Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in pet cats in Henan Province, central China.

Cryptosporidium spp. and G. duodenalis often infect humans, cats, and other mammals, causing diarrhea and being responsible for numerous outbreaks of waterborne and foodborne infections worldwide. The rapid increase in the number of pet cats poses a substantial public health risk. However, there were few reports about the infection of Cryptosporidium spp. and G. duodenalis infections in pet cats in Henan Province, central China. Thus, to understand the prevalence and genetic distribution of Cryptosporidium spp. and G. duodenalis in pet cats, and to evaluate the zoonotic potential, possible transmission routes and public health implications of isolates, fecal samples (n = 898) were randomly collected from pet cats in 11 cities in Henan Province, central China. Nested PCR based on the SSU rRNA gene and bg gene was used to the prevalence of Cryptosporidium spp. and G. duodenalis, respectively. The prevalence was 0.8 % (7/898) and 2.0 % (18/898) for Cryptosporidium spp. and G. duodenalis respectively. Additionally, the Cryptosporidium spp. positive isolates were identified as C. parvum subtype IIdA19G1 by gp60 gene. In the present study, the IIdA19G1 subtype was discovered in pet cats for the first time in China, enriching the information on the host type and geographical distribution of Cryptosporidium spp. in China. For G. duodenalis, a total of 18 G. duodenalis positive samples were identified, belonging to four assemblages: a zoonotic assemblage A1 (4/898), three host-specific assemblages C (8/898), D (5/898), and F (1/898). Interestingly, we found that pet cats infected with Cryptosporidium spp. and G. duodenalis are more likely to experience emaciation symptoms compared to the negative group. More importantly, the prevalence of Cryptosporidium spp. and G. duodenalis detected in the present study were low, but the subtype IIdA19G1 of Cryptosporidium spp. and the assemblages A1, C, D, and F of G. duodenalis have the potential for zoonotic transmission. Thus, we should focus on preventing and controlling the risk of cross-species transmission that may occur in pet cats in Henan Province.

Gut microbiota and Tritrichomonas foetus infection: A study of prevalence and risk factors based on pet cats.

Tritrichomonas foetus (T. foetus) is a protozoal pathogen that infects cats and constitutes a significant cause of chronic colitis and diarrhea. Perturbations in the gut microbiota (GM) are affected by Trichomonas infection. Furthermore, dysregulation of the host GM enhances Trichomonas pathogenicity. However, it remains unclear whether the occurrence of diarrhea is associated with a dysregulation in GM following T. foetus infection in cats. Hence, the primary objective of this investigation was to explore the correlation between T. foetus infection and dysregulation in GM by analyzing fecal samples obtained from pet cats in Henan Province, central China. We randomly collected 898 fecal samples from pet cats living in 11 prefectural cities within Henan Province, and T. foetus was screened with polymerase chain reaction (PCR) amplification based on the 18 S rRNA gene. Subsequently, six T. foetus-positive and six T. foetus-negative samples underwent analysis through 16 S rRNA gene sequencing to evaluate the gut microbiota's composition. The overall prevalence of T. foetus infection among the collected samples was found to be 6.01% (54/898). Notably, a higher prevalence of infection was observed in young, undewormed, unimmunized, and diarrheic pet cats. T. foetus infection was found to significantly alter the composition of the pet cat fecal microbiota, leading to dysfunctions. Moreover, it resulted in a substantial increase in the abundance of Bacteroidetes, Proteobacteria, and Phascolarctobacterium spp., while decreasing the ratio of Firmicutes to Bacteroidetes (F/B) and the abundance of Actinobacteria, Clostridiaceae_Clostridium spp., Phascolarctobacterium spp., SMB53 spp., and Blautia spp. We constructed ROC curves to assess the diagnostic value of specific bacterial taxa in discriminating T. foetus infection. The analysis revealed that Proteobacteria and Clostridiaceae_Clostridium spp. were the most reliable single predictors for T. foetus infection. This finding suggests that alterations in the GM may be strongly associated with T. foetus infections.

Molecular epidemiology of Enterocytozoon bieneusi from foxes and raccoon dogs in the Henan and Hebei provinces in China.

