Biofilm characterisation of Mycoplasma bovis co-cultured with Trueperella pyogenes

Mycoplasma pneumonia, caused by Mycoplasma bovis (Mycoplasmopsis bovis; M. bovis), is linked with severe inflammatory reactions in the lungs and can be challenging to treat with antibiotics. Biofilms play a significant role in bacterial persistence and contribute to the development of chronic lesions. A recent study has shown that polymicrobial interactions between species are an important factor in biofilm formation, yet the precise mechanism of biofilm formation in M. bovis remains unknown. By assuming multiple pathogen infections in the bovine respiratory disease complex (BRDC), this study examined the characterisation of the polymicrobial relationship between M. bovis and Trueperella pyogenes (T. pyogenes) during biofilm formation. Autopsies were performed on four Holstein calves (two chronic Mycoplasma pneumonia calves and two control calves). Bacterium-like aggregation structures (> 10 μm), which were assumed to be biofilms of M. bovis in vivo, were observed adhering to the cilia in calves with Mycoplasma pneumonia. M. bovis released an extracellular matrix to connect with neighbouring bacteria and form a mature biofilm on the plate. Biofilm formation in the co-culture of M. bovis and T. pyogenes (strain T1: 1 × 105 and 1 × 106 CFU/well) significantly increased (p  40 μm), composed of M. bovis and T. pyogenes, were observed. The morphological characteristics of this biofilm were similar to those observed in vivo compared to a single culture. In conclusion, the polymicrobial interaction between M. bovis and T. pyogenes induces biofilm formation, which is associated with increased resistance to antimicrobial agents, and this exacerbates the progression of chronic Mycoplasma pneumonia

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

Extended-spectrum β-lactamase (ESBL)- and AmpC β-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.Citation: Sukmawinata, E.; Uemura, R.; Sato, W.; Thu Htun, M.; Sueyoshi, M. Multidrug-Resistant ESBL/AmpC-Producing Klebsiella pneumoniae Isolated from Healthy Thoroughbred Racehorses in Japan. Animals 2020, 10, 369. https://doi.org/10.3390/ani1003036

Comprehensive serotyping of Mannheimia haemolytica by a PCR system using the diversity of capsule biosynthesis genes

The bovine respiratory disease complex (BRDC) is a global issue affecting dairy and beef farms and is of major concern due to the high morbidity and mortality rates in calves, as well as decreased production it causes, resulting in significant economic losses. Mannheimia haemolytica is one of the secondary pathogens associated with BRDC. M. haemolytica is classified into 12 serotypes based on capsular antigens. In addition to the prevalent serotypes A1, A2, and A6, strains belonging to other serotypes also cause respiratory diseases in cattle and other ruminants, necessitating a method for their rapid and easy identification. In this study, we organized the capsule biosynthesis genes based on genome information from all serotype strains and designed 11 PCR primer pairs targeting serotype-specific genes, which could individually identify serotypes A14/A16, which possess homologous genes, as well as all other serotypes. Additionally, we developed two multiplex PCR kits that include these serotype-specific and M. haemolytica species-specific primers. Specificity testing using reference strains confirmed that these kits can simultaneously and clearly identify both the species and their serotypes. The PCR-based system described here could be a valuable tool for subtyping M. haemolytica strains in epidemiological studies and surveillance efforts in cattle and other reservoir animals. This study also carefully compared and discussed the differences between the capsule synthesis genes of A8 and A14 from previously published and those obtained in this study

Infectious endophthalmitis associated with umbilical infection in Japanese black calf: a case report

A 3-day-old Japanese black calf presented with a swollen and tender umbilical cord and diffusely cloudy and keratoconus eyes. Abdominal ultrasonography confirmed mild enlargement of both umbilical arteries and the urachus with a hyperechoic lumen. Additionally, a hyperechogenic structure suggestive of pus was noted near the abdominal wall. Fluorescein staining revealed corneal epithelial injury, whereas slit lamp examination identified corneal edema, increased corneal thickness, and keratitis with vascularization of the corneal stroma. Based on these findings, diagnoses of omphaloarteritis, omphalourachitis, and bullous keratitis were made. Both umbilical arteries and the urachus were surgically removed; both ocular globes were covered with a third eyelid flap, which was released 30 days postoperatively. On the follow-up, ocular ultrasonography indicated bleeding or fibrin deposits in the vitreous body of the right ocular globe. Because intraocular inflammation was suspected, anterior aqueous humor was collected from the right ocular globe, and bacterial examination was performed with the umbilical artery abscess, urachal abscess, and intraabdominal pus collected intraoperatively. Escherichia coli was isolated from the umbilical artery abscess, urachal abscess, intraabdominal pus, and aqueous humor, and all isolates exhibited identical genotypes. These findings suggest that endophthalmitis occurred as a result of the hematogenous spread of bacteria originating from septic umbilical cord remnants and that ocular ultrasonography is useful for assessing intraocular pathologies

