RHD Science Seminar RHD     One Year On

3 September 1998

A seminar to discuss the results of research that has taken place during the year since rabbit haemorrhagic disease (RHD) was detected in New Zealand

Held at the international Antarctic Centre, Orchard Road, Christchurch.

This seminar is hosted by the Ministry of Research, Science and Technology, P0 Box 5336 WellIngton. Phone (04)4726400


Antibody status of predators, scavengers and hares following RHD epidemics

Richard Heyward1, John Parkes2 and Grant Norbury1.

Landcare Research: PO Box 282, Alexandra; P0 Box 69, Lincoln.

Feral cats, ferrets, harrier hawks, and to a lesser extent hedgehogs, use rabbits as a food source either by scavenging or predation By eating rabbits that have died of, or are infected with, RHD they may produce antibodies in response to the virus, as occurs in foxes (e.g., Leighton at at 1995, Journal of Wildlife Diseases 31: 541-44) Hares, a close relative of the rabbit, may be at risk from cross-infection. Our objective was to determine whether any predators, scavengers, or hares produced an antibody response when exposed to rabbits with RHD.

We collected serum samples from predators and scavengers, from an area of mass biociding in the Mackenzie Basin and from spot-baited areas in North Canterbury, during Febmary and May 199,8. The samples were tested for RHD antibodies using a competition ELI SA test at' 1:40 diluton. We also tested small numbers of cats, ferrets, and hares from areas without RHD.

Fifty-three percent (n=51) of cats, 10% (n=51) of ferrets, 11% (n=18) of hawks, and 3%(n=30) of hedgehogs were seropositive (Those with greater than 50% inhibition).

There was a bimodal distribution of antibody levels for all animals except cats. The proportion of seropositive animals was higher in February than in May.

Although about equal numbers of male and female ferrets were sampled, only female adults were seropositive. No juveniles were seropositive, which suggests ferrets had to be alive during the epidemic and that RHD was not active at these sites in 1998.

No hares (n=34) from the Mackenzie Basin, where RHD had occurred, were seropositive.

In areas where RHD had apparently not occurred, no predators or scavengers were seropositive. However, one of five hares sampled from inland Canterbury was seropositive (75% inhibition at the 1:40 dilution).

This work was funded by MAF. We thank Canterbury Regional Council and Mr K. Prime and Ngati Hine for providing some of the samples.

Impacts of RCD in the Mackenzie Basin: Prey switching, wrybills and black-fronted terns.

Elaine Murphy, Science & Research Unit, Department of Conservation, PB 68-908, Newton, Auckland.

A major concern to Doc from the introduction of RCD was that if rabbit numbers were reduced dramatically, than rabbit predators in these areas would eat more of other species- The level of impact this would have on threatened species in these areas is unknown. We need to determine whether predator-prey switching poses a risk for threatened species and whether this threat can be alleviated by predator control The aims of this study are to: 1) quantify predation rates on wrybill & black-fronted tern in the Tekapo & Tasman Rivers, 2) compare predation rates on wrybill & black-fronted terns with and without mammalian predator control, 3) determine efficiency of control operations on resident mammalian predators, monitor re-invasion and whether any predator guild changes occur and 4) analyse the diet of predators caught to determine if prey switching has occurred.

Wrybill productivity in the untrapped upper Tekapo site was low, and there was an indication that adults may have been killed. More nests and chicks need to be marked to increase sample sizes, Black-fronted terns had a high nesting success in both the trapped and un-trapped areas

The guts from 208 predators caught from the Tekapo and Tasman Rivers were analysed (111 ferrets, 76 cats & 21 stoats). Ferrets ate similar numbers of birds in spring (23%, 6126; Sep-Nov) and summer (24%, 10142; Dec-Jan). Cats however, ate significantly more birds in summer (59%, 32154), than spring (19%: 412i). Stoats were only caught during summer and 56% (10118) contained bird remains. 50% (10120) of bird remains that could be identified further in cats were not passerines, compared to only 8% (1112) in ferrets and 14%(117) in stoats. The high occurrence of bird remains in cats caught during summer (Cf to other studies in a similar area), indicates that prey switching by cats did occur.

