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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Leukocyte responses in two breeds of layer chicken that differ in susceptibility to induced amyloid arthropathy

Amyloid arthropathy in chicken can be induced by intravenous inoculation of an arthropathic and amyloidogenic Enterococcus faecalis in susceptible breeds. The commercial brown layer hybrids (BL) are more susceptible to the disease compared to their white counterparts (WL). The precursor of amyloid-A protein, which is serum amyloid-A (SAA), is identical in WL and BL. To investigate the factors involved in the breed-restricted susceptibility to amyloid arthropathy, we studied the type of leukocyte response and inflammatory reactions in E. faecalis-induced disease. In the BL, a significant dose dependent peripheral leukocytosis mainly by heterophils, and plasma cell infiltration in arthritic joints was found. In contrast, secondary lymphoid nodular aggregates in the synovial membrane were prominent in the WL. The aggregates consisted mainly of CD8+ T cells. The high number of circulating leukocyte and prolific plasma cell responses in the BL predict extensive humoral and acute phase reactions. This is in agreement with literature data on suppressed T-cell function in casein-induced amyloid-susceptible mice strains. The difference in leukocyte response and type of inflammation between WL and BL, when arthropathic and amyloidogenic bacteria induce infection, in conjunction with susceptibility to amyloid arthropathy, is discussed in view of the murine T-helper responses.     

Modulation of cutaneous inflammation induced by ticks in contrasting phenotypes of infestation in bovines

Tick saliva contains molecules that are inoculated at the site of attachment on their hosts in order to modulate local immune responses and facilitate a successful blood meal. Bovines express heritable, contrasting phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus: breeds of Bos taurus indicus are significantly more resistant than those of Bos taurus taurus. Tick saliva may contain molecules that interfere with adhesion of leukocytes to endothelium and resistant hosts may mount an inflammatory profile that is more efficient to hamper the tick's blood meal. We show in vitro that adhesion of peripheral blood mononuclear cells to monolayers of cytokine-activated bovine umbilical endothelial cells was significantly inhibited by tick saliva. The inflammatory response to bites of adults of R. microplus mounted by genetically resistant and susceptible bovine hosts managed in the same pasture was investigated in vivo. The inflammatory infiltrates and levels of message coding for adhesion molecules were measured in biopsies of tick-bitten and control skin taken when animals of both breeds were exposed to low and high tick infestations. Histological studies reveal that cutaneous reactions of resistant hosts to bites of adult ticks contained significantly more basophils and eosinophils compared with reactions of the susceptible breed. Expression of the adhesion molecules – intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin – was higher in adult-infested skin of susceptible hosts undergoing low infestations compared to resistant hosts; when host was exposed to high infestations expression of these adhesion molecules was down-regulated in both phenotypes of infestations. Expression of leukocyte adhesion glycoprotein-1 (LFA-1) was higher in skin from susceptible hosts undergoing low or high infestations compared to resistant hosts. Conversely, higher levels of E-selectin, which promotes adhesion of memory T cells, were expressed in skin of resistant animals. This finding may explain the resistant host's ability to mount more rapid and efficient secondary responses that limit hematophagy and infestations. The expression profiles observed for adhesion molecules indicate that there are differences in the kinetics of the inflammatory reactions mounted by resistant and susceptible hosts and the balance between tick and host is affected by the number of tick bites a host receives. We show that the contrasting phenotypes of infestations seen in bovines infested with R. microplus are correlated with differences in the cellular and molecular composition of inflammatory infiltrates elicited by bites with adult ticks.     

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.     

Immunohistochemical localisation of rabbit haemorrhagic disease virus VP-60 antigen in early infection of young and adult rabbits

This study evaluated the time course distribution of rabbit haemorrhagic disease virus (RHDV) structural protein VP60 in tissues from experimentally infected rabbits from three different age groups. Viral VP60 antigen could not be detected in tissue samples from animals under four weeks, and only a few hepatocytes (0.01 to 0.2 per cent) were stained in the 6-week-old animals. A 6-week-old rabbit euthanised at 72 hpi showed VP60-labelling in hepatocytes and macrophages close to areas of inflammation. Viral VP60 antigen was detected as early as 12 hpi in a few hepatocytes (0.03 per cent) from adult animals. Within this age group, the extent of hepatocyte labelling considerably increased at 18 (3.0 per cent), 24 (25.5 per cent), 36 (50 per cent) and 48 (60 per cent) hpi. Extrahepatic viral VP60 antigen was also detected at 36 and 48 hpi in spleen macrophages and lymphocytes from adult rabbits. These findings support the hypothesis that the hepatocyte is the only cell type in the liver able to support RHDV replication almost immediately after viral infection.     

