Efficacy of treating bacterial bioaerosols with weakly acidic hypochlorous water: A simulation chamber study.

The COVID-19 pandemic highlighted the dangers of airborne transmission and the risks of pathogen-containing small airborne droplet inhalation as an infection route. As a pathogen control, Weakly Acidic Hypochlorous Water (WAHW) is used for surface disinfection. However, there are limited assessments of air disinfection by WAHW against airborne pathogens like bioaerosols. This was an empirical study evaluating the disinfection efficacy of WAHW in an atmospheric simulation chamber system against four selected model bacteria. The strains tested included Staphylococcus aureus (SA), Escherichia coli (EC), Pseudomonas aeruginosa (PA), and Pseudomonas aeruginosa (PAO1). Each bacterial solution was nebulized into the chamber system as the initial step, and bioaerosol was collected into the liquid medium by a bio-sampler for colony forming units (CFU) determination. Secondly, the nebulized bacterial bioaerosol was exposed to nebulized double distilled water (DDW) as the control and nebulized 150 ppm of WAHW as the experimental groups. After the 3 and 30-min reaction periods, the aerosol mixture inside the chamber was sampled in liquid media and then cultured on agar plates with different dilution factors to determine the CFU. Survival rates were calculated by a pre-exposed CFU value as a reference point. The use of WAHW decreased bacterial survival rates to 1.65-30.15% compared to the DDW control. PAO1 showed the highest survival rates and stability at 3 min was higher than 30 min in all experiments. Statistical analysis indicated that bacteria survival rates were significantly reduced compared to the controls. This work verifies the bactericidal effects against Gram-positive/negative bioaerosols of WAHW treatment. As WAHW contains chlorine in the acid solution, residual chlorine air concentration is a concern and the disinfection effect at different concentrations also requires investigation. Future studies should identify optimal times to minimize the treated time range and require measurements in a real environment.

Protective effects of probiotics against tannin-induced immunosuppression in broiler chickens.

Tannins (TAs) are an anti-nutritional substance commonly used as a natural feed additive for livestock. However, our previous study described the dose-dependent adverse effects of TA on immune responses and growth in chickens. In this study, we evaluated the protective effects of a probiotic preparation (BT) consisting of three different bacteria (Bacillus mesenteric, Clostridium butyricum, and Streptococcus faecalis) against TA-induced immunosuppression in chickens. Forty chicks were divided into 4 groups as follows: the CON group (basal diet), BT group supplemented with 3 g BT/kg diet, tannic acid (TA) group supplemented with 30 g TA/kg diet, and BT+TA group supplemented with 3 g BT/kg diet + 30 g TA/kg diet. The feeding trial lasted for 35 days. Lymphocyte subset, macrophage phagocytosis, cytokine mRNA expression, and primary and secondary IgY immune responses were evaluated. BT supplementation significantly improved TA-induced reductions in final body weight, body weight gain, feed intake, and relative weights of lymphoid organs compared with the TA group. Furthermore, in the spleen and cecal tonsil (CT), the relative populations of CD4+, CD8+, and CD4+CD8+ cells in the BT+TA group were significantly ameliorated compared with the TA group. Additionally, comparison with the TA group showed that the chickens in the BT+TA group had an improved relative population of B cells in the CT and that macrophage phagocytosis in the spleen was significantly increased. Chickens in the BT+TA group showed significant increases in IFN-γ and IL-4 mRNA expression in the spleen compared with the TA group. The primary and secondary IgY responses were significantly improved. These results revealed that supplementation with BT protects against TA-induced immunosuppression in chickens.

Effects of Probiotics on Growth and Immunity of Piglets.

