Molecular detection of Mycoplasma haemomuris subspecies using dnaK-targeted real-time PCR with SYBR Green I and melting curve analysis.

Hemoplasmas cause severe infections in mammals, but these pathogens are difficult to detect and identify at the species and subspecies level because of the need for time-consuming sequence based methods. Here, we used real-time PCR with SYBR Green I targeting of the dnaK gene followed by standard melting curve analysis to achieve rapid detection and differentiation of the Mycoplasma haemomuris subspecies 'Candidatus Mycoplasma haemomuris subsp. musculi' and 'Candidatus M. haemomuris subsp. ratti'. The melting temperatures of the PCR products, 84.63 ± 0.14 °C for 'Candidatus M. haemomuris subsp. musculi', and 80.72 ± 0.16 °C for 'Candidatus M. haemomuris subsp. ratti', provided clear differentiation between them. Murine hemoplasma DNA samples, which were used as references, were confirmed for species by an analysis of 16S rRNA sequences. The protocol described herein provides a new rapid detection and identification method suitable for use with two recognized subspecies of M. haemomuris.

Proposal for 'Candidatus Mycoplasma haemomuris subsp. musculi' in mice, and 'Candidatus Mycoplasma haemomuris subsp. ratti' in rats.

Mycoplasma haemomuris is causative of infectious anaemia or splenomegaly in rodents. We examined the nucleotide sequences of the non-ribosomal genes, rnpB and dnaK, in strains of the species M. haemomuris detected in small field mice and black rats. rnpB nucleotide sequences in strains of the species M. haemomuris isolated from small field mice and black rats had only 89 % sequence similarity, suggesting their separation into two distinct subgroups. dnaK had a nucleotide sequence similarity of 84 % between the subgroups. These results support the classification of M. haemomuris into two genetically distinct subgroups. Here we propose the establishment of these subgroups as 'Candidatus Mycoplasma haemomuris subsp. musculi', detected in small field mice (Apodemus argenteus), and 'Candidatus Mycoplasma haemomuris subsp. ratti', detected in black rats (Rattus rattus).

Vertical transmission of Mycoplasma wenyonii in cattle, supported by analysis of the ribonuclease P RNA gene - Short communication.

The vertical transmission of Mycoplasma (M.) wenyonii was investigated in beef cattle raised on a farm in Japan by analysing the ribonuclease P RNA (rnpB) gene sequence using PCR. Peripheral blood samples from 17 dams infected with M. wenyonii and from their neonatal calves were collected and colostrum samples were taken from cows immediately after parturition, and subsequently the blood samples of calves were monitored continuously for three months. At birth on day 0, although no rnpB gene was detected in the colostrum of any of the dams, four (23.5%) of the 17 calves born were positive. At three months after delivery, the number of positive calves decreased to three. Although horizontal transmission by blood-feeding arthropod vectors has been basically accepted as the most common route of haemoplasma infection, these findings suggest that vertical transmission is, at least in part, another most likely route of M. wenyonii infection in cattle.

Salivary nitrate and nitrite may have antimicrobial effects on Desulfovibrio species.

The antibacterial effects of salivary nitrate/nitrite on the growth of three Desulfovibrio species were examined. The bacteria did not grow on plates with ≥ 0.2 mM nitrate or ≥ 1.0 mM nitrite. They were also incubated in filter-sterilized saliva. D. desulfuricans was reduced on the order of >10(2) compared with the control solution (phosphate-buffered saline) in nine out of the 10 participants.

Seroepidemiological survey of sheep flocks from Northern Japan for Mycoplasma ovipneumoniae and Mycoplasma agalactiae.

Sheep flocks from Hokkaido, Iwate and Aomori, three northern prefectures of Japan, were screened for antibodies to Mycoplasma ovipneumoniae and Mycoplasma agalactiae by ELISA. Sixty four animals out of 246 (26%) were seropositive to M. ovipneumoniae, with positive results obtained from all three prefectures. None of the sera tested were serologically positive to M. agalactiae.

Acidic environments induce differentiation of Proteus mirabilis into swarmer morphotypes.

