Using White-tailed Deer (Odocoileus virginianus) in Infectious Disease Research.

Between 1940 and 2004, more than 335 emerging infectious disease events were reported in the scientific literature. The majority (60%) of these events involved zoonoses, most of which (72%) were of wildlife origin or had an epidemiologically important wildlife host. Because this trend of increasing emerging diseases likely will continue, understanding the pathogenesis, transmission, and diagnosis of these diseases in the relevant wildlife host is paramount. Achieving this goal often requires using wild animals as research subjects, which are vastly different from the traditional livestock or laboratory animals used by most universities and institutions. Using wildlife in infectious disease research presents many challenges but also provides opportunities to answer questions impossible to address by using traditional models. Cervid species, especially white-tailed deer (Odocoileus virginianus), elk (Cervus canadensis), and red deer (Cervus elaphus), are hosts or sentinels for several important pathogens, some of which are zoonotic. The long history of infectious disease research using white-tailed deer, conducted at ever-increasing levels of sophisticated biosecurity, demonstrates that this type of research can be conducted safely and that valuable insights can be gained. The greatest challenges to using wildlife in infectious disease research include animal source, facility design, nutrition, animal handling, and enrichment and other practices that both facilitate animal care and enhance animal wellbeing. The study of Mycobacterium bovis infection in white-tailed deer at the USDA's National Animal Disease Center serves to illustrate one approach to address these challenges.

Survival of Mycobacterium bovis on feedstuffs commonly used as supplemental feed for white-tailed deer (Odocoileus virginianus).

Mycobacterium bovis, the causative agent of bovine tuberculosis, has become established in free-ranging white-tailed deer Odocoileus virginianus in northeastern Michigan. The practice of supplemental feeding of white-tailed deer during the winter is believed to contribute to transmission of M. bovis between deer. The current study was conducted to determine the ability of M. bovis to survive on various feedstuffs commonly used as supplemental feed for deer in northeast Michigan (i.e., apples, corn, carrots, sugar beets, potatoes, and hay) and the effect of maintenance at -20 C, 8 C, and 23 C on survival. Mycobacterium bovis survived on all feedstuffs at all temperatures tested for at least 7 days. At 23 C, M. bovis could still be isolated from samples of apples, corn and potatoes at 112 days. This study suggests that contamination of feedstuffs by M. bovis-infected deer could act as a source of indirect transmission between deer because M. bovis is able to survive in temperatures similar to those recorded during winter months in northeastern Michigan. Current efforts to ban or control supplemental feeding of deer should have a positive effect on decreasing transmission of M. bovis among deer.

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., approximately 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.

Evaluation of an in vitro blood-based assay to detect production of interferon-gamma 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-gamma (IFN-gamma) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-gamma 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-gamma production to PWM did not differ significantly between infected and control deer. Measurement of IFN-gamma production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae.

The sensitivity of gross necropsy, caudal fold and comparative cervical tests for the diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTb) was diagnosed in 22 cattle herds in the northeast comer of Michigan's lower peninsula. Of these 22 herds, 494 animals in 7 herds were examined by gross necropsy, histopathologic exam, mycobacterial culture, and polymerase chain reaction (PCR) assay performed only on samples that were histologically compatible for bTb. Results of culture and PCR assay interpreted in parallel were used as the reference test for calculation of the sensitivity of 1) the caudal fold test (CFT), 2) the caudal fold and comparative cervical skin tests used in series (CFTCCTSER), and 3) gross necropsy. Mycobacterium bovis was isolated from 43 animals. Using all 7 herds, the sensitivities of the CFT, the CFTCCTSER, and gross necropsy were 93.02%, 88.37%, and 86.05%, respectively. When the data were stratified by low- and moderate-prevalence herds, the sensitivities were 83.33%, 75.0%, and 83.33% in low-prevalence herds and 96.77%, 93.55%, and 87.10% in moderate-prevalence herds. The sensitivities of the 2 skin tests were slightly higher when 2 or more gross lesions were present, and the sensitivity of gross necropsy was significantly higher (P = 0.049). The sensitivity of the CFT was found to be notably higher than most estimates in other studies; however, a direct comparison was not possible because the amount of purified protein derivative and the reference methods were different in this study compared with other published studies. Although the sensitivities are high, 2 of the 7 herds (29%) would have had 1 or more positive animals left in the herd if a test-and-removal program had been used. This suggests that when positive herds are identified, selective culling of skin test reactors is a less acceptable disease control strategy than is complete depopulation.

