Outbreaks of bovine mastitis caused by specific Mycoplasma bovis strains recurring at multi-year intervals.

Mycoplasma bovis is a major cause of bovine mastitis. Intermittent shedding of the organism for many months is a feature of cows with intramammary infection. A dairy farm in Japan experienced a mastitis outbreak caused by M. bovis in 2016, as well as 2 additional outbreaks and 1 case in 2020-2021. The causative strains in the 3 outbreaks shared a common and identical genetic feature, the insertion of a transposase gene at the same site within the phosphate acetyltransferase-2 gene. Additionally, all isolates were genotyped to closely related sequence types (ST21 and ST141) by multilocus sequence typing, and had similar pulsopatterns by pulsed-field gel electrophoresis. Our results indicate that infection with the same causative strain remained in this herd and environment for 4 y. Treatment with fluoroquinolones, guided by antimicrobial susceptibility test results, eliminated M. bovis from 16 of 20 M. bovis-infected cows, as confirmed by culture and somatic cell counts. However, mastitis caused by other bacteria occurred in 9 M. bovis-free cows within 2 mo of the last treatment.

Roles of mannosylerythritol lipid-B components in antimicrobial activity against bovine mastitis-causing Staphylococcus aureus.

Mannosylerythritol lipid-B (MEL-B), which comprises ester-bonded hydrophilic ME and hydrophobic fatty acids, is a bio-surfactant with various unique properties, including antimicrobial activity against most gram-positive bacteria. The gram-positive Staphylococcus aureus is a causative pathogen of dairy cattle mastitis, which results in considerable economic loss in the dairy industry. Here, we demonstrate the efficacy of MEL-B as a disinfectant against bovine-derived S. aureus and elucidate a mechanism of action of MEL-B in the inhibition of bacterial growth. The growth of bovine mastitis causative S. aureus BM1006 was inhibited when cultured with MEL-B above 10 ppm. The activity of MEL-B required fatty acids (i.e., caprylic and myristoleic acids) as ME, the component of MEL-B lacking fatty acids, did not inhibit the growth of S. aureus even at high concentrations. Importantly, ME-bound fatty acids effectively inhibited the growth of S. aureus when compared with free fatty acids. Specifically, the concentrations of ME-bound fatty acids and free caprylic and myristoleic acids required to inhibit the growth of S. aureus were 10, 1442, and 226 ppm, respectively. The involvement of ME in the antimicrobial activity of MEL-B was confirmed by digestion of MEL-B with alkali, which dissociated ME and fatty acids. These results indicated that a mechanism of action of MEL-B in inhibiting the growth of S. aureus could be explained by the effective transporting of antimicrobial fatty acids to the bacterial surface via hydrophilic ME.

Relationship between Antimicrobial Susceptibility and Multilocus Sequence Type of Mycoplasma bovis Isolates and Development of a Method for Rapid Detection of Point Mutations Involved in Decreased Susceptibility to Macrolides, Lincosamides, Tetracyclines, and Spectinomycin.

