Molecular Investigation of Zoonotic Intestinal Protozoa in Pet Dogs and Cats in Yunnan Province, Southwestern China.

Giardia duodenalis, Enterocytozoon bieneusi and Cryptosporidium spp. are common enteric pathogens that reside in the intestines of humans and animals. These pathogens have a broad host range and worldwide distribution, but are mostly known for their ability to cause diarrhea. However, very limited information on prevalence and genotypes of G. duodenalis, E. bieneusi and Cryptosporidium spp. in pet dogs and cats are available in China. In the present study, a total of 433 fecal samples were collected from 262 pet dogs and 171 pet cats in Yunnan province, southwestern China, and the prevalence and the genotypes of G. duodenalis, E. bieneusi and Cryptosporidium spp. were investigated by nested PCR amplification and DNA sequencing. The prevalence of G. duodenalis, E. bieneusi and Cryptosporidium spp. was 13.7% (36/262), 8.0% (21/262), and 4.6% (12/262) in dogs, and 1.2% (2/171), 2.3% (4/171) and 0.6% (1/171) in cats, respectively. The different living conditions of dogs is a risk factor that is related with the prevalence of G. duodenalis and E. bieneusi (p < 0.05). However, there were no statistically significant difference in prevalence of three pathogens in cats. DNA sequencing and analyses showed that four E. bieneusi genotypes (PtEb IX, CD9, DgEb I and DgEb II), one Cryptosporidium spp. (C. canis) and two G. duodenalis assemblages (C and D) were identified in dogs; two E. bieneusi genotypes (Type IV and CtEb I), one Cryptosporidium spp. (C. felis) and one G. duodenalis assemblage (F) were identified in cats. Three novel E. bieneusi genotypes (DgEb I, DgEb II and CtEb I) were identified, and the human-pathogenic genotypes/species Type IV C. canis and C. felis were also observed in this study, indicating a potential zoonotic threat of pet dogs and cats. Our results revealed the prevalence and genetic diversity of G. duodenalis, E. bieneusi and Cryptosporidium spp. infection in pet dogs and cats in Yunnan province, southwestern China, and suggested the potential threat of pet dogs and cats to public health.

First report of Neospora caninum infection in pigs in China.

Neospora caninum is a protozoan parasite which can infect many mammals and birds with a worldwide distribution. However, no molecular data are available about the occurrence of N. caninum in pigs. In this study, the serological and molecular prevalence of N. caninum infection in farmed pigs were investigated in Hunan province, China, between January 2017 and December 2018. A total of 1,500 serum samples collected from 10 herds in Hunan province were evaluated using a competitive-inhibition enzyme-linked immunoassay assay (cELISA). The overall seroprevalence of N. caninum in the examined pigs was 1.9%. The seroprevalence of N. caninum ranged from 0.3% to 4.6% among different regions in Hunan province of China (p < .05). DNA was extracted from brain samples, and the Nc-5 gene and ITS-1 region were amplified and then sequenced. Three (0.5%) of the examined 600 brain tissues were found to contain N. caninum DNA. Our phylogenetic analyses indicated that N. caninum samples were classified into two distinct groups. Although the prevalence is low within the pig groups investigated, our results revealed the emergence of N. caninum infection in pigs in China. The finding of the present study provides molecular evidence that the pigs are the natural intermediate host of N. caninum and may have major epidemiological importance.

Novel genotypes and multilocus genotypes of Enterocytozoon bieneusi in two wild rat species in China: potential for zoonotic transmission.

Enterocytozoon bieneusi is an opportunistic pathogen in immunodeficient patients. Although this pathogen has been reported in many domestic animals, few data are available about the occurrence of E. bieneusi in wild rats. In the current study, a total of 228 fecal samples from two wild rat species (Leopoldamys edwardsi and Berylmys bowersi) in China were examined by a nested PCR-based sequencing approach employing the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The overall prevalence of E. bieneusi in wild rats was 33.3% (76/228), with 35.1% (39/111) in L. edwardsi and 31.6% (37/117) in B. bowersi. Ten E. bieneusi genotypes (including four known and six novel genotypes) were identified, with the novel CQR-2 (n = 15) as the predominant genotype. Phylogenetic analysis indicated that ten genotypes in the present study belong to zoonotic group 1, which contains many genotypes in humans. Furthermore, multilocus sequence typing (MLST) analysis showed that 19 ITS-positive samples were successfully amplified at three microsatellites and one minisatellite, forming 18 multilocus genotypes (MLGs). This is the first report of E. bieneusi infection in the wild rats L. edwardsi and B. bowersi. Our findings suggest that wild rats could be a significant source of human infection, including contaminated food and water.