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 10(6) CFUs (high dose) or 5 x 10(3) CFUs (low dose) of M. bovis were each administered orally to 4 calves; as positive controls, 2 calves received M. bovis (1.7 x 10(5) 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 10(5) CFUs (phase I) or 7 x 10(5) 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.

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 10(5) colony forming units [CFU]), five daily oral doses of M. bovis (ranging from 10 to 1 x 10(5) CFU), or a single intravenous (i.v.) dose of 1 x 10(5) 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 i.v. inoculated group. In oral 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 i.v. 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 wide-spread tuberculosis among raccoons is unlikely.

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 2 x 10(5) to 1 x 10(6) colony forming units (CFU) (high close, n = 4) of M. bovis or 6 x 10(2) to 1.6 x 10(3) CFU (low dose, n = 8) of M. bovis. Tuberculous lesions were more widely disseminated in (leer 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.

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 naïve deer through the sharing of feed, four deer were intratonsillarly inoculated with 4 x 10(5) colony-forming units of M. bovis. On a daily basis, feed not consumed by inoculated deer after approximately 8 hr was offered to four naïve deer maintained in a separate pen, where direct contact, aerosol transmission, or transmission through personnel were prevented. After 150 days, naïve deer were euthanized and examined. All naïve 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.

Prevalence of Mycobacterium bovis infection in cervids on privately owned ranches.

OBJECTIVE: To determine prevalence of tuberculosis caused by infection with Mycobacterium bovis in cervids on privately owned ranches in northeastern lower Michigan. DESIGN: Epidemiologic survey. ANIMALS: Cervids on 96 privately owned ranches. PROCEDURES: A combination of slaughter and skin tuberculin testing was used to collect data. Infection with M. bovis was confirmed by use of standard necropsy and bacteriologic culture techniques. RESULTS: Cervids with tuberculosis were detected on 1 of the 96 ranches. The apparent prevalence of tuberculosis in cervids from the 96 ranches was 1.1 cases/100 cervids (21 cases/1,867 cervids tested). For the ranch with infected cervids, prevalence of infection with M. bovis was 12.1 cases/100 cervids (21 cases/174 cervids tested). No obvious gross lesions were seen in 8 of 21 white-tailed deer and 1 coyote with culture-confirmed M. bovis infection. CONCLUSIONS AND CLINICAL RELEVANCE: The lack of visible lesions in a substantial proportion of infected animals should be taken into consideration in studies involving detection and prevalence of tuberculosis.

High-impact animal health research conducted at the USDA's National Animal Disease Center.

Commissioned by President Dwight Eisenhower in 1958 and opened with a dedication ceremony in December 1961, the USDA, Agricultural Research Service (ARS), National Animal Disease Center (NADC) celebrated its 50-year anniversary in November 2011. Over these 50 years, the NADC established itself among the world's premier animal health research centers. Its historic mission has been to conduct basic and applied research on selected endemic diseases of economic importance to the U.S. livestock and poultry industries. Research from NADC has impacted control or management efforts on nearly every major animal disease in the United States since 1961. For example, diagnostic tests and vaccines developed by NADC scientists to detect and prevent hog cholera were integral in the ultimate eradication of this costly swine disease from the U.S. Most major veterinary vaccines for critical diseases such as brucellosis and leptospirosis in cattle, porcine respiratory and reproductive syndrome (PRRS), porcine parvovirus and influenza in swine had their research origins or were developed and tested at the NADC. Additional discoveries made by NADC scientists have also resulted in the development of a nutritional approach and feed additives to prevent milk fever in transition dairy cattle. More recently, NADC's archive of historic swine influenza viruses combined with an established critical mass of influenza research expertise enabled NADC researchers to lead an effective national research response to the pandemic associated with the novel 2009 H1N1 influenza virus. This review commemorates some of the key animal health contributions in NADC's first 50 years, recaps the newly completed modernization of the center into new facilities, and offers highlights of the ongoing research that will define NADC's mission going forward.

