Importation of dogs into the United States: risks from rabies and other zoonotic diseases

The importation of dogs into the United States poses a risk for the introduction of rabies and other zoonotic diseases. Federal regulations (42 CFR 71.51) currently require proof of valid rabies vaccination for imported dogs, but allow the importation of some unvaccinated dogs, including dogs less than 3 months of age, provided certain requirements for confinement are met until the dog is vaccinated. Although there are no accurate surveillance data on the number of dogs imported each year, it is estimated based on extrapolated data that over 287,000 dogs were imported into the United States during 2006. Of these, approximately 25% were either too young to be vaccinated or lacked proof of valid rabies vaccination. Import trends suggest that an increasing number of unvaccinated puppies are being imported into the United States, many through commercial resale or rescue operations. Since 2004, foreign canine rabies virus variants have been documented in at least two imported puppies. Federal regulations are currently being reviewed by the Centers for Disease Control and Prevention to determine if they can be updated to address current import trends and disease risks, such as requiring a health screen and valid rabies vaccinations for all dogs prior to entry.     

Zoonotic pathogens isolated from wild animals and environmental samples at two California wildlife hospitals

During 2010, 48 states and Puerto Rico reported 6,154 rabid animals and 2 human rabies cases to the CDC, representing an 8% decrease from the 6,690 rabid animals and 4 human cases reported in 2009. Hawaii and Mississippi did not report any laboratory-confirmed rabid animals during 2010. Approximately 92% of reported rabid animals were wildlife. Relative contributions by the major animal groups were as follows: 2,246 raccoons (36.5%), 1,448 skunks (23.5%), 1,430 bats (23.2%), 429 foxes (6.9%), 303 cats (4.9%), 71 cattle (1.1 %), and 69 dogs (1.1 %). Compared with 2009, number of reported rabid animals decreased across all animal types with the exception of a 1 % increase in the number of reported rabid cats. Two cases of rabies involving humans were reported from Louisiana and Wisconsin in 2010. Louisiana reported an imported human rabies case involving a 19-year-old male migrant farm worker who had a history of a vampire bat (Desmodus rotundus) bite received while in Mexico. This represents the first human rabies case reported in the United States confirmed to have been caused by a vampire bat rabies virus variant. Wisconsin reported a human rabies case involving a 70-year-old male that was confirmed to have been caused by a rabies virus variant associated with tri-colored bats (Perimyotis subflavus).     

检测狂犬病病毒抗原的夹心间接Dot—ELISA的建立和应用研究

本研究建立了检测狂犬病病毒抗原的夹心间接斑点酶联免疫吸附试验(SI-Dot-ELISA).本法检出狂犬病病毒抗原的最低浓度为:0.01IU/mL的标准抗原,1:100000 (相当于20 LD_(50))的狂犬病病毒CVS株和鹿8202株的鼠脑悬液,1:400(相当于7906TCID_(50)/mL)的狂犬病病毒SAG株的细胞培养物.对多种健康动物的组织、健康细胞培养物以及犬瘟热病毒、犬腺病毒等检测均为阴性.用SI—Dot—ELISA、夹心间接ELISA(SI—ELISA)和小鼠脑内接种3种方法,检测了388份材料,检出的阳性份数分别为173、171和179,经统计学处理,3种方法在狂犬病病毒抗原的检测上,可以相互代替.甲醛灭活病毒不影响本方法的检测,而加入氢氧化铝胶后的病毒悬液则不能用本法检测.本法适用于狂犬病的诊断、流行病学调查、疫苗效价的测定和实验研究中病毒抗原的测定.     

Cell-mediated immune response to rabies virus in dogs following vaccination and challenge

Peripheral blood lymphocytes (PBL) from non-vaccinated dogs and from dogs either vaccinated intramuscularly (IM) or subcutaneously (SC) with an inactivated rabies virus vaccine (Rabguard-TC, Norden Laboratories, Lincoln, NE) or intramuscularly with an attenuated rabies virus vaccine (Endurall-R, Norden Laboratories, Lincoln, NE) were exposed in vitro to rabies virus. Blastogenesis of PBL was measured by incorporation of 3H-thymidine into the DNA of proliferating cells in the presence of a suboptimal concentration of phytohemagglutinin (PHA). Following the first vaccination, there was no difference in the blastogenic response of lymphocytes from dogs vaccinated IM with either the inactivated or attenuated rabies virus vaccines. The inactivated rabies vaccine stimulated as great or greater blastogenic response when it was given SC. The PBL from non-vaccinated control dogs were not stimulated by rabies virus. Dogs vaccinated with the inactivated vaccine developed a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus. Nonvaccinated control dogs did not develop a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus.     

