Proceedings of the First Symposium on Marsupials in New Zealand
Leptospirosis in the Possum Trichosurus Vulpecula
The common brushtail possum has been established as a primary host reservoir for Leptospira balcanica (subgroup Hebdomadis) in New Zealand, and evidence of infection was present in over 80% of mature adults in this survey. The presence of balcanica has not been established in domestic stock or man in New Zealand to date, however with definitive reference laboratory typing of isolates, Hebdomadis infections diagnosed in these species may be found to be mainly due to balcanica.
Extensive wildlife surveys in East Europe and throughout the world have failed to identify balcanica in wildlife species. Therefore, the possum is the only reported wildlife reservoir of this organism in the world.
Leptospiral organisms are classified by division into serogroups and within serogroups, serovars, on an international basis. There are twelve main serogroups and contained within these groups are more than 130 serovars. In New Zealand, six leptospiral serovars have been isolated, viz.: ballum, pomona, copenhageni, tarassovi, hardjo and balcanica (Table 1). Of these, hardjo and balcanica, which are antigenically very similar, belong to the Hebdomadis serogroup.
Leptospiral infections are very common in domestic stock in New Zealand, with several serovars being represented. Hebdomadis serogroup infections are most relevant to this discussion and have been demonstrated by serology in cattle, sheep and horses. Serovar hardjo has been isolated from cattle (Lake 1973), and it has been assumed that this serovar has been the most prevalent of the Hebdomadis organisms infecting domestic stock.
The per capita prevalence of leptospirosis in humans in New Zealand is amongst the highest in the world (20 cases: 100,000 c.f. U.K. 0.1: 100,000 and U.S.A. 0.02: 100,000) and Hebdomadis serogroup infections are the most common. Serovar hardjo has been isolated from humans by Christmas et al. (1974).
Recently, hardjo was reported as being isolated from the common brushtail possum Trichosurus vulpecula in New Zealand (Brockie 1975; de Lisle et al. 1975).page 158
|SEROGROUP||SEROVAR||HOST SPECIES||FIRST REFERENCE|
In 1975 the author made a single isolation of balcanica from a possum in the Pohangina Valley. Serovar balcanica had only previously been isolated from a man in Bulgaria (Janev, cit. Babudieri and Mateev 1961) and from cattle and a pig in Russia (Semenova 1965).
Serovars hardjo and balcanica are within the same antigenic subgroup as determined by factor sera analysis (E. Kmety pers. comm.) and as such the only means of differentiating between these organisms is by isolation of the organism in pure culture and examination by cross-absorption studies on specific antisera of all serovars within the Hebdomadis serogroup. This is a complicated procedure and is performed at W.H.O. reference laboratories. Consequently, the agglutination test used routinely on sera in this country will identify infection due to organisms from a particular serogroup, but will not identify page 159 specific serovars. Similarly, any isolated cultures from animals with leptospirosis can only be identified as to serogroup without definitive typing.
Bearing these facts in mind, a survey was undertaken in the southern half of the North Island to determine the prevalence of Hebdomadis serogroup infections in pastoral possum populations, and to identify the specific serovars involved.
Materials And Methods
Field samples were collected by night shooting and trapping from four pastoral localities where possums were of moderate to high population density. Following bleeding, sex and age-group were determined and the animal tagged for identification. Animals were divided into juvenile, transitional-stage adult and mature adult groupings. Differentiation was made on sexual maturity parameters: juveniles had testes lengths < 18 mm (males) or had undeveloped pouches (females); transitional-stage adults were sexually mature but weighed less than 2.4 kg and lacked the conformation of fully grown adults; mature adults weighed 2.4 kg or more and had fully mature head and body conformation.
Sera were tested by the microscopic agglutination test (M.A.T.) against reference antigens ballum, pomona, copenhageni, tarassovi, hardjo and balcanica. The reciprocal of the final dilution of serum that agglutinated 50% of the test antigen was taken as the titre of that serum.
Kidneys were aseptically removed within 3 hours of death and cultured. A 'Coleworth Stomacher1' was used to homogenate whole kidneys and three serial ten fold dilutions were inoculated into EMJH2 semisolid media.
A high prevalence of Hebdomadis serogroup reactions was revealed in the pastoral possum populations sampled, with 60 of 127 sera (47%) positive to Hebdomadis antigens. Serovar hardjo antigen gave a consistently higher titre when compared with balcanica antigen in all sera tested. No cross-reactions were seen with antigens from other serogroups.page 160
There was a marked age differentiation in serological prevalence with mature adults having the highest prevalence, and only one juvenile animal being positive at a low titre (Table 2).
|SUBGROUP||NO. COLLECTED||NO. SEROPOSITIVE TO HEBDOMADIS ANTIGEN (%)||NO CULTURE POSITIVE (%)||NO. SERO. AND/OR CULTURE POSITIVE (%)|
|Trans. stage male||15||7||(47)||4||(27)||7||(47)|
|Trans. stage female||18||5||(28)||3||(17)||5||(28)|
Leptospiral isolations were made from 48 of 127 animals sampled (38%). All were typed against standard antisera in our laboratory as belonging to the Hebdomadis serogroup. Eight isolates were forwarded to the W.H.O. Reference Laboratory in Atlanta, U.S.A. for serovar identification. All eight isolates were found to be serovar balcanica.
