Ryo Harasawa

Comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens

A polymerase chain reaction (PCR) assay, which specifically amplifies the capsid gene of canine parvovirus (CPV), was compared as a diagnostic method for detecting CPV in faeces, with virus isolation (VI) on Crandell feline kidney (CRFK) or Madin-Darby canine kidney (MDCK) cells, and a faecal haemagglutination (HA) assay confirmed by inhibition with a CPV-specific antiserum. Although a false-negative result was obtained in one of 59 faecal samples (1.7 per cent) tested by the PCR assay, it was as sensitive as the VI assay using MDCK cells, and more sensitive than the VI assay using CRFK cells or the HA assay. These results indicate that the PCR assay may be useful as a routine diagnostic method for detecting CPV in faecal specimens.

Demonstration and Genotyping of Pestivirus RNA from Mammalian Cell Lines

We examined 20 cell lines of various animal origins for the presence of pestivirus contamination by the reverse transcription‐polymerase chain reaction (RT‐PCR), and found 15 (75%) cell lines were positive. The RT‐PCR products of the 5′ untranslated region (UTR) of pestivirus genome were sequenced and subjected to genotyping. Stem‐loop structures at three variable regions in the 5′ UTR render genotyping of the contaminated pestiviruses. Bovine cell lines tested were all contaminated with genotypes I, II, or III of bovine diarrhea virus (BVDV). Cell lines of canine, feline, and primate origin were contaminated with genotype II of BVDV. Cell line Ch1Es of caprine origin was contaminated with border disease virus (BDV).

Adventitious pestivirus RNA in live virus vaccines against bovine and swine diseases

Live virus vaccines against bovine and porcine diseases were examined for the presence of adventitious pestivirus RNA or pestiviruses by reverse transcription-polymerase chain reaction (PCR). Pestivirus RNA was detected in the live virus vaccines against Akabane disease, Ibaraki disease, infectious bovine rhinotracheitis, porcine parvovirus infection, transmissible gastroenteritis and Japanese encephalitis. Pestivirus RNA or pestivirus in the fetal bovine serum used to grow the host cells used to prepare the bovine and swine viral vaccines is a likely source of the contamination. Nucleotide sequence analysis of the PCR products suggests that modified live virus vaccines being used for immunization of cattle against bovine viral diarrhoea was not responsible for the contamination of the vaccines examined.

Identification of Phosphocholine-Containing Glycoglycerolipids Purified from Mycoplasma fermentans-Infected Human Helper T-Cell Culture as Components of M. fermentans

Previously, we have reported the occurrence of novel phosphocholine‐containing glycoglycerolipids (GGPLs: GGPL‐I and GGPL‐III) in human helper T‐cell culture (MT‐4 cell line) (Matsuda et al, Glycoconjugate J. 10: 340). However, the GGPLs disappeared from the MT‐4 cells after treatment with an antimycoplasma agent. This disappearance suggested the involvement of microorganisms in the GGPL expression. In this paper, we show that the novel lipids are components of Mycoplasma fermentans itself. The supernatant fluid of the antimycoplasma agent‐untreated MT‐4 cell culture produced mycoplasma‐like colonies on PPLO agar plates, and PCR and immunological methods revealed the presence of M. fermentans. GGPLs were expressed again in the treated MT‐4 cells after infection with the isolated M. fermentans. The isolated M. fermentans had glycoglycerolipids corresponding to GGPL‐I and GGPL‐III. Thin‐layer chromatography‐mass spectrometry and immunological analyses showed that these glycoglycerolipids which were derived from the isolated M. fermentans were identical with GGPL‐I and GGPL‐III previously obtained. This is the first report that shows mycoplasma has phosphocholine‐containing glycoglycerolipids.

PCR: Application of nested PCR to detection of mycoplasmas

This chapter describes the application of nested polymerase chain reaction (PCR) to detection of mycoplasmas. In a typical protocol for the nested PCR, a first-round PCR is performed with a pair of outer primers. The nested PCR is currently the most sensitive means of detecting the mycoplasmas in cell cultures or in clinical materials because a single copy of target sequence can be detected. The second-round PCR serves to confirm the specificity of the first-round PCR. The nested primer pairs recommended in this chapter consist of sequences of the 16S-23S ribosomal RNA intergenic spacer regions. High-titerd mycoplasma cultures inhibit PCR because intracellular mycoplasmal components inhibit the activity of Taq DNA polymerase. Although nested PCR may increase the risk of cross-contamination with product DNA because of its high sensitivity, it does provide a faster means of detecting low-titer infections. Materials required for the isolation of DNA from mycoplasma-contaminated biological materials are illustrated in the chapter.