Molecular prevalence of Tetratrichomonas gallinarum and Trichomonas gallinae in three domestic free-range poultry breeds in Anhui Province, China.

Tetratrichomonas gallinarum and Trichomonas gallinae can colonize the alimentary tract of domestic birds. However, little information is available on the epidemiology of the two trichomonad species in domestic free-range poultry in China. In this study, the occurrence and genetic characteristic of T. gallinarum and T. gallinae among free-range chickens, ducks, and geese in Anhui Province, China, were investigated. The 1910 fecal samples collected from 18 free-range poultry farms throughout Anhui Province were examined for the presence of T. gallinarum and T. gallinae by PCR and sequence analysis of the small subunit (SSU) rRNA gene of T. gallinarum and ITS1-5.8S-ITS2 sequence of T. gallinae. The overall occurrence of T. gallinarum in poultry was 1.2% (22/1910), with infection rates of 2.1% (17/829) in chickens, 0.2% (1/487) in ducks, and 0.7% (4/594) in geese. The constructed phylogeny tree using the concatenated ITS1-5.8S-ITS2 region and SSU rRNA indicated the T. gallinarum isolates detected in this study were closely related to previously defined genogroups A, D, and E, respectively. Nine (0.5%) fecal samples were positive for T. gallinae, with infection rates of 0.8% (7/829) in chickens, 0.4% (2/487) in ducks, and 0% (0/594) in geese. Sequence and phylogenetic analysis showed that four T. gallinae ITS1-5.8S-ITS2 sequences obtained from chicken feces and one duck fecal sample belonged to genotype ITS-OBT-Tg-1. This is the first report of the prevalence and genetic characterization of T. gallinarum and T. gallinae in free-range chickens, ducks, and geese in China.

Molecular prevalence and characterization of Cryptosporidium in domestic free-range poultry in Anhui Province, China.

Free-range chickens might mediate the spread of Cryptosporidium oocysts to humans and other animals. Few studies have evaluated the prevalence of Cryptosporidium species in domestic free-range poultry in China. Here, we characterized the prevalence and distribution of species and genotypes of Cryptosporidium in domestic free-range chickens, ducks, and geese in Anhui Province, China. A total of 1910 fresh fecal samples from three poultry species were examined from 18 free-range poultry farms by nested PCR and analysis of the Cryptosporidium SSU rRNA gene. The overall prevalence of Cryptosporidium species was 2.9% (55/1910), with infection rates of 1.3% (11/829) in chickens, 7.3% (36/487) in ducks, and 1.4% (8/594) in geese. C. baileyi (0.6%), C. meleagridis (0.2%), C. galli (0.2%), and C. xiaoi-like genotype (0.2%) were identified in chickens, and only C. baileyi was identified in ducks and geese, with infection rates of 7.4% and 1.3%, respectively. C. baileyi was the most prevalent species. Sequencing of the GP60 gene revealed that the C. meleagridis isolates belonged to the IIIbA26G1R1b subtype. This is the first study to document C. galli and C. xiaoi-like genotype in domestic free-range chickens in China. These findings expand the range of avian hosts known for Cryptosporidium and highlight the need for additional studies to characterize the diversity of Cryptosporidium in avian species.

Prevalence of Tetratrichomonas buttreyi and Pentatrichomonas hominis in yellow cattle, dairy cattle, and water buffalo in China.

The trichomonad species Tetratrichomonas buttreyi and Pentatrichomonas hominis have been reported in the bovine digestive tract in only a few studies, and the prevalence and pathogenicity of these two protists in cattle herds remain unknown. In this study, the prevalence of T. buttreyi and P. hominis in yellow cattle, dairy cattle, and water buffalo in Anhui Province, China, was determined with a PCR analysis of the small subunit ribosomal RNA genes. The overall infection rates for T. buttreyi and P. hominis were 8.1% and 5.4%, respectively. Double infections were found in 15 (1.6%) samples from four farms. The prevalence of P. hominis in cattle with abnormal feces was significantly higher than that in cattle with normal feces (χ2 = 13.0, p < 0.01), and the prevalence of T. buttreyi in the northern region of Anhui Province was also significantly higher than that in the mid region (χ2 = 16.6, p < 0.01). Minor allelic variations were detected in the T. buttreyi isolates from cattle in this study, as in other hosts in previous studies. Morphological observations, together with the PCR analysis, demonstrated that the trichomonads isolated in this study were P. hominis. The presence of T. buttreyi and P. hominis indicated that cattle are natural hosts of these two trichomonads and could be a potential source of P. hominis infections in humans and other animal hosts.

