Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection. METHODS: Possums were inoculated with 5 x 106 colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7),orally (n=10), via the endobronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 106 cfu and half of which received 5 x 107 cfu of BCG). The possums were humanely killed between 1-4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically. RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endobronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i. CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intradermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection.

METHODS: Possums were inoculated with 5 x 10⁶ colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7), orally (n=10), via the endo-bronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 10⁶ cfu and half of which received 5 x 10⁷ cfu of BCG). The possums were humanely killed between 1–4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically.

RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endo-bronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i.

CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intra-dermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

Influence of contact heterogeneity on TB reproduction ratio R0 in a free-living brushtail possum Trichosurus vulpecula population

Social network analyses were used to investigate contact patterns in a free-living possum Trichosurus vulpecula population and to estimate the influence of contact on R(0) for bovine tuberculosis (TB). Using data collected during a five-year capture-mark-recapture study of a free-living possum population, observed estimates of R(0) were computed and compared with R(0) computed from random networks of similar size that approximated a random mixing process. All networks displayed a heterogeneous pattern of contact with the average number of contacts per possum ranging from 20 to 26 per year. The networks consistently showed small-world and single-scale features. The mean estimates of R(0) for TB using the observed contact networks were 1.78, 1.53, 1.53, 1.51, and 1.52 times greater than the corresponding random networks (P <0.05). We estimate that TB would spread if an average of between 1.94 and 1.97 infective contacts occurred per year per infected possum, which is approximately half of that expected from a random network. These results have implications for the management of TB in New Zealand where the possum is the principal wildlife reservoir host of Mycobacterium bovis, the causal agent of bovine TB. This study argues the relevance of refining epidemiological models used to inform disease management policy to account for contact heterogeneity.     

Lipopigments in the brushtail possum, Trichosurus vulpecula

Two lipopigments have been found in the hepatic and lymphoid tissues of the brushtail possum, Trichosurus vulpecula. One pigment was present in hepatocytes of five out of 15 (33%) possums from a site in the Wairarapa region in the North Island of New Zealand, but was absent from the livers of 34 possums from a site in the Manawatu. The pigment is possibly of dietary origin. An extra-hepatic lipopigment was found within macrophages in the lymph nodes of three out of 12 (25%) possums from the Wairarapa site and of five out of 14 (36%) possums from the Manawatu site. It was also found in periportal macrophages in the livers of two of the possums with hepatocellular pigmentation from the Wairarapa site. This latter pigment is ceroid, and probably reflects the degeneration of lipid.     

Adiaspiromycosis in suspected cases of pulmonary tuberculosis in the common brushtail possum Trichosurus vulpecula)

Pulmonary adiaspiromycosis was diagnosed in 15 of 17 common brushtail possums (Trichosurus vulpecula) examined for bovine tuberculosis. Emmonsia crescens was isolated from two of the affected animals. This is the first reported isolation of this dimorphic fungus in New Zealand. The infections varied from light to heavy with sparsely distributed, focal, 1–2 mm diameter, grey-white spherical granulomas in the lightly infected cases to innumerable foci throughout the lungs in the heavily infected cases. Histologically, the lesions were characterised by a central fungal adiaspore consistent in morphology with E. crescens located in alveolar spaces or bronchioles. These were surrounded by a cuff of granulomatous inflammation which varied in density and extent in proportion to the degree of degeneration exhibited by the organism.

The lesions in most cases of adiaspiromycosis in the possum are sufficiently distinctive to allow their differentiation from tuberculosis by gross examination, but where any doubt exists over the possibility of dual infection by Emmonsia and Mycobacterium species differentiation should be made on the basis of a histological examination of the tissue and culture for mycobacterial organisms.     