BACKGROUND: Enterocytozoon bieneusi is a zoonotic pathogen widely distributed in animals and humans. It can cause diarrhea and even death in immunocompromised hosts. Approximately 800 internal transcribed spacer (ITS) genotypes have been identified in E. bieneusi. Farmed foxes and raccoon dogs are closely associated to humans and might be the reservoir of E. bieneusi which is known to have zoonotic potential. However, there are only a few studies about E. bieneusi genotype identification and epidemiological survey in foxes and raccoon dogs in Henan and Hebei province. Thus, the present study investigated the infection rates and genotypes of E. bieneusi in farmed foxes and raccoon dogs in the Henan and Hebei provinces. RESULT: A total of 704 and 884 fecal specimens were collected from foxes and raccoon dogs, respectively. Nested PCR was conducted based on ITS of ribosomal RNA (rRNA), and then multilocus sequence typing (MLST) was conducted to analyze the genotypes. The result showed that infection rates of E. bieneusi in foxes and raccoon dogs were 18.32% and 5.54%, respectively. Ten E. bieneusi genotypes with zoonotic potential (NCF2, NCF3, D, EbpC, CHN-DC1, SCF2, CHN-F1, Type IV, BEB4, and BEB6) were identified in foxes and raccoon dogs. Totally 178 ITS-positive DNA specimens were identified from foxes and raccoon dogs and these specimens were then subjected to MLST analysis. In the MLST analysis, 12, 2, 7 and 8 genotypes were identified in at the mini-/ micro-satellite loci MS1, MS3, MS4 and MS7, respectively. A total of 14 multilocus genotypes were generated using ClustalX 2.1 software. Overall, the present study evaluated the infection of E. bieneusi in foxes and raccoon dogs in the Henan and Hebei province, and investigated the zoonotic potential of the E. bieneusi in foxes and raccoon dogs. CONCLUSIONS: These findings expand the geographic distribution information of E. bieneusi' host in China and was helpful in preventing against the infection of E. bieneusi with zoonotic potential in foxes and raccoon dogs.

Assessment of potential zoonotic transmission of Giardia duodenalis from dogs and cats.

Giardia duodenalis is one of the major causes of diarrhea among humans, especially in young children. Statistical analysis revealed that the pooled prevalence of G. duodenalis in humans, dogs, and cats was 9.72% (10,921/112383), 15.60% (7510/48140), and 14.53% (1125/7740), respectively. Unquestionably, the canine-specific assemblages C and D and the feline-specific assemblage F were the dominant genotypes in dogs and cats, respectively. Additionally, the prevalence of zoonotic G. duodenalis assemblages (A and B) in dogs and cats was 23.07% (875/3792) and 41.42% (169/408), respectively, implying that the potential transmission of G. duodenalis from dogs and cats to human infection cannot be ignored. The highest frequency of potentially zoonotic assemblages was found among working dogs (3.55%, 25/705) and the 1-5 age group (22.92%, 11/48). In summary, dogs and cats have a significant role in the zoonotic transmission of G. duodenalis due to their close contact with humans and the higher frequency presence of zoonotic assemblages. Further studies are necessary to explore the presence of G. duodenalis among humans and animals and in environmental samples. Researchers should adopt a one-health approach to gain a deeper understanding of G. duodenalis in dogs and cats and potential transmission routes to humans.

Prevalence of Pentatrichomonas hominis in foxes and raccoon dogs and changes in the gut microbiota of infected female foxes in the Hebei and Henan Provinces in China.

Pentatrichomonas hominis (P. hominis) is a zoonotic parasite that affects a wide range of hosts, causing gastrointestinal diseases. The present study aimed to evaluate the prevalence of P. hominis among caged foxes and raccoon dogs and the effect of P. hominis on the gut microbiota in female foxes. A total of 893 fresh fecal samples were collected from the Hebei and Henan Provinces in China. P. hominis was screened based on 18S rRNA gene expression via nested PCR. The difference in the gut microbiota between nine P. hominis-positive and nine P. hominis-negative samples was investigated by 16S rRNA gene sequencing. The total prevalence of P. hominis infection in foxes and raccoon dogs was 31.7% (283/893). The prevalence rates of P. hominis infection were 28.2% (88/312) and 33.6% (195/581) in foxes and raccoon dogs, respectively. Phylogenetic analysis revealed that all P. hominis strains detected in foxes and raccoon dogs in the present study were the zoonotic genotype CC1. Moreover, compared with those in the P. hominis-negative group, the diversity of the gut microbiota in the P. hominis-positive group was lower, and the abundance of Firmicutes and the ratio of Firmicutes/Bacteroidetes (F/B) in the P. hominis-positive group were lower than those in the P. hominis-negative group. We speculate that these differences may be due to indigestion and diarrhea in infected female foxes. Overall, the present study evaluated the prevalence of P. hominis in foxes and raccoon dogs in the Henan and Hebei Provinces and revealed that P. hominis infection interrupted the diversity of the gut microbiota in female foxes.

