Evaluation of blood assays for detection of Mycobacterium bovis in white-tailed deer (Odocoileus virginianus) in Michigan.

Surveillance and control activities related to bovine tuberculosis (TB) in free-ranging, Michigan white-tailed deer (Odocoileus virginianus) have been underway for over a decade, with significant progress. However, foci of higher TB prevalence on private lands and limited agency ability to eliminate them using broad control strategies have led to development and trial of new control strategies, such as live trapping, testing, and culling or release. Such strategies require a prompt, accurate live animal test, which has thus far been lacking. We report here the ability of seven candidate blood assays to determine the TB infection status of Michigan deer. Our aims were twofold: to characterize the accuracy of the tests using field-collected samples and to evaluate the feasibility of the tests for use in a test-and-cull strategy. Samples were collected from 760 deer obtained via five different surveys conducted between 2004 and 2007. Blood samples were subjected to one or more of the candidate blood assays and evaluated against the results of mycobacterial culture of the cranial lymph nodes. Sensitivities of the tests ranged from 46% to 68%, whereas specificities and negative predictive values were all >92%. Positive predictive values were highly variable. An exploratory analysis of associations among several host and sampling-related factors and the agreement between blood assay and culture results suggested these assays were minimally affected. This study demonstrated the capabilities and limitations of several available blood tests for Mycobacterium bovis on specimens obtained through a variety of field surveillance methods. Although these blood assays cannot replace mass culling, information on their performance may prove useful as wildlife disease managers develop innovative methods of detecting infected animals where mass culling is publicly unacceptable and cannot be used as a control strategy.

Human Mycobacterium bovis infection and bovine tuberculosis outbreak, Michigan, 1994-2007.

Mycobacterium bovis is endemic in Michigan's white-tailed deer and has been circulating since 1994. The strain circulating in deer has remained genotypically consistent and was recently detected in 2 humans. We summarize the investigation of these cases and confirm that recreational exposure to deer is a risk for infection in humans.

Estimating the true prevalence of Mycobacterium bovis in free-ranging elk in Michigan.

Although relatively small, Michigan's elk (Cervus elaphus nelsoni) herd is highly valued by both hunters and the general public. Elk and red deer (Cervus elaphus elaphus) are highly susceptible to infection with Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), and outbreaks have been documented worldwide. The Michigan elk range lies entirely within counties where TB is known to be enzootic in white-tailed deer (Odocoileus virginianus). Consequently, a project was undertaken to estimate the true prevalence of TB in Michigan's free-ranging elk herd. All elk harvested by licensed hunters during 2002-2004, and all nonharvest elk mortalities examined by the Michigan Department of Natural Resources Wildlife Disease Laboratory from November 2002-May 2005, were screened for gross lesions of TB with samples of cranial lymph nodes and palatine tonsils collected for histopathology and mycobacterial culture. In all, 334 elk were included in the study. Twenty-three elk with gross lesions were considered TB suspects; all were culture-negative for M. bovis. However, M. bovis was cultured from two elk without gross lesions. The sensitivity, specificity, and negative predictive value of the current TB surveillance protocol were 0%, 100%, and 99.4%, respectively, while the apparent prevalence and true prevalence calculated directly from the sample were 0% and 0.6%, respectively. The positive predictive value and the estimated true prevalence of the population were undefined. The poor sensitivity of current surveillance was likely an artifact of its application to a relatively small sample, in order to detect a disease present at very low prevalence. The low prevalence of TB in Michigan elk, and the early stage of pathogenesis of the few infected animals, does not suggest elk are maintenance hosts at the present time.

Managing the wildlife reservoir of Mycobacterium bovis: the Michigan, USA, experience.

