MYCOBACTERIUM AVIUM PARATUBERCULOSIS (MAP, JOHNES) DISEASE IN FLORIDA BEEF CATTLE.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0210324
• Project Start Date: Jan 1, 2007
• Project End Date: Jan 1, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
College of Veterinary Medicine

Non Technical Summary
An important disease agent impacting the performance of cattle state-wide is Mycobacterium avium subsp. paratuberculosis (MAP). It is a bacterium that infects ruminants worldwide. It causes chronic, thickening of the gut, Johne's disease. The disease is characterized by chronic diarrhea and weight loss. There is no known cure for the disease and it is eventually fatal. The organism can be isolated from the cow's colostrum and milk and is transmitted primarily by these or a fecal-oral route to their calves early in life. Animals tend to become more resistant as age advances. There is a long incubation period. An animal rarely shows clinical signs until two years of age or more. Control of the disease is difficult. Currently there are no reliable tests for detecting early infection. Detection in older animals is problematic due to low test sensitivity. Using available diagnostic testing modalities and herd measures of performance within the cow-calf herd, different aspects of this project will be assessed. This project aims to identify and characterize a number of factors associated with Mycobacterium avium paratuberculosis (MAP) disease. We will look at how it impacts cow performance and how management influences disease within the herd. We will focus on the affect on performance of beef cattle in the state of Florida, including performance and morbidity monitoring, genetic selection and genetic marker identification for disease resistance, and application of biosecurity principles in a commercial cattle setting.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3310	1080	20%
311	3310	1040	10%
311	3310	1100	50%
311	3310	1170	20%

Knowledge Area
303 - Genetic Improvement of Animals; 311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 1080 - Genetics; 1170 - Epidemiology;

Keywords

mycobacterium avium paratuberculosis (map)

johnes' disease

paratuberculosis

beef cattle

genetic markers

biosecurity

risk assessment

Goals / Objectives
To evaluate the influence of Mycobacterium avium paratuberculosis (MAP) associated infection on the innate response of affected cattle populations and their performance among cohorts of [beef] cattle in the state of Florida. 1. To assess cow performance as measured by parameters such as, nutritional status measured by body condition, pregnancy, and calf weight gains, retrospectively as it is associated with likelihood of MAP infection. 2. To assess birth cohorts of calves (by growth, morbidity and mortality factors) born to dams with a greater or lesser likelihood of MAP infection. 3. To evaluate the influence of implemented biosecurity practices on clinical and subclinical cases of MAP, cow performance and environmental contamination. 4. To evaluate the presence/absence of genes associated susceptibility/resistance in multiple breeds of cattle with an aim to identify genetic selection tools for management of MAP. 5. To assess and quantify the effects of the MAP infectious disease process on the Florida calf crop by development of epidemiological models.

Project Methods
Objective 1. To assess cow performance Dam and calf genetic and environmental factors have been evaluated for their association with enzyme-linked immunosorbent assay (ELISA) s/p ratio scores for paratuberculosis (MAP) in a multibreed beef cattle population. The analysis used 359 ELISA s/p ratio scores from 340 dams. Dams with high ELISA s/p ratio scores produced smaller calves, gained less weight (or lost weight) during the preweaning season, and produced less milk, which in turn may have caused calves to have smaller preweaning gains. Factors identified here as associated with ELISA s/p ratio scores could help cattle producers with culling decisions related to paratuberculosis control and eradication efforts in beef cattle. We will continue this evaluation process. Objective 2. To assess birth cohorts of calves. Genetic evaluation for production traits assume that records come from healthy animals. Records from animals suffering from chronic diseases with long subclinical incubation periods may be difficult to identify, thus likely to be included in genetic evaluations, and disease effects not accounted for; this is applicable to MAP infection. A common diagnostic test for paratuberculosis is ELISA. Regression estimates of four cow and two calf traits on ELISA scores were obtained. Cows with greater ELISA scores tended to stay open longer, have larger weight losses, and have calves with lower birth and weaning weights than cows with lesser ELISA scores. We will continue this evaluation process. Objective 3. To evaluate the influence of implemented biosecurity practices on clinical cases of MAP. Biosecurity practices are essential for the successful eradication of MAP in a herd of cattle. Management plus testing is expected to produce a more rapid reduction in prevalence of disease and environmental burden of microbes. Changes in risk of disease, by use of a risk assessment tool, will assess the relative value of biosecurity practices. Objective 4. To evaluate the presence/absence of genes associated susceptibility/resistance in multiple breeds of cattle. There is evidence that resistance to infectious disease in animals has a genetic basis and that genetic variation exists among animals in their response to various infectious challenges. Johnes disease has been demonstrated to have a genetic component. This will be a candidate gene case-control association study. The project consists in determining the alleles present in one candidate gene in a population of infected cows (cases) and of non-infected controls to find a resistance allele that could be useful in selection. Objective 5. To assess and quantify the effects of the MAP infectious disease process on the Florida calf crop. Infectious disease processes can have dramatic effects on beef cattle production. Assessment of the effects of disease, and its economic impact, can be estimated by the development of suitable epidemiological models. A disease model will be developed for MAP infection.

