Electron microscopic observation of Mycobacterium avium subsp. paratuberculosis in cattle feces after a novel purification using liquid paraffin.

We developed a novel method for purifying acid-fast bacteria from feces. The method enabled the observation of characteristic clumps of Mycobacterium avium subsp. paratuberculosis (MAP) under electron microscopy by removing contaminants and other bacteria. Further refinement of this method will contribute to efficient and effective MAP detection.

Verification of different methods used for isolating Salmonella enterica serovar Dublin from cattle feces.

Salmonella enterica serovar Dublin is a cattle-adapted serovar, and some infected cattle can become asymptomatic carriers. Identification of carrier animals is important for preventing the spread of infection within a farm, but low diagnostic sensitivity of the fecal culture method is problematic. In this study, we investigated isolation methods of four S. enterica Dublin strains. Selective enrichment using the tetrathionate broth showed better performance than Rappaport-Vassiliadis R10 broth, but one of the strains was not detectable. Since isolation of such strains by selective enrichment can be difficult, we designed a method using immuno-plates that concentrates S. enterica Dublin by antigen-antibody reaction. Our method is able to detect approximately 200 clony-forming units of S. enterica Dublin in 0.1 g of cattle feces. If S. enterica Dublin was isolated from cattle with clinical signs, the method to identify carriers in the farm should be based on the growth kinetics of the target S. enterica Dublin strain.

The association between detection of Mycobacterium avium subsp. paratuberculosis DNA in feces and histopathological classification.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic infectious disease that causes intractable diarrhea in ruminants. To control the occurrence of JD in cattle, a national surveillance is conducted in Japan. Since 2013, real-time quantitative PCR has been used for definite diagnosis. In this study, we compared the amount of fecal MAP DNA with histopathological classification of ileocecal lesions. Multinomial logistic regression models enabled us to predict the probability of finding the histopathological classification from the amount of fecal MAP DNA. These results suggest that shedding level of MAP DNA could act as an indicator of JD progression.

Identification and phase inversion of Salmonella flagellar antigens, using immuno-discs.

Disc immuno-immobilization is a simple method for typing the flagellar phase of Salmonella enterica. We re-examined this method using commercial antisera, which contains the preservative sodium azide. Originally prepared motility agar activates bacterial motility and renders S. enterica resistant to sodium azide, resulting in the formation of immuno-immobilization lines around reactive immuno-discs. Though disc immuno-immobilization serves both serotyping and phase inversion, this method is insufficient for the strains in which phase variation rarely occurs. Here, we devised a novel immuno-disc phase inversion method, and all S. enterica strains tested were identically typed. These methods would drastically simplify the task of S. enterica typing in clinical laboratories.

Growth kinetics of Salmonella enterica in Hajna tetrathionate broth, Rappaport broth and modified semisolid Rappaport agar.

To determine the appropriate method for isolating Salmonella enterica, we compared the growth of S. enterica serovars using three selective enrichment media. S. enterica was more successfully isolated from artificially contaminated fecal samples after enrichment in Hajna tetrathionate broth or modified semisolid Rappaport agar than in Rappaport broth. Since most bacteria (other than motile S. enterica) do not migrate on modified semisolid Rappaport agar, the growth characteristics of S. enterica can be interpreted easily and quickly. Two S. enterica isolates did not migrate on modified semisolid Rappaport agar, but did grow in Hajna tetrathionate broth, which suggests that the combined use of these selective enrichment media is appropriate for isolating S. enterica.

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.

Detection of hemotropic mycoplasmas in free-living brown sewer rats (Rattus norvegicus).

The prevalence of hemotropic mycoplasmas in wild rodents is largely unknown. Here, we report the presence of hemoplasmas in blood samples collected from brown sewer rats (Rattus norvegicus) trapped during rodent control around an animal hospital in Morioka, Japan. We examined nine rats using real-time PCR and end-point PCR, and found one rat (11.1%) that was positive for a hemoplasma infection. The 16S rRNA gene and 16S to 23S rRNA intergenic spacer region of the hemoplasma detected in a wild-caught rat were amplified using PCR. The nucleotide sequences of the PCR products were further determined and compared to those of other hemoplasmas. Our examinations revealed the presence of a hemoplasma that has not previously been described in rodents. The pathogenic traits of this hemoplasma remain unexplored.

