Infectious Oocysts Research Articles

Corrigendum: Journal of Water and Health 10(1), 147–160: Total and infectious Cryptosporidium oocyst and total Giardia cyst concentrations from distinct agricultural and urban contamination sources in Eastern Canada, Cindy Lalancette, Mylène Généreux, Jacinthe Mailly, Pierre Servais, Caroline Côté, Aubert Michaud, George D. Di Giovanni and Michèle Prévost

Corrigendum: Journal of Water and Health 10(1), 147–160: Total and infectious Cryptosporidium oocyst and total Giardia cyst concentrations from distinct agricultural and urban contamination sources in Eastern Canada, Cindy Lalancette, Mylène Généreux, Jacinthe Mailly, Pierre Servais, Caroline Côté, Aubert Michaud, George D. Di Giovanni and Michèle Prévost

Phagocyte Responses to Protozoan Infection and How Toxoplasma gondii Meets the Challenge

Phagocyte Responses to Protozoan Infection and How Toxoplasma gondii Meets the Challenge

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

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.

Assessing the risk of infectious Cryptosporidium in drinking water

Treated drinking water from 14 surface water treatment plants serving a combined population of approximately 9 million people was monitored for infectious Cryptosporidium oocysts using immunofluorescence microscopy to detect infections in cell culture. No infectious oocysts were detected in 370 samples with a total filtered volume of 349,053 L. The calculated risk of waterborne Cryptosporidium infection for the communities served by the 14 sampled plants was < 1 in 10,000. However, more accurate risk assessments of the threat to public health posed by Cryptosporidium in drinking water are still needed. This study demonstrated that routine infectivity monitoring of large volumes of treated drinking water is feasible. Consequently, testing finished water for infectious Cryptosporidium should be considered during periods of regulatory monitoring such as the second round of the Long Term 2 Enhanced Surface Water Treatment Rule.

The life cycle and fitness domain of gregarine (Apicomplexa) parasites

Theoretical demographic models with accompanying experimental programs provide an important framework to study the life history of organisms. In this paper we examine the fitness characteristics of gregarine parasites (Apicomplexa) and how these evolutionary long-lived parasites are shaped by their own life cycle stages inside and outside a definitive insect host. Although gregarines have been investigated in experimental works, their fitness and population characteristics have not been subject to modeling efforts to help understand their longevity or interactions with their host species. We develop a dynamic, mechanistic population model represented by a system of two differential equations for two of the parasite stages: the mature parasite, or trophont, inside a definitive insect host, and the infectious oocyst stage in the water environment of the host. In contrast to many classical macroparasite models, the force of infection between oocysts and hosts is of sigmoid type. Inside the host, production of the water borne infectious state is modeled by linear production rate in the trophont population with a density-independent trophont mortality. We examine stability of model's equilibria for different parameter values and different host populations. This leads to the definition of a fitness parameter that acts as a bifurcation parameter for the model. The model shows good cause for the establishment and long-time persistence of this common, widespread parasite. It is parameterized by extensive data gathered at Cedar Point Biological Station, and numerical calculations based on those parameters illustrate the dynamics. Possible applications include parasite control in aquacultures.

Eimeripain, a Cathepsin B-Like Cysteine Protease, Expressed throughout Sporulation of the Apicomplexan Parasite Eimeria tenella

The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites.

Effect of the granulometric characteristics of monensin sodium on controlling experimental coccidiosis in broiler chickens

The aim of the present study was to investigate the efficacy of monensin sodium grain size on controlling coccidiosis in broiler chickens. Three hundred and fifty Cobb 700 chicks were infected experimentally with 8 × 10(4) sporulated oocysts of Eimeria spp. The birds were fed with diets supplemented with powdered (n = 150) or granulated (n = 150) monensin, with particle sizes of ≤ 100 µm and 450-650 µm, respectively. The remaining chicks (n = 50) were used as an unmedicated control group. The birds' weights and number of oocysts per gram of litter in each group were recorded weekly. A total of 96 birds were randomly selected and culled during the trial. Their intestinal oocyst counts and lesion scores were determined. No significant differences in body weights were found between the groups at the end of the study. The percentages of infected animals, oocyst counts and lesion scores were significantly higher in the control group than in the other two groups; the two treated groups did not show any appreciable differences to each other. Enteritis was observed in 14 birds, all in the group supplemented with granulated monensin. These results suggest that powdered and granulated monensin have comparable efficacy in controlling coccidiosis in broiler chickens. However, treatments with monensin sodium of high grain size may be subject to contraindications relating to enteritis.

