Experimental infection of Artibeus intermedius with a vampire bat rabies virus

Experimental infection of Artibeus intermedius, the great fruit-eating bat, was performed with vampire bat rabies isolates. Bats (n = 35) were captured in the wild and quarantined prior to experimental infection. No rabies antibodies were detected by rapid fluorescent focus inhibition test (RFFIT) prior to infection. Three doses of rabies virus (RV) and three different routes of infection were used. One out of 35 bats died without showing any clinical signs at day 14 and was positive for rabies. None of the 34 other bats showed clinical signs for rabies, but high antibody titers were detected post-inoculation, suggesting either innate immune response to the vampire bat rabies virus or possible pre-exposure to RV and inoculation leading to a booster effect. Rabies virus was detected by hemi-nested RT-PCR (hnRT-PCR) in the brain (n = 3), stomach (n = 1) of bats that were negative by immunofluorescence and that survived rabies infection. The bat that died on day 14 was positive by hnRT-PCR on the brain, heart and liver. These results suggest that either previous non-lethal exposure to RV or natural low susceptibility to vampire bat viruses somehow protected Artibeus intermedius from clinical rabies infection leading to a marginal lethality effect on this bats species population in the wild.     

湖南湘西地区外观健康犬携带狂犬病病毒的调查研究

为了解湖南湘西地区外观健康犬携带狂犬病病毒的情况,我们于2005年9月从湘西狂犬病疫点采集38份扑杀的外观健康的犬脑组织,并于2006年1月在湘西农贸市场采集了168份外观健康的犬脑组织。RT-PCR和直接荧光抗体试验(FAT)检测表明共有5份犬脑标本为阳性,并用乳鼠脑内接种的方法分离得到了5株狂犬病病毒毒株,分子流行病学分析表明这5株病毒均为野毒。在这5份阳性标本,疫点有4份,检出率为10.5%,市场1份,检出率为0.59%。我们的调查结果表明疫点的外观健康犬携带狂犬病病毒的检出率与国内的其它报道接近,而从市场采集的标本的检出率却要低很多。     

狂犬病快速诊断方法的建立和应用

根据狂犬病病毒核蛋白基因保守区序列设计1对引物，建立了用于狂犬病病毒特异性核酸检测的RT-PCR技术。该技术可从狂犬病病毒CVS株、8202株和SRV9株的含毒细胞培养物及鼠脑组织中。扩增出443bp的核酸片段，检出核酸的敏感性约为3Pg。对30份不同种动物脑组织的检测结果与小鼠脑内接种试验（MIT）的测定结果完全吻合，但前者可在3h内直接对组织匀浆进行诊断，具有快速、简便和敏感的优点。     

Pathogenesis studies with Australian bat lyssavirus in grey-headed flying foxes (Pteropus poliocephalus)

OBJECTIVE: To examine the susceptibility of the grey-headed flying fox (Pteropus poliocephalus) to Australian bat lyssavirus (ABL), and to provide preliminary observations on the pathogenesis of the disease in flying foxes. PROCEDURE: Ten flying foxes were inoculated intramuscularly with ABL, and four with a bat-associated rabies virus. Inoculated animals were observed daily, and clinical samples collected every 9 to 14 days. Animals with abnormal clinical signs were euthanased, and samples collected for histological, serological, virological and immunohistological examinations. At 3 months post inoculation (PI), all survivors were euthanased, and each submitted to a similar examination. RESULTS: Three ABL-inoculated flying foxes, and two rabies-inoculated animals developed abnormal clinical signs between 15 and 24 days PI. All three ABL-inoculated animals had histological lesions consistent with a lyssavirus infection, and lyssaviral antigen was identified in the central nervous system (CNS) of each. Virus was isolated from the brain of two affected animals. Of the rabies-inoculated flying foxes, both had histological lesions and viral antigen in the CNS. Virus was recovered from the brain of only one. None of the five affected flying foxes developed anti-lyssavirus antibodies, but, by 3 months PI, five of the seven ABL-inoculated survivors, and one of the two rabies virus-inoculated survivors, had seroconverted. The dynamics of the immune responses were quite variable. CONCLUSIONS: The response of flying foxes to ABL, administered by a peripheral route of inoculation, was similar to that of bats inoculated peripherally with bat-derived rabies viruses.     

