to 390 Cryptosporidium spp. oocysts per liter and from 79 to 2469 G. duodenalis cysts per liter. The distribution of results per season revealed that in all plants, the highest number of (oo)cysts were detected in spring and summer. Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The risk of contamination of water courses by Cryptosporidium spp. and G. duodenalis is therefore considerable. It is important that wastewater treatment authorities reconsider the relevance of the levels of contamination by both parasites in wastewater, and develop adequate countermeasures.

Transmission-blocking activity is determined by transmission-reducing activity and number of control oocysts in Plasmodium falciparum standard membrane-feeding assay.

PubMed

Miura, Kazutoyo; Swihart, Bruce J; Deng, Bingbing; Zhou, Luwen; Pham, Thao P; Diouf, Ababacar; Burton, Timothy; Fay, Michael P; Long, Carole A

2016-07-29

Malaria transmission-blocking vaccines (TBVs) are potentially helpful tools for malaria eradication. The standard membrane-feeding assay (SMFA) is considered one of the "gold standard" assays for TBV development. However, lack of consensus in reporting results from SMFA has made it very challenging to compare results from different studies. Two main readouts, % inhibition in mean oocyst count per mosquito (TRA) and % inhibition in prevalence of infected mosquitoes (TBA), have been used widely. In this study, we statistically modeled the oocyst data in SMFA using data from 105 independent feeding experiments including 9804 mosquitoes. The model was validated using an independent data set that included 10,790 mosquitoes from 110 feeding studies. The model delineates a relationship between TRA, the mean oocyst count in the control mosquitoes (mo-contl), and TBA. While TRA was independent from mo-contl, TBA values changed depending on mo-contl. Regardless of monoclonal or polyclonal antibodies tested, there were strong concordances between observed TBA and predicted TBA based on the model using mo-contl and observed TRA. Simulations showed that SMFA with lower true control means had increased uncertainty in TRA estimates. The strong linkage between TBA, TRA and mo-contl inspired creation of a standardized TBA, a model-based TBA standardized to a target control mean, which allows comparison across multiple feeds regardless of mo-contl. This is the first study showing that the observed TBA can be reasonably predicted by mo-contl and the TRA of the test antibody using independent experimental data. This study indicates that TRA should be used to compare results from multiple feeds with different levels of mo-contl. If a measure of TBA is desired, it is better to report standardized TBA rather than observed TBA. These recommendations support rational comparisons of results from different studies, thus benefiting future TBV development. Published by Elsevier Ltd.

Transmission-blocking activity is determined by transmission-reducing activity and number of control oocysts in Plasmodium falciparum standard membrane-feeding assay

PubMed Central

Deng, Bingbing; Zhou, Luwen; Pham, Thao P.; Diouf, Ababacar; Burton, Timothy; Fay, Michael P.; Long, Carole A.

2016-01-01

Malaria transmission-blocking vaccines (TBVs) are potentially helpful tools for malaria eradication. The standard membrane-feeding assay (SMFA) is considered one of the “gold standard” assays for TBV development. However, lack of consensus in reporting results from SMFA has made it very challenging to compare results from different studies. Two main readouts, % inhibition in mean oocyst count per mosquito (TRA) and % inhibition in prevalence of infected mosquitoes (TBA), have been used widely. In this study, we statistically modeled the oocyst data in SMFA using data from 105 independent feeding experiments including 9,804 mosquitoes. The model was validated using an independent data set that included 10,790 mosquitoes from 110 feeding studies. The model delineates a relationship between TRA, the mean oocyst count in the control mosquitoes (mo-contl), and TBA. While TRA was independent from mo-contl, TBA values changed depending on mo-contl. Regardless of monoclonal or polyclonal antibodies tested, there were strong concordances between observed TBA and predicted TBA based on the model using mo-contl and observed TRA. Simulations showed that SMFA with lower true control means had increased uncertainty in TRA estimates. The strong linkage between TBA, TRA and mo-contl inspired creation of a standardized TBA, a model-based TBA standardized to a target control mean, which allows comparison across multiple feeds regardless of mo-contl. This is the first study showing that the observed TBA can be reasonably predicted by mo-contl and the TRA of the test antibody using independent experimental data. This study indicates that TRA should be used to compare results from multiple feeds with different levels of mo-contl. If a measure of TBA is desired, it is better to report standardized TBA rather than observed TBA. These recommendations support rational comparisons of results from different studies, thus benefiting future TBV development. PMID:27372156

Identification and genotyping of Giardia spp. and Cryptosporidium spp. isolates in aquatic birds in the Salburua wetlands, Álava, Northern Spain.

PubMed

Cano, Lourdes; de Lucio, Aida; Bailo, Begoña; Cardona, Guillermo A; Muadica, Aly Salimo Omar; Lobo, Luis; Carmena, David

2016-05-15

Aquatic birds are known to be suitable hosts for a number of avian-specific species and genotypes of the enteric protozoan parasites Giardia and Cryptosporidium. Waterbirds have also been reported as sporadic carriers of species of both pathogens from human or domestic animal origin via environmental contamination. Because aquatic birds can shed substantial amounts of infective Giardia and Cryptosporidium (oo)cysts to the environment including surface waters intended for human consumption, this situation may pose a potential risk of waterborne zoonotic disease. A total of 265 waterbird faecal samples were collected from May 2014 to June 2015 at Salburua (Álava), one of the most valued continental wetlands in northern Spain. The detection of Giardia oocyst and Cryptosporidium oocysts was carried out by direct fluorescence microscopy and molecular (PCR and sequence analysis) methods targeting the small subunit ribosomal RNA gene of the parasites. Typing of Giardia duodenalis isolates at the sub-assemblage level was based on the specific amplification and sequencing of a partial fragment of the glutamate dehydrogenase gene. Overall, Giardia cysts and Cryptosporidium oocysts were detected in 22 (8.3%) and 6 (2.3%), respectively, of the 265 faecal samples analysed. The two only Giardia isolates characterized (one novel, one known) were assigned to the sub-assemblage BIV of G. duodenalis, none of them previously reported in Spanish human isolates. This finding raises doubts about the actual origin of the infection and whether waterbirds may serve as potential source of infective Giardia cysts to humans via waterborne transmission or through direct contact. The six Cryptosporidium isolates obtained were characterized as avian genotype III (n=4), duck genotype b (n=1), and goose genotype Id (n=1), all considered avian-specific and therefore of negligible risk of zoonotic infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Oocyst-Derived Extract of Toxoplasma Gondii Serves as Potent Immunomodulator in a Mouse Model of Birch Pollen Allergy.

PubMed

Wagner, Angelika; Schabussova, Irma; Drinic, Mirjana; Akgün, Johnnie; Loupal, Gerhard; Kundi, Michael; Joachim, Anja; Wiedermann, Ursula

2016-01-01

Previously, we have shown that oral infection with Toxoplasma gondii oocysts prevented type I allergy in mice. Here we investigated whether the application of a T. gondii oocyst lysate antigen (OLA) could also reduce allergy development. BALB/c mice were immunised twice with OLA followed by sensitisation with the major birch pollen (BP) allergen Bet v 1 and an aerosol challenge with BP extract. First, we tested OLA in vitro. Stimulation of splenocytes and bone marrow-derived dendritic cells (BMDC) with OLA led to the production of pro-inflammatory and regulatory cytokines such as IL-6, IFN-γ and IL-10. Moreover, BMDC exposed to OLA upregulated the maturation markers CD40, CD80, CD86, and MHCII. Furthermore, OLA was recognised by TLR2-transfected human embryonic kidney cells. Immunisation of mice with OLA induced high levels of Toxoplasma-specific IgG antibodies in sera along with increased production of IFN-γ and IL-10 in Toxoplasma-antigen restimulated splenocytes. OLA reduced allergic airway inflammation as manifested by significant reduction of eosinophils in bronchoalveolar fluids, decreased cellular infiltrates and mucus production in the lungs. Accordingly, Bet v 1-specific IgE was decreased in OLA-pretreated mice. The reduced allergic immune responses were accompanied by increased numbers of CD4+CD25highFoxp3+ regulatory T cells in spleens as well as by increased numbers of granulocytic myeloid-derived suppressor cells in lungs when compared to sensitised controls suggesting that these two cell populations might be involved in the suppression of the allergic immune responses. Our data demonstrate that pretreatment with the oocyst extract can exert anti-allergic effects comparable to T. gondii infection. Thus, the immunomodulatory properties of the parasite extract indicate that this extract and in the future defined molecules thereof might serve as immunomodulatory adjuvants in allergy treatment and prophylaxis.

Detection and characterization of diverse coccidian protozoa shed by California sea lions.

PubMed

Girard, Yvette A; Johnson, Christine K; Fritz, Heather M; Shapiro, Karen; Packham, Andrea E; Melli, Ann C; Carlson-Bremer, Daphne; Gulland, Frances M; Rejmanek, Daniel; Conrad, Patricia A

2016-04-01

Tissue-cyst forming coccidia in the family Sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed Southern sea otter (Enhydra lutris). California sea lions (Zalophus californianus), whose coastal habitat overlaps with sea otters, are definitive hosts for coccidian protozoa provisionally named Coccidia A, B and C. While Coccidia A and B have unknown clinical effects on aquatic wildlife hosts, Coccidia C is associated with severe protozoal disease in harbor seals (Phoca vitulina). In this study, we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free-ranging California sea lions on the Pacific Coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture. Coccidia A and B were shed in similar frequency, particularly by yearlings. Oocysts shed by one free-ranging sea lion sampled at Año Nuevo State Park in California were previously unidentified in sea lions and were most similar to coccidia infecting Guadalupe fur seals (Arctocephalus townsendi) diagnosed with protozoal disease in Oregon (USA). Sporulated Coccidia A and B oocysts did not replicate in three strains of mice or in African green monkey kidney cells. However, cultivation experiments revealed that the inoculum of fecally-derived Coccidia A and B oocysts additionally contained organisms with genetic and antigenic similarity to Sarcocystis neurona; despite the absence of detectable free sporocysts in fecal samples by microscopic examination. In addition to the further characterization of Coccidia A and B in free-ranging and hospitalized sea lions, these results provide evidence of a new role for sea lions as putative mechanical vectors of S. neurona, or S. neurona-like species. Future work is needed to clarify the distribution, taxonomical status, and pathogenesis of these parasites in sea lions and other marine mammals that share their the near-shore marine environment.

Detection and characterization of diverse coccidian protozoa shed by California sea lions

PubMed Central

Girard, Yvette A.; Johnson, Christine K.; Fritz, Heather M.; Shapiro, Karen; Packham, Andrea E.; Melli, Ann C.; Carlson-Bremer, Daphne; Gulland, Frances M.; Rejmanek, Daniel; Conrad, Patricia A.

2015-01-01

Tissue-cyst forming coccidia in the family Sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed Southern sea otter (Enhydra lutris). California sea lions (Zalophus californianus), whose coastal habitat overlaps with sea otters, are definitive hosts for coccidian protozoa provisionally named Coccidia A, B and C. While Coccidia A and B have unknown clinical effects on aquatic wildlife hosts, Coccidia C is associated with severe protozoal disease in harbor seals (Phoca vitulina). In this study, we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free-ranging California sea lions on the Pacific Coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture. Coccidia A and B were shed in similar frequency, particularly by yearlings. Oocysts shed by one free-ranging sea lion sampled at Año Nuevo State Park in California were previously unidentified in sea lions and were most similar to coccidia infecting Guadalupe fur seals (Arctocephalus townsendi) diagnosed with protozoal disease in Oregon (USA). Sporulated Coccidia A and B oocysts did not replicate in three strains of mice or in African green monkey kidney cells. However, cultivation experiments revealed that the inoculum of fecally-derived Coccidia A and B oocysts additionally contained organisms with genetic and antigenic similarity to Sarcocystis neurona; despite the absence of detectable free sporocysts in fecal samples by microscopic examination. In addition to the further characterization of Coccidia A and B in free-ranging and hospitalized sea lions, these results provide evidence of a new role for sea lions as putative mechanical vectors of S. neurona, or S. neurona-like species. Future work is needed to clarify the distribution, taxonomical status, and pathogenesis of these parasites in sea lions and other marine mammals that share their the near-shore marine environment. PMID:27141438

[Optimization of Cryptosporidium and Giardia detection in water environment using automatic elution station Filta-Max xpress].