Total and infectious Cryptosporidium oocyst and total Giardia cyst concentrations from distinct agricultural and urban contamination sources in Eastern Canada

Cryptosporidium and Giardia (oo)cyst concentrations are frequently used for assessing drinking water safety. The widely used USEPA Method 1623 provides total counts of (oo)cysts, but may not be accurate for human health risk characterization, since it does not provide infectivity information. The total counts and infectious fraction of Cryptosporidium oocysts and the total counts of Giardia cysts were assessed in major fecal pollution sources. Fresh calf and cow feces, their manure, and the discharge point were sampled in a small rural sub-watershed (n = 20, 21, 10, 10). Median concentrations for total (oo)cysts were higher in calves (333 oocysts g(-1); 111 cysts g(-1)) than in cows (52 oocysts g(-1); 7 cysts g(-1)). Infectious oocysts were found in 17 (7%) of the samples and none were found in manure or at the discharge point. Urban sources were sampled in the influent and effluent (n = 19, 18) of two wastewater treatment plants. Peak concentrations were 533 oocysts L(-1) and 9,010 cysts L(-1) for influents and 89 oocysts L(-1) and 472 cysts L(-1) for effluents. Infectious oocyst fractions varied from below the detection limit to 7-22% in the effluent and influent respectively. These infectious fractions are significantly lower than those currently used for quantitative microbial risk assessment estimates.

Assessing the effects of tertiary treated wastewater reuse on a Mediterranean river (Llobregat, NE Spain), part III: pathogens and indicators

Need, coupled with advances in water treatment technology, is motivating a growing interest in augmenting drinking water supplies with reclaimed water. Using reclaimed water to increase the flow of the Llobregat River upstream the water catchment site of the complex multi-step drinking water treatment plant of Sant Joan Despí has been considered. The impact of reclaimed water discharges on the load of E. coli, spores of sulphite-reducing clostridia, somatic coliphages, cytopathogenic enteroviruses, and total and infectious Cryptosporidium oocysts in the Llobregat River water was assessed to gain information for funded decisions in potential future emergencies. Enterovirus and Cryptosporidium oocysts were concentrated from great water volumes prior to enumeration, whereas indicators were enumerated directly from the samples. Both indicators and pathogens were enumerated by cultural techniques that determine infectious microbes. Densities of both indicators and pathogens in reclaimed water, despite that it was disinfected by UV irradiation alone or by UV irradiation plus chlorination, were significantly lower than their densities in the river water, both upstream and downstream the reclaimed water release site in the river. Results gathered indicate that discharging reclaimed water into the river does not increment the load of indicators and pathogens of the river water. Then, in emergency situations due to severe water shortages after prolonged droughts, at least from the infectious diseases point of view, the risks of augmenting drinking water supplies with reclaimed water can be satisfactorily and safely managed.

Isolation of Viable Toxoplasma gondii from Feral Guinea Fowl (Numida meleagris) and Domestic Rabbits (Oryctolagus cuniculus) from Brazil

Toxoplasma gondii was isolated from a feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctologus cuniculus) from Brazil for the first time. Serum and brains from 10 guinea fowl and 21 rabbits from Brazil were examined for T. gondii infection. Antibodies to T. gondii were found in 2 of 10 fowl and 2 of 21 rabbits by the modified agglutination test (titer 1∶25 or higher). Viable T. gondii (designated TgNmBr1) was isolated from 1 of the 2 seropositive fowl by bioassay in mice but not from the 8 seronegative fowl by bioassay in cat. Viable T. gondii was isolated from both seropositive rabbits (designated TgRabbitBr1, TgRabbitBr2) by bioassay in mice from 1 and by bioassay in cat from the other. The TgRabbitBr1 strain was highly virulent for out-bred mice; mice fed 1 infective oocyst died of acute toxoplasmosis. The remaining 2 isolates were relatively avirulent for mice; lethal dose for mice was 10,000 oocysts. All 3 isolates were grown in cell culture, and tachyzoite-derived DNA were genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The TgNmBr1 was found to be clonal Type II, a rare finding in Brazil in any host. The rabbit isolates were atypical, similar to isolates from cats from Brazil (TgRabbitBr1 was identical to TgCatBr5, and TgRabbitBr2 was identical to TgCatBr1, a common genotype in Brazil denoted type BrII). This is the first genetic characterization of T. gondii isolates from the rabbits and guinea fowl in Brazil and the first host record for T. gondii in the guinea fowl.

