Isolation of Mycobacterium bovis and other mycobacterial species from ferrets and stoats

As part of wildlife surveillance for bovine tuberculosis, pooled lymph nodes from 21,481 ferrets, 1056 stoats and 83 weasels were cultured for mycobacteria. A total of 268 isolates of Mycobacterium bovis were obtained from ferrets, 2 from stoats and none from weasels, demonstrating the presence of a wildlife reservoir of infection in ferrets. DNA typing by restriction endonuclease analysis (REA) of 48 selected isolates of M. bovis revealed 23 REA types. Twenty-one of these types had previously been isolated from cattle and farmed deer, demonstrating a complex cycle of infection involving wildlife and domestic animals. Apart from M. bovis, a further 208 mycobacterial isolates were obtained, the majority of which (178) were members of the M. avium complex. Speciation of the remaining 30 mycobacterial isolates by DNA sequencing of the 16s rRNA gene, identified half the isolates as M. triplex. Other species identified included M. fortuitum, M. florentinum, M. interjectum, M. intracellulare, M. holsaticum, and M. septicum/M. peregrinum.     

Epidemiology of Mycobacterium bovis infection in feral ferrets (Mustela furo) in New Zealand: II. Routes of infection and excretion

Detailed necropsies of 228 ferrets captured from eight areas in the North and South Islands provided material for an investigation into the epidemiology of tuberculosis in wild ferrets. Seventy-three of the 228 (32%) animals examined were diagnosed as tuberculous, by culture of pooled lymph nodes and detailed histopathological examination. The prevalence of bovine tuberculosis was 96% in 24 ferrets taken from areas in which tuberculous possums were common. None of 35 animals under 4 months of age were found to be infected, and the prevalence of infection was shown to rise with age, such that for each 6 month age increment there was a 2.8 times greater risk of becoming infected. The most common route of infection appeared to be via the alimentary tract, as 79% of 38 animals, in which the initial lesions could be reasonably determined, had these lesions associated with the digestive tract. Samples from potential sites of excretion from infected ferrets were submitted for culturing. The most common route of excretion was via the oral cavity, with M. bovis recovered from 15 of 64 (23%) oral swabs. Mycobacterium bovis was also isolated from four of 64 (6%) tracheobronchial lavage samples, ten of 63 (16%) faecal samples, two of 29 (7%) urine samples and one of 8 (12.5%) mammary glands. The disease in ferrets appears to be principally maintained by ingestion of tuberculous carrion. Although a moderate number of ferrets excrete M. bovis orally, there appears to be only minor intraspecific transmission by bite wounding. The findings provided no evidence to support the occurrence of pseudo-vertical transmission.     

Isolation of Mycobacterium bovis from brushtail possums with non-visible lesions

AIM: To determine the prevalence of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) that did not have macroscopic lesions of bovine tuberculosis, and to evaluate culture of pooled tissues from multiple possums as a method for determining the M. bovis-infection status of wildlife populations in New Zealand.

METHODS: Pools of selected tissues were collected from possums from four different populations known to be infected with M. bovis. Tissue pools from individual animals, and combined pools from multiple animals, were cultured for M. bovis.

RESULTS: In the four populations investigated, the prevalence of possums with macroscopic lesions confirmed by culture to be infected with M. bovis ranged from 1 to 19 (mean 31/283; 10.9)%. The prevalence of possums with non-visible lesions that were culture positive for M. bovis in the same populations ranged from 4 to 10 (mean 24/283; 8.5)%. The mean of the log10 cfu of M. bovis of the macroscopic lesions and of the culture-positive samples that did not have visible lesions was 3.85 (SE 0.26) and 1.46 (SE 0.26) log₁₀ cfu, respectively (p<0.01). Mycobacterium bovis was cultured from pools of 30–50 animals in the four populations studied.

CONCLUSIONS: The finding of M. bovis infection in possums with non-visible lesions identified a potential deficiency of declaring possum populations free of M. bovis on the basis of absence of macroscopic lesions. The culturing of pools of selected tissues from multiple animals without visible lesions can be used to reduce laboratory costs of possum surveys without a major reduction in the ability to detect M. bovis infection.     