A marked age differentiation was seen in cultural prevalences with all isolations being from sexually mature adults (Table 2). Isolates were obtained from 41 of 64 mature adults (64%) and from 7 of 33 transitional-stage adults (21%).
These results demonstrate that leptospiral infections of pastoral possums in New Zealand are very common with over 80% of mature adults showing serological and/or cultural evidence of infection at the time of collection. The recovery and definitive identification of L. balcanica from possums establishes this animal as a primary host reservoir for this organism. The origin of this infection in possums in New Zealand is unknown as this organism page 161 has only previously been isolated from domestic stock and man in East Europe. Other wildlife species including rats, mice, hedgehogs, Mustelidae, pukekos and ducks have been investigated in New Zealand for Hebdomadis infections (unpublished data) but no cultural or serological evidence has so far been found.
Isolations were made in all cases from sexually mature adults, and serological reactions were also restricted to this group. This pattern has been reported in other species of wildlife in leptospiral surveys overseas (Ferris et al. 1961; Wolf and Bohlander 1965; Shotts et al. 1975) and may be a feature of endemic host-adapted leptospiral infection in wild animal populations.
Two earlier reports of leptospiral infections in possums (Brockie 1975; de Lisle et al. 1975) provisionally diagnosed the organism involved as being hardjo. Without the use of cross-absorption tests, it is not possible to distinguish between balcanica and hardjo. In light of the results of the present survey it is more likely that the organisms isolated were in fact balcanica. The fact that possums infected with balcanica give a higher serological titre against hardjo antigen compared with homologous antigen is being further investigated.
I would like to thank Mr Noel Lee, Supervisor of the South Hawkes Bay Pest Destruction Board for invaluable assistance in collection of samples, and Miss Sandra Roxbourgh for preparation of media.
Babudieri, B. & Mateev, D. 1961. Studio serologico di alcuni ceppi bulgari di Leptospire. Rendiconti Instituto Superiore di Sanita 24: 614-622.
Brockie, R.E. 1975. Isolation of Leptospira hardjo from the opossum (Trichosurus vulpecula). N.Z. Veterinary Journal 23: 215-216.
Christmas, B.W. , Till, D.G. & Bragger, J.M. 1974. Dairy farm fever in New Zealand: isolation of Leptospira pomona and Leptospira hardjo from a local outbreak. N.Z. Medical Journal 79: 904-906.
Ferris, D.H. , Rhoades, H.E. , Hanson, L.E. , Galton, M. & Mansfield, M.E. 1961. Research into the nidality of Leptospira ballum in campestral hosts including the hognosed snake (Heterodon platyrhinus). Cornell Veterinarian 51: 405-419.
Lake, D.E. 1973. Bovine leptospirosis. N.Z. Veterinary Journal 21: 52.page 162
De Lisle, G.W. , Almand, K.B. , Julian, A.F. & Wallace, J. 1975. Leptospirosis in the opossum (Trichosurus vulpecula). N.Z. Veterinary Journal 23: 215-216.
Semenova, L.P. 1965. Leptospiry gruppy hebdomadis Soobsceniel II obnazuzenic V Sovetskom Sojuze podtopa L. sejroe balcanica . Journal of Microbiology, Epidemiology and Immunology 4: 61.
Shotts, E.V., Jr., Andrews, C.L. , & Harvey, T.W. 1975. Leptospirosis in selected wild mammals of the Florida Panhandle and South Western Georgia. Journal of the American Veterinary Medical Association 167: 587-589.
Wolf, J.W. & Bohlander, H.J. 1965. Leptospiral infections of hedgehogs of the Netherlands. Tropical and Geographical Medicine 1: 9-16.
MIERS. Do you get any regional differences in the infection rate of possums?
HATHAWAY. I have only sampled in the southern half of the North Island. However we have differentiated between different habitats such as pastoral, forest margin and deep forest. We found leptospiral infection in forest up to 5 miles from the forest margin so it seems capable of residing in the deep forest populations.
B.D. BELL. Have you examined isolated populations, such as the possums on Kapiti Island?
HATHAWAY. Mr A.J. White has been collecting urine from possums in the Orongorongo Valley forest and the organisms appear to occur through the year at a rate of about 10–20% of the possums examined.
KEBER. Could you speculate on how this serotype has become so infective in the possum in New Zealand?
HATHAWAY. I can speculate. In New Zealand all our ground mammals were introduced, and these included species in which the infection could have evolved. So you could speculate that the organism was introduced to possums after European settlement in New Zealand, or was introduced with the brushtail possum from Australia and that in New Zealand we have a specific ecological situation which has allowed balcanica to become endemic.
1 Coleworth Stomacher 400, A.J. Seward and Co. Ltd., 6 Stamford Street, London, SE 19 UG, England.
2 Bacto-Leptospira Medium Base EMJH, Difco Laboratories, Detroit, U.S.A.
* Not subjected to cross-absorption identification procedures