Evaluation of cytokines concentration and percentage of survival of rabies virus-infected mice submitted to anti-rabies Vero-cell propagated vaccine and P. acnes

Previously, survival of rabies infection was shown to correlate with low IL-6 serum concentration in mice subjected to post-exposure treatment with the Fuenzalida Palacios rabies vaccine in conjunction with the immunomodulator Propionibacterium acnes, previously Corynebacterium parvum. Considering the substitution of the Fuenzalida Palacios rabies vaccine by the Vero cell raised anti-rabies vaccine in almost all countries, the objective of this work was to evaluate the survival and cytokine serum concentration of rabies virus-infected mice treated with P. acnes in conjunction with or the anti-rabies-VERO vaccine. For this, Swiss mice were experimentally infected with street rabies virus and subjected to vaccine and/or P. acnes following infection. Animals were killed at different times and serum was collected to evaluate cytokines. The greatest survival was observed in animals given one or two does of P. acnes in the absence of vaccination. Animals given anti-rabies VERO vaccine alone or with three doses of P. acnes had the second highest survival rate. The group that had the highest percentage of mortality also had the highest IL-6 concentration on the 10th day, a time correlating with clinical symptoms of the animals. The results reinforce the inefficacy of anti-rabies vaccine in only one dose as a post-exposure treatment irrespective of the type of vaccine used, the immunomodulation activity of P. acnes in rabies post-exposure treatment and suggest a role for IL-6 in rabies virus pathogenesis.     

重庆市2009-2010年动物狂犬病检测及流行病学调查

用RT-PCR方法检测重庆地区2009—2010年发生的36份临床疑似狂犬病脑组织样品和969份临床健康犬唾液拭子样品,并利用流行病学调查方法对阳性样品进行疫情溯源。结果从20份临床疑似狂犬病样品中检测出狂犬病毒,流行病学调查显示未免疫的流浪犬是主要的传染源,而农民(55.95%)和儿童(30.95%)是最容易受到狂犬攻击的人群。从969份临床健康犬唾液拭子和犬脑组织样品中未检出阳性样品。因此重庆地区狂犬病防控应加强对犬的免疫及流浪犬的管理,实行强制性疫苗免疫,加强对儿童和农民的狂犬病知识宣传和安全防护工作。     

鼬獾群体中狂犬病中和抗体调查与分析

采用荧光抗体病毒中和试验（FAVN）即固定病毒一稀释血清的中和试验方法,对从我国浙江、江西、安徽和广东地区采集的外观健康鼬獾的215份血清进行了狂犬病中和抗体的检测。结果显示,鼬獾体内存在比例较高（38％）的狂犬病中和抗体,但是自不同地区或群体采集的血清样品,狂犬病中和抗体阳性率不同,有些群体样品中完全没有抗体,有些群体样品中狂犬病中和抗体阳性比例100％,但是中和抗体的水平普遍不高,说明鼬獾种群感染狂犬病几率较高,并可能存在狂犬病的一过性或隐性感染。     

Comparison of enzyme-linked immunosorbent assay and RT-PCR for the detection of porcine epidemic diarrhoea virus

Porcine epidemic diarrhoea (PED) is a contagious enteric disease of pigs caused by a coronavirus. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) based on the use of monoclonal antibodies was developed for the detection of porcine epidemic diarrhoea virus (PEDV). The DAS-ELISA was compared with RT-PCR in the examination of 506 specimens collected during 2006-2007 from pigs originating from different farms located in the Po valley. Both faecal samples obtained directly from the rectum of live animals showing clinical signs and intestinal samples collected from the caecum of deceased pigs were included in the study. The correlation between the two methods was higher when testing faecal samples (K = 0.97, 95% CI: 0.94-1.00) than testing intestinal samples (K = 0.62, 95% CI: 0.35-0.89). The use of ELISA technology provided an efficient and effective mean of evaluating the presence of coronavirus PED antigen in field samples and indicates that this procedure is a very useful tool in epidemiological studies.     

Increased interleukin-10 associated with low IL-6 concentration correlated with greater survival rates in mice infected by rabies virus vaccinated against it and immunomodulated with P. acnes

Macrophage activity, cytokines serum concentration, serum neutralizing antibodies and lethality by rabies were evaluated in swiss mice experimentally infected with street rabies virus and submitted or not to antirabies vaccination and immunomodulation with P. acnes. Animals were killed at different times and serum was collected in order to evaluate cytokines concentration; peritonial and splenic macrophages were collected for macrophage activity evaluation. Greater survival rates higher IL-10 and low IL-6 serum concentration were observed in vaccinated animals treated using P. acnes.     

Molecular evidence of bovine herpesvirus 1 and 5 in cattle with suspected rabies in Rio Grande do Sul state,Brazil

Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.     