PubMed

Matuszewska, Renata; Szczotko, Maciej; Krogulska, Bozena

2012-01-01

The presence of parasitic protozoa in drinking water is mostly a result of improperly maintened the water treatment process. Currently, in Poland the testing of Cryptosporidium and Giardia in water as a part of routine monitoring of water is not perform. The aim of this study was the optimization of the method of Cryptosporidium and Giardia detection in water according to the main principles of standard ISO 15553:2006 and using Filta-Max xpress automatic elution station. Preliminary tests were performed on the samples contaminated with oocysts and cysts of reference strains of both parasitic protozoa. Further studies were carried out on environmental samples of surface water sampled directly from the intakes of water (21 samples from Vistula River and 8 samples from Zegrzynski Lake). Filtration process and samples volume reducing were performed using an automatic elution system Filta-Max xpress. Next, samples were purified during immunomagnetic separation process (IMS). Isolated cysts and oocysts were stained with FITC and DAPI and than the microscopic observation using an epifluorescence microscope was carried out. Recovery of parasite protozoa in all contaminated water samples after 9-cycles elution process applied was mean 60.6% for Cryptosporidium oocysts and 36.1% for Giardia cysts. Studies on the environmental surface water samples showed the presence of both parasitic protozoa. Number of detected Giardia cysts ranged from 1.0/10 L up to 4.5/10 L in samples from Zegrzynski Lake and from 1.0/10 L up to 38.9/10 L in samples from Vistula River. Cryptosporidium oocysts were present in 50% of samples from the Zegrzynski Lake and in 47.6% of samples from the Vistula River, and their number in both cases was similar and ranged from 0.5 up to 2.5 oocyst/10 L. The results show that applied procedure is appropriate for detection the presence of parasitic protosoan in water, but when water contains much amount of inorganic matter and suspended solids test method have to be modified like subsamples preparation and filtration process speed reduction. The applied method with the modification using Filta-Max xpress system can be useful for the routine monitoring of water. Detection of Cryptosporidium and Giardia in all samples of water taken from the intakes of surface water shows the possibility oftransfering of the protozoan cysts into the water intended for the consumption, therefore the testing of Cryptosporidium and Giardia should be included into the monitoring of water.

Occurrence of Cryptosporidium spp. in Antillean manatees (Trichechus manatus) and Amazonian manatees (Trichechus inunguis) from Brazil.

PubMed

Borges, Joāo Carlos Gomes; Alves, Leucio Câmara; Faustino, Maria Aparecida da Gloria; Marmontel, Miriam

2011-12-01

Infections by Cryptosporidium spp. in aquatic mammals is a major concern due to the possibility of the waterborne transmission of oocysts. The aim of the present study was to report the occurrence of Cryptosporidium spp. in Antillean manatees (Trichechus manatus) and Amazonian manatees (Trichechus inunguis) from Brazil. Fecal samples were collected and processed using Kinyoun's method. Positive samples were also submitted to the direct immunofluorescence test. The results revealed the presence of Cryptosporidium spp. oocysts in 12.5% (17/136) of the material obtained from the Antillean manatees and in 4.3% (05/115) of the samples from the Amazonian manatees. Cryptosporidium spp. infection was more prevalent in captive animals than in free-ranging specimens.

Life cycle of Hepatozoon canis (Apicomplexa: Adeleorina: Hepatozoidae) in the tick Rhipicephalus sanguineus and domestic dog (Canis familiaris).

PubMed

Baneth, Gad; Samish, Michael; Shkap, Varda

2007-04-01

The life cycle of the apicomplexan protozoon Hepatozoon canis in its natural hosts Rhipicephalus sanguineus (tick) and Canis familiaris (domestic dog) was studied in an experimental infection. Tick nymphs were fed on a naturally infected dog, or they were infected by percutaneous injection of blood. Dogs were inoculated by ingestion of adult ticks containing mature oocysts. Gamonts were in syzygy 24 hr after percutaneous injection of ticks. Early oocysts were detected 96 hr after nymph repletion, and mature oocysts in adult ticks were infective to dogs 40 days postmolt. Merogony was detected in dog bone marrow from 13 days postinoculation (PI) and included meronts containing 20-30 micromerozoites, and a second type with 2-4 macromerozoites. Monozoic cysts were observed in the spleen in conjunction with merogony. Gamontogony with infection of leukocytes by micromerozoites occurred from 26 days PI, and gamont parasitemia, which completed the life cycle, was detected 28 days PI. The length of the life cycle from nymphal attachment to parasitemia in dogs was 81 days. Increased body temperatures were evident from 16 to 27 days PI and paralleled the time of intensive bone marrow merogony. Skeletal pain and recumbency were manifested in 2 dogs. This study further elucidates the life cycle of H. canis and provides a sequential morphologic description of H. canis merogony, gamontogony, and sporogony.

Epidemiology of equine Cryptosporidium and Giardia infections.

PubMed

Xiao, L; Herd, R P

1994-01-01

Prevalence and infection patterns of Cryptosporidium and Giardia infections in horses were studied by a direct immunofluorescence staining method. Faecal examinations of 222 horses of different age groups revealed Cryptosporidium infection rates of 15-31% in 66 foals surveyed in central Ohio, southern Ohio and central Kentucky, USA. Only 1 of 39 weanlings, 0 of 46 yearlings, and 0 of 71 mares were positive. Giardia infection was found in all age groups, although the infection rates for foals were higher (17-35%). Chronological study of infection in 35 foals showed that foals started to excrete Cryptosporidium oocysts between 4 and 19 weeks and Giardia cysts between 2 and 22 weeks of age. The cumulative infection rates of Cryptosporidium and Giardia in foals were each 71%. Some foals were concurrently infected with both parasites and excretion of oocysts or cysts was intermittent and long-lasting. The longest duration of excretion was 14 weeks for Cryptosporidium and 16 weeks for Giardia. Excretion of Cryptosporidium oocysts stopped before weaning, while excretion of Giardia cysts continued thereafter. Infected foals were considered the major source of Cryptosporidium infection in foals, whereas infected mares were deemed the major source of Giardia infection in foals. The high infection rate of Giardia in nursing mares suggested a periparturient relaxation of immunity. The results indicated that Cryptosporidium and Giardia infections are common in horses.

[Investigation of Cryptosporidium sp. in workers of the Van municipality slaughterhouse and in slaughtered animals].

PubMed

Ciçek, Mutalip; Körkoca, Hanifi; Gül, Abdurrahman

2008-01-01

This study was carried out in order to investigate the prevalence of Cryptosporidium sp. in slaughtered animals and workers of the Van municipality slaughterhouse in Van. Animals slaughtered at different times and workers who had been working in different departments of the slaughter house were included in the study for three months. A total of 309 fecal specimens from animals including 167 sheep, 56 goats and 86 cattle and 87 fecal specimens from workers were examined for Cryptosporidium sp. oocysts. In slaughtered animals, the modified acid-fast staining method was used to determine the oocysts of Cryptosporidium sp. The fecal samples of slaughter workers were examined by using RIDA (R) Quick Cryptosporidium Strip Test (R-Biopharm, Germany) and the modified acid-fast staining method. Fecal samples found to be positive by stripe test were also confirmed with the ELISA method (R-Biopharm, Germany). Oocysts of Cryptosporidium sp. were found in fecal specimens of 22 sheep (13.17%), 6 goats (10.71%) and 7 cattle (8.13%). Intestinal parasites were observed in 34 fecal specimens of workers (39.08%). Cryptosporidium sp., Hymenolepis nana, Chilomastix mesnili, Endolimax nana, Iodamoeba bütschlii were found in the specimen of one worker (1.14%), Entamoeba coli in 4 workers (4.59%), Blastocystis hominis (9.19%) in 8 workers, and Giardia intestinalis (19.54%) in 17 workers.

Molecular characterization of Cryptosporidium from animal sources in Qinghai province of China.

PubMed

Karanis, Panagiotis; Plutzer, Judit; Halim, Norhaliza Abdul; Igori, Khatanbaatar; Nagasawa, Hideyuki; Ongerth, Jerry; Liqing, Ma

2007-11-01

The presence of Cryptosporidium oocysts in 20 zoo animals of the Xining Zoo, 16 farm yaks and 42 farm goats in Qinghai province, China was investigated by an immunofluorescence test (IFT). The species and/or genotypes were determined by nested polymerase chain reaction (PCR) and sequence analysis of a fragment of the small subunit (SSU) rRNA gene. Cryptosporidium oocysts were found in 16 zoo animals, 2 yaks, and 15 goats by IFT. The IFT positive samples were further investigated by PCR, and 16 of them were found to be positive by that method also. Sequence analysis of the PCR products derived from Cryptosporidium oocysts from Black leopard (Panthera pardus), Heijing He (Grus nigricollis), Barbary sheep (Ammotragus lervia), Takin (Budorcas taxicolor), Lesser panda (Ailurus fulgens), and White-eared pheasant (Crossoptilon crossoptilon) fecal samples matched that of Cryptosporidium parvum mouse genotype. Sequence analyses of other PCR products were consistent with cervine genotype Cryptosporidium from Ibex (Capra ibex), a novel Cryptosporidium genotype from a wild yak (Bos mutus), C. bovis-like genotype from one goat sample and also a novel Cryptosporidium genotype from one other separate goat sample. The present work reports the first data on Cryptosporidium infections in animals from the Qinghai province of mountainous central western China and the first findings of the 'cervine' genotype in Capra ibex, C. bovis-like genotype and the new Cryptosporidium spp. in farm goat and in wild yak.

Comparison of different solar reactors for household disinfection of drinking water in developing countries: evaluation of their efficacy in relation to the waterborne enteropathogen Cryptosporidium parvum.

PubMed

Gómez-Couso, H; Fontán-Sainz, M; Navntoft, C; Fernández-Ibáñez, P; Ares-Mazás, E

2012-11-01

Solar water disinfection (SODIS) is a type of treatment that can significantly improve the microbiological quality of drinking water at household level and therefore prevent waterborne diseases in developing countries. Cryptosporidium parvum is an obligate protozoan parasite responsible for the diarrhoeal disease cryptosporidiosis in humans and animals. Recently, this parasite has been selected by the WHO as a reference pathogen for protozoan parasites in the evaluation of household water treatment options. In this study, the field efficacy of different static solar reactors [1.5 l transparent plastic polyethylene terephthalate (PET) bottles as well as 2.5 l borosilicate glass and 25 l methacrylate reactors fitted with compound parabolic concentrators (CPC)] for solar disinfection of turbid waters experimentally contaminated with C. parvum oocysts was compared. Potential oocyst viability was determined by inclusion/exclusion of the fluorogenic vital dye propidium iodide. The results demonstrate that static solar reactors fitted with CPCs are an excellent alternative to the conventional SODIS method with PET bottles. These reactors improved the efficacy of the SODIS method by enabling larger volumes of water to be treated and, in some cases, the C. parvum oocysts were rendered totally unviable, minimising the negative effects of turbidity. Copyright © 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

The occurrence of an abdominal fauna in an articulated tapir (Tapirus polkensis) from the Late Miocene Gray Fossil Site, Northeastern Tennessee.

PubMed

McCONNELL, Shannon M; Zavada, Michael S

2013-03-01

The analysis of samples recovered from the abdominal area of an articulated tapir (Tapirus polkensis) from the Late Miocene (4.5-7 million BP) Gray Fossil Site (GFS) revealed a rich palyno-fauna comprised of about 94% egg/oocyst-like structures and 6% pollen and other palynomorphs. In addition, a group of 6 hickory nuts (Carya) was recovered from the same area suggesting that the samples represent the abdominal contents. The analysis of a sample from immediately outside the tapir produced a sample with 98% pollen and less than 0.5% egg/oocyst-like structures. The size, shape, and general morphology of egg/oocyst-like structures were analyzed with light and scanning electron microscopy and were compared to a variety of intestinal parasites found in extant ungulates, and the Perissodactyla in particular. We also compared fossil structures to the numbers and kind of intestinal parasites recovered from fecal samples from the Baird's tapir (T. bairdii) in Costa Rica and from samples collected from the lowland tapir (T. terrestris) from Ecuador to assess their similarity to our fossil sample. Based on these data, we discuss what role parasites may have played in the biology of T. polkensis during the Late Miocene-Early Pliocene. © 2012 Wiley Publishing Asia Pty Ltd, ISZS and IOZ/CAS.