Growth promoting antibiotics are used in modern animal husbandry to promote growth and avoid infections. Negative effects of these antibiotics on human health are a big concern and they need to be replaced. Probiotics are expected to be a good replacement for growth promoting antibiotics. In this study, we evaluated effects of probiotics on growth and immune status of liver and secondary lymphoid organs of piglets. Body weights of probiotic-fed piglets were heavier than those of control piglets (p < 0.05) at days 30 and 45 of the experiment. Relative populations of CD4+ and IgM+ cells isolated from the liver were significantly increased (p < 0.05 and p < 0.01, respectively) in probiotic-fed piglets compared to control piglets. CD4+CD8+ T cells were significantly decreased (p < 0.05) in jejunal Peyer's patches of treated piglets. Phagocytosis of MHC class II+ cells isolated from the liver of probiotic-fed piglets was significantly higher (p < 0.05) than that of control piglets. Phagocytosis of granulocytes isolated from the liver and peripheral blood of probiotic-fed piglets were also higher than those of control piglets. These results indicate excellent effects on growth and immune status of piglets. In conclusion, probiotics have beneficial effects on the growth and health of piglets and could be good replacement for growth promoting antibiotics.

Optimizing antimicrobial use by improving medication adherence among pig producers.

In Japan, many pig producers buy antimicrobials using veterinarians' prescriptions. Even if veterinarians prescribe antimicrobials for treatments and metaphylaxis, producers may be using them for prophylaxis on farms with a high incidence of chronic diseases. To reduce the frequency of antimicrobial use, we applied the concept of medication adherence and launched an intervention in eight farrow-to-finish farms. For the intervention farms, we attempted to implement measures for preventing the repeated onset of chronic infections that interfered with adherence, confirmed the need to continue medications, and discontinued medications if possible. For each farm, these interventions began at different time points after 2017 and continued until 2020. We evaluated the effectiveness of this intervention based on the amount of active ingredients/population correction unit and the total number of defined daily doses for the weight group/slaughter pig. The ratio of antimicrobial use for both indicators in the year 2020 compared with the start of the intervention decreased on all farms. Among the eight non-intervention farms, six showed an increase in both indicators. These results suggest that interventions for chronic diseases administered by producers and veterinarians lead to high adherence to prescriptions and responsible and prudent use of antimicrobials on pig farms.

Interferon gamma, lipopolysaccharide, and modified-live viral vaccines stimulation alter the mRNA expression of tumor necrosis factor α, inducible nitric oxide synthase, and interferon ß in bovine alveolar macrophages.

To understand the pathogenesis of bovine respiratory disease (BRD), it is necessary to elucidate the mechanisms of alveolar macrophage regulation by cytokines and pathogen-associated molecular patterns (PAMPs). Moreover, "non-specific effects (NSEs)" an innate immune regulatory mechanism in response to vaccines containing PAMPs, has recently attracted attention. It may be applied to BRD control, but there is limited knowledge in bovine. To investigate this, we stimulated alveolar macrophages in vitro with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid sodium salt (Poly I:C), interferon gamma (IFN-γ), and modified-live viral (MLV) vaccines, respectively, and analyzed changes in tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), and interferon beta (IFN-ß) mRNA expression levels. mRNA expression levels of TNF-α, iNOS, and IFN-ß were significantly increased in bovine alveolar macrophages stimulated by IFN-γ and MLV vaccine; LPS, IFN-γ, and MLV vaccine; and MLV vaccine only, respectively. Additionally, all MLV vaccine-stimulated mRNA expression increases were observed in a concentration-dependent manner. These results revealed in part, the mechanism of bovine alveolar macrophage regulation by cytokines and PAMPs. Understanding the regulatory mechanisms of alveolar macrophages will contribute to understanding the pathogenesis of BRD and preventive and therapeutic BRD management based on NSEs.

Antibiotic-resistant Escherichia coli isolated from dairy cows and their surrounding environment on a livestock farm practicing prudent antimicrobial use.

On a livestock farm where antimicrobial administration and its history had been managed for prudent use of antimicrobials, we surveyed antibiotic-resistant Escherichia coli strains isolated from cow feces and the surrounding environment (i.e., rat and crow feces, and water samples from a drainage pit and wastewater processing tank) every month for 1 year. Two strains (1.7%) in cow feces were resistant to tetracycline, whereas all other strains were susceptible to all other antimicrobials. Among 136 strains isolated from cows and wild animals, only one ampicillin-resistant strain was identified. The antibiotic resistance rate in the drainage from the barn was 8.3% (10/120), and all strains showed susceptibility for 8 months of the year. Tetracycline resistance was common in all resistant strains isolated from animal feces and water samples; all tetracycline-resistant strains carried tetA. These results strongly support the proper use and management of antibiotics on farms to minimize the outbreak and spread of antibiotic-resistant bacteria.