分子軌道法による原子炉用ジルコニウム合金の耐食機能設計

現在の主要な発電用原子炉において、その炉心部分にはジルコニウム合金が使われている。このジルコニウム合金の表面には、局部腐食(いわゆるノジュラ-腐食)が起こり問題となっている。さらに、ウラン資源の有効利用を目的として進められている核燃料の高燃焼度化の要請とあいまって、腐食ジルコニウム合金の開発が強く望まれている。本研究はこのような背景のもとに計画されたものであり、分子軌道法による電子状態の計算とX線光電子分光(XPS)などの実験を併用して、高耐食ジルコニウム合金の設計と開発の指針を得ることを目標にしている。ジルコニウム合金の表面にできる腐食生成物であるZrO_2(ジルコニア)については、これまでいろいろな方法で調べられてきたが、XPSによる表面皮膜の解析は新しく、その化学成分や電子状態について興味ある情報を得ることができた。例えば、 XPSスペクトルは耐食性の良い合金と悪い合金とでは違っていることがわかった。合金元素の周期表の位置に従って耐食性が変わることも明らかになった。さらに、ジルコニウア皮膜自体、決して均一なものではなく、膜厚方向に合金元素の濃度勾配があることもわかった。また、DV-Xα分子軌道計算からによるジルコニウムおよびジルコニア中の合金元素の振舞いを明らかにした。そして電子論に基づくジルコニウム合金の腐食メカニズムを初めて提案した。本研究を通し、ジルコニウムの腐食を新しい視点から捉えることができた。その結果、新しい高耐食性ジルコニウム合金を開発するときに有用な指針が得られた。科学研究費補助金 研究種目:研究(A)(1) 課題番号:07555500 研究代表者:森永 正彦 研究期間:1995-1996年度research repor

A highly sensitive method for detecting African swine fever virus in oral fluids from naturally infected pigs in Northern Vietnam

Early detection and early slaughter through quarantine are essential to prevent the spread of the African swine fever virus (ASFV). Pooled oral fluids testing is a practical approach for pathogen monitoring, but compared to blood, the virus concentration is lower, increasing false negatives. We developed a highly sensitive method for ASFV detection in pig oral fluids using a pretreatment-based concentration protocol. We collected oral fluids from sub-clinical raised pigs in northern Vietnam and conducted a field evaluation using the developed method. A spike test showed up to 100-fold greater sensitivity than a reference method. For performance evaluation, 68 pooled oral fluid samples were collected, of which 63 originated from raised pigs in northern Vietnam and 5 were obtained from healthy pigs in Japan as negative controls. Using real-time PCR, 9/68 (13.2%) were positive by the reference method, while 23/68 (33.8%) were positive by the developed method. Real-time LAMP detected 1/68 (1.5%) and 6/68 (8.8%) respectively. The developed method therefore improved the diagnostic performance and enabled early diagnosis prior to disease onset. These results suggest enhanced sensitivity and feasibility for early ASF diagnosis, potentially contributing to more effective outbreak control. The developed method offers a simple and sensitive tool for rapid ASFV screening in both affected and at-risk farms

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

Abstract 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.Citation: Toya R, Sasaki Y, Uemura R, Sueyoshi M. 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. J Vet Med Sci. 2021 Mar 5;83(2):322-328. doi: 10.1292/jvms.20-0436. Epub 2020 Dec 21. PMID: 33342965; PMCID: PMC7972880

IncI1 Plasmid Associated with blaCTX-M-2 Transmission in ESBL-Producing Escherichia coli Isolated from Healthy Thoroughbred Racehorse, Japan

In our previous study, extended spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) were isolated from healthy Thoroughbred racehorse feces samples in Japan. Some ESBL genes were predicted to be located on the conjugative plasmid. PCR-based replicon typing (PBRT) is a useful method to monitor and detect the association of replicons with specific plasmid-borne resistant genes. This study aimed to evaluate the plasmid replicon associated with ESBLEC isolated from healthy Thoroughbred racehorses at Japan Racing Association Training Centers in Japan. A total of 24 ESBLECs isolated from 23 (10.8%) individual Thoroughbred racehorse feces samples were used in this study. ESBL gene transfer was performed using a conjugation assay. Then, replicon types of ESBLEC isolates and their transconjugants were determined using PBRT. Pulsed-field gel electrophoresis (PFGE) was performed to look at the clonality of the ESBLECs isolates. ESBLECs were detected from 10.8% of healthy Thoroughbred racehorses. The blaCTX-M-2 was identified as the dominant type of ESBL gene, followed by blaCTX-M-1 and blaTEM-116. In this study, only the blaCTX-M-2 and the IncI1 plasmid were transferred to transconjugants. The PFGE results showed that ESBL genes were distributed in diversity of ESBLECs. This finding suggested that the IncI1 plasmid was associated with the dissemination of blaCTX-M-2 in Thoroughbred racehorses in Japan.Citation: Sukmawinata, E.; Uemura, R.; Sato, W.; Mitoma, S.; Kanda, T.; Sueyoshi, M. IncI1 Plasmid Associated with blaCTX-M-2 Transmission in ESBL-Producing Escherichia coli Isolated from Healthy Thoroughbred Racehorse, Japan. Antibiotics 2020, 9, 70. https://doi.org/10.3390/antibiotics902007

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.Citation: Naik MV, Kirino Y, Uemura R, Kim S, Inoue Y, Hidaka Y. Radiographic diagnosis and surgical management for successful outcomes for osteochondrosis in Japanese Black calves. J Vet Med Sci. 2021 Jan 21;83(1):151-157. doi: 10.1292/jvms.20-0310. Epub 2020 Dec 28. PMID: 33390374; PMCID: PMC7870393

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

application/pdfOn 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