定量测定羊关联性恶性卡他热病毒DNA的竞争性PCR方法的建立

此项研究中建立了用于定量测定羊关联性恶性卡他热 (Sheep- associated malignant catarrhal fever,SA- MCF)病毒 DNA的竞争性聚合酶链反应 (Comppetitive polymerase chain reaction,c PCR)方法。该分析中采用同一引物 ,分别将一固定但未知含量的待测目的 DNA(target DNA)与一系列已知含有不同拷贝数量的竞争性 DNA(com petitive DNA)同时扩增。经4 0次扩增后 ,在 30～ 30 0 0 0 0 DNA拷贝范围内 ,目的 DNA与竞争性 DNA之间其扩增产物具有明显的线性竞争关系 (r=0 .98)     

Outbreak of wildebeest-associated malignant catarrhal fever in Ankole cattle

During an outbreak of malignant catarrhal fever in a herd of Ankole cattle in a zoological collection, two adult cows and one adult bull from a herd of 15 died or were euthanased between July and November 2004. The clinical, gross postmortem and histological findings were typical of the disease in uk native domestic cattle. The diagnosis was confirmed by serology in two animals, and by pcr in all three; the pcr provided evidence of alcelaphine herpesvirus type 1 infection in all three animals and also of ovine herpesvirus type 2 infection in one.     

Suspected sheep-associated malignant catarrhal fever in a zero-grazed dairy herd in Kenya

An outbreak of a disease characterised by very high mortality occurred in a group of nine calves (1B4 months old) in a zero-grazing unit 2-3 weeks after an introduction of an apparently healthy alien sheep into the calf pen. Five of the six calves which contracted the disease died. The main clinical signs observed were marked depression, persistently high body temperature (40,5-41,5 degrees C), copious mucopurulent nasal and ocular discharges, dyspnoea, bilateral keratoconjunctivitis with corneal opacity, enlargement of the superficial lymph nodes and marked erythema and/or superficial erosions of the buccal mucosae. At necropsy there were lesions in the upper respiratory and digestive tracts, lymph nodes, brain, eyes, liver, kidneys and the urinary bladder.The lesions were histopathologically characterized by fibrinoid vasculitis which was accompanied by lymphocytic infiltration in the parenchyma of the affected tissues. Based on the evidence of contact between the calves and the recently introduced foreign sheep, the characteristic clinical signs and histopathological findings, a diagnosis of sheep-associated malignant catarrhal fever was made.     

Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples.

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.     

Studies concerning etiology of sheep-associated malignant catarrhal fever in Europe.

The etiological agent of sheep-associated malignant catarrhal fever (MCF) in Europe has not been isolated directly from sheep. The occurrence of antibodies against the African bovine herpesvirus (BHV-3, WC 11) in cattle and sheep was examined using recently modified indirect immunofluorescence test (IIFT) and neutralization test (NT) methods. Studies revealed that sheep and cattle sera in Europe were free from neutralizing antibodies of BHV-3. The IIFT could not establish the presence of antibodies to African BHV-3 in cattle but revealed that about 22.4% of sheep sera reacted to it. Apart from the well known ovine herpesvirus (BHV-5), occurrence of another herpesvirus in Europe had been expected. This virus is not identical with the WC 11 strain, but it is in antigenic relationship to it. We had for the first time substantial serological evidence to the effect that sheep-associated MCF in Europe is a herpesvirus related to the African strain.     

Quantitation of sheep-associated malignant catarrhal fever viral DNA by competitive polymerase chain reaction.

A single-step, competitive polymerase chain reaction technique was developed to quantitate sheep-associated malignant catarrhal fever (SA-MCF) viral DNA. The assay employed coamplification of a fixed quantity of target DNA with graded amounts of a competitor, generated by truncation of the target sequence lying between the 2 primer binding sites. The assay yielded a linear response (r = 0.98) for DNA measurement within the range of 30-300,000 copies. Amplification efficiency analysis by coamplification of target and competitor in equal copy numbers for various numbers of cycles showed that the relative abundance of the coamplified products remained constant with increasing cycle numbers up to 40. Reproducibility was assessed by repetitively assaying a set of blind-coded samples from a variety of animals and tissues. Results indicated that the assay is reliable and reproducible for quantitation of SA-MCF viral DNA in samples from asymptomatically infected sheep and from animals with clinical SA-MCF.     

