An outbreak of rabbit hemorrhagic disease (RHD) caused by Lagovirus europaeus GI.2/rabbit hemorrhagic disease virus 2 (RHDV2) in Ehime, Japan

A total of ten 1–2-year-old rabbits died within 2 weeks at a facility in Ehime prefecture in May 2019. Necropsy revealed liver discoloration and fragility, hemorrhage of some organs and blood coagulation failure. On histopathologic examination, necrotizing hepatitis was a common finding, together with fibrin thrombi in the small vessels and hemorrhage in some organs. Rabbit hemorrhagic disease (RHD) virus gene was detected in liver samples, and viral particles of approximately 32 nm in diameter were found in the cytoplasm of degenerated hepatocytes by electron microscopy. Phylogenetic analysis based on the partial VP60 gene sequence classified it as Lagovirus europaeus GI.2/RHDV2. This is the first confirmed outbreak of RHD caused by globally emerging GI.2/RHDV2 in Japan.     

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.     

Liver Enzymes and Ultrastructure in Rabbit Haemorrhagic Disease (RHD)

Rabbit haemorrhagic disease (RHD) is caused by a calicivirus infection that kills most adult rabbits 24–72 h after viral inoculation. Two liver enzymes (AST, aspartate aminotransferase, and ALT, alanine aminotransferase) were monitored in blood samples of calicivirus-infected rabbits during the short course of RHD. Values of AST were used to differentiate three stages of hepatocellular degeneration in RHD: mild (up to 20-fold increase in AST), moderate (150–200-fold elevation of AST) and severe (more than 1000-fold elevation in AST). Liver samples of rabbits from these three biochemical stages of hepatocellular degeneration of RHD were studied by transmission electron microscopy to define the fine structure of the hepatocytes. In the mild hepatocellular degeneration there was proliferation (microvesiculation) of the smooth endoplasmic reticulum and swelling of mitochondria into spheroid bodies with loss of cristae. In moderate hepatocellular degeneration, vacuolization of cytoplasm and mitochondrial damage continued to be present, and there was also formation of autophagic vesicles. In the severe hepatocellular degeneration of RHD, the altered mitochondria also showed loss of density of their matrix; rupture of cytoplasmic vacuoles led to the formation of large vesicles. Marked depletion of liver glycogen was also found in this late stage of RHD. These data offer a correlation between biochemical and cytological features of the liver during the hepatocellular degeneration of RHD.     

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.     

Liver disease in young rabbits infected by calicivirus through nasal and oral routes

Calicivirus infection causes rabbit haemorrhagic disease (RHD) that kills more than 90% of adult animals, whereas young rabbits are naturally resistant to this viral disease. It has been proposed that the different response of adult and young rabbits to calicivirus infection is due to absence of viral receptors in respiratory and digestive systems of young animals. We have searched for liver disease in 4-week-old rabbits inoculated with a calicivirus suspension by intranasal and oral routes. These young rabbits showed cell damage and mononuclear infiltration of the liver. The hepatic lesions were associated with mild to moderate increase in circulating transaminases. We conclude that the previously reported reduction of viral receptors in the epithelium of respiratory and digestive systems of young rabbits does not inhibit calicivirus from inducing liver disease in these hosts.     

Liver disease in young rabbits infected by calicivirus through nasal and oral routes

Calicivirus infection causes rabbit haemorrhagic disease (RHD) that kills more than 90% of adult animals, whereas young rabbits are naturally resistant to this viral disease. It has been proposed that the different response of adult and young rabbits to calicivirus infection is due to absence of viral receptors in respiratory and digestive systems of young animals. We have searched for liver disease in 4-week-old rabbits inoculated with a calicivirus suspension by intranasal and oral routes. These young rabbits showed cell damage and mononuclear infiltration of the liver. The hepatic lesions were associated with mild to moderate increase in circulating transaminases. We conclude that the previously reported reduction of viral receptors in the epithelium of respiratory and digestive systems of young rabbits does not inhibit calicivirus from inducing liver disease in these hosts.     

Etiology of rabbit haemorrhagic disease spontaneously occurring in Korea.

Causative agent of rabbit haemorrhagic disease (RHD) was purified by CsCl density gradient centrifugation from the liver homogenate of rabbits infected with RHD virus which originated from Korea. The viral particles were 35-40 nm in diameter, and had hollow depressions on their surface. Protein A-gold immunoelectron microscopy clearly showed that the convalescent antisera of diseased rabbits reacted specifically with the virus particles. SDS-PAGE and Western blot analyses demonstrated that the structural protein of the virus was composed of a single major polypeptide of 63 kD. These findings indicate that the causative agent of RHD, tentatively named as picornavirus in Korea, belongs to calicivirus.     