Long-Term Transport of Cryptosporidium Parvum

NASA Astrophysics Data System (ADS)

Andrea, C.; Harter, T.; Hou, L.; Atwill, E. R.; Packman, A.; Woodrow-Mumford, K.; Maldonado, S.

2005-12-01

The protozoan pathogen Cryptosporidium parvum is a leading cause of waterborne disease. Subsurface transport and filtration in natural and artificial porous media are important components of the environmental pathway of this pathogen. It has been shown that the oocysts of C. parvum show distinct colloidal properties. We conducted a series of laboratory studies on sand columns (column length: 10 cm - 60 cm, flow rates: 0.7 m/d - 30 m/d, ionic strength: 0.01 - 100 mM, filter grain size: 0.2 - 2 mm, various solution chemistry). Breakthrough curves were measured over relatively long time-periods (hundreds to thousands of pore volumes). We show that classic colloid filtration theory is a reasonable tool for predicting the initial breakthrough, but it is inadequate to explain the significant tailing observed in the breakthrough of C. parvum oocyst through sand columns. We discuss the application of the Continuous Time Random Walk approach to account for the strong tailing that was observed in our experiments. The CTRW is generalized transport modeling framework, which includes the classic advection-dispersion equation (ADE), the fractional ADE, and the multi-rate mass transfer model as special cases. Within this conceptual framework, it is possible to distinguish between the contributions of pore-scale geometrical (physical) disorder and of pore-scale physico-chemical heterogeneities (e.g., of the filtration, sorption, desorption processes) to the transport of C. parvum oocysts.

Further characterisation of two Eimeria species (Eimeria quokka and Eimeria setonicis) in quokkas (Setonix brachyurus).

PubMed

Austen, J M; Friend, J A; Yang, R; Ryan, U M

2014-03-01

The identification and characterisation of novel Eimeria species has largely been based on sporulated oocyst and sporocyst morphology, the host species and the geographical range. Variation in the size and shape of Eimeria oocysts across their host range however, make the identification and characterisation of novel species using traditional methodologies alone problematic. The use of molecular markers and phylogenetic analysis has greatly advanced our ability to characterise Eimeria species and has recently been applied to understand evolutionary relationships among Eimeria species from Australian marsupials. In the present study, Eimeria species isolated from quokkas (Setonix brachyurus) captured from Two Peoples Bay, Bald Island and Rottnest Island, Western Australia, were morphologically identified as Eimeria quokka and Eimeria setonicis. Both Eimeria species were identified as being polymorphic in nature with regards to sporulated oocyst and sporocyst morphometrics. Phylogenetic analysis using 18S rRNA and COI (cytochrome c oxidase subunit 1) genes, grouped E. quokka and E. setonicis within the Eimeria marsupial clade together with Eimeria trichosuri from brushtail possums, Eimeria macropodis from tammar wallabies (Macropus eugenii) and several unidentified macropod Eimeria species from western grey kangaroos (Macropus fuliginosus). This study is the first to characterise E. quokka and E. setonicis by molecular analysis, enabling more extensive resolution of evolutionary relationships among marsupial-derived Eimeria species. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of different inoculation routes on the parasitic sites of Cryptosporidium baileyi infection in chickens.

PubMed

Yuan, Lin; Yan, Wenchao; Wang, Tianqi; Qian, Weifeng; Ding, Ke; Zhang, Longxian; Han, Lifang; Shao, Xiaodong

2014-10-01

Cryptosporidiosis is prevalent in domesticated, caged, and wild birds. Cryptosporidium baileyi, an ascendant species of avian Cryptosporidium, is an important pathogen. It causes respiratory disease in chickens, especially chickens younger than 50 days. In this study, SEM, histological, semi-quantitative PCR, and nested PCR techniques were used to explore the impact of different inoculation routes on sites of C. baileyi infection in chickens. Results showed that inoculation with sporozoites or oocysts via the rectum was an effective means of causing infection. This may provide an important reference for the development of the transfection system of C. baileyi in chickens. Numerous endogenous stages of C. baileyi were observed in the bursas of Fabricius (BF) and cloacas of chickens inoculated with sporozoites or oocysts via the rectum, but no parasite was seen in the tracheas of any of these chickens. In chickens infected with oocysts via the crop, the number of parasites in the BF was approximately 23-fold more than in the trachea. All blood samples collected after inoculation were negative for C. baileyi. These data show that C. baileyi was not transferred by blood circulation between the BF and respiratory tract. Different routes of inoculation were here found to distinctly affect sites of parasitism in chickens. These findings may facilitate further understanding of the biology of C. baileyi and efforts to control avian cryptosporidiosis. Copyright © 2014 Elsevier Inc. All rights reserved.

A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology.

PubMed

Miller, Christopher N; Jossé, Lyne; Brown, Ian; Blakeman, Ben; Povey, Jane; Yiangou, Lyto; Price, Mark; Cinatl, Jindrich; Xue, Wei-Feng; Michaelis, Martin; Tsaousis, Anastasios D

2018-03-01

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Susceptibility of three laboratory strains of Anopheles albimanus (Diptera: Culicidae) to coindigenous Plasmodium vivax circumsporozoite protein phenotypes in southern Mexico.

PubMed

Gonzalez-Ceron, L; Rodriguez, M H; Santillan, F V; Hernandez, J E; Wirtz, R A

2000-05-01

The susceptibility to two coindigenous Plasmodium vivax Grassi & Feletti phenotypes VK210 and VK247 of three colonized Anopheles albimanus Wiedemann strains (white-striped, green and brown) from southern Mexico was investigated. Mosquitoes of the three strains were simultaneously fed with P. vivax-infected patient blood and examined 1 wk later for the presence of oocysts. The circumsporozoite protein phenotype type (VK210 and VK247) was determined by immunoflorescence of salivary gland sporozoites using monoclonal antibodies. The proportions of specimens infected and the number of oocyst per mosquito indicated that all mosquito strains were more susceptible to the phenotype VK210 than to VK247, but the white-striped strain was more susceptible to both parasite phenotypes than the other two strains.

Eimeria pipistrellus n. sp. from Pipistrellus kuhlii (Chiroptera: Vespertilionidae) in Saudi Arabia

PubMed Central

Al-Dakhil, Mohamed; Al-Shawa, Yaser

1999-01-01

Fecal samples from 12 Pipistrellus kuhlii captured at Shagrah, Saudi Arabia, were examined for coccidia and three (25%) found to harbor a undescribed eimerian, herein described as Eimeria pipistrellus n. sp. Sporulated oocysts were subspherical, 24.8×23.2 (22-27×20-25) µm, with a bilayered and smooth wall. The micropyle was absent, but a large oocyst residuum and a single polar granule were present. Sporocysts were ovoid, 11.6×8.3 (10.5-13×7.5-9) µm, with a prominent Stieda body, but without a substiedal body; sporozoites lay head to tail in sporocysts and contained one large posterior refractile body. Eimeria pipistrellus n. sp. is the 3rd species of the genus Eimeria found from bats of the genus Pipistrellus. PMID:10188376

Control of Cryptosporidium with wastewater treatment to prevent its proliferation in the water cycle.

PubMed

Suwa, M; Suzuki, Y

2003-01-01

The outbreak of Cryptosporidiosis in 1996 in Japan is thought to have been enlarged by the proliferation of Cryptosporidium in the water cycle from wastewater to drinking water through the river system. From this experience, the wastewater system must have functions to remove Cryptosporidium oocysts effectively. Efficiencies of wastewater treatment processes to remove oocysts were investigated using pilot plants receiving municipal wastewater. An activated sludge process and a following sand filter showed removal efficiencies of 2 log and 0.5 log, respectively. Poly-aluminium chloride dosage improved the efficiencies by 3 log for the activated sludge process and by 2 log for the sand filter. Chemical precipitation of raw wastewater with poly-aluminium chloride could achieve 1 to 3 log removal according on the coagulant concentration.

Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria, protozoa, and viruses from water.

PubMed

Morales-Morales, Hugo A; Vidal, Guadalupe; Olszewski, John; Rock, Channah M; Dasgupta, Debanjana; Oshima, Kevin H; Smith, Geoffrey B

2003-07-01

The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10(5) to 10(6) CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72%. An E. coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When E. coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 x 10(5) and 2.4 x 10(3) per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of E. coli XL1-Blue (9 x 10(5) CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (10(5) PFU/liter), and phage PP7 (10(5) PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.

Two new species of Isospora (Apicomplexa: Eimeriidae) from skinks Emoia spp. (Sauria: Scincidae), from Fiji and Papua New Guinea

USGS Publications Warehouse

McAllister, Chris T.; Duszynski, Donald W.; Fisher, Robert N.

2013-01-01

Between September and October 1991 and again during September 1992, skinks (Emoia spp.) were collected from various localities on Fiji and Papua New Guinea (PNG) and examined for coccidians. One of 4 (25%) De Vis' emo skinks (Emoia pallidiceps) from PNG harbored an undescribed species of Isospora in its feces. Oocysts of Isospora grinbikpelapalai n. sp. were ellipsoidal to subspheroidal, 18.1 × 14.9 (17–20 × 14–16) μm, with a bilayered wall and a length/width index (L/W) of 1.2. Both micropyle and oocyst residuum were absent, but a prominent polar granule was present. Sporocysts were ovoidal, 10.7 × 7.6 (10–11 × 7–8) μm, with a L/W index of 1.4. Stieda and sub-Stieda bodies were present, but para-Stieda bodies were absent. The sporocyst residuum consisted of large scattered globules dispersed between sporozoites. Sporozoites were elongate with spheroidal anterior and posterior refractile bodies. Isospora grinbikpelapalai was also found in 1 of 2 (50%) Pope's emo skinks (Emoia popei) from PNG. One of 13 (8%) white-bellied copper-striped skinks (Emoia cyanura), from Fiji, was passing another undescribed species of Isospora in its feces. Oocysts of Isospora casei n. sp. were elongate, 31.8 × 21.3 (28–35 × 18–24) μm, with a bilayered wall and a L/W index of 1.5. Micropyle, oocyst residuum, and polar granule were all absent. Sporocysts were ovoidal, 15.3 × 10.6 (14–16 × 10–12) μm, with a L/W index of 1.4. Stieda and sub-Stieda bodies were present, but para-Stieda bodies were absent. The sporocyst residuum consisted of scattered globules among sporozoites or as a cluster surrounding sporozoites. Sporozoites were elongate with spheroidal anterior and posterior refractile bodies. Isospora casei was also found in 1 of 2 (50%) Fiji slender treeskinks (Emoia concolor) from Fiji. This represents the first report of coccidia from Emoia spp. and, to our knowledge, the initial documentation of reptilian coccidia from herpetofauna from Papua New Guinea.

Two new species of Isospora (Apicomplexa: Eimeriidae) from skinks, Emoia spp. (Sauria: Scincidae), from Fiji and Papua New Guinea.

