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.

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 study explored the use of a biosecure, static composting structure to inactivate MAP. In Experiment #1, it was concluded that composting is unlikely to achieve temperatures necessary to inactivate MAP associated with cattle mortalities and that M. smegamatis is an unlikely surrogate for MAP. This study also used the same system to explore changes in the microbial community in mortality compost after exposure to thermophilic temperatures. As high-throughput sequencing technologies advance, it is possible to characterize microbial communities in environments with a high degree of resolution. In Experiment #2, as members of Clostridia were present at temperatures > 55°C, it appears that anaerobic conditions existed within regions of the compost. Extreme temperatures and non-homogeneous high moisture conditions resulted in spatial distribution of temperature in a biosecure, static composting system, which failed to meet conditions necessary for complete composting and pathogen reduction.

This study explored the use of a biosecure, static composting structure to inactivate MAP. In Experiment #1, it was concluded that composting is unlikely to achieve temperatures necessary to inactivate MAP associated with cattle mortalities and that M. smegamatis is an unlikely surrogate for MAP. This study also used the same system to explore changes in the microbial community in mortality compost after exposure to thermophilic temperatures. As high-throughput sequencing technologies advance, it is possible to characterize microbial communities in environments with a high degree of resolution. In Experiment #2, as members of Clostridia were present at temperatures > 55°C, it appears that anaerobic conditions existed within regions of the compost. Extreme temperatures and non-homogeneous high moisture conditions resulted in spatial distribution of temperature in a biosecure, static composting system, which failed to meet conditions necessary for complete composting and pathogen reduction.

Paratuberculosis represents a major problem in farmed ruminants and at the present is considered a potential zoonosis. The disease is caused by Mycobacterium avium subsp. paratuberculosis, and susceptibility to infection is suspected to have a genetic component. Caspase recruitment domain 15 (CARD15) gene encodes for a cytosolic protein implicated in bacterial recognition during innate immunity. Crohn's disease (CD) is an idiopathic inflammatory bowel disease in humans comparable in many features to bovine paratuberculosis involving an abnormal mucosal immune response. The association between mutations in the CARD15 gene and increased risk of Crohn's disease has been described. The objective of this candidate gene case-control study was to characterize the distribution of three polymorphisms in the bovine CARD15 gene and test their association with paratuberculosis infection in cattle. Three previously reported single nucleotide polymorphisms (E2[-32] intron 1; 2197/C733R and 3020/Q1007L) were screened for the study population (431 adult cows). The statistical analysis resulted in significant differences in allelic frequencies between cases and controls for SNP2197/C733R (P < 0.001), indicating a significant association between infection and variant allele. In the analysis of genotypes, a significant association was also found between SNP2197/C733R and infection status (P < 0.0001); cows with the heterozygous genotype were 3.35 times more likely to be infected than cows with the reference genotype (P = 0.01). Results suggest a role for CARD15 gene in the susceptibility of cattle to paratuberculosis infection. These data contribute to the understanding of paratuberculosis, suggest new similarities with Crohn's disease and provide new information for the control of bovine paratuberculosis.

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.

No description available.

Genes modulating innate immunity in mammals are generally considered the first line of defense with respect to invading pathogens and therefore it has become important to characterize naturally occurring genetic variation, and subsequently determine whether this variation is likely to be benign, beneficial, or detrimental to the host. Relevant to this study, the mammalian Toll-like receptor proteins (TLR), encoded by members of the TLR gene family, have the capacity to recognize a wide variety of pathogen ligands, and mutations within these genes have been shown to influence disease susceptibility or resistance within mammalian species. Two studies which sought to determine the frequency and distribution of naturally occurring genetic variation within the bovine and equine TLR genes revealed a large number of discrete point mutations, which were subsequently used to reconstruct haplotypes for each investigated gene across a large number of samples. Detailed analyses of haplotypes provided evidence for extensive haplotype sharing among specialized breeds, subspecies, and even divergent species. Classical and new tests of selection provided evidence for significant deviations from a strictly neutral model of molecular evolution for both cattle as well as equids, with some of the same TLR genes deviating from a strictly neutral model among divergent species. As a first step toward determining whether naturally occurring bovine TLR variation is likely to be benign, beneficial, or detrimental, we tested validated variation from bovine TLR genes capable of recognizing components of Mycobacteria for associations with Mycobacterium avium subspecies paratuberculosis (MAP) infection in dairy cattle, and found several SNPs that were nominally associated with disease status, thereby providing evidence for small-effect loci potentially influencing risk for differential susceptibility to Johne's disease.