Leukocyte-hepatocyte interaction in calicivirus infection: differences between rabbits that are resistant or susceptible to rabbit haemorrhagic disease (RHD)

Calicivirus infection is lethal for adult rabbits, whereas young rabbits (less than 8-weeks-old) are resistant to the same infectious agent. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to rabbit haemorrhagic disease (RHD); this is in contrast with the mild and transient hepatitis observed in infected young rabbits. We have used electron microscopy to compare liver leukocyte infiltrates between young (resistant) and adult (susceptible) rabbits, 36-48 h after inoculation of the animals with caliciviruses. In adult rabbits, liver infiltrates were made up mostly of heterophils, and they were located near hepatocytes showing severe cellular damage. In contrast, liver leukocyte infiltrates of RHD-resistant young rabbits were dominated by lymphocytes that depicted membrane contacts with the cell surface of undamaged hepatocytes. We conclude that: (i) the cellular inflammatory response of the liver to calicivirus infection is different in rabbits that are susceptible (adult) or resistant (young) to RHD; (ii) leukocyte infiltration of the adult liver by heterophils is probably directed at the removal of dead hepatocytes, whereas the liver lymphocytic infiltration of young rabbits suggests the expression of viral antigens on the surface of liver cells of the RHD-resistant animals.     

A simple and rapid method for isolation of caliciviruses from liver of infected rabbits

Rabbit Haemorrhagic Disease Virus (RHDV), a member of the Caliciviridae family, is the etiologic agent of Rabbit Haemorrhagic Disease (RHD); this viral disease is highly contagious and kills more than 90% of infected adult rabbits. Research on experimental calicivirus infection uses inocula obtained from livers of rabbits dying from calicivirus infection. This implies that caliciviruses have to be purified from liver homogenates. Current methods to isolate caliciviruses from rabbit livers are time consuming. We propose here a new procedure for fast purification of rabbit caliciviruses from liver homogenates that uses centrifugation through an iodixanol gradient. This method offers in approximately 2 h a sample with a high degree of calicivirus purity, as shown by its biochemical and immunocytochemistry analysis, which is also able to kill adult rabbits from RHD within 48 h of inoculation.     

Early acute depletion of lymphocytes in calicivirus-infected adult rabbits

Rabbit Haemorrhagic Disease (RHD) is a lethal infection caused by calicivirus that kills 90% of the infected adult rabbits within 3 days. The calicivirus replicates in the liver and causes a fulminant hepatitis. Most studies on the pathology of RHD have been focused on the fulminant liver disease. This may not be the only mechanism in the pathogenesis of RHD: calicivirus infection may also induce leukopenia in the infected adult rabbits. We show now by flow cytometry analysis that the calicivirus induces an early decrease in B and T cells, in both spleen and liver. The depletion of B and T cells was associated with apoptosis labelled by annexin V. These changes occurred in rabbits before they showed enzymatic evidence of liver damage and persisted after liver transaminase values were very high. We conclude that depletion of lymphocytes caused by the calicivirus infection precedes or attends liver damage. The relative contribution of this lymphocyte depletion for the pathogenesis of the fatal calicivirus infection of rabbits remains to be investigated.     

Severe leukopenia and liver biochemistry changes in adult rabbits after calicivirus infection

Calicivirus infection is the major cause of the severe decrease in the stocks of wild and farm rabbits that has occurred worldwide during the last two decades. Adult rabbits (10-weeks-old) were experimentally infected with a calicivirus inoculum that killed all animals by causing rabbit haemorrhagic disease (RHD) within 24-62 h of infection. The rabbits were used to evaluate blood cell numbers and serum biochemistry every 6h, starting 12h after the inoculation of the caliciviruses. No significant changes in blood parameters were observed in most of the rabbits up to 18 h of infection. Severe leukopenia was seen 6h before death of the infected rabbits; both heterophils and lymphocytes contributed to the decrease in circulating white blood cells. Platelets were also severely decreased in number. Marked enhancement in liver enzymes was seen 6-12 h before death of the infected rabbits. There was also evidence both for cholestasis, as expressed by the elevated levels of direct (conjugated) bilirubin, and for hypoglycemia, an alteration that it is likely to contribute for the seizures that rabbits show during the late stages of RHD. Liver ultrastructure of rabbits that died from RHD revealed extensive hepatocyte vacuolization, severe changes in mitochondrial structure, and depletion of glycogen granules. We conclude that: (i) severe leukopenia characterizes the final hours of calicivirus-induced RHD; (ii) hypoglycemia and cholestasis precede death of rabbits from RHD; (iii) the kinetics of liver enzymes allows an accurate prediction of the time of death of rabbits from calicivirus-induced RHD.     