PubMed

McAllister, Chris T; Duszynski, Donald W; Fisher, Robert N

2013-08-01

Between September and October 1991 and again during September 1992, skinks (Emoia spp.) were collected from various localities on Fiji and Papua New Guinea (PNG) and examined for coccidians. One of 4 (25%) De Vis' emo skinks (Emoia pallidiceps) from PNG harbored an undescribed species of Isospora in its feces. Oocysts of Isospora grinbikpelapalai n. sp. were ellipsoidal to subspheroidal, 18.1 × 14.9 (17-20 × 14-16) μm, with a bilayered wall and a length/width index (L/W) of 1.2. Both micropyle and oocyst residuum were absent, but a prominent polar granule was present. Sporocysts were ovoidal, 10.7 × 7.6 (10-11 × 7-8) μm, with a L/W index of 1.4. Stieda and sub-Stieda bodies were present, but para-Stieda bodies were absent. The sporocyst residuum consisted of large scattered globules dispersed between sporozoites. Sporozoites were elongate with spheroidal anterior and posterior refractile bodies. Isospora grinbikpelapalai was also found in 1 of 2 (50%) Pope's emo skinks (Emoia popei) from PNG. One of 13 (8%) white-bellied copper-striped skinks (Emoia cyanura), from Fiji, was passing another undescribed species of Isospora in its feces. Oocysts of Isospora casei n. sp. were elongate, 31.8 × 21.3 (28-35 × 18-24) μm, with a bilayered wall and a L/W index of 1.5. Micropyle, oocyst residuum, and polar granule were all absent. Sporocysts were ovoidal, 15.3 × 10.6 (14-16 × 10-12) μm, with a L/W index of 1.4. Stieda and sub-Stieda bodies were present, but para-Stieda bodies were absent. The sporocyst residuum consisted of scattered globules among sporozoites or as a cluster surrounding sporozoites. Sporozoites were elongate with spheroidal anterior and posterior refractile bodies. Isospora casei was also found in 1 of 2 (50%) Fiji slender treeskinks (Emoia concolor) from Fiji. This represents the first report of coccidia from Emoia spp. and, to our knowledge, the initial documentation of reptilian coccidia from herpetofauna from Papua New Guinea.

Comparative efficacy of diclazuril (Vecoxan®) and toltrazuril (Baycox bovis®) against natural infections of Eimeria bovis and Eimeria zuernii in French calves.

PubMed

Philippe, P; Alzieu, J P; Taylor, M A; Dorchies, Ph

2014-12-15

A blinded, randomized, controlled, multi-centric field study was conducted on French dairy farms (n = 9) to evaluate the long term efficacy of metaphylactic, single oral treatments with either 1 mg/kg body weight (BW) of diclazuril (Vecoxan®), or 15 mg/kg BW of toltrazuril (Baycox®) against natural infections with Eimeria zuernii and/or Eimeria bovis, compared to untreated control animals. A total of 199 calves from nine commercial farms aged between 21 and 55 days old at the start of study were included and randomly allocated to one of three groups. Calves on all farms were observed for a period of 78 days post treatment, using both parasitological (oocyst excretion), and clinical parameters (faecal score and body weight). The assessment of efficacy was based on both control of oocyst excretion, and on the average daily weight gains throughout the study. During the whole study period, the mean number of days with diarrhoea (≥ 2) was similar (0.7 days) between treated groups. Excretion in the untreated group peaked at 21 days after treatment. In both the diclazuril and toltrazuril-treated groups, mean oocyst excretion decreased dramatically in the five days following treatment. Thereafter, particularly towards the end of the study period, oocyst counts and percentage levels of E. zuernii were highest in the toltrazuril-treated group. In pooled data from all trial sites, the average daily weight gain was significantly (p = 0.01) higher (+ 0.057 kg/day) in the diclazuril group when compared to the toltrazuril group, and the average body weight gain of the diclazuril treated group was 4.4 kg higher than the toltrazuril group. On eight of the nine trial sites, the average daily gain was greater in the diclazuril group than in the toltrazuril group. This study demonstrates that, over an extended observation period of 78 days, metaphylactic treatment with both diclazuril and toltrazuril reduces the impact of coccidiosis, but greater performance benefits based on average daily weight gains, were achieved following the use of diclazuril. Copyright © 2014 Elsevier B.V. All rights reserved.

Eimeriosis in Danish dairy calves--correlation between species, oocyst excretion and diarrhoea.

PubMed

Enemark, Heidi Larsen; Dahl, Jan; Enemark, Jörg M Dehn

2013-08-01

The study collected up-to-date data on prevalence and importance of Eimeria infections in Danish dairy calves with suspected clinical eimeriosis and analysed correlation between Eimeria spp., oocyst excretion and diarrhoea. From October 2010 through August 2011, veterinarians collected faecal samples from dairy herds (n = 52) with > 50 cows and a history of diarrhoea in young stock. Individual faecal samples were collected 3–4 weeks following re-housing to common pens from calves (n = 453) aged 3 weeks to 6 months. Faecal consistency and total number of oocysts per gram of faeces (opg) were determined, along with opg values for the specific Eimeria spp. Association between opg and faeces consistency was evaluated in a multinomial, logistic regression model. Overall prevalence of Eimeria spp. was 96.2 % with a prevalence of 60.9 % in individual calves. E. zuernii and/or E. bovis were detected in 88.5 % of the herds and 41.5 % of the calves. Mean opg was 2,040 (range 0–114,000) in the calves, of which 18.1 % had opg values ≥ 1,000. A total of 12 Eimeria spp. was found with the following calf prevalences: E. ellipsoidalis (37 %), E. zuernii (32 %), E. bovis (28 %), E. cylindrica (23 %), E. auburnensis (23 %), E. canadensis (10 %), E. subspherica (8 %), E. alabamensis (7 %), E. bukidnonensis (3 %), E. wyomingensis (1 %), E. pellita (0.2 %), E. brasiliensis (0.2 %). Mixed infections were present in all but one Eimeria-positive herds. Diarrhoea was seen in 35.9 % of the calves, and a significant (p = 0.003) positive correlation was detected between diarrhoea and total opg as well as diarrhoea and oocyst excretion for E. zuernii (p = 0.03), E. bovis (p = 0.05) and E. cylindrica (p = 0.04). No such relationship could be detected for E. ellipsoidalis (p = 0.87), E. subspherica (p = 0.54) or E. auburnensis (p = 0.10). Further studies should focus on possible synergistic effects of multiple Eimeria spp. infections as well as interaction between Eimeria spp. and other enteric pathogens.

Class B Alkaline Stabilization to Achieve Pathogen Inactivation

PubMed Central

Bean, Christine L.; Hansen, Jacqueline J.; Margolin, Aaron B.; Balkin, Helene; Batzer, Glenda; Widmer, Giovanni

2007-01-01

Liming is a cost-effective treatment currently employed in many Class B biosolids production plants in the United States. A bench scale model of lime stabilization was designed to evaluate the persistence of viral, bacterial and parasitic pathogens. The survival of fecal coliforms, Salmonella, adenovirus type 5, rotavirus Wa, bacteriophage MS-2, Cryptosporidium parvum oocysts, Giardia lamblia cysts, and Ascaris lumbricoides ova was evaluated under lime stabilization conditions in a water matrix. Fecal coliforms and Salmonella were undetectable following 2 hours of lime stabilization, demonstrating a 7-log reduction. Adenovirus, MS-2 and rotavirus were below detectable levels following 2 h of liming, demonstrating a 4-log reduction. G. lamblia cysts were also inactivated. A. lumbricoides ova remained viable following 72 hours of liming as did C. parvum oocysts. While this study confirmed that Ascaris ova are resistant to liming, their scarcity in sludge and low recovery efficiencies limit their use as indicator. The persistence of C. parvum oocysts after exposure to lime, suggests that this parasite would be a better choice as indicator for evaluating biosolids intended for land application. The studies done with adenovirus Type 5, rotavirus Wa and male specific bacteriophage provided preliminary data demonstrating similar inactivation rates. Monitoring anthropogenic viruses is a time consuming, labor intensive and expensive process. If further studies could demonstrate that phage could be used as an indicator of other enteric viruses, enhanced monitoring could result in greater acceptance of land application of biosolids while demonstrating no increased public health threat. PMID:17431316

Influence of land use and watershed characteristics on protozoa contamination in a potential drinking water resources reservoir.

PubMed

Keeley, Ann; Faulkner, Barton R

2008-05-01

Relative changes in the microbial quality of Lake Texoma, on the border of Texas and Oklahoma, were investigated by monitoring protozoan pathogens, fecal indicators, and factors influencing the intensity of the microbiological contamination of surface water reservoirs. The watershed serves rural agricultural communities active in cattle ranching, recreation, and is a potential drinking water source. A total of 193 surface water samples were tested over a 27-month period to determine levels of parasite contamination. The overall occurrence of Cryptosporidium oocysts was higher in both frequency and concentration than Giardia cysts. Cryptosporidium oocysts were found in 99% and Giardia cysts in 87% of the samples. Although Cryptosporidium and Giardia occurrence were significantly but not strongly correlated, all other correlation coefficients including turbidity and total dissolved solids were non-significant. Statistically supportable seasonal variations were found suggesting that Cryptosporidium and Giardia were higher in summer and fall than in other seasons of the year. While Cryptosporidium levels were correlated with rainfall, this was not the case with Giardia. The maximum numbers for both protozoan parasites were detected from a site impacted by cattle ranching during calving season. Restriction fragment length polymorphism analysis was used for confirmation of Cryptosporidium in surface waters influenced by agricultural discharges. As we had expected, oocysts were of the bovine type indicating that the Cryptosporidium parvum detected in surface waters perhaps came from cattle living in the watershed.

Efficacy of selected coccidiostats in sandhill cranes (Grus canadensis) following challenge

USGS Publications Warehouse

Carpenter, J.W.; Novilla, M.N.; Hatfield, J.S.

2005-01-01

The anticoccidial efficacy of amprolium, clazuril, and monensin were studied in sandhill cranes (Grus canadensis) infected with a mixture of Eimeria spp. oocysts. Five groups of four 1-day-old sandhill crane chicks were maintained on a crumbled ration containing no coccidiostat, amprolium at 2.2 ppm, clazuril at 1.1 ppm, clazuril at 5.5 ppm, or monensin at 99 ppm. After 2 wk on their respective feeding regimens, birds in each of the five groups were administered 25 ? 103 pooled sporulated Eimeria spp. oocysts per os and observed for another 3 wk. A sixth group of four chicks served as nonmedicated, nonchallenged control during the study. Clinical signs and lesions consistent with disseminated visceral coccidiosis were observed in all challenged controls and birds fed amprolium and clazuril. Birds in these groups died 9?10 days after challenge. In contrast, only one monensin-medicated bird had clinical signs of disseminated visceral coccidiosis, and it died 13 days after challenge (DAC). This and an asymptomatic bird that were necropsied at study termination had less-severe gross and microscopic lesions of disseminated visceral coccidiosis. Two of three monensin-treated birds that survived challenge passed from 50 to 500 coccidial oocysts 11 to 18 DAC but were negative at study termination. Of the coccidiostats tested, monensin, at the dietary level of 99 ppm, was the only anticoccidial drug that provided protection against experimentally induced disseminated visceral coccidiosis in sandhill cranes.

In vivo screening of four phytochemicals/extracts and a fungal immunomodulatory protein against an Eimeria acervulina infection in broilers.

PubMed

Peek, H W; Halkes, S B A; Mes, J J; Landman, W J M

2013-01-01

Besides the anticoccidial drug resistance problem, increasing consumer concerns about food safety and residues have propelled the quest for alternative prevention and control strategies amongst which phytotherapy has gained appeal due to a renewed interest in natural medicine. The objective was in vivo screening of four phytochemicals/extracts and a fungal immunomodulatory protein (FIP) against an Eimeria acervulina infection in broilers. Four phytochemicals/extracts (extract from Echinacea purpurea, betaine (Betain™), curcumin, carvacrol (two different doses)), and a recombinant FIP from Ganoderma lucidum cloned and expressed in Escherichia coli were investigated for their anticoccidial potential. The experiment was conducted in a battery cage trial with 54 cages of eight birds each. Broilers infected with E. acervulina (a low and high infection dose of 10(4) and 10(5) sporulated oocysts, respectively) and treated with the phytochemicals/extracts or the FIP were compared with broilers treated with the anticoccidial salinomycin sodium (Sacox®) and with an untreated uninfected and an untreated infected control group. Coccidiosis lesion scores, body weight gains and oocyst shedding were used as parameters. The results showed a coccidiosis infection dose effect on the mean coccidiosis lesion scores. The phytochemicals/extracts and the FIP failed to reduce coccidiosis lesion scores and oocyst shedding, while salinomycin efficiently controlled the E. acervulina infection and enabled significantly higher body weight gains. In conclusion, the selected phytochemicals/extracts and the FIP did not reduce the lesions of an experimentally induced E. acervulina infection.