Derivation of a DNA clone corresponding to the viral agent of sheep-associated malignant catarrhal fever

Malignant catarrhal fever is a fatal lymphoproliferative and degenerative disease of ruminants. One causative agent is the gammaherpesvirus alcelaphine herpesvirus 1 (AHV-1), which produces no disease in its natural host, the wildebeest (Connochaetes species). Epidemiological evidence implicates sheep as the carrier of a similar virus. However, attempts to culture this virus from sheep or from animals affected with sheep-associated malignant catarrhal fever (SA-MCF) have failed. Lymphoblastoid cells have been propagated from cattle, deer and rabbits with SA-MCF. Although these cells show no evidence of viral particles or antigens, hybridisation experiments now show that they contain DNA sequences homologous to those of AHV-1. A genomic library was constructed from one of these lymphoblastoid cell lines and a clone identified which hybridised to cloned AHV-1 DNA. The authors believe that this clone contains part of the SA-MCF viral genome, and that the SA-MCF virus and AHV-1 are closely related gammaherpesviruses.     

Shedding of malignant catarrhal fever virus by wildebeest calves

Malignant catarrhal fever virus (MCFV) was isolated from nasal and ocular secretions of wildebeest calves up to 3 months old. The virus was also isolated from explant cultures of cornea and nasal turbinates. It is suggested that MCFV replicates in the cornea and turbinates of young wildebeest calves less than 4 months old.MCFV was not isolated from secretions of calves older than 3 months, but virus neutralizing antibodies were found in their nasal secretions. The appearance of antibodies in the nasal secretions coincided with the cessation of virus shedding. The failure of calves over 3 months old to shed MCFV might explain the seasonal nature of bovine MCFV infection.     

Diagnosis of malignant catarrhal fever by PCR using formalin-fixed, paraffin-embedded tissues.

A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp fragment of ovine herpesvirus 2 [OHV-2] genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic laboratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for >45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old.     

Cloning and characterization of a genomic probe for malignant catarrhal fever virus

A genomic probe specific for malignant catarrhal fever (MCF) virus was cloned by using purified viral DNA from MCF-virus strain WCll. Restriction endonuclease analysis of the purified viral DNA was used to identify the cloned viral genomic fragment. Dot blot hybridization by use of the genomic probe (pRP-5) indicated that the probe hybridized specifically with WCll-MCF virus, as well as with one other isolate of MCF-associated herpesvirus. Hybridization also was observed to a non-MCF virus strain of bovine herpesvirus.     

Concurrent malignant catarrhal fever and bovine virus diarrhoea virus infection in a dairy herd

An account is given of an outbreak of malignant catarrhal fever which occurred in a 98-cow dairy herd. Ten animals died or were slaughtered and the disease was confirmed by clinical and histological examination. Serological tests for malignant catarrhal fever virus were positive in three of four animals. The diagnosis of malignant catarrhal fever was complicated by the presence of bovine virus diarrhoea virus infection in three of the early cases. The initial cases of malignant catarrhal fever occurred in a group of nine-month-old calves which were housed in an old milking parlour with 19 pedigree Suffolk ewes at lambing time. Later cases occurred in two adult cows and in two heifers. Investigations of the remainder of the herd for evidence of bovine virus diarrhoea virus did not reveal the presence of any persistently infected cattle. Serological examinations for antibody to malignant catarrhal fever and bovine virus diarrhoea virus were carried out on the 19 Suffolk ewes. Six of them had neutralising antibody titres to malignant catarrhal fever virus and three were positive in the indirect immunofluorescence test. The possible roles of bovine virus     

Experimental transmission of sheep-associated malignant catarrhal fever from sheep to Japanese deer (Cervus nippon) and cattle