Mycoplasma bovis isolates belonging to the sequence type 5 (ST5) group, the dominant group in Japan since 1999, were low susceptible to 16-membered macrolides and tetracyclines and were confirmed to have a guanine-to-adenine transition mutation at position 748 in the 23S rRNA gene (rrl) and adenine-to-thymine transversion mutations at positions 965 and 967 in the 16S rRNA gene (rrs) (Escherichia coli numbering). Moreover, isolates of ST93 and ST155, members of the ST5 group, were low susceptible to lincosamides and azithromycin and showed an adenine-to-guanine transition mutation at position 2059 of rrl Isolates of ST93 were additionally low susceptible to spectinomycin and showed a cytosine-to-adenine transversion mutation at position 1192 of rrs Strains of the ST5 group seem to spread to Japan and Europe from North America with imported cows, while strains of ST93 and ST155 originated in Japan. Melting curve analysis using hybridization probes revealed the existence of point mutations involved in decreased susceptibility to macrolides, lincosamides, and spectinomycin, as demonstrated by changes in the melting curve shape and/or decreases in the melting peak temperature, so the susceptibility to these antimicrobials can be assessed on the same day. For decreased susceptibility to fluoroquinolones to exist, nonsynonymous mutations in the DNA gyrase gene (gyrA) and topoisomerase IV gene (parC) had to coexist. The combination of amino acid substitutions of serine at position 83 in gyrA and serine at position 80 in parC resulted in particularly low susceptibility to fluoroquinolones.IMPORTANCEMycoplasma bovis is the main causal species of bovine mycoplasmal disease and leads to significant economic losses because of its severe symptoms, strong infectivity, and refractoriness. As for mastitis, culling cows with intramammary infections is a general countermeasure to prevent spreading. The conventional antimicrobial susceptibility test for mycoplasma is time-consuming and troublesome, but no quick and easy method for grasping the antimicrobial susceptibility of the causal strain exists at present. Treatment without antimicrobial susceptibility information may be one reason why M. bovis infection is refractory. Detecting a mutation involved in decreased susceptibility to antimicrobial agents of the causal strain makes it possible to easily select suitable antimicrobials for treatment, and this technique will help improve the cure rate and prevent the overuse of ineffective antimicrobial agents. In this study, we developed a technique to quickly and easily assess antimicrobial susceptibility based on the genetic characteristics of M. bovis strains in Japan.

Mutations associated with change of susceptibility to lincosamides and/or macrolides in field and laboratory-derived Mycoplasma californicum strains in Japan, and development of a rapid detection method for these mutations.

Five mutations involved in changing of susceptibility to lincosamides and/or macrolides were investigated in field isolates of Mycoplasma californicum in Japan, and reconfirmed in laboratory-derived mutants. In addition, a quick and easy detection method for these mutations was established. Guanine at position 748 (Escherichia coli numbering) of the 23S rRNA gene (rrl) was shown to be involved with decreased susceptibility to 16-membered macrolides, and adenines at positions 2059 and 2062 of rrl were involved with decreased susceptibility to both lincosamides and macrolides. Both guanine at position 2576, and change from cytosine to thymine at position 2611 of rrl were found to be involved with decreased susceptibility to lincosamides, and the latter mutation also increased the susceptibility to erythromycin. These mutations were easily induced by several to approximately 30 passages in a medium containing the respective antimicrobial, but they did not return after their initial appearance. The melting curve analysis using hybridization probes revealed the existence of these mutations by the change in the melting curve shape and/or decrease in the melting peak temperature. The detection limit in milk samples with a somatic cell count up to 716 × 103 cell/mL was 133 cfu/mL, but an excessive increase in the cell count in milk or storage of the milk sample at chilling or freezing temperature decreased the sensitivity. This method requires only a few hours, so field veterinarians can make a same-day determination of susceptibility to macrolides and lincosamides, which are first-line antibiotics for bovine mycoplasmal mastitis.

Identification of a novel mechanism of action of bovine IgG antibodies specific for Staphylococcus aureus.

Staphylococcus aureus is a major pathogen that causes subclinical mastitis associated with huge economic losses to the dairy industry. A few vaccines for bovine mastitis are available, and they are expected to induce the production of S. aureus-specific antibodies that prevent bacterial adherence to host cells or promote opsonization by phagocytes. However, the efficacy of such vaccines are still under debate; therefore, further research focusing on improving the current vaccines by seeking additional mechanisms of action is required to reduce economic losses due to mastitis in the dairy industry. Here, we generated S. aureus-specific bovine IgG antibodies (anti-S. aureus) that directly inhibited bacterial growth in vitro. Inhibition depended on specificity for anti-S. aureus, not the interaction between Protein A and the fragment crystallizable region of the IgG antibodies or bacterial agglutination. An in vitro culture study using S. aureus strain JE2 and its deletion mutant JE2ΔSrtA, which lacks the gene encoding sortase A, revealed that the effect of anti-S. aureus was sortase-A-independent. Sortase A is involved in the synthesis of cell-wall-associated proteins. Thus, other surface molecules, such as membrane proteins, cell surface polysaccharides, or both, may trigger the inhibition of bacterial growth by anti-S. aureus. Together, our findings contribute insights into developing new strategies to further improve the available mastitis vaccine by designing a novel antigen on the surface of S. aureus to induce inhibitory signals that prevent bacterial growth.

