The fliU and fliV genes are expressed as a single ORF in Salmonella choleraesuis.

A DNA fragment carrying flagellar genes was cloned from Salmonella choleraesuis. Compared to the corresponding DNA fragment of Salmonella muenchen, this fragment contained three ORFs instead of four shown in S. muenchen. The DNA sequence data showed that there was an insertion of nucleotide C in the ORF of the S. choleraesuis fliU gene, which resulted in the disappearance of a termination codon downstream. The recombinant plasmid pFU11 containing the coding region of the fliU gene made by PCR on S. choleraesuis genomic DNA was constructed and expressed in Escherichia coli in the presence of IPTG. As expected, a 45 kDa protein band was observed on a SDS-PAGE gel, in contrast to two with each having about a half of the molecular weight. These results demonstrated that the DNA sequence encoding one protein (FliU) in S. choleraesuis corresponded to the DNA sequence encoding two proteins (FliU and FliV) in S. muenchen. The protein encoded by this single ORF might carry out the functions of two separated proteins by folding in such a way that its conformation could function like two interdependent protein subunits.

Identification of the sigma C-encoded gene of avian reovirus by nested PCR and restriction endonuclease analysis.

A nested reverse transcription (RT)-polymerase chain reaction with subsequent restriction endonuclease analysis was developed for identification of the sigma C-encoded gene of avian reoviruses (ARV). PCR products derived from the sigma C-encoded gene of all tested ARVs resulted in a specific DNA band of 1023 bp, indicating that there were no apparent insertions or deletions in this region. Amplification with the nested primer pairs S1M-S1N and S1P-S1N generated 330 and 239 bp, respectively. PCR products amplified from the sigma C-encoded of all tested ARVs isolates were further confirmed by Southern blot hybridization and restriction endonuclease analysis. PCR amplified cDNA fragment (1023 bp) cleaved with Pst I generated two fragments of 565 and 458 bp. The amplified sigma C-encoded gene of ARV was subcloned into PQE 32 vector for further study of its antigenicity and immunogenicity. The sensitivity of RT-PCR was examined on nucleic acids from the ARV infected cell cultures. The detection limit was 10(0) to 10(-1) TCID50 of ARV in a ethidium bromide stained gel and could be increased further to 10(-1) to 10(-2) TCID50 of ARV by Southern blot hybridization using a digoxigenin-labeled cDNA probe. The sensitivity increased approximately 10(3) to 10(4) folds when the cDNA was reamplified with two sets of nested primers.

Naturally-occurring adenovirus-associated gastrointestinal lesions in Coturnix (Coturnix coturnix) quail.

Farm-reared Coturnix quail less than 3 weeks old showed depression, ruffled feathers, diarrhoea and high mortality. Histological examination revealed basophilic intranuclear inclusion bodies mainly in the digestive tract, and rarely in the liver, kidney, nasal epithelium, conjunctiva and columnar epithelial cells within the mucosa of the bursa of Fabricius. The inclusions were more numerous in the caeca than in the small intestine. Ultrastructurally, they contained many adenovirus-like particles approximately 60 nm in diameter. This is the first evidence of adenoviral inclusions in the glandular epithelium of the gizzard, conjunctiva, plical epithelium of bursa of Fabricius, and mucosal epithelium of small and large intestines in Coturnix quail.

Epidemiology of swine toxoplasmosis in Taiwan.

From July 1987 to June 1988, serum samples from 3,880 pigs from eight geographic locations in Taiwan were examined for Toxoplasma gondii antibodies using the latex agglutination test (LA test) and IgM-enzyme-linked immunosorbent assay (IgM-ELISA). A total of 1,073 samples (27.65%) were positive by the LA test. The percentage of positive reactions varied by location as follows: Taoyuan 44.44% (128/288), Taichung 27.60% (183/663), Tainan 22.28% (119/534), Kaohsiung 19.60% (98/500), Pingtung 17.92% (86/480), Hualien 33.95% (163/480), Ilan 31.66% (152/480), Taitung 31.64% (144/455). In the IgM-ELISA 1,828 of 3,880 samples (47.11%) were positive and the distribution of positive reactions were: Taoyuan 59.02% (170/288), Taichung 53.69% (356/663), Tainan 52.24% (279/534), Kaohsiung 54.60% (273/500), Pingtung 18.95% (91/480), Ilan 47.50% (228/480), Hualien 42.70% (205/480), Taitung 49.67% (226/455). On one farm, 20 of 120 sows experienced abortion and stillbirths due to Toxoplasma gondii. Lesions and T. gondii were found in lungs, liver, kidneys, heart, and placenta of one of the aborted fetuses.

Simple hemagglutination inhibition test for the diagnosis of toxoplasmosis.

A simple hemagglutination inhibition (HI) test for the serological diagnosis of toxoplasmosis has been developed and evaluated. A total of 84 human and 120 mouse serum samples were tested by the newly developed HI test and compared with an immunoglobulin G-indirect fluorescent antibody test. Statistical analysis of serum titers obtained by using the HI test and the immunoglobulin G-indirect fluorescent antibody test showed a correlation coefficient of 0.89. The diagnostic efficacy of HI when compared with the immunoglobulin G-indirect fluorescent antibody diagnostic test results was 96.43% for human sera and 100% for mouse sera. The unique hemagglutination antigen, derived from Toxoplasma gondii (Rh strain) exotoxin, spontaneously binds with mouse or rat erythrocytes, causing the hemagglutination reaction. In this study, 2, 4, or 8 hemagglutinating units of T. gondii exotoxin was used with Swiss/Webster mouse erythrocytes as an indicator for the HI assay. The results indicate that 8 hemagglutinating units is optimal because this concentration has the least unexplained variability. T. gondii exotoxin was stable for at least 18 months at -70 degrees C. The Toxoplasma HI test we report in this paper is shown to be a fast, easy, highly specific, and sensitive test for the diagnosis of toxoplasmosis.

Toxoplasma gondii: growth in ovine fetal kidney cell cultures.

Serial, in vitro passage of Toxoplasma gondii (Rh strain) was successfully performed in a cell line derived from ovine fetal kidney cells. Invasion of this parasite into the kidney cells was easily discernible 1 hr after inoculation. The subsequent proliferation of the parasite was followed in the cytoplasm of the kidney cells. Very active endodyogeny and rosette formations, as many as 13 in a cell, were observed in the cytoplasm of the kidney cells 48 hr postinoculation. After 96 hr of incubation, the parasite population had increased about 132-fold. The virulence of T. gondii against mice was not attenuated after 2 years of in vitro growth which represented 100 serial passages through the kidney cell cultures. Although no "exotoxin" was produced by T. gondii grown in vitro, a Toxoplasma sp. agar gel immunodiffusion test antigen was isolated from the cell-free supernatant fluid of the kidney cell cultures which was identical to an antigen isolated from "toxogenic" organisms harvested from infected mice.

Preparation of a Toxoplasma gondii agar gel immunodiffusion test antigen from ovine fetal kidney cell cultures.

A toxoplasma gondii antigen prepared from the cell-free supernatant of ovine fetal kidney (OFK) cell cultures produced a sharp precipitation line against anti-Toxoplasma serum in an agar-gel immunodiffusion (AGID) test. Results obtained from the AGID and indirect hemagglutination (IHA) tests on 46 human and nine rabbit sera were compared. Thirty-three percent of the serum samples, which were negative for the Toxoplasma IHA test, were positive when assayed by the AGID method. The AGID test appears to be superior to the IHA test for detecting low titers of antibodies against T. gondii. After 70 generations of serial passages through the OFK cell cultures, T. gondii still produced the AGID test antigen.