Use of the intradermal tuberculin test in a herd of captive elk (Cervus elaphus nelsoni) naturally infected with Mycobacterium bovis.

In the United States, tuberculosis of captive cervids, caused by Mycobacterium bovis, attracted attention in 1991 when investigations, prompted by the identification of a tuberculous elk (Cervus elaphus nelsoni) of U.S. origin exported to Canada, revealed tuberculosis in 10 different elk herds in 8 different states. Based on methods used in cattle, official regulations pertaining to testing and eradication of tuberculosis in captive cervids were added to the U.S. Department of Agriculture bovine tuberculosis eradication effort in 1994. However, little published information exists on the accuracy of intradermal tuberculin testing in naturally infected cervids. Evaluation of a captive herd of 71 animals in Wisconsin included postmortem examination and tissue sample collection from both tuberculin test responders and nonresponders. Within this captive herd, of admittedly small size, results showed the single cervical test to have a sensitivity of 88% and a specificity of 69%. Evaluation of diagnostic tests in the species of interest is important, as extrapolation of data obtained from other species may not be appropriate.

Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis.

Mycobacterium avium complex strains and Mycobacterium paratuberculosis are closely related intracellular pathogens affecting humans and animals. M. avium complex infections are a leading cause of morbidity and mortality in AIDS patients, and M. paratuberculosis is the agent of Johne's disease in ruminants. Genetic manipulation of these micro-organisms would facilitate the understanding of their pathogenesis, the construction of attenuated vaccine strains and the development of new drugs and treatment methods. This paper describes the replication of mycobacterial shuttle phasmids and plasmids, and the expression of the firefly luciferase reporter gene in M. avium complex and M. paratuberculosis. The mycobacteriophage TM4 propagated on M. smegmatis or M. paratuberculosis plaqued at the same efficiency on these two mycobacterial hosts. Screening of M. avium complex and M. paratuberculosis clinical isolates with TM4-derived luciferase reporter phages demonstrated that the majority of these isolates were susceptible to TM4. Conditions for introduction of DNA were determined by transfection of M. paratuberculosis with TM4 DNA and applied to isolate kanamycin-resistant transformants of M. avium complex and M. paratuberculosis with Escherichia coli-Mycobacterium shuttle plasmids. Recombinant plasmids were recovered from transformants without apparent loss of DNA sequences. These results provide the basis for the genetic manipulation of these pathogenic mycobacterial species.

CD80 and CD86, but not CD154, augment DNA vaccine-induced protection in experimental bovine tuberculosis.

DNA vaccination is known to elicit robust cellular and humoral responses to encoded antigen. The co-administration of costimulatory molecules CD80 (B7-1), CD86 (B7-2) and CD154 (CD40L) has been shown to enhance immune responses in several murine models. The role of specific costimulatory molecules in non-rodent species remains incompletely characterized. In these studies, we demonstrate that the co-administration of CD80 and CD86, but not CD154, to an existing candidate subunit DNA vaccine (ESAT-6) against bovine tuberculosis, enhances protection after aerosol challenge with virulent Mycobacterium bovis. Additionally, we have shown that vaccination with M. bovis BCG is protective against tuberculosis following aerosol challenge in cattle. Two independent trials were conducted in cattle to determine the adjuvant effect of encoded antigen + CD80/CD86 and directly compare the adjuvant activities of CD80/CD86 to those of CD154. Co-administration of either CD80/CD86 or CD154 enhanced ESAT-6-specific IFN-gamma responses as compared to animals vaccinated with ESAT-6 DNA alone. However, following aerosol challenge, only animals vaccinated with CD80/CD86 possessed decreased pathology of the lungs and associated lymph nodes, as measured by gross examination, radiographic lesion morphometry and bacterial recovery. Collectively, these results demonstrate that the co-administration of costimulatory molecules with a protective antigen target enhances bovine immune responses to DNA vaccination, and that CD80/CD86 is superior to CD154 in augmenting DNA vaccine-induced protection in experimental bovine tuberculosis.