Production of granulomas in Mycoplasma bovis infection associated with meningitis-meningoencephalitis, endocarditis, and pneumonia in cattle.

Mycoplasma bovis, the most important primary pathogen in the family Mycoplasmataceae, causes pneumonia, arthritis, otitis media, and mastitis in cattle. Histopathologic pulmonary changes associated with M. bovis infection have been characterized as suppurative-to-caseonecrotic bronchopneumonia; infection in other organs has been reported in only a few studies that examined caseonecrotic endocarditis and suppurative meningitis. Granulomatous lesions associated with M. bovis infection have been reported only rarely. We studied the granulomatous inflammation associated with M. bovis infection in several organs of 21 Japanese Black cattle. M. bovis was detected by isolation and loop-mediated isothermal amplification methods; other bacteria were detected using culture on 5% blood sheep agar and a MALDI-TOF MS Biotyper. Tissues were examined by histopathology and by immunohistochemistry (IHC) using anti-M. bovis, anti-Iba1, anti-iNOS, and anti-CD204 antibodies. All 21 cases, which included 2 cases of meningitis-meningoencephalitis, 8 cases of endocarditis, and 11 cases of bronchopneumonia, had caseonecrotic granulomatous inflammation associated with M. bovis infection. The IHC for macrophages revealed a predominance of iNOS-labeled (M1) macrophages in the inner layer of the caseonecrotic granulomas associated with meningitis-meningoencephalitis, endocarditis, and bronchopneumonia in Japanese Black cattle naturally infected with M. bovis.

Eimeria pragensis infection alters the gut microenvironment to favor extrinsic shiga toxin-producing Escherichia coli O157:H7 colonization in mice.

We examined the effects of Eimeria pragensis infection on intestinal peristalsis, goblet cell proliferation and intestinal flora in C57BL/6 mice. Intestinal peristalsis was evaluated by radiography using barium at 7 days post-infection (p.i.). The intestinal peristalsis of E. pragensis-infected mice was significantly suppressed compared with uninfected control mice. Twenty-three mice were divided into 5 groups of 4 or 5 mice each; 2 groups of mice were infected with E. pragensis and the others were kept uninfected. At 7 days p.i., E. pragensis-infected and -uninfected mice were sacrificed to examine goblet cell numbers in the intestines, and significant decreases were observed only in the infected mice. Shiga toxin-producing Escherichia coli (STEC) O157:H7 was inoculated orally in mice both infected and uninfected with E. pragensis at 7 days p.i., with the remaining mice used as uninoculated controls. When mice were sacrificed at 2 days after STEC inoculation, STEC was only detected in the intestines of E. pragensis-infected mice. Colonization of STEC was also confirmed by immunohistochemistry on the surface of epithelial cells in concurrently infected/inoculated mice. Also, an overgrowth of residential E. coli was observed only in E. pragensis-infected mice. These results suggest that E. pragensis induces the suppression of intestinal peristalsis and modifies the intestinal environment to facilitate artificially introduced STEC colonization and multiplication, in addition to residential E. coli overgrowth.

The presence of adhesion factors NOX, α-enolase, TrmFO, P27, and VpmaX in Mycoplasma bovis wild isolates in Japan.