Adiaspiromycosis in suspected cases of pulmonary tuberculosis in the common brushtail possum (Trichosurus vulpecula)

Pulmonary adiaspiromycosis was diagnosed in 15 of 17 common brushtail possums (Trichosurus vulpecula) examined for bovine tuberculosis. Emmonsia crescens was isolated from two of the affected animals. This is the first reported isolation of this dimorphic fungus in New Zealand. The infections varied from light to heavy with sparsely distributed, focal, 1-2 mm diameter, grey-white spherical granulomas in the lightly infected cases to innumerable foci throughout the lungs in the heavily infected cases. Histologically, the lesions were characterised by a central fungal adiaspore consistent in morphology with E. crescens located in alveolar spaces or bronchioles. These were surrounded by a cuff of granulomatous inflammation which varied in density and extent in proportion to the degree of degeneration exhibited by the organism. The lesions in most cases of adiaspiromycosis in the possum are sufficiently distinctive to allow their differentiation from tuberculosis by gross examination, but where any doubt exists over the possibility of dual infection by Emmonsia and Mycobacterium species differentiation should be made on the basis of a histological examination of the tissue and culture for mycobacterial organisms.     

Anthelmintics for the control of nematode infections in the brushtail possum (Trichosurus vulpecula)

AIMS: To determine the efficacy of eprinomectin, doramectin and a combination of albendazole and levamisole in suppressing or eliminating nematode infections or faecal egg counts (FEC) in possums naturally or experimentally infected with Parastrongyloides trichosuri, Paraustrostrongylus trichosuri and Trichostrongylus colubriformis. METHODS: To establish an effective dose of eprinomectin, groups of naturally infected possums were treated with 0, 0.5, 2.5, 5.0 or 7.5 mg/kg liveweight (LW) eprinomectin pour-on (n=6 possums/group) and changes in FEC and nematode worm counts at necropsy determined, 18 days later. Efficacy of the 7.5 mg/kg dose was re-examined in a second group of naturally infected possums (n=12) by monitoring FEC weekly for 28 days post-treatment. Persistence of the anthelmintic effect of doramectin injection was tested using nematode-free possums treated with 0, 0.2, 0.4, 0.6 or 0.8 mg/kg LW (n=3 possums/ group), which were experimentally infected 14 days later with T. colubriformis, Paraustrostrongylus trichosuri and Parastrongyloides trichosuri infective larvae. Response to treatment was assessed by FEC and nematode worm counts at necropsy, 42 days posttreatment. Efficacy of a 1.0 mg/kg dose of doramectin was subsequently examined using naturally infected possums (n=11) by monitoring FEC weekly for 28 days post-treatment. To determine the efficacy of a levamisole-albendazole combination drench, possums with naturally acquired nematode infections (n=6) were treated orally with 37.5 mg/kg LW levamisole plus 23.75 mg/kg LW albendazole on 2 occasions, 7 days apart, and response to treatment was assessed by monitoring FEC for 57 days. RESULTS: Eprinomectin 7.5 mg/kg LW reduced Paraustrostrongylus trichosuri worm counts by 98 % (p<0.05). Doramectin 0.6 mg/kg LW reduced Parastrongyloides trichosuri and Trichostrongylus spp worm counts by 99% (p<0.05) and 0.8 mg/kg LW reduced Paraustrostrongylus trichosuri by 100% (p<0.05), in possums challenged with larvae 14 days after treatment. Treating possums with a levamisole-albendazole combination orally, twice, 7-days apart, reduced FEC by 99%. CONCLUSIONS: The doses of anthelmintics required to effectively control nematodes in possums were higher than those recommended for animals for which they are currently registered. Possums tolerate the high dose rates of anthelmintics used in this study without apparent adverse effects.     

Polymeric nanoparticles as an oral delivery system for biocontrol agents for the brushtail possum (Trichosurus vulpecula)

Abstract

AIM: To investigate polymeric nanoparticles as an oral delivery system for protein biocontrol agents for control of the brushtail possum, Trichosurus vulpecula.

METHODS: Insulin-loaded poly(ethyl 2-cyanoacrylate) (PECA) nanoparticles were prepared using interfacial polymerisation, and characterised for size, zeta potential, and efficiency of encapsulation of insulin. In-vitro release of insulin-loaded PECA nanoparticles was quantified using reverse-phase highpressure liquid chromatography (HPLC). The in-vivo pharmacokinetics of insulin in PECA nanoparticles was investigated following I/V administration, and when injected directly into the caecum alone or in conjunction with the permeation enhancer EDTA. Blood samples were collected at intervals from ?5 to 180 minutes, and the concentration of insulin in plasma was quantified using a radioimmunoassay (RIA) validated for possum plasma.

