Letter to the editor: Suspected discrepancies due to inadequate quality of an in vitro analysis revealed by an in silico analysis.

The author's in vitro analysis results were compared with those of an in silico structure analysis of the relevant sequences obtained from the author's entire genome sequence data. It was speculated that the in silico results together with author's phylogenetic results demonstrate problems in the authors' published in vitro data, which were presumably due to an inadequate accuracy of an in vitro assay. It was fortuitously suggested that alignment images to an excess repeat structure of the same locus provide a improved speculation of a number of repeats and that accurate in vitro assays are expected to complementarily provide reliable insights in the era of whole genome sequencing.

Comparison of a variable-number tandem-repeat (VNTR) method for typing Mycobacterium avium with mycobacterial interspersed repetitive-unit-VNTR and IS1245 restriction fragment length polymorphism typing.

Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, a variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of the IS1245-restriction fragment length polymorphism (IS1245-RFLP) typing method and a mycobacterial interspersed repetitive-unit (MIRU)-VNTR typing method reported previously (V. C. Thibault, M. Grayon, M. L. Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, J. Clin. Microbiol. 45:2404-2410, 2007). Seventy clinical isolates identified as M. avium from human immunodeficiency virus-negative patients with MAC infections were used. MATR-VNTR typing using 15 loci distinguished 56 patterns of different allele profiles, yielding a Hunter-Gaston discriminatory index (HGDI) of 0.990. However, IS1245-RFLP and MIRU-VNTR typing yielded HGDIs of 0.960 and 0.949, respectively, indicating that MATR-VNTR has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. Moreover, concomitant use of the MATR-VNTR method and IS1245-RFLP typing increased the HGDI to 0.999. MATR-VNTR typing is inexpensive and easy to perform and could thus be useful in establishing a digital multifacility database that will greatly contribute to the clarification of the transmission route and pathophysiology of M. avium infections.

[Usefulness of variable numbers of tandem repeats typing in clinical strains of Mycobacterium avium].

OBJECTIVES: We evaluated the usefulness of Variable Numbers of Tandem Repeats (VNTR) analysis, which was recently reported as a new typing method of Mycobacterium avium strains of animal origin, for strain differentiation of clinical isolates of M. avium in comparison with the standard IS1245-RFLP typing method. In addition, forty M. avium isolates recovered from sputum samples of same patient in different times were analyzed with VNTR typing method. SUBJECTS AND METHODS: The subjects were twenty-four clinical isolates of M. avium stocked at Higashi Nagoya National Hospital and discriminatory power was evaluated with Hunter Gaston Discriminatory Index (HGDI). Furthermore, forty M. avium isolates recovered from sputum samples of one patient obtained at four different times were analyzed by using this VNTR typing method. RESULTS: VNTR typing showed better discriminatory power for twenty-four clinical isolates than IS1245-RFLP method (HGDI: 0.975 vs 0.866). In the second study, polyclonal infection of four genotype strains with different allele profiles were detected. The ratio of mixture of the four different genotype strains varied during clinical course. CONCLUSION: We considered that VNTR typing method was very useful for discriminatory examination of M. avium.

[Molecular epidemiology of tuberculosis by the use of IS6110 restriction fragment length polymorphism: a study from 2001 to 2003].

OBJECTIVE: To analyze the situation of tuberculosis infection by DNA fingerprinting in the middle and eastern part of Osaka, Japan. DESIGN: We performed IS6110 restriction fragment length polymorphism (RFLP) on 1200 isolates from tuberculosis patients who visited our hospital from January 2001 to December 2003. A cluster was defined as a series of isolates with more than 90% similarity by IS6110 RFLP and those with the same drug-susceptibility pattern. The isolates with fewer than six copies of IS6110 were considered to be clustered if the IS6110 RFLP patterns and the variable numbers of tandem repeats with 16 regions of ETR and MIRU "allele profile" were identical. RESULTS: The number of samples in incremental study periods was 422 in 2001, 817 between 2001 and 2002 and 1200 between 2001 and 2003. The percentage of clustered cases was 27.8% in 2001, 19.1% in 2002 and 19.5% in 2003. The cumulative percentage of clustered cases was 27.8% in the first year, 29.7% over two years and 32.6% over three years. The percentage of clustered cases of isolates with a drug resistance was significantly lower (25.0%) than that of drug susceptible isolates (33.7%). Next, we investigated the clustered cases by gender and age. The percentage of clustered cases with isolates from young males and females (0-19 years old) was 23.8%. In contrast, the percentage of clustered cases with isolates from 20-59 year-old females gradually decreased from 14.7% to 4.4%. Conversely, the percentage of clustered cases from young and middle aged male (20-59 years old) was higher (20.2%-32.4%) than that of females. CONCLUSION: The sharp increase in the cumulative cluster formation rate was curbed by the decline in the tuberculosis incidence rate in Osaka, Japan, after the first year of examination. We thought that this phenomenon suggests the success of the anti-tuberculosis measure in Japan.

The artAB genes encode a putative ADP-ribosyltransferase toxin homologue associated with Salmonella enterica serovar Typhimurium DT104.