Background: Mycoplasma bovis causes various diseases such as bronchopneumonia, otitis media, arthritis, and mastitis in cattle. Mycoplasma bovis is often isolated from the deep pharynges of healthy cattle and is generally considered not to cause clinical symptoms while in the upper respiratory tract. In mycoplasma infections, adhesion to the host cells is a crucial step. In recent years, five new adhesins, NOX, α-enolase, TrmFO, P27, and VpmaX, have been reported in M. bovis strains from pneumonia cases. However, the presence of these adhesins in wild isolates has not been established. Aim: This study aimed to investigate the presence of these adhesin genes in wild isolates isolated from cattle nasal cavities and lesion sites (pneumonia, otitis media, arthritis, and mastitis) in various regions in Japan and clarify the relationship between adhesion and the symptoms caused by M. bovis infection. Methods: A total of 141 M. bovis wild isolates isolated from nasal cavities (healthy or sick cattle), lungs with pneumonia, ears with otitis media, joint fluids of arthritic animals, and milk of mastitic animals. Mycoplasma bovis type strain PG45 was also used. Specific polymerase chain reaction reactions were performed to detect nox, α-enolase, trmFO, P27, and vpmaX, which are adhesins of M. bovis. Results: This study reports 139 M. bovis wild isolates were positive for nox, α-enolase, trmFO, P27, and vpmaX, while two isolates each lacked α-enolase or P27 genes. Mycoplasma bovis PG45 also had all five adherens genes. Conclusion: Almost all M. bovis wild isolates possessed all nox, α-enolase, trmFO, P27, and vpmaX genes regardless of the lesion site or region of origin. This means no relationship was found between the presence of the five adhesins and lesion sites in M. bovis and M. bovis isolated from the nasal cavities of asymptomatic cattle have the same numbers and types of adhesins as isolates from symptomatic lesion sites (pneumonia, otitis media, arthritis, and mastitis). This suggests that not only M. bovis isolates from pulmonary lesions, but also M. bovis existing in the nasal cavity has the potential to causes symptoms in the host.

Neutrophil and M2-polarized Macrophage Infiltration, Expression of IL-8 and Apoptosis in Mycoplasma hyopneumoniae Pneumonia in Swine.

Mycoplasma hyopneumoniae (Mhp) is the primary pathogen of porcine enzootic pneumonia (PEP). Consolidated lung tissue from the cranioventral lung lobes of 15 pigs with PEP was collected for quantitative polymerase chain reaction, histopathology and immunohistochemistry. Histopathology revealed the co-existence of bronchial-associated lymphoid tissue hyperplasia with intra-alveolar neutrophils and macrophage infiltration in lesions of suppurative bronchopneumonia. Immunolabelling of infiltrated macrophages with CD163/CD204 indicated the presence of M2-polarized macrophages. Mhp antigen was detected on respiratory epithelial cells and in phagocytosed neutrophils. The intensity of Mhp immunolabelling and number of CD163/CD204-positive macrophages were correlated with the Mhp load in lung tissue (r = 0.87, 0.56, P <0.05). IL-8 immunolabelling was mainly found in neutrophils and correlated with Mhp load, Mhp immunolabelling and histological lesion score (r = 0.70, 0.66, 0.64, P <0.05), respectively. Apoptosis was seen in intra-alveolar cells and was correlated with Mhp load (r = 0.62, P <0.05). It is postulated that IL-8 attracts neutrophils to the lesions, while M2-polarized macrophages are a major source of IL-10 and promote a Th2-type immune response.

Mycoplasma bovis May Travel Along the Eustachian Tube to Cause Meningitis in Japanese Black Cattle.

Mycoplasma bovis (M. bovis) is a common inhabitant of the upper and lower respiratory tracts of cattle and is considered to be the main aetiological agent of otitis media in calves. The eustachian tube appears to be the most common portal for pathogens to enter the middle ear. We investigated the transmission route of M. bovis causing otitis media that progressed to meningitis or meningoencephalitis in Japanese Black cattle. M. bovis was detected in 10 cases by a loop-mediated isothermal amplification method or by immunohistochemistry. One case of caseonecrotic granulomatous meningoencephalitis, one case of caseonecrotic granulomatous meningitis, one case of suppurative meningoencephalitis, eight cases of eustachitis, nine cases of tonsillitis and six cases of suppurative bronchopneumonia were identified by histopathological examination. M. bovis antigen was detected in the eustachian tubes of eight cases. In nine cases, M. bovis was also detected in tonsillar epithelial crypts and lumina, in intraluminal inflammatory cells and in the epithelial cells of minor salivary glands located around the eustachian tubes and tonsils. The results suggest that M. bovis can infect and colonize the tonsils and enter the eustachian tubes, causing otitis media, which, in cases of chronic infection, can progress to meningitis.

