Search

This study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.

Introduction Chronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death‐1 (PD‐1). The inhibitory signal is produced by the interaction between PD‐1 and its PD‐ligand 1 (PD‐L1) and impairs the effector functions of T cells. However, the expression dynamics of PD‐L1 and the immunological functions of the PD‐1/PD‐L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD‐L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD‐1/PD‐L1 pathway. Methods In the initial experiments, anti‐bovine PD‐L1 monoclonal antibodies (mAbs) were tested for cross‐reactivity with swine PD‐L1. Subsequently, immunohistochemical analysis was conducted using the anti‐PD‐L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti‐PD‐L1 mAb. Results Several anti‐PD‐L1 mAbs tested recognized swine PD‐L1‐expressing cells. The binding of swine PD‐L1 protein to swine PD‐1 was inhibited by some of these cross‐reactive mAbs. In addition, immunohistochemical analysis revealed that PD‐L1 was expressed at the site of infection in chronic infections of pigs. The PD‐L1 blockade increased the production of interleukin‐2 from swine PBMCs. Conclusions These findings suggest that the PD‐1/PD‐L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways., Immunohistochemical staining of PD‐L1 in tissues of pigs with chronic infections. Each section was stained using anti‐bovine PD‐L1 mAb (6C11‐3A11)

A bacterial culture of milk is the most common test to determine the presence of mastitis-causing pathogens, which informs appropriate treatment. However, a certain proportion of clinical mastitis milk shows no growth of any mastitis-causing pathogens. We hypothesized that bacterial culture-negative clinical mastitis milk is associated with the activity of antimicrobial components contained in the milk. In this study, the differences in antimicrobial components (lactoferrin, transferrin, lysozyme, lactoperoxidase, and lingual antimicrobial peptide [LAP]) between bacterial culture-positive and culture-negative bovine clinical mastitis milk were investigated using Holstein cows. Our results showed that 37 out of 71 samples of clinical mastitis milk had negative bacterial cultures. The LAP concentration in bacterial culture-negative milk was lower than that in positive milk (31.95 ± 1.64 nM vs. 42.85 ± 4.01 nM). In contrast, the lysozyme concentration in bacterial culture-negative milk was higher than that in positive milk (0.76 ± 0.15 μg/ml vs. 0.42 ± 0.06 μg/ml). In conclusion, the concentration of antimicrobial components was different between bacterial culture-positive and culture-negative bovine clinical mastitis milk, which suggests that antimicrobial components are related to bacterial culture results.

The aim of this study was to develop a pair of primers for detecting ruminant mycoplasma pathogens. We designed a set of primers based on the most similar sequences within 16sRNA regions of seven Mycoplasma spp. These primers have high sensitivity for detecting Mycoplasma dispar, M. arginine, M. canadense, M. bovis, M. alkalescens, M. californicum, and M. bovisgenitalium within the annealing temperature range of 46°C to 48°C. The minimum amount of DNA that can be detected using the protocol is 250 ng, which is equivalent to 2,000 cfu/mL. The primers can detect mycoplasma from DNA extracted directly from milk samples. The common bovine mastitis pathogens of Staphylococcus aureus coagulase-negative staphylococci, Escherichia coli, Streptococcus uberis, Klebiella pneumonia, Korucia rosea, and Acholeplasma spp. were not detected by the primers. We believe the high sensitivity and specificity of these primers make them useful for detecting infection with seven Mycoplasma species in ruminants, allowing the primers to be used in clinical settings.

Bovine Mycoplasma arthritis (MA) is caused by Mycoplasma bovis and exhibits severe clinical symptoms. However, the pathophysiology of bovine MA is incompletely understood. In this study, we examined the cytokine mRNA expression of synovial fluid (SF) cells and cytokine concentrations in the SF of MA calves. The SF was isolated from five clinically healthy (control) and seven MA calves. mRNA and protein levels of interleukin (IL)-1β, IL-6, IL-8, IL-12, and IL-17 in the SF from MA calves were significantly higher than those from control calves. Our results indicate that SF cells produce inflammatory cytokines, which mainly contribute to the severe inflammatory response in the joints of the MA calves.