The first report of Cryptosporidium testudinis in Chinese alligators (Alligator sinensis) in China.

Several Cryptosporidium species that infect reptiles, especially squamates, are well described, but there is limited data about Cryptosporidium species infecting crocodilians. In this study, we assess the occurrence of intestinal parasites using traditional microscopic examination and describe the prevalence and Cryptosporidium species in the captive-bred Chinese alligators (Alligator sinensis) in eastern China using molecular methods. The results of microscopic examination showed that no intestinal parasites were detected among the 491 fecal samples examined from the Chinese alligators. The overall prevalence for Cryptosporidium was 0.41% (2/491) by PCR detection using the SSU rRNA locus. Sequence and phylogenetic analysis of the SSU rRNA, COWP, and actin genes revealed the presence of Cryptosporidium testudinis, which has been isolated primarily from chelonians. This is the first detection of the specific DNA of C. testudinis in the feces of the Chinese alligator. This study expands our knowledge of the Cryptosporidium species involved in crocodiles, and more extensive studies are necessary to confirm the validity of C. testudinis in crocodiles.

Prevalence and Molecular Characterization of Intestinal Trichomonads in Pet Dogs in East China.

The trichomonad species Tritrichomonas foetus and Pentatrichomonas hominis were recently detected in the feces of dogs with diarrhea. However, little information is available on the prevalence and pathogenicity of these parasites in the canine population. Therefore, the aim of this study was to determine the prevalence and molecular characterization of trichomonads infecting pet dogs in Anhui and Zhejiang provinces, east China. In total, 315 pet dogs, with or without diarrhea, from 7 pet hospitals were included in this epidemiological survey. Microscopy and PCR detected P. hominis in 19.7% (62/315) and 31.4% (99/315) of fecal samples, respectively. T. foetus infection was detected in 0% (0/315) of samples with microscopy and in 0.6% (2/315) with PCR. The prevalence of P. hominis was significantly higher in young dogs (≤12 months) than in adult dogs (>12 months), and was significantly higher in diarrheic dogs (50.6%) than in non-diarrheic dogs (24.3%; P<0.05). Infection with T. foetus did not correlate with any risk factors evaluated in this study. A sequence analysis of the P. hominis PCR products showed minor allelic variations between our sequences and those of P. hominis strains from other hosts in different parts of the world. Type CC1 was the most common strain in dogs in east China. The internal transcribed spacer 1 (ITS1)-5.8S rRNA gene sequences from the 2 T. foetus isolates detected in this study displayed 100% identity and were homologous to the sequences of other strains isolated from domestic cats in other countries.

[Blastocystis Infection in Pet Dogs in Selected Areas of Anhui and Zhejiang Provinces].

Objective: To determine the prevalence and subtypes of Blastocystis in pet dogs in Anhui and Zhejiang Provinces. Methods: Pet dog fecal samples were collected from veterinary clinics in Baohe District of Hefei City, Xuanzhou District of Xuancheng City, Fengyang County and Mingguang City of Chuzhou City, Longzihu District of Bengbu City, Si County of Suzhou City in Anhui Province, as well as in Yuhang District of Hangzhou City in Zhejiang Province from April to December, 2013. Blastocystis was detected by microscopic examination as well as nested PCR and single-step PCR based on small subunit ribosomal DNA sequences. The PCR products were sequenced, and Blastocystis subtypes were assigned. Results: A total of 315 pet dog fecal samples were collected. The incidence of Blastocystis infection determined by microscopic examination, nested PCR and single-step PCR was 1.3%（4/315）, 1.9%（6/315） and 1.9%（6/315）, respectively. In addition, the two PCR methods both revealed Blastocystis infection only in Chuzhou City（3.4%, 1/29） and Hefei City（5.6%, 4/72）. The pet dogs in Chuzhou City carried ST1 and ST2 subtypes while those in Hefei carried only ST1. There was no significant difference in infection rate between adult and young dogs and between male and female dogs. Conclusion: Blastocystis infection exists in pet dogs in Chuzhou City and Hefei City.