Progress 01/01/07 to 01/01/12

Outputs
OUTPUTS: Paratuberculosis represents a major and chronic disease problem in farmed ruminants. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Our laboratory continues to examine several aspects of this organism and disease. 1) There is not an effective vaccine or treatment for the disease. Therefore, prevention via management practices is currently the only effective control measure. Methods have focused on purchasing animals from Johne's negative herds, separating new born calves from infected cows, separating and culling infected animals, minimizing exposure to manure contaminated food and water sources and ensuring hygienic manure management practices. Strategies that interrupt the anticipated ecologic cycle of fecal-oral transmission are being examined as a control of the disease. 2) Importantly, diagnosis of paratuberculosis is challenging especially in the early stages of the disease and laboratory tests used today do not show satisfactory efficacy when applied before the onset of visible signs. The lymphatic system plays a major role in the defence of infection and is an important part of the immune system. The superficial lymph vessels of the udder are accessible for collection of lymphatic fluid in lactating cows by lymph vessel puncture. To date, lymphatic fluid from cows have been collected only for a few research purposes. Our studies are a first attempt to use the assumed early presence of the bacteria and specific antibodies in the lymphatic fluids for diagnostic purposes. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Study outcomes are positive and have been reported. Continued results are pending for the above described projects. The results of current study shows that the puncture of lymphatic vessels at the bovine udder is well tolerated by the majority of cows and can easily be performed on farm. The isolation of MAP from lymph fluid by PCR indicates that this approach might be of use for the early detection of MAP in cattle. Intramural grant proposals have been prepared and submitted to granting agencies to support this project.

