Transcriptomic, 16S ribosomal ribonucleic acid and network pharmacology analyses shed light on the anticoccidial mechanism of green tea polyphenols against Eimeria tenella infection in Wuliangshan black-boned chickens.

BACKGROUND: Eimeria tenella is an obligate intracellular parasitic protozoan that invades the chicken cecum and causes coccidiosis, which induces acute lesions and weight loss. Elucidating the anticoccidial mechanism of action of green tea polyphenols could aid the development of anticoccidial drugs and resolve the problem of drug resistance in E. tenella. METHODS: We constructed a model of E. tenella infection in Wuliangshan black-boned chickens, an indigenous breed of Yunnan Province, China, to study the efficacy of green tea polyphenols against the infection. Alterations in gene expression and in the microbial flora in the cecum were analyzed by ribonucleic acid (RNA) sequencing and 16S ribosomal RNA (rRNA) sequencing. Quantitative real-time polymerase chain reaction was used to verify the host gene expression data obtained by RNA sequencing. Network pharmacology and molecular docking were used to clarify the interactions between the component green tea polyphenols and the targeted proteins; potential anticoccidial herbs were also analyzed. RESULTS: Treatment with the green tea polyphenols led to a reduction in the lesion score and weight loss of the chickens induced by E. tenella infection. The expression of matrix metalloproteinase 7 (MMP7), MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2 was significantly altered in the E. tenella infection plus green tea polyphenol-treated group and in the E. tenella infection group compared with the control group; these genes were also predicted targets of tea polyphenols. Furthermore, the tea polyphenol (-)-epigallocatechin gallate acted on most of the targets, and the molecular docking analysis showed that it has good affinity with interferon induced with helicase C domain 1 protein. 16S ribosomal RNA sequencing showed that the green tea polyphenols had a regulatory effect on changes in the fecal microbiota induced by E. tenella infection. In total, 171 herbs were predicted to act on two or three targets in MMP7, MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2. CONCLUSIONS: Green tea polyphenols can directly or indirectly regulate host gene expression and alter the growth of microbiota. The results presented here shed light on the mechanism of action of green tea polyphenols against E. tenella infection in chickens, and have implications for the development of novel anticoccidial products.

Molecular Epidemiological Investigation of Cyclospora spp. in Holstein Cattle in Partial Areas of the Yunnan Province, China.

Cyclospora spp. is a food-borne intestinal protozoan, which is widely distributed in the world and poses the risk of zoonosis. In order to reveal the prevalence of Cyclospora spp. in Holstein cattle in partial areas of the Yunnan Province, 524 fresh fecal samples of Holstein cattle were collected from Dali, Kunming, Chuxiong, and Qujing in Yunnan Province. A nested PCR amplification of the small subunit (SSU) rRNA gene of Cyclospora spp. was carried out, and the products of the nested PCR were further analyzed by restriction fragment length polymorphism (RFLP) using Bsp E Ⅰ. The results of the present study showed that 13 samples were positive for Cyclospora spp., and the total infection rate of Cyclospora sp. was 2.48%. The infection of Cyclospora spp. was detected in Dali, Qujing, and Chuxiong. Chuxiong showed the highest infection rate (5.71%), and infection rate in Dali and Qujing was 2.19% and 3.16%, respectively. Interestingly, the infection of Cyclospora spp. was not detected in Kunming. The infection of Cyclospora spp. showed no significant differences among different regions (p > 0.05). Cyclospora sp. infection was detected in all ages and sexes, but the differences were not significant (p > 0.05). Sequencing and phylogenetic analysis showed that five Cyclospora spp. samples were closely related to the Cyclospora spp. of humans, and the others were closely related to the Cyclospora spp. of bovines. The results of the present study suggested that there was an infection of Cyclospora spp. in Holstein cattle in the Yunnan Province, and the Cyclospora spp. showed a risk of zoonosis. Thus, the prevention and control of Cyclospora spp. should be strengthened in the Yunnan Province, China. The results of this investigation provide data references for the further research of Cyclosporiasis in Holstein cattle in the Yunnan Province.

Phytochemical Analysis and Anti-Ascaris suum Activity of Different Zanthoxylum Species In Vitro and In Vivo.