Efficacy of selected coccidiostats in sandhill cranes (Grus canadensis) following challenge.

PubMed

Carpenter, James W; Novilla, Meliton N; Hatfield, Jeff S

2005-09-01

The anticoccidial efficacy of amprolium, clazuril, and monensin were studied in sandhill cranes (Grus canadensis) infected with a mixture of Eimeria spp. oocysts. Five groups of four 1-day-old sandhill crane chicks were maintained on a crumbled ration containing no coccidiostat, amprolium at 2.2 ppm, clazuril at 1.1 ppm, clazuril at 5.5 ppm, or monensin at 99 ppm. After 2 wk on their respective feeding regimens, birds in each of the five groups were administered 25 x 10(3) pooled sporulated Eimeria spp. oocysts per os and observed for another 3 wk. A sixth group of four chicks served as nonmedicated, nonchallenged control during the study. Clinical signs and lesions consistent with disseminated visceral coccidiosis were observed in all challenged controls and birds fed amprolium and clazuril. Birds in these groups died 9-10 days after challenge. In contrast, only one monensin-medicated bird had clinical signs of disseminated visceral coccidiosis, and it died 13 days after challenge (DAC). This and an asymptomatic bird that were necropsied at study termination had less-severe gross and microscopic lesions of disseminated visceral coccidiosis. Two of three monensin-treated birds that survived challenge passed from 50 to 500 coccidial oocysts 11 to 18 DAC but were negative at study termination. Of the coccidiostats tested, monensin, at the dietary level of 99 ppm, was the only anticoccidial drug that provided protection against experimentally induced disseminated visceral coccidiosis in sandhill cranes.

Evaluation of fenbendazole for treatment of Giardia infection in cats concurrently infected with Cryptosporidium parvum.

PubMed

Keith, Carey L; Radecki, Steven V; Lappin, Michael R

2003-08-01

To determine whether fenbendazole effectively eliminates Giardia organisms from chronically infected cats that have a concurrent Cryptosporidium parvum infection. 16 clinically normal cats. Eight cats with chronic concurrent Giardia and C parvum infections received fenbendazole (50 mg/kg, PO, q 24 h) for 5 days (treatment-group cats). Feces from each cat were collected and processed 3 days weekly for 23 days after treatment. By use of an immunofluorescent assay for detection of Giardia lamblia cysts and C parvum oocysts, organism numbers were counted and scored. Fecal results from treatment-group cats were compared with those of 8 untreated cats with Giardia infection but no C parvum infection (control-group cats). Four of 8 treatment-group cats had consistently negative results for Giardia infection after treatment. These 4 cats had consistently positive results for C parvum oocysts prior to treatment and consistently negative results after treatment. One treatment-group cat had positive results for cysts on all fecal samples, and 3 treatment-group cats had 1 to 3 negative results and then resumed shedding large numbers of cysts; each of these cats had consistently positive results for C parvum oocysts. When compared with control-group cats, treatment-group cats shed less Giardia cysts during week 1 after treatment but not during week 2. Administration of fenbendazole decreases Giardia cyst shedding to less than detectable numbers in some cats. In our study, persistent C parvum infection may have been associated with failure of fenbendazole to eliminate Giardia infection.

Microbiological indicators of water quality in the Xochimilco canals, Mexico City.

PubMed

Juárez-Figueroa, Luis Alfredo; Silva-Sánchez, Jesús; Uribe-Salas, Felipe Javier; Cifuentes-García, Enrique

2003-01-01

To quantify microbiology indicators of fecal contamination in the effluents of two waste water treatment plants and in samples collected in several canals in Xochimilco. A cross sectional study was performed. Ten sites, 5 from plant effluents and 5 from canals, were selected for sampling during November and December 2001. Fecal coliforms and enterococci were quantified by membrane filtration, male specific (F+) and somatic coliphages by double agar layer technique, and Cryptosporidium oocysts and Giardia cysts by concentration with Envirocheck filter followed by immunofluorescence microscopy quantification. The average of organisms counts from effluents and canal water were compared with t Student test. Treated water discharge in canals showed a low count of Fecal Coliforms (average 40.4/100 ml), enterococci (average 58.8/100 ml) and Cryptosporidium oocysts (average 13.2/100 l), while coliphages and Giardia cyst rendered higher counts (average 1467.5/100 ml and 1199.8/100 l, respectively) suggesting the water treatment methods could fail to remove these agents. A significant lower count of Giardia cysts (average 45/100 l) and no Cryptosporidium oocysts were found in irrigation canals, which suggests a natural clearance of these pathogens. Strains of Escherichia coli isolated in one of the canals contaminated with sewage had antimicrobial multi-resistance that was transferred by conjugation suggesting that resistance is encoded in a plasmid potentially transferable to other pathogenic bacteria. Cost effective and culturally acceptable waste treatment methods will require careful planning and consultation if they are to be adopted and mantained by local populations.

Construction and immunological evaluation of recombinant Lactobacillus plantarum expressing SO7 of Eimeria tenella fusion DC-targeting peptide.

PubMed

Yang, Guilian; Yao, Jiayun; Yang, Wentao; Jiang, Yanlong; Du, Jinfen; Huang, Haibin; Gu, Wei; Hu, Jingtao; Ye, Liping; Shi, Chunwei; Shan, Baolong; Wang, Chunfeng

2017-03-15

The coccidiosis caused by Eimeria tenella (coccidian) and other species is a serious parasitic disease that affects the global poultry breeding industry. Lactobacillus strains exhibit a number of properties that make them attractive candidates as delivery vehicles for presentation to the mucosa of compounds with pharmaceutical interest, particularly vaccines. Here, the recombinant Lactobacillus plantarum (co-expressing SO7 and DCpep gene) was constructed, and its efficacy against E. tenella challenge was evaluated in this study. Broiler chickens were orally immunized with live recombinant L. plantarum NC8-pSIP409-SO7-DCpep for two weeks and were then challenged with 5×10 4 E.tenella sporulated oocysts per chicken. During the experiment, body weight gains, cecum lesion scores, fecal oocyst shedding and antibody responses in serum and intestinal washes were assessed as measures of protective immunity. The results indicated that chickens immunized with live recombinant L. plantarum can increase body weight gains and serum antibody responses compared to the control groups. Meanwhile, fecal oocyst shedding in the immunized group was significantly reduced (p<0.01). Moreover, recombinant L. plantarum can significantly relieve pathological damage in cecum, according to lesion scores and histopathologic cecum sections (p<0.01). Therefore, these results indicate that recombinant L. plantarum NC8-pSIP409-SO7-DCpep could become a promising oral vaccine candidate against E. tenella infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Eimeria ninakohlyakimovae Yakimoff & Rastegaieff, 1930 emend. Levine, 1961 in experimentally infected goats (Capra hircus).

PubMed

Vieira, L S; Lima, J D; Rosa, J S

1997-12-01

The endogenous development and prepatent and patent periods of Eimeria ninakohlyakimovae were studied in 43 1-3-wk-old coccidia-free kids inoculated with 5.0 x 10(4), 1.5 x 10(5), 2.0 x 10(5), or 9.0 x 10(5) sporulated oocysts/kg. Twenty-five kids were killed at 24- or 48-hr intervals, 2-18 days after inoculation (DAI). Two generations of meronts, gamonts, gametes, and oocysts were found in sections stained with hematoxylin and eosin and examined using under light microscopy. The first generation of meronts developed in the endothelium of the lacteals, in the lamina propria, and in the lymphatic vessels of the ileum submucosa. Mature, first-generation meronts, 165.5 x 123.6 microm, were first found 10 DAI. Second-generation merogony developed in the crypt epithelial cells of the cecum and colon; mature meronts, 16.8 x 11.6 microm, were first seen 12 DAI. Gametogenesis occurred in the cecum and colon epithelium; mature microgamonts (16.1 x 13.0 microm), microgametes, macrogametes (14.7 x 12.5 microm), and oocysts (18.3 x 13.3 microm) were seen at 13 DAI. The course of the infection was followed in 18 kids examined every day until 24 DAI. The prepatent period was 14.7 (13-17) days and the patent period 6.8 (4-10) days. The sporulation time at 30 C, with constant aeration, was 2-3 days.

OZONE BYPRODUCT FORMATION

EPA Science Inventory

The use of ozone for water treatment has been increasing as ozone has great potential for degrading water pollutants and inactivating viruses, Giardia cysts, and Cryptosporidium oocysts. Although it appears that ozone generates less undesirable disinfection by-products (DBPs) th...

[Results of parasitological examinations of faecal samples from horses, ruminants, pigs, dogs, cats, hedgehogs and rabbits between 1998 and 2002].

PubMed

Epe, C; Coati, N; Schnieder, T

2004-06-01

The results of coproscopical examinations in horses, ruminants, pigs, dogs, cats, hedgehogs and rabbits between 1998 and 2002 are presented. In 4399 samples from horses 37.4% stages of strongylids, 1.4% anoplocephalids, 1.3% Strongyloides westeri, 0.9% Parascaris equorum, 0.04% Oxyuris equi, 0.04% Eimeria sp. and 0.04% Fasciola hepatica were found. In 998 samples of cattle 22.1% stages of strongylids, 11.2% of Eimeria spp., 3.5% of cryptosporidium, 2.9% of Moniezia spp., 1.3% of Trichuris spp., 0.7% of Dictyocaulus sp., 0.6% of Fasciola hepatica, 0.6% of Strongyloides sp., 0.5% of Nematodirus spp. and 0.4% of Capillaria sp. could be detected. In 524 samples of sheep 60.7% eggs of strongylids, 43.1% oozysts of Eimeria spp., 11.1% stages of Nematodirus spp., 9.5% of Moniezia spp., 7.8% of Trichuris spp., 6.7% of Strongyloides sp., 1.7% of Fasciola hepatica, 1% of Capillaria spp., 0.4% of protostrongylidae, 0.2% of Skrjabinema sp. and 0.2% of Dictyocaulus sp. were found. 33.9% of the 118 samples of goats that were examined were positive for oocysts of Eimeria spp., 30.5% for eggs of strongylids, 6.8% for Nematodirus spp., 4.2% for Trichuris spp., 3.4% for Moniezia spp., 0.8 for protostrongylids and 0.8% for Strongyloides sp. 5.7% of 1427 samples of pigs contained stages of strongylids, 1.5% of Ascaris suum, 0.4% of Isospora, 0.3% of Eimeria spp., 0.3% of Trichuris sp., 0.1% of Giardia sp., 0.1% of cryptosproidium as well as 0.1% of metastrongylids. In 1281 of the samples of dogs 2.3% Giardia sp., 2.3% Isospora sp., 2.2% Toxocara canis, 1.4% ancylostomids, 0.8% taeniids, 0.6% larvae of Crenosoma sp., 0.2% Capillaria sp, 0.2% Trichuris vulpis and 0.2% Hammondia-like oocysts were found. In 441 samples of cats 10.7% stages of Isospora sp., 3.9% eggs of Toxocara cati, 1.6% of ancylostomids, 1.4% of taeniids, 1.1% of Giardia sp., 0.7% of Toxoplasma-like oocysts, 0.7% of Aelurostrongylus abstrusus, 0.5% of Toxascaris leonina and 0.2% of Capillaria spp. were found. Furthermore 0.2% of the samples contained proglottids of Mesocestoides and 0.2% stages of Dipylidium sp. Eggs of Capillaria sp. were found in 33% of the 106 samples of hedgehogs, larvae of Crenosoma striatum in 27.4%, oocysts of Isospora sp. in 5.7% of the cases. In 232 samples of rabbits 56.9% oocysts of Eimeria sp., 4.8% stages of Passalurus ambiguus, 1.3% of strongylids, 0.9% of Strongyloides sp., 0.4% of trematodes were found.

Protozoan Infections

DTIC Science & Technology

1989-01-01

an incidental host, acquire toxoplasmosis transplacentally, by ingestion of oocysts excreted by felines, or by ingestion of tissue cysts from under...cause illness. Encephalitis is the most common manifestation of acute toxoplasmosis in such individ- uals (Ruskin and Remington, 1976). The most

Gastrointestinal parasite egg excretion in young calves in periurban livestock production in Mali.

