Johne’s disease is a chronic and debilitating disease of cattle caused by infection with Mycobacterium avium subsp. paratuberculosis (Mptb). This disease affects both dairy and beef cattle, though it is more commonly recognized in dairy cattle. Mptb is able to persist in the environment of cattle for extended periods of time; therefore the distribution of the disease depends on the presence of infected animals and environmental conditions. Three surveillance systems were used to describe the spatial distribution of Johne’s disease seropositivity in Texas cattle. These three systems were hypothesized to describe different spatial patterns. These systems involved sampling, 1) herds throughout Texas, 2) market cattle from four markets each month (one each from northern, southern, eastern, and western regions of Texas) and 3) sick animals submitted by veterinarians throughout Texas. Samples were tested for Johne’s disease at the Texas Veterinary Medical Diagnostic Laboratory using serum ELISA. Spatial distributions were estimated by kriging the sample-to-positive control ratios (S/P). Sera were evaluated for Mptb antibodies from 2358 cattle with 1084 animals in system 1, 1200 from system 2 and 74 from system 3. Total number of positive ELISA results was 51, with 25, 19 and 7 positve ELISA results for systems one, two and three, respectively. Results showed an overall prevalence of 2.16%, and prevalence’s of 2.31%, 1.58% and 9.46% for systems one, two and three, respectively. Differences in the spatial distribution of Johne’s disease seropositvity, based on the three surveillance systems, confirmed our hypothesis that estimation of disease distribution is dependant upon the source of surveillance samples.

The first objective of this study was to evaluate the utility of bulk tank tests to detect the presence of Mycobacterium avium subspecies paratuberculosis (MAP) antibody in dairy herds for the purpose of Johne’s disease surveillance. Individual cow milk samples were collected by CanWest Dairy Herd Improvement customer service representatives in herds across Ontario, Canada. These samples, along with bronopol preserved bulk tank samples were collected from herds participating in the Ontario Johne’s Education and Management Assistance Program (OJEMAP), a producer funded Johne’s control scheme. Overall, there were 309 farms tested, with herd size from 15 to 986 milking cows. The relative sensitivity and specificity of the bulk tank ELISA test when a positive herd was defined as 1 or more positive cows was 54.7% and 90.6%, respectively. The second objective was to determine the effect of milk storage temperature and duration on the Johne’s milk ELISA test result. When herd level factors were considered in a logistic model, average monthly protein (%) and the percent of positive milk contributed to the bulk tank by milk ELISA positive cows were found to be significantly (p<0.05) associated with the probability of a herd testing positive on the bulk tank Hyper ELISA protocol. Positive and negative MAP milk samples were stored for varying times and under different temperature conditions. In a mixed linear model, time was found to be significantly (<0.001) associated with the log transformed ELISA optical density. When the results were dichotomized into positive and negative by the cut-off of 0.10 and cross classified, the amount of misclassification was considered biologically negligible.

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Paratuberculosis is an intestinal condition in ruminants infected with Mycobacterium avium subspecies paratuberculosis (MAP) and precedes Johne's disease, a chronic enteric disorder in ruminants caused by MAP infection. Necropsy with histopathology provides definitive diagnosis of Johne's disease and positive culture of MAP from tissues provides definitive diagnosis of paratuberculosis. To determine assay sensitivity, 85 formalin-fixed paraffin-embedded (FFPE) tissues from ruminants diagnosed with Johne's disease were tested with a commercial paratuberculosis quantitative polymerase chain reaction (qPCR) assay and had a sensitivity of 92%. To determine assay specificity, 21 FFPE tissues from animals without gastrointestinal disease combined with 13 FFPE tissues from non-ruminant animals (frog, dove, turtle, dog, and 2 cats) with non-paratuberculosis mycobacterial diseases were tested with the commercial qPCR assay and had a specificity of 100%. Slides prepared from the FFPE tissue blocks were stained with hematoxylin & eosin (H&E) and Ziehl-Neelsen's (acid fast stain), then examined for granulomatous inflammation and scored on a scale from 0-4 based on the quantity of acid fast bacteria (AFB). Digital microscopy and morphometric software were used to compute an acid fast bacteria area index (AFBAI) to evaluate a more precise correlation with the qPCR results. The quantity of AFB in tissue slides showed medium to strong correlation with the appropriate qPCR results. The results indicate that the commercial qPCR assay can be used on FFPE tissues with good results and the qPCR results have medium-strong correlation with quantitative acid fast histopathology. / Master of Science

Through a binding partner the CARD15 gene activates NF-kB, a molecule with a role in the initiation of the inflammatory immune response. The gene is highly conserved in both structure and function in human and mouse and has recently been implicated as a disease resistance gene in Crohn's disease and Blau Syndrome in human. The gene's relationship to disease and its conservation between species suggests that it may also have a conserved role in bovine disease resistance. To elucidate the potential role of bovine CARD15 in disease resistance, the gene was characterized in cattle. Bovine CARD15 is located 4.2 cR5000 telomeric to ADCY7 on chromosome 18. It spans ~30 kb and is comprised of 12 exons, 11 of which are coding. Bovine CARD15 is expressed in many tissues, but is most abundant in peripheral blood leukocytes. An extensive comparative analysis between the bovine, mouse and human CARD15 genes revealed high levels of inter-species conservation in sequence, genomic structure and protein domains. Conserved putative regulatory motifs were identified in the three species comparison of the 5'UTR, 3'UTR and the intronic sequences flanking exons. Additionally, diverse regulatory motifs were identified in each of the species indicating an evolutionary divergence in the mechanisms of regulation of gene expression. To assess the extent of genetic diversity within bovine CARD15, 41 individuals from nine breeds representing two subspecies were sequenced and screened for polymorphisms. Thirty-six single nucleotide polymorphisms (SNPs) were identified including 26 within the gene transcript. Haplotypes were estimated for each individual and parsimonious SNP sets were identified with which the multi-locus Bos taurus and Bos indicus haplotypes may be reconstructed. There was a significantly higher rate of substitutions within Bos indicus than in Bos taurus. A significantly higher rate of nonsynonymous to synonymous substitutions was found in Bos taurus indicating that positive Darwinian selection is acting on the gene within this subspecies. Association analyses were performed between these SNP loci and haplotypes with Johne's disease. No overwhelming evidence for a simple causal relationship was detected. Assays are provided to screen populations of cattle for variation in the CARD15 gene.

