An epidemiological study of the spread of rabbit haemorrhagic disease virus amongst previously non-exposed rabbit populations in the North Island of New Zealand

AIM: To monitor the initial releases of rabbit haemorrhagic disease virus (RHDV) into previously unexposed rabbit populations in the North Island of New Zealand. METHODS: The study programme consisted of pre-release spotlight counts of rabbits on the study farms, pre-release serological samples to check for prior exposure to RHDV, a farmer-completed questionnaire and post-release spotlight counts to measure any change in rabbit numbers following the release of RHDV. In total, 23 sites within the lower North Island where RHDV was released during the period November 1997 to June 1998, were monitored. The most common release method involved the spreading of chopped carrot bait laced with a solution of virus-infected material obtained from dead rabbits. RESULTS: Eighty percent of farmers thought that the disease had spread away from the release sites to areas where virus had not been liberated, although only 27% reported finding dead rabbits more than 300 m away from release locations. Seventy-three percent of farmers were satisfied with the overall effectiveness of rabbit haemorrhagic disease (RHD) as a means of reducing rabbit numbers, but 56% indicated they would modify the way they released the virus in the future. Average pre-release night spotlight counts per property ranged from 2.2 rabbits/km to 36.9 rabbits/km, the median being 12.8 rabbits/km. The time interval from initial release to when the first dead rabbit was seen which the farmer believed to have died from RHD varied from 3 to 21 days, the mean being 7.4 days and the median 7 days. The median change in night spotlight counts per site at 3 weeks after release, expressed as a percentage relative to pre-release counts, was -15.5% (range +18.9% to -76.9%) and at 6 weeks was -49.7% (range 0% to -76.9%). The time of the estimated peak of the disease epidemic ranged from 1 to 7 weeks after release of RHDV, the mean being 3.1 and the median 3 weeks. CONCLUSION: Rabbit haemorrhagic disease reduced rabbit numbers on the majority of farms where the virus was released, and appears to be an effective measure for controlling rabbit populations in New Zealand.     

A serial cross-sectional study of the prevalence of rabbit haemorrhagic disease on three farms in the lower North Island of New Zealand

AIM: To estimate over a 3-year period following the first release of rabbit haemorrhagic disease virus (RHDV) the prevalence of rabbit haemorrhagic disease (RHD) and the abundance of rabbits (Oryctolagus cuniculus) in an area that historically had low rabbit densities.

METHODS: Three farms grazing predominantly sheep and beef cattle, located close together and with low initial rabbit densities, were selected for study. RHDV had been deliberately released on all farms in December 1997. Farms were visited 2–3 times per year between June 1998 and April 2001. At each visit, rabbits were shot with the aid of spotlights at night and blood samples were collected for detection of RHDV antibodies. Rabbit carcasses were necropsied and the age of the animals was determined. Rabbit abundance on each property was measured throughout the study using spotlight night counts. Logistic regression was used to identify factors associated with the risk of carcasses being seropositive for RHDV.

RESULTS: Rabbit density differed initially between farms (8.2, 9.9, 2.3 rabbits per spotlight km in June 1998), and declined on all three properties over time (1.2, 2.4, 1.1 rabbits per spotlight km in November 2000). Highest antibody titres to RHDV were initially evident on the farm on which rabbits were most abundant. The average prevalence of seropositive rabbits overall was 21% (95% CI=15–28%). Female rabbits tended to be less likely to be seropositive for RHDV than males (OR=0.47; 95% CI=0.21–1.02). The odds of becoming seropositive were reduced for rabbits born in the breeding season of 1999–2000 (OR=0.17; 95% CI=0.05–0.64).

CONCLUSIONS: The temporal pattern of outbreaks measured by peaks of seroprevalence differed between closely-spaced farms when they had different rabbit densities, but were similar when rabbit densities were similar. Microclimate and vegetation influencing abundance of insect vectors for RHDV and intrinsic population-related factors like rabbit breeding behaviour are also likely to be involved in local patterns of spread.     

