C.G. Mackintosh

Mycobacterial diseases of deer

Abstract The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johnes disease (paratuberculosis), caused by Mycobacterium avium subsp paratuberculosis (basonym M. paratuberculosis), and avian tuberculosis, caused principally by M. avium subsp avium. The first case of M. bovis infection in farmed deer was identified in New Zealand in 1978. In 1983, a voluntary scheme was introduced in New Zealand to control tuberculosis in farmed deer, followed by a compulsory tuberculosis control scheme in 1990. The primary control measure is the slaughter of infected animals, detected by skin testing and blood testing, together with movement control and vector control. The number of infected deer herds peaked in the mid 1990s at over 160 herds, but by 30 June 2002 this had been reduced to 79 (1.45%), and to 67 (1.23%) by June 2003. Deer-to-deer transmission occurs, but the majority of herd breakdowns are believed to be from infected vectors. Factors likely to affect the susceptibility of deer include age, environment, population density, exposure and genetics. Avian tuberculosis occasionally causes clinical disease in wild, captive and farmed deer in New Zealand and overseas. Mycobacterium intracellulare, and subspecies of M. avium other than M. paratuberculosis, are widespread throughout New Zealand and are thought to be largely responsible for the high level of sensitisation to avian purified protein derivative (PPD), which is used for comparison purposes in tuberculosis skin testing of deer in this country. Infections with these organisms are usually subdinical in farmed deer, although M. avium subsp avium commonly causes lesions in retropharyngeal, mesenteric and ileocaecal lymph nodes. These lesions cause problems because of their gross and microscopic similarity to those due to M. bovis infection. Birds and domestic animals are most likely to become infected via environmental contamination of food, water, bedding litter or soil, while carnivores or scavengers may also become infected by ingesting infected carcasses. Johnes disease has been reported in deer in the wild and in zoos, especially in North America, the United Kingdom (UK) and Europe. Since first being confirmed in farmed deer in New Zealand in 1979, the incidence of Johnes disease has increased steadily. To date, M. paratuberculosis has been identified in >600 farmed deer on 300 properties. The majority of cases have been identified from suspected tuberculous lesions submitted from deer slaughter plants. Clinically, Johnes disease in deer is similar to the disease in sheep and cattle, with typical signs of loss of weight and condition, and diarrhoea. However, outbreaks of Johnes disease frequently occur in young red deer, 8–15 months of age, whereas the clinical disease in sheep and cattle is sporadic and usually affects adults 3–5 years of age. The disease is characterised by a chronic granulomatous enteritis and lymphadenitis, especially affecting the jejunum and ileum and the mesenteric lymph nodes. Deer affected subclinically may have lesions in these lymph nodes at slaughter, which are grossly indistinguishable from those due to bovine tuberculosis. Because of the antigenic similarity between M. intracellulare and all the subspecies of M. avium, including M. paratuberculosis, the diagnostic tests for Johnes disease lack sensitivity and specificity, making control difficult.

Genetic Resistance to Experimental Infection with Mycobacterium bovis in Red Deer (Cervus elaphus)

ABSTRACT Tuberculosis (Tb) caused by Mycobacterium bovis is a worldwide threat to livestock and humans. One control strategy is to breed livestock that are more resistant to Mycobacterium bovis. In a 3-year heritability study 6 farmed red deer stags were selected from 39 on the basis of their differing responses to experimental challenge via the tonsillar sac with approximately 500 CFU of M. bovis. Two stags remained uninfected, two were moderately affected, and two developed serious spreading Tb. Seventy offspring, bred from these six stags by artificial insemination using stored semen, were similarly challenged withM. bovis. The offspring showed patterns of response toM. bovis challenge similar to those of their sires, providing evidence for a strong genetic basis to resistance to Tb, with an estimated heritability of 0.48 (standard error, 0.096; P< 0.01). This is the first time the heritability of Tb resistance in domestic livestock has been measured. The breeding of selection lines of resistant and susceptible deer will provide an ideal model to study the mechanisms of Tb resistance in a ruminant and could provide an additional strategy for reducing the number and severity of outbreaks of Tb in farmed deer herds. Laboratory studies to identify genetic and immunological markers for resistance to Tb are under way. Preliminary studies showed no associations between NRAMP or DRB genes and resistance to Tb in deer. Patterns of immune responses seen in resistant animals suggest that both innate and acquired pathways of immunity are necessary to produce the resistant phenotype.

Animal models of protective immunity in tuberculosis to evaluate candidate vaccines

While the etiology of tuberculosis is well understood, the nature of the protective immune response to the causative mycobacteria has remained a mystery. There is an urgent need to define protective immunity critically, and to develop alternative animal models to evaluate the efficacy of new-generation vaccines against tuberculosis in a cost-effective way.

