Efficacy of fenbendazole and ivermectin in treating gastrointestinal nematode infections in an Ontario cow-calf herd.

The objective of this randomized clinical trial was to compare performance of cow-calf pairs in southern Ontario treated with fenbendazole or ivermectin, or not treated, for gastrointestinal nematode infections. Treatments were administered to 128 cow-calf pairs over 2 years. Weights, body condition score, and fecal egg counts (FEC) were collected at treatment and at 28-day intervals. Treating calves with an anthelmintic was significantly advantageous compared with not treating, and there was no significant difference between treatment with fenbendazole or ivermectin. Neither treatment nor calf FEC had a significant effect on calf weaning weight. This could be the result of time of treatment, low initial FEC, or lack of power. Treatment affected cow FEC (P = 0.003). Cows in the ivermectin groups had the lowest FEC (P < 0.05), but because FEC were all low, biological significance is questionable. Additional work is needed to provide recommendations on when an anthelmintic should be used.

Efficacité du fenbendazole et de l'ivermectin pour traiter les infections à nématodes gastrointestinaux dans un troupeau de vaches-veaux en Ontario. L'objectif de cet essai clinique randomisé était de comparer les performances de paires de vaches-veaux dans le sud de l'Ontario traitées avec du fenbendazole ou de l'ivermectin, ou non-traitées, pour des infections à nématodes gastro-intestinaux. Les traitements furent administrés à 128 paires de vaches-veaux sur une période de 2 ans. Le poids, le pointage de l'état corporel, et le dénombrement des oeufs dans les fèces (FEC) furent colligés au moment du traitement et à des intervalles de 28 jours. Traiter des veaux avec un anthelmintique était significativement avantageux comparativement à ne pas les traiter, et il n'y avait pas de différence significative entre un traitement au fenbendazole ou à l'ivermectin. Ni l'un ou l'autre des traitements ou les FEC n'avaient un effet significatif sur le poids au sevrage des veaux. Ceci pourrait être dû au moment du traitement, un FEC initial peu élevé, ou un manque de puissance. Les traitements ont affecté les FEC des vaches (P = 0,003). Les vaches dans le groupe ivermectin avaient les plus bas FEC (P < 0,05), mais étant donné que tous les FEC étaient bas, la signification biologique est questionnable. Du travail supplémentaire est requis pour fournir des recommandations sur le moment où un anthelmintique devrait être utilisé.(Traduit par Dr Serge Messier).

Development of an ante-mortem diagnostic test for enzootic nasal tumor virus and detection of neutralizing antibodies in host serum.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the nasal mucosa of sheep and goats and is associated with enzootic nasal tumour virus (ENTV). As ENA is a common disease in North America and there are no vaccines against ENTV-1, diagnostic tests that can identify infected animals and assist with eradication and disease surveillance efforts are greatly needed. In this study, we endeavoured to develop a novel, non-invasive diagnostic tool that could be used not only to validate clinical signs of ENA but also to detect ENTV-1 infection prior to the onset of disease signs (i.e. pre-clinical diagnosis). Cytology, serology and reverse transcription (RT)-PCR-based diagnostic methods were investigated. Although the cytology-based assay was able to detect ENTV-1 infection in some animals, it had poor sensitivity and specificity and thus was not developed further as an ante-mortem diagnostic method. Three different assays, including ELISA, Western blotting and virus neutralization, were developed to detect the presence of ENTV-1-specific antibodies in sheep serum. Whilst a surprisingly large number of sheep mounted an antibody-mediated immune response against ENTV-1, and in some cases neutralizing, correlation with disease status was poor. In contrast, RT-PCR on RNA extracted from nasal swabs reliably detected exogenous ENTV-1 sequences, did not amplify endogenous ovine betaretroviral sequences, demonstrated high concordance with immunohistochemical staining for ENTV-1 envelope protein, and had perfect sensitivity and specificity. This report describes a practical and highly specific RT-PCR technique for the detection of clinical and pre-clinical ENA that may prove beneficial in future control or eradication programmes.

Pilot project to investigate over-wintering of free-living gastrointestinal nematode larvae of sheep in Ontario, Canada.