Zanthoxylum plants (ZPs), including multiple Chinese prickly ash species, are dual-purpose functional foods favored by the general population around the world in foods, cosmetics, and traditional medicines and have antipruritic, insecticidal, and fungicidal bioactivities. For the first time, the anti-roundworm bioactivity of ZPs and the active ingredients were compared and investigated. Through nontarget metabolomics following targeted quantitative analysis, qinbunamides, sanshools, sanshooel, asarinin, and sesamin were found to be the main different components of Zanthoxylum species. Coincidentally, the 12 chemical components were also the dominant anti-roundworm ingredients of ZP extracts. The extracts of three species of Chinese prickly ash (1 mg/mL) decreased the hatchability of roundworm eggs significantly, and the ChuanJiao seed killed roundworms (insecticidal rate 100%) and alleviated the symptoms of pneumonia in mice. Furthermore, retention time-accurate mass-tandem mass spectrometry-ion ratio (RT-AM-MS/MS-IR) were modeled by assaying 108 authentic compounds of ZP extracts, and 20 metabolites were confidently identified in biological samples from ZP extract-treated mice by analyzing the m/z values and the empirical substructures. This study provides a good reference for the proper application of ZPs.

Seroprevalence and risk factors of Toxoplasma gondii and Neospora caninum infection in black goats in Yunnan Province, Southwestern China.

Toxoplasma gondii and Neospora caninum are two obligate intracellular protozoan parasites that can cause reproductive failure and production losses. To date, there is no data of T. gondii and N. caninum seroprevalence in black goats in Yunnan Province, southwestern China. In the present study, a total of 734 serum samples were collected from black goats in four different counties of Yunnan Province. 734 and 590 serum samples were examined for antibodies against T. gondii and N. caninum by using MAT and indirect ELISA, respectively. A total of 123 and 76 samples were T. gondii-positive and N. caninum-positive, respectively. The overall seroprevalence of T. gondii in black goats was 16.76% (123/734, 95% CI: 14.06-19.46) with the titer ranged from 1:25 to 1:3200. The seroprevalence of N. caninum was 12.88% (76/590, 95% CI: 10.18-15.58). There was significant difference in seroprevalence of N. caninum in different regions (P < 0.01, χ2 = 30.63) and age groups (P < 0.05, χ2 = 11.85). Significant differences in seroprevalence of T. gondii were observed in different regions (P < 0.05, χ2 = 9.21) and different gender groups (P < 0.01, χ2 = 12.29). Results of seroprevalence of T. gondii and N. caninum indicated that T. gondii and N. caninum were prevalent parasites in black goats in Yunnan Province. This is the first report of seroprevalence of T. gondii and N. caninum in black goats in Yunnan Province. The results of this study indicated that some measures should be taken to control these two parasites and to reduce economic losses to the livestock industry in Yunnan Province.

First report of haemosporidia and associated risk factors in red junglefowl (Gallus gallus) in China.

BACKGROUND: Avian haemosporidia infect both domestic and wild birds, causing anemia, acute tissue degeneration, and depopulation in wild birds. Poultry and wild birds have been reported as common reservoirs of haemosporidia, but limited information is available for red junglefowl (Gallus gallus) in China. The present study investigated the prevalence and molecular characterization of haemosporidia in red junglefowl. METHODS: Blood samples were collected from 234 red junglefowl from Jinghong City of Yunnan Province, and genomic DNA was extracted from these samples. The prevalence of haemosporidia was determined by nested PCR targeting the mitochondrial cytochrome b (cytb) gene. Molecular characterization was investigated based on phylogenetic analysis of cytb sequences, and associated risk factors were analyzed using the Chi-square (χ2) test. RESULTS: The overall prevalence of haemosporidia was 74.8% (175/234), and three species were identified, namely Haemoproteus enucleator, Leucocytozoon californicus, and Plasmodium juxtanucleare. The prevalence of haemosporidia in adult fowl (81.1%, 107/132) was significantly higher (χ2 = 6.32, df = 1, P = 0.012) than that in juveniles (66.7%, 68/102). Three novel haemosporidian lineages were revealed. CONCLUSIONS: This study examined the prevalence and identified species of avian haemosporidians in red junglefowl, providing new information on the molecular epidemiology and geographical distribution of haemosporidian parasites. Our results indicated high prevalence and diverse species distribution of these haemosporidians in red junglefowl. To the best of our knowledge, this is the first record of haemosporidian infection in red junglefowl in China.