Although swarmer morphotypes of Proteus mirabilis have long been considered to result from surfaced-induced differentiation, the present findings show that, in broth medium containing urea, acidic conditions transform some swimmer cells into elongated swarmer cells. This study has also demonstrates that P. mirabilis cells grown in acidic broth medium containing urea enhance virulence factors such as flagella production and cytotoxicity to human bladder carcinoma cell line T24, though no significant difference in urease activity under different pH conditions was found. Since there is little published data on the behavior of P. mirabilis at various hydrogen-ion concentrations, the present study may clarify aspects of cellular differentiation of P. mirabilis in patients at risk of struvite formation due to infection with urease-producing bacteria, as well as in some animals with acidic or alkaline urine.

Mycoplasma ovis detected in free-living Japanese serows, Capricornis crispus.

Nineteen blood samples collected from free-ranging wild Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the hemoplasma infection by real-time PCR targeting the 16S rRNA gene. Five (26.3%) out of the 19 samples were positive in real-time PCR with an average melting temperature at 85.18 °C. The positive samples in the real-time PCR were reconfirmed by conventional PCR, and one of them was successful for direct DNA sequencing. The nucleotide sequence of the 16S rRNA gene of the representative stain was identical to that of Mycoplasma ovis. This was the first demonstration of hemotropic mycoplasma infections among the free-living Japanese serows in Japan.

Molecular detection of filarial nematode parasites in Japanese black bears (Ursus thibetanus japonicus) from Iwate Prefecture, Japan.

This study aimed to detect filarial parasites in blood samples of Japanese black bears (Ursus thibetanus japonicus) collected from Iwate Prefecture, Japan. Positive amplicons were obtained from 26 out of 30 samples by nested PCR targeting 18S ribosomal RNA gene and first internal transcribed spacer regions. DNA sequences of Mansonella sp. close to M. ozzardi and Dirofilaria sp. were detected for eight and 11 positive amplicons, respectively. Co-infection was detected for the remaining seven amplicons. Dirofilaria sp. was identified as D. ursi by further genetic analysis of 5S ribosomal RNA gene sequence. The results of this study will contribute to further investigations of Japanese black bears for monitoring their risk as a reservoir of possible zoonotic filarial parasites.

Nitric oxide causes anoikis through attenuation of E-cadherin and activation of caspase-3 in human gastric carcinoma AZ-521 cells infected with Mycoplasma hyorhinis.

Mycoplasma hyorhinis (M. hyorhinis) infection leads cultured cells to various biological alterations in cell metabolism including apoptosis. Apoptosis induced by M. hyorhinis has mainly been considered to be due to mycoplasmal endonucleases. We previously reported that apoptosis in a human carcinoma cell line AZ-521 infected with M. hyorhinis was enhanced by addition of L-ascorbic acid to cell cultures. Since both L-ascorbic acid addition and M. hyorhinis infection activated cellular iNOS, we examined the hypothesis that nitric oxide (NO) exerts an apoptotic effect on M. hyorhinis-infected cells and down-regulates E-cadherin. In this study, we showed that M. hyorhinis infection activates iNOS mRNA synthesis, NO production, and caspase-3 activity and attenuates E-cadherin mRNA synthesis by quantitative real-time PCR, Griess assay and fluorescence caspase-3 detection. L-NAME decreased the numbers of apoptotic cells through inhibition caspase-3 activity. Our results indicate that NO causes anoikis throughout attenuation of E-cadherin and activation of caspase-3 in human gastric carcinoma cell line AZ-521 cells infected with M. hyorhinis.

Differential detection of hemotropic Mycoplasma species in cattle by melting curve analysis of PCR products.

We developed a real-time PCR procedure followed by melting curve analysis using the green fluorescence dye SYBR Green I for rapid detection and differentiation of hemplasmas in cattle. Analysis of the melting temperature (Tm) of the PCR products allowed for differentiation of the 2 bovine hemoplasmas, Mycoplasma wenyonii and a provisional species, ;Candidatus Mycoplasma haemobos' (a synonym of ;Candidatus M. haemobovis'). The Tm (mean +/- S.E.) of the PCR products from the bovine hemoplasmas were 86.98 +/- 0.12 degrees C for M. wenyonii and 82.04 +/- 0.27 degrees C and 86.98 +/- 0.12 degrees C, respectively; [corrected] Candidatus M. haemobos' in the melting experiments. The protocol described in the present study can decrease the time to results by simultaneous detection and differentiation of the two hemoplasmas in cattle. By using this protocol, we examined hemoplasma prevalence in 109 cattle in Miyagi Prefecture and found that 67 (61.5%) were infected with M. wenyonii, 25 (22.9%) were infected with ;Candidatus M. haemobos' and 14 (12.8%) were infected with both.