A systematic study of Tupaia as a model for human acute hepatitis B infection

The molecular features of hepatitis B virus (HBV) infection, eradication, and pathogenesis are poorly understood, partly due to the lack of an adequate animal model that faithfully reproduces the course of infection. Although Tupaia belangeri were previously recognized as HBV-susceptible animals, the course of infection in adult tupaias remains obscure. Herein, we performed a longitudinal study and demonstrated that adult tupaias were efficiently infected (90% infection rate) with 10⁸ copies of the HBV genome. HBV replicated vigorously, produced high levels of covalently closed circular DNA (cccDNA) in hepatocytes, and released hepatitis B surface antigen (HBsAg), hepatitis Be antigen (HBeAg), and HBV DNA into the serum at day 9 post-inoculation (p.i.), which then decreased on day 15 p.i. The kinetics were consistent with the expression of liver HBsAg and HBeAg, as determined with immunohistochemistry. The viral products in serum at day 9 and 15 p.i. represented de novo synthesized viral products, as treatment with a viral entry inhibitor completely abolished these products from the serum. Viral clearance and serological conversion occurred at day 21 p.i. and were accompanied by elevated alanine transaminase (ALT) levels and liver pathology, such as inflammatory infiltration and hepatocyte ballooning degeneration. Although ALT levels eventually returned to normal levels by day 42 p.i., the liver pathology persisted until at least day 120 p.i. The HBV infection process in tupaia, therefore, exhibits features similar to that of human acute HBV infection, including viral replication, viral eradication, ALT elevation, and liver pathology. Thus, adopting the tupaia model to study host-HBV interactions presents an important advance which could facilitate further investigation and understanding of human HBV infection, especially for features like cccDNA that current small-animal models cannot effectively model.     

Upregulation of major histocompatibility complex class II antigens in hepatocytes in Doberman hepatitis

Major histocompatibility complex (MHC) class II antigen expression in hepatocytes and its correlation with mononuclear cell infiltration into the liver were studied using immunohistochemical techniques in 38 Dobermans with Doberman hepatitis (DH). Liver biopsy samples were obtained from 18 dogs at the subclinical stage. Autopsy samples were taken from 6 DH dogs euthanized for a reason other than DH, from 14 dogs euthanized because of advanced liver failure and from 6 control Dobermans. Upon examination of the control liver samples, no expression of MHC class II antigens was detected in hepatocytes. By contrast, in 15 of the 18 DH biopsies (83%) and in all 20 DH autopsy liver samples, hepatocytes expressed MHC class II molecules. MHC class II expression was either cytoplasmic or membranous and occurred in conjunction with lymphocyte infiltration. A correlation between the inflammatory reaction and the expression of MHC class II in hepatocytes suggests that the aberrant expression of MHC class II in hepatocytes is induced by cytokines. Hepatocytes presenting a putative MHC class II molecule-associated autoantigen could thus become the target of an immune attack mediated by CD4+ T cells. In addition, corticosteroid treatment was observed to significantly decrease MHC class II expression in DH hepatocytes. Inappropriate MHC class II expression in hepatocytes and mononuclear cell infiltration are suggesting an autoimmune nature for chronic hepatitis in Dobermans.     

Giant cell hepatitis in two young cats

A very rare case of the liver lesion characterized by formation of multinucleated giant hepatocytes with inflammatory cell infiltration were observed in two young (1.5 years and 2 years old) cats bearing thymic malignant lymphoma. Histopathological features of this liver lesion were very similar to giant cell hepatitis (GCH) in human neonates and infants. Therefore, the lesion was diagnosed as feline GCH.