Background Enterocytozoon bieneusi is one of the most prevalent causative species of diarrhea and enteric diseases in various hosts. E. bieneusi has been identified in humans, mammals, birds, rodents and reptiles in China, but few studies have reported E. bieneusi in horses. Therefore, the present study was conducted to assess the prevalence, molecular characteristics and zoonotic potential of E. bieneusi among horses in southwestern China.FindingsThree hundred and thirty-three fecal specimens were collected from horses on five farms in the Sichuan and Yunnan provinces of southwestern China. The prevalence of E. bieneusi was 22.5 % (75/333), as determined by nested polymerase chain reaction and sequencing analysis of the internal transcribed spacer region of the ribosomal RNA gene of E. bieneusi. Altogether, 10 genotypes were identified among the 75 E. bieneusi-positive samples: four of these genotypes were known (horse1, horse2, SC02 and D) and six were novel (SCH1-4 and YNH1-2). Multilocus sequence typing using three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) revealed three, two, three and three genotypes at these four loci, respectively. In phylogenetic analysis, all the genotypes of E. bieneusi obtained in this study were clustered into three distinct groups: D, SC02 and SCH1-3 were clustered into group 1 (zoonotic potential); SCH4 was clustered into group 2 (cattle-hosted); whereas horse2, YNH1 and YNH2 were clustered into group 6 (unclear zoonotic potential).ConclusionsThis is the first report of E. bieneusi among horses in southwestern China. This is also the first multilocus genotyping analysis using microsatellite and minisatellite markers of E. bieneusi in horses. The presence of genotype D, which was previously identified in humans, and genotypes SC02 and SCH1-3, which belong to potential zoonotic group 1, these results indicate that horses are a potential source of human E. bieneusi infections in China.

Nocardia seriolae is the causative agent of nocardiosis in both marine and freshwater fish. Here, we report on multiple outbreaks of nocardiosis associated with elevated mortality (23-35%) in farmed largemouth bass in Sichuan, China, from 2017 to 2018. A total of 9 strains isolated from diseased largemouth bass were identified as N. seriolae by phenotypic characterization, 16S rRNA and hsp65 gene sequence analysis. The clinical signs of infected largemouth bass included hemorrhage, skin ulcers and prominent tubercles varying in size in the gill, liver, spleen and kidney. Experimental infection indicated that these isolates were the pathogens responsible for the mortalities. In vitro antibacterial activities of 12 antibiotics against N. seriolae isolates were determined as minimum inhibitory concentrations. Histopathological observation of diseased fish infected with N. seriolae showed necrotizing granulomatous hepatitis, nephritis, splenitis, epithelial hypertrophy and hyperplasia with degenerative changes of the epithelium in the gill. Large quantities of bacterial aggregates were found in the necrotic area of the granuloma by Lillie-Twort Gram stain and immunocytochemistry. Our findings indicated that N. seriolae is a serious threat to the largemouth bass Micropterus salmoides industry in Southwest China.

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Ranaviruses are important emerging pathogens of ectothermic vertebrates that threaten aquaculture and wildlife worldwide. A mortality event occurred in a cultured population of catfish‐like loach (Triplophysa siluorides) in Sichuan Province, China. Gross clinical signs of the affected fish included skin lesions and haemorrhagic ulcers, which are often associated with ranaviruses. Inoculation of liver, kidney and spleen tissue homogenates in epithelioma papulosum cyprini (EPC) cells at 25°C resulted in cytopathic effect within 24 hr. Transmission electron microscopy of infected EPC cells revealed hexagonal viral arrays in the cytoplasm and icosahedral geometry of the virions. Following exposure of T. siluroides to the isolated virus, similar clinical signs were observed and the fish experienced 40% and 90% mortality after 21 days at 103.58 and 107.8 TCID50/0.1 ml doses, respectively, providing evidence the isolated virus was the main causative agent of the mortality event. Diagnostic PCR of the major capsid protein gene of ranavirus showed that all samples of diseased fish and isolated virus were positive. Phylogenetic analysis revealed that the isolated virus, designated as FYLl40220, was associated with the Common Midwife Toad Virus (CMTV)‐like ranavirus clade. To our knowledge, this case represents the first report of CMTV‐associated mortality in a fish species. Collectively, these results suggest that the host range of CMTV‐like ranaviruses is greater than previously thought, and this clade of ranaviruses could have significant economic and biodiversity impacts.