Experimental Deer-To-Deer Transmission of Mycobacterium bovis

Objective—To determine whether Mycobacterium bovis can be transmitted from experimentally infected deer to uninfected in-contact deer. Animals—Twenty-three 6-month-old white-tailed deer. Procedure—On day 0, M bovis (2 X 108 colony-forming units) was administered by intratonsillar instillation to 8 deer; 3 control deer received saline (0.9% NaCl) solution. Eight in-contact deer were comingled with inoculated deer from day 21. On day 120, inoculated deer were euthanatized and necropsied. On day 180, 4 in-contact deer were euthanatized, and 4 new incontact deer were introduced. On day 360, all in-contact deer were euthanatized. Rectal, oral, and nasal swab specimens and samples of hay, pelleted feed, water, and feces were collected for bacteriologic culture. Tissue specimens were also collected at necropsy for bacteriologic culture and histologic analysis. Results—On day 90, inoculated and in-contact deer developed delayed-type hypersensitivity (DTH) reactions to purified protein derivative of M bovis. Similarly, new in-contact deer developed DTH reactions by 100 days of contact with original in-contact deer. Tuberculous lesions in in-contact deer were most commonly detected in lungs and tracheobronchial and medial retropharyngeal lymph nodes. Mycobacterium bovis was isolated from nasal secretions and saliva from inoculated and in-contact deer, urine and feces from in-contact deer, and hay and pelleted feed. Conclusions and Clinical Relevance—Mycobacterium bovis is efficiently transmitted from experimentally infected deer to uninfected in-contact deer through nasal secretions, saliva, or contaminated feed. Wildlife management practices that result in unnatural gatherings of deer may enhance both direct and indirect transmission of M bovis.

Humoral Immune Responses of White-Tailed Deer (Odocoileus virginianus) to Mycobacterium bovis BCG Vaccination and Experimental Challenge with M. bovis

Monitoring of the kinetics of production of serum antibodies to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and the efficacy of intervention strategies in several species. The humoral immune responses to multiple M. bovis antigens by white-tailed deer vaccinated with BCG orally via a lipid-formulated bait (n = 5), orally in liquid form (n = 5), and subcutaneously (n = 6) were evaluated over time after vaccination and after experimental challenge with virulent M. bovis and were compared to the responses by unvaccinated deer (n = 6). Antibody responses were evaluated by using a rapid test (RT), a multiantigen print immunoassay (MAPIA), a lipoarabinomannan enzyme-linked immunosorbent assay (LAM-ELISA), and immunoblotting to whole-cell sonicate and recombinant antigen MPB83. MAPIA and RT detected minimal to no antibody responses over those at the baseline to multiple M. bovis antigens in vaccinated white-tailed deer after challenge. This was in contrast to the presence of more readily detectable antibody responses in nonvaccinated deer with more advanced disease. The LAM-ELISA results indicated an overall decrease in the level of production of detectable antibodies against lipoarabinomannan-enriched mycobacterial antigen in vaccinated animals compared to that in nonvaccinated animals after challenge. Immunoblot data were inconsistent but did suggest the occurrence of unique antibody responses by certain vaccinated groups to Ag85 and HSP70. These findings support further research toward the improvement and potential use of antibody-based assays, such as MAPIA, RT, and LAM-ELISA, as tools for the antemortem assessment of disease progression in white-tailed deer in both experimental and field vaccine trials.

Wildlife-Cattle Interactions in Northern Michigan: Implications for the Transmission of Bovine Tuberculosis