Bovine pneumonia is a disease that causes significant economic losses in livestock industries and is vital for animal welfare. The whole-genome sequence of Pasteurella multocida strain Pm1, isolated from a calf suffering from pneumonia in Japan, is reported here.

This study evaluated the health status of the mammary glands and milk composition of dairy goats. The California mastitis test (CMT) score, somatic cell counts (SCCs), somatic cell type, electrical conductivity (EC), N-acetyl-β-d-glucosaminidase (NAGase) activity, mastitis-causing pathogens, and milk composition in 121 udder-half milk samples from 62 crossbreed goats (1-3 years old) at 23-45 days postpartum were compared in four categories with SCCs of200 × 10

The objective of this study was to evaluate effects of butyrate supplementation on plasma concentration of glucagon-like peptide-2 (GLP-2), apparent total-tract digestibility, and responses to a grain challenge of lactating dairy cows fed diets differing in starch content. Eight Holstein cows averaging 58.6 ± 9.96 d in milk (4 primiparous cows fitted with rumen cannula and 4 multiparous intact cows) were blocked by parity and assigned to one of two 4 × 4 Latin squares balanced for carryover effects with a 2 × 2 factorial arrangement of treatments. Treatments were dietary starch content [20.6 vs. 27.5%, respectively, for low starch (LS) and high starch (HS)] and butyrate supplementation (butyrate vs. control) with 21-d periods. Butyrate was provided as Gustor BP70 WS (Norel, S.A., Madrid, Spain), containing 70% sodium butyrate and 30% fatty acid mixture, at 2% of dietary dry matter (providing butyrate at 1.1% of dietary dry matter), and control premix contained 70% wheat bran and 30% fatty acid mixture. Feeds, orts, and fecal samples were collected from d 17 to 19 to determine apparent total-tract nutrient digestibility. Blood and rumen fluid samples were collected on d 19. The baseline of dry matter intake (DMI) was determined as average DMI from d 17 to 19 for each cow, and cows were feed-restricted at 60% of the baseline DMI on d 20, and a grain challenge was conducted by providing steam-flaked corn grain at 0.6% of body weight, on an as-fed basis, in addition to each treatment diet on d 21, and blood and ruminal fluid samples were collected. The interaction of dietary starch content by butyrate supplementation was significant for plasma GLP-2 concentration, being greater for cows fed butyrate with the HS diet than those fed the other 3 diets. Cows fed butyrate increased n-butyrate concentration in the ruminal fluid and tended to increase dry matter and organic matter digestibility compared with the control. During the grain challenge, rumen endotoxin concentration increased over time and was higher for cows fed the HS diets compared with those fed LS diets. However, response variables related to inflammation were not affected by the grain challenge. However, serum haptoglobin, lipopolysaccharide-binding protein, and serum amyloid-A concentrations were greater for cows fed butyrate with the LS diet, but not for those fed the HS diet. These results indicate that butyrate supplementation may increase plasma GLP-2 concentration for cows fed HS diets, and total-tract digestibility regardless of dietary starch content. However, butyrate supplementation did not mitigate inflammation in this study.

This study aimed to evaluate innate immune responses of mammary glands induced by intramammary infusion of Bifidobacterium breve in dairy cows. Somatic cell counts in quarters of cows showed a marked increase following B. breve infusion on days 1 and 2. Opsonized-stimulated chemiluminescence response in quarter milk was significantly (P

The immune related factors of peripheral blood mononuclear cells (PBMC) were analyzed in the clinical cases with Mycoplasma (M.) bovis infection. Seventy-eight Holstein calves in one farm were used. These calves were divided into three groups; the calves with M. bovis infection of poor outcome after treatment (Non-Recovery Group), the calves with M. bovis infection recovered (Recovery Group) and clinically healthy calves (Control Group). Blood samples were collected at days of the first medical treatment and the final treatment or euthanasia. IL-17A levels in the Non-Recovery Group were higher than those in the Recovery Group on both days. Our result suggested that the IL-17A of PBMC is an important factor to affect outcome of the calves with M. bovis infection.

