Isolation and propagation of infectious bursal disease virus using the ovine kidney continuous cell line.

Twenty-six samples known to contain infectious bursal disease virus (IBDV) were examined by virus-isolation attempts on ovine kidney (OK) cell line, Vero cell line, and chicken embryo fibroblast (CEF) cultures. Virus was isolated from two of 26 samples, three of 26 samples, and three of 25 samples on OK, Vero, and CEF cultures, respectively. However, in contrast to IBDV replication in Vero and CEF cultures, isolated virus was unable to induce serially sustained cytopathic effects (CPE) during successive passages in the OK cell line, unless cell lysates were treated with chloroform between every other passage. The cytopathogenicity of the untreated virus passaged in OK cells was revived and maintained upon passage in Vero cells. An initial single passage of laboratory or field material in OK cells followed by further passages in Vero cells resulted in virus isolation from six of 26 samples, which was better virus recovery than when either cell line was used alone or when CEF cultures were used. Twenty of the 26 test samples were originally positive when examined by nucleic acid hybridization with radiolabeled IBDV cDNA, indicating that some of the samples that were negative upon virus isolation using OK and Vero cells may have contained inactivated virus.     

Replication of infectious bursal disease virus in continuous cell lines

Three mammalian continuous cell lines--MA-104, Vero, and BGM-70--were tested for their ability to support replication of infectious bursal disease virus (IBDV). Selected tissue-culture-adapted vaccine strains and tissue-culture-adapted field isolates of IBDV replicated in the MA-104, Vero, and BGM-70 cells; cytopathic effects were most pronounced in the BGM-70 cells. The cytopathic effects of the viruses in BGM-70 cells and chicken embryo fibroblast (CEF) cultures were similar. Virus-neutralization titers of selected serum samples determined in BGM-70 cultures compared well with those obtained from CEF cultures.     

Vacuolating cytotoxin produced by avian pathogenic Escherichia coli

The purpose of this study was to determine whether avian pathogenic Escherichia coli produced cytotoxic activity. Culture supernatants of 20 E. coli strains isolated from cellulitis lesions in chickens, five E. coli strains from avian septicemia, five from swollen head syndrome, and five from the feces of healthy chickens were incubated with primary chicken embryo fibroblast (CEF) cells, primary chicken kidney (PCK) cells, a quail fibroblast cell line (QT-35), and four mammalian cell lines (human epithelioid cervical carcinoma, African green monkey kidney, Chinese hamster ovary, and human larynx epidermoid carcinoma). Cytotoxicity was observed with supernatants from the 30 avian pathogenic strains on the two primary chicken cells (CEF and PCK). The highest dilution of culture supenatant that induced cytotoxic changes in 50% of the cells was 1/64. Supernatants from the five strains from normal feces were noncytotoxic, and none of the supernatants was cytotoxic for the QT-35 or the four mammalian cell lines. The cytotoxic effect, which was observed as early as 2 hr after exposure of the cells, was maximal at 6 hr and was evident as vacuolation, morphologically indistinguishable from that previously reported for culture supernatants of Helicobacter pylori. Like the activity in H. pylori, the cytotoxicity of the avian pathogenic strains was destroyed by heating at 70 C for 30 min and by exposure to proteolytic enzymes and was retained by filtration with a 100,000 molecular weight cut-off ultrafilter. Supernatants of two vacuolating cytotoxin-positive cultures of H. pylori failed to induce vacuolation of the CEF and PCK cells but caused the characteristic vacuolation in HeLa and Vero cells. The observations suggest that avian pathogenic E. coli produce a cytotoxin that is similar to the cytotoxin of H. pylori but may be specific for avian cells.     

Isolation of infectious bursal disease virus from turkeys with arthritic and respiratory symptoms in commercial farms in Quebec.

Infectious bursal disease viruses (IBDVs) were isolated from turkeys showing symptoms of arthritis and respiratory disease in commercial poultry farms in the province of Quebec, Canada. Synovial fluids collected from hock joints of arthritic birds and peripheral blood leukocytes obtained from the birds with respiratory problems were used for virus isolation in embryonated chicken eggs, and Vero and BGM-70 cell cultures. The infected cells were evaluated for the presence of IBDV by indirect immunofluorescence assay using monoclonal antibodies. The viruses were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral genome and by electron microscopy. Although one of these turkey isolates tested was neutralized by serotype 1-specific commercial chicken antisera, preliminary results indicated that there are antigenic differences between the Quebec isolate, IBDV QT-1, and the existing strains of IBDV belonging to serotype 1.     