狂犬病病毒BD-06株基质蛋白的原核表达、纯化及其多克隆抗体的制备

本研究旨在获得抗狂犬病病毒M蛋白的多克隆抗体,为进一步研究M蛋白功能提供材料。本试验以狂犬病病毒BD06毒株为模板克隆M基因序列,将其克隆至原核表达载体pET28a;经酶切和测序鉴定,M蛋白基因序列已正确插入表达载体pET28a;以表达载体pET28a-M转化E.coli Rosetta株,并以0.5 mmol/L IPTG诱导,结果表达出27 ku左右的M蛋白;将诱导表达的蛋白质回收纯化,以纯化的M蛋白多次免疫兔子制备多克隆抗体;Western blotting分析和间接免疫荧光分析结果表明,制得的抗体与病毒能够反应,运用制得的多克隆抗体定位了基质蛋白在感染狂犬病病毒的BHK-21细胞中的位置。结果表明成功制得了抗狂犬病病毒M蛋白的多克隆抗体,为研究狂犬病病毒M蛋白功能奠定了基础。     

狂犬病病毒流行病学特征与实验室诊断技术的研究进展

狂犬病是由狂犬病病毒引起的以中枢神经系统感染为特征的一种人兽共患病。狂犬病病毒具有嗜神经性,能在人和多种哺乳动物中引起致死性感染,病死率几乎为100%。除常规的临床诊断外,狂犬病的确诊依赖于实验室诊断方法,方法主要有病原学诊断、血清学诊断和分子生物学诊断方法等。狂犬病作为一种人兽共患烈性传染病,呈全球分布的态势,中国每年因狂犬病病毒感染而导致死亡的人数居全球第2位。因此,加强对该病的病原学、流行病学特征的认识、加快实验室诊断方法的研究对其综合防控工作具有十分重要的意义。作者主要介绍了目前国内外狂犬病流行病学特征和诊断方法的研究进展,并比较了各种方法的优点和缺点,为狂犬病的临床快速诊断和深入研究提供科学依据。     

抗狂犬病毒单克隆抗体的纯化及标记

对一株稳定、特异、高效的单克隆抗体（mAb）进行了纯化和荧光素（FITC）的标记,利用标记产物与已有的抗狂犬病毒核蛋白单克隆抗体标记物相比较,效果良好,二者可以结合（或单独）使用;应用标记好的荧光抗体与FITC-抗狂犬病毒免疫球蛋白比较进行血清样品效价测定实验,来确定纯化、标记后的单克隆抗体的灵敏度,结果表明本实验制备的单克隆抗体与FITC-抗狂犬病毒免疫球蛋白具有相同的灵敏度,该单克隆抗体测得的血清样品效价结果与FITC-抗狂犬病毒免疫球蛋白测得的结果相同。因此,此荧光抗体可应用于狂犬病病毒检测（FAT）、病毒毒力测定（TCID50）、中和抗体实验（FAVN）等方面的检测实验,此荧光抗体可为狂犬病的相关研究提供有力的保障。     

Mokola virus infection: description of recent South African cases and a review of the virus epidemiology

Five cases of Mokola virus, a lyssavirus related to rabies, are described. The cases occurred in cats from the East London, Pinetown and Pietermaritzburg areas of South Africa from February 1996 to February 1998. Each of the cats was suspected of being rabid and their brains were submitted for laboratory confirmation. Four of the cases were positive, but with atypical fluorescence, and 1 was negative. Mokola virus infection was identified by anti-lyssavirus nucleocapsid monoclonal antibody typing. As in rabies cases, the predominant clinical signs were of unusual behaviour. Aggression was present, but only during handling. Four of the 5 cats had been vaccinated for rabies, which is consistent with other studies that show that rabies vaccination does not appear to protect against Mokola virus. Since Mokola may be confused with rabies, the incidence of Mokola virus may be more common in Africa than is currently reported. As human infections may be fatal, the emergence of this virus is a potential threat to public health.     

狂犬病病毒分子流行病学研究进展

介绍了狂犬病病毒的流行特点、基因型和检测方法 ,综述了狂犬病病毒核蛋白和糖蛋白基因的研究进展 ,并简要概述了我国学者对狂犬病病毒N基因和G基因的序列分析比较结果 ,表明我国狂犬病病毒的街毒存在着地域差异。     

Nested PCR检测狂犬病病毒方法的建立及病毒在小鼠体内的移行动态

应用建立的Nested PCR特异地检出狂犬病病毒株CVC、HEP-Flury、ERA、RC-HL、1008、Komatsug-awa的RNA,但对类狂犬病病毒Lagos bat、Duvenhage、Mokola及水泡性口膜炎病毒、轮状病毒、犬瘟热病毒均为阴性。该法敏感性很高,能检出3 TCID50或0.8pg的狂犬病病毒RNA。用该法测定了小鼠脑内感染CVS株后的病毒增殖和移行动态,对感染小鼠的主要内脏器官进行了病毒RNA检测,结果发现小鼠脑内感染CVS 5 d以后在其心、肝、脾、肺等内脏器官均检出了病毒RNA。     