This study investigated the overwintering survival and infectivity of free-living gastrointestinal nematode (GIN) stages on pasture. The presence of GIN larvae was assessed on 3 sheep farms in Ontario with a reported history of clinical haemonchosis, by collecting monthly pasture samples over the winter months of 2009/2010. The infectivity of GIN larvae on spring pastures was evaluated using 16 tracer lambs. Air and soil temperature and moisture were recorded hourly. Free-living stages of Trichostrongylus spp. and Nematodirus spp. were isolated from herbage samples. Gastrointestinal nematodes were recovered from all tracer lambs on all farms; Teladorsagia sp. was the predominant species. Very low levels of Haemonchus contortus were recovered from 1 animal on 1 farm. The results suggest that Haemonchus larvae do not survive well on pasture, while Teladorsagia sp., Trichostrongylus spp. and Nematodirus spp. are able to overwinter on pasture in Ontario and are still infective for sheep in the spring.

Projet pilote pour faire enquête sur l'hivernage des larves de nématodes gastro-intestinaux libres chez les moutons en Ontario, au Canada. Cette étude a examiné la survie à l'hivernage et le pouvoir infectieux des stades des nématodes gastro-intestinaux (NGI) libres dans les pâturages. La présence de larves de NGI a été évaluée en recueillant des échantillons mensuels dans le pâturage pendant les mois de l'hiver 2009­2010 dans 3 fermes ovines en Ontario avec des antécédents documentés d'hémonchose clinique, tandis que le pouvoir infectieux des larves de NGI sur les pâturages du printemps a été évaluée en utilisant 16 agneaux sentinelles. La température et l'humidité de l'air et du sol ont été notées toutes les heures. Les stades libres de Trichostrongylus spp. et de Nematodirus spp. ont été isolés d'échantillons d'herbage. Les NGI ont été récupérés de tous les agneaux sentinelles dans toutes les fermes et Teladorsagia sp. était l'espèce prédominante. De très faibles taux d'Haemonchus contortus ont été récupérés chez 1 animal dans 1 ferme. Les résultats suggèrent que les larves d'Haemonchus ne survivent pas bien dans le pâturage, tandis que Teladorsagia sp., Trichostrongylus spp. et Nematodirus spp. peuvent survivre l'hiver dans le pâturage de l'Ontario et être toujours infectieux pour les moutons au printemps.(Traduit par Isabelle Vallières).

Experimental transmission of enzootic nasal adenocarcinoma in sheep.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the secretory epithelial cells of the nasal mucosa of sheep and goats. It is associated with the betaretrovirus, enzootic nasal tumor virus (ENTV), but a causative relationship has yet to be demonstrated. In this study, 14-day-old lambs were experimentally infected via nebulization with cell-free tumor filtrates derived from naturally occurring cases of ENA. At 12 weeks post-infection (wpi), one of the five infected lambs developed clinical signs, including continuous nasal discharge and open mouth breathing, and was euthanized. Necropsy revealed the presence of a large bilateral tumor occupying the nasal cavity. At 45 wpi, when the study was terminated, none of the remaining infected sheep showed evidence of tumors either by computed tomography or post-mortem examination. ENTV-1 proviral DNA was detected in the nose, lung, spleen, liver and kidney of the animal with experimentally induced ENA, however there was no evidence of viral protein expression in tissues other than the nose. Density gradient analysis of virus particles purified from the experimentally induced nasal tumor revealed a peak reverse transcriptase (RT) activity at a buoyant density of 1.22 g/mL which was higher than the 1.18 g/mL density of peak RT activity of virus purified from naturally induced ENA. While the 1.22 g/mL fraction contained primarily immature unprocessed virus particles, mature virus particles with a similar morphology to naturally occurring ENA could be identified by electron microscopy. Full-length sequence analysis of the ENTV-1 genome from the experimentally induced tumor revealed very few nucleotide changes relative to the original inoculum with only one conservative amino acid change. Taken together, these results demonstrate that ENTV-1 is associated with transmissible ENA in sheep and that under experimental conditions, lethal tumors are capable of developing in as little as 12 wpi demonstrating the acutely oncogenic nature of this ovine betaretrovirus.

Identifying hepatic genes regulating the ovine response to gastrointestinal nematodes using RNA-Sequencing.