Publications

• Khol JL, Pinedo PJ, Buergelt CD, Neuman LM, Baumgartner W, Rae DO. Lymphatic vessel puncture at the bovine udder: feasibility for and detection of Mycobaterium avium subsp. paratuberculosis in the lymphatic fluid by PCR. J Vet Diagn Invest (2012) 24:23-31.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Paratuberculosis represents a major and chronic disease problem in farmed ruminants. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Our laboratory continues to examine several aspects of this organism and disease. 1) There is not an effective vaccine or treatment for the disease. Therefore, prevention via management practices is the only effective control measure. Methods have focused on purchasing animals from Johne's negative herds, separating new born calves from infected cows, separating and culling infected animals, minimizing exposure to manure contaminated food and water sources and ensuring hygienic manure management practices. Consumption of forages contaminated directly with infective manure is an important avenue for transmission. Can infected cows shed MAP in their manure and contaminate irrigation waters and thus crops raised to feed other animals? This hypothesis is being pursued. Can MAP cycle from cow to manure to water slurry to irrigated crops to harvested feeds and be the source of infection in this hypothesized ecologically cycle? This is being tested in a series of experimental trials looking at recovery rates in plants exposed to MAP on its leaves or in the soil. Simultaneously, strategies that interrupt the anticipated ecologic cycle of fecal-oral transmission are being examined as an effective control of the disease. Ensiled crops hold promise in this regard and are being studied. They have been shown to reduce the numbers of organisms as compared to dry-stored feeds (hay). Studies under US dairy and crop management systems are being developed to test this hypothesis and determine factors that might reduce the survival of the pathogen in ensiled forages or in the rumen. 2) Diagnosis of paratuberculosis is challenging especially in the early stages of the disease and laboratory tests used today do not show satisfactory efficacy when applied before the onset of visible signs. The lymphatic system plays a major role in the defence of infection and is an important part of the immune system. The superficial lymph vessels of the udder are accessible for collection of lymphatic fluid in lactating cows by lymph vessel puncture. To date, lymphatic fluid from cows have been collected only for a few research purposes. Our studies are a first attempt to use the assumed early presence of the bacteria and specific antibodies in the lymphatic fluids for diagnostic purposes. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Veterinarians and livestock producers working with cattle and species susceptible to Johne's Disease. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Study outcomes are pending for the above described projects. The results of current study shows that the puncture of lymphatic vessels at the bovine udder is well tolerated by the majority of cows and can easily be performed on farm. The isolation of MAP from lymph fluid by PCR indicates that this approach might be of use for the early detection of MAP in cattle. Intramural and extramural grant proposals have been prepared and submitted to granting agencies to support each of these two described projects.

Publications

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Paratuberculosis represents a major and chronic disease problem in farmed ruminants. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Our laboratory is currently examining two aspects of this disease. 1) There is no effective cure for the disease; therefore prevention via various management practices is currently the only effective control measure. Methods have focused on purchasing animals from Johne's negative herds, separating new born calves from infected cows, separating and culling infected animals, disinfection of water, liming to inhibit bacterial survival in the soil, and ensuring hygienic manure management practices. Little attention has been paid to inhibiting transmission of MAP from forages irrigated with contaminated manure to the consuming cows. Yet, consumption of contaminated forage is an important avenue for transmission- infected cows shed MAP in their manure, contaminate irrigation waters used to raise crops. Consequently, strategies that interrupt the fecal-oral transmission may be particularly effective in controlling the disease. Ensiled crops have been shown to reduce the numbers of organisms as compared to dry-stored feeds (hay). Studies under US dairy and crop management systems are needed to validate this hypothesis. Studies are currently being performed to determine factors that reduce the survival of the pathogen in ensiled forages or in the rumen. 2) Diagnosis of paratuberculosis is challenging especially in the early stages of the disease and laboratory tests used today do not show satisfactory efficacy when applied before the onset of visible signs. The lymphatic system plays a major role in the defence of infection and is an important part of the immune system. The superficial lymph vessels of the udder are accessible for collection of lymphatic fluid in lactating cows by lymph vessel puncture. To date, lymphatic fluid from cows have been collected only for a few research purposes. Our studies are a first attempt to use the assumed early presence of the bacteria and specific antibodies in the lymphatic fluids for diagnostic purposes. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Study outcomes are pending for the above described projects.

Publications

• No publications reported this period

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: 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). In a companion study, several other genetic markers were examined. Interferon gamma is an inducible cytokine with a crucial role in the innate host response to intracellular bacteria. Toll-like receptors are trans-membrane structures responsible for coordination of innate and adaptive immune responses. The solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1) gene plays an important role in innate immunity, preventing bacterial growth in macrophages during the initial stages of infection. The objective of this candidate gene case-control study was to characterize the distribution of polymorphisms in three candidate genes related to the immune function; interferon gamma (BoIFNG), toll-like receptor 4 (TLR4), and SLC11A1 genes and to test their role as potential risk factors for paratuberculosis infection in cattle. The statistical analysis demonstrated significant differences in allelic frequencies between cases and controls for BoIFNG-SNP12781 and SLC11A1 microsatellites, indicating a significant association between infection and variant alleles. In the analysis of genotypes, a significant association was also found between infection status and BoIFNG-SNP12781 and SLC11A1-275-279-281 microsatellites. However, when variables such as breed and age were included in the multivariate logistic regression analysis, a tendency toward statistical significance for the effect of polymorphisms in the odds of infection was only found for alleles SLC11A1-275 and 279. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Information from the on-going studies have been shared with livestock (cattle) producers and veterinarians serving these producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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. The companion study did not demonstrate a definite association between paratuberculosis infection in cattle and the proposed candidate genes [interferon gamma (BoIFNG), toll-like receptor 4 (TLR4), and SLC11A1 genes], the analyses suggested a potential connection between polymorphisms in SLC11A1 gene and the risk of paratuberculosis infection. Further analysis including a larger population and new polymorphisms in this gene is warranted.