Ecological consequences of RHD

Grant Norbury, Landcare Research, P0 Box282, Alexandra

Rabbits are the main food of three predator species (ferrets, cats1 and harrier hawks)-Increased consumption of native prey of secondary importance in predators' diets is commonly observed after declines in rabbit abundance. This is corroborated by studies of predation on banded dotterels in braided riverbeds. The proportion of banded dotterel eggs lost to predators shortly after 1080 poisoning of rabbits was 52+ 7% (averaged from 4 sites + 95% confidence interval, calculated from studies reported in Rebergen et at 1998, New Zealand Journal of Ecology 22: 33~1). This compared with only 23 + 4% egg loss (averaged from 12 sites) during subsequent breeding seasons when no rabbit control was conducted. Preliminary data from the breeding season during the 1997 RHD epidemic indicated that 56 + 10% (averaged from 4 sites) of eggs were lost to predators where rabbit abundance was originally high (up to 50 rabbits per spotlight km) and population declines were pronounced (up to 90%). This is a similar predation rate to that reported after rabbit poisoning. The longer-term implications for dotterel populations are unknown. Continued monitoring during subsequent breeding seasons will quantify the longer-term effects of RHD on native bird populations.

Rabbits are also important consumers of vegetation. Rabbits, other introduced herbivores, and humans have had such pervasive effects on New Zealand's short tussock grasslands that any reduction of rabbit numbers by itself wilt have variable outcomes. Rabbit reductions in the longer term (provided stock grazing has been removed) will most likely result in a succession towards perennial grasses and shrubland as demonstrated by European and New Zealand studies. The general opinion in New Zealand (amid a wide lack of data) seems to be that the overall effects of less rabbits will benefit native flora, especially palatable species such as blue tussock, native broom, and pingao. Greater pastoral production and less erosion of coastal dunes will also result from fewer rabbits. However, there are risks associated with reduced herbivory, especially in heavily modified seral habitats. Some unpalatable slow-growing native shrubs, such as kanuka, may be out~competed by faster-growing palatable weeds released from grazing. Some rare ephemerals and sedges may be suppressed by an increase in palatable weeds such as sweet briar. A similar sequence of events was witnessed in Britain after rabbit numbers were reduced by myxomatosis. Some small annual plants became locally extinct or rare, and the once floristically rich vegetation became dominated by a few grass and shrub species. More recent studies have emphasised the importance of climate and the presence of invading species, rather than rabbit graring1 on changes in species composition in degraded tussock grasslands.

One cannot point to overseas examples to find consistent effects of rabbits on native species. Needless to say, each ecosystem is unique and each requires its own programme of research. New Zealand ecosystems are no exception.

This work is funded by FRST under programmes C09640 and 009801, and by the Departrnent of Conservation, which also provided some of the baseline data.

Serology and Virology of RHDV in Central Otago

J S O’Keefe, AgResearch Wallaceville

Wallaceville RCD studies have addressed the origin of NZ RHDV, serological status of rabbits prior to and after the initial RHDV epizootics and efficacy of methods of initiating RHDV epizootics.

In collaboration with the Otago Regional Council (ORC) the livers of five rabbits were obtained from properties near Cromwell in September 1997. The presence of virus in these samples was confirmed by antigen detection ELISA (MAF GAHL) and PCR Sequencing of PCR derived DNA confirmed that the virus was most similar to the Czech V351 strain described in Australia (O'Keefe et at. NZVJ 46, 4243, 1998)

Prior to the introduction of RHDV to New Zealand a collaborative effort by the Applicant Group and MAF resulted in a large number of rabbit serum being tested in Italy at the OIE Reference Laboratory These serum originated from Southland, Otago, Canterbury1 Marlborough and Hawkes Bay. Between 65 and 80% of these gave positive results when tested for RHDV related antibodies (cross-reactive amongst members of the lagovirus genus). The sera from the Otago region was retested by MAF and results were comparable. These results support the contention that a RHDV related virus was present in New Zealand rabbits prior to RHDV introduction.