Identification of a sporozoite-specific antigen from Toxoplasma gondii.

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. 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 (T. 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; 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- T. gondii 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- T. gondii 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 T. gondii infection and implicates oocysts as the agent of infection.

Disseminated visceral coccidiosis in Eurasian cranes (Grus grus) in the UK

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....

Insecticide resistance and malaria transmission: infection rate and oocyst burden in Culex pipiens mosquitoes infected with Plasmodium relictum.

BackgroundThe control of most vectors of malaria is threatened by the spread of insecticide resistance. One factor that has been hitherto largely overlooked is the potential effects of insecticide resistance on the ability of mosquitoes to transmit malaria: are insecticide-resistant mosquitoes as good vectors of Plasmodium as susceptible ones? The drastic physiological changes that accompany the evolution of insecticide resistance may indeed alter the ability of vectors to transmit diseases, a possibility that, if confirmed, could have major epidemiological consequences.MethodsUsing a novel experimental system consisting of the avian malaria parasite (Plasmodium relictum) and its natural vector (the mosquito Culex pipiens), two of the most common mechanisms of insecticide resistance (esterase overproduction and acetylcholinesterase modification) were investigated for their effect on mosquito infection rate and parasite burden. For this purpose two types of experiments were carried out using (i) insecticide-resistant and susceptible laboratory isogenic lines of Cx. pipiens and (ii) wild Cx. pipiens collected from a population where insecticide resistant and susceptible mosquitoes coexist in sympatry.ResultsThe isogenic line and wild-caught mosquito experiments were highly consistent in showing no effect of either esterase overproduction or of acetylcholinesterase modification on either the infection rate or on the oocyst burden of mosquitoes. The only determinant of these traits was blood meal size, which was similar across the different insecticide resistant categories in both experiments.ConclusionsInsecticide resistance was found to have no effect on Plasmodium development within the mosquito. This is the first time this question has been addressed using a natural mosquito-Plasmodium combination, while taking care to standardize the genetic background against which the insecticide resistance genes operate. Infection rate and oocyst burden are but two of the factors that determine the vectorial capacity of mosquitoes. Other key determinants of parasite transmission, such as mosquito longevity and behaviour, or the parasite's incubation time, need to be investigated before concluding on whether insecticide resistance influences the ability of mosquitoes to transmit malaria.

Inhibitory effect of S-nitroso-glutathione on Eimeria tenella oocysts was mainly limited to the early stages of sporogony

The objective of this study was to evaluate the action of S-nitroso-glutathione (GSNO) on the sporogony and the mRNA expression for EtCRK2 (a cyclin-dependent kinase related protein-2 in Eimeria tenella) during oocyst sporulation. Meanwhile, the effects of GSNO on the sporozoite excystability and infectivity to chickens were also investigated. The results showed that within 10 h after the initial sporulation, the sporulation process could be interrupted by GSNO treatment, and most of the oocysts failed to continue to sporulate. However, if the sporulation lasted more than 12 h, the sporogony process could not be blocked by GSNO. RT-PCR assay suggested that in the early stage of sporulation, the mRNA expression of EtCRK2 was completely inhibited by GSNO. For the infective oocysts or sporocysts treated by GSNO, the proportion of sporozoite excystation decreased significantly by about 20% and 40%, respectively. However, clinical coccidiosis could be observed in all experimental broilers inoculated with the oocysts or sporocysts treated by GSNO or not. The animal test showed that GSNO treatment reduced the infectivity of sporocysts, whereas it did not alleviate that of oocysts to broilers. Taken together, the data indicated that the viability of sporulated E. tenella oocysts could not be inactivated in vitro by GSNO. The inhibiting action of GSNO on oocysts was mainly limited to the early stages of sporulation, and this may partially associate with the inhibition of EtCRK2 mRNA expression.

Metam Sodium Reduces Viability and Infectivity of Eimeria Oocysts

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.