Intramammary infusion of Bifidobacterium breve (B. breve)-induced somatic cell (SC) counts, chemiluminescent response (CL), lactoferrin (LF) concentrations and mastitis-causing pathogens from quarters with subclinical mastitis were measured to evaluate innate immune response of mammary glands in dairy cows at 3 to 4 weeks before drying off. SC counts in 7 quarters of 7 control cows and 5 quarters of 6 cows with mastitis increased markedly on day 1 and SC values in control cows were significantly (P

Pseudomonas aeruginosa causes various opportunistic infections in animals. Here, we report the complete genome sequence of P. aeruginosa strain Pa12, a fluoroquinolone-resistant isolate from a canine skin lesion. To expand the molecular antimicrobial characteristics of the isolate, the whole Pa12 genome was sequenced and assembled via long- and short-read platforms.

Mycoplasma arthritis that caused by Mycoplasma bovis exhibit severe lameness. This disease is difficult to cure with antibiotics, but the detailed pathological mechanisms have not been fully clarified. In this study, we examined the effects of intra-articular inoculation with M. bovis on immunological responses in calf joints. We inoculated three calves each with M. bovis or phosphate buffer saline (control) into the right stifle joint and dissected them at 15 days postinoculation. Mycoplasma bovis-inoculated calves exhibited swelling of the stifle joint, increases in synovial fluid, fibrin deposition, and cartilage thinning. Intracellular M. bovis was detected in synovial tissues analyzed by immunohistochemistry and transmission electron microscopy. Messenger RNA expressions of interleukin (IL)-1β, IL-6, IL-8, IL-12p40, and IL-17A in synovial fluid cells and synovial tissues from M. bovis-inoculated calves were significantly higher than those from control calves. Protein levels of these cytokines in synovial fluid from M. bovis-inoculated calves were markedly higher than those from control calves. Our study clarified that inoculation with M. bovis into the stifle joint induced the production of inflammatory cytokines by synovial fluid cells and synovial tissues, causing a severe inflammatory response in joints. Additionally, M. bovis could invade cells in synovial tissues, which may have aided it in evading antibiotics and host immune surveillance.

Mycoplasma bovis(M. bovis) is a significant worldwide pathogen of cattle. Neutrophils have an important role in the innate immune response during infection withM. bovis. However, even though neutrophils accumulate inM. bovisinfection, the interaction ofM. bovisand neutrophils has not been fully elucidated. We attempted to elucidate the innate immune response of neutrophils stimulated withM. bovisand evaluate the transcriptome and functional analysis of bovine neutrophils stimulated withM. bovis. Proinflammatory cytokines, such as inducible nitric oxide (iNOS), which was the most increased gene in transcriptome analysis, were increased in quantitative polymerase chain reaction analysis of bovine neutrophils stimulated with live or heat-killedM. bovis. Nitric oxide and intracellular reactive oxygen species production of neutrophils stimulated withM. boviswas significantly increased. Neutrophils stimulated withM. bovisshowed an increased ratio of nonapoptotic cell death compared to unstimulated controls. We demonstrated that neutrophil extracellular traps (NETs) formation was not recognized in neutrophils stimulated with liveM. bovis. However, heat-killedM. bovisinduced NETs formation. We also showed the interaction withM. bovisand bovine neutrophils regarding proinflammatory cytokine gene expression and functional expression related to NETs formation. Live and killedM. bovisinduced innate immune responses in neutrophils and had the potential to induce NETs formation, but liveM. bovisescaped NETs.

In recent years, antimicrobial-resistant Pseudomonas aeruginosa strains have increased in the veterinary field. Therefore, phage therapy has received significant attention as an approach for overcoming antimicrobial resistance. In this context, we isolated and characterized four Pseudomonas bacteriophages. Phylogenetic analysis showed that the isolated phages are novel Myoviridae Pbunavirus PB1-like phages with OR12 belonging to a different clade compared with the other three. These phages had distinct lytic activity against 22 P. aeruginosa veterinary isolates. The phage cocktail composed from the PB1-like phages clearly inhibited the occurrence of the phage-resistant variant, suggesting that these phages could be useful in phage therapy.