PubMed

Wymann, Monica Natalie; Traore, Koniba; Bonfoh, Bassirou; Tembely, Saïdou; Tembely, Sékouba; Zinsstag, Jakob

2008-04-01

To acquire the information needed to improve parasite control in periurban cattle production in Mali, repeated sampling of faeces of 694 calves kept around Bamako was done in 2003/2004. The effects of season, age, breed, management type, parasite control and presence of sheep on egg and oocyst counts were determined. A Bayesian model was used with a negative binomial distribution and herd and individual effects, to account for the clustering of calves in herds and the repeated sampling. Interviews were conducted to report the current control strategies. We found eggs of Strongyloides papillosus (Age class 0-1 month: prevalence 39%, 2-3 months: 59%, 5-6 months: 42%), strongyles (14%, 24%, 36%), coccidian oocysts (37%, 68%, 64%) and at low prevalence eggs of Toxocara vitulorum, Moniezia sp., Trichuris sp. and Paramphistomum sp. Season and age effects occurred. Reported utilisation of parasite control was high (92%) but monthly recorded use was significantly lower (61%).

Spinacia oleracea L. Leaf Stomata Harboring Cryptosporidium parvum Oocysts: a Potential Threat to Food Safety ▿ †

PubMed Central

Macarisin, Dumitru; Bauchan, Gary; Fayer, Ronald

2010-01-01

Cryptosporidium parvum is a cosmopolitan microscopic protozoan parasite that causes severe diarrheal disease (cryptosporidiosis) in mammals, including humans and livestock. There is growing evidence of Cryptosporidium persistence in fresh produce that may result in food-borne infection, including sporadic cases as well as outbreaks. However, drinking and recreational waters are still considered the major sources of Cryptosporidium infection in humans, which has resulted in prioritization of studies of parasite etiology in aquatic environments, while the mechanisms of transmission and parasite persistence on edible plants remain poorly understood. Using laser scanning confocal microscopy together with fluorescein-labeled monoclonal antibodies, C. parvum oocysts were found to strongly adhere to spinach plants after contact with contaminated water, to infiltrate through the stomatal openings in spinach leaves, and to persist at the mesophyll level. These findings and the fact that this pathogenic parasite resists washing and disinfection raise concerns regarding food safety. PMID:19933348

Disseminated visceral coccidiosis in Eurasian cranes (Grus grus) in the UK.

PubMed

O'Brien, M F; Brown, M J; Stidworthy, M F; Peirce, M A; Marshall, R N; Honma, H; Nakai, Y

2011-02-26

Clinical disease and mortalities due to disseminated visceral coccidiosis were identified for the first time in a group of captive juvenile Eurasian cranes (Grus grus) in the UK during 2008. Presumptive diagnosis was made from the finding of granulomatous nodules in the liver, spleen and other organs at gross postmortem examination, and confirmed histologically by the presence of intracellular coccidial stages within lesions. The species of coccidian was determined to be Eimeria reichenowi on the basis of faecal oocyst morphology and sequencing of 18S rDNA by PCR. A further outbreak of clinical disease occurred in the same enclosure in 2009, affecting a new group of juvenile Eurasian cranes and demoiselle cranes (Anthropoides virgo) and indicating the persistence of infective oocysts in the environment. Clinical sampling of birds during both years demonstrated positive results from examination of both faecal samples and peripheral blood smears.

Detection and molecular identification of Cryptosporidium species in laboratory rats (Rattus norvegicus) in Ibadan, Nigeria

PubMed

Ayinmode, Adekunle Bamidele; Ogbonna, Nkeiruka Fortunate; Widmer, Giovanni

To study the occurrence of Cryptosporidium infection in laboratory rats (Rattus norvegicus) raised for experimental usage, 134 faecal samples were obtained from two rearing houses in Ibadan and examined for the presence of Cryptosporidium oocyst using the modified acid fast staining technique. Cryptosporidium species in 2 samples positive for microscopy were further characterized by a nested polymerase chain reaction (PCR) amplifying the 18S rRNA gene. Two of 134 samples were positive for the Cryptosporidium oocysts. Sequencing of the small-subunit rRNA amplicons identified the species in the two PCR positive samples as Cryptosporidium andersoni and Cryptosporidium rat genotype. These findings showed that laboratory rat is a potential reservoir for diverse Cryptosporidium species and suggests that laboratory rats should be screened for Cryptosporidium infection prior to experiments, especially where pathogen free animals are not available. This the first report to identify Cryptosporidium species infecting laboratory rats in Nigeria.

Effect of pathogen concentrations on removal of Cryptosporidium and Giardia by conventional drinking water treatment.

PubMed

Assavasilavasukul, Prapakorn; Lau, Boris L T; Harrington, Gregory W; Hoffman, Rebecca M; Borchardt, Mark A

2008-05-01

The presence of waterborne enteric pathogens in municipal water supplies contributes risk to public health. To evaluate the removal of these pathogens in drinking water treatment processes, previous researchers have spiked raw waters with up to 10(6) pathogens/L in order to reliably detect the pathogens in treated water. These spike doses are 6-8 orders of magnitude higher than pathogen concentrations routinely observed in practice. In the present study, experiments were conducted with different sampling methods (i.e., grab versus continuous sampling) and initial pathogen concentrations ranging from 10(1) to 10(6) pathogens/L. Results showed that Cryptosporidium oocyst and Giardia cyst removal across conventional treatment were dependent on initial pathogen concentrations, with lower pathogen removals observed when lower initial pathogen spike doses were used. In addition, higher raw water turbidity appeared to result in higher log removal for both Cryptosporidium oocysts and Giardia cysts.

High occurrence of cyclosporiasis in Istanbul, Turkey, during a dry and warm summer

PubMed Central

2010-01-01

We evaluated the incidence of Cyclospora cayetanensis in immunocompetent, diarrheic patients during the summers of 2006-2009 in Istanbul. Stools from 1876 patients were examined using microscopic techniques. Cyclospora oocysts were observed in wet preparations by light and epifluorescence microscopy and in fecal smears that were stained by Kinyoun's modified acid-fast stain. Characteristic Cyclospora oocysts were observed in 2 patients in 2006, 17 in 2007, and one in 2009. Samples positive for Cyclospora were further analyzed by a single step polymerase chain reaction (PCR) with Cyclospora-specific primers from the ITS-1 region of the genome. The majority of the Cyclospora positive cases (15) were clustered during about 15 days in June 2007, indicating an unusual incidence of cyclosporiasis in this time period. The climatic characteristics of 2007 could have played a role in this high occurrence rate. PMID:20416057

Dual wavelength multiple-angle light scattering system for cryptosporidium detection

NASA Astrophysics Data System (ADS)

Buaprathoom, S.; Pedley, S.; Sweeney, S. J.

2012-06-01

A simple, dual wavelength, multiple-angle, light scattering system has been developed for detecting cryptosporidium suspended in water. Cryptosporidium is a coccidial protozoan parasite causing cryptosporidiosis; a diarrheal disease of varying severity. The parasite is transmitted by ingestion of contaminated water, particularly drinking-water, but also accidental ingestion of bathing-water, including swimming pools. It is therefore important to be able to detect these parasites quickly, so that remedial action can be taken to reduce the risk of infection. The proposed system combines multiple-angle scattering detection of a single and two wavelengths, to collect relative wavelength angle-resolved scattering phase functions from tested suspension, and multivariate data analysis techniques to obtain characterizing information of samples under investigation. The system was designed to be simple, portable and inexpensive. It employs two diode lasers (violet InGaN-based and red AlGaInP-based) as light sources and silicon photodiodes as detectors and optical components, all of which are readily available. The measured scattering patterns using the dual wavelength system showed that the relative wavelength angle-resolved scattering pattern of cryptosporidium oocysts was significantly different from other particles (e.g. polystyrene latex sphere, E.coli). The single wavelength set up was applied for cryptosporidium oocysts'size and relative refractive index measurement and differential measurement of the concentration of cryptosporidium oocysts suspended in water and mixed polystyrene latex sphere suspension. The measurement results showed good agreement with the control reference values. These results indicate that the proposed method could potentially be applied to online detection in a water quality control system.

Effect of nutritional plane on health and performance in dairy calves after experimental infection with Cryptosporidium parvum.

PubMed

Ollivett, Theresa L; Nydam, Daryl V; Linden, Thomas C; Bowman, Dwight D; Van Amburgh, Michael E

2012-12-01

To evaluate the effect of nutritional plane on health and performance of dairy calves after infection with Cryptosporidium parvum. Randomized, controlled trial. 20 Holstein bull calves. Calves were assigned to a higher plane of nutrition (HPN; 0.30 Mcal intake energy/kg of metabolic body weight using a 28% protein-20% fat milk replacer) or conventional nutrition (CN; 0.13 Mcal intake energy/kg of metabolic body weight using a 20% protein-20% fat milk replacer). Calves were inoculated with C parvum oocysts at 3 days old. Fecal and health scores, oocyst counts, weight gain, dry matter intake, and hematologic variables were measured for 21 days. Data were analyzed with nonparametric and regression methods. Results-Body weight (day 1), serum total protein concentration (day 3), and PCV (day 3) were not different between groups. Oocyst shedding was not different between groups. The PCV was higher in the CN group (40%), compared with the HPN group (32%) at the end of the study. Fecal scores (FS) improved faster in the HPN group (median, -0.1 FS/feeding), compared with the CN group (median, -0.06 FS/feeding). The HPN calves had better average daily gain (ADG) than did CN calves (median, 433 g/d vs -48 g/d, respectively). Feed efficiency (ADG:dry matter intake ratio) was better for HPN calves than CN calves (median, 131.9 g/kg vs -31.4 g/kg). After a pathogen challenge, calves maintained hydration, had faster resolution of diarrhea, grew faster, and converted feed with greater efficiency when fed a higher plane of nutrition.

Protection Efficacy of Multivalent Egg Yolk Immunoglobulin against Eimeria tenella Infection in Chickens

PubMed Central

Xu, JJ; Ren, CZ; Wang, SS; Liu, DD; Cao, LQ; Tao, JP

2013-01-01

Background To control avian coccidiosis with drug-independent strategy effectively and safely, multivalent hyperimmune egg yolk immunoglobulin (IgY) was prepared and its ability to protect against Eimeria tenella infection was evaluated. Methods Hens were orally immunized with live oocysts of 5 species of Eimeria for six times, antibody titers in serum and yolk were monitored by indirect enzyme-linked immunosorbent assay. The specific IgY was isolated, purified and lyophilized. IgY powder was orally administrated as dietary supplement in newly hatched chicks at various dosages. Birds were orally challenged with 10000 sporulated oocysts of E. tenella at 10 days of age, weighed and killed at 8 days post challenge, and the protective effect was assessed. Results The averge yeid of IgY was 9.2 mg/ml yolk, the antibody titer of IgY reached to 1:163840 per mg with the purity up to 98%. Chickens fed IgY resulted in reduced mortality, increased body weight gain (BWG), reduced oocyst shedding, reduced caecal lesion score and increased anti-coccidial index. In terms of BWG and caecal lesion, IgY significantly enhanced the resistance of bird at ≥ 0.05% of IgY in the diet when compared with the challenged control group (P<0.05). No significant difference was observed at dosage ≥ 0.5% and 1.0% when BWG and caecal lesion were compared with the sodium salinomycin control group, respectively (P>0.05). Conclusion Supplementing newly hatched chicks with Eimeria-specific IgY represents a promising strategy to prevent avian coccidiosis. PMID:24454440

Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies.

PubMed

Lemos, Vanessa; Graczyk, Thaddeus K; Alves, Margarida; Lobo, Maria Luísa; Sousa, Maria C; Antunes, Francisco; Matos, Olga

2005-12-01

In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the beta-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.

Spatial distribution of soil contamination by Toxoplasma gondii in relation to cat defecation behaviour in an urban area.