Bovine tuberculosis (Mycobacterium bovis) was discovered in northern Michigan white-tailed deer (Odocoileus virginianus) in 1994, and has been known to exist in Michigan cattle herds since 1998. Despite efforts to eradicate the disease in cattle, infection and re-infection of farms continues to occur, suggesting transmission among cattle, deer, or other wildlife reservoirs. The goals of this study were to document wildlife activity on farms and evaluate the possible role wildlife play in the ecology of bovine tuberculosis (TB) in Michigan. Visual observations were conducted on farms in a 5-county area of northern Michigan to document direct wildlife-cattle interactions (i.e., <5 m between individuals) and indirect interactions (e.g., wildlife visitations to food stores and areas accessible to cattle). Observations were conducted primarily during evening and early morning hours between January and August, 2002, and on a 24-hour schedule between January and August, 2003. Total observation time accumulated through the duration of the study was 1,780 hours. Results indicated that direct interaction between deer and cattle was a rare event; no direct interactions were observed during the first year, and only one direct interaction was observed during the second year. However, through the duration of the study 21 direct interactions were documented between cattle and turkey, and 11 direct interactions were documented between cattle and mammals other than deer. In total, 273 indirect interactions by deer, 112 indirect interactions by turkeys, and 248 indirect interactions by mammals other than deer were observed during the 2 field seasons combined. These data supported the hypothesis that indirect interactions among wildlife and cattle are a potential mechanism for the transmission of TB in Michigan. If direct interactions were important mechanisms of TB transmission to cattle in northern Michigan, my data suggested that feral cats were the species of most concern, even though there were more observations between turkey and cattle. Unlike cats, which can become infected with and transmit TB, there is no evidence for such pathogenesis in turkey.

Advances in Bovine Tuberculosis Diagnosis and Pathogenesis: What Policy Makers Need to Know

The mainstay of tuberculosis diagnosis in cattle and deer has been the tuberculin skin test. Recent advances have allowed the incorporation of blood based assays to the diagnostic arsenal for both cattle and deer. Use of defined and specific antigens has allowed for improved specificity of cell mediated assays in both cattle and deer and advances in antibody tests for tuberculosis have potential for use in free-ranging and captive cervid populations. Combined use of blood-based assays with skin testing will require further understanding of the effect of skin testing on the accuracy of blood based assays. Models of experimental infection of cattle have allowed for increased understanding of natural disease pathogenesis. Differences likely exist; however, between cattle and deer in both disease distribution and primary route of inoculation in naturally infected animals.

Shared Feed as a Means of Deer-to-Deer Transmission of Mycobacterium bovis

To determine the ability of experimentally inoculated white-tailed deer (Odocoileus virginianus) to transmit Mycobacterium bovis to naive deer through the sharing of feed, four deer were intratonsillarly inoculated with 4x105 colony-forming units of M. bovis. On a daily basis, feed not consumed by inoculated deer after approximately 8 hr was offered to four naıve deer maintained in a separate pen, where direct contact, aerosol transmission, or transmission through personnel were prevented. After 150 days, naıve deer were euthanized and examined. All naıve deer had lesions consistent with tuberculosis and M. bovis was isolated from various tissues. The most commonly affected tissues were lung, tracheobronchial lymph nodes, and mediastinal lymph nodes. This study demonstrates the potential for indirect transmission of M. bovis through the sharing of feed. Intentional or unintentional feeding of deer by wildlife or agricultural interests in regions where M. bovis infection is endemic should be avoided because both direct and indirect transmission through sharing of feed are enhanced.

Investigation of the Transmission of Mycobacterium bovis from Deer to Cattle Through Indirect Contact

Objective—To investigate the infection of calves with Mycobacterium bovis through oral exposure and transmission of M bovis from experimentally infected white-tailed deer to uninfected cattle through indirect contact. Animals—24 11-month-old, white-tailed deer and 28 6-month-old, crossbred calves. Procedure—In the oral exposure experiment, doses of 4.3 X 106 CFUs (high dose) or 5 X 103 CFUs (low dose) of M bovis were each administered orally to 4 calves; as positive controls, 2 calves received M bovis (1.7 X 105 CFUs) via tonsillar instillation. Calves were euthanatized and examined 133 days after exposure. Deer-to-cattle transmission was assessed in 2 phases (involving 9 uninfected calves and 12 deer each); deer were inoculated with 4 X 105 CFUs (phase I) or 7 X 105 CFUs (phase II) of M Bovis. Calves and deer exchanged pens (phase I; 90 days’ duration) or calves received uneaten feed from deer pens (phase II; 140 days’ duration) daily. At completion, animals were euthanatized and tissues were collected for bacteriologic culture and histologic examination. Results—In the low- and high-dose groups, 3 of 4 calves and 1 of 4 calves developed tuberculosis, respectively. In phases I and II, 9 of 9 calves and 4 of 9 calves developed tuberculosis, respectively. Conclusions and Clinical Relevance—Results indicated that experimentally infected deer can transmit M bovis to cattle through sharing of feed. In areas where tuberculosis is endemic in free-ranging white-tailed deer, management practices to prevent access of wildlife to feed intended for livestock should be implemented.