Novel hemoplasma species detected in free-ranging sika deer (Cervus nippon).

Hemoplasma infections in wild ungulates have not been reported yet in Japan. We examined presence of hemoplasmas in blood samples collected from 147 sika deer (Cervus nippon) in the Iwate prefecture by real-time PCR, and found 13 (9%) were positive. Almost entire region of the 16S rRNA gene of the representative strains from positive samples was amplified by conventional PCR. The nucleotide sequences of the 16S rRNA gene were further determined and compared with those of other hemoplasmas. Our examinations 1st revealed the presence of 2 distinct hemoplasma species in sika deer, which are previously not described. One of them was closely related to M. ovis by the 16S rRNA sequence analysis, but was found distinct by comparison of the RNase P RNA gene sequences. Pathogenicity of these two hemoplasma species in sika deer is currently unknown.

Variation of genes encoding GGPLs syntheses among Mycoplasma fermentans strains.

The information of the biosynthesis pathways of Mycoplasma fermentans specific major lipid-antigen, named glycoglycerophospholipids (GGPLs), is expected to be some of help to understand the virulence of M. fermentans. We examined primary structure of cholinephosphotransferase (mf1) and glucosyltransferase (mf3) genes, which engage GGPL-I and GGPL-III synthesis, in 20 strains, and found four types of variations in the mf1 gene but the mf3 gene in two strains was not detected by PCR. These results may have important implications in virulence factor of M. fermentans.

Bovine viral diarrhea virus 2 infection activates the unfolded protein response in MDBK cells, leading to apoptosis.

Bovine viral diarrhea virus 2 (BVDV-2) strains are divided into cytopathic and non-cytopathic biotypes based on the ablity to induce cytopathic effects in cultured cells. The mechanism of cytopathogenicity of BVDV-2 is not well understood. We examined cytopathogenesis in MDBK cells resulting from BVDV-2 infections by microscopic examinations and microarray analysis. We found that BVDV-2 activates endoplasmic reticulum (ER) stress signaling pathways that contribute to apoptosis of infected cells. We also monitored the expression of ER stress marker gene by RT-PCR during BVDV-2 infection and demonstrated that infection of MDBK cells with a cytopathic strain of BVDV-2 induces glucose-regulated protein 78 expression. Infection with BVDV-2 also induces DNA-damage-inducible transcript 3 expression and downregulates the lectin-galactoside-binding soluble 1 level. These results show that cytopathic strains of BVDV-2 induce an ER stress response resulting in apoptosis.

Effects of chelating reagents on colonial appearance of Paenibacillus alvei isolated from canine oral cavity.

A bacterial strain isolated from the oral cavity of a healthy dog revealed an unusual colony formation in nebular appearance on agar plates. The isolated bacterial strain was Gram-positive, spore-forming rod with peritrichous flagella, and grown under aerobic conditions, but unable to grow at 45 degrees C. The strain was tentatively classified as Paenibacillus alvei according to the biochemical properties and the 16S rRNA gene sequence. The isolate exhibits collective locomotion on solid agar plates. The bacterial motility was inhibited with EDTA and was restored by adding magnesium. We concluded that magnesium ion is essential for collective locomotion of P. alvei. This suggests that EDTA is useful for inhibition of biofilm formation.

Phylogenetic analysis of the 16S rRNA gene of Anaplasma species detected from Japanese serows (Capricornis crispus).

Nineteen blood samples collected from free-living Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the anaplasma infection by PCR amplification of a part of the 16S rRNA gene. Ten (52.6%) out of the 19 samples produced a visible band in electrophoresed agarose gels. Positive PCR products were subjected to DNA sequencing. We found the nucleotide sequences were identical. Almost entire length of the 16S rRNA gene for a representative stain was then sequenced. We found the nucleotide sequence of the 16S rRNA gene distinct from previously established Anaplasma species in phylogenetic analysis. Our data first indicated that anaplasma infection occurred continuously among the free-living Japanese serow populations in northern Japan.

