Genetic analysis based on next generation sequencing of Streptococcus equi subsp. equi isolated from horses imported into Japan.

Strangles is a globally widespread, commonly diagnosed and important infectious disease of equids caused by Streptococcus equi subsp. equi. We performed whole genome sequencing of 19 S. equi isolates collected from imported horses at the Japanese border. Of these isolates, 15 isolates were obtained from clinical cases and 4 were from subclinical cases. The 19 isolates were grouped into 3 Bayesian analysis of population structure (BAPS) groups by the core genome single nucleotide polymorphism analysis corresponding to exporting country, SeM typing, or exporter of the horses. The 19 isolates possessed same pathogenic genes regardless of clinical status in imported horses and no antimicrobial resistance genes. The disease status of the horses may rather reflect the prior exposure of animals with sub-clinical infection to S. equi.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from pigs in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of infection in hospitalized patients and can be prevalent in humans and various animal species. In European countries, MRSA isolates belonging to clonal complex 398 have been detected at high rates in pigs. However, the prevalence of MRSA in pigs and farm environments in Japan remains unclear. MRSA isolates were obtained from pigs in slaughterhouses, diseased pigs on farms, imported breeding pigs, and farm dust. We conducted whole-genome sequencing (WGS) and analyzed the molecular epidemiological relationship between these MRSA isolates using core genome multilocus sequence typing (cgMLST). The prevalence rates of MRSA among pigs in slaughterhouses, diseased pigs on farms, imported breeding pigs, and farm dust were 5.2 %, 3.4 %, 28.8 %, and 0.06 %, respectively. ST 398 isolates that classified as ST398/t034 were isolated from pigs from all sources. The results of cgMLST showed that ST398/t034 isolates originating from domestic pigs clustered into the same cluster as the isolates from imported breeding pigs. However, some clusters only included isolates of domestic pig origin. Most MRSA isolates in this study carried resistance genes for aminoglycosides, ß-lactams, macrolides, tetracyclines, and zinc. None of the MRSA isolates in this study harbored Panton-Valentine leukocidin toxin genes. Molecular epidemiological analysis suggested a relationship between isolates from slaughter pigs and imported breeding pigs and the presence of MRSA isolates of domestic origin. However, more data are needed for elucidation of the origin of these MRSA variants in the pig industry in Japan.

Genetic analysis of Streptococcus equi subsp. equi isolated from horses imported into Japan.

Strangles is a commonly diagnosed and important infectious disease of equids worldwide, caused by Streptococcus equi subsp. equi. We determined the SeM genotypes of S. equi isolated from imported horses at the Japanese border within the past 8 years, which allowed us to classify 12 strains isolated from these horses from each exporter into four allelic groups. These alleles were different from the alleles of past isolates found in Japan. Furthermore, four strains classified into the same allele were isolated from horses from one exporter over several years. In this study, S. equi isolates from different exporters had different SeM alleles. Attention to the hygiene status of farms will be necessary to prevent the incursion of strangles.

Systemic Staphylococcus pseudintermedius infection in an arctic fox (Vulpes lagopus) with severe multifocal suppurative meningoencephalitis and nephritis.

A 2-year-female arctic fox (Vulpes lagopus) developed anorexia, dehydration, and emaciation during the quarantine period for importation from Norway, and died 17 days later. At necropsy, a fistula was observed on the left gluteal region, and the left eye, left brain, and kidneys were discolored. Histologically, severe diffuse suppurative meningoencephalitis and renal abscesses were detected. Numerous Gram-positive cocci were detected in these lesions. Multidrug-susceptible Staphylococcus pseudintermedius were isolated from the lesions. These results suggest that S. pseudintermedius can cause severe multifocal suppurative meningoencephalitis and nephritis in foxes. This is the first report of multidrug-susceptible S. pseudintermedius meningoencephalitis and nephritis in a fox.

Septicemic invasive Klebsiella pneumoniae infection in a cynomolgus monkey (Macaca fascicularis) with severe diffused suppurative meningoencephalitis.

A 2-year-old male cynomolgus monkey (Macaca fascicularis) showed neurological symptoms during quarantine for importation into Japan, and was euthanized due to poor prognosis. Gross anatomical examination revealed a hemorrhagic lesion around the lateral ventricle in the cerebrum. Histologically, severe diffused suppurative meningitis and ventriculitis were detected with numerous Gram-negative bacilli in the cerebrum. Immunohistochemically, the bacilli were positively stained with an antibody against Klebsiella pneumoniae. The bacterium was isolated from the liver, and it was confirmed to be K. pneumoniae by 16S rDNA sequencing. The isolate displayed a hypermucoviscosity phenotype, was positive for the rmpA and k2A genes, and demonstrated multidrug resistance. These results suggest that invasive K. pneumoniae can cause septicemic infection, characterized by severe diffused suppurative meningoencephalitis in monkeys.

Three Cases of Escherichia coli Meningitis in Chicks Imported to Japan.

Three outbreaks of colibacillosis have occurred in chicks during the quarantine period after importation to Japan. All three were derived from three different countries without epidemiologic relevance. Some birds from each infected flock were examined pathologically and bacteriologically. The characteristic histologic finding common to all three cases was severe bacterial meningitis in the central nervous system. Pericarditis, perihepatitis, and omphalitis with bacterial colonies were also observed. The bacterial colonies observed histologically were immunohistochemically positive for Escherichia coli antigens. Escherichia coli was isolated from the organ samples from each outbreak. At least two E. coli isolates were serotyped as O18 and O161, which differed from the popular serotypes in Japan. These results suggest that avian pathogenic E. coli of uncommon serotypes can be imported from outside countries by infected chicks. Colibacillosis should be included in the differential diagnosis when meningitis is histologically observed in chicks.

Multifocal suppurative granuloma caused by Actinobacillus lignieresii in the peritoneum of a beef steer.

An imported crossbred Angus beef steer aged eight to twelve months died suddenly on the eighth day of a quarantine period in Japan. Gross examination showed the peritoneum and mesentery consisted of numerous nodules of various sizes. Histological examination revealed chronic suppurative granulomatous peritonitis with eosinophilic rosettes surrounding colonies of Gram-negative bacilli. The bacteria isolated from the nodules were confirmed to be Actinobacillus lignieresii based on the results of 16S rRNA gene sequencing and immunohistochemistry. Antibiotic sensitivity testing showed that the isolate was resistant to penicillin. Thus, a diagnosis of atypical actinobacillosis caused by A. lignieresii was made.

Systemic Streptococcus dysgalactiae subspecies equisimilis infection in a Yorkshire pig with severe disseminated suppurative meningoencephalomyelitis.

An 18-week-old female breeding Yorkshire pig displayed symptoms of astasia and subsequently died. Histologically, severe disseminated suppurative meningoencephalomyelitis was detected, as were numerous myocardial microabscesses. Gram-positive cocci were detected in these suppurative lesions, and these cocci reacted with an antibody against Streptococcus C group species. Gram-positive cocci were isolated from the liver, spleen, kidney, heart, lungs, pleural abscess and articular fluid of the right tarsal joint. The isolates were ß-hemolytic, categorized into Lancefield group C and were identified as Streptococcus dysgalactiae subspecies equisimilis by analysis of the 16S ribosomal DNA sequence. This is the first report of systemic S. equisimilis infection in a pig with severe disseminated suppurative meningoencephalomyelitis.