A serial cross-sectional study of the prevalence of rabbit haemorrhagic disease on three farms in the lower North Island of New Zealand

AIM: To estimate over a 3-year period following the first release of rabbit haemorrhagic disease virus (RHDV) the prevalence of rabbit haemorrhagic disease (RHD) and the abundance of rabbits (Oryctolagus cuniculus) in an area that historically had low rabbit densities. METHODS: Three farms grazing predominantly sheep and beef cattle, located close together and with low initial rabbit densities, were selected for study. RHDV had been deliberately released on all farms in December 1997. Farms were visited 2-3 times per year between June 1998 and April 2001. At each visit, rabbits were shot with the aid of spotlights at night and blood samples were collected for detection of RHDV antibodies. Rabbit carcasses were necropsied and the age of the animals was determined. Rabbit abundance on each property was measured throughout the study using spotlight night counts. Logistic regression was used to identify factors associated with the risk of carcasses being seropositive for RHDV. RESULTS: Rabbit density differed initially between farms (8.2, 9.9, 2.3 rabbits per spotlight km in June 1998), and declined on all three properties over time (1.2, 2.4, 1.1 rabbits per spotlight km in November 2000). Highest antibody titres to RHDV were initially evident on the farm on which rabbits were most abundant. The average prevalence of seropositive rabbits overall was 21% (95% CI=15-28%). Female rabbits tended to be less likely to be seropositive for RHDV than males (OR=0.47; 95% CI=0.21-1.02). The odds of becoming seropositive were reduced for rabbits born in the breeding season of 1999-2000 (OR=0.17; 95% CI=0.05-0.64). CONCLUSIONS: The temporal pattern of outbreaks measured by peaks of seroprevalence differed between closely-spaced farms when they had different rabbit densities, but were similar when rabbit densities were similar. Microclimate and vegetation influencing abundance of insect vectors for RHDV and intrinsic population-related factors like rabbit breeding behaviour are also likely to be involved in local patterns of spread.     

Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease

A novel, recombinant myxoma virus-rabbit haemorrhagic disease virus (RHDV) vaccine has been developed for the prevention of myxomatosis and rabbit haemorrhagic disease (RHD). A number of laboratory studies are described illustrating the safety and efficacy of the vaccine following subcutaneous administration in laboratory rabbits from four weeks of age onwards. In these studies, both vaccinated and unvaccinated control rabbits were challenged using pathogenic strains of RHD and myxoma viruses, and 100 per cent of the vaccinated rabbits were protected against both myxomatosis and RHD.     

云南省楚雄州部分地区猪瘟血清学调查

为了解楚雄州部分地区的猪瘟免疫情况,利用酶联免疫法(ELISA)对楚雄市、南华县和禄丰县随机采取的393份血清进行猪瘟抗体检测,并对各县(市)的调查数据加以比较,了解猪瘟在楚雄州部分地区的免疫情况。结果显示,楚雄州部分地区均有较高的猪瘟抗体阳性率,各县(市)的猪瘟抗体阳性率都在80%以上,有的县(市)猪瘟抗体甚至达到了100%。说明楚雄州部分地区的猪瘟免疫效果较好,猪瘟免疫成功。     

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.     

Crypt abscesses in the caeca of feral rabbits in a rabbit haemorrhagic disease endemic region of New Zealand

AIM: To describe the pathology of crypt abscesses in the caeca of feral rabbits in the Manawatu region of New Zealand, and to examine the possible relationship between their prevalence and rabbit haemorrhagic disease (RHD) virus (RHDV) infection.

METHODS: During the course of a 3-year study of RHD, 173 wild rabbits (Oryctolagus cuniculus) were shot on three pastoral livestock farms in the Manawatu region of New Zealand. All rabbits were necropsied, and tissue samples of the caeca were examined histologically. The age of each animal was determined, and blood samples collected for the detection of RHDV antibodies. Logistic regression was used to model the odds of rabbits having crypt abscesses.

RESULTS: At necropsy, 63/173 (36.4%) rabbits were found to have small circular black nodules on the mucosal surface of their appendix caecum and/or sacculus rotundus. Microscopically, these were identified as small crypt abscesses composed of dilated sacs at the base of the mucosa that were often lined by a thin layer of attenuated epithelial cells. They usually contained large amounts of concentrically-laminated mucopolysaccharide material that was sometimes pigmented, inflammatory debris, and were often the site of a moderate multifocal appendicitis. Although RHDV was active in the study area, no association was found between RHDV antibodies in serum and the presence of lesions. The lesions were more common in older individuals and those born in summer or autumn.

