- Programme Title:Possum parasite immunology
- Programme Title:Possum parasite vectors
- Programme Title:Possum viruses and bacterial pathogens in New Zealand
- Programme Title:An analysis of samples of possums from a broad range of habitats and geographic areas in Australia to obtain data on the prevalence of pathogens and their potential as biological control agents
- Programme Title:Mammary viruses in the possum
3.2 Microbiology/Parasitology
Studies investigating possum parasites and diseases are being conducted in both New Zealand and Australia. These studies seek to identify parasites and diseases which could have an adverse effect on possums such asnematode worms and "Wobbly Possum Disease". There is no expectation that a single disease causing agent will solve the problem but it is believed that a number of different organisms could, collectively, adversely affect possums and afford some level of control - especially when added to other environmental stresses.
The surveys are also seeking candidate vector (disease transmitter) organisms which could, possibly, be genetically manipulated in order to enable them to spread disruptive genes throughout the possum population. These genes would be expressed as biochemicals which would interfere with possum reproduction or health.
3.2.1
Programme Title: Possum parasite immunologyResearch Leader: Dr D Heath Institution:AgResearch |
Summary
This programme aims to examine the host/parasite relationship at the immunological level between possums and the two possum-specific nematodes, Parastrongyloides trichosuri and Paraustrostrongylus trichosuri and determine whether there is density-dependent immunological regulation of parasite numbers in possums, and what pathological effects the specific parasites might have at the level of the gastrointestinal mucosa (lining of the digestive system). This objective is closely linked to FRST-funded work - "Potential Pathogenic Parasites of Possums".
The life-cycles of the two parasites were established in the laboratory and possums were successfully infected from larvae grown from eggs produced by the implanted parasites.
Results to date suggest that Parastrongyloides trichosuri and Paraustrostrongylus trichosuri may be good candidates for acting as a vector of DNA.
Description: Host/parasite relationship in possums
To continue the investigation of the host/parasite relationship between possums and the two possum-specific nematodes, Parastrongyloides trichosuri and Paraustrostrongylus trichosuri, at the gastrointestinal level, using native possums obtained from the South Island by:
- determining the immunohistopathology of pure infections with each of the species;
- using chemically-truncated infections to attempt to induce immunity to Paraustrostrongylus;
- monitoring systematic antibody production;
- recording faecal egg-production dynamics from single infections; and
- determining the immune status to the two parasites in possums of various ages from an endemic area, using serological studies and experimental infections.
Approach & Outcomes
This programme sought to understand the host-parasite relationship at the immunological level between the possum and the two possum-specific nematodes present in New Zealand and to determine whether there is density-dependent immunological regulation of parasite numbers in possums, and what pathological effects the specific parasites might have at the level of the gastrointestinal mucosa.
The free-living styles of the two parasites (Parastrongyloides trichosuri and Paraustrostrongylus trichosuri) were established in the laboratory and possums were successfully infected from larvae grown from eggs produced by the implanted parasites.
Parastrongyloides trichosuri and Paraustrostrongylus trichosuri have been found to infect most possums in the hyperendemic regions. The parasites can be effectively transmitted and parts of their life-cycles lend themselves to experimental transfection with DNA.
The immunity-systemic antibody status of possums with respect to Paraustrostrongylus has yet to be determined. However, surgically-implanted worms produced similar numbers of eggs each week for 13 weeks, indicating no evidence of loss of worms over that period. Four possums that received oral infections with 4,500L3 of Paraustrostrongylus all produced approximately 10,000 e.p.g. for 7 weeks, at which time they were euthanased because of inappetance and bloody diarrhea. All four possums were found to be parasitized by several thousand worms.
A specific antigen of P. trichosuri was identified. It appeared to be secreted by infective larvae where they penetrated the skin of the possum, and a strong systemic antibody response was detected after a second and third exposure to infection. The presence of this specific antibody was used to determine the epidemiology of Parastrongyloides in three different areas.
The epidemiology of P. trichosuri, which is the most common in possums, was elucidated by the antibody test. It appears that a density-dependent immune constraint can be activated in areas where the parasite is hyperendemic.
Publications
Heath, D.D.; Flanagan, J.U.; Jowett, G.; Stankiewicz, M. and Cowan, P.E. (1996): Comparison of necropsy and seology for epidemiological studies of Parastrongyloides trichosuri in the brushtail possum, Trichosurus vulpecula in New Zealand. International Journal for Parasitology, (in prep).