Historical, social and economic factors combined to provide a focus where bovine tuberculosis has become established in free-ranging wildlife in northeastern lower Michigan. White-tailed deer, the primary reservoir and maintenance host of tuberculosis, are highly valued by the public, and particularly hunters, for cultural and economic reasons. Since 1995, significant progress has been made in defining and reducing the reservoir of tuberculosis in deer. As yet, no other wildlife species has been shown to play an epidemiologically important role in the disease cycle. The importance of deer and deer hunting to Michigan has uniquely shaped tuberculosis control policies, and poses ongoing challenges as wildlife managers strive to maintain momentum for broad control strategies, and develop focused strategies that are publicly acceptable. Even if momentum and funding can be maintained, tuberculosis will likely continue to be present for a decade or longer. Thus, cattle producers waiting for tuberculosis to be eradicated from wildlife to eliminate risks to their herds and markets face disappointment for the foreseeable future. Such unrealistic expectations also place Michigan's federal tuberculosis accreditation status at perpetual risk. Accredited free status is unlikely to be regained without accompanying changes in cattle management. In Michigan, management of tuberculosis has clearly demonstrated that social issues and public approval are likely to be the critical limiting factors in control.

Patterns of antimicrobial susceptibility in Michigan wildlife and bovine isolates of Mycobacterium bovis.

The state of Michigan has recognized the presence of Mycobacterium bovis in its free-ranging white-tailed deer population since 1994. This endemic infection is primarily located in a 12-county area in the northeastern lower peninsula of Michigan. A statewide surveillance and eradication program of the disease has been in effect since 1994. Worldwide, Mycobacterium tuberculosis complex organisms have a known predilection toward development of antimicrobial resistance. The objective of this study was to investigate the antimicrobial susceptibility of M. bovis isolates from white-tailed deer in Michigan and detect any changes in susceptibility over time. M. bovis isolates from 2 fall hunting seasons (1999 and 2004) were used in this study. The fall season of 2004 marked the first documented case of direct transmission of M. bovis from a wild deer to a human in Michigan. Since M. bovis is a zoonotic disease, knowledge of susceptibility can expedite treatment options in humans. M. bovis isolates were obtained from 58 deer, 4 coyotes, 3 cattle, 2 raccoons, and 1 human case from the 2 years combined. Methods of susceptibility testing included 1% proportion agar plates and Bactec radiometric broth testing. M. bovis was found to be uniformly resistant to the antibiotic pyrazinamide; this resistance is common to all M. bovis isolates. No other antimicrobial resistance was found in any of the tested M. bovis isolates, which may be, in part, attributed to the lack of any significant treatment pressure in wildlife.

Epidemiology of Mycobacterium bovis in free-ranging white-tailed deer, Michigan, USA, 1995-2000.

An endemic area of bovine tuberculosis (TB) (Mycobacterium bovis) currently affecting wild white-tailed deer (Odocoileus virginianus) in northern lower Michigan, USA, constitutes the first self-sustaining outbreak of the infection in free-ranging North American cervids. Given this precedent, epidemiologic insights gained from the outbreak afford the opportunity to guide not only current surveillance and intervention but also control efforts for future outbreaks involving wildlife reservoirs. Our specific objectives were to evaluate retrospective data from field surveillance conducted from 1995 to 2000 to determine apparent prevalence, trends in apparent prevalence, and the effects of various factors on the odds of being M. bovis positive. Data were gathered from post-mortem examinations of 62,560 wild deer collected from all 83 Michigan counties. Records of survey method, sex, age, geographic area and infection status as determined by mycobacterial culture were subjected to trend analysis and multivariable logistic regression. Apparent prevalence for the period was 0.54% (336/62,560) statewide. Prevalence varied widely with geographic area, but significantly decreased since 1995 in the core area of the outbreak-which coincided with implementation of control strategies. Significant risk factors were geographic area, sex, age, and the sex-by-age interaction. The survey method by which deer were obtained for testing was not a predictor of infection. Our results to date suggest an outbreak characterized by broad areas of very low prevalence surrounding focal areas where prevalence is sometimes orders-of-magnitude higher (e.g., deer originating from the core area were up to 147 times more likely to be TB positive than deer from other areas). Our results also identify older male deer as most likely to be M. bovis positive (OR=11.3, 95% CI 3.2, 40.3 for bucks > or =5 years vs. does < or =1.5 years)-an observation consistent with the biology and behavior of the species. Synthesizing these results with those of other ongoing investigations, we hypothesize a two-stage model of disease transmission where TB is maintained at very low prevalence in matriarchal groups, with primary dissemination of the disease attributable to the dispersal and movements of bucks (as well as to the large aggregations of animals created by human activities).