Gastrointestinal nematode (GIN) infections are considered the most important disease of grazing sheep and due to increasing anthelmintic resistance, chemical control alone is inadequate. Resistance to Gastrointestinal nematode infection is a heritable trait, and through natural selection many sheep breeds have higher resistance. Studying the transcriptome from GIN-exposed and GIN-unexposed sheep using RNA-Sequencing technology can provide measurements of transcript levels associated with the host response to Gastrointestinal nematode infection, and these transcripts may harbor genetic markers that can be used in selective breeding programs to enhance disease resistance. The objective of this study was to compare liver transcriptomes of sheep naturally exposed to Gastrointestinal nematode s, with either high or low parasite burdens, to GIN-unexposed control sheep in order to identify key regulator genes and biological processes associated with Gastrointestinal nematode infection. Differential gene expression analysis revealed no significant differentially expressed genes (DEG) between sheep with a high or low parasite burden (p-value ≤0.01; False Discovery Rate (FDR) ≤ 0.05; and Fold-Change (FC) of > ±2). However, when compared to the control group, low parasite burden sheep showed 146 differentially expressed genes (64 upregulated and 82 downregulated in the low parasite burden group relative to the control), and high parasite burden sheep showed 159 differentially expressed genes (57 upregulated and 102 downregulated in the low parasite burden group relative to the control) (p-value ≤0.01; FDR ≤0.05; and FC of > ±2). Among these two lists of significant differentially expressed genes, 86 differentially expressed genes (34 upregulated, 52 downregulated in the parasited group relative to the control) were found in common between the two parasite burden groups compared to the control (GIN-unexposed sheep). Functional analysis of these significant 86 differentially expressed genes found upregulated genes involved in immune response and downregulated genes involved in lipid metabolism. Results of this study offer insight into the liver transcriptome during natural Gastrointestinal nematode exposure that helps provide a better understanding of the key regulator genes involved in Gastrointestinal nematode infection in sheep.

Correlation of salivary antibody to carbohydrate larval antigen (CarLA) with health and gastrointestinal nematode parasitism in sheep under Ontario grazing conditions.

Leveraging mucosal immunity is a promising method for controlling gastrointestinal nematode (GIN) parasitism in sheep. Salivary antibody to carbohydrate larval antigen (sCarLA), a heritable measure of immunity to third-stage GIN larvae (L3), has been successfully applied to genetic improvement programs in New Zealand. However, sCarLA levels wane in the absence of ongoing GIN exposure. New Zealand's temperate climate permits year-round exposure to L3, but cold winters in boreal regions such as Ontario, Canada interrupt exposure for five months or more. This study investigated associations between sCarLA levels, GIN parasitism, and indicators of overall health in sheep grazing under Ontario conditions. A commercial flock of 140 Rideau cross ewe lambs were followed from approximately 30 days of age in May 2016 until November 2017, including lambing and lactation in the spring of 2017. Every 6-8 weeks during the grazing season and at mid-gestation in March 2017, fecal egg counts were performed, blood collected to assess serum albumin, globulin, and hematocrit, and pasture samples obtained to confirm exposure to infective larvae. Measurements of sCarLA level were performed at the beginning, middle, and end of each grazing season, and at mid-gestation. Spearman's rank correlation coefficients were calculated to compare sCarLA levels over time, and general linear mixed models created to evaluate associations between sCarLA levels, GIN fecal egg count, hematocrit, serum albumin, and serum globulin. Levels of sCarLA followed a similar seasonal pattern to GIN fecal egg counts with a 6-8 week delay; much higher sCarLA levels were observed in the second grazing season. The proportion of the flock with detectable sCarLA (≥ 0.3 units/mL) was 68.3 % by the end of the first grazing season, declined over winter to 43.9 % at lambing, and approached 100 % after 3 months of grazing in the second grazing season. Correlations between sCarLA levels over time were consistently positive, of weak to moderate strength, and significant (p < 0.05). At all time points, sCarLA level was significantly (p < 0.001) and negatively associated with fecal egg counts. The flock displayed minimal variability in hematocrit, serum albumin, and serum globulin; none of which were significantly associated with sCarLA levels. These results suggest that sCarLA can be maintained over winter and is a useful measure of immunity to GINs in sheep under Ontario grazing conditions.

Correlation of subclinical gastrointestinal nematode parasitism with growth and reproductive performance in ewe lambs in Ontario.