The assumption that sheep carry ovine herpesvirus-2 (OvHV-2), the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), is widely accepted, albeit OvHV-2 has not been isolated. We attempted experimental contact transmission of MCF from Japanese sheep persistently infected with OvHV-2 to Japanese deer (Cervus nippon) and cattle. In Experiment 1, a deer was kept in close quarters with an infected ewe. In Experiment 2, a second deer was kept with the same ewe. In Experiment 3, two cows were each kept with two infected wethers. In Experiment 1, the deer developed clinical signs at 138 days after first contact and then died. OvHV-2 genes by polymerase chain reaction (PCR) and fluorescent antibodies to Alcelaphine herpesvirus-1 were detected in the affected deer. Moreover, sequences of PCR products (423bp), obtained by amplification of materials from the sheep and from the affected deer, coincided. These results clearly confirmed that the sheep was a carrier of OvHV-2, and that this virus had induced SA-MCF in a deer. In other experiments, no OvHV-2 infection occurred in deer and cattle during the 6-18 months periods of contact, though viral genes were detected in the nasal swabs and white blood cells of the sheep. To our knowledge, this is the first report on successful experimental transmission of MCF from OvHV-2-infected sheep to deer.     

Target cells for malignant catarrhal fever virus in rabbits

Lymphocytes from lymph nodes and spleens of malignant catarrhal fever virus (MCFV) infected rabbits were tested for MCFV infectivity in secondary calf thyroid cell monolayers. Most infectivity was demonstrated in the lymphocytes. Some infectivity was also detected in macrophages/monocytes. It was thus concluded that lymphocytes form the major target cell for the herpesvirus of malignant catarrhal fever in rabbits.     

牛附红细胞体单管套式PCR检测方法的建立

为快速、准确地检测出牛附红细胞体,根据GenBank上发表的牛温氏附红细胞体(Mycoplasma wenyonii)l6S rRNA基因序列(登录号AF016546)设计合成内外2对引物,建立了牛附红细胞体单管套式PCR检测方法,并进行了特异性、敏感性及应用试验。结果:建立的牛附红细胞体单管套式PCR检测方法扩增的片段大小为361 bp,与GenBank中相应序列同源性为98%,该方法扩增不出牛瑟氏泰勒虫、新孢子虫、布氏杆菌及牛无乳链球菌等基因片段,检测出标准模板DNA的最小量为1.22 fgμ/L。通过对吉林省延边地区66份血液样本的临床检测显示,单管套式PCR检出率28.8%,高于常规PCR的21.2%和鲜血压片镜检的12.1%,具有准确、特异、敏感等优点,是检测牛附红细胞体的一种新型、可靠的诊断技术。     

Infectivity of cell-free malignant catarrhal fever virus in rabbits and cattle

Malignant catarrhal fever was induced in cattle and rabbits inoculated intranasally with cell-free wildebeest-calf nasal and ocular secretions. This method of reproducing the disease is similar to that occurring in nature and should prove useful as a means of challenging experimentally induced immunity.     

Detection of African malignant catarrhal fever virus antigens in cell cultures by immunofluorescence

A fluorescent antibody (FA) test for antigens of African bovine wildebeest-derived malignant catarrhal fever virus was developed. Serum from one of the few survivors of the experimental disease in steers was used to prepare the conjugate. Both a virulent and an attenuated strain of malignant catarrhal fever virus were used to infect bovine thyroid cell cultures. Cells infected with both strains were readily detected by FA staining as early as 24 h post infection, whereas cytopathic effect could be observed by bright-field microscopy only after days 5 or 6 post infection. Controls consisting of normal bovine thyroid cells or infected cells treated with conjugated normal globulins did not show autofluorescence. The reaction was blocked by treatment of infected cells with homologous positive antisera but not by treatment with normal bovine serum or antisera to foot-and-mouth disease, rinderpest, bovine virus diarrhea, Ibaraki, infectious bovine rhinotracheitis, or bovine herpes mammilitis viruses. Treated with African malgnant catarrhal fever virus conjugate did not react.