Hemotropic mycoplasma infection in wild black bears (Ursus thibetanus japonicus).

This is the first report on Mycoplasma infection in wild bears. We report a novel hemotropic Mycoplasma (also called hemoplasma) detected in a free-ranging black bear (Ursus thibetanus japonicus) in Japan. We then used real-time PCR to look for hemoplasma DNA in blood samples collected from 15 bears and found that eight (53%) were positive. Among these eight PCR samples, seven showed a melting temperature of around 85.5°C, while the remaining one showed a single peak at 82.26°C. Almost the entire region of the 16S rRNA gene as well as the 16S-23S rRNA intergenic transcribed spacer (ITS) region from the sample that showed a melting temperature of 82.26°C was successfully amplified by means of end-point PCR. The nucleotide sequences of the 16S rRNA gene and the ITS region were then determined and compared with those of authentic Mycoplasma species. Our examinations revealed the presence of a novel hemoplasma in Japanese black bears.

Two genotypes among 'Candidatus Mycoplasma haemobos' strains based on the 16S-23S rRNA intergenic spacer sequences.

'Candidatus Mycoplasma haemobos', sometimes causative of bovine infectious anemia at various extents, has been demonstrated throughout the world. Here, we show two distinct types of 'Ca. M. haemobos' are distributed among cattle in Japan, by examining the primary and secondary structures of the 16S-23S rRNA intergenic spacer region that has been shown to be a stable genetic marker for mycoplasma species. Our results may explain differences in severity of anemic condition as well as provide a genetic marker for an epidemiological study of bovine hemoplasma infections.

Two clusters among Mycoplasma haemomuris strains, defined by the 16S-23S rRNA intergenic transcribed spacer sequences.

Mycoplasma haemomuris is a causative organism of infectious anemia or splenomegaly in rodents. Here, we report two distinct genetic groups among M. haemomuris strains detected from rats and mice, respectively, by examining the nucleotide sequences of the 16S-23S rRNA intergenic transcribed spacer region that has been shown to be a stable genetic marker for mycoplasma species. Our results may reveal host-tropism of each cluster of M. haemomuris strains, and suggest an idea to distinguish M. haemomuris into two different genetic clusters.

Prevalence of swine hemoplasmas revealed by real-time PCR using 16S rRNA gene primers.

Hemoplasma is a tribal name for epierythrocytic mycoplasmas including Mycoplasma suis and M. parvum which are currently recognized in pigs as causative of porcine hemoplasmosis. Here, we report a real-time PCR assay for differential detection of these swine hemoplasma species by using allelic primers in the16S rRNA gene, and its application to survey for hemoplasma infections in pigs. Universal primers and species-specific primers were designed and evaluated by using swine blood samples positive in hemoplasmas. Mycoplasma suis and M. parvum infections were both confirmed by universal primers, and mixed infections were clearly distinguished by species-specific primers. Further, we applied this real-time PCR assay to 120 swine blood specimens from clinically healthy pigs in eleven farms in Japan, and found six (5.0%) were positive for M. suis and 18 (15.0%) were positive for M. parvum, and three (2.5%) were mixed infection by both hemoplasma species.

Examination of the 16S-23S rRNA intergenic spacer sequences of 'Candidatus Mycoplasma haemobos' and Mycoplasma haemofelis.