Virulence-Associated Gene Profiles of Escherichia coli Isolated from Chickens with Colibacillosis in Japan and Their Correlation with Pathogenicity in Chicken Embryos.

Colibacillosis, an infectious disease of chickens, is caused by avian pathogenic Escherichia coli (APEC); however, in addition to APEC, other pathogens are also frequently isolated from chickens affected with colibacillosis. Therefore, experimental infections in chickens are necessary to evaluate the pathogenicity of APEC isolates. Recent studies have shown that embryo lethality assays can be used as an alternative method to evaluate the pathogenicity of E. coli. In this study, to determine the important virulence genes associated with the pathogenicity of E. coli, 67 strains of E. coli that possessed different combinations of eight representative virulence genes (cva/cvi, vat, tsh, iucD, papC, irp2, iss, and astA) were isolated from broilers with colibacillosis in Japan, and the chicken embryo lethal assay was conducted. The genes vat, papC, and irp2 showed strong correlation with the level of virulence in E. coli. Our study provides useful information about the important virulence-associated genes in relation to the pathogenicity of E. coli in Japanese chickens.

Nota de investigación- Perfiles de genes asociados a la virulencia de Escherichia coli aislada de pollos con colibacilosis en Japón y su correlación con la patogenicidad en embriones de pollo. La colibacilosis, una enfermedad infecciosa de los pollos, es causada por Escherichia coli patógena aviar (APEC); sin embargo, además de E. coli patógena aviar, también se aíslan con frecuencia otros patógenos de los pollos afectados por colibacilosis. Por lo tanto, las infecciones experimentales en pollos son necesarias para evaluar la patogenicidad de los aislamientos de E. coli patógena aviar. Estudios recientes han demostrado que los ensayos de letalidad embrionaria se pueden utilizar como método alternativo para evaluar la patogenicidad de E. coli. En este estudio, para determinar los genes de virulencia importantes asociados con la patogenicidad de E. coli, 67 cepas de E. coli que poseían diferentes combinaciones de ocho genes de virulencia representativos (cva/cvi, vat, tsh, iucD, papC, irp2, iss, y astA) fueron aisladas de pollos de engorde con colibacilosis en Japón y se llevó a cabo el ensayo de letalidad de embriones de pollo. Los genes vat, papC e irp2 mostraron una fuerte correlación con el nivel de virulencia en E. coli. Este estudio proporciona información útil sobre los genes importantes asociados a la virulencia en relación con la patogenicidad de E. coli en pollos japoneses.

Radiographic diagnosis and surgical management for successful outcomes for osteochondrosis in Japanese Black calves.

Osteochondrosis (OC) is not well recognized in cattle. Japanese Black (JB) cattle are valued for their beef and studies regarding OC are scarce. Hence, the aim of this study was to retrospectively discuss clinical and radiographical findings, diagnosis and treatment options for OC in JB cattle for successful outcomes. Medical records of 11 calves affected with OC were reviewed, retrospectively. Clinical and radiographic findings, treatment and outcomes and postmortem examination findings were analyzed. All calves had lameness. Stifle (4/14) and tarsal (4/14) were the most affected joints. Subchondral bone lucencies were observed in all joints (14/14) and accompanied with sclerosis (10/14). Radiographic grading was performed in calves. Calves were treated surgically (8/11) or conservatively (1/11). Surgically treated calves were auctioned (5/8). Postmortem examination was performed (2/11). Gross findings revealed ulcerative lesion at articular surface. Histopathologically, cartilage layer was defected at lesion. Inflammation (2/2) and granulation tissue (1/2) was also observed. In conclusion, OC in JB cattle can be diagnosed on the basis of radiography with history and clinical signs in clinical setting. Surgery should be performed in mild cases for successful outcomes. A modified radiographic grading criteria for clinical cases may hold potential in evaluating prognosis and outcomes.

Indications and patterns of antimicrobial use in pig farms in the southern Kyushu, Japan: large amounts of tetracyclines used to treat respiratory disease in post-weaning and fattening pigs.

