Stephanie L. Schaaf

Prevalence of Giardia sp., Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in 109 dairy herds in five counties of southeastern New York

Abstract A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites.

Risk factors associated with Cryptosporidium parvum infection in dairy cattle in southeastern New York State.

An observational analytical epidemiologic study was carried out to identify factors associated with the risk of infection with Cryptosporidium parvum in dairy herds in southeastern New York state. A random sample of 2943 cattle on 109 farms was selected from the target population. Fecal samples were collected from animals in three different age groups and examined for the presence of C. parvum using a quantitative centrifugation concentration flotation method. Data on intrinsic, preweaning, postweaning, maternity, and general management factors were collected and evaluated for their association with the risk of infection with C. parvum. Indices for each of these categories of management were developed from factors significantly associated with the risk of infection with C. parvum. Significant factors were identified using the logistic regression statistical technique. A final analysis, including the indices, age, and season, was performed to identify factors significantly associated with the risk of infection with C. parvum while simultaneously controlling for the effect of other factors. The farm effect was evaluated using a mixed effect model. Preweaning factors found to be significantly associated with a decreased risk of infection were: use of ventilation in calf rearing areas, daily addition of bedding, feeding of milk replacer, daily disposal and cleaning of bedding, and use of antibiotics. Postweaning factors such as moving of the animals after weaning, cleaning of soiled bedding, and use of antibiotics and ionophores as preventive measures were significantly associated with the decreased risk of an infection with C. parvum. Consideration of maternity management factors showed that winter housing of cows individually within 2 months of calving, use of fresh colostrum to feed calves, and having a concrete floor in the calving area were significantly associated with decreased risk of C. parvum infection. The total number of dairy cattle, total number of other species of agricultural animals on the farm, and the distance of the barn water source from the septic system were found to be significantly associated with increased risk of C. parvum infection. In the final analysis, the risk of infection with C. parvum was significantly decreased with an increased value of the maternity management index score. The general management significantly affected the risk of infection with C. parvum where the risk increased with the increase of the value of the index. The risk of infection significantly decreased with increase in the age of the animal.

Epidemiologic study of Giardia sp. infection in dairy cattle in southeastern New York State

To identify animal and management factors associated with the risk of Giardia sp. infection in dairy cattle in southeastern New York State, an observational analytical epidemiologic study was conducted. A random sample of 2943 animals in 109 dairy herds located in five counties of southeastern New York was selected from the target population. Fecal samples were collected from animals in the study population and examined for the presence of Giardia sp. using a quantitative centrifugation concentration flotation technique. Data on each animal, and on general management, maternity, preweaning, and postweaning practices were collected by personal interview with the farmer or farm manager. The significance of association of these factors, within each management practice group, with the risk of infection was evaluated using the logistic regression analysis. Weighted indices for each of these four groups were developed, and were evaluated together with the age of the animal and season of sampling for their significance of association with the risk of infection using mixed effect logistic regression analysis. Only the maternity management practices, age of the animal, and the season of sampling were significantly associated with the risk of infection with Giardia sp. Summer housing of bred heifers, on pasture or in tie stalls, was associated with increased risk of infection. Calves that were fed fresh colostrum or separated immediately after birth from the dam were at decreased risk of infection. The risk of infection decreased with the age of the animal and varied by the season of sampling. There was no significant extra binomial variation in the risk of infection in this data. If consideration is to be given to reducing the risk of infection with this parasite, management must be a top priority, especially in younger animals.

CRYPTOSPORIDIUM SPP. FROM SMALL MAMMALS IN THE NEW YORK CITY WATERSHED

The objective of this study was to assess the potential role that wildlife plays in environmental degradation of watersheds through the contamination of the water supply with zoonotic genotypes of Cryptosporidium. Cryptosporidium isolates recovered from wildlife in the New York City (NYC) watershed were examined to determine genotype using a polymerase chain reaction protocol targeting the 18-Small Subunit (SSU) rRNA locus. Seventy-seven DNA samples recovered from 12 wildlife host species captured in the NYC watershed were amplified and sequenced. Data on risk factors associated with the perpetuation of these genotypes also were collected and analyzed. Although many genotypes appeared to be host-specific, 38% of the samples examined were identified as Cryptosporidium parvum, indicating the presence of zoonotic Cryptosporidium. Adult animals were more likely to shed the zoonotic strains of Cryptosporidium spp. Animals captured in the fall and winter were more likely to be infected with C. parvum than those captured in spring and summer.