Growth inhibition of human melanoma cells by a recombinant arginine deiminase expressed in Escherichia coli.

We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361.

Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis.

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient's tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-alpha and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.

L-ascorbic acid enhances apoptosis in human gastric carcinoma cell line AZ-521 cells infected with Mycoplasma hyorhinis.

Mycoplasma hyorhinis (M. hyorhinis) exerts multiple effects on cell metabolisms including apoptosis mediated by their endonucleases and nitric oxide production in vitro. Although AsA is preferable to health in general because of its reactive oxygen species scavenging activity, we found that in a human carcinoma cell line AZ-521 infected with M. hyorhinis, apoptosis was enhanced by addition of L-ascorbic acid (AsA) to the cell cultures. No significant differences were evident between the AZ-521 cells with and without AsA (AsA-) after 24 hr of incubation in the mitochondrial fluorescence. M. hyorhinis-infected AZ-521 cells treated with AsA (AsA +) have developed distinct DNA ladders as compared to the control cells AsA- after 24 hr of incubation. Marked cytopathic effects were rather apparent in AsA-treated cells than in control cells AsA- after 24 hr. Our data demonstrate that AsA addition to cell cultures enhances apoptosis induced by M. hyorhinis infection. We suggest that the presence of another external apoptotic pathway by M. hyorhinis infection.

Genoepidemiological evaluation of Bovine viral diarrhea virus 2 species based on secondary structures in the 5' untranslated region.

Bovine viral diarrhea virus 2 (BVDV-2) strains demonstrated in cattle, sheep, and adventitious contaminants of biological products have been evaluated by the palindromic nucleotide substitutions (PNS) method at the three variable loci (V1, V2 and V3) in the 5' untranslated region (UTR), to determine their taxonomical status. Variation in conserved genomic sequences was used as parameter for epidemiological evaluation of the species in relation with geographical distribution, animal host and virulence. Four genotypes, BVDV-2a, BVDV-2b, BVDV-2c, and BVDV-2d have been identified within the species. Taxonomical segregation corresponded to geographical distribution of genotype variants. Genotype 2a was present worldwide, and was the only circulating also in sheep, in addition to cattle. Genotypes 2b, 2c and 2d were restricted to South America. Contamination of biological products was related to genotypes 2a and 2d. Genetic variation could be related with chronological diffusion of the BVDV-2 species variants in different geographic areas. Chronologically, the species emerged in North America in 1978, spreading in UK and Japan, continental Europe, South America and New Zealand. Correlation between clinical features related with isolation of BVDV-2 strains and genetic variation indicated that subgenotype 1, variant 4 of genotype 2a was related with hemorrhagic syndrome. These observations suggest that evaluation of genomic secondary structure, by identifying markers for expression of virus biological activities and species evolutionary history, may be applied as useful tool for epidemiological evaluation of the BVDV-2 species, and possibly for other species of the genus Pestivirus.

Salivary nitrate-nitrite conversion capacity after nitrate ingestion and incidence of Veillonella spp. in elderly individuals.

Dietary nitrate has several beneficial effects, including blood pressure reduction and improved oxygen consumption efficiency, but in order to do so it must first be reduced to nitrite by oral bacteria. Veillonella spp., a strictly anaerobic group, are the most prevalent nitrate-reducing bacteria in the oral cavity. In response to some early studies that have hinted at inter- and intra-individual variation in salivary nitrate-nitrite conversion capacity, the purpose of the present study was to investigate the incidence of and variation in the Veillonella species V. atypica, V. dispar, and V. rogosae by direct PCR and to assess salivary nitrate-nitrite conversion capacity and its reproducibility after dietary nitrate consumption in 24 elderly individuals. V. atypica, V. dispar, and V. rogosae were detected in 10 (41.7%), 24 (100%), and 14 (58.3%) participants, respectively. The coefficients of correlation between the first and second experiments on increased nitrate/nitrite were 0.637 and 0.583, respectively, both of which were statistically significant (P < 0.01). In both experiments, 6 participants produced relatively low levels of nitrite (<0.5 mM Δ nitrite) while 7 produced relatively high levels (>1.0 mM Δ nitrite). The data suggested that V. dispar was the most prevalent species, being present even in individuals producing low levels of salivary nitrite.