Occurrence and Molecular Characterization of Cryptosporidium spp. in Dairy Cattle and Dairy Buffalo in Yunnan Province, Southwest China.

Cryptosporidium spp. are important foodborne and waterborne pathogens in humans and animals, causing diarrheal diseases. Cattle are one of the reservoirs of Cryptosporidium infection in humans. However, data on the occurrence of Cryptosporidium spp. in cattle in Yunnan Province remains limited. A total of 700 fecal samples were collected from Holstein cows (n = 442) and dairy buffaloes (n = 258) in six counties of Yunnan Province. The occurrence and genotypes of Cryptosporidium spp. were analyzed using nested PCR and DNA sequencing. Furthermore, the C. andersoni isolates were further analyzed using multilocus sequence typing (MLST) at four gene loci (MS1, MS2, MS3, and MS16), and the C. parvum isolate was subtyped by 60-kDa glycoprotein (gp60) loci. The occurrence of Cryptosporidium spp. in Holstein cows and dairy buffaloes was 14.7% (65/442) and 1.1% (3/258), respectively. Of these positive samples, 56 Holstein cow samples represented C. andersoni, four Holstein cow samples represented C. bovis, three Holstein cow samples represented C. ryanae, and one represented C. parvum. Meanwhile, only three dairy buffalo samples represented C. ryanae. MLST analysis of subtypes of C. andersoni detected four subtypes, including A5A4A2A1 (n = 7), A4A4A4A1 (n = 7), A1A4A4A1 (n = 2), and A4A4A2A1 (n = 1). One C. parvum isolate was identified as the IIdA18G1 subtype. These results revealed the high occurrence and high genetic diversity of Cryptosporidium spp. in Holstein cows in Yunnan Province, enriching the knowledge of the population genetic structure of Cryptosporidium spp. in Yunnan Province.

Temporal transcriptomic changes in long non-coding RNAs and messenger RNAs involved in the host immune and metabolic response during Toxoplasma gondii lytic cycle.

BACKGROUND: Long non-coding RNAs (lncRNAs) are important regulators of various biological and pathological processes, in particular the inflammatory response by modulating the transcriptional control of inflammatory genes. However, the role of lncRNAs in regulating the immune and inflammatory responses during infection with the protozoan parasite Toxoplasma gondii remains largely unknown. METHODS: We performed a longitudinal RNA sequencing analysis of human foreskin fibroblast (HFF) cells infected by T. gondii to identify differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs), and dysregulated pathways over the course of T. gondii lytic cycle. The transcriptome data were validated by qRT-PCR. RESULTS: RNA sequencing revealed significant transcriptional changes in the infected HFFs. A total of 697, 1234, 1499, 873, 1466, 561, 676 and 716 differentially expressed lncRNAs (DElncRNAs), and 636, 1266, 1843, 2303, 3022, 1757, 3088 and 2531 differentially expressed mRNAs (DEmRNAs) were identified at 1.5, 3, 6, 9, 12, 24, 36 and 48 h post-infection, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DElncRNAs and DEmRNAs revealed that T. gondii infection altered the expression of genes involved in the regulation of host immune response (e.g., cytokine-cytokine receptor interaction), receptor signaling (e.g., NOD-like receptor signaling pathway), disease (e.g., Alzheimer's disease), and metabolism (e.g., fatty acid degradation). CONCLUSIONS: These results provide novel information for further research on the role of lncRNAs in immune regulation of T. gondii infection.

Molecular Detection and Genotyping of Enterocytozoon bieneusi in Black Goats (Capra hircus) in Yunnan Province, Southwestern China.

