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.     

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.     

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.     

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.     

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。     

The Efficacy of a Bivalent Vaccine Against Pasteurellosis and Rabbit Haemorrhagic Disease Virus

Rabbit haemorrhagic disease virus (RHDV) and Pasteurella multocida bacteria cause severe losses among rabbit populations. The efficacy of a recently developed bivalent vaccine against pasteurellosis and RHDV was investigated. Doses exceeding 2 haemagglutinating units (HU) of viral antigen were sufficient to protect rabbits against infection with RHDV. The bivalent vaccine appeared to be safe for use in all age groups of rabbits, including pregnant females, even after treatment with 20 times the normal vaccine dose. Rabbits injected with 8 or 4 HU of bivalent vaccine showed high antibody titres against both organisms for 9 months after inoculation. The antibody levels against RHDV in young rabbits at 30 days of age were elevated when they originated from mothers with high antibody titres. The most suitable period for vaccination of offspring appeared to be around 50 days of age. The bivalent vaccine against pasteurellosis and RHDV combined speed and longevity of the immune response. Immune protection against pasteurellosis and RHDV can thus be achieved with only one manipulation.     

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.     

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.     

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.     

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.     

Persistence of viral RNA in rabbits which overcome an experimental RHDV infection detected by a highly sensitive multiplex real-time RT-PCR

An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.     

中国首例兔出血症病毒2型(RHDV2/b/GI.2)的鉴定及病理学观察

本试验旨在鉴定四川金堂某兔场疑似兔出血症病毒2型（RHDV2）感染疫情的病原,并分析病兔的病理组织学变化。利用血凝试验和RT-PCR检测病死兔内脏组织中的病原,取病变组织制作病理切片,观察分析各组织的病理组织学变化,同时应用病兔肝脏悬液感染幼兔,分析该毒株的致病力。血凝试验结果显示,所采集病死兔肝脏样品能凝集人"O"型血红细胞;RT-PCR扩增及测序结果显示,多对引物均能从样品中扩增出RHDV2特异性条带;病理组织学观察结果显示,病兔多脏器严重出血、肿胀,淋巴细胞和中性粒细胞大量浸润,气管黏膜、肝脏、肺脏出血尤为严重;动物试验结果显示,该毒株毒力较强,含毒肝脏悬液能在24 h内迅速致死幼兔。本研究经临床诊断、核酸检测及测序证实了此次疫情确由RHDV2感染引起,动物试验和病理组织学观察表明该毒株毒力较强,可引发脏器严重出血,造成病兔急性死亡,RHDV2的出现提示病毒的跨境传播情况不容乐观,应引起更大的重视。     

基因重组抗原酶联免疫法检测兔出血症病毒抗体及其标准化

以重组兔出血症病毒(RHDV)VP60蛋白为抗原，建立了RHDV抗体间接ELISA检测方法。优化的试验反应务件为：重组VP60的包被质量浓度为1．0mg／L，用10％牛血清封闭，以大肠杆菌提取物稀释被检血清以消除非特异性反应。将所建立的ELISA与现行血凝抑制(HI)试验比较发现，不同免疫状态的兔血清的RHDV ELISA抗体与HI抗体均呈正相关。对11个RHD免疫兔场1130份血清样品的抗体检测表明，各免疫兔群血清RHDV抗体水平不完全一致，D值在1．09～1．76之间，显著高于非免疫兔(0．05)及SPF兔(0．02)，低于高免兔(2．34)。在此基础上，研制了RHDV抗体酶联免疫检测试剂盒，测定了其主要指标，制定了各成分的质量控制标准，为兔群进行免疫学监测及评价疫苗的免疫效果提供了便利。     

兔出血症病毒分子生物学研究进展

兔出血症是由兔出血症病毒引起的一种急性、高度致死性传染病，病死率可高达100％，给养兔业带来了巨大的经济损失。近年来，随着对其研究的不断深入，在病毒分子生物学方面取得了很大的进展。文章主要从基因组结构及功能、编码的结构蛋白与非结构蛋白、基因疫苗等方面系统阐述了兔出血症病毒分子生物学方面的研究进展，同时，提出了存在的问题和展望。为进一步研制兔出血症病毒基因工程疫苗及诊断试剂提供理论基础，以期能更有效地防治此病。     

Risk factors for an acute expression of Epizootic Rabbit Enteropathy syndrome in rabbits after weaning in French kindling-to-finish farms

Epizootic Rabbit Enteropathy (ERE) is a severe clinical syndrome of rabbits causing high economic losses for farmers. ERE first appeared in France in 1996. A retrospective case–control survey was carried out to identify the risk factors of acute expression of ERE, after weaning, in 96 kindling-to-finish rabbit farms in western France during 2001 and 2002. Farm status was defined according to the expression of clinical signs of ERE and mortality rates in the last five broiler rabbit batches. Comparisons of structural characteristics, rearing conditions and herd management showed that the risk factors for acute ERE expression were late weaning (rabbit age at weaning ≥ 35 days, RR = 4.44, 95% CI [1.36–21.71]), transfer of young rabbits at weaning (young rabbit transfer or combined practice RR = 2.83, 95% CI [1.16–9.33]), and high volume of the fattening room (air volume/rabbit weight in fattening room at weaning ≥ 0.14m³/kg, RR = 2.98, 95% CI [1.29–8.42]) and a high mortality rate in young rabbits before weaning (i.e. rate ≥ 10.5%; RR = 2.18, 95% CI [1.20–3.53]).     

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.     

应用单克隆抗体夹心酶联免疫吸附试验检测兔出血症病毒

应用建立的单克隆抗体夹心酶联兔疫吸附试验（ＭｃＡｂ－ＥＬＩＳＡ）,检测了人工感染兔出血症病毒（ＲＨＤＶ）ＤＪＲＫ细胞毒、肝毒的兔以及自然感染ＲＨＤＶ的兔的组织样品。结果表明,感染死亡兔的肝、脾、肾、骨髓样品病毒抗原的检出率为１００％,淋巴结和肌肉的检出率分别为９７．５％和７９．５％。ＭｃＡｂ－ＥＬＩＳＡ能检出肌肉中血凝试验不能检出的ＲＨＤＶ抗原。此外,还用ＭｃＡｂ－ＥＬＩＳＡ检测了肝毒人工感染兔血中ＲＨＤＶ的动态,并对１０份兔出血症脏器灭活苗的效价作了滴定。     

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.