[Prevalence of Neospora caninum Infection in the Intestine of Pet Dogs in Some Areas of Anhui and Zhejiang].

OBJECTIVE: To investigate the prevalence of Neospora caninum infection in the intestine of pet dogs in areas of Anhui and Zhejiang. METHODS: A total of 315 fecal samples from pet dogs were collected in pet clinics from April to December 2013 in Baohe District in Hefei city, Xuanzhou District in Xuancheng city, Fengyang County in Chuzhou city, Longzihu District in Bengbu city, and Si County in Suzhou City in Anhui Province, as well as in Yuhang District in Hangzhou city of Zhejiang province. All samples underwent nested-PCR targeting Neospora-specific gene NCLI-004830. The results were further confirmed by PCR amplification of N. caninum ITS1 followed by sequence analysis. RESULTS: The rate of N. caninum infection in the 315 samples was 1.59% (5/315). The infection rate in Chuzhou and Bengbu was 3.37% and 6.45%, respectively, and no N. caninum infection was found in the remaining areas. There was no association between the infection rate and the sex or age of the dogs. CONCLUSION: N. caninum infection is prevalent in pet dogs in Chuzhou and Bengbu of Anhui.

[Molecular Detection of Giardia lamblia and Cryptosporidium Species in Pet Dogs].

OBJECTIVE: To determine the prevalence of Giardia lamblia and Cryptosporidium species infection in pet dogs, and identify the G. lamblia assemblages and Cryptosporidium species. METHODS: A total of 315 fresh fecal samples were collected from pet clinics in five counties of Anhui Province and in Hangzhou of Zhejiang Province. Hemi-nested-PCR targeting the GDH gene of G. lamblia and nested-PCR targeting the SSU rRNA gene of Cryptosporidium were performed in all the fecal samples. The PCR products were sequenced and analyzed using bioinformatics methods to identify the G. lamblia assemblages and Cryptosporidium species. RESULTS: The positive rates of G. lamblia and Cryptosporidium spp. infections in the 315 fecal samples were 3.2% (10/315) and 1.6% (5/315), respectively. Specifically, the two indicators were both significantly higher in dogs ≤12 months (17.8% and 11.1%, respectively) than in adult dogs (0.7% and 0.0%)(P<0.05). However, there was no significant difference in the two indicators between male and female dogs. In addition, two G. lamblia assemblages were identified, assemblages B (n=6) and D (n=4). Sequence analysis of PCR products of the SSU rRNA gene showed that the five Cryptosporidium isolates were C. canis (n =5). CONCLUSION: The prevalences of G. lamblia and Cryptosporidium infection in pet dogs in Anhui and Zhejiang Provinces were 3.2 % and 1.6 %, respectively. The assemblages of G. lamblia in this study are of types B and D.

[In vitro effect of osthole on ultrastructure of Giardia lamblia].

Giardia lamblia trophozoites were cultivated axenically in TYI-S-33 modified medium containing 1.345 mg/ml of osthole (24 h IC50). The parasites were observed by scanning and transmission electron microscopes after treated with osthole for 24 h. The surface of the trophozoites treated with osthole was rough. The surface of ventral sucker and median body had obvious lesions, the cell membrane was damaged and the content spilled out. There were a lot of vacuoles in the cytoplasm. And the nuclear was severely deformed with a serrated edge and marginated nuclear chromatin. The microtubules of sucker had partially disintegrated.

[Prevalence and molecular characterization of Giardia lamblia isolates from goats in Anhui Province].