The scavenging behaviour of ferrets (Mustela furo), feral cats (Felis domesticus), possums (Trichosurus vulpecula), hedgehogs (Erinaceus europaeus) and harrier hawks (Circus approximans) on pastoral farmland in New Zealand: Implications for bovine tuberculosis transmission

Abstract Aims: To identify species that scavenge carcasses in pastoral habitats in New Zealand; to determine whether there were interspecific or intraspecific differences in scavenging behaviour and; to document any interspecific or intraspecific interactions occurring at carcasses. Methods: Scavenging by ferrets (Mustela furo), feral cats (Felis domesticus), possums (Trichosurus vulpecula), hedgehogs (Erinaceus europaeus) and harrier hawks (Circus approximans) was studied from autumn to midwinter on pastoral farmland near Palmerston (45S, 170E), Otago, New Zealand. Time-lapse video recorders and camera lens mounted with infra-red light illumination were used to monitor carcasses of 10 ferrets, 12 possums, 2 hedgehogs and 7 rabbits (Oryctolagus cuniculus) until they were totally scavenged. Results: Ferrets scavenged 5/8 ferret carcasses, 8/9 possum carcasses and 6/7 rabbit carcasses encountered. Feral cats scavenged 3/8 ferret carcasses, 5/7 rabbit carcasses, and 3/8 possum carcasses encountered. Possums scavenged 1/2 ferret carcasses and 3/4 rabbit carcasses encountered. The proportion of encounters resulting in feeding on ferret carcasses differed between ferrets (45.7%) and possums (6.3%), and between possums and cats (29.7%). Similarly, for possum carcasses, differences were found between ferrets (76.6%) and possums (0%), ferrets and cats (60.6%) and possums and cats. No interspecific differences were found in the proportion of encounters that resulted in feeding on rabbit carcasses between ferrets (85.2%), possums (75%) and cats (73.1%). In 8/12 instances of ferrets coming into contact with other ferrets whilst feeding, ferrets fed together at the carcass. On 1 occasion, 4 ferrets were recorded feeding together. In 7/8 instances where cats and ferrets came into contact over carrion, ferrets maintained possession or displaced the cat from the carcass. Conclusions: Communal carrion feeding by ferrets may facilitate intraspecific and interspecific transmission of bovine tuberculosis (caused by Mycobacterium bovis) by the consumption of contaminated carrion, fighting, or close-contact activities. Cannibalism may be one mechanism by which tuberculosis is transmitted within ferret populations. Our results also suggest that possums may acquire infection from carrion, despite being mainly herbivorous.

Sensitivity, specificity, and confounding factors of novel serological tests used for the rapid diagnosis of bovine tuberculosis in farmed red deer (Cervus elaphus).

ABSTRACT In this study, novel serological tests were used to detect tuberculosis (TB) in groups of farmed red deer (Cervus elaphus) varying in disease status or possible confounding factors. Groups of deer naturally or experimentally infected with Mycobacterium bovis and animals vaccinated against paratuberculosis were studied, as were uninfected animals and animals naturally or experimentally infected with Mycobacterium avium subsp. paratuberculosis. Sera were assayed using two rapid lateral-flow tests, Chembios CervidTB STAT-PAK and DPP VetTB tests, and results were compared to those from tuberculin skin tests. Both serological tests had a high sensitivity, but specificity was adversely affected after animals had received a vaccine against paratuberculosis and were subsequently skin tested. The specificity of the DPP VetTB test was higher than that of the CervidTB STAT-PAK test, with natural infection with M. avium subsp. paratuberculosis adversely affecting the specificity of only the CervidTB STAT-PAK test. The sera from M. avium subsp. paratuberculosis-infected deer that produced false-positive reactions in the CervidTB STAT-PAK test were retested with a multiantigen print immunoassay (MAPIA), and some of these sera were shown to react with the MPB83 antigen. Combining the results from the serological tests and the skin tests showed only a slight increase in the sensitivity of detection of M. bovis-infected animals. It is concluded that both the CervidTB STAT-PAK and DPP VetTB tests offer rapid, convenient, and easy detection of bovine tuberculosis in deer, albeit with significant interference from paratuberculosis vaccination status and subsequent skin testing. The latter finding illustrates one of the limitations of currently available vaccines against paratuberculosis.