Evaluation of gut microbiota composition to screening for potential biomarker in AFB1-exposed sheep.

Aflatoxin B1 (AFB1) is an inevitable contaminant in animal feed and agricultural products, which seriously threatens the health of animals. However, there is currently no better diagnostic tool available than depending on clinical symptoms, pathophysiology, biochemical indicators, etc. Here, we profiled the fecal microbiomes of sheep exposed to and not exposed to AFB1 to identify potential non-invasive biomarkers of AFB1 intoxication by 16S rRNA gene sequencing technology, while measuring serum biochemical indexes. The results showed that the sheep exposed to AFB1 had significantly higher levels of the liver function indicators ALT (alanine transaminase) and AST (aspartate aminotransferase), and their microbial profiles were different from those of the CON (Control) group. In detail, the relative abundance of seven phyla and three genera were overrepresented in the AFB1 group from top 10 relative abundance. Importantly, we found that Prevotella and Bifidobacterium were significantly different in the CON and AFB1 groups (p = 0.032 and p = 0.021, respectively) based on linear discriminant analysis effect size (LEfSe) and random forest analysis. Additionally, the area under curve (AUC) of ALT was 1 (95% CI 1.00-1.00; p < 0.001) and that of Bifidobacterium was 0.95 (95% CI 0.81-1.00; p = 0.0275), suggesting that Bifidobacterium correlated with ALT (r = 0.783, p < 0.01) may be a potential biomarker for AFB1 exposure in sheep.

Aflatoxin B1 Exposure in Sheep: Insights into Hepatotoxicity Based on Oxidative Stress, Inflammatory Injury, Apoptosis, and Gut Microbiota Analysis.

The widespread fungal toxin Aflatoxin B1 (AFB1) is an inevitable pollutant affecting the health of humans, poultry, and livestock. Although studies indicate that AFB1 is hepatotoxic, there are few studies on AFB1-induced hepatotoxicity in sheep. Thus, this study examined how AFB1 affected sheep liver function 24 h after the animals received 1 mg/kg bw of AFB1 orally (dissolved in 20 mL, 4% v/v ethanol). The acute AFB1 poisoning caused histopathological injuries to the liver and increased total bilirubin (TBIL) and alkaline phosphatase (AKP) levels. AFB1 also markedly elevated the levels of the pro-inflammatory cytokines TNF-α and IL-6 while considerably reducing the expression of antioxidation-related genes (SOD-1 and SOD-2) and the anti-inflammatory gene IL-10 in the liver. Additionally, it caused apoptosis by dramatically altering the expression of genes associated with apoptosis including Bax, Caspase-3, and Bcl-2/Bax. Notably, AFB1 exposure altered the gut microbiota composition, mainly manifested by BF311 spp. and Alistipes spp. abundance, which are associated with liver injury. In conclusion, AFB1 can cause liver injury and liver dysfunction in sheep via oxidative stress, inflammation, apoptosis, and gut-microbiota disturbance.

Prevalence and genetic diversity of Giardia duodenalis in pet dogs from Zhengzhou, central China and the association between gut microbiota and fecal characteristics during infection.

