Detection of Mycobacterium avium subsp. paratuberculosis in skeletal muscle and blood of ewes from a sheep farm in New Zealand

Abstract

AIM: To determine whether viable Mycobacterium avium subsp. paratuberculosis (Map) is present in skeletal muscle and blood in ewes with and without Johne's disease confirmed histologically.

METHODS: A total of 51 mixed-aged ewes in poor body condition from a farm with a history of clinical Johne's disease were culled and examined at necropsy. BACTEC radiometric culture was performed on samples of skeletal muscle from the biceps femoris, mononuclear cells in peripheral blood (hereafter referred to as blood), and ileum. Histological sections and Ziehl-Neelsen (ZN)-stained impression smears of terminal ileum and mesenteric lymph nodes were examined. Ewes were defined as having confirmed Johne's disease if there was histopathological evidence typical of the disease within the ileum and adjacent lymph nodes.

RESULTS: Eighteen of 21 (86%) ewes with confirmed clinical Johne's disease were culture-positive for Map from sites peripheral to the alimentary tract, comprising 15 from skeletal muscle and 13 from blood. Five of 30 (17%) ewes that did not have Johne's disease were culture-positive, with four from skeletal muscle and one from blood. The likelihood that ewes with confirmed Johne's disease had systemic Map infection compared with ewes without was determined as OR=30 (95% CI=6.3–142.0; p<0.001).

CONCLUSION: The prevalence of Map infection of skeletal muscle and blood in ewes with confirmed Johne's disease was 71% and 62% respectively, and in unaffected ewes was 13% for muscle and 3% for blood.

CLINICAL RELEVANCE: Skeletal muscle and blood are potential sources of exposure of humans to Map, and the risk appears higher from sheep with Johne's disease.     

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.     

Toxoplasmosis as a cause of ovine perinatal mortality

Aims. To determine temporal and spatial patterns of bovine tuberculosis (Tb) in a population of brushtail possums (Trichosurus vulpeculu) free from commercial and recreational hunting and living contiguously with livestock, and to relate these patterns to measures of possum health and density.

Methods. Possums were trapped on 11 lines located on a forest-rough grazing margin in the Ahaura Valley in Westland in 1979-80 and each August from 1992 to 1996, and were examined post mortem for gross lesions indicative of Tb. Infection levels in possums were compared with Th test data from cattle grazing in the same area.

Results. Tuberculosis was identified from the trapped sample of possums in 1980. Trapping of further possums on the same trap lines confirmed the presence of the disease each year from 1992 to 1996, and suggested a clustering of infection in time and space. The prevalence of Th in possums declined with increasing possum population size and well being, and decreasing age. The prevalence of Tb in possums was highest in 1980 and 1992, and appeared to roughly coincide with similar upsurges in Tb in nearby cattle.

Conclusions. Our results suggest that the exceptional levels of Tb recorded in possums in 1992 had a direct effect on possum numbers, and gave rise to very low trap catches in 1993-94. They also suggest thatTb can persist for several years in possum populations existing well below the 5% trap catch targeted by regipnal councils forTb possum control.     

Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n=1532), white-tailed deer (n=463), brushtail possums (n=129), and wild boar (n=177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.     

Lung worm (Marsupostrongylus spp.) infection in common brushtail possums (Trichosurus vulpecula)

Marsupostrongylus spp. are the metastrongyloid nematodes most commonly associated with verminous pneumonia in Australian marsupials. Currently, there is a scarcity of information regarding this parasite in the common brushtail possum (Trichosurus vulpecula). Thirty-four free-living possums submitted to two wildlife hospitals in Sydney, Australia, between 2008 and 2015 were diagnosed with verminous pneumonia on postmortem examination. The majority of possums presented ill with multiple comorbidities. However, only five cases had clinical signs of respiratory disease. Necropsy and histopathology revealed extensive lung lesions characterised by diffuse, mixed interstitial infiltrates of macrophages, lymphocytes and plasma cells with mild to marked concentrations of eosinophils. Bronchopneumonia, pulmonary oedema, interstitial fibrosis, atelectasis and type II pneumocyte hyperplasia were also present in most cases. Adult nematodes, first-stage larvae and embryonating eggs were present in the large airways and alveolar spaces. The parasites were definitively identified as Marsupostrongylus spp. in eight cases with presumptive diagnoses based on histopathological characteristics reached in a further 26 cases. Twenty-nine of the 34 affected possums were adults with no sex predisposition. A review of the brushtail possum records at Taronga Wildlife Hospital from 1999 to 2015 revealed no lungworm infections were reported in the 45 possums examined before 2008. However, between 2008 and 2015, 30 of 47 possums (63.8%) examined were diagnosed with metastrongyloid lungworms. This case series is the first detailed report of Marsupostrongylus nematodes in common brushtail possums and highlights the clinical and pathological features, along with epidemiological findings.     