Experimental Infection Model for Johne's Disease in Sheep

ABSTRACT Johnes disease in ruminants results in chronic enteritis caused by the pathogenic bacterium Mycobacterium avium subsp. paratuberculosis. This study examined two M. avium subsp. paratuberculosis strains (JD3 and W), using different doses and routes of infection, to establish the optimal time postchallenge when predictable levels of infection, gut lesions, and clinical disease occur in a large proportion of sheep. While a small proportion (25%) of sheep challenged with a low-passage-number laboratory culture of M. avium subsp. paratuberculosis (strain W) became infected, no infection was found in animals exposed to a high-passage-number culture isolate of strain W. In contrast, a primary tissue homogenate of M. avium subsp. paratuberculosis (JD3) resulted in high (90%) infection rates and gut histopathology following oral or intratonsillar challenge. The optimal conditions necessary to produce Johnes disease involve oral inoculation of 3-month-old lambs with four doses of 5 × 108 CFU of M. avium subsp. paratuberculosis isolated directly from the gut lymphatic tissues of clinically affected sheep. This resulted in consistent gut histopathology at 9 months and the onset of clinical disease by 11 months postchallenge.

An in vivo comparison of bacillus Calmette–Guérin (BCG) and cytokine-secreting BCG vaccines

A recombinant bacillus Calmette–Guérin (BCG) vaccine has been developed, which constitutively secretes interleukin (IL)‐2. Groups of deer were immunized with either normal BCG (Pasteur 1173 P2 strain) or recombinant BCG (rBCG/IL‐2) and their immune responses were monitored over 3 months. Animals gained weight over this period and showed no signs of adverse reactions to either vaccine. Lymphocyte transformation responses did not differ significantly between the two groups. No antibody that was specific for BCG was detected in any animal. Intradermal skin‐test responses to BCG antigens showed that the rBCG/IL‐2 induced a smaller delayed‐type hypersensitivity response than the normal BCG. Cytokine transcription was determined by reverse transcription–polymerase chain reaction (RT–PCR). While IL‐2 and interferon‐γ (IFN‐γ) levels did not differ significantly between the two groups, the level of IL‐4 was found to be lower in the group given rBCG/IL‐2. This resulted in a strong interferon‐γ:IL‐4 ratio, suggesting a skewing of the immune response towards a Type 1 response. The rate at which the vaccine was eliminated from the host was the same regardless of whether BCG or rBCG was used. At autopsy (3 months after vaccination) 99·99% of the organisms had been eliminated. The small number of organisms isolated from the draining lymph node of animals given rBCG/IL‐2 were grown in antibiotic‐containing media. They were shown to still contain the shuttle plasmid and to secrete biologically active IL‐2, indicating that the plasmid was stably maintained despite the host’s immune response and in the absence of antibiotic selection.

Leptospirosis in farmed deer in New Zealand: A review

Abstract Current knowledge of leptospirosis in farmed deer in New Zealand is reviewed. Over the past 25 years, leptospirosis has been reported to occur in individual cases as well as in herd outbreaks in farmed deer and in human cases linked to farmed deer. Serological studies and evidence from bacterial culture suggest infection is widespread. Mixing of young stock from several sources appears to be a significant risk factor for outbreaks. The culture of Leptospira interrogans Hardjobovis, Pomona and Copenhageni has been reported. Infection with serovar Hardjobovis had the highest prevalence, either individually or mixed with serovar Pomona. Infection with serovar Copenhageni appears uncommon and its pathogenicity in deer is unproven. Titres to serovars Australis, Ballum, Balcanica and Tarassovi have been reported. Deer appear to be maintenance hosts for serovar Hardjobovis, incidental or accidental hosts and probably a maintenance population for serovar Pomona, since some infections persist for several months, and accidental hosts for serovar Copenhageni. Serovar Pomona appears to produce clinical and probably subclinical disease, whereas serovar Hardjobovis appears to cause only subclinical disease, although the relative risk of disease causation has not been determined. Clinical disease is usually manifested by haemolysis, jaundice, renal lesions, haemoglobinuria and often by sudden death. Renal lesions are commonly observed at slaughter and many are associated with leptospiral infections. Occupationally, slaughterhouse workers appear to beat greatest risk of contracting the disease from deer. Vaccination produces serological responses, but its effectiveness in protecting against disease, and prevention or reduction of shedding in urine, has not yet been confirmed in deer. More robust knowledge of the epidemiology of leptospiral infections in deer, and the effectiveness of vaccines and vaccination regimes, is needed to assist the deer industry to develop a strategy to manage this disease.