As a common zoonotic intestinal parasite, Giardia duodenalis could infect humans and various mammals worldwide, including pet dogs, leading to giardiasis. This study detected the infection of G. duodenalis in asymptomatic pet dogs in Zhengzhou, and evaluated the possibility of zoonosis and the relationship between gut microbiota and fecal characteristics. We randomly collected 448 fresh fecal samples from Zhengzhou, and G. duodenalis was screened based on the beta-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) genes. The difference of gut microbiota between five G. duodenalis-positive and five G. duodenalis-negative samples was investigated by 16S rRNA gene sequencing. The overall prevalence of G. duodenalis was 7.1% (32/448) based on bg, gdh, and tpi locus, two G. duodenalis assemblages (C = 13, D = 14) and five (15.6%) mixed infection (C + D) were identified. Moreover, compared with the G. duodenalis-negative group, the diversity of gut microbiota increased in G. duodenalis-positive group. The decrease of Lactobacillus spp. and considerable increase of Prevotella spp. were associated with the fecal characteristics. These results show that the transmission of zoonotic giardiasis between humans and pet dogs is rare in Zhengzhou, central China, and support the use of Lactobacillus spp. as a potential probiotic agent to improve intestinal health in dogs, or even humans, by treating G. duodenalis. Therefore, the public health significance of G. duodenalis to humans, companion animals, and the environment should be further evaluated from One Health perspective.

Aflatoxin B1 causes oxidative stress and apoptosis in sheep testes associated with disrupting rumen microbiota.

Aflatoxin B1 (AFB1) is an unavoidable environmental pollutant commonly found in feed and foodstuffs. It is the most toxic one of all the aflatoxins, which can cause severe impairment to testicular development and function. Yet, the underlying mechanisms of reproductive toxicity in rams sheep remain inconclusive. The study was designed to explore the effects of AFB1 on sheep testes through rumen-microbiota, oxidative stress and apoptosis. Six-month-old male Dorper rams (n = 6) were orally administrated with 1.0 mg/kg AFB1 (dissolved in 20 mL 4% ethanol) 24 h before the experiment. At the same time, rams in the control group (n = 6) were intragastrically administrated with 20 mL 4% ethanol. It was observed that acute AFB1 poisoning had significant (p < 0.05) toxin residue in the testis and could cause testicular histopathological damage. AFB1 stimulated the secretion of plasma testosterone level through regulating testosterone synthesis-related genes (StAR, 3ß-HSD, CYP11A1, and CYP17A1), which are accompanied by the increase of oxidative stress and testicular apoptosis that had a close relationship with the regulation of testosterone secretion. Interestingly, we observed rumen dysbacteriosis and decreased the abundances of Prevotella, Succiniclasticum, CF231, Ruminococcus, and Pseudobutyrivibrio in AFB1-exposed sheep, which were negatively correlated to the testosterone synthesis-related gene levels. Taken together, our findings indicated that AFB1 induced testicular damage and testicular dysfunction, which is related to testicular oxidative stress and apoptosis involved in rumen dysbacteriosis in sheep.

Molecular detection and genotyping of Enterocytozoon bieneusi in captive foxes in Xinxiang, Central China and its impact on gut bacterial communities.

Enterocytozoon bieneusi is a common gastrointestinal parasite that has a broad range of hosts, including birds, mammals, and even humans. The changes of gut bacterial communities have been demonstrated during the course of E. bieneusi. This study aimed to examine the infection status, and assess the zoonotic potential and influence of E. bieneusi on gut bacterial communities of captive foxes in Xinxiang, central China. Fecal samples were collected from 216 captive foxes, and amplification of the internal transcribed spacer (ITS) gene of each sample was performed by nested polymerase chain reaction. E. bieneusi was detected in 48 (22.2%) samples, and five previously reported genotypes CHN-F1 (n = 25), D (n = 18), BEB6 (n = 3), NCF2 (n = 1), and CHN-DC1 (n = 1) were identified, with CHN-F1 being the dominant genotype (25/48, 52.1%). Phylogenetic analysis further revealed that CHN-F1, D, NCF2, and CHN-DC1 genotypes belonged to the zoonotic group 1 of E. bieneusi, whereas BEB6 belonged to group 2. Moreover, sequencing and bioinformatics analysis of the V3-V4 region of 16S rRNA gene in five E. bieneusi-positive and five negative samples showed that gut microbial diversity was higher in the infected animals. In both groups, Firmicutes and Bacteroidetes were the two most abundant phyla, but the Firmicutes/Bacteroidetes ratio was lower in E. bieneusi-positive foxes (7.9:1) as compared with E. bieneusi-negative foxes (5:1). More importantly, at the phylum level, "beneficial bacteria" such as Firmicutes (1.6%) and Bacteroides (5.8%) increased, whereas "opportunistic pathogens" such as Fusobacteria (8.2%) decreased. Similarly, at the genus level, the pathogenic Clostridiaceae_Clostridium (10.0%) decreased, whereas the "beneficial bacteria" Lactococcus (6.6%) increased. These interesting phenomena warrant further investigation.