Infection with gastrointestinal nematode parasites (GINs) is an important cause of productivity loss on sheep farms in Ontario and worldwide. However, efforts to quantify the effect of GIN infection on growth have demonstrated mixed results. Furthermore, there has been limited investigation of their effect on reproductive performance. This study evaluated the effect of subclinical GIN parasitism on growth and reproductive performance of ewe lambs under Ontario grazing conditions. Rideau cross ewe lambs (n = 140) born in spring 2016 on a farm in central Ontario were followed for two years from before weaning through to November 2017, including their first lambing and lactation. These animals grazed from May to November of each year and were sampled every 6-8 weeks during both grazing seasons and once at mid-gestation in March 2017. At each sampling the ewe lambs were weighed, body condition scores assigned, fecal egg counts (FECs) performed, and pasture samples collected to assess number of infective GIN larvae. Study animals with a FEC of 500 eggs per gram or higher were selectively treated with anthelmintics to prevent morbidity and mortality. Fecal samples were cultured to determine infecting GIN species, and climate data were obtained from a weather station 26 km away from the farm. Precipitation levels and numbers of infective larvae on pasture were low during the first grazing season but were more typical of Ontario conditions in the second grazing season. The three most common GIN species were Haemonchus contortus, Teladorsagia circumcincta, and Trichostrongylus spp. General linear mixed models were generated for weight change over time, litter size at lambing, and weaning weights of offspring. Despite moderate peak GIN burdens in both grazing seasons, FEC was not significantly associated with weight change or litter size, apart from periparturient egg rise in study ewe lambs with larger litters (p = 0.05). Significant positive quadratic and negative linear associations were identified between late lactation FECs and offspring weaning weights; the association between FECs and weaning weights changed from negative to positive at a FEC of 361 eggs per gram. These results indicate that when GIN burdens are moderate as evidenced by fecal egg counts and infection is subclinical, there appears to be low to no impact on growth and reproductive performance in ewe lambs in the first 18 months of life. This suggests that when GIN parasitism is regularly monitored and controlled using targeted selective treatment, animal performance is minimally affected.

Comparison of ITS-2 rDNA nemabiome sequencing with morphological identification to quantify gastrointestinal nematode community species composition in small ruminant feces.

Mixed gastrointestinal nematode (GIN) infections are a common and significant cause of financial loss for small ruminant producers. Morphologic examination of third-stage larvae (L3) can be used to identify species composition in feces but has limitations due to the requirement for specialized expertise and the extensive time (8-15 d depending on method used) and labour involved. Moreover, differential development and survival of larvae during coproculture to the third stage often occurs. Deep amplicon sequencing of the ITS-2 rDNA locus of first-stage larvae (L1) allows for higher throughput with reduced specialist labour and reduces the risk of misidentification. Harvesting of L1 soon after hatching is also faster and further reduces labour as well as biases that can occur due to differential larval development and survival. This study compares the results of morphologic examination of L3 with those of ITS-2 rDNA deep amplicon sequencing of L1 from a set of pooled fecal samples. The proportions of eggs that were successfully recovered as larvae following culture to L3 and L1 were also compared. Larval recovery rate was significantly lower from L3 cultures than from L1 cultures (p < 0.001); eggs were 238.7 times less likely to develop to L3 than to L1 (95 % confidence interval for odds ratio 80.0-712.0). Significantly lower proportions of Teladorsagia circumcincta (odds ratio = 3.1, p = 0.008) and higher proportions of Trichostrongylus spp. (p = 0.009) were identified using morphologic examination of L3 compared with deep amplicon sequencing of L1 on the same samples. This is consistent with previous reports of differential survival of these species in L3 cultures. These results indicate that deep amplicon sequencing of L1 may reduce bias introduced by differential GIN survival to L3 in small ruminants.

Survey of the status of copper, interacting minerals, and vitamin E levels in the livers of sheep in Ontario.

Livers from cull ewes and market lambs raised in Ontario were obtained to determine the status of specific minerals and vitamin E. Values for copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) obtained by atomic absorption and inductively coupled plasma--atomic emission spectroscopy (ICP-AES) were found to be statistically different but sufficiently biologically similar to allow the use of ICP-AES for screening groups of samples for deficient or toxic levels of those minerals. Toxic levels of cadmium were not found. Toxic levels of aluminum were found in 1 cull ewe and 1 market lamb. A significant proportion of both market lamb samples (40.0%) and cull ewe samples (50.0%) had high to toxic levels of Cu. In market lambs, Fe, Mn, molybdenum (Mo), selenium (Se), and Zn were not found to be important determinants of Cu level. In cull ewes, Fe, Mn, and Zn play a moderate role in the variability of liver Cu levels. Selenium was found to be present at marginal levels in 3.3% of cull ewe samples and in 42.6% of market lamb samples. Vitamin E was found to be low to deficient in 10.0% of cull ewe samples and in 90.0% of market lamb samples. In market lambs, only Mo was associated with Se levels, and no minerals were associated with vitamin E levels. In cull ewes, there was a strong association between Se and vitamin E. This survey demonstrates that marked nutritional imbalances of Cu, Se, and vitamin E exist in cull ewes and market lambs in Ontario.

Development of a Taenia ovis transmission model and an assessment of control strategies.