This thesis summarizes an investigation of Mycobacterium avium subspecies paratuberculosis (Map) as a pathogen within the cow-calf industry in Canada. The specific objectives of this project were to describe the distribution of this pathogen in this industry provincially, as well as at the individual farm level in wildlife species, and in the environment. Secondary objectives of this project were to identify on-farm management risk factors that are associated with this disease and to examine potential options for herd level diagnostic capabilities. Nationally, 0.8% (95%CI = 0.4-1.1%) of the cows in the cow-calf industry were seropositive for Map with 11.7% (95%CI=7.0-16.5%) of the herds sampled having a minimum of one positive test result or 4.5% (95%CI=1.4-7.5%) of the herds having a minimum of two positive test results. The true cow prevalence was estimated as 1.8% (95%CI= 0.4 3.1). No Map was detected in any of the non-ruminant wildlife species sampled on cow-calf operations suggesting that these species were not of primary concern when dealing with the management of this disease. In a study not focussed on a cow-calf operation, Map was detected in one cluster of trapped coyote samples in a region with cow-calf production. The prevalence of Map infection in this cluster of coyotes was calculated to be 9.1% (CI: 5.7-12.5). The prevalence of infection in coyotes including all sites, ignoring the effect of clustering, was calculated to be 3.7% (CI: 2.3-5.1). The use of a commercial colostrum replacement on farm (Odds Ratio =3.96; 95% CI = 1.1014.23, p=0.035) and the presence of wild deer interacting with the cattle (Odds Ratio = 14.32; 95% CI = 1.13181.90, p=0.040) were positively associated with being a herd infected with paratuberculosis. The use of rotational grazing practices was protective (Odds Ratio = 0.20; 95% CI = 0.040.93, p=0.039). It was possible to detect environmental contamination with Map on cow-calf farms using bacterial culture and PCR for confirmation. No water samples were positive to Map; however, 6.2% of the non-water environmental samples were positive. The use of an environmental sampling protocol had a herd sensitivity of 29.6%. This finding led to a simulation modelling study to evaluate how various testing methods would compare in the broader population of cow-calf herds. The final mean risk of selecting a herd infected with Map that was not identified as positive via the herd screen test strategy was 12.9%, 9.8%, 9.6%, and 6.1% for no herd screen test, environmental sampling, ELISA serology, and pooled fecal culture strategies, respectively.

This thesis summarizes an investigation of Mycobacterium avium subspecies paratuberculosis (Map) as a pathogen within the cow-calf industry in Canada. The specific objectives of this project were to describe the distribution of this pathogen in this industry provincially, as well as at the individual farm level in wildlife species, and in the environment. Secondary objectives of this project were to identify on-farm management risk factors that are associated with this disease and to examine potential options for herd level diagnostic capabilities. Nationally, 0.8% (95%CI = 0.4-1.1%) of the cows in the cow-calf industry were seropositive for Map with 11.7% (95%CI=7.0-16.5%) of the herds sampled having a minimum of one positive test result or 4.5% (95%CI=1.4-7.5%) of the herds having a minimum of two positive test results. The true cow prevalence was estimated as 1.8% (95%CI= 0.4 3.1). No Map was detected in any of the non-ruminant wildlife species sampled on cow-calf operations suggesting that these species were not of primary concern when dealing with the management of this disease. In a study not focussed on a cow-calf operation, Map was detected in one cluster of trapped coyote samples in a region with cow-calf production. The prevalence of Map infection in this cluster of coyotes was calculated to be 9.1% (CI: 5.7-12.5). The prevalence of infection in coyotes including all sites, ignoring the effect of clustering, was calculated to be 3.7% (CI: 2.3-5.1). The use of a commercial colostrum replacement on farm (Odds Ratio =3.96; 95% CI = 1.1014.23, p=0.035) and the presence of wild deer interacting with the cattle (Odds Ratio = 14.32; 95% CI = 1.13181.90, p=0.040) were positively associated with being a herd infected with paratuberculosis. The use of rotational grazing practices was protective (Odds Ratio = 0.20; 95% CI = 0.040.93, p=0.039). It was possible to detect environmental contamination with Map on cow-calf farms using bacterial culture and PCR for confirmation. No water samples were positive to Map; however, 6.2% of the non-water environmental samples were positive. The use of an environmental sampling protocol had a herd sensitivity of 29.6%. This finding led to a simulation modelling study to evaluate how various testing methods would compare in the broader population of cow-calf herds. The final mean risk of selecting a herd infected with Map that was not identified as positive via the herd screen test strategy was 12.9%, 9.8%, 9.6%, and 6.1% for no herd screen test, environmental sampling, ELISA serology, and pooled fecal culture strategies, respectively.