PREVALENCE AND MOLECULAR CHARACTERISTICS OF BLASTOCYSTIS SP. FROM PEAFOWL (PAVO CRISTATUS) IN CHINA.

This study is the first description of Blastocystis infection in peafowls in China. In total, 143 fecal specimens collected from a peafowl breeding farm in Henan Province were tested for Blastocystis infection by PCR assay targeting the small subunit ribosomal RNA (SSU rRNA) gene, and a total of 50 specimens (35.0%) were positive. Based on sequences and phylogenetic analysis, 2 genetically distinct subtypes (STs) were determined: ST9 and ST7. ST9 was the predominant subtype, accounting for 82% (41/50). The rare zoonotic subtype ST7 was also identified in peafowls, with the infection rate of 18% (9/50). Altogether, the present study is the first report of the prevalence and molecular characteristics of Blastocystis in peafowls in central China. The presence of zoonotic subtypes in peafowls suggests the potential risk of zoonotic transmission of Blastocystis to workers at peafowl farms.

Aflatoxin B1 alters meat quality associated with oxidative stress, inflammation, and gut-microbiota in sheep.

Aflatoxin B1 (AFB1) is an unavoidable contaminant in animal feed and agricultural products. AFB1 has been found to impair the liver and kidney function of sheep. However, few data are available, which explain the toxic damage of AFB1 exposure on meat quality. In the study, male Dorper RAMS sheep (6-month-old) were orally administrated with AFB1 at the dose of 1 mg/kg body weight once. The body temperature, serum biochemistry, meat quality-related parameters, oxidation indicators in meat and serum, the mRNA expression of pro-inflammatory cytokines and anti-inflammatory, and microbiota composition of feces were measured 24 h after AFB1 exposure. The results showed that the body temperature was slightly increased, the mental state of mutton sheep was suppressed, and biochemical indicators were significantly changed after AFB1 exposure. AFB1 impaired mutton quality reflected by the structure of muscle fibers was changed, and increased muscle drip loss and lightness (L*), and decreased muscle redness (a*). Moreover, we found that AFB1 caused changes in the oxidative stress indicators T-SOD, T-AOC, MDA, GSH level, and GSH/GSSG ratio, and inflammation damage of mutton reflected by increasing pro-inflammatory TNF-α and reducing anti-inflammatory IL-10 mRNA levels, disrupts the secretion of inflammatory factors, and changed the composition of gut microbiota reflected by significantly increased Firmicutes/Bacteroidetes ratio and decreased the abundances of Butyrivibrio, which are related to the quality of the mutton. In summary, gut microbiota participates in AFB1 to damage mutton quality, which may be co-mediated by oxidative stress, inflammation, and gut microbiota.

Molecular identification and subtyping of Blastocystis sp. in hospital patients in Central China.

Blastocystis is a common enteric protist that inhabits the gastrointestinal tract of approximately 1 billion people worldwide. In this study, a total of 1,070 patients from two hospitals in Zhengzhou, Central China were enrolled to know molecular characteristics of Blastocystis sp. The microorganism was identified and subtyped with a PCR amplification and sequencing of the small subunit ribosomal deoxyribonucleic acid (SSU-rDNA). The overall minimum prevalence of Blastocystis sp. in participants was 3.1% (33/1070). Although there were no significant differences on Blastocystis sp. infections among study sites, age groups, and gender, the higher infection was observed in the patients with gastrointestinal diseases (8.8%, 15/170). Sequence analysis of the 33 isolates revealed three known subtypes, such as ST1 (n = 7), ST3 (n = 23), and ST7 (n = 3). Among them, ST3 was the dominant subtype being detected in 23 isolates (69.7%), followed by ST1 (21.2%, 7/33) and ST7 (9.1%, 3/33). The phylogenetic analysis demonstrated that three subtypes (ST1, ST3 and ST7) were clustered with their reference sequences with good bootstrap support. The subtype determination of Blastocystis sp. isolates by the phylogenetic analysis was well supported by online platform. The present study provides the first molecular report of Blastocystis sp. infections in hospital patients in Central China.

First report of Blastocystis infection in Pallas's squirrels (Callosciurus erythraeus) in China.