RESULTS: Poly(ethyl 2-cyanoacrylate) nanoparticles were produced with a uniform particle size of 200–300 nm, and the mean entrapment of insulin was 78%. In-vitro release of insulin from the PECA nanoparticles was controlled, although incomplete, and approximately 30% of the insulin remained entrapped. The bioavailability of insulin when administered in a PECA nanoparticulate formulation injected directly into the caecum was <1%, and was not increased by addition of the permeation enhancer.

CONCLUSIONS: The nanoparticulate formulations investigated as part of this study resulted in low bioavailability of the peptide insulin in the brushtail possum.     

Temporal patterns in bovine tuberculosis in a brushtail possum population contiguous with infected cattle in the Ahaura Valley, Westland

AIMS: To determine temporal and spatial patterns of bovine tuberculosis (Tb) in a population of brushtail possums (Trichosurus vulpecula) 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 Tb 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 that Tb can persist for several years in possum populations existing well below the 5% trap catch targeted by regional councils for Tb possum control.     

A neurological syndrome in a free-living population of possums (Trichosurus vulpecula)

AIMS: To determine the clinical and pathological features of a neurological disease syndrome in a free-living possum population in New Zealand and to compare this syndrome with wobbly possum disease. METHODS: An outbreak of a neurological disease in possums in the Rotorua district was investigated in 1994. A variety of tissues was collected and investigated microbiologically and histopathologically. Tissues stored from clinically affected possums were homogenised, clarified and inoculated into healthy possums by the intra-peritoneal route. The clinical signs and histopathological lesions in naturally-infected and in experimentally-inoculated possums were assessed and compared with those of possums affected with wobbly possum disease. RESULTS: Histopathological investigation of three of the naturally-affected possums revealed non-suppurative encephalitis with perivascular cuffing, diffuse non-suppurative meningitis and focal non-suppurative myocarditis. These lesions were suggestive of a viral infection. No pathogenic bacteria were recovered and no viruses were isolated in tissue culture. A neurological disease, indistinguishable from wobbly possum disease, was reproduced in five out of the eight experimentally inoculated possums. In two experimental cases the clinical signs were very mild and, in most cases of the natural and experimental disease, histopathological lesions in the central nervous system were mild in comparison with wobbly possum disease. Possums which did not develop clinical signs of neurological disease or have lesions in the central nervous system did have infiltrations of mononuclear inflammatory cells in the liver and kidney. CONCLUSIONS: This neurological disease, reported for the first time in a free-living population, closely resembles and may be the same as wobbly possum disease. The milder nature of this disease could suggest there may be more than one strain of the aetiological agent.     

Identification of a novel nidovirus associated with a neurological disease of the Australian brushtail possum (Trichosurus vulpecula)

A novel, fatal neurological disease of the Australian brushtail possum (Trichosurus vulpecula) was first identified in 1995 in a research facility and subsequently in free-living possums in New Zealand and termed wobbly possum disease (WPD). The results of previous transmission studies suggested that the aetiological agent of WPD is most likely a virus. However, the identity of the presumed viral agent had not been elucidated. In the current report, we describe identification of a novel virus from tissues of WPD-affected possums using a combination of next generation sequencing and traditional molecular methods. The proportion of possums positive for the novel virus by PCR was significantly higher (p<0.0001) among animals with WPD than clinically healthy possums, strongly suggesting an aetiological involvement of the virus in WPD. Analysis of the partial genomic sequence of the putative WPD virus indicated that it is a novel nidovirus, most closely related to the current members of the family Arteriviridae.     