The metacestode stage of the tapeworm, Taenia ovis, causes cystic lesions in the skeletal and cardiac muscle of sheep, which can result in the condemnation of the entire carcass. In recent years, Canadian farms have seen a marked increase in the number of condemnations due to T. ovis. Mathematical transmission models provide a useful tool for predicting parasite transmission and for evaluating the efficacy of potential control options. To date, no model has been developed exclusively for T. ovis. In the work described here, a compartmental, deterministic transmission model was developed to better understand the transmission dynamics of T. ovis on Canadian sheep farms. The model was intended to be practical, and represent the transmission of infection burdens in lambs that result in carcass condemnation, or transmission to canids. All transmission parameters were obtained from the literature or, when unavailable, expert opinion. The model incorporated each stage of the parasite lifecycle using the most probable transmission route on Canadian sheep farms; including definitive host (guard dogs), intermediate host (pastured lambs), and environment. Based on literature, the model performed as expected, and provided a reasonable estimate of parasite prevalence in lambs. In addition, modeling allowed the efficacy of potential control options to be evaluated and compared. Model simulations suggested that infection risk in market lambs could be eliminated through the regular treatment of guardian dogs every fifth week with an appropriate cestocide, or through eliminating carcass consumption by guardian dogs.

Genetic characterization of small ruminant lentiviruses circulating in naturally infected sheep and goats in Ontario, Canada.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are related members of a group of small ruminant lentiviruses (SRLVs) that infect sheep and goats. SRLVs are endemic in many countries, including Canada. However, very little is known about the genetic characteristics of Canadian SRLVs, particularly in the province of Ontario. Given the importance of surveillance and eradication programs for the control of SRLVs, it is imperative that the diagnostic tests used to identify infected animals are sensitive to local strains of SRLVs. The aim of this work was to characterize SRLV strains circulating in Ontario and to evaluate the variability of the immunodominant regions of the Gag protein. In this study, the nearly complete gag sequence of 164 SRLVs, from 130 naturally infected sheep and 32 naturally infected goats from Ontario, was sequenced. Animals belonged to distantly located single and mixed species (sheep and goats) farms. Ovine lentiviruses from the same farm tended to cluster more closely together than did caprine lentiviruses from the same farm. Sequence analysis revealed a higher degree of heterogeneity among the caprine lentivirus sequences with an average inter-farm pairwise DNA distance of 10% and only 5% in the ovine lentivirus group. Interestingly, amplification of SRLVs from ELISA positive sheep was successful in 81% of cases, whereas amplification of SRLV proviral DNA was only possible in 55% of the ELISA positive goat samples; suggesting that a significant portion of caprine lentiviruses circulating in Ontario possess heterogeneity at the primer binding sites used in this study. Sequences of sheep and goat SRLVs from Ontario were assembled into phylogenetic trees with other known SRLVs and were found to belong to sequence groups A2 and B1, respectively, as defined by Shah et al. (2004a). A novel caprine lentivirus with a pairwise genetic difference of 15.6-25.4% relative to other group B subtypes was identified. Thus we suggest the designation of a novel subtype, B4, within the caprine lentivirus-like cluster. Lastly, we demonstrate evidence of recombination between ovine lentiviruses. These results emphasize the broad genetic diversity of SRLV strains circulating in the province of Ontario and show that the gag region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

Distribution of, and risk factors associated with, sheep carcass condemnations due to Cysticercus ovis infection on Canadian sheep farms.

Cysticercus ovis, the intermediate stage of a canine tapeworm, Taenia ovis, produces cystic lesions in the skeletal and cardiac muscle of sheep which, if numerous, will result in the condemnation of an entire carcass. In 2007 and 2008, the number of carcass condemnations due to C. ovis rose dramatically across Canada, suggesting that the prevalence of this infection on sheep farms was increasing. Trace-back of 237 carcasses condemned at Ontario provincially inspected abattoirs, between March 2009 and March 2011, revealed they originated from 133 farms across Canada. A case-control study was performed (n=40 cases, 56 controls) to identify farm-level risk factors associated with carcass condemnations due to C. ovis. Participating farms, located in Alberta, Saskatchewan, Manitoba and Ontario, were asked to answer a short questionnaire which collected information about each farm's geographic location and management practices. A multivariable logistic regression model revealed that farm dogs scavenging deadstock (OR=4.04; 95% CI: 1.16-14.04) and failing to dispose of deadstock (OR=11.78; 95% CI: 2.93-47.40) were significantly associated with condemnations (p ≤ 0.05).