Conferences
Flanagan, J.U.; Heath, D.D.; Stankiewicz, M. and Cowan, P. (1995): The brushtail possum, Trichosurus vulpecula: specific detection of past and present infections with Parastrongyloides trichosuri using immunoblotting. Proceedings of the joint meeting of the Australian and New Zealand Societies of Parasitology, Adelaide, A71.
3.2.2
Programme Title: Possum parasite vectorsResearch Leader: Mr S Bisset Institution:AgResearch |
Summary
This programme aims to develop methods to incorporate foreign DNA into a selected nematode parasite of possums, which could interfere with the fecundity or life-span of parasitized possums. A free-living nematode, Caenorhabditis elegans was used as a model.
The techniques for transforming C. elegans were established using the rol 6 marker gene. Studies on the effect of culture medium and population density on the maintenance of the free-living life cycle of Parastrongyloides trichosuri under laboratory conditions were largely completed. To date, attempts to achieve satisfactory transformation of Parastrongyloides trichosuri with rol-6 have been unsuccessful.
Description: Incorporation of foreign DNA into possum parasites
To develop methods to incorporate foreign DNA into the genome of selected nematode parasites of possums, using C. elegans as a model, in order that they may be used as vectors to express proteins which could interfere with the fecundity (or life span) of possum populations, by:
- adapting microinjection techniques established for C. elegans for use with Parastrongyloides trichosuri;
- assessing whether marker genes commonly used to verify successful transformation of C. elegans (e.g. rol 6) are expressed in progeny of microinjected free living Parastrongyloides trichosuri; C. elegans as a control;
- determining patterns of inheritance of extrachromosomal arrays of injected DNA in free living stages of Parastrongyloides trichosuri in relation to those expected in C. elegans;
- identifying factors involved in triggering Parastrongyloides trichosuri to switch from the free living to parasitic life cycle under laboratory conditions; and
- determining whether Parastrongyloides trichosuri transformed with reporter genes as free-living individuals will continue to express inserted genes in their parasitic phase.
Approach & Outcomes
At present, neither of the nematode species, Parastrongyloides trichosuri and Paraustrostrongylus trichosuri appears to be exerting a major influence on the mortality or fecundity of possums in the wild. However, it may be possible to genetically modify nematodes so that they express hormones or other proteins which could interfere with their host’s fecundity or longevity. Nematodes may have some advantages over viral or bacterial vectors, from the point of view of biosecurity in relation to genetically modified organisms.
The successful transformation of C. elegans with the marker gene rol 6 can now be achieved routinely. The procedure used to genetically transform C. elegans involves microinjection of small amounts of cloned DNA containing genes of interest (in appropriate plasmid or cosmid vectors) into the ovaries of recipient worms. Generally the mixture injected also contains DNA which codes for a dominant marker gene such as the mutant collagen gene rol 6 (su 1006). This allows successfully transformed individuals to be readily identified. Animals carrying the rol 6 gene for example (usually as an extra-chromosomal array) exhibit a helically twisted cuticle and body and this results in them showing a characteristic rolling movement as they move around on the culture medium.
The effects of the type of culture medium, used to support the growth of the free living cycle on agar plates, and population density on triggering the worms, Parastrongyloides trichosuri, to switch from free-living rhabdoid cycle to infective filariform larvae were investigated. Free-living cultures can now be maintained in the laboratory for an indefinite period or alternatively be triggered to develop infective larvae for reinfection of a possum host.
Work was undertaken to identify the development of the reproductive system in Parastrongyloides trichosuri in order to identify most appropriate developmental stage and injection sites. As a result of some initial attempts to test the micro-injection procedures developed for C. elegans on Parastrongyloides trichosuri, some modifications were necessary.
To date, attempts to achieve satisfactory transformation of Parastrongyloides trichosuri with rol-6 have been unsuccessful. Many different approaches have been taken including changing injection concentrations and/or injecting the nuclei of unfertilised eggs further down the oviduct.
Publications
A paper entitled "Maintenance of the free-living life cycle of the possum parasite Parastrongyloides trichosuri in the laboratory" has been prepared for presentation at the meeting of the New Zealand Society for Parasitology.