Five hundred and six fresh fecal samples were collected from Lu'an, Fuyang, Suzhou, Chizhou, Wuhu, Chuzhou and Bozhou in Anhui Province, and detected firstly by direct smear microscopy. The microscopy-positive samples were amplified by nested PCR targeting the triosephosphate isomerase (TPI) and glutamate dehydrogenase(GDH) genes. The positive PCR products were sequenced in both directions. The sequences were analyzed by ClustalX 1.81 for sequence alignment and the neighbor-joining trees were constructed by Mega 5.05. Thirty-two out of 506 fecal specimens were diagnosed as Giardia-positive by microscopy with an infection rate of 6.3%. 23 and 16 of the samples were typed as assemblage E by the TPI (530 bp) and GDH (450 bp) genes, respectively. These findings indicated that there was a different distribution of subtypes of assemblage E in different areas. The zoonosis genotypes such as assemblage A or B was not found in the present study.

First Detection and Molecular Identification of Entamoeba bovis in Farm-Raised Sika Deer from Anhui Province, China.

BACKGROUND: Protozoans of Entamoeba spp. are one of the most common enteric parasites that infect humans and diverse animals including deer. PURPOSE: However, data regarding the prevalence and species/genotypes of Entamoeba spp. in deer in China is scarce. This study investigated the prevalence and species distribution of Entamoeba spp. in sika deer (Cervus nippon) in Anhui Province. METHODS: In our survey, 336 fecal samples were collected from five sika deer farms in different regions of Anhui Province. All samples were examined for the presence of Entamoeba spp. by PCR and phylogenetic analysis of the conserved 18S rRNA gene. RESULTS: 106/336 (31.5%) fecal samples were positive for Entamoeba spp. A statistically significant difference in the prevalence of Entamoeba spp. infection was observed between sampling farms (p < 0.001), and the prevalence of Entamoeba spp. in male and female sika deer showed no significant difference (p > 0.05). Sequence and phylogenetic analysis revealed the single species, E. bovis, was identified in this study. CONCLUSION: This is the first report about the identification of E. bovis in farm-raised sika deer in China, and these results expand our understanding of host range and species distribution of Entamoeba spp. in ruminants.

Detection and Genotyping Study of Enterocytozoon bieneusi in Sheep and Goats in East-central China.

BACKGROUND: Enterocytozoon bieneusi, the dominant zoonotic species of microsporidia, has been reported in various animals and humans. However, relatively little information is available concerning the worldwide prevalence and genotypes of E. bieneusi in ruminants, such as sheep and goats. The present study was conducted to evaluate the prevalence and genetic characteristics of E. bieneusi in sheep and goats in east-central China. METHODS: Fresh fecal samples from 832 sheep and 781 goats were evaluated for the presence of E. bieneusi using PCR and sequencing of the ribosomal internal transcribed spacer. RESULTS: 28 sheep specimens (3.4%) and 32 goat specimens (4.1%) were positive for E. bieneusi. A total of 12 ITS genotypes were identified, including 7 known genotypes (COS-I, OEB1, BEB6, CHG1, CHG3, COS-II, and CHC8) and 5 novel genotypes (named AHS1, AHS2, JSS1, AHG1, and AHG2). The dominant genotypes in sheep and goats were BEB6 and CHG3, respectively. Phylogenetic analysis revealed that all genotypes obtained in this study belonged to the so-called "cattle-specific" Group 2. CONCLUSIONS: These findings show a relatively low occurrence and genetic diversity of E. bieneusi in sheep and goats in east-central China. Further studies are required to elucidate the role of sheep and goats in the epidemiology of microsporidia.

Genotypes of Enterocytozoon bieneusi in Dogs and Cats in Eastern China.