The intergenic spacer region between the 16S and 23S rRNA genes of mycoplasmas has been used for a genetic marker for identification of the species. Here we show the intergenic spacer regions of two hemotropic mycoplasmas, Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos (synonym: 'C. M. haemobovis')' are also useful for classification of this particular group of mycoplasms. The spacer region of M. haemofelis and `C. M. haemobos' consisted of 209 and 210 base pairs, respectively, and both lacked the spacer tRNA genes. Phylogenetic analysis suggested a monophyletic relationship among hemoplasmas and M. fastidiosum. A hypothetical secondary structure predicted in the spacer regions tentatively assigned the boxA and boxB motifs peculiar to the members of the genus Mycoplasma. M. haemofelis and 'C. M. haemobos' possessed a stem-loop structure in common, despite the presence of a palindromic nucleotide substitution in the stem region.

Rapid identification of hemoplasma species by palindromic nucleotide substitutions at the GAAA tetraloop helix in the specificity domain of ribonuclease P RNA.

We examined secondary structures of the ribonuclease P RNA sequences obtained from DNA databases, and identified a determinative prototype of the P12 helix peculiar to each species of hemoplasmas. This key structure will provide a rapid means for species identification of these uncultivable pathogens without making a phylogenetic tree based on alignments of nucleotide sequences. This procedure based on palindromic nucleotide substitutions at the stem portion of the P12 helix provide clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. In conclusion, the PNS analysis is based on the evaluation of only the strategic and highly conserved genomic region in the specificity domain of RNase P RNA.

Two genetic clusters in swine hemoplasmas revealed by analyses of the 16S rRNA and RNase P RNA genes.

Only two hemoplasma species, Eperythrozoon parvum and Mycoplasma suis, have been recognized in pigs. Here we demonstrate the genetic variations among six hemoplasma strains detected from pigs, by analyzing the 16S rRNA and RNase P RNA (rnpB) genes, and propose a novel hemoplasma taxon that has not been described previously. Phylogenetic trees based on the nucleotide sequence of the 16S rRNA gene indicated that these six hemoplasmas were divided into two clusters representing M. suis and a novel taxon. We further examined the primary and secondary structures of the nucleotide sequences of the rnpB gene of the novel taxon, and found it distinct from that of M. suis. In conclusion, we unveiled a genetic cluster distinct from M. suis, suggesting a new swine hemoplasma species or E. parvum. Our findings also suggest that this novel cluster should be included in the genus Mycoplasma.

Prevalence of hemoplasma infection among cattle in the western part of Japan.

We have examined for hemoplasma infection among cattle in the Hiroshima and Miyazaki prefectures by using a sensitive real-time PCR, with SYBR Green I and with melting curve analysis, which allow to distinguish the two bovine hemoplasma species, Mycoplasma wenyonii and 'Candidatus M. haemobos'. We found 69.4% of 36 cattle in Hiroshima and 93.8% of 32 cattle in Miyazaki infected with either of these two hemoplasma species. High morbidity in western part of Japan may reflect the activity of arthropod vectors for hemoplasma transmission. We also demonstrated neonatal calves less than three months old affected with hemoplasmas without grazing in summer, suggesting a possibility of vertical transmission.

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.

Molecular identification of `Candidatus Mycoplasma haemovis' in sheep with hemolytic anemia.

We examined the presence of hemoplasmas, hemotropic mycoplasmas, among 11 sheep (Ovis aries) with regenerative and hemolytic anemia and found six of them were positive by real-time PCR. The positive samples were then subjected to conventional PCR for direct sequencing of the 16S rRNA gene. Nucleotide sequences of all the positive samples were identified as the 16S rRNA gene of `Candidatus Mycoplasma haemovis' by phylogenetic analysis, demonstrating the infections with this particular hemoplasma species in Japan.

A feline hemoplasma, 'Candidatus Mycoplasma haemominutum', detected in dog in Japan.

We examined for 'Candidatus Mycoplasma haemominutum' infection in 167 blood samples collected from domestic dogs between 2008 and 2009 in the Tohoku area, Japan, and found 5 (3.0%) were positive by PCR assay. This is the first demonstration of 'Candidatus Mycoplasma haemominutum', a feline haemotropic mycoplasma, in the dogs raised in Japan.

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.