PubMed

Afonso, Eve; Lemoine, Mélissa; Poulle, Marie-Lazarine; Ravat, Marie-Caroline; Romand, Stéphane; Thulliez, Philippe; Villena, Isabelle; Aubert, Dominique; Rabilloud, Muriel; Riche, Benjamin; Gilot-Fromont, Emmanuelle

2008-07-01

In urban areas, there may be a high local risk of zoonosis due to high densities of stray cat populations. In this study, soil contamination by oocysts of Toxoplasma gondii was searched for, and its spatial distribution was analysed in relation to defecation behaviour of cats living in a high-density population present in one area of Lyon (France). Sixteen defecation sites were first identified. Cats were then repeatedly fed with marked food and the marked faeces were searched for in the defecation sites. Of 260 markers, 72 were recovered from 24 different cats. Defecation sites were frequented by up to 15 individuals. Soil samples were also examined in order to detect the presence of T. gondii using real-time PCR. The entire study area was then sampled according to cat density and vegetation cover type. Only three of 55 samples were positive and all came from defecation sites. In a second series of observations, 16 defecation sites were sampled. Eight of 62 samples tested positive, originating in five defecation sites. Laboratory experiments using experimental seeding of soil showed that the inoculated dose that can be detected in 50% of assays equals 100-1000oocysts/g, depending on the strain. This study shows that high concentrations of oocysts can be detected in soil samples using molecular methods and suggests that spatial distribution of contamination areas is highly heterogeneous. Positive samples were only found in some of the defecation sites, signifying that at-risk points for human and animal infection may be very localised.

Fluorescent microspheres as surrogates in evaluating the efficacy of riverbank filtration for removing Cryptosporidium parvum oocysts and other pathogens

USGS Publications Warehouse

Harvey, Ronald W.; Metge, David W.; Sheets, Rodney A.; Jasperse, Jay

2011-01-01

A major benefit of riverbank filtration (RBF) is that it provides a relatively effective means for pathogen removal. There is a need to conduct more injection-and-recovery transport studies at operating RBF sites in order to properly assess the combined effects of the site heterogeneities and ambient physicochemical conditions, which are difficult to replicate in the lab. For field transport studies involving pathogens, there is considerable interest in using fluorescent carboxylated microspheres (FCM) as surrogates, because they are chemically inert, negatively charged, easy to detect, available in a wide variety of sizes, and have been found to be nonhazardous in tracer applications. Although there have been a number of in-situ studies comparing the subsurface transport behaviors of FCM to those of bacteria and viruses, much less is known about their suitability for investigations of protozoa. Oocysts of the intestinal protozoan pathogen Cryptosporidium spp are of particular concern for many RBF operations because of their ubiquity and persistence in rivers and high resistance to chlorine disinfection. Although microspheres often have proven to be less-than-ideal analogs for capturing the abiotic transport behavior of viruses and bacteria, there is encouraging recent evidence regarding use of FCM as surrogates for C. parvum oocysts. This chapter discusses the potential of fluorescent microspheres as safe and easy-to-detect surrogates for evaluating the efficacy of RBF operations for removing pathogens, particularly Cryptosporidium, from source waters at different points along the flow path.

A comparative survey of the prevalence of human parasites found in fresh vegetables sold in supermarkets and open-aired markets in Accra, Ghana.

PubMed

Duedu, Kwabena O; Yarnie, Elizabeth A; Tetteh-Quarcoo, Patience B; Attah, Simon K; Donkor, Eric S; Ayeh-Kumi, Patrick F

2014-11-25

Consuming raw vegetables offers essential nutrients that one may not get when such vegetables are usually cooked. However, eating them raw may pose a great risk for transmissions of pathogens. Such risks may be influenced by the sources of the vegetables and washing techniques used. The aim of the study was to compare the prevalence and diversity of parasitic pathogens associated with vegetables sold at the two types of markets in Ghana and compare effectiveness of various washing techniques. We purchased two batches of samples of cabbage, sweet bell pepper, carrot, lettuce, tomato and onion within a two week interval. The vegetables were washed by three methods and the wash solution was concentrated and analyzed for parasites. The prevalent parasites detected were Strongyloides stercoralis larvae (43%) and Cryptosporidium parvum oocyst (16%). Others present were Hookworm ova, Entamoeba histolytica cysts, Giardia lamblia cysts, Cyclospora cayetanensis oocysts, Entamoeba coli cysts, Trichuris trichiuria ova, Enterobius vermicularis ova, Isospora belli oocysts and Fasciolopsis buski ova. Contamination was highest in lettuce (61%) and cabbage and the least contaminated was tomato (18%). Contamination of vegetables sold at the open-aired markets was about ten-times that of the supermarkets. In Ghana, the large open-aired markets are the most patronized and serve as a supply point for most corner shops and stalls. The results thus highlight the potential of fresh vegetables serving as a major source of food-borne disease outbreaks and the contribution of open-aired markets to their transmission. Urgent public education on handling of fresh vegetables is recommended.

Occurrence, Source, and Human Infection Potential of Cryptosporidium and Giardia spp. in Source and Tap Water in Shanghai, China▿

PubMed Central

Feng, Yaoyu; Zhao, Xukun; Chen, Jiaxu; Jin, Wei; Zhou, Xiaonong; Li, Na; Wang, Lin; Xiao, Lihua

2011-01-01

Genotyping studies on the source and human infection potential of Cryptosporidium oocysts in water have been almost exclusively conducted in industrialized nations. In this study, 50 source water samples and 30 tap water samples were collected in Shanghai, China, and analyzed by the U.S. Environmental Protection Agency (EPA) Method 1623. To find a cost-effective method to replace the filtration procedure, the water samples were also concentrated by calcium carbonate flocculation (CCF). Of the 50 source water samples, 32% were positive for Cryptosporidium and 18% for Giardia by Method 1623, whereas 22% were positive for Cryptosporidium and 10% for Giardia by microscopy of CCF concentrates. When CCF was combined with PCR for detection, the occurrence of Cryptosporidium (28%) was similar to that obtained by Method 1623. Genotyping of Cryptosporidium in 17 water samples identified the presence of C. andersoni in 14 water samples, C. suis in 7 water samples, C. baileyi in 2 water samples, C. meleagridis in 1 water sample, and C. hominis in 1 water sample. Therefore, farm animals, especially cattle and pigs, were the major sources of water contamination in Shanghai source water, and most oocysts found in source water in the area were not infectious to humans. Cryptosporidium oocysts were found in 2 of 30 tap water samples. The combined use of CCF for concentration and PCR for detection and genotyping provides a less expensive alternative to filtration and fluorescence microscopy for accurate assessment of Cryptosporidium contamination in water, although the results from this method are semiquantitative. PMID:21498768

Point-of-Use Removal of Cryptosporidium parvum from Water: Independent Effects of Disinfection by Silver Nanoparticles and Silver Ions and by Physical Filtration in Ceramic Porous Media.

PubMed

Abebe, Lydia S; Su, Yi-Hsuan; Guerrant, Richard L; Swami, Nathan S; Smith, James A

2015-11-03

Ceramic water filters (CWFs) impregnated with silver nanoparticles are a means of household-level water treatment. CWFs remove/deactivate microbial pathogens by employing two mechanisms: metallic disinfection and physical filtration. Herein we report on the independent effects of silver salt and nanoparticles on Cryptosporidium parvum and the removal of C. parvum by physical filtration in porous ceramic filter media. Using a murine (mouse) model, we observed that treatment of oocysts with silver nitrate and proteinate-capped silver nanoparticles resulted in decreased infection relative to untreated oocysts. Microscopy and excystation experiments were conducted to support the disinfection investigation. Heat and proteinate-capped silver-nanoparticle treatment of oocysts resulted in morphological modifications and decreased excystation rates of sporozoites. Subsequently, disk-shaped ceramic filters were produced to investigate the transport of C. parvum. Two factors were varied: sawdust size and clay-to-sawdust ratio. Five disks were prepared with combinations of 10, 16, and 20 mesh sawdust and sawdust percentage that ranged from 9 to 11%. C. parvum removal efficiencies ranged from 1.5 log (96.4%) to 2.1 log (99.2%). The 16-mesh/10% sawdust had the greatest mean reduction of 2.1-log (99.2%), though there was no statistically significant difference in removal efficiency. Based on our findings, physical filtration and silver nanoparticle disinfection likely contribute to treatment of C. parvum for silver impregnated ceramic water filters, although the contribution of physical filtration is likely greater than silver disinfection.

ANIMAL SOURCE IDENTIFICATION USING A CRYPTOSPORIDIUM DNA CHARACTERIZATION TECHNIQUE

EPA Science Inventory

This document summarizes the application of a particular molecular method to improve detection and differentiation of species and genotypes of Cryptosporidium oocysts found in environmental samples. Of particular interest is the method's potential for determining the source anim...

Molecular Genotyping of Viable Cryptosporidium Oocysts

EPA Science Inventory

Cryptosporidium is a chlorination-resistant protozoan parasite that causes a self-limiting diarrheal disease in the immunocompetent or severe chronic diarrhea in the immunocompromised. Two species, C. parvum and C. hominis, cause most cases of cryptosporidiosis in humans, while C...

CRYPTOSPORIDIUM INACTIVATION AND REMOVAL RESEARCH

EPA Science Inventory

Bench- and pilot-scale tests were performed to assess the ability of conventional treatment, ozonation and chlorine dioxide to remove and inactivate Cryptosporidium oocysts. The impacts of coagulant type, coagulant dose, raw water quality, filter loading rates and filter media w...

Morphological and molecular characterization of Isospora manorinae n. sp. in a yellow-throated miner (Manorina flavigula wayensis) (Gould, 1840).

PubMed

Yang, Rongchang; Brice, Belinda; Jian, Fuchun; Ryan, Una

2016-04-01

A new Isospora (Apicomplexa:Eimeriidae) species is described from a single yellow-throated miner bird (Manorina flavigula) (subspecies M. f. wayensis) in Western Australia. Sporulated oocysts (n = 32) of this isolate are spherical to subspherical, 22.8 (20.3-23.8) × 18.3 (17.7-18.7) μm, with a shape index (length/width) of 1.25 (1.2-1.3); and a smooth and bilayered oocyst wall, 1.3 μm thick (outer layer 0.9 μm, inner 0.4 μm). A polar granule is present, but the micropyle and oocyst residuum are absent. The sporocysts are lemon-shaped, 15.5 (14.6-15.8) × 9.5 (9.5-10.2) μm, with a shape index of 1.6. Stieda and substieda bodies are present, the Stieda body being knob-like and the substieda body being subspherical-shaped. A sporocyst residuum is present and composed of numerous granules of different size scattered among the sporozoites, a spheroid or subspheroid refractile body is present in the sporozoite. Morphologically, the oocysts from this isolate are different from those of all known valid Isospora spp. Molecular analysis was conducted at 3 loci; the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, this new isolate exhibited 99.2% similarity to Isospora gryphoni and three other Isospora spp. Further analysis of a subgroup of 300 bp long 18S sequences (8), including Isospora anthochaerae was conducted. This new isolate grouped in a clade with I. anthochaerae and exhibited 99.3% similarity. At the 28S locus, this new isolate grouped with I. anthochaerae with which it shared 99.1% similarity. At the COI locus, this new isolate exhibited 96.8% similarity to Isospora sp. JCI-2015 from a spectacled warbler (Sylvia conspicillata) in Spain. Further analysis from a subgroup of shorter COI sequences (n = 13) was performed and this new isolate exhibited 99.1% similarity to I. anthochaerae. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora manorinae n. sp. after its host, the yellow-throated miner (Manorina flavigula wayensis). Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

FINGERPRINTING OF C. PARVUM BY MATRIX ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY

EPA Science Inventory

The oocysts of Cryptosporidium parvum, an enteric protozoan pathogen, are responsible for the worst microbial waterborne outbreak of gastroenteritis in recent history. The 1993 outbreak in Milwaukee, WI, sickened approximately 403,000 individuals, resulting in the hospitalizatio...

Removal Efficiencies and Attachment Coefficients for Cryptosporidium in Sandy Alluvial Riverbank Sediment

EPA Science Inventory

Riverbank filtration has been shown to be effective at removing viable Cryptosporidium parvum oocysts and, therefore, drinking water systems that employ riverbank filtration may receive additional treatment credits beyond that which they can obtain using traditional engineering a...