BACKGROUND: Enterocytozoon bieneusi is a common opportunistic pathogen found in both humans and animals. As companion animals live in close contact with human being, they may act as a zoonotic reservoir and play an important role in transmitting this parasite to humans. We evaluated the prevalence, genotypic diversity and zoonotic potential of E. bieneusi in dogs and cats in eastern China during Apr to Dec 2013. METHODS: Fecal specimens from 315 dogs and 143 cats from veterinary hospitals in eastern China were examined in 2015 by internal transcribed spacer (ITS)-based PCR. RESULTS: E. bieneusi was detected in 8.6% of canine and in 1.4% of feline samples. Seven genotypes of E. bieneusi were identified, including four known genotypes (PtEb IX, EbpC, Type IV and D) and three novel genotypes, named CHD1, CHD2 and CHD3. The dominant genotype in dogs was PtEbIX (59.3%; n=16/27). Five (CHD1, EbpC, CHD2, D and Type IV) of the seven genotypes were in the so-called zoonotic group 1, whereas genotypes PtEbIX belonged to the dog-specific group and genotypes CHD3 were placed in group 2. CONCLUSION: Dogs are predominately infected with host-specific genotypes of E. bieneusi, and the finding of several zoonotic genotypes in dogs and cats reminds us of potentially zoonotic transmission of microsporidiosis.

Survey and Molecular Characterization of Trichomonads in Pigs in Anhui Province, East China, 2014.

BACKGROUND: In pigs, several different trichomonad species such as Tritrichomonas foetus, Tetratrichomonas buttreyi, and Pentatrichomonas hominis have been described as inhabiting the digestive tract. However, little information is available on the epidemiology of these neglected parasites in the Chinese pig population. METHODS: The prevalence of T. suis, T. buttreyi and P. hominis among 500 fecal specimens from pigs at seven pigs farms in Anhui Province in China between Oct and Dec 2014, was determined by PCR and DNA sequence analysis of the small subunit ribosomal RNA (SSU rRNA) genes. RESULTS: The prevalence rates for T. suis, T. buttreyi, and P. hominis were 2.8% (14/500), 42.0% (210/500) and 7.8% (39/500), respectively. Mixed infections of two or three trichomonads were detected in 24 samples. The prevalence of the three trichomonads differed significantly between some age groups, with higher infection rates of T. suis and T. buttreyi in nursery pigs and P. hominis in preweaned pigs. The SSU rRNA sequences from T. suis and P. hominis showed 100% homology with their respective homologous database sequences. However, we observed minor allelic variations in the SSU rRNA sequences from T. buttreyi, and the five representative sequences identified were named firstly as types 1, 2, 3, 4 and 5. Moreover, type 1 was found to be dominant in the present study. CONCLUSION: These findings highlight the potential risk posed by pigs in the transmission of trichomonad infections to humans and other animals.

First report on the occurance of intestinal Entamoeba spp. In pigs In China.

Three Entamoeba spp. including E. suis, zoonotic E. polecki, and E. histolytica, have been described in pigs to date. However, little is known about the molecular epidemiology of these neglected parasites in pigs globally. In this study we surveyed the occurrence and molecular epidemiology of porcine Entamoeba spp. in pigs in eastern China and evaluated their zoonotic potential. Five hundred fresh fecal samples, collected from seven pig farms in Anhui province, eastern China,were examined for the presence of E. histolytica, E. suis, and E. polecki ST1 and ST3 infections by a combination of nested PCR targeting the small subunit ribosomal DNA gene and subsequent sequencing.The overall occurrence of Entamoeba spp. was 45.8% (229/500). Infection with E. polecki ST1 (38.2%; 191/500) was the most common, followed by E. polecki ST3 (10.0%; 50/500), and E. suis (0.8%; 4/500). No E. histolytica infection was detected. Double infections with E. polecki ST1 and E. suis, and with E. polecki ST1 and ST3 were found in two (0.4%) and 14 (2.8%) samples, respectively. No age predisposition to infection with Entamoeba spp. was observed. PCR and subsequent sequencing confirmed the validity and feasibility of the nested PCR method used in this study in identifying species/subtypes of porcine Entamoeba spp.This is the first report to describe the occurrence and molecular epidemiology of Entamoeba species in pigs in China. The presence of two zoonotic E. polecki subtypes implies that pigs can be reservoirs for human E. polecki infections. More studiess are needed to better understand the transmission and public health significance of porcine Entamoeba spp.