Blastocystis, an intestinal anaerobic protist with high genetic diversity, inhabits a variety of hosts worldwide, including rodents. However, there have been few studies on squirrel Blastocystis infections in China to date. Herein, 171 fecal samples from Pallas's squirrels (Callosciurus erythraeus) sold as pets were collected to investigate the prevalence and genetic characteristics of Blastocystis. A total of 10 Blastocystis-positive samples (10/171, 5.9%) were obtained by PCR amplification and DNA sequencing of the barcode region of the SSU rRNA gene. Blastocystis subtype analysis revealed four known subtypes, namely, ST1, ST3, ST5 and ST6, with ST5 and ST6 being predominant. Phylogenetic analysis was performed to identify each subtype. To our knowledge, this study is the first to explore Blastocystis infection in Pallas's squirrels, expanding the host range of this parasite. Moreover, multiple zoonotic subtypes were found in Pallas's squirrels, suggesting that these animals may serve as reservoirs for pathogens of human Blastocystis infections.

Molecular Identification of Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in Captive Pet Birds in Henan Province, Central China.

Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis are common enteric pathogens that are capable of infecting humans and animals. Total of 1,005 fecal samples from captive pet birds were collected from seven locations in Henan Province, China. The results demonstrated that 9.9% (99/1,005) of the captive birds were infected with one of these three pathogens. Enterocytozoon bieneusi was the most prevalent species among the birds (45/1,005, 4.5%) followed by G. duodenalis (33/1,005, 3.3%) and Cryptosporidium spp. (21/1,005, 2.1%). Five Cryptosporidium species were identified, namely, C. baileyi (10), C. galli (5), C. meleagridis (4), C. andersoni (1), and C. parvum (1). Two known E. bieneusi genotypes were identified: Peru 6 (44) was identified in pigeons (34) and European turtle doves (10); whereas, the genotype PtEb I (1) was only identified in a pigeon. Only G. duodenalis assemblage E (33) was identified in some pet birds. To the best of our knowledge, this study is the first to undertake the molecular identification of G. duodenalis in birds in China. The identification of potentially zoonotic species/genotypes of the pathogens suggests that exposure to the excreta of these birds, either directly or via food and water, may pose a threat to human health.

Occurrence of a Cryptosporidium xiaoi-like genotype in peafowl (Pavo cristatus) in China.

The aim of this study was to survey the Cryptosporidium species in peafowls (Pavo cristatus) in Henan Province, China. A total of 143 fecal specimens collected from a breeding farm were tested for Cryptosporidium by nested PCR targeting the small subunit rRNA (SSU rRNA), 70-kDa heat shock protein (HSP70), and actin genes of Cryptosporidium followed by sequence analysis. Only one isolate from an asymptomatic host was obtained, and the isolate differed from a new C. xiaoi-like genotype by one nucleotide and from C. xiaoi or C. bovis at the SSU rRNA locus by six nucleotides. Likewise, the actin gene shared 99% identity with the C. xiaoi-like genotype, accompanied by four nucleotide mutations. A complete sequence of the HSP70 gene was obtained, and exhibited 96% similarity with that from C. xiaoi and differed by one nucleotide from that with the C. xiaoi-like genotype. Phylogenetic analysis of the current isolate revealed genetic relatedness to the C. xiaoi-like genotype and distinction from C. xiaoi and C. bovis. Therefore, our results provided the first documentation of avian infection with a C. xiaoi-like genotype in China and further insight into the diversity of Cryptosporidium spp. in avians.

Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Rabbits in Xinjiang, China.

A total of 321 rabbit fecal samples were collected from 10 farms in the Xinjiang Uyghur Autonomous Region, China. The prevalence of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in the samples was 3.4% (11/321), 1.9% (6/321), and 2.8% (9/321), respectively. Small subunit ribosomal RNA (SSU rRNA) gene sequence analysis identified all 11 Cryptosporidium-positive samples as C. cuniculus. Further subtyping based on the 60-kDaglycoprotein locus (gp60) identified five of the C. cuniculus isolates as subtype VbA24. G. duodenalis genotypes were determined by multilocus sequence typing of the SSU rRNA, triosephosphate isomerase, ß-giardin and glutamate dehydrogenase loci, which confirmed that six G. duodenalis isolates belonged to subtype BIV of assemblage B. Analysis of the internal transcribed spacer region, showed that five, three, and one E. bieneusi isolates belonged to genotypes J, BEB8, and Type IV, respectively. These results suggest that Cryptosporidium spp., G. duodenalis, and E. bieneusi isolates from rabbits in China have zoonotic potential.