The effect of fertility control on the transmission of bovine tuberculosis in wild brushtail possums

AIM: To determine the effect of fertility control on the rate of transmission of bovine tuberculosis (Tb), caused by Mycobacterium bovis, in brushtail possums (Trichosurus vulpecula).

METHODS: At two study sites with a history of Tb infection in the resident possum population, a sample of adult male and female possums (n=50), determined by palpation to be Tb-free, was surgically sterilised by gonadectomy. A sample of untreated Tb-free male and female possums (n=54) served as controls. Each study site was trapped every 2 months over a 3-year period, and the Tb status of the individuals in the trial assessed. At the conclusion of the trial, all remaining experimental individuals were killed, necropsied and examined for characteristic lesions typical of Tb. The rate of transmission of Tb infection was estimated using the incidence of tuberculous cases in the experimental animals and comparing it between sites, sex and sterilisation treatment.

RESULTS: Sterilisation by gonadectomy resulted in a reduction in the rate of transmission of Tb in male possums by 53%, but a corresponding increase in sterilised females for reasons that are still unclear. However, this interaction was statistically weak (p=0.10). When the sexes were combined, there was no overall effect of sterilisation on the rate of transmission of Tb (p=0.43). Sterility treatment notwithstanding, there was a highly significant difference in the rate of transmission between the sexes (p=0.01), being almost one order of magnitude higher in untreated males compared with untreated females.

CONCLUSIONS: Although lacking strong statistical support, these results suggest that fertility control that targets endocrine control of fertility may provide some additional benefits for disease control over that achieved by reductions in population density, by reducing the rate of transmission of M. bovis in male possums. However, additional studies are needed to confirm the validity of these results. The large difference in the rate of transmission of M. bovis in untreated males compared with untreated females suggests that transmission of Tb among males may be an important driver of the dynamics of Tb infection in possums.     

Longevity of Mycobacterium bovis in brushtail possum (Trichosurus vulpecula) carcasses,and contact rates between possums and carcasses

Abstract

AIM: To determine, for a variety of environmental conditions, how long Mycobacterium bovis might remain viable inside the carcass of a brushtail possum (Trichosurus vulpecula) that died of bovine tuberculosis (Tb), and to measure the rate of contact between free-ranging possums and possum carcasses.

METHODS: Lesions of M. bovis were simulated by inoculating excised spleens weighing 0.5–1 g with 0.2 mL liquid culture containing approximately 5 x 10⁷ cfu M. bovis/mL. Simulated lesions were inserted into possum carcasses (n=48) at the peripheral lymph nodes. Carcasses were placed in the field at two sites (a tussock grassland and a podocarp-broadleaved forest site) and in two seasons (summer and winter) for up to 62 days. Survival rates of M. bovis were estimated by sampling the simulated lesions over time, and culturing the recovered lesion to determine if any viable M. bovis bacteria were present.

The time taken for a free-ranging possum to first encounter a dead possum in its home range was estimated by live-trapping possums and fitting them with proximity loggers (n=13). A ‘contact’ was recorded if these possums came within 40–50 cm of proximity loggers fitted to possum carcasses.

RESULTS: There were strong seasonal and site effects in the survival rate of M. bovis in possum carcasses. In the grassland habitat, no viable bacilli were cultured from any carcass after 3 days in summer, whereas in winter all samples were culture-positive for the first 20 days, and some were still positive after 27 days. The survival rates for forest habitat were intermediate between the results for grassland, and there were no culture-positive carcasses after 9 days in summer or 27 days in winter.

In summer, infected carcasses (n=6) were first encountered by possums a mean 1.9 (range 0.4–6.7) days after placement.

CONCLUSIONS: Possum carcasses were contacted by free-ranging possums within the period that viable M. bovis were shown to survive in a carcass. The risk of such infection is likely to be most significant in winter or in areas with microhabitats where the survival of M. bovis is high. However, the generally low survival rate of M. bovis in possum carcasses and the low frequency of possum-to-carcass contacts indicate this route of transmission alone could not maintain Tb in a possum population.     

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis.

METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 × 10⁵ colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50–51 days after challenge were euth anised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis.

RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05).

CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.     

Endoparasites of brushtail possums (Trichosurus vulpecula) from the South Island,New Zealand

As part of a study to assess whether endoparasites could assist in the biological control of brushtail possums in New Zealand, we investigated the composition and distribution of possum endoparasites in the South Island. Possums were collected near five of the original release areas in the South Island: Banks Peninsula, Hokitika, Nelson, Dunedin and Invercargill.

Among the nematodes, those most frequently encountered were Trichostrongylus spp., which were present in possums from all five study areas. Trichostrongylus species from possums in the Invercargill area comprised 4.5% T. colubriformis, 0.9% T. vitrinus and 11.3% T. retortaeformis. Paraustrostrongylus trichosuri and Parastrongyloides trichosuri were found only in the Invercargill area, where they infected 1.4% and 14% of possums respectively. The cestode Bertiella trichosuri was present in possums from all locations except Dunedin. The protozoan Eimeria spp. occurred in all areas.

These are the first records of Parastrongyloides trichosuri, Paraustrostrongylus trichosuri, T. vitrinus, T. retortaeformis and Eimeria spp. in South Island possums.

The prevalence of endoparasites and the intensity of infection was very low compared to the lower North Island of New Zealand. Endoparasites at the existing levels in the South Island probably have very little effect on possum populations.     

Distribution of leptospirosis in possums from New Zealand and its offshore islands

Extract

As part of a larger project investigating methods of brushtail possum (Trichosurus vulpecula) biocontrol, possum sera were collected from 15 sites throughout New Zealand and its offshore islands. These sites were selected to cover the original release sites of possums in New Zealand.     

Haematological and serum biochemical reference ranges for brushtail possums

Extract

I am writing with reference to the recent article by O'Keefe and Wickstrom (Routine blood values for New Zealand brushtail possums (Trichosurus vulpecula) at capture. New Zealand veterinary Journal 46, 41–2,1998).     

Interferon gamma responses to proteome-determined specific recombinant proteins: Potential as diagnostic markers for ovine Johne's disease

Johne's disease (JD), or paratuberculosis is a fatal enteritis of animals caused by infection with Mycobacterium avium subspecies paratuberculosis (Map). There may be a long subclinical phase with no signs of clinical disease.     

Intra-uterine transmission of Mycobacterium avium subsp paratuberculosis in subclinically affected red deer (Cervus elaphus)

AIM: To determine the rate of transmission of Mycobacterium avium subsp paratuberculosis (M. ptb) from hind to fetus in utero, and the risk of transmission from dam to fawn via infected colostrum and milk in subclinically affected red deer hinds.

METHODS: Hinds were sourced from farms in Otago or Southland and selected for the study if they were positive to the immunoglobulin G1 (IgG1) modified enzyme-linked immunosorbent assay (ELISA) (Paralisa) and exhibited no clinical signs of Johne's disease. The hinds (n=35) were sent to a deer slaughter premises (DSP; n=31) or were killed on-farm (n=4). All post-mortem samples were collected from the fetus first and then from the dam, taking care to avoid cross contamination between samples. Fresh samples (n=185) were collected for culture, and tissue samples (n=72) were collected from 24 hinds and their fetuses for histopathological examination.

RESULTS: A total of 24/35 hinds selected were suitable for inclusion in the study. Eighteen of these pregnant hinds were culture-positive for M. ptb, and 14 of these had culture-positive fetuses, representing a transmission rate of 78% (95% confidence interval (CI) =0.58–0.98) from dam to fetus. Of the 16 mammary glands sampled, 11 (69%) were culture-positive for M. ptb while 12/15 (80%) mammary lymph nodes sampled were also culture-positive.

CONCLUSIONS: This study demonstrated a high rate of transmission of M. ptb from dam to fetus in red deer, and a potential risk of transmission to fawns suckling from mothers that are subclinically affected with Johne's disease.     

Potential wildlife sources of Yersinia pseudotuberculosis for farmed red deer (Cervus elaphus)

During 1982 and 1983 15 serotype I,6 serotype II, 1 serotype III and 3 untyped strains of Yersinia pseudotuberculosis were isolated from 675 apparently normal small mammals and birds from the Invermay farm and nearby rubbish tip with the following prevalence rates: feral cats 27.8%, Norway rats 8.6%, mice 5.5%, hares 3.8%, rabbits 1.9%, ducks 5.3%, sparrows 2.3%, seagulls 2.3% and starlings 1.7%. For rabbits a significantly higher prevalence of infection was found in the autumn/winter period (4.8%) than the spring/summer period (0%).