Enterocytozoon bieneusi is a fungus-like protist that can parasitize in the intestines of humans and various animals causing a threat to public health. However, there has been no data for E. bieneusi prevalence and genotypes in black goats in Yunnan Province, Southwestern China. In this study, 907 fecal samples were collected from black goats in 5 counties from Yunnan Province. The prevalence and genotypes of E. bieneusi were examined by nested PCR amplification targeting the nuclear internal transcribed spacer (ITS). Multi-locus sequence typing (MLST) was used to further examine the potential occurrence of genetic segregation. The overall prevalence of E. bieneusi in black goats in Yunnan Province was 10.3% (93/907). Statistical analysis revealed that E. bieneusi prevalence was significantly associated with the region, age and gender of black goats (p < 0.001). Four new genotypes (CYG-1, CYG-2, CYG-3, CYG-4) and 11 known genotypes (CHG1, CHG2, CHG3, CHG5, CHG28, J, D, BEB6, Wildboar3, CD6, SDD1) of E. bieneusi were identified. At the microsatellite and minisatellite loci, 15, 2, 17, and 33 sequences were obtained, respectively, forming one new multi-locus genotype (MLG27). Phylogenetic analysis revealed that all 15 genotypes were clustered into group 1 and group 2, with zoonotic potential. This is the first report of E. bieneusi prevalence and genotypes in black goats in Yunnan Province, China. Effective control strategies and measures should be taken to reduce the risk of E. bieneusi transmission between black goats, other animals, and humans.

Prevalence and Novel Genotypes Identification of Enterocytozoon bieneusi in Dairy Cattle in Yunnan Province, China.

Enterocytozoon bieneusi is a fungus-like protist parasite that can cause diarrhea and enteric diseases. The infection of E. bieneusi has been reported in many host species, including cattle and humans. However, information on prevalence and genotype distribution of E. bieneusi in dairy cattle in Yunnan province in China is still absent. In this study, 490 Holstein Cows and 351 dairy buffalo fecal samples were collected from three regions in Yunnan province, China. By using nest-PCR that targets the internal transcribed spacer (ITS), we found that the prevalence of E. bieneusi was 0.59% (5/841). DNA sequence analysis showed that five E. bieneusi genotypes were identified in this study, including two novel genotypes, YNDCEB-90 and YNDCEB-174, and three known genotypes (I, J, BEB4). Phylogenetic analysis revealed that two novel genotypes, YNDCEB-90 and YNDCEB-174, were clustered into Group 1, representing the zoonotic potential. The remaining genotypes I, J, and BEB4, which are the most frequent genotypes of E. bieneusi infection in cattle and lead to E. bieneusi infection in humans, belonged to Group 2. Although the lower prevalence of E. bieneusi was detected in dairy cattle in Yunnan province, it indicates that dairy cattle should be considered to be one of the potential hosts for transmitting E. bieneusi to humans. These findings are important for the development of effective prevention strategies for microsporidiosis.

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.

Transcriptional profiling of buffalo mammary gland with different milk fat contents.

Milk fat is the most important energy substance in milk and contributes to its quality and health benefits. Water buffalo milk is well known for its high milk quality with higher fat contents compared with cattle milk. Dehong buffalo is a unique local swamp breed in Yunnan Province with higher milk fat and excellent milk quality which provides a good model for the investigation of the molecular mechanisms of milk fat deposition. In this study, we used RNA-seq to obtain mammary tissue transcriptomics of buffalo with different milk fat phenotypes including high(H), medium (M)and low (L) fat content groups. Comparative analyses of buffalo among three groups yielded differentially expressed genes (DEGs). Analyzing the number of different genes among H_VS_L, H_VS_M, and M_VS_L showed the same expression pattern between H_VS_M. The increasing expression levels of genes including CSN1S1, BTN1A1, LALBA, ALDH1L2, SCD and MUC15, and down-regulated expression levels of genes containing CCL2, CRABP2, ADTRP, CLU and C4A in H_VS_L and M_VS_L were found. GO and KEGG enriched pathways revealed these DEGs involved in milk protein and fat as well as immune response. The findings would enhance the understanding of the interplay between the milk composition and immune response, which suggests that the immune capacity should be considered when we tried to improve the milk quality.

First report of the prevalence and genetic characterization of Giardia duodenalis and Cryptosporidium spp. in Yunling cattle in Yunnan Province, southwestern China.