3.2.3
Programme Title: Possum viruses and bacterial pathogens in New ZealandResearch Leader: Dr D Heath Institution:AgResearch (collaboration with Central Animal Health Laboratory) |
Summary
This programme aims to find pathogens that may be useful in biological control of possums, through completing a pathogen survey and the investigation of previously identified pathogens that have biocontrol potential. The emergence of "Wobbly Possum Disease" in April of 1995 led to a shift in emphasis towards investigation of this disease and its potential for biocontrol.
The serological (blood-related) and pathological (disease-related) effects of "Wobbly Possum Syndrome" (WPS) has been studied. A range of tissues including brain, spleen, liver, blood and urine from the acute stage disease and homogenised possum itch mites was shown to contain the WPS virus. The disease was found to be experimentally transmitted by a variety of routes (gastric, tracheal, intra-dermal and intra-peritoneal).
A survey of possums from ten sites throughout New Zealand and the culture of their tissues was completed. Although no viruses were isolated, several virus families were detected by electron microscopy and two new possum cell lines were developed which may prove useful in future studies.
A papillomavirus found in 1994 has been sequenced. It is significantly different from human papillomaviruses and known animal papillomaviruses.
Description: Viruses and bacterial pathogens of possums in New Zealand
To continue attempts to find specific viruses and bacterial pathogens of possums in New Zealand by:
- examining live possum tissues from the Auckland region and from the Shannon region;
- screening the 1995/96 possum serum bank for antibodies to Chlamydia spp. and Leptospira balcanica;
- completing in vitro studies on all possum tissues collected so far, to attempt to isolate viruses in cell culture;
- extending the lymphocyte/monocyte studies to determine if there is a possum retrovirus in the multinucleate cell cultures;
- attempting further transmission studies with the identified papilloma virus, and completing the molecular characterisation;
- establishing the nature of the agent involved in the neurological disease known as "Wobbly Possum Syndrome": (in collaboration with Central Animal Health Laboratory).
- applying the molecular virus detection methods developed in the past year to collected tissues, and analysing in detail any tissue samples positive for viral DNA; and
- investigating the methodology for identifying and recording natural outbreaks of disease in possums and for isolating causative agents.
Approach & Outcomes
This objective aims to find pathogens that may be useful in biological control of possums, through completing a pathogen survey and the investigation of previously identified pathogens that have biocontrol potential. The emergence of "Wobbly Possum Disease" in April of 1995 developments, led to a shift in emphasis towards investigation of this disease and its potential for biocontrol.
AgResearch and the MAF Quality Management Central Animal Health Laboratory researched Wobbly Possum Disease (WPD). Following an outbreak of WPD in a colony of brushtail possums, experimental passage of thedisease was established in captive possums using intra-peritoneal inoculation with tissue homogenates. The disease was characterised by docility, uncoordination, loss of balance and wasting. Fifteen of sixteen infected animals required euthanasia due to the severity of symptoms.
A range of tissues including brain, spleen, liver, blood and urine from the acute stage disease and homogenised possum itch mites was shown to contain the WPD virus. The disease can be experimentally transmitted by a variety of routes (gastric, tracheal, intra-dermal and intra-peritoneal).
Due to the lack of transmission to non-contact control possums and the lack of clinical or histological signs for respiratory tract involvement, the respiratory route would seem an unlikely natural route of transmission. From findings, possible natural routes of transmission include the urine/oral route, urine/sexual transmission, faecal/oral route, mite/oral (allogrooming), mite/intra-dermal and blood transfer (fighting, arthropod vector).
The planned survey of possums from ten sites throughout New Zealand and the culture of their tissues has been completed. Although no viruses were isolated, several virus families were detected by electron microscopy and two new possum cell lines were developed which may prove useful in future studies.
The prevalence of Leptospira balcanica was found to be high in all mainland North Island areas except Northland. No antibodies were detected in South Island areas or on Kawau, Stewart or the Chatham Islands. The findings suggest the possums in these areas may be free of L. balcanica infection.
The isolation of retroviruses was attempted by the establishment of peripheral blood mononuclear cell (PBMC) cultures. Large multi-nucleated cells were observed in cultures from 4 of 13 possums.
In 1994 a papillomavirus was found on a single possum. To establish that the papillomavirus was a new type and specific to possums the amplimer from the PCR reaction was sequenced. The sequence was compared to other known sequences (Table 1). The PPV is a new papillomavirus specific to the possum.