The relationship between prevalence of Mycobacterium bovis infection in feral ferrets and possum abundance

AIM: To determine the relationship between the prevalence of macroscopic Mycobacterium bovis infection in feral ferrets (Mustela furo) and the abundance of brushtail possums (Trichosurus vulpecula). METHODS: The predictive power of a previously reported positive association between the prevalence of macroscopic M. bovis infection in ferrets and possum abundance was examined by undertaking surveys of M. bovis infection in ferrets at sites of low and high possum abundance. The association was then tested by a manipulative experiment that measured changes in the prevalence of macroscopic M. bovis infection in feral ferrets after reducing possum abundance. RESULTS: The positive relationship between the prevalence of macroscopic M. bovis infection in ferrets and possum abundance remained valid for new survey data, although the goodness of fit of the relationship was reduced. Experimentally reducing possum abundance reduced the odds of macroscopic M. bovis infection in ferrets by 80% in the years immediately following possum control (Odds Ratio=0.23, p=0.003). CONCLUSIONS: There is a causal link between possum abundance and the prevalence of macroscopic M. bovis infection in feral ferrets in areas in which M. bovis infection is known to occur in ferret populations. This suggests that possum-to-ferret transmission of disease occurs and accounts for most of the disease evident in ferret populations, though does not determine whether ferrets are spillover or maintenance hosts of M. bovis. Management to reduce the prevalence of M. bovis infection in ferrets should consider reducing possum abundance as a control tactic. KEY WORDS: Mustela furo, ferret, Trichosurus vulpecula, brushtail possum, Mycobacterium bovis, bovine tuberculosis.     

The distribution of gross lesions of tuberculosis caused by Mycobacterium bovis in feral ferrets (Mustela furo) from Otago, New Zealand

The distribution of gross lesions of Mycobacterium bovis was examined in 94 tuberculous feral ferrets (Mustela furo) collected from 1992 to 1995 from areas of Otago endemic for bovine tuberculosis. Overall, 56.4% of tuberculous ferrets had single-site lesions, 24.5% had multiple infections and 19.1% had generalised infections. The mesenteric lymph node was the most common site of infection (34.5% of all lesions), with the retropharyngeal (17%) and the prescapular lymph nodes (16.4%) also frequently infected. Only 2.9% of lesions involved the respiratory tract. Of single-site lesions, 60.4% were in the mesenteric lymph node. The high proportion of lesions in the alimentary tract suggests that the ingestion of infectious material, possibly carrion or prey, is an important source of infection. Peripheral lymph nodes contributed to 24.5% of all infections, suggesting that within species transmission by social contact such as fighting and mating also occurs. Open and respiratory lesions were found in 11.7% of tuberculous ferrets, which suggests that ferrets are potentially infectious and therefore may be involved in the transmission of bovine tuberculosis to domestic stock and other mammals. The distribution of gross M. bovis lesions in ferrets is compared to those observed in possums (Trichosurus vulpecula) and badgers (Meles meles).     

The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n = 215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).     

Broad-scale possum and ferret correlates of macroscopic Mycobacterium bovis infection in feral ferret populations

AIMS: To examine the relationships between the prevalence of macroscopic Mycobacterium bovis infection (bovine tuberculosis) in feral ferrets (Mustela furo), the abundance of ferrets, and the abundance of brushtail possums (Trichosurus vulpecula) . METHODS: Data on the prevalence of macroscopic M. bovis infection in ferrets, abundance of ferrets, and abundance of possums were analysed from 12 comparable independent broad-scale surveys. RESULTS: The prevalence of macroscopic M. bovis infection in ferrets was positively correlated with possum abundance but unrelated to ferret abundance, suggesting that possums are an important source of M. bovis infection in ferrets. The lack of any positive relationship between the prevalence of M. bovis infection in ferrets and ferret abundance does not support the hypothesis that per capita transmission rates, and therefore disease prevalence, should be higher at higher ferret abundance. CONCLUSION: The results support the hypothesis that tuberculous possums are the major underlying source of M. bovis infection for feral ferrets in New Zealand.     

Evaluation of the interferon-gamma (IFN-γ) assay to diagnose Mycobacterium bovis infection in pigs

Mycobacterium bovis recognizes as hosts a wide spectrum of animal species. In particular epidemiological situations, high prevalence of infection is found also in pigs. In the present study, we evaluated the capability of the interferon-gamma (IFN-γ) assay to identify pigs infected with M. bovis. The results of the immune-diagnosis were correlated to the findings of the post mortem inspection and the bacterial culture of lymph nodes. Blood samples of 146 pigs, belonging to a local breed of Sicily reared in free or semi-free roaming conditions, were collected to assess the specificity and the sensibility of the IFN-γ assay. Thirty-one pigs, from M. bovis free herds, did not react to the IFN-γ assay, yielding a specificity of 100%. The IFN-γ assay identified 15 out of 19 animals positive to the bacterial culture and 22 out of 26 animals with tuberculous lesions, with a sensibility of 78.9-84.6%, respectively. Out of 26 reactors to the test, 15 pigs (57.7%) confirmed to be infected after the bacterial culture and 22 (84.6%) had tuberculous lesions. The IFN-γ assay was able to reveal 4 animals with no visible lesions (NVL). Together, these findings support the feasible use of the IFN-γ assay as an intra vitam tool for the surveillance and management of M. bovis infection in swine populations.     

An outbreak of tuberculosis by Mycobacterium bovis in coatis (Nasua nasua)

Mycobacterium tuberculosis complex, which includes Mycobacterium bovis, infrequently causes severe or lethal disease in captive wildlife populations. A dead coati from a wildlife triage center showing pulmonary lesions compatible with tuberculosis had raised suspicion of a potential disease caused by mycobacteria species and was further investigated. Four native coatis (Nasua nasua) with suspected mycobacterial infection were sedated, and bronchoalveolar lavages and tuberculin skin tests (TSTs) were performed. All animals tested positive upon TST. Mycobacterial culturing, Ziehl-Neelsen staining, and genetic testing were performed on postmortem samples and the etiologic agent was identified as M. bovis. Molecular genetic identification using a polymerase chain reaction panel was crucial to achieving a definitive diagnosis.     

Epidemiology of Mycobacterium bovis infections of pigs and wild boars using a molecular approach

A molecular epidemiological approach was applied to establishing a possible role for the wild boar as a natural reservoir of Mycobacterium bovis in Sierra de Villuercas, Western Spain; an area free of farmed cattle and wild deer populations. Spoligo and VNTR typing were used over a three year period to study the epidemiological relationship between the occurrence of bovine tuberculosis (TB) in extensively bred Iberian pigs and indigenous wild boar. The 37 sampled wild boar showed different degree of calcified granulomatous lesions in retropharyngeal, mediastinal and pulmonary lymph nodes. The 25 sampled Iberian pigs showed calcified lesions, mainly in the respiratory tract. Lesions located in the mesenteric lymph nodes appeared secondarily. M. bovis was isolated from all affected animals. Twenty-five and 37 isolates of M. bovis were obtained from domestic pigs and wild boar, respectively. Our findings provide evidence that supports the possibility of cross infection between wild boar and domestic pig populations. This is contrary to the generally held belief that swine represent an epidemiological dead end host and play no role in the epidemiology of M. bovis.     

Epidemiology of Mycobacterium bovis infection in feral ferrets (Mustela furo) in New Zealand: I. Pathology and diagnosis

Necropsies from 228 ferrets captured from eight areas in the North and South Islands provided material for an investigation into the epidemiology of tuberculosis in feral ferrets. Mycobacterial culture of pooled lymph nodes (retropharyngeal, respiratory and jejunal) identified the prevalence of infection to be much higher than that estimated from gross lesions only. Seventy-three of the 228 animals examined (32%) were diagnosed as tuberculous. Fifty-three culture-positive ferrets and 18 seemingly uninfected animals were subjected to detailed histopathological examination. The outcomes of these investigations, including the characteristics of the disease, distribution of lesions and aids to diagnosis, are presented. Of the feral carnivores found in New Zealand, the disease persists at high prevalence only in ferrets, and is probably the maintained principally by ingestion of tuberculous carrion. The course of the disease may be prolonged in some ferrets, but tuberculosis eventually causes death of many infected animals. Microscopic hepatic granulomas may be considered pathognomonic of the disease, and have potential to be used as a rapid diagnostic tool in ferrets with no gross lesions.     

A study of bovine tuberculosis in domestic animals and wildlife in the MacKenzie Basin and surrounding areas using DNA fingerprinting

The MacKenzie Basin, an area of about 5150 km2 in the South Island of New Zealand, was free of bovine tuberculosis prior to 1980. During the next 13 years, the majority of the cattle and deer herds in this area became infected with Mycobacterium bovis. The history of infection in the MacKenzie Basin has all the characteristics of a newly developed region of endemic tuberculosis with a wildlife reservoir of M. bovis. Tuberculous possums and ferrets were found in the MacKenzie Basin and both may have been a source of infection for domestic animals. DNA fingerprinting of 125 isolates of M. bovis from domestic animals and wildlife by restriction endonuclease analysis revealed two major groups of isolates. The same groups were identified using IS6110 as a DNA probe. Restriction endonuclease analysis enabled one group to be subdivided into seven restriction types and the other group into eight types. Mycobacterium bovis isolates with the most common restriction types were present in both domestic animals and wildlife, indicating that infection had spread between these two groups of animals. DNA fingerprinting also revealed that M. bovis was introduced into the MacKenzie Basin from at least two distinct sources. Furthermore, DNA finger-printing was able to identify probable sources of infection.     

Epidemiology of Mycobacterium bovis infection in feral ferrets (Mustela furo) in New Zealand: I. Pathology and diagnosis

Necropsies of 228 ferrets captured from eight areas in the North and South Islands provided material for an investigation into the epidemiology of tuberculosis in feral ferrets. Mycobacterial culture of pooled lymph nodes (retropharyngeal, respiratory and jejunal) identified the prevalence of infection to be much higher than that estimated from gross lesions only. Seventy-three of the 228 animals examined (32%) were diagnosed as tuberculous. Fifty-three culture-positive ferrets and 18 seemingly uninfected animals were subjected to detailed histopathological examination. The outcomes of these investigations, including the characteristics of the disease, distribution of lesions and aids to diagnosis, are presented.

Of the feral carnivores found in New Zealand, the disease persists at high prevalence only in ferrets, and is probably maintained principally by ingestion of tuberculous carrion. The course of the disease may be prolonged in some ferrets, but tuberculosis eventually causes the death of many infected animals. Microscopic hepatic granulomas may be considered pathognomonic of the disease, and have potential to be used as a rapid diagnostic tool in ferrets with no gross lesions.     

Naturally occurring tuberculosis in white-tailed deer

OBJECTIVE: To determine the distribution of lesions and extent of tissues infected with Mycobacterium bovis in a captive population of white-tailed deer. DESIGN: Cross-sectional study. ANIMALS: 116 captive white-tailed deer. PROCEDURE: Deer were euthanatized, and postmortem examinations were performed. Tissues with gross lesions suggestive of tuberculosis were collected for microscopic analysis and bacteriologic culture. Tissues from the head, thorax, and abdomen of deer with no gross lesions were pooled for bacteriologic culture. Tonsillar, nasal, oral, and rectal swab specimens, fecal samples, and samples of hay and pelleted feed, soil around feeding sites, and water from 2 natural ponds were collected for bacteriologic culture. RESULTS: Mycobacterium bovis was isolated from 14 of 116 (12%) deer; however, only 9 of 14 had lesions consistent with tuberculosis. Most commonly affected tissues included the medial retropharyngeal lymph node and lung. Five of 14 tuberculous deer had no gross lesions; however, M bovis was isolated from pooled tissue specimens from the heads of each of these deer. Bacteriologic culture of tonsillar swab specimens from 2 of the infected deer yielded M bovis. Mean (+/- SEM) age of tuberculous deer was 2.5 +/- 0.3 years (range, 0.5 to 6 years). Mycobacterium bovis was not isolated from feed, soil, water, or fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Examination of hunter-killed white-tailed deer for tuberculosis commonly includes only the lymph nodes of the head. Results of such examinations may underestimate disease prevalence by as much as 57%. Such discrepancy should be considered when estimating disease prevalence.     