CONCLUSIONS: This is the first report of crypt abscesses with inflammation and necrotic debris in the caeca of rabbits. No association between the occurrence of crypt abscesses and RHDV infection was identified. Wild rabbits born in a particular season were presumably exposed very early in life to conditions that caused the crypt abscesses to develop. Alternatively, association with season of birth may represent rabbits that were of similar age in a later stage of their lives, when they became exposed to the cause of the lesions, which remains unidentified.     

A cross-sectional study of risk factors affecting the outcome of rabbit haemorrhagic disease virus releases in New South Wales

OBJECTIVE: To determine what factors governed the extent of outbreaks of rabbit haemorrhagic disease (RHD) following releases in New South Wales. DESIGN: Retrospective cross-sectional study. PROCEDURE: Information from the data set of official releases was subjected to two preliminary analyses. More comprehensive information on a subsample of official RHD releases, sites and animals was gathered by telephone survey of Rural Lands Protection Board staff and farmers. Data were analysed using multivariate techniques to determine which factors were associated with rabbit mortality within one month of RHDV release, within several months of release and in affecting the proportion of the population killed. RESULTS: A strong association was found between the presence of heavy flea infestation (odds ratio 2.7), breeding in rabbits and outbreaks of RHD. For each week following breeding there was an 8% decline in the odds of an outbreak. Low temperatures also promoted outbreaks. Less important effects included the prior presence of RHD at the release site, which reduced the likelihood and severity of outbreaks. The presence of cattle and proximity to the nearest water body were associated with increased severity and likelihood of outbreaks respectively. CONCLUSION: Both breeding of rabbits and associated high flea numbers may act together or independently in promoting outbreaks of RHD. Stresses involved with rabbit reproduction and low environmental temperatures also appear to influence the likelihood of outbreaks. The effects of proximity to cattle and water suggests that both flies and mosquitoes may have a minor role in local transmission.     

Rabbit haemorrhagic disease: advantages of cELISA in assessing immunity in wild rabbits (Oryctolagus cuniculus)

Rabbit haemorrhagic disease (RHD) is an acute fatal disease of domestic and wild European rabbits (Oryctolagus cuniculus) caused by RHD virus (RHDV). Accurate assessment of immunity is of great importance for the conservation and control of wild rabbits. We evaluated a competitive ELISA (cELISA) against isotype ELISAs for assessing the protective immunity against the disease by challenging 50 wild-caught rabbits with a lethal dose of RHDV. Death or survival to the challenge was used as a criterion to determine the performance characteristics of the assay for the assessment of immunity in rabbits. At 1:10 dilution, a serum exhibiting ≥ 25% inhibition (1:10(25)) was regarded as the presence of RHDV-specific antibodies. Eleven of 16 (68.8%) rabbits with antibodies at 1:10(25) (<1:40) died of RHD. When the cut-off was moved from 25% to 50% inhibition (1:10(50)) at 1:10 serum dilution, the assay sensitivity, specificity and accuracy for the protective immunity were improved from 84%, 54.2% and 69.4% to 84%, 100% and 91.8%, respectively. We also demonstrated at the epitope amino acid sequence level why the presence of the RHDV-cross reactive benign rabbit calicivirus, which interfered with isotype ELISAs, had little impact on the specificity of the cELISA for the diagnosis of RHDV infection. The presence of RHDV-specific antibody at 1:10(50) by the cELISA is a reliable indicator for the protective immunity. In contrast to isotype ELISAs, the cELISA is a valuable specific tool for monitoring the herd immunity to RHD for the conservation and management of wild rabbits in the field.     

Possible interaction between myxomatosis and calicivirosis related to rabbit haemorrhagic disease affecting the European rabbit

Serological data on myxoma virus, rabbit haemorrhagic disease (RHD) virus and RHD-like viruses in juvenile rabbits (Oryctolagus cuniculus) trapped in 1995, 1996 and 1997 in two areas of France were analysed. For each disease, the effects of bodyweight, year, month and seropositivity for the other disease were modelled by using logistic regressions. In one area, a model including RHD seropositivity was selected to explain the myxoma virus seropositivity. Models including myxoma virus seropositivity were selected to explain the RHD seropositivity in both areas, and the odds of a rabbit being seropositive to both viruses were 5.1 and 8.4 times higher than the odds of a rabbit being seronegative to myxoma virus and seropositive to RHD. The year and bodyweight had significant effects for myxomatosis in one area and for RHD in both areas.     