Rabies surveillance in the United States during 2006

During 2006, 49 states and Puerto Rico reported 6,940 cases of rabies in animals and 3 cases in humans to the CDC, representing an 8.2% increase from the 6,417 cases in animals and 1 case in a human reported in 2005. Approximately 92% of the cases were in wildlife, and 8% were in domestic animals. Relative contributions by the major animal groups were as follows: 2,615 raccoons (37.7%), 1,692 bats (24.4%), 1,494 skunks (21.5%), 427 foxes (6.2%), 318 cats (4.6%), 82 cattle (1.2%), and 79 dogs (1.1%). Compared with numbers of reported cases in 2005, cases in 2006 increased among all groups except cattle. Increases in numbers of rabid raccoons during 2006 were reported by 11 of the 20 eastern states where raccoon rabies was enzootic, and reported cases increased by 3.2% overall, compared with 2005. On a national level, the number of rabies cases in skunks during 2006 increased by 6.1% from the number reported in 2005. Once again, Texas reported the greatest number (n = 351) of rabid skunks and the greatest overall state total of animal rabies cases (889). No cases of rabies associated with the dog/coyote rabies virus variant were reported. The last identified case of this canine rabies virus variant was identified in March 2004, along the US/Mexico border. With 2006 marking the second year of no apparent transmission of the dog/coyote variant, these findings from surveillance data support the contention that the canine rabies virus variant is no longer in circulation in the United States. Total number of cases of rabies reported nationally in foxes increased 13.6%, compared with 2005. Increases in the number of reported rabid foxes were attributable to greater numbers of foxes reported with the Arctic fox rabies virus variant in Alaska, the Texas gray fox rabies virus variant in Texas, and the raccoon rabies virus variant in Virginia. The 1,692 cases of rabies reported in bats represented a 14.5% increase, compared with numbers reported in 2005, making bats the second most reported rabid animal behind raccoons. Cases of rabies in cats, dogs, horses and mules, and sheep and goats increased 18.2%, 3.9%, 12.8%, and 22.2%, respectively, whereas cases reported in cattle decreased 11.8%. In Puerto Rico, reported cases of rabies in mongooses increased 9.2%, and rabies in domestic animals, presumably attributable to spillover infection from mongooses, increased 20%. Three cases of human rabies were reported from Texas, Indiana, and California during 2006. The cases in Indiana and Texas were attributed to bat rabies virus variants, whereas the case in California was attributed to an exposure to a dog in the Philippines.     

Distribution of rabies virus in infected mice,vaccinated and submitted to P. acnes as immunomodulator

The lethality and distribution of rabies virus were evaluated in swiss mice experimentally infected with street rabies virus, vaccinated and submitted to immunomodulation by P .acnes (formerly Corynebacterium parvum). The animals were sacrificed at different times,when the different tissues were collected and submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT). The group submitted to vaccination and P. acnes treatment presented a percentage of survival superior to that observed in infected mice only treated with P. acnes. Control infected animals had the lowest survival rates.The distribution of rabies virus in spleen of infected mice, vaccinated and submitted to P. acnes was superior to that verified in infected mice not treated with P.acnes. The increased survival correlated with the distribution of rabies virus in lymphoid tissues, could be interpreted as the consequence of P. acnes activity on macrophages. The results suggest the role of macrophages against rabies virus infection in mice and the importance of vaccination in the post expositive treatment of rabies.     