Outbreak of Mycobacterium bovis infection in a wild animal park

An outbreak of tuberculosis due to Mycobacterium bovis occurred in a wild animal park. Three pot-bellied pigs (Sus scrofa vittatus), one red deer (Cervus elaphus), one buffalo (Bison bonasus) and two European lynxes (Lynx lynx) were affected and showed clinical signs including weight loss, enlarged lymph nodes and paralysis of the hindlimbs. Postmortem examinations revealed multifocal granulomatous lesions in various organs, including the lymph nodes, lungs, intestines, kidneys and the central nervous system. Acid-fast organisms were demonstrated in various organs histologically and bacteriologically. Spoligotyping of 17 isolates from various organs of the affected animals confirmed an infection by M bovis and revealed an identical pattern indicating a common origin. The spoligotype was different from the pattern of M bovis recorded in the cattle population in Germany between 2000 and 2006. Investigations of sentinel animals such as an aged silver fox (Vulpes vulpes), a badger (Meles meles), a ferret (Mustela putorius) and rodents, and tuberculin skin tests of the animal attendants and randomly collected faecal samples from the enclosures were all negative for M bovis.     

Farm and slaughter survey of bovine tuberculosis in captive deer in Switzerland

In 1998, a survey was conducted by postal questionnaire to gather basic knowledge about the management, health and productivity of captive deer in Switzerland. In addition, lymph nodes were collected from slaughtered deer from 124 of the 262 holdings surveyed, and tested for Mycobacterium bovis and Mycobacterium tuberculosis. The total farmed deer population was 8389 animals kept on 485 holdings; 87 per cent were fallow deer, 8 per cent red deer, 4 per cent sika deer, and there were small numbers of other species. The median herd sizes were 12 for fallow deer and eight for red deer. Few owners had handling facilities or crushes. In none of the lymph nodes examined were lesions typical of bovine tuberculosis observed, and neither M bovis nor M tuberculosis was cultivated from any of the samples.     

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation of Mycobacterium bovis: I. Pathology and bacteriology

The aim was to develop an endobronchial infection procedure for the study of Mycobacterium bovis infection in badgers. The badgers were anaesthetised and a cannula was passed per os to the tracheal bifurcation. When in place 1 ml of M. bovis suspension was inoculated. Three concentrations of M. bovis suspension were used; <10 colony forming units (cfu), approximately 10(2) cfu and approximately 3 x 10(3) cfu. The badgers were examined at three weekly intervals for clinical signs of disease and a tracheal aspirate was collected at each examination. The badgers were euthanased 17 weeks post infection (pi) and at the post mortem examination a wide range of tissues were examined for gross and histopathological lesions of tuberculosis and cultured for M. bovis. A sample of bronchial alveolar lavage (BAL) fluid was collected at post mortem for culture. At post mortem examination 17 weeks after infection, gross and histopathological lesions of tuberculosis were observed in all badgers inoculated with the high and medium dose and 1/3 inoculated with the low dose. M. bovis was recovered from all inoculated badgers. Infection in the high dose group was more widely disseminated than in the other groups. The number of sites with gross and histopathological lesions increased with increasing dose of M. bovis. All tracheal aspirates were negative on culture and only one BAL, collected from a badger of the high dose group, was positive on culture. No clinical signs due to the experimental infection were observed. The endobronchial route of inoculation is an effective route for establishing experimental infection, and could be used for studies of tuberculosis pathogenesis, immunology of M. bovis infection in badgers and for challenging badgers in vaccine protection studies. Badgers appeared to be very susceptible to infection by this procedure even with a dose of < 10 cfu but appear to control and limit the resulting infection.     