Manure compost has been thought of as a potential important route of transmission of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) from livestock to humans. To clarify the abundance of ARB and ARGs, ARB and ARGs were quantitatively determined in tetracycline-resistant Escherichia coli (harboring the tetA gene)-spiked feces in simulated composts. In the simulated composts, the concentration of spiked E. coli decreased below the detection limit at day 7. The tetA gene remained in manure compost for 20 days, although the levels of the gene decreased. Next, to clarify the field conditions of manure compost in Japan, the quantities of tetracycline-resistant bacteria, tetracycline resistance genes, and residual tetracyclines were determined using field-manure-matured composts in livestock farms. Tetracycline-resistant bacteria were detected in 54.5% of tested matured compost (6/11 farms). The copy number of the tetA gene and the concentrations of residual tetracyclines in field manure compost were significantly correlated. These results suggest that the use of antimicrobials in livestock constitutes a selective pressure, not only in livestock feces but also in manure compost. The appropriate use of antimicrobials in livestock and treatment of manure compost are important for avoiding the spread of ARB and ARGs.

The present study assessed the effects of intramammary infusion of Bifidobacterium breve (B. breve) on mastitis-causing pathogens and on the somatic cell counts (SCC) in lactating cows with chronic subclinical mastitis. The bacteriological cure rates of 42 quarters from 42 cows infected with Staphylococcus aureus, Corynebacterium bovis, coagulase-negative staphylococci, and environmental streptococci were 18.2% (2/11), 14.3% (1/7), 58.8% (10/17), and 28.6% (2/7), respectively, on day 14 after B. breve infusion. In a second trial, B. breve was infused into 18 quarters from 18 cows with chronic subclinical mastitis from which pathogens had not been isolated; the rates of quarters showing SCC > 50 × 104 cells/ml prior to B. breve infusion that decreased to < 30 × 104 cells/ml after infusion were significantly (p < .01) increased to 61.1% (11/18) on day 14 compared to that prior to infusion (0/18). The intramammary infusion of B. breve appears to be a non-antibiotic approach for elimination of minor pathogens and decreasing SCC in quarters with chronic subclinical mastitis in dairy cows.

The scavenger receptor CD163 is widely used as a cell signature for alternatively active “M2” macrophages in mammals. In this study, we generated two monoclonal antibodies, ABM-1A9 and ABM-2D6, against the extracellular region of bovine CD163. Conventional Western blotting using the antibodies yielded immunoreactive bands of bovine CD163 at 130 and 150 kDa in non-reduced and reduced spleen lysates, respectively. The minimum limit of detectable concentration of both antibodies was relatively lower (5.0 ng/mL) than that of the anti-human CD163 monoclonal antibody AM–3 K (>1.0 μg/mL), which has been used previously for the detection of bovine CD163. An immunohistochemical study using formalin-fixed paraffin-embedded sections revealed that ABM-1A9 and ABM-2D6 clearly stained some Iba1+ macrophages in the lymph nodes of cattle with mastitis. Moreover, the CD163-stained macrophages were frequently observed engulfing leukocytes. ELISA using ABM-2D6 distinguished levels of circulating soluble CD163 in healthy cattle (less than 16.9 pmol/mL) and cattle with mastitis (more than 33.7 pmol/mL). These new monoclonal antibodies can be used in the diagnosis and evaluation of inflammatory disease prognosis in cattle with immunohistological analyses and blood test applications.

Mycoplasma mastitis is a dairy herd health problem with growing concern in Japan. To complement the lack of epidemiological knowledge of the disease, we conducted estimation of herd-level prevalence and risk factor analysis for Mycoplasma bovis (M. bovis) mastitis using data collected from Tokachi region, one of the nation's largest milk producing area, in Hokkaido Prefecture in 2015. The herd-level prevalence was estimated at 3.8% (95% confidence interval (CI): 2.6%, 5.4%) across the region with municipality-specific prevalence ranging from 0% to 14.3%. In this study, identified risk factors for the presence of with M. bovis infection on farms were corporation-type farms and purchased cattle, after controlling for the herd size. Corporation-type farms may reflect higher frequencies of moving cows and fomites to and from other farms, which increases the risk of pathogen introduction. Purchased cattle was considered as one of the major pathways of the disease incursion, and this finding highlighted the importance of more stringent separation or quarantine protocols when introducing cattle from outside in Tokachi region. Due to the limited information available in this study and inherent nature of the study designs, these results should be interpreted with caution and further research is needed.

