Detection of rabbit haemorrhagic disease virus antigen in tissues by immunohistochemistry

Formalin fixed liver, spleen, kidney, heart, lung, duodenum and appendix tissues from nine rabbits, experimentally infected with rabbit haemorrhagic disease virus (RHDV), were investigated for evidence of RHDV antigen by the direct avidin-biotin peroxidase complex immunohistochemical method. In all the rabbits examined, RHDV antigen was detected in degenerative and necrotic hepatocytes of the liver tissues. The area involved coincided with histopathological lesions on serial liver sections. The RHDV antigen was expressed in the cytoplasm of the hepatocytes, suggesting that RHDV replicated in these cells. RHDV antigen was also detected in the spleen. The results of immunohistochemistry were supported by the demonstration of RHDV protein by Western blot analysis and of RHDV particles by protein A-gold immunoelectron microscopy in the liver homogenate from all the rabbits that were examined.     

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.     

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.     

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.     

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.     

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.     

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。     

Adenovirus-based oral vaccine for rabbit hemorrhagic disease

Vaccine antigens for rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from the livers of experimentally infected rabbits or from several recombinant immunogens. However, the application of these vaccine antigens has been restricted because of biosecurity and immunity characteristics. In the current study, a recombinant adenovirus expressing the RHDV capsid protein (VP60) was constructed and the expression of the recombinant protein was identified through western blot analysis using RHDV-positive rabbit sera. Eighteen rabbits were immunized by injection, direct oral instillation, or using bait. They were challenged with RHDV isolate three weeks after boost immunization. In all cases, the rabbits immunized with the recombinant adenovirus developed RHDV-specific antibodies and cell immune response. The rabbits injected with the recombinant adenovirus were completely protected against RHDV challenge. The adenovirus expression system may provide a strategy for the immunization of rabbits, particularly for the control of RHDV in wild rabbits.     

兔出血症病毒两种感染途径的致死性比较研究

为提高猪瘟活疫苗的外源性兔出血症病毒(RHDV)的检测敏感性,进行了RHDV对家兔感染途径的致死性比较试验,以便筛选出具有较高致死性的感染途径。将RHDV通过皮下、静脉两种途径感染家兔,比较了家兔两种感染途径的半数致死量(LD_(50)),同时对1批污染RHDV的脾淋源猪瘟活疫苗采用皮下、静脉两种途径注射家兔进行RHDV检测。结果表明,静脉途径对家兔的LD_(50)是皮下途径的126倍;污染疫苗的检测应用也表明家兔感染的静脉途径比皮下途径更加敏感,提高了猪瘟活疫苗外源性RHDV污染的检出率;RT-PCR证实了注苗死亡家兔的肝脏组织含有RHDV。本试验可为《中国兽药典》的修订提供数据参考。     

应用单克隆抗体夹心酶联免疫吸附试验检测兔出血症病毒

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

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。     

Growth of Tyzzer's organisms in preneoplastic hepatocytes of rats.

Tyzzer's disease in rats carrying preneoplastic or neoplastic lesions in the liver was studied histopathologically and immunohistochemically. The formation of necrotic foci and the growth of organisms were detected within both glutathione S-transferase placental type (GST-P) positive preneoplastic or neoplastic hepatocyte areas and GST-P negative areas. However, plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) values and number of necrotic foci of infected animals having preneoplastic lesions in the liver were smaller than those of infected animals receiving no carcinogenic procedure. These results suggest that the environment consisted of preneoplastic hepatocytes was unsuitable for the growth of organisms as compared with that of intact hepatocytes though the organism could grow within the preneoplastic hepatocytes.     

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

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

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.     

Ultrastructural pathology of nasal and tracheal mucosa of rabbits experimentally infected with Pasteurella multocida serotype D:1

Sixteen 8- to 9-week-old Pasteurella multocida-free New Zealand White rabbits were divided into two equal groups. The first group was inoculated intranasally with P multocida serotype D:1 strain and the second group that was inoculated with phosphate-buffered saline (PBS) only was used as a control group. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits in group 1 and from tracheal swabs of seven rabbits in this group. Four rabbits in group 1 died with clinical signs of septicaemia, two rabbits had mucopurulent nasal discharge and pneumonic lesions and the other two did not show any clinical signs or gross lesions. The ultrastructural changes detected were deciliation or clumping of cilia of ciliated epithelium, cellular swelling, vacuolation and sloughing. The subepithelial capillaries showed congestion, intravascular fibrin deposition, platelets aggregation and endothelial injury. Pasteurella multocida was observed attached to the injured endothelial cells. Heterophils, mast cells, vacuolated monocytes and macrophages infiltrated the lamina propria and between the degenerated epithelial cells.     

Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration

Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.     

Apoptosis of peripheral blood leukocytes from rabbits infected with non-haemagglutinating strains of rabbit haemorrhagic disease virus (RHDV)

The report demonstrates that the induction of apoptosis in peripheral blood granulocytes and lymphocytes of rabbits infected with three non-haemagglutinating RHDV strains (English Rainham, German Frankfurt, and Spanish Asturias) is a crucial determinant of the pathogenesis of rabbit haemorrhagic disease. Apoptosis was measured by flow cytometric detection of caspase activity. These studies demonstrated that the investigated RHDV (rabbit haemorrhagic disease virus) viral strains affected leukocyte apoptosis to varying degrees. Enhanced leukocyte apoptosis was detected between 4 and 36h after infection and was more pronounced in lymphocytes than in granulocytes. The data presented here thus provide a preliminary understanding of the kinetics of apoptosis in leukocytes of rabbits infected with RHDV.     

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.     

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.     

Experimental infection of domestic swine with Baylisascaris procyonis from raccoons

Six 8-week-old ascarid-naive pigs which were experimentally infected with 72,000 embryonated Baylisascaris procyonis eggs of raccoon origin developed lesions limited to the intestines and liver. Intestinal lesions consisted of multifocal areas of inflammation by macrophages, eosinophils, and lymphocytes in the mucosa and submucosa, in association with Baylisascaris larvae; similar lesions were seen in the mesenteric lymph nodes. Typical white, granulation type, multifocal interstitial hepatitis ("milk-spots"), 1 to 5 mm in diameter, were seen in the livers by 7 days, with resolution by 47 days. Microscopically, these consisted of multifocal areas of marked periportal and interlobular edema, and influx of eosinophils, and large intralobular aggregates of eosinophils. At 47 days, hundreds to thousands of small white granulomas were seen on the serosa of the intestines; microscopically, they were discrete collections of macrophages, lymphocytes, and eosinophils in the submucosa and muscle layers surrounding nonviable remnants of Baylisascaris larvae. Larvae or lesions were not seen in other tissues, including the brain. These experiments indicated that B procyonis will undergo limited migration in swine and can produce typical white spots in the liver. The larvae were killed by cellular reactions in the intestinal wall and liver, and, unlike the situation in most other animals infected with this parasite, no somatic migration or CNS disease occurred after infection.