Identification of Cryptosporidium viatorum XVa subtype family in two wild rat species in China.

BACKGROUND: Cryptosporidium viatorum is a minor Cryptosporidium pathogen in humans. Currently, there is limited information regarding the prevalence and genotypes of C. viatorum in animals in China. METHODS: In this study, 228 faecal samples were collected from two wild rat species (Leopoldamys edwardsi and Berylmys bowersi) in Chongqing Municipality and Guangdong Province, China. These specimens were analyzed for C. viatorum and then subtyped it using PCR and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) and 60-kilodalton glycoprotein (gp60) genes, respectively. RESULTS: A total of 25 (11.0%) faecal samples were tested positive for C. viatorum by SSU rRNA assay. Of these samples, 4 (3.6%) came from L. edwardsi and 21 (18.0%) from B. bowersi. Of the 25 C. viatorum-positive samples, 17 were successfully amplified at the gp60 gene locus, which represented four subtypes belonging to two subtype families, including XVa (XVaA6, XVaA3g, XVaA3h) and XVc (XVcA2G1). Phylogenetic analysis based on the gp60 amino acid sequences indicated that all of the C. viatorum isolates grouped together, supporting the conclusion that C. viatorum from the wild rats represent two subtype families. CONCLUSIONS: These results indicate an occurrence of C. viatorum XVa subtype family from rats which is genetically identical to those found in humans. Our findings suggest that wild rats may be a potential source of human cryptosporidiosis.

Seroprevalence and Risk Factors of Toxoplasma gondii Infection in Growth Stages of Pigs in Hunan Province, Subtropical China.

Toxoplasma gondii infection is prevalent in humans and animals worldwide. In the present study, a total of 612 serum samples were examined using indirect hemagglutination test (IHAT) for T. gondii infection. Antibodies to T. gondii were detected in 49 (8.0%, confidence interval [95% CI]: 5.9-10.2) serum samples (IHA titer ≥1:16). The T. gondii seroprevalence ranged from 4.4% (95% CI: 0-10.5) to 14.3% (95% CI: 0-40.2) among different regions in Hunan province of subtropical China. The highest seroprevalence was found in breeding sows (18.8%). The T. gondii seroprevalence was higher in winter (18.3%, 95% CI: 8.5-28.1) and spring (10.9%, 95% CI: 5.7-16.1) than in summer (6.4%, 95% CI: 2.8-10.1) and autumn (4.9%, 95% CI: 2.2-7.7), and the differences were statistically significant excepting summer. In addition, developmental stage and season were identified as risk factors for T. gondii infection. Our findings revealed the seroprevalence of T. gondii in growth stages of pigs in the Hunan province of subtropical China, indicating that it may cause public health and economic problems. To our knowledge, this is the first report of the comprehensive survey of T. gondii seroprevalence in pigs in the Hunan province of subtropical China.

Molecular Detection and Genotyping of Toxoplasma gondii in Edward's Long-Tailed Rats (Leopoldamys edwardsi).