In Japan, reducing the use of antimicrobials in pig production is a significant issue. However, there are no published reports concerning porcine disease treatment, as related to the age of the pigs and the indications (e.g., organ system) in Japan. In this study, we analyzed the prescription records of 17 farrow-to-finish farms from 2014 to 2018 in southern Kyushu, Japan. The farms' antimicrobial usage was calculated as the active ingredient per population correction unit (PCU) or the number of treated pigs per PCU using the defined daily dose. All data were analyzed according to the indications and production stages (sows, suckling pigs, post-weaning pigs, and fattening pigs). In terms of active ingredients/PCU, tetracyclines were the most commonly used (43.2-59.3%), and the largest amounts of antimicrobials administered through feeds were for treating the respiratory organs of fattening pigs. In terms of the number of treated pigs/PCU, tetracyclines were most frequently used (16.3-31.1%), and a high frequency of antimicrobials administered through feeds was used for the treatment of respiratory organs in post-weaning pigs. In this study, it was confirmed that tetracyclines were used frequently as a herd treatment for respiratory diseases in post-weaning and fattening pigs in southern Kyushu, Japan. The findings suggest that it is necessary to improve the treatment and prevention of respiratory diseases in post-weaning and fattening pigs in order to reduce the frequencies of antimicrobial treatments.

Antimicrobial resistance profiles and phylogenetic groups of Escherichia coli isolated from healthy Thoroughbred racehorses in Japan.

In this study, we investigated the occurrence of antimicrobial resistance in commensal Escherichia coli isolated from healthy Thoroughbred (TB) racehorses in Japan. A total of 212 fecal samples were individually collected from TB racehorses from March 2017 to August 2018 at Japan Racing Association training centers. E. coli was isolated by using selective agar media, deoxycholate-hydrogen sulfide-lactose (DHL) and eosin methylene blue (EMB). A total of 417 E. coli isolates were examined against 10 antimicrobial agents by using the broth microdilution method. The 417 E. coli isolates were phylogenetically grouped using a multiplex polymerase chain reaction. The highest proportion of resistance was observed for streptomycin (30.9%, 129/417) followed by ampicillin (19.4%, 81/417), trimethoprim (15.8%, 66/417), tetracycline (8.4%, 35/417), chloramphenicol (2.6%, 11/417), kanamycin (1.2%, 5/417), nalidixic acid (0.5%, 2/417), cefazolin (0.2%, 1/417), colistin (0.2%, 1/417), and gentamycin (0%). Multidrug-resistant (MDR) E. coli was detected in 7.9% (33/417) of isolates. The proportions of resistance against ampicillin, streptomycin, kanamycin, and chloramphenicol and of multidrug-resistant phenotypes in E. coli belonging to phylogenetic group B2 were significantly higher than those of other groups. This study clarified the distribution of antimicrobial-resistant (AMR) E. coli in Japanese racehorses. A continuous monitoring program for antimicrobial resistance is required to control the spread of AMR bacteria in racehorses.

Assessment of reproductive and growth performance of pigs on commercial swine farms in southern Kyushu, Japan.

The objective of the present study was to assess the productivity of pigs to investigate the time-dependent change in productivity and compare productivity according to breeding company group and porcine reproductive and respiratory syndrome (PRRS) status on commercial swine farms in southern Kyushu, Japan. Data used in the present study were 245 annual productivity records from 2014 to 2018 obtained from 72 farms. Average sow inventory was 481.2 sows. Mean numbers of pigs weaned per sow per year and market pigs sold per sow per year were 23.7 and 21.6, respectively. Pigs born alive per litter increased from 11.1 to 11.9 pigs from 2014 to 2018 (p < .05). Farms using domestic breeding companies had similar numbers in 2016 to those using international breeding companies, but fewer pigs in 2014, 2015, 2017, and 2018 (p < .05). Farms with an unknown or unstable PRRS status had fewer pigs born alive per litter and pigs weaned per sow per year than those with stable or negative PRRS statuses (11.2 ± 0.06 vs. 11.8 ± 0.08 pigs and 22.6 ± 0.38 vs. 25.0 ± 0.22 pigs, respectively; p < .05). These results can be used to establish feasible targets and standards of performance to identify problem areas and improve production.