Apoptosis of peripheral blood leukocytes from rabbits infected with non-haemagglutinating strains of rabbit haemorrhagic disease virus (RHDV)

The report demonstrates that the induction of apoptosis in peripheral blood granulocytes and lymphocytes of rabbits infected with three non-haemagglutinating RHDV strains (English Rainham, German Frankfurt, and Spanish Asturias) is a crucial determinant of the pathogenesis of rabbit haemorrhagic disease. Apoptosis was measured by flow cytometric detection of caspase activity. These studies demonstrated that the investigated RHDV (rabbit haemorrhagic disease virus) viral strains affected leukocyte apoptosis to varying degrees. Enhanced leukocyte apoptosis was detected between 4 and 36h after infection and was more pronounced in lymphocytes than in granulocytes. The data presented here thus provide a preliminary understanding of the kinetics of apoptosis in leukocytes of rabbits infected with RHDV.     

Crypt abscesses in the caeca of feral rabbits in a rabbit haemorrhagic disease endemic region of New Zealand

AIM: To describe the pathology of crypt abscesses in the caeca of feral rabbits in the Manawatu region of New Zealand, and to examine the possible relationship between their prevalence and rabbit haemorrhagic disease (RHD) virus (RHDV) infection. METHODS: During the course of a 3-year study of RHD, 173 wild rabbits (Oryctolagus cuniculus) were shot on three pastoral livestock farms in the Manawatu region of New Zealand. All rabbits were necropsied, and tissue samples of the caeca were examined histologically. The age of each animal was determined, and blood samples collected for the detection of RHDV antibodies. Logistic regression was used to model the odds of rabbits having crypt abscesses. RESULTS: At necropsy, 63/173 (36.4%) rabbits were found to have small circular black nodules on the mucosal surface of their appendix caecum and/or sacculus rotundus. Microscopically, these were identified as small crypt abscesses composed of dilated sacs at the base of the mucosa that were often lined by a thin layer of attenuated epithelial cells. They usually contained large amounts of concentrically-laminated mucopolysaccharide material that was sometimes pigmented, inflammatory debris, and were often the site of a moderate multifocal appendicitis. Although RHDV was active in the study area, no association was found between RHDV antibodies in serum and the presence of lesions. The lesions were more common in older individuals and those born in summer or autumn. CONCLUSIONS: This is the first report of crypt abscesses with inflammation and necrotic debris in the caeca of rabbits. No association between the occurrence of crypt abscesses and RHDV infection was identified. Wild rabbits born in a particular season were presumably exposed very early in life to conditions that caused the crypt abscesses to develop. Alternatively, association with season of birth may represent rabbits that were of similar age in a later stage of their lives, when they became exposed to the cause of the lesions, which remains unidentified.     

No virus replication in domestic cats fed with RHDV-infected rabbit livers

Previous studies have shown that feral cats (Felis catus) from rabbit haemorrhagic disease (RHD) epidemic areas in New Zealand had antibodies against RHD Virus (RHDV) and RHDV RNA was identified by nested RT-PCR from one seropositive feral cat liver. To assess whether RHDV replicates and produces clinical consequences in cats following the consumption of RHDV-infected rabbit, a challenge trial was conducted by feeding cats RHDV-infected rabbit livers. Antibodies against RHDV were detected by immunoassay from sera of cats collected 10 days after the consumption of RHDV-infected livers. Animals fed four times with RHDV-infected livers, had higher antibody titres than animals fed only once. RHDV RNA was detected by nested RT-PCR from mesenteric lymph nodes, tonsil, spleen and liver of cats fed with RHDV-infected livers. RHDV anti-genomic RNA was also detected by nested RT-PCR from mesenteric lymph nodes collected from one animal 2 days after the fourth feed. RHDV was detected by antigen ELISA from cat faeces 1-2 days after the consumption of RHDV-infected livers. Even though a large amount of RHDV has been used, cats did not show any signs of disease. Although abortive RHDV replication could not be ruled out, active RHDV replication was not demonstrated.     