Toxoplasma gondii is an important zoonotic parasite infecting humans and various animals with a worldwide distribution. However, limited information is available on T. gondii infection in wild rats. The present study aimed to examine the prevalence and characterize the genotypes of T. gondii in wild rats in two regions of China. Brain tissues were collected from 111 Edward's long-tailed rats (Leopoldamys edwardsi) and 117 Bower's white-toothed rats (Berylmys bowersi) between November 2017 and January 2018. Genomic DNA was extracted and amplified by PCR targeting the T. gondii B1 gene. B1 gene-positive samples were genotyped at 10 genetic markers (SAG1, SAG2 [5', 3'] and [alternative], SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using multilocus nested polymerase chain reaction/restriction fragment length polymorphism. Six (5.41%, 6/111) Edward's long-tailed rats from Chongqing Municipality were positive for T. gondii B1 gene, whereas no T. gondii infection was detected in Bower's white-toothed rats (n = 117) from Guangdong province. T. gondii prevalence in female and male rats was 1.77% (2/113) and 3.48 (4/115), respectively. Four of the six positive DNA samples were completely genotyped at 10 genetic loci and were identified as ToxoDB#20. The present study revealed the occurrence of T. gondii infection in Edward's long-tailed rats. These findings raised public health concerning about T. gondii infection in wild rats. These results provide reference data for understanding the distribution of T. gondii genotypes in wild rats in China.

Molecular detection of Neospora caninum from naturally infected four passeriforme birds in China.

Neospora caninum is an intracellular protozoan infecting many domestic and wild animals. In the present study, the brain tissues of wild birds collected in Hunan province of China were examined by N. caninum specific nested PCR, targeting the Nc-5 gene and the internal transcribed spacer 1 (ITS-1) region of the nuclear ribosomal DNA. The prevalence of N. caninum was detected in 15.5% (37/239) of wild birds, including 20.5% (9/44) of the examined olive-backed pipit Anthus hodgsoni, 18.3% (24/131) of the examined tree sparrows Passer montanus, 7.9% (3/38) of the examined chestnut bunting Emberiza rutila and 3.8% (1/26) of the examined yellow-breasted bunting E. aureola. Phylogenetic analyses showed that N. caninum from different hosts and geographical origins are genetically diverse and can be further classified into two distinct groups. Our findings indicated that wild birds are potential source of N. caninum for other animals. To our knowledge, this is the first report of N. caninum infection in wild birds in China, which provides a foundation for the prevention and control of this parasite in China and elsewhere.

Molecular Prevalence and Genetic Characterization of Toxoplasma gondii in Wild Birds in Hunan Province, China.

Toxoplasma gondii is a ubiquitous apicomplexan parasite of warm-blooded animals and humans. However, limited information is available about T. gondii infection in wild birds. In this study, 239 wild birds were collected from Hunan province of China, including 38 chestnut bunting, 44 olive-backed pipit, 26 yellow-breasted bunting, and 131 tree sparrows. Genomic DNA of brain tissues were extracted and assayed by B1 gene, and the positive samples were genotyped at 10 genetic markers [SAG1, SAG2 (5'+3' SAG2, alter. SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico] using multilocus nested-PCR-restriction fragment length polymorphism technology. The results showed that 13 (5.51%) of the 239 wild birds were positive for T. gondii. Among them, three samples have completely genotyped at all loci, and were identified as ToxoDB #10. Our results have indicated that wild birds can carry and potentially disseminate the T. gondii. This is the first report of the molecular prevalence and genetic characterization of T. gondii in wild birds in Hunan province, China. Further research should be investigated to understand weather T. gondii can be transmitted from wild birds to other animals or humans.

Molecular Detection and Genotyping of Toxoplasma gondii in Pigs for Human Consumption in Hunan Province, China.

Toxoplasma gondii is one of the most important sources of foodborne diseases. In this study, the molecular prevalence and genotypes of T. gondii were investigated in pigs in Hunan province, China. A total of 339 brain tissue samples of pigs were collected from April 2015 to December 2017 in Hunan province and were used to detect the T. gondii B1 gene. Of these, 34 (10%; 95% confidence interval: 8.7-12.6) samples were tested positive for the T. gondii B1 gene. Positive samples were genotyped at 10 genetic markers (SAG1, SAG2 [5' + 3' SAG2, alter. SAG2], SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using polymerase chain reaction-restriction fragment length polymorphism technology. Moreover, one sample was identified as genotype ToxoDB#10 (Type I), and another sample was suspected to be unusual genotype ToxoDB#61 that has never been reported in China. This study showed that T. gondii is prevalent in pigs in Hunan province, posing a food safety threat to the public health in the investigated areas. Our result has implications for better understanding the genetic diversity of T. gondii infections in animals in China.