Yunling cattle is an unique cattle breed distributed in Yunnan Province, southwestern China. It is yet to know whether Yunling cattle are infected with Giardia duodenalis and Cryptosporidium spp.. The objectives of the present study were to investigate the prevalence and characterize the assemblages of G. duodenalis and species of Cryptosporidium spp. in Yunling cattle in Yunnan province. The overall prevalence of G. duodenalis and Cryptosporidium spp. were 10.49% (41/391) and 0.77% (3/391), respectively. The age was considered as the risk factor for Yunling cattle infection with G. duodenalis (χ2 = 8.082, OR = 2.56, P = 0.004). Two assemblages of G. duodenalis, assemblage A (n = 1) and assemblage E (n = 40), were identified by amplification of the ß-giardin (bg) and glutamate dehydrogenase (gdh) gene loci using the nested PCR methods. Furthermore, Cryptosporidium andersoni (n = 1) and Cryptosporidium ryanae (n = 2) were detected by nested PCR targeting the small subunit (SSU) rRNA gene. This is the first report of G. duodenalis and Cryptosporidium spp. in Yunling cattle in China, which provided baseline date for further studies of the prevalence, genetic identity, and public health potential of these parasites in Yunling cattle.

Molecular detection and subtype distribution of Blastocystis in farmed pigs in southern China.

Blastocystis is one of the most common causative agents of intestinal diseases, which can cause enteric diseases in animals and humans. However, limited data is available on the prevalence or subtypes of Blastocystis infections in farmed pigs in southern China. In this study, a total of 396 fecal samples were collected from farmed pigs in three provinces in southern China in 2016, and screened for Blastocystis by PCR amplification of the small subunit rRNA (SSU rRNA) gene fragment. One hundred and seventy (42.93%) of the examined fecal samples were detected Blastocystis-positive, and two known zoonotic subtypes ST1 and ST5 were identified, with ST5 being the predominate subtype. Moreover, gender, age and region were considered as risk factors that associated with Blastocystis infection in farmed pigs. The present study revealed the prevalence and subtypes of Blastocystis infections in farmed pigs in southern China, which provided essential data for the control of Blastocystis infections in pigs, other animals and humans in China.

First Report of Chlamydia Seroprevalence and Risk Factors in Domestic Black-Boned Sheep and Goats in China.

The Gram-negative bacteria of the genus Chlamydia cause a wide range of diseases in humans and animals. The seroprevalence of Chlamydia in domestic black-boned sheep and goats in China is unknown. In this survey, a total of 481 serum samples were collected randomly from domestic black-boned sheep and goats from three counties in Yunnan province, southwest China, from July to August 2017. The sera were examined by an indirect hemagglutination assay (IHA). Antibodies to Chlamydia were detected in 100/481 [20.79%, 95% confidence interval (CI), 17.16-24.42] serum samples (IHA titer ≥1:64). The Chlamydia seroprevalence ranged from 12.21% (95% CI, 7.81-16.61) to 30.89% (95% CI, 22.72-39.06) across different regions in Yunnan province, and the differences were statistically significant (P < 0.01). The seroprevalence in male domestic black-boned sheep and goats (28.64%; 95% CI, 22.36-34.92) was significantly higher than that in the females (15.25%; 95% CI, 11.05-19.45) (P < 0.01). However, there was no statistically significant difference in Chlamydia seroprevalence in domestic black-boned sheep and goats between ages and species (P > 0.05). To our knowledge, this is the first report of Chlamydia seroprevalence in domestic black-boned sheep and goats in Yunnan Province, southwest China. These data provide baseline information for future implementation of measures to control Chlamydia infection in these animals.

Prevalence, molecular epidemiology and zoonotic risk of Entamoeba spp. from experimental macaques in Yunnan Province, southwestern China.