Rabies surveillance in the United States during 2007

SUMMARY: During 2007, 49 states and Puerto Rico reported 7,258 cases of rabies in animals and 1 case in a human to the CDC, representing a 4.6% increase from the 6,940 cases in animals and 3 cases in humans reported in 2006. Approximately 93% of the cases were in wildlife, and 7% were in domestic animals. Relative contributions by the major animal groups were as follows: 2,659 raccoons (36.6%), 1,973 bats (27.2%), 1,478 skunks (20.4%), 489 foxes (6.7%), 274 cats (3.8%), 93 dogs (1.3%), and 57 cattle (0.8%). Compared with numbers of reported cases in 2006, cases in 2007 increased among dogs, bats, foxes, and skunks while decreases were reported among cattle, cats, and skunks. Increases in numbers of rabid raccoons during 2007 were reported by 11 of the 20 eastern states where raccoon rabies was enzootic, and reported cases increased by 1.7% overall, compared with 2006. On a national level, the number of rabies cases in skunks during 2007 decreased by 1.1% from the number reported in 2006. Texas reported the greatest number (n = 362) of rabid skunks and the greatest overall state total of animal rabies cases (969). No cases of rabies associated with the dog/coyote rabies virus variant were reported. The United States remains free of dog-to-dog transmission of canine rabies virus variants. The total number of cases of rabies reported nationally in foxes increased 14.5%, compared with 2006. Increases in the number of reported rabid foxes were attributable to greater numbers of foxes reported with the Arctic fox rabies virus variant in Alaska, the Texas gray fox rabies virus variant in Texas, and the raccoon rabies virus variant in Virginia. The 1,973 cases of rabies reported in bats represented a 16.6% increase over numbers reported in 2006. Cases of rabies in dogs and in sheep and goats increased 17.7% and 18.2%, respectively, whereas cases reported in cattle, cats, and horses and mules decreased 30.5%, 13.8%, and 20.8%, respectively. In Puerto Rico, reported cases of rabies in mongooses decreased 51.5%, and rabies in domestic animals, presumably attributable to spillover infection from mongooses, increased 25%. One human rabies case was reported from Minnesota during 2007. Although typing of the rabies virus variant in this case was not possible, an investigation of this case indicated a bat as the most likely source of exposure.     

Cerebrospinal fluid analysis of rabid and vaccinia-rabies glycoprotein recombinant, orally vaccinated raccoons (Procyon lotor)

Cerebrospinal fluid obtained from clinically normal free-ranging raccoons was analyzed and compared with CSF obtained from raccoons vaccinated orally with vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine and subsequently challenged peripherally with street rabies virus, and CSF from naive, rabies virus challenge-exposed control raccoons. Significant differences were not found in CSF of free-ranging or V-RG recombinant virus vaccine recipient raccoons, and there was no evidence of CNS invasion by V-RG virus. The CSF of naive, rabies challenge-exposed control raccoons contained high numbers of lymphocytes and monocytes, compatible with rabies virus encephalitis. Although V-RG orally vaccinated challenge-exposed raccoons were protected from lethal rabies virus infection, a mild lymphocytic pleocytosis was evident at 90 days after challenge exposure.     

三株广西狂犬病病毒NS基因和M基因的克隆与序列分析

本研究设计了一对特异性引物NSM1/NSM2,对三株广西狂犬病病毒NS和M基因同时进行了RT_PCR扩增、克隆和测序。同源性分析表明,三株广西野毒NS基因核苷酸同源性为87.2%~98.4%,M基因核苷酸同源性为90.1%~99.7%;与固定毒和狂犬病相关病毒比较,NS基因分别为79.9%~82.8%和69.7%~71.0%;M基因的分别为82.8%~87.8%和75.0%~77.8%。三株野毒NS基因氨基酸同源性为93.3%~98.7%,M基因氨基酸同源性分别为97.5%~100%。表明广西各地毒株之间亲缘关系不同,但最为相近;与狂犬病固定毒株亲缘关系较远;与狂犬病相关病毒亲缘关系最远。     

Adsorption of Wild Rabies Virus and Neurotropic-Adapted Viruses by Activated Attapulgite

The adsorption by attapulgite (a naturally occurring clay) of wild rabies virus in naturally rabid dogs was investigated. Attapulgite selectively absorbed rabies virus in the brain tissue of naturally rabid dogs and rejected rabies virus in extracts from the submaxillary salivary glands of the rabid dogs. Attapulgite rejected the neurotropic-adapted viruses lymphocytic choriomeningitis, Eastern equine and Western equine encephalitis, Japanese B encephalitis, and St. Louis encephalitis in the brain tissue of suckling mice.     

Infection of Bergmann glia in the cerebellum of a skunk experimentally infected with street rabies virus.

Rabies virus is a highly neuronotropic virus and glial cell infection is not prominent in the central nervous system (CNS). Paraffin-embedded tissues from the cerebella of skunks experimentally infected with either a skunk salivary gland isolate of street rabies virus or the challenge virus standard (CVS) strain of fixed rabies virus were examined with immunoperoxidase staining for rabies virus antigen by using an anti-rabies virus nucleocapsid protein monoclonal antibody. A skunk infected with street rabies virus showed prominent infection of Bergmann glia. Although infected Purkinje cells were observed, they usually demonstrated a relatively small amount of antigen in their perikarya. A CVS-infected skunk showed many intensely labeled Purkinje cells and a relatively small number of infected Bergmann glia. These findings indicate that although rabies virus is a highly neuronotropic virus, street rabies virus strains do not always demonstrate strict neuronotropism in the central nervous system.     

Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line.

The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes.