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 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.     

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.     

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.     

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.     

Immunosuppression abrogates resistance of young rabbits to Rabbit Haemorrhagic Disease (RHD)

Rabbit Haemorrhagic Disease (RHD) is caused by a calicivirus (RHDV) that kills 90% of infected adult European rabbits within 3 days. Remarkably, young rabbits are resistant to RHD. We induced immunosuppression in young rabbits by treatment with methylprednisolone acetate (MPA) and challenged the animals with RHDV by intramuscular injection. All of these young rabbits died within 3 days of infection due to fulminant hepatitis, presenting a large number of RHDV-positive dead or apoptotic hepatocytes, and a significant seric increase in cytokines, features that are similar to those of naïve adult rabbits infected by RHDV. We conclude that MPA-induced immunosuppression abrogates the resistance of young rabbits to RHD, indicating that there are differences in the innate immune system between young and adult rabbits that contribute to their distinct resistance/susceptibility to RHDV infection.     

RABBIT HEMORRHAGIC DISEASE VIRUSES DETECTED IN PET RABBITS IN A COMMERCIAL LABORATORY IN EUROPE

Rabbit hemorrhagic disease (RHD) is an important cause of disease and mortality in wild and domestic European rabbits (Oryctolagus cuniculus) throughout the world. Testing for 2 distinct RHD virus types (RHDV/RHDVa and RHDV2) was carried out on samples collected from 684 rabbits submitted from veterinary practices and private owners throughout Europe between January 2015 and June 2017. Four (0.6%) were positive for RHDV/RHDVa and 257 (37.4%) were positive for RHDV2. RHDV/RHDVa was detected in individual samples from Germany and the Netherlands, while RHDV2 was found in animals from Germany, Great Britain, Luxembourg, The Netherlands, Spain, Switzerland, Poland, Belgium, Austria, Sweden, and Finland.     

Clinical and pathologic findings in an outbreak in rabbits of natural infection by rabbit hemorrhagic disease virus 2 in the northwestern United States

Rabbit hemorrhagic disease virus 2 (RHDV2) causes an often-fatal disease of rabbits that has resulted in outbreaks in rabbitries in Europe, Africa, Australia, and Asia. RHD has historically been characterized as a foreign animal disease in the United States. In July 2019, RHDV2 was detected in rabbits on Orcas Island along the northwestern coast of Washington (WA) State following reports of deaths in multiple feral and domestic rabbits. We document and highlight here the unique clinical presentation and gross and histologic lesions observed in this recent WA outbreak. Affected rabbits died without premonitory signs or displayed hyporexia and/or lethargy for ≤1 d prior to death. The most consistent pathologic finding was random, multifocal hepatocellular necrosis, often with concurrent multifocal-to-diffuse splenic necrosis. The lack of significant clinical signs in conjunction with the random distribution of hepatic necrosis in the WA outbreak contrasts with previous reports of RHDV2 disease progression.     

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.     

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。     

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.     

应用免疫组化PAP法检测兔出血症病毒抗原的动态分布

应用免疫组化PAP法检测了人工感染成年患兔、30～40日龄患兔以及自然感染患兔体内兔出血症病毒（RHDV）抗原的动态分布。结果表明,在成年患兔的肝、肾、脾、胃、十二指肠、睾丸以及幼兔的肝、肾、脾中检出了RHDV抗原;无论在成年或幼龄患兔,RHDV抗原主要位于受侵害细胞胞浆中,少部分位于核中;在成年患兔,RHDV抗原阳性细胞的数量随病程发展而增加,而在幼龄患兔,这种增加趋势不明显。本文还分析了RHDV抗原在患兔体内的动态分布与病变形成之间的关系。     

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.     

Regulatory T cells are decreased in acute RHDV lethal infection of adult rabbits

Rabbit hemorrhagic disease virus (RHDV) is the etiologic agent of rabbit hemorrhagic disease (RHD), an acute lethal infection that kills 90% of adult rabbits due to severe acute liver inflammation. Interestingly, young rabbits are naturally resistant to RHDV infection. Here, we have compared naturally occurring CD4(+)Foxp3(+) regulatory T cells (Tregs) between young and adult rabbits after infection by RHDV. The number and frequency of Tregs was decreased in the spleen of adult rabbits 24h after the RHDV infection; this was in contrast with the unchanged number and frequency of splenic Tregs found in young rabbits after the same infection. Also, serum levels of IL-10 and TGF-β were enhanced in the infected adult rabbits whereas no alteration was observed in infected young rabbits. However, this increase is accompanied by a burst of pro-inflammatory cytokines, but seems not able to prevent the death of the animals with severe acute liver inflammation in few days after infection. Since Tregs downregulate inflammation, we conclude that their decrease may contribute to the natural susceptibility of adult rabbits to RHDV infection.     

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.     

Emergence of a new lagovirus related to Rabbit Haemorrhagic Disease Virus

Since summer 2010, numerous cases of Rabbit Haemorrhagic Disease (RHD) have been reported in north-western France both in rabbitries, affecting RHD-vaccinated rabbits, and in wild populations. We demonstrate that the aetiological agent was a lagovirus phylogenetically distinct from other lagoviruses and which presents a unique antigenic profile. Experimental results show that the disease differs from RHD in terms of disease duration, mortality rates, higher occurrence of subacute/chronic forms and that partial cross-protection occurs between RHDV and the new RHDV variant, designated RHDV2. These data support the hypothesis that RHDV2 is a new member of the Lagovirus genus. A molecular epidemiology study detected RHDV2 in France a few months before the first recorded cases and revealed that one year after its discovery it had spread throughout the country and had almost replaced RHDV strains. RHDV2 was detected in continental Italy in June 2011, then four months later in Sardinia.     

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.