Amebiasis is a worldwide parasitic zoonosis, with symptoms of abdominal discomfort, indigestion, diarrhea, and even death. However, limited information about the prevalence of Entamoeba spp. in experimental nonhuman primates (NHPs) in southwestern China is available. The objective of the current study was to investigate the frequency and species identity of Entamoeba to evaluate potential zoonotic risk factors for Entamoeba spp. infection in experimental NHPs. A total of 505 fecal samples were collected from NHPs (macaques) and analyzed by PCR analysis the small subunit rRNA (SSU rRNA) gene of Entamoeba spp. Forty-seven specimens were positive for Entamoeba spp., and the prevalence of Entamoeba spp. was 9.31% (47/505). Significant differences in the prevalence rates among the three breeds (P = 0.002 < 0.01, df = 2, χ2 = 12.33) and feed types (P = 0.001 < 0.01, df = 1, χ2 = 10.12) were observed. Altogether, four Entamoeba species, including E. dispar (57.44%), E. chattoni (29.78%), E. histolytica (6.38%), and E. coli (6.38%), were identified by DNA sequence analysis. The results suggested a low prevalence but high diversity of Entamoeba species in experimental NHPs in Yunnan Province, southwestern China. Results of this study contribute to the knowledge of the genetic characteristics of Entamoeba spp. in NHPs.

First report of Cryptosporidium spp. infection and risk factors in black-boned goats and black-boned sheep in China.

Cryptosporidium is an opportunistic protozoan parasite that can inhabit in the gastrointestinal tract of various hosts. Cryptosporidium infection in black-boned goats and black-boned sheep may pose a threat to the survival and productivity, causing considerable economic losses to the livestock industry. However, it is yet to know whether black-boned goats and black-boned sheep in China are infected with Cryptosporidium. Thus, the objective of the present study was to investigate the prevalence and associated risk factors of Cryptosporidium infection in black-boned goats and black-boned sheep in Yunnan province, China. A total of 590 fecal samples were obtained from black-boned goats and black-boned sheep from five counties in Yunnan province, and the prevalence and species distribution of Cryptosporidium were determined by amplification of the 18S rDNA fragment using the nested PCR. The overall Cryptosporidium prevalence was 13.2% (78/590), with 18.0% (55/305) in black-boned goats and 8.1% (23/285) in black-boned sheep. The age and sampling site were identified as main factors that result in significant differences in Cryptosporidium prevalence. Three species, namely C. muris, C. xiaoi, and C. ubiquitum, were identified in black-boned goats and black-boned sheep in the present study, with C. muris (46/78) as the predominant species. This is the first report of Cryptosporidium infection in black-boned goats and black-boned sheep in China, and the findings will facilitate better understanding, prevention, and control of Cryptosporidium infection in black-boned goats and black-boned sheep in China.

Prevalence and Genotyping of Toxoplasma gondii in Cats, Rats, and Chickens in Border Areas of Yunnan Province, China.

The objective of the present study was to determine the characterization of Toxoplasma gondii in cats, rats, and chickens in the border areas of Yunnan Province. A total of 259 samples was collected from 10 border areas in Yunnan Province including 94 cats, 58 rats, and 107 chickens. Samples were screened by a nested polymerase chain reaction (PCR) assay and the positive products were analyzed by multilocus PCR-restriction fragment length polymorphism (RFLP) to determine the genotypes. Toxoplasma gondii deoxyribonucleic acid (DNA) was detected from 15.96% of 94 cats, 15.52% of 58 rats, and 6.54% of 107 chickens, respectively, and the average infection rate is 11.97%. Using the multilocus PCR-RFLP, we found that the genotype of T. gondii in cats and rats was ToxoDB#9. Because of low DNA concentration, no genotype was determined from chickens. These results fill the gaps of knowledge in the prevalence and genotype of T. gondii in the border areas of Yunnan Province and have implications for the better control of T. gondii infection in humans and animals.

Assessment of the subtypes and the zoonotic risk of Blastocystis sp. of experimental macaques in Yunnan province, southwestern China.