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis infection in red deer (Cervus elaphus): An histopathological grading system, and comparison of paucibacillary and multibacillary disease

Abstract AIM: To describe a grading system for evaluating lesions in the small intestine and mesenteric lymph nodes of red deer (Cervus elaphus) infected with Mycobacterium avium subsp. paratuberculosis (MAP), and report the distribution of granulomas and findings seen in paucibacillary and multibacillary forms of the disease. METHODS: Tissues were examined from red deer either experimentally (n=300) or naturally (n=131) infected with MAP. A disease severity score developed previously was expanded to help provide more sensitivity in assessing severity of disease. The distribution of granulomatous, paucibacillary and multibacillary lesions in sections of jejunum, ileocaecal valve and mesenteric lymph nodes was compared between sites and between animals with mild (severity score ≤7) and severe (severity score ≥8) forms of the disease. RESULTS: Based on the results of three published studies, the severity score related well with the clinical severity and gross lesions associated with the disease. Paucibacillary lesions tended to have smaller macrophages and increased numbers of Langhans giant cells than multibacillary forms, but this was not a consistent finding. The multibacillary form of the disease had Langhans giant cells, containing numerous acid-fast organisms (AFO), and in one form sheets of epithelioid-like macrophages with prominent vacuolated cytoplasm and few Langhans giant cells. In deer experimentally infected with MAP, granulomatous lesions were more evident in mesenteric lymph nodes than in intestinal tissue, especially in the mild form of the disease. In mild cases, granulomas were significantly more evident in Peyers patches than in the intestinal mucosa, but in severe cases, the difference was not significant. Paucibacillary forms of the disease were more evident in deer with the mild disease, and multibacillary forms were more evident in deer with the severe disease. CONCLUSIONS: The severity score provides an objective measure of the severity of Johnes disease, and is useful for comparing individuals and groups of deer in studies involving experimental or natural infection with MAP. In mild disease, lesions were more evident in mesenteric lymph nodes than in jejunum and ileocaecal tissue, and Langhans giant cells were present in both paucibacillary and multibacillary forms of the disease. The post erior jejunum and ileocaecal-valve lymph nodes were the best sites for detecting mild lesions, while intestinal samples from the posterior jejunum and ileocaecal valve had a lesser but useful role.

Experimental infections in young red deer (Cervus elaphus) with a bovine and an ovine strain of Mycobacterium avium subsp paratuberculosis.

Abstract AIMS: To compare the virulence of a ‘bovine’ and an ‘ovine’ strain of Mycobacterium avium subsp paratuberculosis (M. ptb) in red deer (Cervus elaphus) after experimental inoculation orally, and to examine the relationship between the dose of the bovine strain given and immunological, clinical and histopathological outcomes in young red deer. METHODS: Newly-weaned 4-month-old male red deer (n=81) were randomly assigned to one of five groups. Three groups (n=16) received high (109 colony forming units (cfu); HB), medium (107 cfu; MB) or low (103 cfu; LB) oral doses of a bovine strain of M. ptb, one group (n=16) received medium (107cfu; MO) doses of an ovine strain of M. ptb, and a Control group (n=17) was not dosed. The HB and Control groups were grazed together, the MB and LB groups were grazed together, and the MO group was grazed alone, in separate small paddocks on a quarantined area of the farm for 45 weeks. Liveweight, clinical signs and immunoglobulin G1 (IgG1) antibody levels were monitored for up to 45 weeks. Deer affected with Johnes disease were euthanised when they showed obvious clinical signs. Unaffected deer were slaughtered at the end of the trial (Week 45), and all deer were necropsied. Faeces and tissue samples were cultured for M. ptb, and fixed tissues were examined for histopathology. RESULTS: Between 21 and 38 weeks post-challenge (pc), 5/16 animals in the HB group developed early signs of Johnes disease and were euthanised. The remaining deer in the five groups were all apparently healthy and reached good liveweights (∼100 kg average), and were euthanised and examined 45 weeks pc. Three deer (two HB and one MB) had small caseous lesions in their jejunal lymph nodes (JJLNs) and one HB animal had a small caseous lesion in a retropharyngeal lymph node. The remaining animals had no grossly-visible lesions. Mycobacterium avium subsp paratuberculosis was cultured from samples from 100% of the HB and MB animals, 50% of the LB group, 69% of the MO group and all Control animals. Thus all Control deer were infected by natural transmission from the HB group but none developed signs of clinical disease. Examination of histological sections of jejunum, ileocaecal valve (ICV) and associated lymph nodes showed a gradation of severity of lesions that was positively correlated (p<0.001) with dose of the bovine strain administered; mean lesion severity scores were 4.8, 2.9 and 0.9 for HB, MB and LB groups, and 2.2 and 0.9 for the Control and MO groups, respectively. IgG1 antibody levels at the time of euthanasia were also correlated with lesion severity scores at slaughter (p<0.001). CONCLUSIONS: The ovine strain of M. ptb used in this study was less virulent for red deer than the bovine strain. The correlation between dose of the bovine strain and the severity of lesions suggests that clinical Johnes disease in yearling red deer likely results from high oral challenge with a bovine strain whilst they are young. The minimum oral infective dose may be close to 103 cfu for this bovine strain.