Evidence of the role of European wild boar as a reservoir of Mycobacterium tuberculosis complex

Bovine tuberculosis (bTB) is caused by Mycobacterium bovis and closely related mycobacteria of the Mycobacterium tuberculosis complex. They have an extensive host range and may cause zoonotic TB. A major obstacle to bTB eradication in livestock is the implication of wildlife in the natural cycle of the pathogen. The identification of wildlife reservoir hosts is crucial for the implementation of effective control measures. The European wild boar (Sus scrofa) is frequently considered a spillover or dead end host rather than a true reservoir, and scientific evidence is conflicting outside Mediterranean Spain. The aim of this review is to update current scientific evidence of the wild boar as a TB reservoir and to underline those aspects that need further research. Evidences supporting that wild boar is a TB reservoir host include: (i) presence of common M. tuberculosis complex genotypes in wild boar, domestic and wild animals and humans, (ii) high prevalence of M. bovis among wild boar in estates fenced for decades in complete absence of contact with domestic livestock, and other wild ungulates (iii) TB lesions are frequently seen in thoracic lymph nodes and lungs, suggesting that respiratory infection and excretion may occur, and (iv) extensive tuberculous lesions in more than one anatomical region occur in a high proportion of juvenile wild boar that probably represents the main source of mycobacterial excretion. Hence, epidemiological, pathological and microbiological evidence strongly suggests that, at least in Spanish Mediterranean ecosystems, wild boar are able to maintain TB infection in the wild and are most probably able to transmit the disease to other species, acting as a true wildlife reservoir. These results expand the list of wildlife species that act as natural reservoirs of TB in different parts of the world and suggest the need to control the infection in wild boar populations for the complete eradication of the disease in Spain.     

Experimental infection of brushtail possums (Trichosurus vulpecula) with Mycobacterium bovis by conjunctival instillation

In New Zealand, the brushtail possum (Trichosurus vulpecula) is the major wildlife reservoir of Mycobacterium bovis. Procedures for experimentally infecting possums are required to study the pathogenesis of the disease and to challenge possums in vaccine efficacy studies. Conjunctival instillation of a suspension of M. bovis was effective in producing bovine tuberculosis in captive possums. The experimental disease progressed slowly with the development of palpable lesions in superficial lymph node lesions, both characteristics of the disease in wild, naturally infected possums. At necropsy there was widespread distribution of macroscopic and microscopic lesions. The proportion of possums that became diseased, the rate of development and severity of lesions, the severity of clinical signs, all increased when the dose of M. bovis was increased. Of the three doses used, the medium dose (1000-2000 colony forming units) produced the disease with the most desired characteristics. As a procedure for exposing possums to infection with M. bovis the conjunctival route has advantages in that it is simple and safe to perform, and possums need only to be sedated for infection.     

Immunological approaches to the control of tuberculosis in wildlife reservoirs

Attempts to eradicate tuberculosis from cattle and farmed deer in some countries have been frustrated by the existence of wildlife reservoirs of Mycobacterium bovis infection. Possum control programmes in New Zealand using poisons have shown clearly that the brushtail possum is an important source of infection for cattle and farmed deer, and the sum of evidence strongly suggests that badgers serve as a source of infection for cattle in the UK. Bovine tuberculosis can only be eradicated from these countries by controlling M. bovis infection in both wildlife and domestic animals. The most promising options for control of M. bovis infection in wildlife in the longer term include the development of a tuberculosis vaccine for wildlife and a strategy for biological control of possums. The aim of this review is to address the problems and approaches involved in the control of wildlife tuberculosis from an immunological perspective.