Aerosol Exposure of White-Tailed Deer (Odocoileus virginianus) to Mycobacterium Bovis

Tuberculosis due to Mycobacterium bovis affects both captive and free-ranging Cervidae in the United States. Various animal models have been developed to study tuberculosis of both humans and animals. Generally, tuberculosis is transmitted by aerosol and oral routes. Models of aerosol exposure of large animals to M. bovis are uncommon. In order to develop a reliable method of aerosol exposure of white-tailed deer (Odocoileus virginianus) to M. bovis, 12 healthy white-tailed deer, aged 8–10 mo, were infected by aerosol exposure to 2x105 to 1x106 colony forming units (CFU) (high dose, n=4) of M. bovis or 6x102 to 1.6 x 103 CFU (low dose, n=8) of M. bovis. Tuberculous lesions were more widely disseminated in deer receiving the high dose, while lesions in deer receiving the low dose were more focused on the lungs and associated lymph nodes (tracheobronchial and mediastinal). Aerosol delivery of M. bovis to white-tailed deer results in a reliable manner of experimental infection that may be useful for studies of disease pathogenesis, immune response, mycobacterial shedding, and vaccine efficacy.

Susceptibility of Raccoons (Procyon lotor) to Infection with Mycobacterium bovis

Tuberculosis due to Mycobacterium bovis infection is endemic in white-tailed deer (Odocoileus virginianus) in the northeastern portion of the lower Michigan peninsula (USA). Various wild carnivores and omnivores, including raccoons (Procyon lotor), are infected with M. bovis within the endemic area. To investigate the pathogenesis of tuberculosis in raccoons and the likelihood of M. bovis transmission from infected raccoons to other susceptible hosts, we experimentally inoculated raccoons with single oral doses of M. bovis (ranging from 30 to 1.7 x 105 colony forming units [CFU]), five daily oral doses of M. bovis (ranging from 10 to 1 x 105 CFU), or a single intravenous (IV) dose of 1 x 105 CFU of M. bovis, from November 1998 through December 2000. Granulomatous lesions consistent with tuberculosis, or tissue colonization with M. bovis, were seen in one of five raccoons in the single low oral dose group, one of five raccoons in the multiple low oral dose group, two of five raccoons in the multiple medium oral dose group, five of five raccoons in the multiple high oral dose group, and five of five raccoons in the IV inoculated group. In orally inoculated raccoons, lesions were most common in the tracheobronchial and mesenteric lymph nodes and lung. Excretion of M. bovis in saliva or nasal secretions was noted in all IV inoculated raccoons and two of five multiple low oral dose raccoons. Mycobacterium bovis was not isolated from urine or feces from any experimentally inoculated raccoons. The need for multiple large oral doses to establish infection, and the low number of orally inoculated raccoons that excreted M. bovis in nasal secretions or saliva, suggest that widespread tuberculosis among raccoons is unlikely.

Lesion Development in White-tailed Deer (Odocoileus virginianus) Experimentally Infected with Mycobacterium bovis

The recent discovery of tuberculosis in free-living white-tailed deer in northeastern Michigan underscores the need for increased understanding of the pathogenesis of tuberculosis in wildlife species. To investigate lesion development in white-tailed deer, 32 deer were experimentally infected by intratonsilar instillation of 300 colony-forming units of Mycobacterium bovis. Three deer each were euthanatized and examined at days 15, 28, 42, and 56 after inoculation, and five deer each were euthanatized and examined at days 89, 180, 262, and 328 after inoculation. Microscopic lesions first were seen in the medial retropharyngeal lymph node and lung 28 and 42 days after inoculation, respectively. Lung lesions were present in 12 (38%) of 32 deer, involving 23 lung lobes. Left caudal and right middle and caudal lobes were involved in 17 (74%) of the 23 affected lung lobes. Lesions in the medial retropharyngeal lymph node first appeared as granulomas composed of aggregates of macrophages and Langhans-type giant cells. Some early granulomas contained centrally located neutrophils. As granulomas developed, neutrophils were replaced with a central zone of caseous necrosis that first showed signs of mineralization 42 days after inoculation. Granulomas increased in size as the zone of caseous necrosis expanded. Peripheral fibrosis, first seen at 56 days after inoculation, progressed to only a thin fibrous capsule by 328 days after inoculation. By the termination of the study, the central necrotic core of the granuloma contained abundant liquefied necrotic material and grossly resembled an abscess. Although tuberculous lesions in white-tailed deer follow a developmental pattern similar to that in cattle, fibrosis is less pronounced and the advanced lesions may liquefy, a change seldom reported in cattle. An understanding of lesion development will aid in the identification of the spectrum of disease that may be seen in this important wildlife reservoir of tuberculosis.