Prevalence of Giardia duodenalis assemblages among dairy herds in the New York City Watershed

A longitudinal herd-level study was carried out to determine the cumulative incidence of Giardia duodenalis infections in dairy cattle in the New York City Watershed. We also sought to assess the changes in infection pattern of animals diagnosed as shedding Giardia over time, determine risk factors that may be associated with G. duodenalis infections, and identify potentially zoonotic infections. A total of 2109 fecal samples were randomly collected from dairy cattle at 34 farms in the New York City Watershed on a seasonal basis. A total of 504 Giardia-positive samples were identified by zinc sulfate flotation. The overall cumulative incidence of G. duodenalis based on flotation results was 23.9% with 73.8% of all infections occurring in animals under 180 days of age (372/504). The intensity of infection ranged from 2 to 563,200 cysts/gram of feces. Cattle shedding Cryptosporidium spp. oocysts were twice as likely to shed G. duodenalis cysts in comparison to the animals that did not shed oocysts (1.81 95% CI 1.26-2.60 p=0.0012). In the multivariate analysis, only the age of the animal and the presence of dogs on the farm were significantly associated with the likelihood of shedding G. duodenalis. DNA was extracted from positive samples and analyzed by polymerase chain reaction (PCR) of the beta-giardin and triosephosphate isomerase genes of Giardia spp. 304 samples were analyzed by PCR of which 131 were sequenced. 22.1% of sequenced samples were identified as assemblage A and 77.9% were identified as assemblage E. Interestingly, 100% of specimens identified as assemblage A were from calves under 84 days of age indicating that younger cattle are important reservoirs for potentially zoonotic assemblages of G. duodenalis.

The Sensitivity of PCR Detection of Cryptosporidium Oocysts in Fecal Samples Using Two DNA Extraction Methods

AbstractBackground: The implementation of cost-effective intervention strategies for zoonotic protozoa relies on the development of sensitive and accurate diagnostic methods. We carried out a study to evaluate the accuracy of a PCR method for the detection of Cryptosporidium spp. oocysts in fecal samples from cattle. Methods: Fecal samples were spiked with different numbers of oocysts and the limit of detection of the method was determined. Two methods of DNA extraction were assessed: glass beads and freeze-thawing using liquid nitrogen. A nested PCR approach was developed targeting the Cryptosporidium SSU rRNA and TRAP-C2 genes. Agreement between the diagnosis of Cryptosporidium spp. at the SSU rRNA and TRAP-C2 loci was quantified using the κ-coefficient. Results: Compared with the freeze-thawing method, the glass beads method was found to be a better way of extracting DNA from Cryptosporidium oocysts (sensitivities were 83 and 100%, respectively). The limits of detection for glass beads and freeze-thaw were low, 1 and 10 oocyst/g fecal samples, respectively. Forty-six percent of the field samples previously classified as negative for Cryptosporidium parvum by the floatation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol. Conclusion: Primers for SSU rRNA are more successful in producing an amplification than primers for the TRAP-C2 gene which makes the former PCR protocol the approach of choice for detecting Cryptosporidium parvum oocysts in field samples.

Detection of Cryptosporidium oocysts in soil samples by enzyme-linked immunoassay.

An ELISA protocol was adapted for detection of Cryptosporidium parvum oocysts in soil samples and the limit of detection of the test was determined. A modified indirect antigen capture ELISA protocol was developed using monoclonal antibodies against the oocyst outer wall. The accuracy of the ELISA was compared to spiked soil samples and measured in terms of sensitivity and specificity of the test. The performance of the ELISA was evaluated in field soil samples and measured using the kappa-statistics. Similarly, the performance of the ELISA was compared to the concentration flotation method, to a modified concentration flotation method and to a commercial ELISA (ProSpecT) in field fecal and soil samples. The limit of detection of the test was selected to be 10,000 oocysts/g. At this limit of detection, the ELISA had a sensitivity of 95% and specificity of 100%. The agreement between the ELISA and the modified flotation-concentration method in detecting Cryptosporidium oocysts in soil samples was 32% (kappa=0.32). The ELISA had the same relative sensitivity (82%) in comparison to both the flotation and ProSpecT in determining Cryptosporidium-infection status of an animal. The kappa-statistics was 0.26 for both tests. The developed ELISA proved to be a valuable diagnostic test for detecting oocysts in soil samples and has a potential application in determining the infection status of animals.

