Antibody responses to rabbit haemorrhagic disease virus in predators, scavengers, and hares in New Zealand during epidemics in sympatric rabbit populations.

AIM: To test for antibodies to rabbit haemorrhagic disease (RHD) virus (RHDV) in sera from mammals and birds associated with rabbit populations infected with RHDV. METHODS: Sera from feral and domestic cats, feral ferrets, stoats, hedgehogs, hares, harrier hawks, and black-backed gulls were taken (apart from some of the hares) from areas in New Zealand where RHD was active among rabbit populations. The presence of antibodies to RHD was investigated using a competition enzyme-linked immunosorbent assay (ELISA). RESULTS: Some individual animals of all species were seropositive. Thirty eight of 71 feral cats, but only 1/80 domestic cats were seropositive at a 1:40 dilution. The latter had not been exposed to RHDV. Also reactive in the ELISA were 2/8 stoats; 11/115 ferrets, with significantly more females having antibodies than males; 4/73 hedgehogs; 2/18 hawks, and 1/30 gulls. Three of 66 hares, comprising 3/14 from one population, were seropositive. CONCLUSIONS: Apart from the hares, all these species are known to prey upon rabbits or scavenge their carcasses, a possible means of exposure to RHDV. The possibility that the positive test reactions were due to cross-reactions with other caliciviruses cannot be ruled out, especially for the hares. Nor could the study differentiate whether the positive results were due to an antigenic reaction to ingestion of RHDV, as suggested by overseas work, or to infection of new species by RHDV. These possibilities are being investigated further.

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis.

AIM: To determine the aetiology of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep. METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp. RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected. CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks. KEYWORDS: Infectious keratoconjunctivitis, Mycoplasma conjunctivae, Chlamydophila pecorum, Branhamella ovis, polymerase chain reaction, ELISA, complement fixation test.

Eradication of Aujeszky's disease from New Zealand pig herds 1976-1997.

Aujeszky's disease was first diagnosed in the North Island of New Zealand in 1976. It has never been reported in the South Island. An industry-funded eradication programme was initiated in 1989 to eradicate the disease from the national pig herd. By using a combination of serological surveys, abattoir surveillance, test and slaughter, depopulation, vaccination and movement restrictions, Aujeszky's disease was eradicated by 1997.

Serology of rabbit haemorrhagic disease virus in wild rabbits before and after release of the virus in New Zealand.

Rabbit haemorrhagic disease virus (RHDV) was illegally released in New Zealand in August 1997. The initial release and spread of the virus was conducted by landholders in an effort to reduce costs associated with more conventional control methods (poisoning and shooting). Serum was collected from wild rabbits throughout the Otago region prior to the release and from 13 sites in the months following the first epizootic. Following the occurrence of the first RHDV epizootic on 13 pastoral farming properties a range of survival rates was found. The major factor influencing the survival rate was found to be the method of release, with widespread use of carrot or oat baits containing RHDV resulting in poor kills. Widespread use of baits also resulted in higher levels of antibody in surviving adult rabbits with a higher proportion of adult females surviving the epizootic, compared with properties where the disease was allowed to spread naturally. A correlation was found between survival rate and the percentage of surviving adults with high levels of antibody. These results suggest that poor kill rates are not due to poor spread of the virus, that the large-scale use of baits resulted in protective immunisation and that rabbit control should in the future be achieved through establishing naturally spreading epidemics rather than widespread use of baits.

A chondropathy of the pinna in rabbits associated with rabbit haemorrhagic disease.

AIMS: To investigate the relationship between loss of parts of the pinna in rabbits and rabbit haemorrhagic disease (MD). METHODS: A case-control study design was employed. Rabbits with ear lesions were shot on farms in various locations in the South Island of New Zealand. For each case, an attempt was made to obtain a sex and size-matched control rabbit 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:lO to 1:640. A selection of affected ears was examined histologically. Odds ratios and their 95% confidence intervals were calculated to assess the relationship between ear loss and RHD antibody status at various serological cut-off levels. RESULTS: 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 052.0 cm above the intertragic 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. CONCLUSION: The loss of the 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.

Evaluation of three tests for the detection of rabbit haemorrhagic disease virus in wild rabbits.

Two double antibody sandwich ELISA kits and the immunoblot were evaluated for the detection of rabbit haemorrhagic disease (RHD) virus in wild rabbits. Either liver alone or liver and spleen separately or a pool of liver and spleen tissues from 106 wild rabbits were tested in all three tests. They produced very similar results, except that ELISA kit A gave three doubtful results which were confirmed as negative by retesting in the same test and by the immunoblot and ELISA kit B. Both ELISA kits can be used for the rapid detection of acute RHD, and ELISA kit A can detect both acute and chronic RHD virus infection. The immunoblot is useful as a confirmatory test when ELISA-positive results do not correlate with gross and/or histopathological findings.