Pseudomonas aeruginosa, which causes chronic infections, has demonstrated rapid acquisition of antimicrobial resistance (AMR). Therefore, bacteriophages have received significant attention as promising antimicrobial agents; however, previous trials have reported the occurrence of phage-resistant variants. P. aeruginosa has lost large chromosomal fragments via evolutionary selection by MutL. Mutants lacking galU and hmgA, located in close proximity, exhibit phage resistance and brown color phenotype since hmgA encodes a homogentisic acid metabolic enzyme and deletion of galU results in a lack of O-antigen polysaccharide and absence of the phage receptor. In the present study, we evaluated this mechanism for controlling phage resistance in P. aeruginosa veterinary isolate Pa12. Phage-resistant Pa12 brown mutants (brmts) with galU and hmgA deletions were isolated. Whole-genome sequencing of the brmts revealed that regions 148-27 kbp upstream and 261-110 kbp downstream of galU were largely deleted from the Pa12 parental chromosome. Furthermore, all of these fluctuating deleted sequences in Pa12 brmts, tentatively designated bacteriophage-induced galU deficiency (BigD) regions, harbor multi-drug efflux system genes (mexXY). Minimum inhibitory concentration (MIC) assays demonstrated that brmts altered sensitivity to antibiotics and exhibited increased levofloxacin sensitivity compared with the Pa12 parent. Orbifloxacin and enrofloxacin also effectively suppressed growth of the Pa12 brmts, suggesting that MexXY, which mediates quinolone efflux and is located in the BigD region, might be associated with restoration of fluoroquinolone sensitivity. Our findings indicate that AMR-related genes in the BigD region could produce trade-off effects between phages and drug sensitivity and thereby contribute to a potential strategy to control and prevent phage-resistant variants in phage therapy.

Introduction Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. Methods In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. Results Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+CD4+ and PD-L1+CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+CD4+ and PD-1+CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. Conclusions These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.

Mycoplasma bovis is a significant pathogen in bovine infections including mastitis, pneumonia, arthritis and otitis media, and is the cause of large economic losses in beef and dairy farms. During infection with M. bovis, recruited neutrophils are not sufficient to eradicate M. bovis from the infection site. The release of neutrophil extracellular traps (NETs) is one of the innate immune responses of neutrophils but the effect of M. bovis on NET formation by bovine neutrophils has not yet been clarified. The objective of our research was to examine the effect of M. bovis on NET formation and the killing activity of bovine neutrophils. We showed that NETs were not detected following stimulation of neutrophils by M. bovis alone or with Phorbol 12-myristate 13-acetat (PMA). Reactive oxygen species production is essential for NET formation but the levels in neutrophils stimulated with M. bovis at multiplicity of infections of 10, 100, and 1000 were similar to those of unstimulated cells. NET formation induced by PMA stimulated neutrophils disappeared following the addition of M. bovis but this phenomenon was not observed when ethylenediaminetetraacetic acid (EDTA) was added. M. bovis colony forming units were significantly decreased by the addition of EDTA in the presence of NETs. Our results suggested that M. bovis infection alone did not induce NETs and that M. bovis nucleases, as hypothesis-based, contributed to resistance against the killing activity of NETs.

Natural infection with larval Echinococcus multilocularis was recognized in one of eight Norway rats, Rattus norvegicus, caught indoors in 2009 in Ebetsu, Hokkaido, northern Japan. Cystic lesions were found in the right median and lateral lobes of the liver, with numerous alveolar cysts in the periphery of the lesions. Protoscolices were formed within large cysts. The laminated layers of the cysts were positive for PAS staining. Nested PCR using the primers specific for Taenia mitochondrial 12S rDNA yielded a 250-bp product, and the sequence of the PCR product matched that of E. multilocularis isolates from Hokkaido and Germany. This is the third natural alveolar hydatidosis in R. norvegicus in Japan.

