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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No description available.

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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

Biosecurity practices for Mycobacterium avium subsp. paratuberculosis (Mptb), the etiologic agent for Johne's disease (JD), are predicated on the fact that fecal-oral is the major route of infection and that Mptb is present in the environment of affected farms. The objectives of these studies were to describe perceived benefits of testnegative Level 4 status in the Voluntary Bovine Johne's Disease Control Program (VBJDCP), describe producer and veterinarian attitudes towards JD relevant biosecurity practices, compare 5 JD control options using a Markov model, determine if tangential flow filtration (TFF) increases the detection sensitivity for Mptb and describe the distribution of environmental predictors for Mptb survival. Twenty-five percent and 39% of beef producers in the VBJDCP reported that they received substantial or marginal benefits (financial and non-financial), respectively, from program participation. Producers suggested increased marketing opportunities to improve the VBJDCP. Producers in a cross-sectional mailed survey of attitudes towards biosecurity practices were more likely than veterinarians to agree that separating JD clinical or suspects from calves or heifers; acquiring replacements or additions from JD low-risk herds, testing for JD every 10 to 14 months and test and culling clinical suspects only were useful for control of JD. A state transition Markov model, with the environment as the source of Mptb, was used to compare 6 alternative control strategies for JD. Management and the probability of Mptb surviving 1 year in the environment were important determinants of the prevalence of subclinical JD on beef farms under the analyzed control strategies. Heterogenous distribution of environmental predictors for Mptb survival was observed in spatial risk maps. In conclusion, although some beef producers experienced gains from participation in the VBJDCP, the perceived program benefits could be improved by increased marketing and education on the advantages of participation. Specific problem areas should be addressed. The length of time Mptb survived in the environment was an important parameter in the Markov chain model. Additionally, due to the heterogenous distribution of environmental predictors, a multiscale approach to sampling and analysis should be useful.

The validity of Johne??s disease herd status programs and on-farm disease control programs that rely on established ??cutpoints?? (e.g., S/P ratios) for ELISA serological tests such as the HerdChek?? (IDEXX Laboratories Inc., Westbrook, Maine) may be susceptible to varied seasonal test accuracy. An observed depression in the proportion of a large central Texas dairy herd classified as ??positive?? during the months of July and August led to our investigation. We hypothesized that there exists a seasonal variability in serological response to Mycobacterium avium subsp. paratuberculosis that is directly related to heat stress. We further hypothesized that a reciprocal response may occur during periods of heat stress that results in a greater risk of fecal shedding in subclinically-infected animals. Starting in October 2002, we invoked a testing regime that included multiple testing of 720 individual adult cows over each of four seasons including spring, summer, fall, and winter. We collected serum on a cyclic, monthly basis from three random groupings of cows, and, based on the ELISA results, collected fecal samples from the 20% of cows with the highest S/P ratios. We continued to sample in this manner for the period of one year and at the end of that period, analyzed the serum en masse. The ELISA outcome values were treated both as categorical and continuous variables (e.g., S/P ratio). The potential lagged effects of heat stress on S/P ratio, as well as the potential for a change in test result (negative to positive or vice versa) due to heat stress were assessed. The results for fecal culture were analyzed on a categorical scale and were compared to the ELISA results to explore the possibility of a reciprocal response. In the present study, we did not observe any of the significant seasonal effects of heat stress on S/P ratios and proportion seropositive to MAP that were observed in the historical (and less valid) cross-sectional time-series data conducted in 2001. In addition, we found no evidence to support a hypothesis linking seasonal heat stress to the risk of fecal culture positivity for the causative bacterium for Johne??s disease.