Monitoring of the first epizootic of RHDV on 13 properties in Central Otago revealed marked differences in mortality rates and residual levels of antibodies in the survivors. Mortalities were uniformly higher in areas where the viral epidemic had been seeded from point sources and allowed to spread naturally. Where the virus had been widely distributed on baits kills were low, with some properties showing no detectable kill. In addition, the average antibody titres were higher amongst biocide survivors than natural epidemic survivors, and the number of surviving females was higher in biocide areas. The results suggested that the ability of the epidemic to spread was not a limiting factor in determining the eventual mortality rate.

We are currently involved in collaborative work with Landcare Research and the Otago Regional Council to investigate the presence of an RHDV related virus and its significance in terms of both efficacy of RHDV for biocontrol and any possible affects on serological testing.

Laboratory Diagnosis of Rabbit Haemorrhagic Disease

Julian Motha

Central Animal Health Laboratory, MAF Quality Management, P0 Box 40063, Wallaceville

Rabbit haemorrhagic disease (RHD), also known as rabbit calicivirus disease, is a highly contagious and acute fatal disease of wild and domestic European rabbits (Oryctolagus cuniculus), caused by a calicivirus. This virus has not been isolated in vitro as no satisfactory growth conditions or sensitive cell substrates have been established. Experimental infection of rabbits remains the only possible method for the production of large quantities of viral antigen for the preparation of diagnostic reagents and vaccines

Several virus detection and serological tests are available for the diagnosis of RHD virus. For virus detection, the test of choice is the double antibody sandwich ELISA. This test is rapid, highly sensitive and specific, and capable of detecting both acute and chronic infections. The disadvantage of this test is that the range of samples that can be tested is limited, since for the detection of RHD virus, this test requires the presence of a very high concentration of virus. For specimens that have a low concentration of virus other tests, like the western blot and RT-PCR, should be used.

For the detection of antibodies, the competitive-ELISA is considered as the reference test. This test is widely used internationally and is highly sensitive and specific. An indirect-ELISA for the detection of antibodies is also available but this test is less specific than the competive-ELlSA. The indirect-ELISA results should be interpreted in conjunction with the competitive-ELISA results.

Factor x and survival in challenged pre-RHD rabbits

John Parkes and Susie Scobie

Landcare Research, P0 Box 69, Lincoln. Email

A non-pathogenic rabbit calicivirus, thought to be the ancestor of pathogenic Rabbit Haemorrhagic Disease (RHD) virus, has been identified in Europe (Capucci et at 1996, J Virology 70: 8614-23), and it appears to impart immunity to the pathogenic virus (Chasey et at 1997, First International Symposium on Caliciviruses, pp 1 56~62). Before the arrival of RHD in New Zealand, the New Zealand Applicant Group conducted a serological survey of wild rabbits using various ELISA tests some of which showed high titres of "factor x", and they concluded that this was evidence of a benign calicivirus being already present in a high proportion of wild rabbits (Lough 1998, unpubl. report, 12 p)' The questions remain (a) whether this conclusion is correct, (b) if so, whether the factor is similar to the European non-pathogenic virus, and (c) whether it imparts any immunity to rabbits challenged with RHD virus.

In March 1998, we captured 64 rabbits from areas of New Zealand where RHD had not been reported. Serum from each was, tested at four dilutions (1:10,1:40, 1:160, and 1:640) using both the competition ELISA specific for RHD and a less-specific indirect "sandwich" ELISA used by the Applicant Group to measure the presence of any caliciviruses. All rabbits were then orally dosed' with 50 LD of the Czech-train of RHD virus, obtained from the Elizabeth MaCArthur Institute in Victoria, and their fate monitored. Serum was taken from each rabbit at death, or after I and 6 months from the survivors. The survivors were rechallenged after 6 months.

