Milk Containing Mycobacterium bovis as a Source of Infection for White-Tailed Deer Fawns (Odocoileus virginianus)

Setting:White-tailed deer represent the first wildlife reservoir of Mycobacterium bovis in the United States. The behavior of does with nursing fawns provides several potential mechanisms for disease transmission. Little information exists concerning transmission between doe and fawn, specifically transmammary transmission. Objective: Determine if fawns can become infected by ingestion of milk replacer containing M. bovis, thus simulating transmission from doe to fawn through contaminated milk. Design: Seventeen, 21-day-old white-tailed deer fawns were inoculated orally with 2x108 CFU (high dose, n=5), 2.5 x 105 to 2.5 x 106 CFU (medium dose, n=5), and 1x104 CFU (low dose, n=5) of M. bovis in milk replacer. Dosages were divided equally and fed daily over a 5-day period. Positive control fawns (n=2) received 1x105 CFU of M. bovis instilled in the tonsillar crypts. Fawns were euthanized and examined 35-115 days after inoculation and various tissues collected for bacteriologic and microscopic analysis. Results: All fawns in the tonsillar, high oral and medium oral dose groups developed generalized tuberculosis involving numerous organs and tissues by 35-84 days after inoculation. Three of five fawns in the low-dose oral group had tuberculous lesions in the mandibular lymph node, and one of five had lesions in the medial retropharyngeal lymph node when examined 115 days after inoculation. Conclusion: White-tailed deer fawns can become infected through oral exposure to M. bovis. Therefore, the potential exists for fawns to acquire M. bovis while nursing tuberculous does.

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.

Tuberculin Skin Testing in White-Tailed Deer (Odocoileus virginianus)

The comparative cervical skin test for antemortem diagnosis of tuberculosis was done 169 times on 116 different white-tailed deer of known Mycobacterium bovis infection status. The sensitivity and specificity were 97 and 81%, respectively. The magnitude of change in skin thickness at test sites was not significantly influenced by dosage of inoculum, dissemination of the disease process, or repeated skin testing. However, the magnitude of change in skin thickness was significantly greater in deer infected for less than 109 days than in deer infected for more than 109 days. As used in the present study, the comparative cervical skin test is a sensitive method of antemortem diagnosis of M. bovis infection in white-tailed deer.

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.

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 220 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., ~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.

Bovine Tuberculosis in Swedish Farmed Deer: Detection and Control of the Disease

Bovine tuberculosis (BTB) was introduced into Swedish farmed deer herds in 1987. Epidemiological investigations showed that 10 deer herds had become infected (July 1994) and a common source of infection, a consignment of 168 imported farmed fallow deer, was identified (I). As trace-back of all imported and in-contact deer was not possible, a control program, based on tuberculin testing, was implemented in July 1994. As Sweden has been free from BTB since 1958, few practicing veterinarians had experience in tuberculin testing. In this test, result relies on the skill, experience and conscientiousness of the testing veterinarian. Deficiencies in performing the test may adversely affect the test results and thereby compromise a control program. Quality indicators may identify possible deficiencies in testing procedures. For that purpose, reference values for measured skin fold thickness (prior to injection of the tuberculin) were established (II) suggested to be used mainly by less experienced veterinarians to identify unexpected measurements. Furthermore, the within-veterinarian variation of the measured skin fold thickness was estimated by fitting general linear models to data (skin fold measurements) (III). The mean square error was used as an estimator of the within-veterinarian variation. Using this method, four (6%) veterinarians were considered to have unexpectedly large variation in measurements. In certain large extensive deer farms, where mustering of all animals was difficult, meat inspection was suggested as an alternative to tuberculin testing. The efficiency of such a control was estimated in paper IV and V. A Reed Frost model was fitted to data from seven BTB-infected deer herds and the spread of infection was estimated (< 0.6 effective contacts per deer and year) (IV). These results were used to model the efficiency of meat inspection in an average extensive Swedish deer herd. Given a 20% annual slaughter and meat inspection, the model predicted that BTB would be either detected or eliminated in most herds (90%) 15 years after introduction of one infected deer. In 2003, an alternative control for BTB in extensive Swedish deer herds, based on the results of paper V, was implemented.