Growth of serotypes I and II and variant strains of infectious bursal disease virus in Vero cells

The growth of five strains of infectious bursal disease virus--three strains of serotype I (SAL, D-78, 2512), one of serotype II (OH), and one variant strain (Variant-A)--were compared in Vero and chicken embryo fibroblast (CEF) cell cultures in order to characterize the replication of different strains of IBDV in Vero cells. For all five virus strains, the latent period in Vero cells ranged from 12 to 18 hr, which was longer than the 4-to-6-hr latent period observed in CEF cultures for strains SAL, D-78, and OH. Virus strains SAL, D-78, and OH, which were examined in both Vero and CEF cultures, also had a more extensive maturation phase and higher yields of virus in Vero than in CEF cultures. Total titers of these viruses of 5.35 to 6.10 log10 TCID50/ml in CEFs occurred 24 to 30 hr postinoculation (PI), although the cytopathic effect (CPE) was not seen until 72 hr PI. By comparison, their total infectious virus titers of 6.85 to 8.35 log10 TCID50/ml in Vero cells occurred from 48 hr PI, coinciding with the appearance of CPE. The growth curve of Variant-A in Vero cells differed from the other viruses by showing steadily rising extracellular and cell-associated virus titers throughout the 72-hr observation period. Only very low titers of Variant-A were obtained in CEF cultures, and thus no growth curve in CEFs was performed.     

Isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate.

Porcine epidemic diarrhea virus (PEDV) was isolated in Vero cell cultures from the small intestine of a piglet experimentally infected with porcine coronavirus 83P-5, that had been isolated during outbreaks of porcine acute diarrhea and passaged in piglets. The isolation of the PEDV was successful only in Vero cells maintained in the maintenance medium (MM) containing trypsin. Infected Vero cell cultures exhibited CPE characterized by cell-fusion and syncytial formation, as well as cytoplasmic fluorescence when examined by the indirect immunofluorescent test using rabbit anti-83P-5 virus serum. The isolate was adapted to serial propagation in Vero cell cultures by adding trypsin to MM. Vero cell-adapted PEDV was successfully propagated in the MA104, CPK and ESK cell lines in the presence of trypsin in MM. Vero cell-adapted PEDV had morphologic and physicochemical characteristics similar to those of other members of the coronaviridae. The isolate differed serologically from porcine transmissible gastroenteritis (TGE) and porcine hemagglutinating encephalomyelitis viruses, and no antigenic relationship between the isolate and TGE virus could be detected by the indirect immunofluorescent test. Attempts to isolate PEDV in 6 types of primary fetal pig cell cultures and 6 of 10 established cell lines resulted in the failure, probably because these cells were damaged by the action of trypsin.     

Isolation of serotype 2 Marek's disease virus from birds belonging to genus Gallus in Japan

Serotype 2 of Marek's disease virus (MDV) was isolated from apparently healthy birds belonging to genus Gallus that had no history of vaccination with MDV or herpesvirus of turkeys (HVT). Buffy-coat cells from these birds were inoculated onto chicken embryo fibroblast (CEF) cultures for primary isolation. Thirteen isolates from one golden pheasant and three white silky fowls, three black silky fowls, three Japanese long crowers, and three Japanese bantams produced herpes-like cytopathic effects (CPE) in the CEF cultures. Using serotype-specific monoclonal antibodies to MDV and HVT, 11 isolates were identified as serotype 2 MDV by indirect fluorescent antibody tests. The other two isolates were complicated with serotypes 1 and 3 of MDV-related viruses. Of 13 isolates, three cloned by the limiting-dilution method were further characterized as serotype 2 MDV biologically, genetically, and serologically. The results showed that the birds of the genus Gallus were naturally infected with serotype 2 MDV. This is the first report ever published about the distribution of serotype 2 MDV among healthy birds of the genus Gallus.     

犬瘟热病毒在不同组织细胞上增殖特性的研究

将犬瘟热病毒(CDV)分别接种于长满单层的鸡胚成纤维细胞(CEF)、非洲绿猴肾细胞系(Ve-ro)、犬肾细胞系(MDCK),观察其病变时间、病变特征,并对其进行RT-PCR鉴定和TCID50测定。结果表明,CDV在这3种细胞上均能增殖,并产生明显的细胞病变(CPE),但在Vero细胞上产生病变的时间短,病变明显,且TCID50最高;RT-PCR检测CDV在不同细胞上的病毒培养液,均能扩增出337bp的片段;CDV在Vero细胞上的增殖滴度明显高于在CEF、MDCK细胞上的增殖滴度。表明本试验所选用的3种细胞中,Vero细胞最适宜培养CDV,所获得的CDV的毒价最高,病变最明显。     

Adaptation of Marek's disease virus to the Vero continuous cell line

Marek's disease virus (MDV) is a highly infectious, cell-associated oncogenic herpesvirus. Production of MD vaccines has been limited to primary chicken and duck embryo fibroblast (CEF and DEF) cultures. These have a limited life span and cannot be readily stored in liquid nitrogen. Moreover, the need to prepare CEF and DEF cells on a regular basis from 10 to 11 day-old embryos derived from a flock that must be tested continuously for the presence of avian pathogens adds to the cost of vaccine production. A continuous cell line that would support MDV replication could have significant advantages for the rapid large-scale preparation of MD vaccines. In this report, we describe the adaptation to growth of CEF-grown preparations of serotype 1 and serotype 3 (herpesvirus of turkeys; HVT) strains of MDV in cells of the Vero continuous cell line. Although both viruses produced typical CPE, higher levels of infectious progeny and more extensive virus-specific immunofluorescence were obtained for HVT than for the serotype 1 virus. PCR and pulsed field electrophoresis (PFE) analysis of the DNA from Vero cells infected with either virus confirmed the presence of virus-specific DNA.     