The objective of this case-control study was to determine the herd- and cow-level risk factors associated with an outbreak of Mycoplasma bovine mastitis in the winter of 2014–2015 in Nemuro, Hokkaido, Japan. Two questionnaire surveys were sent to all 40 Mycoplasma-infected farms in the area and 73 non-infected farms for the farm-level analysis. Infected cows were matched to twice the number of non-infected cows in the same herds by parity and days after calving. Movement records, dairy herd test records, and clinical records of infected cows and matched non-infected cows were collected for the cow-level analysis. Risk factors for Mycoplasma infection were explored by multivariable analyses at both levels. In the herd-level analysis, tie stall housing for milking cows (odds ratio [OR] = 0.20, 95 % confidence interval [CI]: 0.07-0.60, p = 0.004), consciously wiping of teat openings before milking (OR = 0.15, 95 % CI: 0.02-0.76, p = 0.030), and use of paper towels to wipe teats (OR = 0.31, 95 % CI: 0.09-0.92, p = 0.045) were identified as preventive factors, whereas introduction of cattle (OR = 3.43, 95 % CI: 1.14–10.86, p = 0.030) was identified as a risk factor. In the cow-level analysis, a history of presence in livestock markets (OR = 10.80, 95 % CI: 1.12–104.38, p = 0.040), higher milk yield 2 months prior to Mycoplasma infection (OR = 1.09, 95 % CI: 1.02–1.18, p = 0.014), and previous diagnosis of acute mastitis without isolation of the causal pathogen (OR = 3.14, 95 % CI: 0.86–11.41, p = 0.082) were identified as risk factors. These results highlight the importance of proper milking hygiene control and quarantine of introduced cattle to prevent Mycoplasma infection.

The aim of this study was to characterize the phenotypes and genotypes of Staphylococcus aureus isolated from raw bovine milk in Hokkaido, Japan. S. aureus isolates were identified in 135 of 436 milk samples from cows with and without signs of mastitis from three farms in Hokkaido. These clinical isolates were characterized for antimicrobial susceptibility patterns, molecular typing using phage-open-reading frame typing (POT), coagulase gene type, virulence genes, and biofilm-associated genes and were evaluated for biofilm-forming ability. Most isolates were susceptible to the antimicrobial agents tested. The highest rate of resistance was to ampicillin. Molecular typing of all S. aureus isolates indicated a predominance of coagulase type VI and 0-17-34 POT type, and virulence genes were highly prevalent in the isolates from all farms. Moreover, a high percentage of the 0-17-34 POT type isolates showed extensive formation of biofilm. These findings will help veterinarians and farmers to understand the epidemiology of S. aureus so that they can monitor the transmission and spread of this pathogen and control it more effectively.

Mycoplasma bovis causes chronic arthritis in cattle, accompanied by a severe inflammatory reaction of the joints. Recent studies demonstrated that M. bovis can invade bovine non-phagocytic cells, but the mechanism of M. bovis internalization in the cells remains unclear. In this study, we examined the mechanism by which M. bovis invades synovial cells, including the pathway of cell invasion. Using fluorescence and electron microscopy, multiple M. bovis were observed to adhere to and be internalized in cultured bovine synovial cells. The number of M. bovis colocalized with clathrin heavy chain (CLTC) per cell was significantly higher than the number of M. bovis colocalized with caveolin-1 (Cav-1). The internalized ratio of M. bovis in synovial cells treated with clathrin-dependent endocytosis inhibitor and small interfering RNA (siRNA) against CLTC was significantly lower than that in control cells. In contrast, the internalized ratio of M. bovis in synovial cells was unaffected by siRNA against Cav-1. These findings provide the first evidence that clathrin-dependent endocytosis is one of the major pathways by which M. bovis invades into synovial cells.

Mycoplasma bovis is a pathogenic bacterium in bovines that causes huge global economic losses. Numerous factors play important roles in M. bovis pathogenesis; however, the host immune response involved in M. bovis infection has not been fully elucidated. We aimed to determine the characteristics of the host immune response to Mycoplasma infection. We evaluated the responsiveness of bovine peripheral blood mononuclear cells (PBMCs) stimulated with M. bovis via microarray analysis. The transcriptional abundance of innate immune-related genes IL-36A, IL-27, IFN-γ, and IL-17 in PBMCs increased after M. bovis exposure. Upon M. bovis infection, there was increased expression of the lymphocyte activated genes basic leucine zipper transcription factor (BATF) and signaling lymphocytic activation molecule family members 1 and 7 (SLAMF 1 and SLAMF 7) in PBMCs compared with that in unstimulated cells. The study revealed that the transcriptional abundance of innate immunity genes in PBMCs increased during M. bovis infection. This induced the activation of PBMCs, giving rise to an immune response, which is followed by the development of the inflammatory response. The results from this study could be used as the basis for the development of novel vaccine candidates against M. bovis.