Only one rabbit of the 64 was seropositive (it was above 50% inhibition at the 1:160 dilution) before challenge it was among 29 of the rabbits exposed to field RHD virus during a preliminary trial. None of the other rabbits were seropositive at the I :40 dilution, although four exceeded 50% inhibition at the 1:10 dilution.

Fourteen rabbits survived challenge including the seropositive rabbit above. One of these did not sero-convert at the first challenge but died at the second challenge. All but one of the survivors was positive to "factor x", the negative survivor was a juvenile. We assumed all those that died did so because of RHD, but post-mortem ELISAs showed some interesting variation in test responses depending on time to death and rabbit age.

The levelof factor x was measured in seven categories (zero, doubtful, and then five positive classes). The % inhibition at 1:40 dilution from the competition ELI SA increased roughly (r2 = 0.21) but significantly (P = 0.001) as "'factor x" increased. However, this relationship did not cause diagnostic problems in identifying rabbits seronegative to RHD at the 1:40 dilution.

All adult rabbits, both survivors and victims of the challenge, were positive to 'factor X', but only 6 of 22 juvenile rabbits, i.e. those born in the previous breeding season, were positive to 'factor x'

'Factor x' clearly does not guarantee immunity to RHD, which means that it will not affect the outcomes resulting from the presence of RHD virus - unless it is a calicivirus and recombines with RHD virus. Lack of cross-immunity is not unexpected given the high prevalence of 'factor X' in the Applicant Group's survey yet high mortality rates during the initial RHO epidemics in New Zealand. The question remains whether 'factor x' is a calicivirus descended from the benign rabbit calicivirus.

This work was funded by FRST and MAF under programme C09645. We thank Northland Regional Council for help in catching the rabbits.

A chondropathy of the pinna in rabbits associated with rabbit haemorrhagic disease - a field study

Gary clark*, Robert Sanson, Jeff Donaldson and Gary Knowles


in areas of endemic rabbit haemorrhagic disease (RHD) in Otago, Canterbury and Marlborough rabbits with loss of parts of the pinna were reported in the late spring early summer of 1997. To investigate the relationship between loss of parts of the pinna in rabbits and RHD a case-control study design was employed. Rabbits with ear lesions were shot on farms in Otago and Marlborough. For each case, a gender and size-matched control rabbit was obtained from the same farm on the same day. Serum samples were collected immediately after shooting The serum samples were tested for RHD titres from 1:10 to I :640 A selection of affected ears was examined histologically. Odds Ratios (OR) and their 95% confidence intervals were calculated to assess the relationship between ear loss and RHD antibody status at various serological cut-off levels.

Affected ears were characterised by firm cartilaginous nodules and ridges, folding of the ear or loss of pinna to form a notch or complete loss of the outer pinna from about 0.5 to 2 cm above the intedragic notch. histological changes in affected ears consisted mostly of focal mineralisation in the auricular cartilage, proliferation of cartilaginous tissue and loss of cartilage. The serological findings showed a signIficant association between rabbits with ear lesions and elevated RHD titres.

The loss of outer pinna in the rabbits under study was due to degenerative and hyperplastic changes in the auricular cartilage with distortion of the pinna, withering and loss of the outer pinna. The serological findings suggests that RHD is a likely factor in the development of the ear lesions.

A more in-depth study of this disease syndrome is required to determine if there is cartilaginous damage in other parts of the body or other pathological changes indicative of recovery from the acute form of the disease. Field observations suggest that rabbits with these ear lesions are more prone to dying.

invermay Animal Health Laboratory, PB 50035, Mosgiel

MAF Quality Managernent, P0 Box Palmerston North

Otago Regional Pest Services, PG 1 9S4. Dunedin

MAF Quality Management. Alexandra

Insect vectors of rabbit haemorrhagic disease virus




New Zealand Pastoral Agricufture Research Institute Ltd., AgResearch, invermay

Agricultural Centre, Private Bag 50034, Mosgiel

2New Zealand Pastoral Agriculture Research Institute Ltd., Wallaceville Animal

Research Centre, P0 Bax 40063, Upper Hutt



A field experiment in Otago, New Zealand, investigated whether healthy rabbits, Otyctolagus cuniculus1 exposed to insects in an area where rabbit haemorrhagic disease (RHD) was present could be infected with the disease. Thirty rabbits were placed individually in cages, half of which were covered with fine mesh All rabbits in the mesh covered cages survived. In the 'open' cages, four rabbits died, three of which tested positively for RHD, and serum samples from surviving rabbits suggested that two may have received a sub-lethal dose of virus. Of the insects trapped in the cages, Hybopygia varia (Walker) (Sarcophagidae) was considered the most likely vector, and in some cases tested positively for RH DV.


