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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Occurrence of 'Candidatus Mycoplasma turicensis' infection in domestic cats in Japan.

We have examined the presence of hemoplasmas, hemotropic mycoplasmas, among domestic cats (Felis catus) in Japan by using a species specific PCR, and found 'Candidatus Mycoplasma turicensis', a recently recognized hemoplasma species. A total of 60 feline blood samples collected in 2004 and 2005 were subjected to PCR amplification for the detection of Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis'. Six blood samples collected from domestic cats were found infected with the 'Candidatus M. turicensis'. All of them are also infected with other species of hemoplasmas, M. haemofelis and/or 'Candidatus M. haemominutum'. This is the first to demonstrate 'Candidatus M. turicensis' infections among cat population in Japan.

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.

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.

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.

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.

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.

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.