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.     

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.     

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.     

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.     

Adult rabbits acquire resistance to lethal calicivirus infection by adoptive transfer of sera from infected young rabbits

Calicivirus infection of adult rabbits induces the so-called rabbit haemorrhagic disease (RHD) that kills 90% or more of the infected animals; in contrast, young rabbits (up to 8-week-old animals) are resistant to the same infectious agent. We report that calicivirus inoculation of young rabbits induced moderate titres of antiviral antibodies. When these rabbits reached adulthood, a second calicivirus inoculation resulted in resistance to RHD and boosting of antibody titres in half of the rabbits. Adoptive transfer of sera from calicivirus-infected young rabbits to na?ve adult rabbits conferred resistance to RHD. We conclude that calicivirus infection of young rabbits induces specific anti-calicivirus antibodies that will protect them from RHD when they reach adulthood.     

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.     

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.     

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.     

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.     

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.     

Hepatic lesions in young rabbits experimentally infected with rabbit haemorrhagic disease virus

Twenty young rabbits (eleven 2-week-old and nine 4-week-old) were experimentally infected with rabbit haemorrhagic disease virus (RHDV) to clarify susceptibility. They were killed chronologically up to 96 hours post-inoculation (PI) and examined for lesions. All inoculated rabbits were clinically normal, but grossly minute white or grey spots were detected throughout the liver. Histologically, the lesions consisted of aggregates of lymphocytes, macrophages and heterophils, with or without acidophilic bodies and necrotic hepatocytes. Immunohistochemically, RHDV antigens were found in the degenerated hepatocytes and in macrophages. The cellular aggregates were considered to be a reaction to necrotic hepatocytes infected with RHDV. It was concluded that some hepatocytes are susceptible to RHDV in young rabbits.     

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。     

Hepatic coccidiosis in two pet rabbits

Hepatic coccidiosis is a frequent disease in rabbits. However, limited information is available pertaining to hematology, biochemistry, and ultrasonography of the liver in naturally infected rabbits. Two young emaciated rabbits presented with diarrhea. Microscopic fecal examination identified ellipsoidal oocysts. Eosinophilia, thrombocytopenia, elevated alkaline phosphatase, alanine aminotransferase, globulin, and gamma-glutamyl transferase, and decreased serum albumin, amylase, blood urea nitrogen, and creatinine were detected. Ultrasonography showed mild ascites, an enlarged liver with heterogeneous parenchyma, dilated hepatic blood vessels, and dilated bile ducts. Despite treatment with trimethoprim-sulfamethoxazole and toltrazuril both rabbits died. Necropsy revealed ascites and an enlarged liver with multiple yellowish nodules. Histologically, distended bile ducts almost completely replaced the hepatic parenchyma, and numerous coccidial gametocytes and oocytes were observed. Microscopic fecal examination, hematology, biochemistry, and ultrasonography of the liver can contribute to diagnose of hepatic coccidiosis.     

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses.     

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。     

Cytometric analysis of lymphocytes T and B in rabbits infected with non-haemagglutinogenic strains of RHD virus (rabbit haemorrhagic disease)--Rainham, Frankfurt and Asturias

The present paper refers to the cytometric analysis of lymphocytes T (with receptor CD5+), Th (with receptor CD4+), Tc/Ts (with receptor CD8+), lymphocytes CD25+ and lymphocytes B with receptor CD19+ in rabbits experimentally infected with strains of RHD virus--Rainham, Frankfurt and Asturias, not having haemagglutinogenic capacities, which makes them unique, as haemagglutinogenic capacity is a classic and typical property of most strains of this virus. The study was performed in the dynamic system, drawing blood samples from animals at hour 0, namely before the administration of the viral antigen, and then at 4, 8, 12, 24 and 36 h after the infection. The study indicated that Rainham and Asturias strains of RHD virus cause a similar amount of changes as the most immunogenic haemagglutinogenic strains CAMP V-561 and CAMP V-562 of the RHD virus do. In contrast, the Frankfurt strain of the RHD virus is characterised with 5-6-fold lower reactivity in this respect and is most similar to the least immunogenic haemagglutinogenic strain CAMP V-558 of the RHD virus.     

Immunohistological diagnosis of rabbit haemorrhagic disease in experimentally infected rabbits in Spain.

The immunoreactivity of routinely processed liver and lung tissue samples obtained from rabbits inoculated with tissue explants from naturally infected animals when antisera directed against parvovirus from different species (canine, feline and porcine) as well as a RHD virus antiserum were employed has been tested by different immunoperoxidase methods. Cross-reactivity between RHD-virus antigens and parvovirus antigens was present. Best results were obtained with RHD and canine parvovirus antisera with the ABC method. The immunoreactivity in the liver was found in hepatocytes, Kupffer and bile duct cells. In the lung, it was exclusively observed in intravascular macrophages.     

Effects of combined parenteral vitamins C and E administration on the severity of anaemia, hepatic and renal damage in Trypanosoma brucei brucei infected rabbits.

Rabbits infected with Trypanosoma brucei brucei (Basa isolate) were intraperitoneally administered with vitamins C and E at 100 mg/kg and 10 mg/kg body weight, respectively, from day 7 before infection to day 12 post-infection (p.i.). Another group of rabbits were similarly infected, but received no vitamin treatment. The uninfected (control) rabbits were either untreated or treated with vitamins like the infected group. Treatment of the infected animals did not affect the onset and level of parasitaemia. On day 12 p.i., the anaemia tended to be ameliorated, but insignificantly, by the treatment. The infection increased (p<0.05) serum urea and creatinine concentrations to similar levels in treated and untreated groups. However, the increase (p<0.05) in alanine and aspartate transaminases in the untreated infected animals was prevented in the treated infected ones. Therefore, it seemed that the treatment with antioxidant vitamins boosted their storage in hepatic cells, but not in erythrocytes and glomeruli, to annul any cellular injury due to infection. It is concluded that this may be an indirect evidence that the hepatic damage may be principally due to oxidative injury.