Detection of Dirofilaria immitis antigen and antibodies against Anaplasma phagocytophilum, Borrelia burgdorferi and Ehrlichia canis in dogs from ten provinces of China.

Despite the fact vector-borne diseases (VBDs) have been increasingly reported in dogs worldwide, there are only limited reports on VBDs in dogs in China with most being based on molecular detection of active infections. To provide further data on the exposure of dogs in China to VBD agents, we used commercial immunochromatographic assays to test plasma from 637 apparently healthy indoor and breeding colony dogs from 21 veterinary clinics in 10 provinces in China and a commercial dog breeding facility for circulating antigen of Dirofilaria immitis, and for circulating antibodies against Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. Overall, we found only low levels of exposure to Ehrlichia spp. (4.7%; 30/637), Anaplasma spp. (1.4%; 9/637), B. burgdorferi (0.9%; 6/637) and D. immitis (0.2%; 1/637) with most of the positive animals coming from the commercial breeding colony (26/103; 25.2%) where ectoparasites were most commonly noted. At least one vector-borne agent was found in dogs from 6 of the 10 provinces investigated. Our results confirm exposure of dogs from around China to a variety of VBDs, even indoor pets seldom observed to harbor ectoparasites.

First confirmed report of outbreak of theileriosis/anaplasmosis in a cattle farm in Henan, China.

Tick-borne diseases (TBDs) impose a significant constraint to livestock production world widely. In this paper, we presented a case of TBD in a cattle farm in Henan, China. 35 blood samples (7 samples sent by veterinarian, 28 samples gathered by our colleagues) were collected from ill, surviving and asymptomatic cattle and microscopic observation and PCR assays were conducted to characterize the pathogens. Genus Ixodes feeding on these cattle were collected and identified. Theileria annulata-like and Anaplasma marginale-like pathogens were observed in the blood smears stained with Giemsa staining under microscope. Furthermore, 5 out of 7 cattle blood samples were found to be positive for T. annulata by PCR. In the 28 blood specimens, three were positive for T. annulata, while A. marginale DNA was detected in nine blood DNA samples. Besides, 56 ticks feeding on cattle were collected from this farm and were all identified as Rhipisephalus microplus, meanwhile, 10 of them were found to be positive for A. marginale. In addition, phylogenetic analysis of the msp4 gene sequences of A. marginale obtained in this study showed that the isolate from cattle (KX840009) fell in the same clade with that of R. microplus (KX904527), sharing 100% similarity. To the best of our knowledge, this is the first confirmed report of outbreak of theileriosis/anaplasmosis in cattle farms in Henan, China.

Prevalence, molecular epidemiology, and zoonotic potential of Entamoeba spp. in nonhuman primates in China.

Amebiasis is a major public-health concern. It has a global distribution, and is listed as the third leading parasitic cause of human mortality. To survey the prevalence and assess the potential zoonotic transmission of Entamoeba spp. in nonhuman primates (NHPs) in China, 2688 fresh fecal specimens were collected from NHPs reared in farms or zoos/parks or free ranging in 13 districts. The overall prevalence of Entamoeba spp. infection determined with microscopy analysis was 39.4% (1059/2688). Higher infection rates were detected in the free ranging group (41.1%, 169/411) and in animals <1year old (58.7%, 556/947). Gene fragments were successfully amplified 463 (87.2%) out of the 531 selected specimens (approximately half of the total microscopy-positive specimens). Polymerase chain reaction (PCR) amplification identified 386 (83.4%) Entamoeba dispar and 287 (62.0%) E. coli infections, and among these, 210 (45.4%) were mixed infections. And that the E. dispar and E. coli had also been detected in humans. In phylogenetic analysis, the E. dispar and E. coli sequences clustered with reference E. dispar and E. coli, respectively. In conclusion, nonhuman primates infected with Entamoeba species, with high prevalence and zoonotic potential, should be considered when evaluating the maintenance of Entamoeba spp. and its transmission between animal reservoirs and humans.