Purification of secretory immunoglobulin A from milk of the brushtail possum (Trichosurus vulpecula)

AIM: To identify and purify secretory immunoglobulin A (sIgA), a key effecter molecule in mucosal immune responses, from milk of the brushtail possum (Trichosurus vulpecula). METHODS: Milk samples were collected from female possums with pouch young, and clarified by centrifugation and precipitation methods. The clarified fraction was purified by gel filtration and affinity chromatography to yield sIgA. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) and immunoblotting techniques were used to assess the purity of the final product, and to identify the heavy (H) chain, light (L) chain and secretory component (SC) of possum sIgA. RESULTS: Immunoblotting, using antibodies raised against cloned possum sIgA SC and H-chain, and a synthetic peptide fragment of the H-chain, confirmed the identity of the purified protein. The N-terminal amino acid sequence of purified possum sIgA showed strong homology to reported sequences of H-chain variable regions of marsupial immunoglobulins. CONCLUSIONS: Milk was shown to be a convenient source of mucosal secretion containing sIgA, and a process involving 2 precipitation and 2 chromatography steps produced purified sIgA. This IgA preparation will prove useful for the generation of sIgA-specific immunological reagents for measurement of immune responses in the development of mucosal-based vaccines for biological control of possums.     

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.     

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.     

Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand

AIMS: To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand.

METHODS: Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000–2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450?nm (OD₄₅₀) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus.

RESULTS: Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD₄₅₀≥0.41 for samples positive, and OD₄₅₀<0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196–0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001).

CONCLUSION: The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to protect possums from disease following experimental infection, as one third of possums from the previous challenge study showed evidence of pre-existing antibody at the time of challenge. These results provide further support for existence of different pathotypes of WPD virus, but the exact determinants of protection against WPD and epidemiology of infection in various regions of New Zealand remain to be established.

CLINICAL RELEVANCE: Availability of the indirect ELISA for detection of WPD virus antibody will facilitate prospective epidemiological investigation of WPD virus circulation in wild possum populations in New Zealand.     

Oral vaccination of brushtail possums (Trichosurus vulpecula) with BCG: immune responses,persistence of BCG in lymphoid organs and excretion in faeces

AIMS: To determine immune responses, and the localisation and persistence of Mycobacterium bovis bacille Calmette-Guérin (BCG) in gut-associated lymphoid tissues (GALT) and other organs in possums vaccinated orally with lipid-formulated BCG vaccine. To determine the duration of excretion and longevity of survival of BCG in the faeces of vaccinated animals.

METHODS: Possums (n=28) were vaccinated with lipid-formulated BCG (1 x 10 ⁸ colony forming units (cfu) of formulated BCG) by the oral route. Control possums (n=17) were fed oral bait pellets containing formulation medium only. Possums were sacrificed at 3 days and at 1, 3, 6 and 8 weeks after vaccination or ingestion of bait. Proliferation responses to bovine purified protein derivative (PPD) were measured in lymphocytes from blood and mesenteric lymph nodes (MLN) and samples of lung, spleen, liver, MLN and Peyer's patches (PP) were cultured for the presence of BCG. The number of BCG organisms excreted in faeces and the duration of excretion were determined in eight vaccinated possums and eight control possums over a 3-week period. In a separate experiment, a further six possums were vaccinated with oral BCG vaccine (5–10 x 10 ⁸ cfu BCG/possum) and their faeces collected over 48–72 h, for culture of BCG. The longevity of survival of BCG in these faeces was determined by storing faecal samples (n=12) under three different conditions: in an incubator (22.5°C), and conditions which simulated the forest floor and open pasture. A proportion (1–2 g) of these faecal samples was collected after storage for 1, 3, 5, 8 or 20 weeks, and cultured for BCG.

RESULTS: Possums vaccinated orally with BCG vaccine showed strong proliferation responses to bovine PPD in peripheral blood lymphocytes at 6–8 weeks post-vaccination (p.v.). Positive lymphocyte proliferation assay (LPA) responses to bovine PPD were first evident in MLN at 3 weeks p.v. BCG was cultured from MLN and PP in a proportion of animals at 3–8 weeks p.v. BCG was not cultured from sections of spleen, lung or liver at any time p.v. BCG was recovered in low to moderate numbers from the faeces of vaccinated possums for up to 7 days, and maximal numbers were cultured in faeces collected 48–72 h p.v. After storage for 1 week, BCG was cultured from all faecal samples placed in the incubator and from a proportion of faeces exposed to conditions similar to those on the forest floor and pasture. With the exception of one faecal sample stored under forest floor conditions which was culture-positive for BCG at 3 and 5 weeks, BCG was not cultured from any other faecal sample stored for more than 1 week.

CONCLUSIONS: Ingestion of oral BCG vaccine by possums was associated with the development of strong cell-mediated immunity in both blood and MLN. Following oral vaccination with BCG, the organisms were localised and persisted in GALT but did not spread to the spleen, liver or lungs. BCG was shed in low to moderate numbers in the faeces for up to 7 days p.v. The viability of BCG excreted in faeces decreased rapidly, particularly when faeces were exposed to an open pasture environment. Oral vaccination of possums with formulated BCG is unlikely to result in undue contamination of the environment with BCG.     

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.     

Characterisation of the kappa light chain of the brushtail possum (Trichosurus vulpecula)

Two full length cDNA sequences encoding the kappa light chain of the Australian marsupial, Trichosurus vulpecula, the brushtail possum, were isolated from a mesenteric lymph node cDNA library. The constant regions (Ckappa) of the two light chains were identical, but the variable (Vkappa) and joining (Jkappa) regions were different. At the amino acid level, possum Ckappa was most similar to Ckappa of an American marsupial, Monodelphis domestica (75%), with similarity to eutherian Ckappa ranging from 47 to 63%. The availability of molecular data will enable the development of immunological reagents for studying immune responses and disease in marsupials, thereby aiding conservation strategies and veterinary medicine.     

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.     

Characterisation of antisera to recombinant IgA of the common brushtail possum (Trichosurus vulpecula)

The purpose of this experiment was to estimate the influence of nonspecific immunostimulation of pregnant sows on the immunological value of colostrum. The studies were done in the fall-winter season on 20 pregnant sows (Polish Landrace) divided into four groups, five animals each. The sows were clinically healthy and unvaccinated. Group I received isoprinosine, group II (TFX), group III (HMB), and group IV served a control. The immunostimulants were applicated 4-6 weeks before the expected date of delivery. The following parameters were determined in colostrum taken from sows after completed delivery: specific gravidity; total proteins, lysozyme activity, IgG level. It was found that the nonspecific immunostimulation of pregnant sows increased in colostrum the level of IgG, total protein content and lysozyme activity.     

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.     

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.