Bovine Tuberculosis: Epidemiology, Diagnosis, and Pathogenesis

NON-TECHNICAL SUMMARY: Since bovine TB control and prevention programs for livestock have significant costs, there is a need to improve the performance of these programs. The proposed research will evaluate current TB control and prevention programs, the reliability of TB tests being used, factors that influence the effectiveness of these tests and programs, and the efficacy of two TB vaccines for animals.

<P>

APPROACH: Project 1, Part A: A case-control study will compare risk factors (farm location, ecological data, herd management practices before and after restocking) between herds diagnosed with TB that were depopulated and repopulated (controls) with repopulated herds that have become reinfected with TB (cases). Project I, Part B: A retrospective case-control study will compare spatial risk factors between herds diagnosed with TB since the beginning of the current disease outbreak (cases) and herds that have not been diagnosed with TB (controls) from the five county area. Case herds will be those. Risk factors include livestock housing location data (located by Global Positioning Satellite (GPS) systems), specific ecological conditions in cattle housing areas, and herd management practices. Project 2, Part A: The effect of infection with M. paratuberculosis on results of the bovine TB caudal fold test (CFT) test and gamma interferon assay will be assessed by comparing the false negative rate for these two assays in Johnes disease high prevalence dairy herds and herds known to be free of Johnes disease. Project 2, Part B: The effect of use of select veterinary vaccines on the reliability of the CFT for TB in cattle will be tested by comparing CFT test results and M. bovis gamma interferon production between groups of calves sensitized to M. bovis that are unvaccinated or vaccinated with a commercially available vaccine containing modified-live bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis (IBR), PI-3 and bovine respiratory syncital virus (BRSV) (Pyramid, Ft. Dodge Animal Health, Fort Dodge, IA). Project 2, Part C: A retrospective study of the role of M. paratuberculosis in false reactor response on the CFT and the comparative cervical test (CCT) will compare levels of M. paratuberculosis between CFT positive/M. bovis culture negative cattle and CFT negative cattle by PCR for M. paratuberculosis from sections of ileum and ileal-cecal lymph nodes. Project 3, Part A: The optimal time of exposure of whole blood to bovine ppd for detection of diagnostic gene targets in caudal fold suspect cattle will be determined by comparing levels of altered expression of 10 genes after 0, 1, 2, 4 or 20 hours of antigen stimulation, between cattle that have been sensitized to M. bovis and cattle that have not been sensitized. Project 3, Part B: Optimal gene targets for detection of bovine tuberculosis will be identified by stimulating blood with phosphate-buffered saline (PBS) (negative control), M. avium purified protein derivative (PPD), and M. bovis PPD, and harvesting total cellular RNA to generate cDNA for use in real-time quantitative polymerase chain reaction (RT-QPCR). Project 4: The efficacy of BCG vaccine and a recombinant vector vaccine (RB-51) for M. bovis in laboratory mice will be tested by comparing lesion development and mycobacterial isolation results from vaccinated and unvaccinated mice challenged with deer-origin M. bovis.
<P>
PROGRESS: 2004/09 TO 2007/08<BR>
Project 1: The predictive risk assessment model for reacquiring TB and proximity to other herds affected by TB, deer habitat, surface water features, and other ecological features in the region has been finalized. Updated information on data from newly infected cattle herds (biosecurity, cattle movement to and from affected herds, cattle feeding practices, cattle housing, and wildlife access to livestock and livestock feed) and additional retrospective data collection was used to refine the existing predictive model, which is undergoing testing. Project 2: Dairy cattle with Johne's disease (JD, infection with M. avium ssp. paratuberculosis [n=11]) and age-matched cattle without JD (n=8) were sensitized with antigen from M. bovis to stimulate an immune response that would cause the cattle to test positive for TB using currently approved testing methods. JD did not affect the result of the caudal fold test (CFT), as all sensitized cattle showed a positive reaction, but did adversely affect the results of the whole blood gamma interferon test in some cattle. Project 3: To identify altered gene expression patterns in cattle that are false positive on current TB tests as potential diagnostic targets for TB detection, whole blood was collected from 60 lesion negative cattle suspect for TB on the CFT and the comparative cervical test (CCT) or the whole blood gamma interferon test, and 19 microarray analyses have been completed for RNA samples from blood pre-stimulated with M. bovis antigen for 4 hrs (n=15; 7 to CFT reactors, 4 double reactors, 4 TB positive) and 0-hr (n=4; all double reactors). Altered gene expression of 5-fold or greater was seen in each group: 10 up- and 2 down-regulated genes from CFT reactors, 223 up- and 5 down-regulated genes from double reactors, and 12 up- and 5 down-regulated genes from TB positives. No genes demonstrating altered expression levels were shared among or between these groups of cattle. The 0-hr microarrays showed only 4 genes with altered expression and none showed altered levels that were greater than 5-fold above or below control values. Project 4: BALBc mice were vaccinated twice with standard BCG or a new recombinant vaccine, then challenged intranasally with live M. bovis. 12 unvaccinated controls and 12 recombinant vaccine mice lost weight, became moribund, and were sacrificed within 4 weeks of challenge. BCG vaccinates maintained activity and body weight, and only 2 of 12 mice were sacrificed 7 weeks post challenge. The recombinant vaccine showed marked reduction in mortality, gross and microscopic lesions compared to controls, which shows promise for development of a vaccine that will not interfere with current tests. A second trial was performed using a modified subunit vaccine:weight loss, lesions development, and mortality were reduced compared to controls, but slightly higher compared to BCG vaccinates. Final summarization of the study is pending on final mycobacterial isolation and titration. A study was conducted on Michigan strains of M. bovis isolates from 1999 and 2004, to look for evidence of antimicrobial resistance, and no evidence of antimicrobial resistance development was seen.
<P>
IMPACT: 2004/09 TO 2007/08 <BR>
Infection of cattle with Johne's disease does not appear to have major influence on the rate of false positive skin TB tests in Michigan, but may cause cattle to test negative on some secondary laboratory diagnostic assays. Results of gene expression profiling showed that there are promising gene targets that may be used for developing diagnostic tools to detect TB in live cattle. The intranasal challenge in BALBc mice is an efficient system for testing efficacy of tuberculosis vaccines. BCG vaccine demonstrated good protection to challenge, and the redesigned recombinant vaccine was much more effective that earlier recombinant vaccines. Lack of antimicrobial resistance in M. bovis from Michigan is important for the treatment of TB if cases in humans occur.