Experimental inoculation of North American opossums (Didelphis virginiana) with Mycobacterium bovis.

Eight North American opossums (Didelphis virginiana) were inoculated with 1 x 10(5) colony forming units of Mycobacterium bovis to investigate their potential as reservoir hosts for bovine tuberculosis in Michigan. Four animals received this dose orally and four were inoculated intramuscularly (i.m.). In each group, two animals were euthanized 1 mo postinoculation (PI) and two at 2 mo PI. Four control animals were housed separately and sacrificed in the same manner as those inoculated. One of four orally inoculated opossums and three of four i.m.-inoculated opossums were positive for M. bovis by culture of tissues obtained at necropsy. The oral recipient had positive cultures from intestine and pooled lymphoid samples. Pooled lymphoid samples were positive in three i.m.-inoculated animals and two of these also had positive liver and lung cultures. One animal with gross and histologic lesions compatible with tuberculosis had negative tissue cultures. The findings suggest that opossums are susceptible to M. bovis infection by multiple routes, although their relative susceptibility compared to true reservoir hosts appears to be low.

Experimental aerosol inoculation of Mycobacterium bovis in North American opossums (Didelphis virginiana).

The goal of this study was to evaluate the susceptibility of North American opossums (Didelphis virginiana) to aerosol inoculation of Mycobacterium bovis at two dose levels in order to gain information on disease pathogenesis, fecal shedding of the organism, and the potential role that opossums play in the spread of this disease in nature. Six opossums received high dose (1 x 10(7) colony forming units (cfu) by aerosol inoculation, six opossums received low dose (1 x 10(3) cfu inoculation, and six opossums were sham-inoculated with sterile water and served as controls. Lungs were the most frequently infected tissues, with nine of 12 inoculated opossums positive for M. bovis on culture. Gross lesions consisted of multifocal pneumonia and enlarged lymph nodes. Microscopically, granulomatous pneumonia and granulomatous lymphadenitis associated with acid-fast bacilli were present in eight of 12 inoculated opossums. Fecal shedding of M. bovis was uncommon at both inoculation doses. While opossums were highly susceptible to aerosol inoculation of M. bovis, they did not become emaciated or develop widely disseminated lesions. From this study, opossums may transmit tuberculosis by aerosol infection to other opossums in close contact and serve as a source of infection to carnivores that feed upon them, however, transmission of the disease to large herbivores by fecal shedding or direct contact may be less likely.

Estimating the true prevalence of Mycobacterium bovis in hunter-harvested white-tailed deer in Michigan.

Apparent prevalence, although useful as a consistent index, may underestimate the true prevalence of disease. In Michigan, the ability to estimate the true prevalence of bovine tuberculosis (TB; caused by Mycobacterium bovis) in free-ranging white-tailed deer (Odocoileus virginianus) will become increasingly important to accurately assess progress towards eradication. Our objectives were threefold: to estimate the true prevalence of M. bovis in free-ranging deer in Michigan, to evaluate the effectiveness of existing TB surveillance methods, and to indirectly assess whether TB epidemiologic data from captive cervid herds can be meaningfully extrapolated to free-ranging populations. The study population consisted of all free-ranging deer submitted for TB testing in 2001 from six townships in northeastern Lower Michigan. Tissue samples of tonsil and cranial lymph nodes were collected bilaterally from all deer eligible for the study that did not have gross lesions suggestive of TB (n = 701). Samples were subjected to histopathologic, acid-fast (AF) staining, mycobacterial culture, and polymerase chain reaction (PCR) testing. Seven deer cultured positive for M. bovis that would not have been detected by current surveillance, yielding apparent and true prevalence estimates (95% confidence limits) of 2.7% (1.6, 3.8) and 3.6% (2.3, 4.9), respectively. The sensitivity, specificity, and positive and negative predictive values of the current surveillance protocol were 75, 100, 100, and 99%, respectively. Histologic lesions were present only in tonsils, and ranged from simple necrosis to caseation, suppuration, and granuloma formation. Acid-fast staining and PCR detected M. bovis in only one of the seven culture-positive deer. Our study provides the first estimate of the true prevalence of M. bovis in Michigan's free-ranging deer population and suggests modest underestimation of that prevalence by current surveillance. This study also suggests that caution is warranted when extrapolating epidemiologic data on TB in captive cervids to free-ranging populations and confirms the pivotal role of the tonsil in early infections.