Publications

• Pinedo PJ, Buergelt CD, Donovan GA, Melendez P, Morel L, Wu R, Langaee TY, Rae DO. Candidate gene polymorphisms (BoIFNG, TLR4, SLC11A1) as risk factors for Paratuberculosis infection in cattle. Prev Vet Med 91 (2009)189-196.
• Pinedo PJ, Buergelt CD, Donovan GA, Melendez P, Morel L, Wu R, Langaee TY, Rae DO. Association between CARD15/NOD2 gene polymorphisms and paratuberculosis infection in cattle. Vet Microbiol. 134 (2009) 346-352.
• Pinedo PJ, Wang C, Li Y, Rae DO, Wu R. Risk haplotype analysis for bovine paratuberculosis. Mamm Genome 20 (2009) 124-129 [DOI 10.1007/s00335-008-9167-0].
• Elzo, MA, Rae DO, Lanhart SE, F. G. Hembry, J. G. Wasdin, and D. J. Driver. 2008. Association between cow reproduction and calf growth traits and ELISA scores for paratuberculosis in a multi-breed herd of beef cattle. Trop Anim Health Prod DOI 10.1007/s11250-008-9262-y.
• Pinedo PJ, Williams JE, Monif GRG, Rae DO, Buergelt CD. Mycobacterium paratuberculosis shedding into milk: Association of ELISA seroreactivity with DNA detection in milk. Intern J Appl Res Vet Med 6 (2008) 137-144.
• Pinedo PJ, Rae DO, Williams JE, Donovan, G.A, Melendez, P, Buergelt, CD: Association among results of serum ELISA, fecal culture, and nested PCR on milk, blood, and feces for the detection of paratuberculosis in dairy cows. Transbound Emerg Dis 55 (2008) 125-133.

Progress 04/01/07 to 12/13/07

Outputs
Association among serum ELISA, fecal culture, and nested PCR on milk, blood, and feces for the detection of paratuberculosis infection in dairy cows was evaluated. The objective was to analyze the association among a serum ELISA, fecal culture, and nested PCR tests on milk, blood, and feces for Mycobacterium avium subsp. paratuberculosis detection in Holstein cows. Feces, blood and milk samples were collected from 328 lactating dairy cows in four dairy herds to detect paratuberculosis infection. Association between two polymorphisms in the bovine CARD15/NOD2 gene and paratuberculosis infection in Florida dairy and beef cattle was studied. Caspase recruitment domain 15 (CARD15/NOD2) is a gene codifying for a cytosolic protein implicated in bacterial recognition by cells of the innate immune system. The objective of this candidate gene case-control study was to characterize the distribution of two polymorphisms in the bovine CARD15/NOD2 gene and test their association with paratuberculosis infection. The study population consisted of 432 adult cows in four herds. Infection status was determined using five diagnostic tests (serum ELISA, milk/blood/fecal nested PCR, and fecal culture); a parallel interpretation of results was used. Two single nucleotide polymorphisms in the gene were determined for study animals by genotyping assay. It was hypothesized that alleles in our candidate gene would be present in higher frequency in controls compared to cases, suggesting a role in resistance to infection. Association between cow reproduction and calf preweaning growth traits and ELISA scores for paratuberculosis in a multibreed herd of beef cattle was assessed. The objective of this study was to assess the association between 4 cow reproductive and weight traits, and 3 preweaning calf traits and ELISA scores for paratuberculosis in a multibreed herd of cows ranging from 100% Angus to 100% Brahman. Cow data included gestation length, time open, calving interval, and weight change for 502 cows. Calf data consisted of birth, weaning, and adjusted weaning weights for 956 calves. Presentations: 9th International Colloquium for Paratuberculosis, Tsukuba, Japan, Oct 29-Nov 2, 2007, 6A-O2, Association between Two Polymorphisms in the Bovine CARD15/NOD2 Gene and Paratuberculosis Infection in Florida Dairy and Beef Cattle. PJ Pinedo, CD Buergelt, R Wu, GA Donovan, JE Williams, PG Melendez, L Morel, and DO Rae, Poster and Oral. Research Summary, 2007 (20 Sep) American Association of Bovine Practitioners, Annual Conference, Vancouver, BC, Genetic Resistance to Johne's Disease in Four Cattle Breeds: A Candidate Gene Case Control Study, Preliminary Results, Poster and Oral. Emerging Pathogens Institute, Fall Research Retreat, University of Florida, Dec 13, 2007, Poster. Association between CARD15/NOD2 Gene polymorphisms and paratuberculosis infection in Florida Cattle, P Pinedo, C Buergelt, A Donovan, P Melendez, DO Rae, R Wu. 2nd Bi-annual Research Emphasis Day, College of Veterinary Medicine, University of Florida, May 11, 2007, Diagnostic development in bovine paratuberculosis, P Pinedo, DO Rae.

