Isolation of citrate-utilizing variants of Escherichia coli has been demonstrated (5), but no genetic study of citrate-utilizing ability (Cit+) has been reported. This paper deals with isolation of Cit+ E. coli strains from pigeons, pigs and cattle, and the instability and transferability of citrate-utilizing ability in the strains, indicating that the ability is controlled by a plasmid.Thirteen of 36 strains of E. coli isolated from cloacal swabs of 22 pigeons in a pigeonry in Sapporo, Hokkaido, grew on Simmons' citrate agar (Eiken) within 1 to 3 days. Twelve of 33 E. coli strains obtained from 33 composite fecal samples of 33 pens in a piggery in the same district utilized citrate. In addition, one of 24 E. coli strains isolated from feces of seven cattle on a farm in Hokkaido was citrate-positive. These three kinds of animals were kept in epidemiologically-unrelated places. In basic biochemical reactions except citrate utilization, the Cit+ strains could not be differentiated from the Cit-strains and a typical E. coli strain (ATCC11775). The Cit+ strains were resistant to three to six antimicrobial agents including chloramphenicol (CM), and carried thermosensitive R plasmids (3). A genetic study of the R plasmids of the representative strains indicated that the R plasmids showed no fertility inhibition and no restriction against phage lambda, and were classified into incompatibility group H (4).Three Cit+ E. coli strains [HT58 from pigeons, carrying R plasmid conferring resistance to tetracycline (TC), CM, streptomycin (SM) and sulfadimethoxine (SA); KE10 from pigs, carrying R plasmid conferring resistance to TC, CM, SM, SA and kanamycin (KM) ; and C53 from cows, carrying R plasmid conferring resistance to TC, CM, SM and SA] were used in this genetic study. Two methionine-requiring F-derivatives [ML1410 resistant to nalidixic acid (NA) and ML1410RFP resistant to rifampicin (RIF)] of E. coli K-12 strain were employed as recipients. In this paper, only the data of the HT58 strain are tabulated.The effect of passaging Cit+ strains in penassay broth (Difco) at 25 C, 37 C and 42 C was investigated. After each passage, the cultures were streaked onto desoxycholate-hydrogen sulfide-lactose agar plates (DHL, Eiken), and more than 100 colonies grown on the plates were impressed on Simmons' citrate agar plates contain-357
Transmissible gastroenteritis virus grown in primary swine kidney cell cultures agglutinated erythrocytes from chicken, guinea pig and cattle but not erythrocytes from mouse and goose. The optimal incubation temperature was at 4 degrees C. The hemagglutination (HA) reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were established. HI antibody titers of individual pig sera showed a significant positive correlation with their neutralizing antibody titers.
Transmissible gastroenteritis virus was readily adsorbed onto chicken erythrocytes at 4 degrees C. The hemagglutinin thus adsorbed could be eluted from the erythrocytes by incubating in phosphate buffered saline at 37 degrees C. The receptor on chicken erythrocytes for the hemagglutinin was inactivated by neuraminidase and potassium periodate, but not by trypsin, 2-mercaptoethanol and formalin. The hemagglutinin was inactivated by trypsin, papain, pepsin, alpha-amylase, phospholipase C, neuraminidase, formalin, 2-mercaptoethanol, potassium periodate, ethyl ether, chloroform, Tween-80 and beta-propiolactone, but not by sodium deoxycholate and trichlorotrifluoroethane, suggesting that the active component of the hemagglutinin involved glycoproteins. The hemagglutinin was stable at 37 degrees C or lower temperatures but not at 60 degrees C or higher temperatures. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 45,000 x g for 60 minutes. In rate zonal centrifugation of the hemagglutinin preparation on a sucrose density gradient, the hemagglutinin activity showed a sharp peak at 1.19 g/ml coinciding with the peak of infectivity. The activity in the peak fraction seemed to be structurally associated with virus particles.
A reversed passive hemagglutination (RPHA) method was developed for the detection of bovine coronavirus in fecal specimens. Sheep erythrocytes fixed with glutaraldehyde, and then treated with tannic acid were coated with anti-bovine coronavirus rabbit antibodies purified by affinity chromatography using bovine coronavirus linked to Sepharose 4B. The RPHA test was carried out by a microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by bovine coronavirus, but not by bovine rotavirus or enterovirus. The reaction was inhibited by antiserum to bovine coronavirus, confirming the specificity of the reaction. The RPHA test detected bovine coronavirus in 13 of 22 fecal specimens (59 per cent), from natural cases of diarrhea, while the positive rates were only 14 per cent (3/22) and 22 per cent (5/22) for immunofluorescent staining of primary cultures of calf kidney cells infected with the specimens, and immune electron microscopy respectively. The advantages of the RPHA method are its simplicity, high sensitivity and rapidity.
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