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

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.

Structural and Functional Characterization of SporoSAG: A SAG2-RELATED SURFACE ANTIGEN FROM TOXOPLASMA GONDII

Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage-specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to large scale outbreaks of toxoplasmosis. SporoSAG (surface antigen glycoprotein) is the dominant surface coat protein expressed on the surface of sporozoites. Using a bioinformatic approach, we show that SporoSAG clusters with the SAG2 subfamily of the SAG1-related superfamily (SRS) and is non-polymorphic among the 11 haplogroups of T. gondii strains. In contrast to the immunodominant SAG1 protein expressed on tachyzoites, SporoSAG is non-immunogenic during natural infection. We report the 1.60 A resolution crystal structure of SporoSAG solved using cadmium single anomalous dispersion. SporoSAG crystallized as a monomer and displays unique features of the SRS beta-sandwich fold relative to SAG1 and BSR4. Intriguingly, the structural diversity is localized to the upper sheets of the beta-sandwich fold and may have important implications for multimerization and host cell ligand recognition. The structure of SporoSAG also reveals an unexpectedly acidic surface that contrasts with the previously determined SAG1 and BSR4 structures where a basic surface is predicted to play a role in binding negatively charged glycosaminoglycans. Our structural and functional characterization of SporoSAG provides a rationale for the evolutionary divergence of this key SRS family member.

Toxoplasma gondii: epidemiology, feline clinical aspects, and prevention

Toxoplasma gondii is a parasite of birds and mammals. Cats are the only definitive host and thus the only source of infective oocysts, but other mammals and birds can develop tissue cysts. Although feline infections are typically asymptomatic, infection during human pregnancy can cause severe disease in the fetus. Cat owners can reduce their pets' exposure risk by keeping all cats indoors and not feeding them raw meat. Humans usually become infected through ingestion of oocyst-contaminated soil and water, tissue cysts in undercooked meat, or congenitally. Because of their fastidious nature, the passing of non-infective oocysts, and the short duration of oocyst shedding, direct contact with cats is not thought to be a primary risk for human infection.

The Effect of Host Foraging Ecology on the Prevalence and Intensity of Coccidian Infection in Wild Passerine Birds

The prevalence and intensity of Infection with isosporan Coccidia parasites in wild passerine birds varies greatly between bird species. Faeces of Infected hosts contain oocysts that are a source of new infections when ingested. As a consequence, we expect that the main route of coccidiosis transmission in the wild is related to the foraging behaviour of the hosts, and that bird species are exposed to infective oocysts, depending upon the way of foraging. We studied how prevalence and intensity of isosporan infection in wild birds are related to foraging stratum, gregariousness, and diet. Our data reveal that a bird's feeding habits play a significant role in the extent and severity of infection by isosporan Coccidia in the wild.

Improved Risk Analysis by Dual Direct Detection of Total and Infectious Cryptosporidium Oocysts on Cell Culture in Combination with Immunofluorescence Assay

The inactivation of Cryptosporidium oocysts is a main driver in the selection of water treatment disinfection strategies, and microbial risk analysis provides a sound basis for optimizing water treatment processes. U.S. Environmental Protection Agency method 1622/23 provides an estimate of the total oocyst count; however, it cannot be used directly for risk assessment, as it does not determine the fraction of infectious oocysts. Improved assessment of the risk for designated sources or in treated water requires evaluation of the total number of oocysts and an estimate of their infectivity. We developed a dual direct detection method using differential immunofluorescent staining that allows detection of both oocysts and cell culture infection foci for each sample. Using Cryptosporidium parvum oocysts, various pH levels, proteases, and gastroenteric compounds and substrates were assessed to determine their abilities to enhance the number of infection foci. The results showed that the key trigger for oocyst stimulation was acidification. Addition of a low concentration of D-glucose (50 mM) to the infection media increased rates of infectivity, while a higher dose (300 mM) was inhibitory. The total number of oocysts in each sample was determined by counting the oocysts remaining on a cell monolayer and the oocysts recovered from cell monolayer washes during processing using a simple filtration technique. With the dual direct detection on cell culture with immunofluorescence assay method, it is now possible to determine the numbers of total and infectious oocysts for a given sample in a single analysis. Direct percentages of infectivity are then calculated, which allows more accurate assessments of risk.