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip.

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.

Complete Genome Sequence of Mycoplasma bovirhinis Strain HAZ141_2 from Bovine Nasal Discharge in Japan.

Mycoplasma bovirhinis, a mycoplasmal species involved in bovine respiratory diseases, is also a commensal microorganism that inhabits the bovine respiratory and reproductive organs. We present the complete 948,039-bp genome sequence of M. bovirhinis strain HAZ141_2, which was isolated from bovine nasal discharge in Japan.

Effect of intramammary infusion of recombinant bovine GM-CSF and IL-8 on CMT score, somatic cell count, and milk mononuclear cell populations in Holstein cows with Staphylococcus aureus subclinical mastitis.

The effect of intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) and interleukin-8 (rbIL-8) on mononuclear cell populations in quarters, somatic cell count (SCC) and the California Mastitis Test (CMT) score were investigated. From the selected cows with naturally occurring Staphylococcus aureus subclinical mastitis, one quarter of each cow were selected for the infusions of rbGM-CSF (400 µg/5 mL/quarter, n = 9), rbIL-8 (1 mg/5 mL/quarter, n = 9), and phosphate-buffered saline (5 mL/quarter, n = 7). The CMT score of both cytokines post infusion temporarily increased between days 0 and 1 and significantly decreased between days 7 and 14 compared to the preinfusion level. The SCC on day 14 after infusions of rbGM-CSF tended to be lower than that of the control group. The percentage of CD14+ cells increased on days 1 and 2 post infusion of rbGM-CSF. The percentage of CD4+ and CD8+ cells also increased on days 2 and 3, suggesting that the infusion of rbGM-CSF enhanced cellular immunity in the mammary gland. In contrast, the percentage of CD14+ cells decreased on days 0.25 and 1 post infusion of rbIL-8. No significant changes in the percentages of CD4+ and CD8+ cells in milk after infusion of rbIL-8 were evident during the experimental period, which suggested that rbIL-8 had little effect on the function of T cells in the mammary gland. These results indicated that rbGM-CSF and rbIL-8 decreased the CMT score by a different mechanism and may have a potential as therapeutic agents for subclinical mastitis.

Complete Genome Sequence of Mycoplasma bovigenitalium Strain HAZ 596 from a Bovine Vagina in Japan.

Mycoplasma bovigenitalium, a mycoplasmal species involved in various bovine diseases, including genital disease and mastitis, is also a commensal microorganism that inhabits the bovine genital organs. We present here the complete 853,553-bp genome sequence of M. bovigenitalium strain HAZ 596, which was isolated from a bovine vagina in Japan.

Photopolymerizable nanocomposite photonic materials and their holographic applications in light and neutron optics.

We present an overview of recent investigations of photopolymerizable nanocomposite photonic materials in which, thanks to their high degree of material selectivity, recorded volume gratings possess high refractive index modulation amplitude and high mechanical/thermal stability at the same time, providing versatile applications in light and neutron optics. We discuss the mechanism of grating formation in holographically exposed nanocomposite materials, based on a model of the photopolymerization-driven mutual diffusion of monomer and nanoparticles. Experimental inspection of the recorded grating's morphology by various physicochemical and optical methods is described. We then outline the holographic recording properties of volume gratings recorded in photopolymerizable nanocomposite materials consisting of inorganic/organic nanoparticles and monomers having various photopolymerization mechanisms. Finally, we show two examples of our holographic applications, holographic digital data storage and slow-neutron beam control.

The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells.

Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.