Spill-over of European bat lyssavirus type 1 into a stone marten (Martes foina) in Germany

European bat lyssavirus type 1 (EBLV-1, genotype 5) is known to endemically circulate in insectivorous bat populations in Germany. In August 2001, a rabies suspect stone marten (Martes foina) was found in the city of Burg (Saxony-Anhalt, Germany) and was sent to the regional veterinary laboratory for routine rabies diagnosis. Whereas brain samples repeatedly tested negative in the fluorescent antibody test for classical rabies virus (genotype 1), the mouse inoculation test and the rabies tissue culture inoculation test yielded positive results. Rabies viral RNA was also detected in the stone marten brain sample both by nested and heminested RT-PCR specific for the nucleoprotein gene and for the nucleoprotein phosphoprotein junction of rabies virus. The amplification products were sequenced to genotype the isolate. Sequence data obtained from the first-round RT-PCR products were analysed and the suspect stone marten isolate was confirmed as a rabies related virus (EBLV-1a). Phylogenetic comparison with sequences from recent genotype five isolates from Germany and Denmark showed that it was closely related to a previous isolate of EBLV-1 from a serotine bat in Saxony-Anhalt obtained in the same year in an area adjacent to the place where the EBLV-1 infected stone marten was found. Both EBLV-1 isolates share a 99.5% identity. This is the first report of an EBLV-1a spill-over from an insectivorous bat into wildlife in Europe.     

A retrospective longitudinal study of animal and human rabies in Botswana 1989-2006

A longitudinal study of animal and human rabies covering 18 years from 1989 to 2006 was retrospectively conducted in order to highlight the epidemiological features and trends of the disease in Botswana. Over the 18-year period, a total of 4 306 brain specimens collected from various species of animals including human beings with clinical signs consistent with rabies were submitted to the National Veterinary Laboratory in Gaborone for confirmatory diagnosis. Of the samples submitted, 2419 cases were found to be positive for lyssavirus antigen; this presents an overall prevalence rate of 56.18 +/- 1.48%. About 85.7% (2 074/2 419) of the cases were from domestic animals, 14.2% (343/2 419) cases were from wild animals and two cases (0.1%) were from human beings. During the first half of the study (1989-1997) the prevalence rate of the disease was estimated at 62.79 +/- 1.85% (1645/2620 positive) whereas during the second half (1998-2006) it was estimated at 45.91 +/- 2.38% (774/1686 positive) and the difference between the two estimates was statistically, highly significant (delta % = 16.88, SE(95) diff % = 3.015, SD = 5.599; P < 0.001). Ruminant rabies accounted for 79.99% (50.92% bovine, 928.40% caprine and 0.67% ovine) whereas canine (domestic dog) and feline (domestic cat) accounted for 16.01 and 0.87%, respectively. Equine rabies accounted for 3.13% with 1.35 and 1.78%, respectively, for horses and donkeys. Jackal rabies accounted for more than 60% of the total cases in wild animals. These findings are discussed in relation to the previous epidemiological situation of the disease (1979-1988), its socio-economic impact, monitoring and control in Botswana.     

The Epidemiological Importance of Bats in the Transmission of Rabies to Dogs and Cats in the State of São Paulo,Brazil, Between 2005 and 2014

In Brazil, rabies control in dogs and cats was pioneered by the state of São Paulo with the adoption of the Pan American Health Organization recommendations for prophylaxis and control, which led to a reduction in rabies cases from 1994 onwards. As a result of these measures, the rabies virus (RABV) genetic lineage associated with dogs has not been found in the state since 1998, and all the cases in domestic animals reported since then have been caused by bat‐associated lineages of RABV. In the light of this, this study sought to investigate rabies cases in dogs and cats in the state of São Paulo between 2005 and 2014 and identify the associated transmission cycles by characterizing the RABV lineages responsible for these cases. Nine samples from dogs (n = 5) and from cats (n = 4) were collected between 2005 and 2014. The tenth animal, a rabid cat, was analysed by a different laboratory. The N gene nucleotide sequences obtained were analysed with the neighbor‐joining algorithm and Kimura 2‐parameter model using the MEGA 6 program. Phylogenetic analysis revealed that the genetic lineages identified in all the samples were those circulating in Brazilian bats. The findings of this study demonstrate that bats play an important role in the transmission of rabies to domestic animals in São Paulo state and that emphasis should be placed on the implementation of public policies to support surveillance of chiropterans for rabies.     

狂犬病病毒荧光定量RT-PCR快速检测试剂盒的评价及应用

基于目前在我国人群和动物中流行的狂犬病病毒（RV）均属于基因Ⅰ型,在本室所建立的基因Ⅰ型RV荧光定量RT-PCR方法的基础上,组装了可以便捷使用的RV快速检测试剂盒。对4 577份临床样品的检测结果表明,该试剂盒的检测灵敏度达4.68个TCID50,与《狂犬病防治技术规范》规定的套式RT-PCR灵敏度相当,而且稳定性良好,可以冷冻保存8个月以上。所研制的荧光定量RT-PCR快速检测试剂盒具有操作简便、快速、特异性强、灵敏度高、重复性好、稳定性强等优点,既适合单个临床样品的确诊,又适合RV流行病学监测的大规模筛查。