A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.     

水貂犬瘟热病毒LD-1株的分离与鉴定

取山东某貂场疑似犬瘟热病毒（canine distemper virus,CDV）感染的水貂肝脏等组织病料,通过RT-PCR检测呈CDV阳性,且无水貂细小病毒存在,将病料接种原代CEF细胞、传代系Vero细胞和DF1细胞3种细胞进行病毒分离,通过优化细胞培养条件,最终在Vero细胞上传代培养成功,出现露珠状典型细胞病变（CPE）。分离毒应用RT-PCR、PCR产物测序、抗体中和试验（SN）、间接免疫荧光试验（IFA）及病毒包涵体检查等多种方法进行了CDV的鉴定,结果显示CDV阳性,表明分离到的病毒为水貂CDV,并将其命名为CDV LD-1株。     

Rapid detection of avian pneumovirus in tissue culture by microindirect immunofluorescence test.

An indirect immunofluorescence (IFA) test with a 96-well, flat-bottomed microplate was developed to detect avian pneumovirus (APV) antigen in Vero cell cultures. Samples of nasal turbinates and swabs from infraorbital sinuses and trachea were collected from 4-week-old poults experimentally inoculated with APV. The APV titers by tissue culture IFA staining were compared with that of visual reading of cytopathic effect (CPE). The ability of IFA staining to detect APV antigen correlated well with visualizing CPE. The use of IFA staining of Vero cell cultures allowed detection of APV in substantially less time than the use of visualizing CPE. In addition, the use of IFA allowed specific identification of the virus in cell culture.     

Profiles of toxin production by thermophilic Campylobacter of animal origin.

Seventy-five strains of Campylobacter jejuni and C. coli, which were isolated from a variety of animal species, primarily poultry, were examined for production of toxin. Polymyxin extracts were tested in in vitro assays using CHO-KI, FCL (foetal calf lung), Vero, HeLa and CEF (chicken embryo fibroblast) cells. The toxic effects observed were cell rounding and death. Extracts from almost all C. jejuni and C. coli strains were toxic to both CHO-KI and FCL cells and 69.0% of C. jejuni isolates and 75% of C. coli isolates were also toxic to CEF cells. 50.7% of C. jejuni extracts were toxic to Vero cells and 46.5% toxic to HeLa cells. None of the C. coli isolates were toxic to either of these cell lines. None of the strains tested produced cytotonic enterotoxin. No differences in toxigenicity patterns were evident between Campylobacter isolated from different sources.     

First isolation of Neospora caninum from the faeces of a dog from Portugal

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of γ-interferon knockout mice with 10² sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.     

三黄鸡临床多发性肿瘤相关的J亚群禽白血病病毒的分离鉴定及其病理学观察

为研究蛋鸡多发性肿瘤的病因,本实验对病鸡肿瘤组织进行病毒分离、培养及PCR检测,均扩增出针对J亚群禽白血病病毒(ALV-J)gp85基因序列的阳性条带;组织病理学观察显示发病鸡呈现髓细胞瘤、血管瘤以及纤维肉瘤等多发性肿瘤的病理变化;肿瘤细胞通过血液转移、浸润,在肝和脾组织形成局灶性或弥漫性肿瘤病灶。免疫组化染色显示肿瘤组织内,部分肿瘤细胞呈现阳性反应,表明只有部分肿瘤细胞存在ALV-J感染,而大部分肿瘤细胞检测结果呈阴性。这些病毒检测为阴性的肿瘤细胞可能是正常细胞转化为肿瘤细胞大量克隆化增殖的结果。     

Evaluation of five different antigens in enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Five different antigens were evaluated in enzyme-linked immunosorbent assay (ELISA) tests for the detection of avian pneumovirus (APV) antibodies. Two of the 5 antigens were prepared from recent APV isolates from Minnesota. The 2 older isolates were passage 63 of a strain currently used as a live, attenuated vaccine and a Colorado strain isolated for the first time in the United States and currently used in an ELISA test. The fifth antigen is based on an APV recombinant N-protein. Basic parameters and positive-negative threshold of the assays were established for all 5 antigens on the basis of data obtained by testing 46 known negative and 46 known positive serum samples. Subsequently, 449 field samples were tested by all 5 ELISAs. The optical density difference (ODD) was calculated by subtracting optical density of the sample in the negative antigen well from that in the positive antigen well. In the current ELISA test based on the Colorado strain, an ODD of 0.2 is considered to be the cutoff value to classify samples as negative or positive. In this study, however, use of different cutoffs, based on ODD of negative control plus 3 SD or values estimated from Receiver operating characteristic analysis, was considered to be more appropriate for the various antigens used. Overall person-to-person and day-to-day variability was found to be large for all tests using either ODD or sample to positive ratio to report results. In addition, results suggest that antigenicity of the APV isolates in the United States has not changed between 1997 and 2000.