Bovine mastitis outbreak in Japan caused by methicillin-resistant Staphylococcus aureus New York/Japan clone.

Many methicillin-resistant Staphylococcus aureus (MRSA) strains are multidrug-resistant; consequently, infectious diseases involving MRSA are recognized as troublesome diseases not only in human health care but also in animal health care. A bovine mastitis case caused by MRSA isolates of the New York/Japan clone (NJC), which occurred in Japan in 2005, was monitored in the current study. Isolates of the NJC are typical of hospital-acquired MRSA in Japan. The genetic backgrounds of these strains differ from those of bovine-associated S. aureus, which are typically of clonal complex (CC)97, CC705, and CC133. Moreover, the NJC isolates in this bovine outbreak possessed a ß-hemolysin-converting bacteriophage and an immune evasion cluster, as found in the NJC isolates from humans, so it is possible that this clone was introduced into the dairy herd by a human carrier. Most bovine intramammary infections (IMIs) caused by the NJC isolates in our study were asymptomatic, and obvious clinical signs were recognized in only the first 3 infected cows. Of a total of 78 cows, 31 cows were MRSA carriers, and these carrier cows were detected by testing the milk of all lactating cows at 1-month intervals. These S. aureus carrier cows were culled or the infected quarter was dried off and no longer milked. Both IMI and mastitis caused by MRSA were completely eradicated after 5 months. Genotyping data suggested that exchanging of the staphylococcal cassette chromosome mec (the determining factor in methicillin resistance) occurred easily between MRSA and methicillin-sensitive S. aureus in the udders of carrier cows. This case study demonstrates an effective procedure against the spread of MRSA in a dairy herd, and highlights the risk of emergence of new MRSA strains in a dairy herd.

Variation and association of fibronectin-binding protein genes fnbA and fnbB in Staphylococcus aureus Japanese isolates.

Fibronectin-binding proteins A and B (FnBPA and FnBPB) mediate adhesion of Staphylococcus aureus to fibrinogen, elastin and fibronectin. FnBPA and FnBPB are encoded by two closely linked genes, fnbA and fnbB, respectively. With the exception of the N-terminal regions, the amino acid sequences of FnBPA and FnBPB are highly conserved. To investigate the genetics and evolution of fnbA and fnbB, the most variable regions, which code for the 67th amino acids of the A through B regions (A67-B) of fnbA and fnbB, were focused upon. Eighty isolates of S. aureus in Japan were sequenced and 19 and 18 types in fnbA and fnbB, respectively, identified. Although the phylogeny of fnbA and fnbB were found to be quite different, each fnbA type connected with a specific fnbB type, indicating that fnbA and fnbB mutate independently, whereas the combination of both genes after recombination is stable. Hence those fnbA-fnbB combinations were defined as FnBP sequence types (FnSTs). Representative isolates of each FnST were assigned distinct STs by multilocus sequence typing, suggesting correspondence of FnST with genome lineage. Linkage disequilibrium (LD) analysis of the A67-B region revealed that subdomains N2, N3 and FnBR1 form a LD block in fnbA, whereas N2 and N3 form two independent LD blocks in fnbB. N2-N3 three-dimensional structural models indicated that not only the variable amino acid residues, but also well-conserved amino acid residues between FnBPA and FnBPB, are located on the surface of the protein. These results highlight a molecular process of the FnBP that has evolved by mingled mutation and recombination with retention of functions.

Complete Genome Sequence of Mycoplasma arginini Strain HAZ 145_1 from Bovine Mastitic Milk in Japan.

Mycoplasma arginini is a species sometimes isolated from bovine specimens, mastitic milk, etc. Its pathogenicity against cows, however, is unspecific, unlike other bovine mycoplasmas. Its whole-genome sequence is needed to comprehend its real image. We present here the 678,592-bp complete genome sequence of M. arginini strain HAZ 145_1.

Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens.

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.

Complete Genome Sequence of Mycoplasma canadense Strain HAZ 360_1 from Bovine Mastitic Milk in Japan.