Wild Boar and Red Deer Display High Prevalences of Tuberculosis-Like Lesions in Spain

We describe the distribution of tuberculosis-like lesions (TBL) in wild boar (Sus scrofa) and red deer (Cervus elaphus) in Spain. Animals with TBL were confirmed in 84.21% of mixed populations (n = 57) of red deer and wild boar and in 75% of populations of wild boar alone (n = 8) in central and southern Spain (core area). The prevalence of TBL declined towards the periphery of this region. In the core area, the prevalence ranged up to 100% in local populations of wild boar (mean estate prevalence 42.51%) and up to 50% in red deer (mean estate prevalence 13.70%). We carried out exploratory statistical analyses to describe the epidemiology of TBL in both species throughout the core area. Prevalence of TBL increased with age in both species. Wild boar and red deer mean TBL prevalence at the estate level were positively associated, and lesion scores were consistently higher in wild boars than in red deer. The wild boar prevalence of TBL in wild boar did not differ between populations that were or were not cohabiting with red deer. Amongst the wild boars with TBL, 61.19% presented generalized lesions, and the proportion of generalized cases was similar between sex and age classes. In red deer, 57.14% of TBL-positive individuals presented generalized lesions, and the percentage of generalized cases increased with age class, but did not differ between the sexes. These results highlight the potential importance of wild boar and red deer in the maintenance of tuberculosis in south central Spain.

Risk Factors Associated With the Prevalence of Tuberculosis-Like Lesions in Fenced Wild Boar and Red Deer in South Central Spain

In recent decades the management of large game mammals has become increasingly intensive in south central Spain (SCS), resulting in complex epidemiological scenarios for disease maintenance, and has probably impeded schemes to eradicate tuberculosis (TB) in domestic livestock. We conducted an analysis of risk factors which investigated associations between the pattern of tuberculosis-like lesions (TBL) in wild boar (Sus scrofa) and red deer (Cervus elaphus) across 19 hunting estates from SCS and an extensive set of variables related to game management, land use and habitat structure. The aggregation of wild boar at artificial watering sites was significantly associated with an increasing risk of detecting TBL in both species, which probably relates to enhanced opportunities for transmission. Aggregation of wild boar at feeding sites was also associated with increased risks of TBL in red deer. Hardwood Quercus spp. forest availability was marginally associated with an increased risk of TB in both species, whereas scrubland cover was associated with a reduced individual risk of TBL in the wild boar. It is concluded that management practices that encourage the aggregation of hosts, and some characteristics of Mediterranean habitats could increase the frequency and probability of both direct and indirect transmission of TB. These findings are of concern for both veterinary and public health authorities, and reveal tuberculosis itself as a potential limiting factor for the development and sustainability of such intensive game management systems in Spanish Mediterranean habitats.

VS - Bovine Tuberculosis (Mycobacterium bovis) Surveillance Standards 11/2001

Bovine tuberculosis (TB) is an infectious and communicable granulomatous disease caused by the acidfast bacilli bacteria of Mycobacterium bovis (M. bovis). It is commonly a chronic, debilitating disease, but occasionally may assume an acute, rapidly progressive course. M. bovisis a widespread zoonosis that is global in magnitude and affects nearly all species of vertebrates (cattle, sheep, goats, pigs, bison, buffalo, and camelids.) Disease is spread by direct contact, inhalation of infected droplets expelled from infected lungs, and ingestion of contaminated feed or milk. In most countries, TB is a notifiable disease. Overall, TB has an important world-wide impact on animal industries and human health. Control measures are based on prevention and eradication. Surveillance is a key element for management of preventions and control programs. Surveillance for TB serves the purpose of enabling Veterinary Services to obtain an accurate picture of the scope of the disease in the US livestock populations; in the event of a disease outbreak, the course TB follows in livestock and wildlife populations for a given area over time; and permits timely intervention if the trend observed deviates from what is expected.

Seasonality of tuberculosis in India: is it real and what does it tell us?

India has a third of the world’s tuberculosis cases. Large-scale expansion of a national program in 1998 has allowed for population-based analyses of data from tuberculosis registries. We assessed seasonal trends using quarterly reports from districts with stable tuberculosis control programs (population 115 million). In northern India, tuberculosis diagnoses peaked between April and June, and reached a nadir between October and December, whereas no seasonality was reported in the south. Overall, rates of new smear-positive tuberculosis cases were 57 per 100 000 population in peak seasons versus 46 per 100 000 in trough seasons. General health-seeking behavior artifact was ruled out. Seasonality was highest in paediatric cases, suggesting variation in recent transmission.

Monitoring of Transmission of Tuberculosis between Wild Boars and Cattle: Genotypical Analysis of Strains by Molecular Epidemiology Techniques

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovisstrains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis.

Bovine Tuberculosis in Free-Ranging White-Tailed Deer From Michigan

A 4.5 yr-old male white-tailed deer (Odocoileus virginianus) killed by a hunter during the 1994 firearm hunting season in northeastern Michigan (USA) had lesions suggestive of tuberculosis and was positive on culture for Mycobacterium bovis the causative agent for bovine tuberculosis. Subsequently, a survey of 354 hunter-harvested white-tailed deer for tuberculosis was conducted in this area from 15 November 1995 through 5 January 1996. Heads and/or lungs from deer were examined grossly and microscopically for lesions suggestive of bovine tuberculosis. Gross lesions suggestive of tuberculosis were seen in 15 deer. Tissues from 16 deer had acid-fast bacilli on histological examination and in 12 cases mycobacterial isolates from lymph nodes and/or lungs were identified as M. bovis. In addition, lymph nodes from 12 deer (11 females and 1 male) without gross or microscopic lesions were pooled into 1 sample from which M. bovis was cultured. Although more male (9) than female (3) deer had bovine tuberculosis infections, this difference was not statistically significant. Mycobacterium bovis culture positive deer ranged in age from 1.5 to 5.5 yr with a mean of 2.7 yr (median 2.5 yr) for males and 3.2 yr (median 3.5 yr) for females. This appears to be the first epidemic occurrence of M. bovis in free-ranging cervids in North America. A combination of environmental (high deer density and poor quality habit) and management-related factors (extensive supplemental feeding) may be responsible for this epizootic.

Risk of Disease from Wildlife Reservoirs: Badgers, Cattle, and Bovine Tuberculosis

Livestock face complex foraging options associated with optimizing nutrient intake while being able to avoid areas posing risk of parasites or disease. Areas of tall nutrient-rich swards around fecal deposits may be attractive for grazing, but might incur fitness costs from parasites. We use the example of dairy cattle and the risks of tuberculosis transmission posed to them by pastures contaminated with badger excreta to examine this trade-off. A risk may be posed either by aerosolized inhalation through investigation or by ingestion via grazing contaminated swards. We quantified the levels of investigation and grazing of 150 dairy cows at badger latrines (accumulations of feces and urine) and crossing points (urination-only sites). Grazing behavior was compared between strip-grazed and rotation-grazed fields. Strip grazing had fields subdivided for grazing periods of <24 h, whereas rotational grazing involved access to whole fields for 1 to 7 d each. A higher proportion of the herd investigated badger latrines than crossing points or controls. Cattle initially avoided swards around badger latrines but not around crossing points. Avoidance periods were shorter in strip- compared with rotation-grazing systems. In rotation-grazing management, latrines were avoided for longer times, but there were more investigative contacts than with strip-grazing management. If investigation is a major route of tuberculosis transmission, the risk to cattle is greatest in extensive rotation-grazing systems. However, if ingestion of fresh urine is the primary method of transmission, strip-grazing management may pose a greater threat. Farming systems affect the level and type of contact between livestock and wildlife excreta and thus the risks of disease.