Chimeric protein A/G conjugate for detection of anti–Toxoplasma gondii immunoglobulin G in multiple animal species

Serological testing for toxoplasmosis diagnosis remains the method of choice in human medicine due to the accessibility of the requisite sample, the difficulty in predicting the parasite’s location in the host for direct detection, and the availability of established commercial methods. In veterinary medicine, although the first 2 conditions are unchanged, there is a need for commercially produced test methods that are validated for Toxoplasma gondii detection across the range of animal species that can serve as intermediate hosts. The development of such a serological method for animals would allow the diagnosis of toxoplasmosis in individual animals and a higher throughput method for population-level toxoplasmosis surveys. The incorporation of a non–species-specific chimeric protein A/G conjugate into an anti–Toxoplasma immunoglobulin G enzyme-linked immunosorbent assay is described. Serum from potential intermediate hosts was reevaluated using this method and compared with earlier testing using an established agglutination procedure. Very good agreement between the 2 tests was noted (κ = 0.81), establishing the method as a useful option for veterinary diagnostic testing.

Incidence of and risks associated with Giardia infections in herds on dairy farms in the New York City Watershed

BackgroundThe primary aims of this study were to determine the incidence of Giardia infections in dairy herds on farms in the New York City Watershed region and to evaluate risk factors associated with infections. Because co-infections of Giardia and Cryptosporidium spp. are common in this population, we also evaluated the effect of herd infection status on Giardia infections.MethodsFarms were grouped into three cohorts based on their prior infection status with Giardia and/or Cryptosporidium spp. The sampling plan included collecting fecal samples from all calves below 30 days of age and proportional sampling of calves, young stock, and adults. A total of 10,672 fecal samples were collected and analyzed for the presence of Giardia cysts using zinc sulfate flotation. Herds enrolled in the study were sampled seasonally for a study period of two years. The probability of shedding cysts past a certain age and the factors that influenced the likelihood of shedding were evaluated using survival analysis. Linear regression was used to evaluate factors that were associated with the intensity of shedding.ResultsThe majority of Giardia infections occurred in calves within their first 180 days of age, with the most number of calves shedding Giardia cysts between 11 and 20 days of age. The incidence of shedding of Giardia cysts ranged from 0.0004 per animal day for cattle in the low risk cohort to 0.0011 per animal day for cattle in the high risk cohort. The likelihood of shedding was influenced by the prior infection status of the herd and the season of collection. Infected animals shed on average 9,658 cysts/gram and the intensity of shedding Giardia cysts varied significantly with the age (p < 0.0001) and the season of collection (p = 0.0151 for Spring).ConclusionGiardia infections are common in dairy herds in the New York City watershed, particularly in calves less than 6 months of age. Seasonality may be an important factor in the perpetuation of infections based on changes in management practices corresponding to weather patterns of a particular season. A dairy herds prior infection status with Cryptosporidium influences the likelihood of infection with Giardia.

Serological detection of exposure to Cryptosporidium parvum in cattle by ELISA and its evaluation in relation to coprological tests

Abstract.We evaluated serum examination as an alternative to fecal analysis for the diagnosis of exposure to Cryptosporidium parvum in cattle. The accuracy of the serum ELISA was compared to the combined results of concentration flotation microscopy and fecal enzyme immunoassay. The expected performance of the serum ELISA at different levels of infection with C. parvum was evaluated using the predicative values positive and negative. Optimal conditions for the serum ELISA can be achieved by diluting the serum samples 1:20 and the conjugate 1:8,000. The serum ELISA had a relatively high sensitivity of 97.5% (95% CI=87–100%) and poor specificity, 4% (95% CI=1–20%). There was a poor agreement between the serum ELISA and the fecal tests (κ=0) on samples collected from adult cows in a high-risk and a low-risk population. Examination of some of these fecal samples using a PCR detection method demonstrated the presence of C. parvum DNA in 10% of the samples.