Control of important causes of infectious abortion in sheep and goats.

This article summarizes control measures for the most common causes of abortion in North America, New Zealand, the United Kingdom, and Europe. When dealing with an abortion outbreak in a flock or herd, diagnostic investigation is critical to assuring that any future control measures are effective and worthwhile. Biosecurity is an important consideration for any abortion control program, and should be promoted regardless of whether an abortion problem exists in the flock. Many of the infectious agents that cause abortion in small ruminants are also zoonotic pathogens, and producers should be educated to avoid risk to themselves and their families.

Antimicrobial resistance and small ruminant veterinary practice.

Antimicrobial resistance (AMR) is recognized as an emerging issue in the practice of veterinary medicine. Although little surveillance and research has been completed on the prevalence of AMR and associated risk factors in small ruminants, evidence of AMR is present in many countries. Furthermore, antimicrobial use (AMU) practices in sheep have been shown to be associated with increased resistance, highlighting the issue of prudent use of these drugs in many countries. Furthermore, AMU practices in sheep have been shown to be associated with increased resistance, highlighting the issue of prudent use of these drugs.

Principles of mastitis treatment in sheep and goats.

This article indicates the principles for treatment of mastitis in ewes/does and explains the reasons why treatment may occasionally fail. It presents the principles for administration of antimicrobial agents at drying off of the animals. Finally, it addresses the risk of antimicrobials present in milk when improper withdrawal periods are used and the issues around testing for inhibitors before putting the milk into in a farm's tank.

Full-length genome sequence analysis of enzootic nasal tumor virus reveals an unusually high degree of genetic stability.

Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the lack of an infectious molecular clone and the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in North America, and only one full-length sequence is available for each of ENTV-1 and ENTV-2. In order to understand the molecular evolution of ENTV-1, the full-length genome sequence of ten ENTV-1 proviruses derived from clinical samples of ENA isolated from conventionally reared sheep in Canada and the United States was determined. The North American ENTV-1 (ENTV-1(NA)) genomes shared greater than 96% sequence identity with the European ENTV-1 sequence (ENTV-1(EU)). Most of the amino acid differences were found in Orf-x, which in the corresponding ENTV-1(EU) genome is truncated by 44 amino acids. Apart from Orf-x, the long terminal repeat (LTR) is where the majority of differences between ENTV-1(NA) and ENTV-1(EU) reside. Overall, there was an unusually high degree of amino acid conservation among the isolates suggesting that ENTV-1 is under stabilizing selection and K(a)/K(s) ratios calculated for each of the viral genes support this hypothesis. The unusually high degree of genetic stability of the ENTV-1 genome enabled us to develop a hemi-nested PCR assay for detection of ENTV-1 in clinical samples. Additionally, multiple nasal tumor cell clones were established and while most had lost the provirus by passage 5; one polyclonal line retained the provirus and attempts are being made to culture these cells. These tumor cells, the first of their kind, may provide a system for studying ENTV-1 in vitro. This work represents an important step in the study of ENTV and sets the foundation for the construction of an infectious molecular clone of ENTV-1.

Transmission of foodborne zoonotic pathogens to riparian areas by grazing sheep.

The objective of this study was to determine if sheep grazing near riparian areas on pasture in Ontario are an important risk factor for the contamination of water with specific foodborne pathogens. Ten Ontario sheep farms were visited weekly for 12 wk during the summer of 2005. Samples of feces, soil, and water were collected and analyzed for the presence of Escherichia coli O157:H7, Salmonella spp., Campylobacter jejuni and C. coli, and Yersinia enterocolitica, by bacteriological identification and polymerase chain reaction (PCR). The data was analyzed as repeated measures over time using mixed models. No samples were positive for Salmonella, and no samples were confirmed positive for E. coli O157:H7 after PCR. Levels of Campylobacter were highest in the soil, but did not differ between soil where sheep grazed or camped and roadside soil that had never been grazed (P = 0.85). Levels of Yersinia were highest in water samples and were higher in soil where sheep had access (P = 0.01). The prevalence of positive Campylobacter and Yersinia samples were not associated with locations where sheep spent more time (Campylobacter P = 0.46, Yersinia P = 0.99). There was no effect of stocking density on the prevalence of Campylobacter (P = 0.30), but as the stocking density increased the levels of Yersinia increased (P = 0.04). It was concluded that although sheep transmit Yersinia to their environment, pastured sheep flocks are not major risk factors for the transmission of zoonotic pathogens into water.