Bovine mycoplasmal mastitis is spreading quickly among cows. Mycoplasma canadense, a causal species of bovine mastitis, reduces milk quality and quantity via the infiltration of numerous inflammatory cells. Presented here is the complete 693,241-bp genome sequence of M. canadense strain HAZ 360_1, which was isolated in Japan.

Molecular epidemiology of cases of Mycoplasma californicum infection in Japan.

Bovine mastitis due to Mycoplasma californicum is often accompanied by huge economic losses, and the disease spreads very quickly. An appropriate molecular epidemiological analysis is needed to prevent and control infectious disease, but molecular epidemiological analysis methods for M. californicum have not yet been reported. Here we developed a combination of multiple-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) methods, which are common genotyping methods for various bacteria, for M. californicum. The MLVA is based on four interspersed repeat units that were found in the M. californicum genome data. The MLVA using these repeat units showed sufficient discriminatory power for a molecular epidemiological analysis; i.e., a Hunter-Gaston diversity index (HGDI) of 0.949, against M. californicum strains in Japan and M. californicum strain ATCC 33461. The PFGE for M. californicum also showed sufficient discriminatory power, with an HGDI of 0.985. Strain ATCC 33461 showed MLVA profiles and pulsotypes that differed greatly from those of strains from Japan. These results indicate that MLVA and PFGE are good tools for identifying M. californicum transmission events more accurately. Our combined MLVA and PFGE analysis suggests the persistence of M. californicum infection among herds in a specific area for a long period of time, as well as the movement of cows and heifers accompanying the expansion of M. californicum infection. Failure to identify asymptomatic infected cows is suspected as one of the central causes of the present M. californicum infection scenario in Japan.

Complete Genome Sequence of Mycoplasma californicum Strain HAZ160_1 from Bovine Mastitic Milk in Japan.

Bovine mycoplasmal mastitis is spreading quickly among cows. It often leads to clinical mastitis outbreaks and often results in huge economic losses. Mycoplasma californicum is an important causal species of bovine mastitis. Presented here is the 799,088-bp complete genome sequence of M. californicum strain HAZ160_1, which was isolated in Japan.

Eosinophilic granuloma with Splendore-Hoeppli material caused by toxigenic Corynebacterium ulcerans in a heifer.

Raised lesions were present on the left nasal vestibule of a 20-month-old Japanese Brown heifer. The largest mass which caused partial nasal obstruction was removed surgically. Corynebacterium ulcerans was identified in the mass. 16S ribosomal RNA and RNA polymerase beta subunit genes were 100% and 98% identical to other C. ulcerans strains. Histologically, multiple foci of eosinophilic granuloma with Splendore-Hoeppli material were seen. Rod-shaped Gram-positive organisms were detected with metachromatic granules, producing diphtheria toxin with 5, 30 and 48 amino acid differences to another C. ulcerans strain, C. diphtheriae or C. pseudotuberculosis, respectively. The toxin is highly cytotoxic and may be responsible for the formation of abundant Splendore-Hoeppli material. The lesion was therefore judged to be an allergic reaction to bacterial antigens or diphtheria toxin.

Molecular-based identification of yeasts isolated from bovine clinical mastitis in Japan.

This study analyzed molecular-based identification of yeasts that associated with bovine clinical mastitis in Japan. Over 3,200 quarter milk samples from Holstein dairy cows collected in 2011 on Hokkaido and Honshu islands were examined. Yeast isolates were characterized by polymerase chain reaction amplification and sequencing of the D1/D2 region of the 26S rDNA. Molecular characterization confirmed that Candida spp. and Pichia spp. were most frequently isolated species. Our molecular analysis of mastitic milk samples demonstrated the prevalence of Pichia kudriavzevii(22/58) and Candida tropicalis(14/58). In addition, we demonstrated that molecular analysis of the D1/D2 region of the 26S rDNA is a rapid and reliable method for identifying clinically significant yeasts in dairy hygiene, including potentially new or emerging pathogenic species.