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.

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.

Characterization of Fasciola samples by ITS of rDNA sequences revealed the existence of Fasciola hepatica and Fasciola gigantica in Yunnan Province, China.

On mainland China, liver flukes of Fasciola spp. (Digenea: Fasciolidae) can cause serious acute and chronic morbidity in numerous species of mammals such as sheep, goats, cattle, and humans. The objective of the present study was to examine the taxonomic identity of Fasciola species in Yunnan province by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from 10 samples representing Fasciola species in cattle from 2 geographical locations in Yunnan Province, by polymerase chain reaction (PCR), and the products were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361-362 base pairs, respectively, for all samples sequenced. Using ITS sequences, 2 Fasciola species were revealed, namely Fasciola hepatica and Fasciola gigantica. This is the first demonstration of F. gigantica in cattle in Yunnan Province, China using a molecular approach; our findings have implications for studying the population genetic characterization of the Chinese Fasciola species and for the prevention and control of Fasciola spp. in this province.