Resolution of Mycobacterium bovis infection in wild brushtail possums (Trichosurus vulpecula)

The brushtail possum (Trichosurus vulpecula) is the principal wildlife reservoir of Mycobacterium bovis in New Zealand (Morris and Pfeiffer 1995). Tuberculosis in possums is a progressive, fatal infection. The complete resolution of infection in possums has not been reported. The purpose of this communication is to present two cases of complete and spontaneous resolution of tuberculosis in wild, naturally infected possums. During an 11-year (1989-2000) longitudinal study of a possum population at Castlepoint in the Wairarapa, 90 bacteriologically-confirmed cases of M. bovis infection were observed, and 88 of the tuberculous possums were either found dead or were presumed to have died when they ceased to be trapped. As the home range of adult possums is stable (Efford et al 2000) it was assumed that if an adult possum could no longer be trapped, it had died. In many cases, the declining physical condition of a tuberculous possum prior to its disappearance supported the assumption. However, two cases survived and were euthanised, one 15 months and the other 30 months after they first showed clinical signs of infection. When examined post mortem, neither showed evidence of M. bovis infection. The case histories are described and the significance of the observations discussed.     

Characterisation of and immunity to the aerobic bacteria found in the pouch of the brushtail possum Trichosurus vulpecula

The bacterial composition of the brustail possum (Trichosurus vulpecula) pouch was characterized throughout the reproductive cycle using brushtails from an Australian captive breeding colony (45 swabs) and a wild population in New Zealand (26 swabs). Gram-positive coccal species predominate throughout the reproductive cycle. Enteric Gram-negative rods, particularly Escherichia coli, were most prevalent when a pouch young was present and was most likely the result of faecal contamination from the pouch young. As culturing is only able to detect a proportion of bacteria present in a particular environment, molecular 16S rDNA sequencing was carried out on DNA extracted from a pouch wash of a female carrying a pouch young to gain a more accurate assessment of the pouch microflora. This approach identified approximately five times the number of bacterial species when compared to culture results. The majority detected were Gram negative rods or most closely related to Gram-negative rods species. Brushtails are immunologically immature at birth yet survive in a pouch colonised with potentially pathogenic bacteria. A haemagglutination assay was used to determine whether antibodies to a frequently isolated bacterium (Klebsiella pneumoniae) were transferred via milk from mother to pouch young. IgG antibodies were detected in maternal serum, milk and pouch young serum. In young over 70 days, antibody titres were significantly higher than those found in maternal serum, suggesting that the young is capable of producing adult type antibodies to pouch bacteria at this time.     

Natural transmission of Mycobacterium bovis infection in captive brushtail possums (Trichosurus vulpecula)

AIMS: To examine natural transmission of bovine tuberculosis (Mycobacterium bovis infection) in captive brushtail possums (Trichosurus vulpecula) and to determine if this mode of transmission could be employed to challenge possums in vaccination studies. METHODS: Three experiments were conducted. In Experiment 1, 11 pairs of possums were housed together in cages, one of the pair having been experimentally infected with M. bovis. Of the in-contact possums 5/11 had been vaccinated with bacille Calmette-Guerin (BCG). In Experiment 2, three susceptible possums were placed in a colony of 19 possums that had been experimentally infected with M. bovis. In Experiment 3, the four most socially active possums in each of two colonies (24 possums in one colony and 23 in the other) were experimentally infected with M. bovis, and 10 of the remaining possums in each colony were vaccinated with BCG. RESULTS: In Experiment 1, transmission of M. bovis infection occurred in only 1/11 pairs. In Experiment 2, none of the three in-contact possums became infected. In Experiment 3, infection was transmitted to 5/20 in-contact possums in one colony and 12/19 in-contact possums in the other. The possums that became infected by natural transmission were significantly more socially interactive than those that remained free of infection (p<0.05). CONCLUSIONS: When susceptible and infected possums were randomly mixed, the rate of transmission of M. bovis was low, but when highly sociable possums were infected the rate of transmission increased markedly. The risk of transmission was dependent on the close proximity of infected and susceptible possums and the frequency and duration of their social interactions. Natural transmission from experimentally infected to incontact possums in a colony would be a useful way of studying the pathogenesis of tuberculosis in this species, and the social behaviour of the possums studied should be taken into account. The high degree of variation in the rate of natural transmission of M. bovis infection between possums makes this mode of transmission unreliable for assessing vaccine efficacy.     

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.