Field studies of RHD during the first year in New Zealand - an overview

Grant Norbury14 John Parkes21 Richard Heyward1.

Landcare Research: P0 Box 282, Alexandra; 2 P0 Box 69, Lincoln.


Landcare Research, AgResearch, Massey University, Auckland University, Rural Futures Trust, and several regional/district councils have collaborated in research on Rabbit Haemorrhagic Disease (RHD) since the disease first appeared in New Zealand 13 Months ago. Estimates of rabbit population mortality and antibody status, and the role of insect vectors1 have contributed to the following general observations on the initial epidemiology of RHD in New Zealand.

1. Mortality rates are highly variable - Mortality rates, based on spotlight counts, varied from 0-94% during the 1997 epidemics, but most were within 60-70%. Much of the variation was due to mass aerial baiting (biociding). Natural epidemics generally caused more consistent declines of 60-85%. Variable mortality is presumably a function of insufficient virus to challenge all rabbits (confirmed by RHD antibody-negative survivors immediately after the epidemic) and some forrn of innate or acquired immunity (confirmed by antibody-positive survivors). There is correlative evidence that high immunity is related to poor handling of virus during biociding.

2. RHD spreads naturally - Flies carrying virus have been shown to infect and kill rabbits. Flies may transmit virus directly from live infected to uninfected rabbits, or they may transmit virus from opened carcasses (therefore scavengers may be important) to uninfected rabbits indirectly via contaminated food. Virus may also be transmitted directly between rabbits.

3. RHD persistence is patchy - in some areas, occasional fresh carcasses have been observed sporadically since the 1997 epidemics. This suggests RHD has continued to challenge rabbits, but probably at a reduced rate. In other areas, antibody levels in samples of rabbits shot during 1998 have fallen suggesting rabbits have not been rechallenged by virus. This is also supported by the lack of seropositive predators among those born after the 1997 epidemics. Apparent reductions or cessation of virus activity may be because we cannot detect very low rates of mortality. Other explanations include: susceptible rabbit populations are below the threshold density needed to sustain an epidemic, their behaviour may have changed to reduce rabbit4o-rabbit contact, the virus may not have persisted in the environment1 there may be climatic effects, or (our pick) the vectors needed for transmission are less active.

4. Rabbit abundance has been suppressed - There has been little or no recruitment of rabbits since the 1997 epidemics1 resulting in greater than usual decreases in rabbit densities during autumn and winter. However, the evidence above suggests that this decline may not be due to RHD. We have no evidence that RHD suppressed fecundity. The simplest explanation is that predators had a good breeding season with fresh rabbit carcasses supplied by RHD, and they have been able to exert additional pressure on rabbit populations during 1998.

5. Proportion of anbbody-positive rabbits has declined The proportion of antibody-positive rabbits was diluted to some extent by recruitment of susceptible young born during the epidemic. It is also possible that proportionally more antibody-positive rabbits died, or that individuals lost antibodies. Our cage trials show that rabbits lose antibodies but retain immunity.

Unanswered questions include: will epidemics recur naturally? If there is a need to release virus, when is the best time to do so, and what proportion of immunity in the population should indicate likely failure of baiting? Will a better understanding of the role of vectors improve predictions of the Uming and intensity of epidemics? How accurate are the diagnostic tools for detecting immunity? And what is the optimum mix of conventional rabbit control with RHD?

This work was funded by FRST and MAF under programmes C09645 and 009803.

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