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The apicomplexan Eimeria ovinoidalis is distributed worldwide. It can cause clinical coccidiosis, which is one of the most pathogenic species in sheep, reducing growth rates and resulting in significant economic losses in the industry. Its principal clinical sign is profuse diarrhoea in young animals. In this study, we established a model of E. ovinoidalis infection in lambs to understand its pathogenicity and evaluate the gut microbiota and fecal metabolite profiles. Specifically, we observed a significant shift in the abundance of bacteria and disrupted metabolism in lambs. Especially during the peak period of excrete oocysts, it promoted the reproduction of some harmful bacteria in Proteobacteria and Actinobacteriota, and reduced the abundance of beneficial bacteria such as Lachnospiraceae and Rikenellaceae. In the later stage of the patent period, the abundance of harmful bacteria in the intestine decreased, the abundance of beneficial bacteria which could produce anti-inflammatory substances began to increase, and the abundance and diversity of intestinal flora also tended to parallel with the control group. Coccidia infection could lead to the increase of differential metabolites and metabolic pathways between infected and control group, but the difference decreased with time. During the peak period of excrete oocysts, although the antimicrobial metabolites such as Lividamine were up-regulated, the excess of these metabolites could still induce the production of endotoxin, while Butanoic acid and other anti-inflammatory metabolites decreased significantly. A metabolomics analysis showed that E. ovinoidalis infection altered metabolites and metabolic pathways, with biosynthesis of unsaturated fatty acids, Teichoic acid biosynthesis and Butanoate metabolism as the major disrupted metabolic pathways. Details of the gut microbiota and the metabolome after infection with E. ovinoidalis may aid in the discovery of specific diagnostic markers and help us understand the changes in parasite metabolic pathways., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cryptosporidium parvum is an important zoonotic pathogen that is studied worldwide. MicroRNAs (miRNAs) act as post-transcriptional regulators and may play a key role in modulating host epithelial responses following Cryptosporidium infection. Our previous study has shown that C. parvum downregulates the expression of miR-181d through the p50-dependent TLRs/NF-κB pathway. However, the mechanism by which miR-181d regulates host cells in response to C. parvum infection remains unclear. The present study found that miR-181d downregulation inhibited cell apoptosis and increased parasite burden in HCT-8 cells after C. parvum infection. Bioinformatics analysis and luciferase reporter assays have shown that BCL2 was a target gene for miR-181d. Moreover, BCL2 overexpression and miR-181d downregulation had similar results. To further investigate the mechanism by which miR-181d regulated HCT-8 cell apoptosis during C. parvum infection, the expression of molecules involved in the intrinsic apoptosis pathway was detected. Bax, caspase-9, and caspase-3 expression was decreased at 4, 8, 12, and 24 hpi and upregulated at 36 and 48 hpi. Interfering with the expression of miR-181d or BCL2 significantly affected the expression of molecules in the intrinsic apoptosis pathway. These data indicated that miR-181d targeted BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to C. parvum infection via the intrinsic apoptosis pathway. These results allowed us to further understand the regulatory mechanisms of host miRNAs during Cryptosporidium infection, and provided a theoretical foundation for the design and development of anti-cryptosporidiosis drugs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The genus Anaplasma comprises eight bacterial species that are obligate intracellular pathogens that affect human and animal health. The zoonotic species A. phagocytophilum is the causative agent of tick-borne fever in ruminants, and of granulocytic anaplasmosis in horses, dogs, and humans. Recently, novel strains related to A. phagocytophilum (A. phagocytophilum-like 1/Japanese variant and A. phagocytophilum-like 2/Chinese variant) have been identified. The aim of this study was to reveal the prevalence and phylogeny of A. phagocytophilum and related stains in small ruminants and ticks in China based on sequences of the 16S rRNA combined restriction fragment length polymorphism (RFLP) and groEL genes. PCR-RFLP and phylogenetic analyses based on the 16S rRNA gene showed the presence of A. phagocytophilum-like 1 and 2 variants in sampled animals from China, with prevalence rates of 22.6% (303/1338) and 0.7% (10/1338), respectively. Only A. phagocytophilum-like 1 DNA was found in Haemaphysalis longicornis. The phylogeny based on the groEL gene showed inclusion of A. phagocytophilum-like 1 and some A. phagocytophilum-like 2 strains in two unique clades distinct from, but related to, Japanese and Chinese strains of related A. phagocytophilum, respectively. One noteworthy result was that the SSAP2f/SSAP2r primers detected Ehrlichia spp. strains. Moreover, the A. phagocytophilum-like 1 and 2 strains should be considered in the differential diagnosis of caprine and ovine anaplasmosis. Further investigations should be conducted to provide additional epidemiological information about A. phagocytophilum and A. phagocytophilum-like variants in animals and ticks., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cryptosporidium spp. can cause diarrhea and even death in humans and animals. Host microRNAs (miRNAs) play an important role in the post-transcriptional regulation of the innate immune response to Cryptosporidium infection. To study host miRNA activity in the innate immune response to C. parvum infection, we examined the expression of miR-181d in HCT-8 cells infected with C. parvum and found that it was significantly downregulated, while TLR2, TLR4, NF-κB, and myD88 involved in the TLR/NF-κB signaling pathway were significantly upregulated at the early stages of C. parvum infection. We transfected cells with short-interfering RNAs (siRNA) as TLR2, TLR4, and NF-κB inhibitors. Analysis by quantitative real-time polymerase chain reaction (qPCR) and