Regulatory T cells (Tregs) regulate immune responses and maintain host immune homeostasis. Tregs contribute to the disease progression of several chronic infections by oversuppressing immune responses via the secretion of immunosuppressive cytokines, such as transforming growth factor (TGF)-β and interleukin-10. In the present study, we examined the association of Tregs with Mycoplasma bovis infection, in which immunosuppression is frequently observed. Compared with uninfected cattle, the percentage of Tregs, CD4+CD25highFoxp3+ T cells, was increased in M. bovis-infected cattle. Additionally, the plasma of M. bovis-infected cattle contained the high concentrations of TGF-β1, and M. bovis infection induced TGF-β1 production from bovine immune cells in in vitro cultures. Finally, we analyzed the immunosuppressive effects of TGF-β1 on bovine immune cells. Treatment with TGF-β1 significantly decreased the expression of CD69, an activation marker, in T cells, and Th1 cytokine production in vitro. These results suggest that the increase in Tregs and TGF-β1 secretion could be one of the immunosuppressive mechanisms and that lead to increased susceptibility to other infections in terms of exacerbation of disease during M. bovis infection.

Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria,Pseudomonas aeruginosasometimes causes rapid corneal corruption and, in some cases, blindness. Antimicrobial resistance can make treatment very difficult. Therefore, new strategies to control bacterial infection are required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is different, we examined the use of phage to treat horse bacterial keratitis. We isolatedMyoviridaeorPodoviridaephages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ΦR18 phage were adsorbed to host cells after 30 s. In our keratitis mouse model, the administration of phage within 3 h also could kill bacteria and suppress keratitis. A phage multiplicity of infection of 100 times the host bacterial number could kill host bacteria effectively. A cocktail of two phages suppressed bacteria in the keratitis model mouse. These data demonstrated that the phages in this study could completely prevent the keratitis caused byP. aeruginosain a keratitis mouse model. Furthermore, these results suggest that phage may be a more effective prophylaxis for horse keratitis than the current preventive use of antibiotics. Such treatment may reduce the use of antibiotics and therefore antibiotic resistance. Further studies are required to assess phage therapy as a candidate for treatment of horse keratitis.IMPORTANCEAntibiotic-resistant bacteria are emerging all over the world. Bacteriophages have great potential for resolution of this problem. A bacteriophage, or phage, is a virus that infects bacteria specifically. As a novel therapeutic strategy against racehorse keratitis caused byPseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work hadin vitroeffectiveness for a broad range ofP. aeruginosastrains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models ofP. aeruginosakeratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

Mycoplasma bovis causes chronic arthritis in calves, presenting as osteolysis in affected joints. Matrix metalloproteinase-3 (MMP-3), an enzyme involved in cartilage degradation, is produced by synovial cells. Production of this proteinase is regulated by interleukin (IL)-1β, which is produced by mononuclear cells. Both factors are known to play important roles in osteolysis in human autoimmune and bacterial arthritis. However, the pathophysiology of Mycoplasma arthritis (MA) has not been elucidated. In this study, we evaluated the levels of MMP-3 and IL-1β in synovial fluid (SF) from MA calves and examined the effect of IL-1β on MMP-3 expression in bovine synovial cells in vitro. Levels of MMP-3 and IL-1β in SF from MA calves were significantly higher than those of clinically healthy calves. Mycoplasma bovis induced significant increases in the expression of IL-1β mRNA and protein in mononuclear cells, compared with cells not exposed to M. bovis. Interestingly, the supernatant of mononuclear cells stimulated with M. bovis contained high levels of IL-1β, which induced higher expression of MMP-3 mRNA and protein in synovial cells than direct stimulation by M. bovis. Recombinant bovine IL-1β also induced increased MMP-3 mRNA and protein expression in synovial cells. Our results indicate that M. bovis induces IL-1β expression by bovine mononuclear cells, and this cytokine then promotes MMP-3 production by synovial cells. These findings suggest that MMP-3 and IL-1β are key factors in the development of osteolysis in MA calves.

Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis. Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis. The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.