Comparative analysis of rabbit hemorrhagic disease virus (RHDV) and new RHDV2 virus antigenicity,using specific virus-like particles

In 2010 a new Lagovirus related to rabbit haemorrhagic disease virus (RHDV) emerged in France and has since rapidly spread throughout domestic and wild rabbit populations of several European countries. The new virus, termed RHDV2, exhibits distinctive genetic, antigenic and pathogenic features. Notably, RHDV2 kills rabbits previously vaccinated with RHDV vaccines. Here we report for the first time the generation and characterization of RHDV2-specific virus-like particles (VLPs). Our results further confirmed the differential antigenic properties exhibited by RHDV and RHDV2, highlighting the need of using RHDV2-specific diagnostic assays to monitor the spread of this new virus.     

Serological evidence for a non-protective RHDV-like virus

The data were recorded during a Rabbit haemorrhagic disease outbreak that occurred in France in 2001 in a wild population of rabbits that we have been monitoring since 2000. These data suggested the existence of non-protective antibodies due to a putative RHDV-like virus. Twenty-one blood and 22 liver samples were taken from the 26 corpses of recently dead rabbits that were found. RHDV was found in all liver samples. A first screening for RHD antibodies, carried out using an ELISA based on the detection of VP60-RHDV antigen, showed that 20 of the rabbits were seropositive. Moreover, we determined antibody titres for 13 of these 20 seropositive samples. All were > or = 1/400. Such titres normally indicate antibody levels sufficient to confer protection to all known RHDV or RHDV-like strains. For 16 samples, we determined whether these rabbits had died of a chronic or an acute form of the disease, by employing monoclonal antibody (Mabs)--based differential ELISA. All had died of an acute form of RHD. Because the antibodies detected by this VP60-ELISA test are known to appear 5-6 days after infection and since acute RHD generally kills the rabbits 2-3 days after infection, we assumed that the detected antibodies must have been present before the exposure to the virus that killed these rabbits. A second detection of antibodies was made with Mabs that are specific for RHDV. The results were negative, showing that the antibodies detected with the VP60 ELISA test were not specific for RHDV. We sequenced a portion of the VP60 gene of viruses isolated in 17 rabbits. All RHDV isolates were very similar to the RHDV strains commonly isolated in France during this period, suggesting that this viral strain was not a putative variant that is not neutralised by antibodies. Therefore we conclude that the detected antibodies were probably due to a RHDV-like virus that induces the production of detectable but non-protective antibodies.     

Detection of rabbit haemorrhagic disease virus antigen in tissues by immunohistochemistry

Formalin fixed liver, spleen, kidney, heart, lung, duodenum and appendix tissues from nine rabbits, experimentally infected with rabbit haemorrhagic disease virus (RHDV), were investigated for evidence of RHDV antigen by the direct avidin-biotin peroxidase complex immunohistochemical method. In all the rabbits examined, RHDV antigen was detected in degenerative and necrotic hepatocytes of the liver tissues. The area involved coincided with histopathological lesions on serial liver sections. The RHDV antigen was expressed in the cytoplasm of the hepatocytes, suggesting that RHDV replicated in these cells. RHDV antigen was also detected in the spleen. The results of immunohistochemistry were supported by the demonstration of RHDV protein by Western blot analysis and of RHDV particles by protein A-gold immunoelectron microscopy in the liver homogenate from all the rabbits that were examined.     

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.     

Attitudes of New Zealand farmers to methods used to control wild rabbits

Four years after the release of Rabbit haemorrhagic disease virus (RHDV) in New Zealand, we conducted a mail survey of farmers to ascertain their attitudes and practices about rabbit control. A multistage sampling frame (stratified by rabbit-proneness and farm type) was used to select 828 farms in eight geographical regions. The useable response proportion of the survey was 69.3%, and 21% of respondents considered rabbits to be a problem on their farms. Although practices for rabbit control had changed from 1995 to 2001, shooting (practised by 85% of respondents) remained the predominant method used (albeit less frequently than in 1995). Ten percent of farmers used RHDV baiting; of those, 90% released the virus relatively infrequently. Farmers perceived shooting to be the most-humane and environmentally safe method, while RHDV was perceived to be the most effective. Perception of the level of competition for grazing between rabbits and livestock was the factor most-strongly associated with the use of shooting and RHDV. Most (60%) respondents considered the introduction of RHDV to have been beneficial.