兔出血症Dot—ELISA双抗体夹心诊断法及其应用的研究

以建立的斑点酶联免疫吸附试验双抗体夹心法检测兔出血症病毒抗原.结果表明,对23只病兔各脏器病毒抗原检测,以肝、脾的检出率为最高.对人工感染兔各脏器检测,肝脏首先出现病毒抗原,其次是脾脏;各脏器病毒抗原含量依次为,肝>脾>肺>肾>心.用该法从人工感染24h后兔的口腔、鼻腔分泌物及粪便中检出了病毒抗原,表明病兔可通过消化道和呼吸道排毒.与血凝试验对比检测兔出血症可疑标本192份,两者符合率为90.6%.     

Possible interaction between myxomatosis and calicivirosis related to rabbit haemorrhagic disease affecting the European rabbit

Serological data on myxoma virus, rabbit haemorrhagic disease (RHD) virus and RHD-like viruses in juvenile rabbits (Oryctolagus cuniculus) trapped in 1995, 1996 and 1997 in two areas of France were analysed. For each disease, the effects of bodyweight, year, month and seropositivity for the other disease were modelled by using logistic regressions. In one area, a model including RHD seropositivity was selected to explain the myxoma virus seropositivity. Models including myxoma virus seropositivity were selected to explain the RHD seropositivity in both areas, and the odds of a rabbit being seropositive to both viruses were 5.1 and 8.4 times higher than the odds of a rabbit being seronegative to myxoma virus and seropositive to RHD. The year and bodyweight had significant effects for myxomatosis in one area and for RHD in both areas.     

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。     

“黄白散”对兔接种RHD疫苗后免疫功能的影响

在注射兔病毒性出血症 (RHD)疫苗后 ,30只兔随机分为 2组 ,试验组兔饲喂含 1 5 %黄白散的饲料。从接种到第 1 80天 ,每组取兔 8只 ,每隔 1 5d采血 1次 ,检测血凝抑制 (HI)抗体 ;在接种前 1天及接种后 5、 1 0、 1 7、 2 4、 31、 41、 5 1d ,每组取兔 1 0只 ,采血检测ANAE+淋巴细胞百分率 ;在 1 80、 2 4 0、 30 0d时 ,每组分别取兔 5只做攻毒保护试验。结果表明 :2组兔HI抗体峰值分别为 7 3log2和 9 8log2 ,差异极显著 (P <0 0 1 ) ;试验组疫苗保护期可延长 60d ;试验组ANAE+率从第 1 0天至 5 1天与对照组比较 ,差异极显著(P<0 0 1 )。所以黄白散可增强兔接种RHD疫苗后的特异性免疫功能 ,增强RHD疫苗的免疫效果。     

Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease

A novel, recombinant myxoma virus-rabbit haemorrhagic disease virus (RHDV) vaccine has been developed for the prevention of myxomatosis and rabbit haemorrhagic disease (RHD). A number of laboratory studies are described illustrating the safety and efficacy of the vaccine following subcutaneous administration in laboratory rabbits from four weeks of age onwards. In these studies, both vaccinated and unvaccinated control rabbits were challenged using pathogenic strains of RHD and myxoma viruses, and 100 per cent of the vaccinated rabbits were protected against both myxomatosis and RHD.     

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.     

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.     

散发性兔病毒性出血症的观察

通过对云南发病较多的12个兔场进行流行病学调查、临床症状及剖检变化观察、细菌培养、HA和HI试验,结果发现：兔病毒性出血症（RHD）可以在免疫过的兔场呈散发性流行;RHD散发性流行时,还会有其他疾病的存在：观察到散发性RHD的主要特点是最急性病例少见,病变出现率低,病变轻微,病程比文献报道的长。结果显示．散发性RHD发生的主要原因是兔群中存在免疫空白和免疫力低下的个体。建议开展RHD免疫程序与抗体水平,仔兔、幼兔母源抗体的消长规律,不同母源抗体水平的免疫效果观察等研究,建立更科学的免疫程序．并根据母源抗体监测结果确定首免日龄。     

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.     

Outbreak of rabbit hemorrhagic disease in domestic lagomorphs

Rabbit hemorrhagic disease (RHD) was diagnosed in domestic lagomorphs on a rabbit farm in Illinois. Clinical signs of RHD in affected rabbits included signs of depression, anorexia, fever, paddling, convulsions, and sudden death. Findings of necropsies and histologic evaluations of specimens of liver and spleen were indicative of RHD. In liver specimens obtained from dead rabbits, RHD viral antigen was detected via hemagglutination assay and viral antigen-detection ELISA. The source of the outbreak was traced to a rabbitry in Utah. As the disease spread, the outbreak involved rabbits in various regions of the United States; > 4,800 rabbits were euthanatized and buried as a result of the depopulation effort in several states. The economic impact of the disease can be considerable; if the disease is suspected, it is imperative that the appropriate state or federal veterinarian's office be contacted immediately.     

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.     

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.     

Immunohistological diagnosis of rabbit haemorrhagic disease (RHD).

In the present study the diagnostic use of a biotinylated serum from an immune rabbit was investigated by means of an Avidin-Biotin-Complex (ABC)-Peroxidase method on paraffin sections. 15 cases of RHD which had been verified histologically and/or by haemagglutination test (HA), 4 suspected cases and 3 cases without history of RHD were included (cases 1 to 22). From 5 prospective cases a wider tissue range was examined (cases 23 to 25 and 29 to 30). Furthermore lungs, liver and placenta of 3 fetuses from a RHD affected dam were investigated (cases 26 to 28). The 20 typical cases had intense intranuclear and diffuse intracytoplasmic immunostaining of hepatocytes, predominantly in the periportal areas. In some cases there was also positive staining of macrophages in the lungs (4 cases), spleen (4 cases) and in lymph nodes (1 case). Positive granular staining in the renal mesangial cells of the glomeruli was observed in 1 case. No positive staining was observed in the 3 negative cases. In contrast to other reports (4), crossreactivity of these antigens to Porcine Parvovirus (PPV) could not be confirmed. Furthermore the RHD virus (RHDV) seems not to crossreact with Feline and Bovine Parvoviruses.     

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.     

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。     

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.     

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.