Impacts
The study of association among serum ELISA, fecal culture, and nested PCR on milk, blood, and feces for the detection of paratuberculosis infection in dairy cows analyzed results to establish the association and the level of agreement between pairs of tests. A total of 61 animals (18.6%) tested positive when all the tests were interpreted in parallel. The agreement between results in different pairs of tests was poor, slight and fair. Fecal culture vs. fecal PCR resulted in the highest kappa coefficient (0.39; fair agreement), with the lowest agreement being for serum ELISA vs. PCR on blood (-0.036; poor agreement). Statistically significant association was found between the following test pairs; ELISA:fecal culture; ELISA:fecal PCR; PCR on milk:fecal PCR, PCR on blood:fecal PCR and fecal culture:fecal PCR. The complementary sensitivity values obtained in this study suggests the potential use of different tests combinations to increase the overall sensitivity for the diagnosis of paratuberculosis infection. A study of association between two polymorphisms in the bovine CARD15/NOD2 Gene and paratuberculosis infection in Florida dairy and beef cattle resulted in significant differences in allelic frequencies between cases and controls for SNP1 indicating a significant association between infection and mutant allele. Significant association was found between SNP1 and infection status. A significant association between allele combinations and infection status was found when both SNPs (1 and 2) were considered in the genotype. The low representation of the variant allele for SNP1 in Holstein and Jersey breeds raises the prospect of a potential confounding role of breed for its connection with infection. However, a significant association between SNP1 and infection was confirmed when tested within the Brahman-Angus sub-population. Preliminary results suggest a role for CARD15/NOD2 gene in the susceptibility of cattle to paratuberculosis infection. Amino acid substitution C733R (SNP1) appears to be associated to paratuberculosis infection in Florida cattle. These results could be the basis for further research to create a rapid method to select for more resistant individuals, genetically contributing to the control of Johne's disease. Association between cow reproduction and calf preweaning growth traits and ELISA scores for paratuberculosis in a multibreed herd of beef cattle found that estimates of differences between cows with non-zero and zero ELISA scores were associated with lower cow fertility (longer TO), lower ability of cows to maintain weight (negative WC), lower calf BWT, and lower calf weaning weights (WWT and WW205). Considering TO, WC, and WWT, and using average market prices of cows and calves, potential losses of income due to subclinical paratuberculosis were estimated to be $62.4 for cows with positive ELISA score. Further research on the effects of subclinical paratuberculosis in beef cattle at regional and national levels seems advisable considering the large potential economic cost of this disease.

Publications

• No publications reported this period