Previous research performed in 1999 by Harris et al. has shown that many varieties of ruminants serve as the host species for Mycobacterium avium subspecies paratuberculosis (MparaTb) infections. Gene sequencing has supported the contention that organisms isolated from different hosts harbor different gene sequences; this has been exemplified by Amonsin et al. in 2004 with the sequencing of the mfd (transcription-repair coupling factor) and by Motiwala et al. in 2005 through sequence analysis of phosphatidylethanolaminebinding proteins which reveal a host-specific correlation of isolates. Some contradicting reports from Bannantine et al. from 2003 have further claimed that MparaTb is a monogenic organism based upon sequence data from regions flanking the origin of replication and the 16s rRNA. One of the drawbacks to the techniques implemented in these reports is the extremely restricted region of the bacterial genome that was analyzed; furthermore, only a select number of isolates were analyzed. In the present studies, amplified fragment length polymorphism (AFLP) was used as a tool for a genome scale comparison of MparaTb isolates from differing isolation types as well as a comparison of MparaTb isolates to the genetically similar yet avirulent Mycobacterium avium subspecies avium isolates. AFLP data reveals the MparaTb genome to be much more plastic and polymorphic than previously thought. These polymorphic regions were identified and characterized and are shown to be unique to the organism when compared to an array of Mycobacterial isolates of differing species. These polymorphic regions were also utilized in polymerase chain reaction (PCR) based diagnostic as well as epidemiologic tests. Furthermore, AFLP comparative analysis of intracellular and fecal MparaTb isolates reveals polymorphic regions unique to each isolate type. While these genomic differences are not based upon differences in the genetic code, they are based upon epigenetic modifications such as DNA methylation. These DNA methylation patterns are unique to intracellular MparaTb isolates as opposed to isolates from fecal material. Furthermore, AFLP comparisons of fecal MparaTb isolates that were passaged through the bovine ileum revealed banding pattern differences as compared to the original inoculum.

Johne’s disease (JD) in cattle is a disease of economic importance caused by Mycobacterium avium subspecies paratuberculosis (MAP). Studies were conducted to estimate the losses due to lower weaning weight of beef calves from MAP test-positive dams, to compare the perceptions of producers and veterinarians on the burden and economic aspects of MAP infection in cow-calf herds, and to evaluate whether testing and culling MAP test-positive cows is economically beneficial. Calves from cows with strong-positive ELISA results were 21.5 kg lighter at weaning compared to calves from ELISA-negative cows. Calves from heavy MAP shedding cows were 58.5 kg lighter, and calves from moderate shedders were 40.8 kg lighter compared to the calves from fecal-culture negative cows. Based on average feeder calf value during 2007 to 2012, these losses corresponded to US $57 per calf for ELISA strong-positive dams, US $157 per calf for heavy fecal shedder dams, and US $109 per calf for a moderate fecal shedder dam. Seedstock producers and the producers enrolled in control programs were more likely to have MAP uninfected herds. The average prevalence reported by producers was 0.8%. Compared to the small herds (<50 head), the average test-positive percentages and estimated prevalences were reported to be higher in medium (50-149) and highest in large (≥150) herds. Veterinarians reported an overall animal level prevalence in their client herds of 5%. Seedstock herds had a lower prevalence and these producers were more likely to enroll in a JD control program. Income lost due to the presence of JD in an infected cattle herd was perceived to be higher by veterinarians. Compared to the veterinarians, seedstock producers were more likely to perceive genetic losses due to culling MAP positive cows. Average annual loss due to JD in a 100 cow herd with a 7% MAP prevalence was $1,644 and $1,747 based on information provided by producers and veterinarians, respectively. Herd level production decreased with increasing prevalence. Compared to test and cull after ELISA or ELISA followed by fecal culture, using fecal culture alone provided the fastest reduction in herd prevalence. Fecal culture was also the least costly alternative based on long-term cumulative costs of an annual test and cull program. Results from the current study suggest that although testing provides faster progress, limiting within herd transmission by sale of all weaned calves and purchasing only low-risk replacements can also reduce prevalence. Results suggest that MAP infection in cows causes significant losses for the calves that are produced. While the knowledge about JD varied between producers and veterinarians, seedstock producers were more enthusiastic about MAP control programs and had lower MAP prevalence in their herds. Overall losses due to MAP infection in the herd might be substantial. It is very costly to control or eliminate MAP once the infection is established in a herd.