Progress towards the eradication of Aujeszky's disease in New Zealand by vaccination with a subunit vaccine.

Attempts to control Aujeszky's disease by vaccination with a glycoprotein-I negative subunit vaccine have been made on nine New Zealand pig farms. Thirty-one to 42 months after the programme of vaccination began, its progress was assessed by measuring the gI-antibody response in pigs from seven of the farms. Three farms had totally eradicated the 'wild' virus infection, one farm was close to achieving complete eradication and the other three farms had made little or no progress. One of the farms which eradicated the 'wild' virus infection achieved this status in two years by combining vaccination with an intensive testing and culling programme; the other two farms had eradicated the 'wild' virus infection by a combination of vaccination and good standards of hygiene without undertaking an intensive culling programme. The farms that had made little or no progress had less satisfactory standards of hygiene and did not practise an intensive testing and culling programme.

Evaluation of ELISA for detection of antibodies to CAEV in milk.

Milk was found to be a suitable alternative specimen for the serological diagnosis of caprine arthritis-encephalitis virus (CAEV) using the ELISA. The relative sensitivity and specificity of testing individual milk samples as compared to individual sera was 96.4 and 97.3% respectively. The overall agreement between the testing of milk and sera was 96.9% and the correlation coefficient between testing sera and milk, 0.94%. The testing of bulk milk could be used to predict approximately the prevalence of CAEV infection in a dairy goat flock. It was estimated that a prevalence of about 1.6% to 7.5% could be detected in the ELISA when bulk milk samples from two infected goat flocks were tested. Either chilled milk samples or milk samples treated with merthiolate were found to be suitable for testing.

Polymerase chain reaction amplification of latent Aujeszky's disease virus in dexamethasone treated pigs.

A polymerase chain reaction (PCR) amplification assay was developed for the detection of Aujeszky's disease virus (ADV) DNA in cell cultures and clinical samples. Pigs vaccinated with commercial ADV vaccines and challenged with a field isolate of ADV were immunosuppressed by dexamethasone treatment. Nasal swabs collected from the pigs at various times post-immunosuppression showed that ADV was excreted for at least four to six days starting from day 8 or day 10 following dexamethasone treatment, by virus isolation and/or PCR. However, PCR only detected latent ADV in the trigeminal ganglia, mandibular lymph node, spleen and tonsils, but not in the brain stem, pons and olfactory lobe of two pigs following dexamethasone treatment, whereas tissue explanation and cocultivation failed to demonstrate the presence of the virus.

gIII antibody responses in pigs following vaccination and challenge to Aujeszky's disease.

Serological responses to a genetically engineered Aujeszky's disease "marker" vaccine (dl gIII + dl tk) were monitored using a blocking-ELISA (B-ELISA), a serum neutralisation test (SNT) and an indirect ELISA (I-ELISA). The B-ELISA is capable of differentiating pigs vaccinated with the above vaccine from natural infection. The SNT and the I-ELISA indicated that the pigs responded to vaccination and challenge. All three tests showed that the controls and the in-contact pigs always reacted negative for antibodies. The B-ELISA was able to detect pigs challenged with a field isolate 24 days post-challenge. These pigs remained positive until 110 days post-challenge when last tested. These findings indicate that the B-ELISA could be used successfully with this vaccine in a control eradication programme. This trial also shows that the vaccine virus did not spread to the in-contact pigs and also the vaccinated and challenged pigs did not transmit the disease to other susceptible pigs when they were introduced 14 days after challenge.

Clinical effects, virological and serological responses in chickens following in-ovo inoculation of reticuloendotheliosis virus.

Six-day-old embryonated specific pathogen free chicken eggs were inoculated with reticuloendotheliosis virus (REV) into the yolk sac and were incubated until they hatched. The hatchability of eggs inoculated with REV was significantly less (P less than 0.025) than that of media-inoculated controls. Although there were no significant differences in the body weights of these chickens at hatching, there were differences (P less than 0.001) at 6, 25 and 51 days of age between the infected and control chickens. Six of 10 chickens hatched from eggs inoculated with REV had feathering defects at 6 days of age. All chickens hatched from infected eggs had cell-free viraemia and antigenaemia, but not precipitating antibodies. Some of these chickens had very low neutralising antibody titres (less than 45) when examined at 25 and 37 days of age, as did all 10 chickens at 51 days of age. A low rate of horizontal transmission was indicated by the detection of antibodies at 37 and 51 days of age in chickens running in contact with the chickens hatched from eggs inoculated with REV.