Mycobacterium bovis–Infected White-Tailed Deer (Odocoileus Virginianus): Detection of Immunoglobulin Specific to Crude Mycobacterial Antigens by ELISA

White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis–infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis–infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded (P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K–digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded (P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., ~11 months). This response also exceeded (P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K–digested M. bovis antigens in the antibody-based assays of tuberculosis.

A New Approach for Managing Bovine Tuberculosis: Veterinary Services’ Proposed Action Plan

Bovine tuberculosis (TB) is a serious disease with animal health, public health, and international trade consequences. The cooperative Federal-State-industry effort to eradicate bovine TB from cattle in the United States has made significant progress since the program’s inception in 1917. However, the goal of eradication remains elusive. This proposed action plan presents Veterinary Services’ (VS’) current thinking about changes we are considering for the TB program to address our current challenges. This action plan will: 1. Reduce the introduction of TB into the U.S. national herd from imported animals and wildlife by: o Applying additional requirements to cattle imports from Mexico o Enhancing efforts to mitigate risks from wildlife 2. Enhance TB surveillance by: o Crafting a comprehensive national surveillance plan o Accelerating diagnostic test development to support surveillance 3. Increase options for managing TB-affected herds by: o Conducting epidemiological investigations and assessing individual herd risk o Applying whole-herd depopulation judiciously and developing alternative control strategies o Applying animal identification (ID) standards to meet animal ID needs 4. Modernize the regulatory framework to allow VS to focus resources where the disease exists 5. Transition the TB program from a State classification system to a science-based zoning approach to address disease risk To succeed, this new approach will require VS’ continued partnership with State animal health and wildlife officials, other Federal agencies, industry, international partners, academia, and other stakeholders. Successful partnerships will allow us to use available resources efficiently to achieve program objectives and protect our nation’s herd. Implementation of the VS proposed action plan will benefit Federal and State animal health officials, the regulated industries, and producers by allowing a more rapid response that employs up-to-date science and can adapt rapidly to changing situations.

Evaluation of an In Vitro Blood-Based Assay to Detect Production of Interferon-γ By Mycobacterium Bovis–Infected White-Tailed Deer (Odocoileus Virginianus)

Tuberculosis due to Mycobacterium bovis in captive Cervidae was identified as an important disease in the United States in 1990 and prompted the addition of captive Cervidae to the USDA Uniform Methods and Rules for eradication of bovine tuberculosis. As well, M. bovis infection was identified in free-ranging white-tailed deer in northeast Michigan in 1995. Tuberculosis in both captive and free-ranging Cervidae represents a serious challenge to the eradication of M. bovis infection from the United States. Currently, the only approved antemortem tests for tuberculosis in Cervidae are the intradermal tuberculin skin test and the blood tuberculosis test (BTB). At present, the BTB is not available in North America. Tuberculin skin testing of Cervidae is time-consuming and involves repeated animal handling and risk of injury to animals and humans. This study evaluated the potential of a new blood-based assay for tuberculosis in Cervidae that would decrease animal handling, stress, and losses due to injury. In addition, a blood-based assay could provide a more rapid diagnosis. Twenty 6–9-month-old white-tailed deer, male and female, were experimentally inoculated by instillation of 300 colony-forming units of M. bovis in the tonsillar crypts. Seven, age-matched uninfected deer served as controls. Blood was collected on days 90, 126, 158, 180, 210, 238, 263, and 307 after inoculation and was analyzed for the production of interferon-γ (IFN-γ) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-g in response to PPDb was significantly greater (P < 0.05) at all time points in samples from M. bovis–infected deer as compared with uninfected control deer, whereas IFN-γ production to PWM did not differ significantly between infected and control deer. Measurement of IFN-γ production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae.

Antibody Responses in Reindeer (Rangifer tarandus) Infected with Mycobacterium bovis

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.