Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells

Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.     

Isolation and trypsin-enhanced propagation of turkey enteric (bluecomb) coronaviruses in a continuous human rectal adenocarcinoma cell line

Turkey enteric coronavirus (TCV) from intestinal contents of diarrheal poults was isolated and serially propagated in HRT-18 cells, an established cell line derived from a human rectal adenocarcinoma. In these cells, TCV induced cytopathic changes, including polykaryocytosis, which depended on trypsin in the medium and incubation at 41 C. Viral antigens could be demonstrated in the cytoplasm by immunofluorescence, and extracellular virus was detected by an ELISA and negative electron microscopy. The cell-free virus had characteristics of TCV: shape, surface projections, buoyant density of 1.18 to 1.20 g/ml in sucrose, and hemagglutination of rat RBC. The one-step growth curve was complete by postinoculation hours 14 to 16, and maximal titers reached 9 to 9.5 log10 TCID50/ml during 5 passages, after which the titer remained stable. Electron microscopic examination of infected cell monolayers revealed budding of typical coronavirus particles through intracytoplasmic membranes and accumulation of complete virus in cytoplasmic vesicles. Late in the infection, aggregated progeny vial particles were detected near the outer surface of infected cells. One-day-old turkey poults inoculated orally with tissue culture-adapted TCV isolates developed mild to severe diarrhea.     

Primary isolation and identification of avian rotaviruses from turkeys exhibiting signs of clinical enteritis in a continuous MA 104 cell line

Avian rotaviruses were isolated from turkeys with enteritis using MA 104 cell line. MA 104 cells were suitable for primary isolation and propagation of avian rotaviruses. Trypsin appeared essential for the enhancement of infectivity and the occurrence of cytopathic effect (CPE). Serum neutralization (SN), electron microscopy (EM), and analysis of genomic RNA were done to identify and confirm the identity of rotaviruses. Electrophoretic migration patterns of genomic RNA from avian rotaviruses were examined, and they were compared with those from mammalian rotaviruses. The migration patterns differed between these groups.     

Campylobacter-induced fetal death in a rhesus monkey

A pregnant 4-year-old rhesus monkey (Macaca mulatta) was presented with a history of acute vaginal bleeding. Physical examination revealed an open cervix. An ultrasound scan demonstrated a viable early third-trimester fetus, approximately 16 weeks of gestational age. Hematology results showed that the monkey was anemic, with a normal leukogram and D?hle bodies. A subsequent cervical culture was positive for Campylobacter fetus. The fetus died 3 days later, and a necropsy of the fetus and placenta was performed. Microscopic examination of the placenta revealed villitis, perivillitis, and deciduitis with S-shaped and gull wing-shaped bacteria. C. fetus was considered the cause of the placental lesions and fetal death; however, the pathogenesis of the infection (hematogenous vs. ascending from the maternal genital tract) was not clear. This is the first report of a Campylobacter-induced fetal death in the rhesus monkey. Because macaques can be asymptomatic carriers and Campylobacter-induced diarrhea is common, this finding has implications for breeding success in nonhuman primate breeding colonies.     

Propagation of avian rotavirus in primary chick kidney cell and MA104 cell cultures

The Ch2 strain of avian rotavirus was propagated in primary chick kidney cell (CKC) and MA104 cell cultures in the presence of trypsin. Cultures were evaluated for the presence of rotavirus by an indirect fluorescent antibody (IFA) assay and by an indirect enzyme-linked immunosorbent assay (ELISA). After two passages, the viral titer was significantly higher in CKC than MA104 cell cultures. Also, the IFA assay was more sensitive than the indirect ELISA for detecting rotavirus-positive cultures.     

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.     

Attachment and infection to MA104 cells of avian rotaviruses require the presence of sialic acid on the cell surface

To determine the characters of receptors on target cells for avian rotaviruses, the receptors on MA104 cells for the pigeon rotavirus PO-13, the turkey rotaviruses Ty-1 and Ty-3, and the chicken rotavirus Ch-1 were analyzed. Pretreatment of MA104 cells with neuraminidase greatly reduced the infection by all of the four avian rotavirus strains. Binding of the cell-attachment protein, purified VP8 expressed in bacteria, of strain PO-13 to MA104 cells was also inhibited by pretreatment of cells with neuraminidase. These findings suggest that avian rotaviruses primarily utilize sialic acid-containing molecules as receptors on MA 104 cells.     

Susceptibility of a new fetal pig kidney cell line (MVPK-1) to foot-and-mouth disease virus.

The Mengeling-Vaughn Porcine Kidney (MVPK-1) cell line, derived in October, 1970, from fetal pig kidneys, is susceptible to all 7 types of foot-and-mouth disease (FMD) virus. A plaque assay was developed for FMD virus that depended on washing MVPK-1 cells in serum-free medium before infection and excluding serum from 0.6% gum tragacanth overlay during plaque formation. The numbers of plaques that formed on MVPK-1 cells by a representative strain of each FMD type were comparable with the numbers of those on primary bovine calf kidney (BK) cells. Virus passaged in BK cell cultures did not have to be adapted to the cell line to obtain these results. The cell line lost susceptibility rapidly at 37 C after confluency was reached but retained susceptibility if maintained at room temperature. The cell line has the potential of replacing BK cells for many diverse purposes.     

A spontaneous uterine sarcoma in a rhesus monkey (Macaca mulatta)

In primates, little has been reported about malignant mesenchymal uterine tumours. A case of a spontaneous metastasising uterine sarcoma in a 17-year-old rhesus monkey is presented. Clinically, transient abdominal pain, spasms, nausea, anaemia, a firm uterus and bloody vaginal discharge were noted. In a diagnostic laparoscopy, both massive adhesions in the lesser pelvis and 10 ml of ascites fluid were detected. In necropsy, in addition to peritonitis with massive adhesions, a cauliflower-shaped, irregular, tough, greyish-white uterine tumour was seen. Two cherry-sized tumour metastases were noticed in the greater omentum. In histology, both in the uterus and the metastases, a sarcoma with a low amount of connective tissue and well-differentiated cell nuclei was identified.     

Continuous in vitro propagation of Cowdria ruminantium (Welgevonden stock) in a canine macrophage-monocyte cell line

The Welgevonden stock of Cowdria ruminantium, aetiologic agent of heartwater, was continuously propagated in DH82 cells, a continuous canine macrophage-monocyte cell line. Cultures of DH82 cells were readily infected provided that the culture medium was supplemented with cycloheximide. Cultures were split at regular 3-day intervals and infection rates ranged between 60% and 95%. Cultures were continuously propagated through more than 125 passages over a period of more than one year.