Transient decrease in blood heterophils and sustained liver damage caused by calicivirus infection of young rabbits that are naturally resistant to rabbit haemorrhagic disease

Young rabbits are naturally resistant to rabbit haemorrhagic disease (RHD) caused by the same calicivirus that kills, within 3 days, nearly all adult animals. We have investigated changes in blood leukocytes, and in the morphology and biochemistry of the liver (the organ where caliciviruses replicate) of young rabbits undergoing benign infection by the RHD virus. Four-week-old rabbits were infected with a calicivirus inoculum having a titre of 2(12) haemagglutination units either sacrificed 18, 24, 48 and 72 h later, or kept for follow-up studies up to 21 days after inoculation. The infection caused an acute and transient decrease in blood heterophils, and sustained enhancement in hepatic transaminases. Inflammatory infiltrates of the liver were seen in all animals after 24 h of infection; they had a predominant midlobular location. Hepatocytes could present different degrees of cell damage, including cell death; these lesions were limited to the liver cells located around the inflammatory infiltrates. Liver transaminases peaked 24-48 h after calicivirus infection; this was the same timing when liver infiltration and hepatocyte damage were more evident. No alterations of other parameters of liver biochemistry were observed. We conclude that calicivirus infection of young rabbits causes a subclinical disorder characterised by an acute and transient decrease in circulating heterophils, and focal liver damage that is expressed by intralobular infiltration by heterophils, initially, and, later on, by mononuclear cells. Our finding of persistence of increased values of liver transaminases suggests chronicity of the infection in young rabbits. We propose that, although resistant to RHD, young rabbits infected by calicivirus may be long-term carriers of the infectious agent and, thus, become a major source of transmission of the virus.     

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.     

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。     

家兔暴发病毒性出血症的病理学诊断

对送检的9只家兔进行病理解剖,对心血、肝、脾及肠系膜淋巴结进行细菌分离培养,并选取心、肝、脾、肺等组织按常规病理制片进行病理组织学检查,应用免疫组化染色(ABC法)检测肝细胞内兔病毒性出血症病毒(RHDV)抗原,结果证实此次家兔自然暴发的传染病即是兔病毒性出血症(RHD)。其临床主要表现为最急性型及急性型,病理变化特点以全身微循环障碍为主;DIC形成,以实质器官淤血、水肿、出血、变性、坏死为主要特点,其中以肝、肾、脾等器官病变较重,尤其肝脏的病变具有特征性。综合病理学检测结果,确定此次家兔暴发的是RHD。     

实验性兔出血症病兔肝脏酶组织化学变化研究

用组织化学染色法,对实验性感染兔出血症(RHD)病毒后不同病期(潜伏期、高热期、濒死期和死亡时)病兔的肝脏碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、三磷酸腺苷酶(ATPase)、琥珀酸脱氢酶(SDH)和乳酸脱氢酶(LDH)的活性变化进行了动态观察。结果表明,肝脏AKP、ACP、和LDH活性在潜伏期至高热期增强,以后各期均减弱;ATPase和SDH在病程各期均明显减弱。从而推论,肝细胞的坏死与ACP活性增强、溶酶体活跃有关;黄疸的发生与ATPase活性减弱及肝细胞线粒体受损有关;病兔的肝脏代谢以无氧酵解为主。     

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.     

獭兔玉米赤霉烯酮中毒的诊断

山东某獭兔场饲养的3 000只母兔发生了一种以流产及青年兔死亡为特征的疾病,通过对发病兔进行细菌培养及兔病毒性出血症病毒的RT-PCR以及饲料中的玉米赤霉烯酮、呕吐毒素及黄曲霉毒素进行鉴别检测,首先排除细菌病及兔病毒性出血症发病的可能,并从饲料中检测3种毒素的含量分别为1 196.1、130.8、6μg/kg,经过对照饲料标准发现玉米赤霉烯酮严重超出标准(标准为300μg/kg)。通过饲喂试验发现这次发病与所饲喂的饲料有关,诊断为玉米赤霉烯酮中毒。