Insufficient numbers of other mammals were obtained to demonstrate any seasonal difference in prevalence. All bird isolations were obtained between March and July (8/158) compared with none from August to October (0/144). It appears that a number of free-living species of small mammal and birds may be reservoir hosts for Y. pseudotuberculosis and potential sources of infection for red deer on the Invermay farm.     

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.     

Haematological and biochemical values for brushtail possums

Extract

As an investigator with an interest in marsupial haematology, I was interested to read the study by O'Keefe and Wickstrom pertaining to haematological and biochemical values for wild-caught brushtail possums (Trichosurus vulpecula) in New Zealand (New Zealand veterinary Journal 46,4l-2,1998). Unfortunately, there are some design and interpretation flaws in this study that detract from its usefulness to researchers in the field of marsupial haematology.     

T lymphocyte responses during early enteric Mycobacterium avium subspecies paratuberculosis infection in cattle

Johne's disease (JD) is a costly intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map), which is transmitted to perinatal calves by the fecal-oral route. Disease control efforts focus on identification and culling of infected cattle from herds; therefore failure to identify animals early is a major obstacle to reducing transmission. Development of host immunity during early JD remain incompletely characterized so detecting subclinical JD using immunologic techniques is a substantial challenge in the field. Development of a test with high sensitivity and specificity is a major research goal with significant implications for the cattle industry. The objectives of this study were to compare early Map-specific T lymphocyte responses in naive, experimentally Map infected and Map vaccinated calves using a subcutaneous matrigel biopolymer-based assay. We examined the phenotype of recruited lymphocytes and local interferon gamma (IFNγ) production within subcutaneously placed matrigel containing Map antigen 30 days after experimentally induced intestinal Map infection or Map vaccination. We show that IFNγ-secreting CD4+ T cells are recruited to matrigel sites in vaccinated but not infected or naïve calves. γδ T cells recruited to matrigel sites of Map-infected calves were mostly WC1-, while γδ T cells recruited to matrigel sites of Map-vaccinated calves were predominantly WC1+. IFNγ at matrigel sites was a discriminating factor between infected calves, naïve calves and vaccinated calves. These data contribute to our understanding of early anti-Map immunity, and may be useful for detecting early intestinal Map infections in calves or for enhancing our ability to discriminate between Map-infected and Map-vaccinated calves.     

Mycobacterium bovis infection in New Zealand hedgehogs (Erinaceus europaeus)

Seventy-nine wild hedgehogs from the Wairarapa, an area of New Zealand in which bovine tuberculosis is endemic, were examined for lesions suggestive of tuberculosis. Sixteen animals with suspicious lesions had samples submitted for culture. Mycobacterium bovis was recovered from four animals with gross pulmonary lesions. The gross and histopathological appearance of the lesions are described and the significance discussed in the light of previously reported experimental and captive animal infections. It is likely that infection arose from the scavenging behaviour of hedgehogs. The moderate prevalence (5%) of tuberculosis in these animals combined with their small home ranges may allow them to be used in wildlife surveys to pinpoint the locality in which tuberculous possums have died.     

Tissue localisation of Mycobacterium avium subspecies paratuberculosis following artificially induced intracellular and naked bacteraemia

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants. While Johne's disease is primarily expressed in the gastrointestinal tract, isolation of MAP from extra-intestinal tissues indicates that microbial dissemination via the haematogenous route may occur during the infection. This study examined the movement of peripheral blood mononuclear cells (PBMCs) infected with MAP and the dissemination of MAP following mycobacteraemia induced by IV inoculation over a time frame of 3 days.     

Experimental infection of the possum (Trichosurus vulpecula) with Leptospira interrogans serovar balcanica I. Characteristics of infection

The serological, bacteriological and histopathological characteristics of experimental infection with serovar balcanica in possums (Trichosurus vulpecula) are described, and the possum is shown to be a potential maintenance host for this organism. Serum agglutination titres were maintained at almost constant levels for longer than a year, and leptospiruria was present in 50% of animals for a similar period. A paradoxical reaction to hardjo antigen was found in sera from all possums infected with balcanica.

Comparative studies were conducted using recently isolated field strains of serovars hardjo and ballum. Young possums seronegative to Hebdomadis group titres were insusceptible to challenge with hardjo, and the pathogenesis of ballum infection was characteristic of leptospiral infection in an accidental host.

The occurrence of antibodies in the urine of possums infected with balcanica is also described.