Table 1. Possum Papillomavirus: Homology with Other Papillomaviruses
| PAPILLOMAVIRUS (PV) | HOMOLOGY WITH POSSUM PV |
| Human PV supergroup B (epidermodysplasia verruciformis) |
61 - 69 % |
| Pygmy chimpanzee PV | 59% |
| Bovine PV type 4 | 58% |
| Human PV supergroup A (genital HPVs) | 55% |
| Bovine PV type 5 | 52% |
Publications
Horner, G. (1996): Serological survey for antibody of Leptospira balcanica. New Zealand Veterinary Journal, 44, p.162.
Rice, M. and Willis, C.R. (1996): Virus and virus-like particles observed in the intestinal contents of the possum, Trichosurus vulpecula. Archives of Virology, 141, pp.945-950.
Conferences
Perrott, M.; Meers, J.; Willis. C.; Lugton, I.; Meekin, G.; Farmer, S. and Okeefe, J. (1995): Possum papillomavirus. Australian Society for Microbiology Annual Conference, Canberra, September.
Perrott, M. (1996): Transmission studies and field cases of wobbly possum disease. New Zealand and Australian Conference of Comparative Veterinary Pathology, Christchurch, June.
3.2.4
Summary
This was the final year for research on the prevalence and abundance of all helminth, protozoan and arthropod parasites encountered in Australian possums. Particular attention was paid to any helminth, protozoan or arthropod species suspected of having significant effects on possums and which therefore might be useful as an agent for biological control. In addition, blood, tissue and organ specimens were collected from possums throughout Australia for examination.
Collections of Trichosurus vulpecula (Australian Possum) were completed for Victoria, Tasmania, South Australia, and Queensland. In addition, collection of Trichosurus caninus (Brush Possum) from New South Wales, Victoria and Queensland was achieved.
A detailed list of parasites found in the specimens so far examined has been compiled. The most promising candidate helminth for biocontrol in New Zealand still remains as Adelonema trichosuri, an oxyurid parasite apparently absent from NZ possums. This organism cannot be regarded as pathogenic in Australian possums but it would at least add to the parasitic burden of New Zealand possums.
Serum samples were analysed for evidence of antibodies to a number of viral, bacterial and protozoan infections. The prevalence of viruses found differed with location.
| Objective 1: | Collection of specimens from possums in Australia | |
| Research Leader: | Dr B Coman | |
| Objective 2: | Examination for helminth & arthropod parasites of possums in Australia | |
| Research Leader: | Dr D Spratt (Collaborator, Dr I Beveridge) | |
| Objective 3: | Examination for microbial disease organisms of possums in Australia | |
| Research Leader: | Dr P Presidente (Co-investigators - Obendorf, Speare, Gibson) | |
Description - Objectives 1 & 2 : Examination for helminth & arthropod parasites
To obtain a further 230 possums throughout Australia from a wide range of habitats and geographical by:
• analysing blood, tissue or organ samples for serological testing and parasitological examination or virus isolation; and
• recording data on the prevalence and abundance of all helminth, protozoan and arthropod parasites encountered in Australian possums.
Approach and Outcomes
Collections of T. vulpecula have been completed for Victoria, Tasmania, South Australia, and Queensland. In addition, the full collection of T. caninus (New South Wales-35, Victoria-45 and Queensland-26) has been achieved. Also, six specimens of T. arnhemensis have been received from the Northern Territory. In addition, researchers have been given sera for 37 possums from Western Australia.
A detailed list of parasites found in the specimens so far examined has been compiled. To date, 169 specimens of T. vulpecula have been processed yielding 9 identified helminth species and 19 identified ectoparasite species. The sample of 130 T. caninus has been examined and 8 helminths and 15 ectoparasites have been recorded.
At this stage, the most promising candidate helminth for biocontrol in New Zealand still remains as A. trichosuri, an oxyurid parasite apparently absent from New Zealand possums. This organism cannot be regarded as pathogenic in Australian possums but it would at least add to the parasitic burden of New Zealand possums. Other helminths recorded in Australia (not this survey) but not recorded in New Zealand include a number of marsupial strongylids in Dromaeostrongylidae, metastrongyles in Angiostrongylidae and filarioids in the Onchocercidae.
The poor showing of ectoparasites in the survey of T. vulpecula (19 out of 36 known) may reflect the fact that material for inspection comes from deep-frozen specimens.