Blastocystis is an enteral eukaryote with an omnipresent existence in animals and humans globally. Animals have been proposed to be a major reservoir for the transmission of Blastocystis to individuals due to their high prevalence and large amount of zoonotic subtypes. However, limited data on Blastocystis infection in experimental macaques in China exists. The objective of the current study was to investigate the frequency and subtypes of Blastocystis infection in macaques in southwestern China. A total of 505 fecal samples were collected from experimental macaques in Yunnan province and were analyzed by nested PCR and phylogenetic analyses on the basis of small subunit rRNA (SSU rRNA) gene fragments. A total of 235 specimens were positive for Blastocystis sp., and the prevalence of Blastocystis sp. was 46.5% (235/505). Significant differences in prevalence were also observed among the various species of macaques (P < 0.0133, df = 2, χ2 = 8.64) and the different feed types (P < 0.0093, df = 1, χ2 = 6.77). Moreover, three zoonotic subtypes, ST1, ST3, and ST5, were identified by DNA sequence analysis. There were mainly single subtype infections with some mixed subtype infections, and the predominant subtype was ST3. The results suggested a high prevalence and diversified subtypes in macaques in Yunnan province, southwestern China. Macaques are likely to be potential reservoirs capable of zoonotic transmission of Blastocystis sp. to humans. To our knowledge, this study is the first large-scale systematic analysis of Blastocystis sp. colonization in Yunnan province in the subtropics of China; these results contribute to the in-depth study of genetic characteristics and the prevention, control, and treatment of Blastocystis sp. in macaques in Yunnan province and other regions.

Prevalence, risk factors and genotype distribution of Toxoplasma gondii DNA in soil in China.

In the present study, we performed a cross-sectional survey to determine the occurrence and genotype distribution of T. gondii DNA in soil samples collected from different sources from six geographic regions in China. Between March 2015 and June 2017, 2100 soil samples were collected from schools, parks, farms and coastal beaches, and examined for T. gondii DNA using three PCR assays targeting 529-bp repeat element (RE) sequence, B1 gene and ITS-1 gene sequences. Also, we investigated whether geographic region, soil source and type, and sampling season can influence the prevalence of T. gondii DNA in the soil. Soil samples collected from farms and parks had the highest prevalence, whereas samples collected from school playgrounds and coastal beaches had the lowest prevalence. PCR assays targeting 529-bp RE and ITS-1 gene sequences were more sensitive than the B1 gene-based assay. Positive PCR products were genotyped using multi-locus PCR-RFLP, and ToxoDB #9 was the predominant genotype found in the contaminated soil samples. Multiple logistic regression identified factors correlated significantly with the presence of T. gondii DNA in the soil to be the source of the soil, including farms (odds ratio 3.10; 95% confidence interval [CI], 1.52 to 6.29; p = 0.002) and parks (2.59; 95% CI 1.28 to 5.27; p = 0.009). These results show that Chinese soil hosts T. gondii of the most prevalent genotype in China (ToxoDB#9) and that the soil type influences infection patterns.

Serological evidence of Toxoplasma gondii and Neospora caninum infection in black-boned sheep and goats in southwest China.

Toxoplasma gondii and Neospora caninum are two closely related protozoan parasites which can cause abortion and significant economic losses in sheep and goats. However, it is yet to know whether black-bone sheep and goats are infected with T. gondii and N. caninum in China. In the present investigation, the seroprevalence and risk factors of T. gondii and N. caninum infections in black-boned sheep and goats were investigated in Yunnan Province, subtropical southwest China between July and August of 2017. A total of 481 serum samples were tested for T. gondii antibodies using the Modified Agglutination Test (MAT), and 468 serum samples were examined for N. caninum antibodies by indirect Enzyme-Linked Immunosorbent Assay (iELISA). The overall seroprevalence of T. gondii in black-boned sheep and goats was 36.80% (177/481, 95% CI 32.49-41.11), and 40 out of 468 serum samples were N. caninum-seropositive (8.55%, 95% CI 6.02-11.08). There was significant difference in the seroprevalence of T. gondii infection in different regions (χ2 = 19.869, df = 2, P<0.01). As for the seroprevalence of N. caninum infection, region (χ2 = 8.558, df = 2, P<0.05), age (χ2 = 16.631, df = 3, P < 0.01), gender (χ2 = 11.219, df = 1, P < 0.01) and species (χ2 = 8.673, df = 1, P < 0.01) were the risk factors. In addition, the seroprevalence of coinfection of T. gondii and N. caninum in black-boned sheep and goats was 3.63% (17/468, 95% CI 1.94-5.32). To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in black-boned sheep and goats in China, which provided base-line data for the execution of control strategies and measures against T. gondii and N. caninum infection in black-boned sheep and goats.