Antimicrobial-Resistant Enterococcus faecium and Enterococcus faecalis Isolated From Healthy Thoroughbred Racehorses in Japan.

In this study, the occurrence of antimicrobial-resistant (AMR) enterococci was evaluated in Thoroughbred (TB) racehorses in Japan. Fecal samples were collected from 212 healthy TB racehorses at the Miho and Ritto Training Centers of the Japan Racing Association from March 2017 to August 2018. Isolation and identification were performed by enterococcus selective medium and confirmed to the species using MALDI-TOF MS. Enterococcus faecium and E. faecalis isolates were subjected to antimicrobial susceptibility test against 11 antimicrobials by minimum inhibitory concentration based on recommendation from Clinical and Laboratory Standards Institute guidelines. Among 583 enterococcus isolates, E. faecium and E. faecalis were identified for 48.2% (281/583) and 7.4% (43/583), respectively. One isolate that was representing E. faecium (153 isolates) and E. faecalis (31 isolates) from each sample was selected for antimicrobial susceptibility test. The highest rate of resistance for E. faecium isolates was observed against enrofloxacin (57.5%; 88/153), followed by streptomycin (32.0%; 49/153), kanamycin (18.3%; 28/153), gentamycin (5.9%; 9/153), erythromycin (5.9%; 9/153), and oxytetracycline (4.6%; 7/153). For E. faecium isolates, the highest resistance was observed against streptomycin (90.3%; 28/31), followed by kanamycin (41.9%; 13/31), gentamycin (29.0%; 9/31), lincomycin (9.7%; 3/31), oxytetracycline (6.5%; 2/31), erythromycin (6.5%; 2/31), tylosin (6.5%; 2/31), enrofloxacin (6.5%; 2/31), and chloramphenicol (3.2%; 1/31). The results indicated that enrofloxacin and aminoglycosides were highly resistant among tested antimicrobials. Continuous monitoring studies are useful to increase the awareness of the potential for AMR bacteria to arise from imprudent use of antimicrobials in TB racehorses in Japan.

Upregulation of PD-L1 Expression by Prostaglandin E2 and the Enhancement of IFN-γ by Anti-PD-L1 Antibody Combined With a COX-2 Inhibitor in Mycoplasma bovis Infection.

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 E2 (PGE2) 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 PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade 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 PGE2 and the PD-1/PD-L1 pathway.

Multidrug-Resistant ESBL/AmpC-Producing Klebsiella pneumoniae Isolated from Healthy Thoroughbred Racehorses in Japan.

Extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase (AmpC)-producing Klebsiella spp. have become a major health problem, leading to treatment failure in humans and animals. This study aimed to evaluate the presence of ESBL/AmpC-producing Klebsiella spp. isolated from racehorses in Japan. Feces samples from 212 healthy Thoroughbred racehorses were collected from the Japan Racing Association Training Centers between March 2017 and August 2018. ESBL/AmpC-producing Klebsiella spp. were isolated using selective medium containing 1 µg/mL cefotaxime. All isolates were subjected to bacterial species identification (MALDI-TOF MS), antimicrobial susceptibility test (disk diffusion test), characterization of resistance genes (PCR), conjugation assay, and genetic relatedness (multilocus sequence typing/MLST). Twelve ESBL/AmpC-producing Klebsiella pneumoniae (ESBL/AmpC-KP) were isolated from 3.3% of horse samples. Antimicrobial resistance profiling for 17 antimicrobials showed all ESBL/AmpC-KP were multidrug-resistant (MDR). Only 1 isolate was confirmed as an ESBL producer (blaCTX-M-2-positive), whereas the other 11 isolates were plasmid-mediated AmpC (pAmpC) producers (blaCMY positive). On the basis of MLST analysis, the ESBL-KP isolate was identified as sequence type (ST)-133 and four different STs among AmpC-KP isolates, ST-145, ST-4830, ST-4831, and ST-4832, were found to share six of the seven loci constituting a single-locus variant. This is the first study to show K. pneumoniae carrying MDR pAmpC isolated from a racehorse.