Description - Objective 3: Examination for microbial disease organisms
To collect from Australian possums whole blood samples from the heart of anaesthetized or recently euthanised animals into an EDTA tube as well as into plain tubes for serum recovery and clotted blood and by:
- using EDTA samples for conducting routine haematological examinations and for checking for haematozoon parasites and microfilariae;
- using serum samples to conduct routine serological tests;
- subjecting specimens to a haemogram to determine any evidence of disease;
- culturing (at the time of necropsy) any gross lesions caused by bacterial or viral agents and fix the associated lesions in formalin;
- conducting histological examination of selected fixed tissue (liver, lungs, heart, kidney, spleen, brain, tongue, muscle); and
- isolating (on a limited number of selected possums) microbial agents from clotted blood and taking frozen tissue samples.
Approach and Outcomes
Serum samples from 53 Brushtail Possums collected in Queensland and 37 animals from two sites in Western Australia were analysed for antibodies to select viral, bacterial and protozoan infections. Antibodies to Ross River Virus, Murray Valley Encephalitis Virus, Barmah Forest Virus, Kunjin Virus, Leptospira interrogans serovars hardjo, Leptospira interrogans serovars pomona, Toxoplasma gondii and Sindbis Virus were tested for by haemagglutination inhibition assay. Results are listed in Table 2.
Table 2. The prevalence of viral antibodies in possums in Queensland, Western Australia and Northern Territory
| Virus | Queensland | Western Australia |
Northern Territory |
| Ross River Virus | 6 | ANC | 2 |
| Murray Valley Encephalitis | 1 | ND | ND |
| Barmah Forest Virus | ND | ANC | ND |
| Kunjin Virus | ND | ND | 2 |
| Leptospira interrogans serovar hardjo | 8 | ND | ND |
| Leptospira interrogans serovar pomona | ND | ND | ND |
| Sindbis Virus | ND | ANC | ND |
| toxoplasma gondii | ND | ND | ND |
KEY: ND = No detection ANC= Analysis not completed
A low but consistent prevalence of an "Eimeria sp" in possums’ faecal specimens was found from most collection sites. The oocysts recovered so far are morphologically similar and probably represent a single species. All oocysts have been detected in low numbers except for a single animal from Victoria.
The only other protozoan organism recovered from faeces is an Entamoeba. Cysts of this organism were recovered from five animals originating in the Townsville area. There appears to be two morphologically distinct types based on size.
Conferences
Obendorf, D. (1996): Disease causing agents in Australian Trichosurid possums. Wildlife Diseases Conference, New Zealand, June.
3.2.5
Programme Title: Mammary viruses in the possumPhD Fellowship:Gregory Bailie Institution:University of Waikato/AgResearch |
Summary
This PhD fellowship aims to investigate mamary specific (retro) viruses and assess their potential as genetically engineered vectors for the biological control of possums.
Product enhanced reverse transcriptase (PERT) assays on blood, liver, and mammary samples from possums have suggested that retroviruses are present in the blood (and possibly other tissues) of possums.
RT-PCR from possum liver and mammary RNA suggested that some retroviral sequences may be expressed in these tissues, although this has not been confirmed by sequencing.
Description
To investigate mammary specific (retro) viruses and assess their ultimate potential as genetically engineered vectors for the biological control of possums by:
- detecting virus particles using molecular biological methods;
- isolating virus genomes;
- sequencing possum specific viral genomes; and
- modifying such genomes with the aim of producing viral vectors specific to the possum.
Approach & Outcomes
A product enhanced reverse transcriptase (PERT) assays on blood, liver, and mammary samples from possums have suggested that retroviruses are present in the blood (and possibly other tissues) of possums.
PCR using degenerate primers derived from regions of the pol gene conserved among all retroviruses indicated the presence of pol sequences in DNA isolated from blood, liver, and mammary tissues. Sequencing of PCR products generated from mammary DNA confirmed their retroviral origin, and revealed high homologies to mouse mammary tumur virus (MMTV) and human endogenous retroviruses (HERVs), as well as low homologies to other human and non-human retroviruses.
RT-PCR from possum liver and mammary RNA suggested that some retroviral sequences may be expressed in these tissues, although this has not been confirmed by sequencing.
Contact for Enquiries
Farm Monitoring Programme Manager
Monitoring and Evaluation
MAF Policy
PO Box 2526
Wellington
NEW ZEALAND
Phone: +64 4 894 0623
Fax: +64 4 894 0741
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