Many bacterial pathogens encode ADP-ribosyltransferase toxins. The authors identified an ADP-ribosyltransferase toxin homologue (ArtA, ArtB) in Salmonella enterica serovar Typhimurium (S. typhimurium) DT104. ArtA is most homologous to a putative pertussis-like toxin subunit present in Salmonella typhi (STY1890) and Salmonella paratyphi A (SPA1609), while ArtB shows homology to a hypothetical periplasmic protein of S. typhi (STY1364) and S. paratyphi A (SPA1188), and a putative pertussis-like toxin subunit in S. typhi (STY1891) and S. paratyphi A (SPA1610). The artA gene was detected from the phage particle fraction upon mitomycin C induction, and the flanking region of artAB contains a prophage-like sequence, suggesting that these putative toxin genes reside within a prophage. Southern blotting analysis revealed that artA is conserved in 12 confirmed DT104 strains and in four related strains which are not phage-typed but are classified into the same group as DT104 by both amplified-fragment length polymorphism and pulsed-field gel electrophoresis. Except for one strain, NCTC 73, all 13 S. typhimurium strains which were classified into different groups from that of DT104 lacked the artA locus. The results suggest that phage-mediated recombination has resulted in the acquisition of art genes in S. typhimurium DT104 strains.

Experimental transmission of ovine herpesvirus-2 in sheep.

Transmission of ovine herpesvirus-2 (OvHV-2) in sheep via natural contact and nasal secretions was examined. OvHV-2-free lambs were produced by separating newborn lambs from their mothers within 5 days of birth and raising them in an isolation facility. Transmission experiments via natural contact were conducted by keeping OvHV-2-free lambs with OvHV-2-infected sheep of different ages. Six of the infected ewes in this experiment were pregnant and gave birth during the experimental period. OvHV-2 was not transmitted from the adult sheep, though viral DNA was consistently detected in their peripheral blood leukocytes (PBL). On the other hand, OvHV-2 was transmitted from recently infected lambs to sheep at 10 or 12 weeks after the onset of contact. In addition, we attempted the experimental transmission of OvHV-2 via nasal secretions, by transferring nasal washings from infected sheep to the nostrils of uninfected sheep. Sheep receiving the nasal washings from infected adult sheep maintained their negative status for 15 months, whereas sheep receiving nasal washings from recently infected lambs acquired OvHV-2 by 8 months. The results of these experiments support that OvHV-2 is more easily transmitted to negative sheep by recently infected lambs than by adult sheep. Further, it is supposed that the nasal cavity is a portal for entry and shedding of infectious OvHV-2 in sheep.

Molecular characterization of a prophage of Salmonella enterica serotype Typhimurium DT104.

Isolates of the Salmonella enterica serotype Typhimurium definitive phage type (DT104) were found to contain the same prophage (designated phage ST104). The complete sequence of the DNA genome of prophage ST104 was determined. The entire DNA sequence consisted of 41,391 bp, including 64 open reading frames, and exhibited high similarity to P22 and to phage type conversion phage ST64T.

Comparison of Salmonella enterica serovar Abortusequi isolates of equine origin by pulsed-field gel electrophoresis and fluorescent amplified-fragment length polymorphism fingerprinting.

Equine paratyphoid is caused by Salmonella enterica serovar Abortusequi, and manifests mainly as abortion in the mare. We compared S. Abortusequi strains isolated in Japan and other countries using pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment length polymorphism (FAFLP) analysis. PFGE analysis of S. Abortusequi strains gave 21-27 fragments ranging in size from 33 to 602kb. Although two PFGE profiles were observed among the 20 S. Abortusequi isolates in Japan, the restriction fragments originating from the chromosome were common between the two profiles. The similarity index of the two profiles was 90.9%, while those between Japanese and five other S. Abortusequi strains were 29.8-37.5%. On the other hand, FAFLP analysis of S. Abortusequi strains generated 64-67 amplified fragments ranging in size from 100 to 400bp. One polymorphic fragment was observed among the 20 S. Abortusequi isolates in Japan. These data indicate the close relation of this agent in Japan. S. Abortusequi strains sharing a common ancestry might have been conserved in Japan.

Development of a shuttle polymerase chain reaction for the detection of bovine herpesvirus 2.

Three different polymerase chain reaction (PCR) protocols were evaluated for their ability to detect bovine herpesvirus 2 (BoHV-2): single-step PCR with 3 reaction stages (denaturation, annealing and extension), 2 reaction stages (denaturation and annealing/extension; shuttle PCR), and semi-nested PCR with 3 reaction stages. All the PCR protocols showed the same sensitivity (detection limit of 0.4 TCID(50)). A non-specific band sometimes appeared in mock cell DNA at annealing temperatures below 64 degrees C. The shuttle PCR was found to be superior to the other protocols under consideration because of the speed of its application. Furthermore, no non-specific band was detected in DNAs of eight other DNA viruses. Thus, the shuttle PCR seems to be an excellent diagnostic tool for BoHV-2 infections.