Management of bovine tuberculosis in Michigan wildlife: current status and near term prospects.

Surveillance and control activities for bovine tuberculosis (bTB) in free-ranging Michigan white-tailed deer (Odocoileus virginianus) have now been underway for over a decade. Significant progress has been made, lowering apparent prevalence in deer in the core area by >60%, primarily via reduction of deer densities through hunting, and restrictions on public feeding and baiting of deer. These broad strategies of the Michigan Department of Natural Resources (MDNR), implemented with the cooperation of Michigan deer hunters, halved the deer population in the bTB endemic area. However, as hunters see fewer deer, their willingness to sustain aggressive harvests has waned, and public resentment of control measures has grown. During the past four years, apparent prevalence in core area deer has held approximately steady just below 2%. After bottoming out in 2004 at an estimated 10-12 deer/km(2), deer numbers have since rebounded by ∼ 30%. Public compliance with baiting and feeding restrictions has been variable. In general, hunters in the core area do not perceive bTB as a problem, in spite of 13 years of MDNR outreach. To date, MDNR has expended more than US$23 million on TB-related activities. Of late, a substantial portion of that funding has been diverted to support other programs which have suffered from budget shortfalls. Livestock herd breakdowns continue to occur sporadically, averaging 3-4 per year 2005 to present. In total, 46 cattle and 4 captive deer herds have been diagnosed bTB positive statewide, the majority yielding only 1 positive animal. Five cattle herds were twice infected, one thrice. Michigan Department of Agriculture (MDA) policy emphasis has shifted towards obtaining producer support for wildlife risk mitigation and farm biosecurity. Funding has proven a limiting factor, with the majority of the US$63 million spent to date devoted to whole herd testing. Nevertheless, some initiatives justify cautious optimism. Promising research to support eventual vaccination of wild deer continues. Some hunters and landowners have begun to recognize the costs of high deer densities and supplemental feeding. A peninsula-wide ban on baiting and feeding was enacted. Some cattle producers, recognizing their precarious circumstances, have begun work to change long-held prevailing opinions among their peers about farm biosecurity. Yet formidable challenges remain, and evidence suggests that eradication of bTB, if it can be achieved, will take decades, and will require greater public and political resolve than has been demonstrated thus far.

Antimicrobial Susceptibility Testing of Mycobacterium bovis Isolates from Michigan White-Tailed Deer during the 2009 Hunting Season.

Michigan has had an ongoing outbreak of endemic Mycobacterium bovis which has been recognized within and sustained by its free-ranging white-tailed deer population since 1994. Worldwide, organisms within the Mycobacterium tuberculosis complex have exhibited the ability to develop resistance to antimicrobial agents, resulting in both the multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of human tuberculosis. Michigan's Bovine Tuberculosis Working Group has conducted active antimicrobial susceptibility testing on wildlife isolates of the endemic M. bovis organism at five-year intervals to detect any emerging drug resistance patterns. The results of 33 white-tailed deer origin isolates collected from the 2009 hunting season are reported here. There continues to be no evidence of any drug resistance except for pyrazinamide resistance. These results are likely due to the lack of antibacterial treatment applied to either wildlife or domestic animals which would provide selection pressure for the development of drug resistance.