Bovine mastitis is an inflammation of the mammary gland caused by bacterial infection in dairy cattle. It is the most costly disease in the dairy industry because of the high use of antibiotics. Staphylococcus aureus is one of the major causative agents of bovine mastitis and antimicrobial resistance. Therefore, new strategies to control bacterial infection are required in the dairy industry. One potential strategy is bacteriophage (phage) therapy. In the present study, we examined the host range of previously isolated S. aureus phages ΦSA012 and ΦSA039 against S. aureus strains isolated from mastitic cows. These phages could kill all S. aureus (93 strains from 40 genotypes) and methicillin-resistant S. aureus (six strains from six genotypes) strains tested. Using a mouse mastitis model, we demonstrated that ΦSA012 reduced proliferation of S. aureus and inflammation in the mammary gland. Furthermore, intravenous or intraperitoneal phage administration reduced proliferation of S. aureus in the mammary glands. These results suggest that broad host range phages ΦSA012 is potential antibacterial agents for dairy production medicine.

Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria, Pseudomonas aeruginosa sometimes 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 isolated Myoviridae or Podoviridae phages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ⌽R18 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 by P. aeruginosa in 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 by Pseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work had in vitro effectiveness for a broad range of P. aeruginosa strains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models of P. aeruginosa keratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

We investigated the contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (enrofloxa-cin, orbifloxacin and danofloxacin) susceptibility in 58 Mycoplasma bovis isolates from dairy cattle in Japan. Fluoroquinolone non-resistant isolates (fluoroquinolone-MICs≤2 µg/ml) possessed no QRDR mutations (19 isolates) or Ser83Leu in GyrA (32 isolates). Fluoroquinolone-resistant isolates (fluoroquinolone-MICs≥4 µg/ml) possessed Ser83Leu in GyrA and Ser81Pro in ParC (3 isolates) or Ser83Phe in GyrA and Ser80Ile in ParC (4 isolates). Laboratory-derived fluoroquinolone-resistant mutants selected from 2 isolates (possessing Ser83Leu in GyrA) had an amino acid substitution in ParC at the same position (Ser80Ile or Ser81Tyr) as fluoroquinolone-resistant isolates, suggesting a concurrent amino acid substitution in ParC (Ser80 or Ser81) is important in fluoroquinolone resistance in M. bovis isolates. Mycoplasma bovis (Mb), a causative agent of bovine respiratory disease, causes high morbidity, and has an economic impact on the cattle industry [3]. Antimicrobials, such as fluoroquinolones, macrolides and tetracyclines, are important for treating Mb infections and preventing its spread in dairy calves. Fluoroquinolones are broad-spectrum anti-microbials, and enrofloxacin (ENR), orbifloxacin (ORB) and danofloxacin (DAN) have been approved for use in cattle in Japan. Fluoroquinolone minimum inhibitory concentrations (MICs) against Mb have previously been investigated [6, 8]. Mb isolated from cattle in Japan showed various ENR MICs (range≤0.125-4 µg/ml) [8]. Ten of eleven ENR-resistant (ENR MIC≥2 µg/ml) Mb isolates derived from cattle in Israel carried 2 missense mutations within quinolone resistance-determining regions (QRDRs), causing the amino acid substitutions (AASs) Ser83Phe in GyrA and Asp84Asn in ParC [6]. Although studies have suggested that QRDR mutations contribute to elevated ENR MICs, the association between QRDR mutations and fluoroquinolone MICs has yet to be confirmed in Mb [6]. We investigated QRDR mutations in Mb isolates and in vitro fluoroquinolone-selected mutants obtained from Mb isolates with various ENR, ORB and DAN MICs to clarify the effect of AAS in GyrA and ParC on fluoroquinolone resistance in Mb isolates from dairy calves in Japan. Fifty-eight Mb isolates were derived from nasal swabs of asymptomatic dairy calves from 51 farms located throughout Hokkaido during 2010-2011. These isolates were provided by 7 branches of the Hokkaido Agricultural Insurance Scheme (Dounan, Iburi-Hidaka, Ishikari-Sorachi, Kitami-Ohotsuku, Kushiro-Nemuro, Souya-Rumoi-Kamikawa and Tokachi) and were cultured in 1 ml NK broth (Kanto Chemical , Tokyo, Japan). After culture on modified Hayflick agar (35.5 g/l of PPLO, 15% equine serum, 2.5% yeast extract and 0.0024% deoxyribonucleic acid; Kanto Chemical) at 37°C in 5% CO 2 for 3 days to 1 week, a single colony was picked, inoculated into 1 ml of NK broth and further cultured at 37°C in 5% CO 2 for 3 days. ENR, ORB and DAN MICs were determined using ...

Culture-independent PCR-DGGE fingerprinting was used to reveal the bacterial composition and diversity associated with raw milk of mastitis cows from Hokkaido, Japan for the first time. All the mastitis milk samples were diagnosed as solely infection by Coliforms using the classical microbiological method based on on-farm culturing. Our results revealed that the bovine mastitis-associated bacteria were host-specific because community structure varied between each sample. Klebsiella pseudomoniae, Lactococcus lactis Staphylococcus aureus and Escherichia sp were found to be the widely distributed species. Furthermore, more than one mastitis-causing pathogen was found to be present in some mastitis samples. These pathogens may not only act as etiology agents but also play a role in disrupting the natural microbial ecology in mastitis bovine. This finding highlights the limitation of the traditional identification and characterization strategy. Therefore, it is suggested that the methodology applied in this study might be a valuable addition to mastitis control and prevention.

IntroductionBovine 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.MethodsIn 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.ResultsExpectedly, 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.ConclusionsThese 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.

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ABSTRACT. A female stillborn Holstein calf with shortened cervical and thoracic regions, protrusion of the tongue, and bilateral symmetric flexural contraction of the anterior limbs was delivered on gestation day 281. Multiple hemivertebrae, fused and misshaped vertebrae, synostosis and scoliosis of cervical, thoracic and lumbar vertebral column were found in the affected calf by radiographic and c omputed tomographic (CT) analysis. Ten pairs of ribs were present and the sternum consisted of 9 sternebrae. Multiple morphologic abnormalities including fusion, malformation, and displacement, were found in the ribs and sternum. Cardiac anomalies, including atrial septal defect and hypertrophy of right ventricle, were observed. DNA-polymerase chain reaction (PCR) analysis demonstrated that amplified product from the liver DNA of the affected calf had identical pattern to that associated with complex vertebral malformation (CVM) of Holstein calves and that her dam was a heterozygous carrier of CVM. The affected calf was diagnosed as having CVM based on the DNA-PCR results and the characteristic findings, and was recorded as a first documentation of CVM confirmed in a Holstein calf in Japan. KEY WORDS: CVM, genetic defect, Holstein calf, malformation of vertebrae, stillborn.

Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E 2 (PGE 2 ) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE 2 in immune dysfunction and the relationship between PGE 2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE 2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE 2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE 2 levels were positively correlated with the proportions of PD-L1 + monocytes in M. bovis-infected cattle. Additionally, plasma PGE 2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE 2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade Goto et al.PGE 2 -Induced Immunodysfunction During Bovine Mycoplasmosis assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE 2 and the PD-1/PD-L1 pathway.