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

EPA Science Inventory

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs res...

MOLECULAR CLONING AND ANALYSIS OF THE CRYPTOSPORIDIUM PARVUM AMINOPEPTIDASE N GENE. (R828035)

EPA Science Inventory

Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules fo...

MOLECULAR CLONING AND ANALYSIS OF THE CRYPTOSPORIDIUM PARVUM AMINOPEPTIDASE N GENE. (R829180)

EPA Science Inventory

Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules fo...

FLOW CYTOMETRIC DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN HUMAN STOOL SAMPLES. (R824759)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

EFFECT OF LACTOBACILLUS AND BIFIDOBACTERIUM ON CRYPTOSPORIDIUM PARVUM OOCYST VIABILITY. (R826138)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

COMPARISON OF FILTRATION METHODS FOR PRIMARY RECOVERY OF CRYPTOSPORIIDUM PARVUM FROM WATER

EPA Science Inventory

Waterborne disease outbreaks from contaminated drinking water have been linked to the protozoan parasite, Cryptosporidium parvum. To improve monitoring for this agent, the USEPA developed Method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 i...

AGING OF CRYPTOSPORIDIUM PARVUUM OOCYSTS STUDIED BY MALDI-TOF MS

EPA Science Inventory

Cryptosporidium parvum is a protozoan parasite, and it causes a potentially fatal gastrointestinal illness. This water borne pathogen has been the subject of several high profile disease outbreaks in the US and abroad. C. parvum presents challenges for both compliance monitorin...

AGING OF CRYPTOSPORIDIUM PARVUM OOCYSTS STUDIED BY MALDI-TOF MS

EPA Science Inventory

Cryptosporidium parvum is a protozoan parasite, and it causes a potentially fatal gastrointestinal illness. This water borne pathogen has been the subject of several high profile disease outbreaks in the US and abroad. C. parvum presents challenges for both compliance monitoring ...

ENHANCED DAPI STAINING FOR CRYPTOSPORIDIUM IN WATER SAMPLES

EPA Science Inventory

The U.S. Environmental Protection Agency's Method 1623 is used to detect and quantify the presence of {ital Cryptosporidium} spp. oocysts in water. The protocol consists of concentrating a sample, staining this concentrate with a fluorescent antibody, and examining the sample mi...

First description of Cryptosporidium parvum in carrier pigeons (Columba livia).

PubMed

Oliveira, Bruno César Miranda; Ferrari, Elis Domingos; da Cruz Panegossi, Mariele Fernanda; Nakamura, Alex Akira; Corbucci, Flávio Sader; Nagata, Walter Bertequini; Dos Santos, Bianca Martins; Gomes, Jancarlo Ferreira; Meireles, Marcelo Vasconcelos; Widmer, Giovanni; Bresciani, Katia Denise Saraiva

2017-08-30

The carrier pigeon and the domestic pigeon are different breeds of the species Columba livia. Carrier pigeons are used for recreational activities such as bird contests and exhibitions. Due to the close contact with humans, these birds may potentially represent a public health risk, since they can host and disseminate zoonotic parasites, such as those belonging to the genus Cryptosporidium (phylum Apicomplexa). The purpose of this work was the detection by microscopic and molecular techniques of Cryptosporidium spp. oocysts in fecal samples of carrier pigeons, and subsequently to sequence the 18S ribosomal RNA marker of positive samples to identify the species. A total of 100 fecal samples were collected individually in two pigeon breeding facilities from Formiga and Araçatuba, cities located in Minas Gerais state and São Paulo state, Brazil, respectively. The age of the birds ranged from one to 12 years; 56 were females and 44 males. Fecal smears were stained with negative malachite green, whereas the molecular characterization was based on the sequence of a ∼800bp fragment of the 18S rRNA gene. Microscopic examination of fecal smears revealed 4% (4/100) oocyst positivity. On the other hand, 7% (7/100) of positivity were found using nested PCR. Three samples were 99% to 100% similar to Cryptosporidium parvum 18S rDNA type A (Genbank AH006572) and the other three samples had 99% to 100% similarity to C. parvum 18S rDNA type B (Genbank AF308600). To our knowledge, this is the first report of C. parvum oocysts in carrier pigeons. Copyright © 2017 Elsevier B.V. All rights reserved.

The occurrence and genetic characterization of Cryptosporidium and Giardia species in foals in Belgium, The Netherlands, Germany and Greece.

PubMed

Kostopoulou, D; Casaert, S; Tzanidakis, N; van Doorn, D; Demeler, J; von Samson-Himmelstjerna, G; Saratsis, A; Voutzourakis, N; Ehsan, A; Doornaert, T; Looijen, M; De Wilde, N; Sotiraki, S; Claerebout, E; Geurden, T

2015-07-30

Faecal samples were collected from foals between the age of 1 week and 6 months in Belgium, The Netherlands, Germany and Greece. A quantitative direct immunofluorescence assay based on the commercial MERIFLUOR Cryptosporidium/Giardia kit was performed to evaluate the presence of (oo) cysts. Parasite positive samples were genotyped, based on the 18S ribosomal DNA gene and the heat shock protein (HSP70) gene for Cryptosporidium and on the β-giardin gene and the triose phosphate isomerase (TPI) gene for Giardia. In total, 134 foals from Belgium, 44 foals from The Netherlands, 30 foals from Germany and 190 foals from Greece were examined. No Cryptosporidium oocysts were identified in faecal samples from foals in Germany and The Netherlands. In Belgium and Greece, 4.5% and 1.1% of the foals examined were Cryptosporidium positive, respectively, all with a low oocyst excretion ranging from 100 to 2450 oocysts per gram of faeces. For Giardia, 14.2%, 11.4%, 10.0% and 11.6% of the foals in Belgium, The Netherlands, Germany and Greece, respectively, were found to excrete cysts, with a range of 50 up to 4,000,000 cysts per gram of faeces. Younger animals secreted significantly more Giardia cysts than older horses (p<0.05), but no significant correlation between Giardia infection and diarrhoea was observed. Most Giardia positive samples belonged to assemblage AI and/or BIV, but also assemblage E was detected in two samples. Together with the identification of Cryptosporidium horse genotype, this suggests only a low risk for zoonotic transmission. Copyright © 2015 Elsevier B.V. All rights reserved.

Histomorphometry and macroscopic intestinal lesions in broilers infected with Eimeria acervulina.

PubMed

Assis, R C L; Luns, F D; Beletti, M E; Assis, R L; Nasser, N M; Faria, E S M; Cury, M C

2010-03-25

This study aimed at measuring intestinal villi and assessing the intestinal absorptive area in broilers infected with Eimeria acervulina under different treatments to control coccidiosis. The experiment was divided into two stages, carried out in successive housings, raised in the same environment (or aviary). In the first stage, on 25 May 2008, fifty 12-day-old birds were orally inoculated with 3 x 10(3) oocysts of E. acervulina. In the second stage, on July 2008, other 50 birds were allocated on litter contaminated by the feces of birds on the first housing (natural infection by oocysts present in the reused litter). The experiment was arranged in a complete randomized design with five treatments and three replicates of 10 chicks per treatment. Broiler chicks were housed at 1 day of age and autopsies were performed at 21 days of age. Three 2-cm-long segments of the duodenum were excised from each bird and fixed in 10% buffered formalin. A total of 30 slides were prepared for each treatment, totaling 150 evaluated histological sections using H&E staining. Villus morphology was carried out by the HL Image 97 software. The intestinal absorptive area was calculated and macroscopic lesions were classified according to standard lesion scores. Results showed that intestinal villus measurements and absorptive area are directly affected by E. acervulina and that there is direct and positive correlation between the macro and microscopic findings observed in intestinal coccidiosis. E. acervulina causes shortening of villi and reduction in the intestinal absorptive area, affecting broiler growth. The prevention method of litter fermentation during the interval between housings and oral administration of Diclazuril can reduce the severity of intestinal lesions by E. acervulina in broilers impairing oocyst virulence or viability.

EVALUATION OF THE ANTIGENICITY AND IMMUNOGENICITY OF Eimeria tenella BY REPRODUCTIVE INDEX AND HISTOPATHOLOGICAL CHANGES OF CECAL COCCIDIOSIS VIRULENT LIVE VACCINE IN BROILER CHICKENS

PubMed Central

Suprihati, Endang; Yunus, Muchammad

2018-01-01

Background: The development of vaccine to control coccidiosis caused by Eimeria tenella (E. tenella) in chickens is intensifying because of the increasing threat of drug resistance to anticoccidial agents. It is important, therefore, to develop a reliable standard method for the assessment of vaccine afficacy particularly antigenicity and immunogenicity become crucial. Evaluation of E. tenella antigenicity and immunogenicity to some low doses can be reflected by reproductive index and histopathological changes. Materials and Methods: The complete random design of research was used in this study. Sixty of two weeks old broilers were divided into four groups and each group composed 15 replications. The group 1 was chicken group without virulent E. tenella oocyst inoculation. The group 2, 3 and group 4 were chicken group inoculated with virulent E. tenella oocyst at doses of 1.0 x 102, 2.0 x 102, 3.0 x 102, respectively. Then all chicken groups were challenged with E. tenella oocyst at doses of 1.0 x 103. Observation of research that represented antigenicity and immunogenicity was clinical sign, reproductive index, histopathological changes. Results: On virulent E. tenella inoculation step, some clinical signs such as appetite, weakness, and diarrhea were very slight on all chicken groups. While on challenge test step, there were no clinical signs of all chicken groups except the group 1. For the reproductive index of virulent E. tenella inoculation step, there were no significantly differences in all chicken groups except the group 1. As reproductive index, the same result pattern was seen for histopathological changes. Conclusion: The low number virulent E. tenella had low reproductive index and few histopathological changes effect that represents a promising strategy to prevent cecal coccidiosis in chickens. PMID:29619439

EVALUATION OF THE ANTIGENICITY AND IMMUNOGENICITY OF Eimeria tenella BY REPRODUCTIVE INDEX AND HISTOPATHOLOGICAL CHANGES OF CECAL COCCIDIOSIS VIRULENT LIVE VACCINE IN BROILER CHICKENS.

PubMed

Suprihati, Endang; Yunus, Muchammad

2018-01-01

The development of vaccine to control coccidiosis caused by Eimeria tenella ( E. tenella ) in chickens is intensifying because of the increasing threat of drug resistance to anticoccidial agents. It is important, therefore, to develop a reliable standard method for the assessment of vaccine afficacy particularly antigenicity and immunogenicity become crucial. Evaluation of E. tenella antigenicity and immunogenicity to some low doses can be reflected by reproductive index and histopathological changes. The complete random design of research was used in this study. Sixty of two weeks old broilers were divided into four groups and each group composed 15 replications. The group 1 was chicken group without virulent E. tenella oocyst inoculation. The group 2, 3 and group 4 were chicken group inoculated with virulent E. tenella oocyst at doses of 1.0 x 10 2 , 2.0 x 10 2 , 3.0 x 10 2 , respectively. Then all chicken groups were challenged with E. tenella oocyst at doses of 1.0 x 10 3 . Observation of research that represented antigenicity and immunogenicity was clinical sign, reproductive index, histopathological changes. On virulent E. tenella inoculation step, some clinical signs such as appetite, weakness, and diarrhea were very slight on all chicken groups. While on challenge test step, there were no clinical signs of all chicken groups except the group 1. For the reproductive index of virulent E. tenella inoculation step, there were no significantly differences in all chicken groups except the group 1. As reproductive index, the same result pattern was seen for histopathological changes. The low number virulent E. tenella had low reproductive index and few histopathological changes effect that represents a promising strategy to prevent cecal coccidiosis in chickens.

Induction of Protective Immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina Infections Using Dendritic Cell-Derived Exosomes

PubMed Central

Gallego, Margarita; Lee, Sung Hyen; Lillehoj, Hyun Soon; Quilez, Joaquin; Lillehoj, Erik P.; Sánchez-Acedo, Caridad

2012-01-01

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting Th1 cytokines, Th2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the Th1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the Th2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible. PMID:22354026