western blot confirmed that C. parvum downregulates miR-181d expression via the p50 subunit-dependent TLR2/TLR4-NF-κB signaling pathway in HCT-8 cells. This study provides a new theoretical foundation to elucidate the regulatory mechanism of host miRNAs against Cryptosporidium infection., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Anaplasma are tick-borne obligate intracellular bacteria that can endanger human and animal health, and until now, there have been few reports on the seasonal dynamics of Anaplasma species in China. In this study, a total of 491 goat blood samples were collected in spring (n = 124), summer (n = 135), autumn (n = 110), and winter (n = 122) from Shaanxi provinces. Single and mixed infections of Anaplasma spp. from warm-temperate regions of China were analyzed according to seasons using a nested PCR method. Positive samples were sequenced to observe the molecular and phylogenetic characteristics of the Anaplasma species, and we determined the co-infection rates of Anaplasma spp. for each season. A molecular survey of Anaplasma phagocytophilum, A. bovis, A. ovis, and A. capra in goats showed average prevalences of 71.6 % (maximum 86.7 % in summer and minimum 48.4 % in winter), 62.2 % (minimum 38.7 % in spring and maximum 94.1 % in summer), 25.5 % (minimum 0% in summer and maximum 51.6 % in spring), and 26.6 % (minimum 8.2 % in winter and maximum 55.6 % in summer), respectively. In the phylogenetic analysis, A. phagocytophilum and A. capra occupied two separate groups, Chinese A. bovis and foreign isolates appeared to be geographically isolated, and all A. ovis isolates were in the same branch as the previously described sequences. The survey indicated that goats in warm-temperate regions of China are frequently exposed to Anaplasma spp. all year round, and thus prevention and treatment efforts for anaplasmosis in the region should be strengthened., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Background: Toxoplasma gondii is widely distributed and can infect many species of warm-blooded animals, including swine. This study aimed to evaluate the prevalence of T. gondii in swines from the central of China. A total of 2798 samples, including 305 hearts, 2086 diaphragms, and 407 sera were collected from different swine in Henan Province, China. The modified agglutination test was used to detect antibodies against T. gondii in sera from jugular vein blood and heart blood (cut-off: 1:25), diaphragm juice (cut-off: 1:10). T. gondii DNA was screened from the digestive fluids of all diaphragm tissue samples and seropositive hearts, and attempt to isolate viable T. gondii strain by bioassay in mice., Results: A total of 9.94% (278/2798) swine tested positive for T. gondii antibodies. Region, but not gender, was associated with T. gondi seropositivity in swine. T. gondii nucleic acid was not found in the tissue digestive fluids (2090 swines). Three groups of mice showed T. gondii antibodies after having been bioassayed with diaphragm samples (n = 81, which came from 2090 swine). No viable T. gondii strain was isolated from muscle of swine., Conclusions: This is the first large-scale survey T. gondi infection in swine from the central of China. Overall, the prevalence of viable T. gondii in swine was low. Nevertheless, T. gondii infection is present in swine from the central of China. Consumers may acquire T. gondii infection from ingestion of raw or undercooked pork., Competing Interests: Declaration of Competing Interest The authors declare no competing interests. None of the authors of this report have financial or personal relationships with other people or organizations that could inappropriately influence its content., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Human giardiasis is a common waterborne/foodborne parasitic disease worldwide, especially in developing countries. Prevalence and molecular identity of Giardia parasites are largely controversial. The present study was conducted to determine the occurrence of Giardia parasites and the genetic profile of circulating assemblage(s) in patients attended the outpatient clinic at Kafrelsheikh University hospital, Kafr El Sheikh Province, Egypt. A total of 318 patients, of different age and sex, referred to the clinic were subjected to fecal examination. Microscopic results revealed that 181/318 (56.9%) were positive for Giardia parasites. Multilocus genotyping by PCR/sequencing of beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh) genes of representative number of positive samples (65) revealed that assemblages A, B and mixed infections (A + B) occurred in 26/65 (40.0%), 32/65 (49.2%) and 10.8% (7/65) of the analyzed isolates, respectively. MLGs analysis indicated that assemblage A sequences clustered in two novel types of AII sub-assemblage. In assemblage B sequences, BIII was the predominant (22/23, 95.7%) sub-assemblage compared to BIV (1/23, 4.3%). Collectively, assemblage B MLGs displayed greater levels of genetic diversity compared to assemblage A. Our data indicate that assemblages A and B of G. duodenalis circulate in humans at Kafr El Sheikh Province, Egypt, and that high genetic diversity exists at the assemblage and/or sub-assemblage levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62°C for 60min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5×10 0 copies/μL, 100 times more than that of conventional PCR (5×10 2 copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017. Published by Elsevier B.V.)

A total of 1040 fecal samples, collected from 12 dairy cattle farms in Hebei and Tianjin, near the Bohai area of China, were screened for Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi by polymerase chain reaction. The overall prevalence for Cryptosporidium, G. duodenalis and E. bieneusi was 1.0% (n=10), 4.7% (n=49) and 19.4% (n=202), respectively. Ten Cryptosporidium-positive samples were identified as C. parvum by DNA sequence analysis of the small subunit rRNA (SSU rRNA) gene. DNA sequencing of the 60-kDa glycoprotein gene revealed that the C. parvum samples were all subtype IIdA19G1. Forty-nine G. duodenalis-positive samples belonged to assemblage E (n=47) and assemblage E mixed with A (n=2), based on the sequenced SSU rRNA, triosephosphate isomerase, and glutamate dehydrogenase genes. Sequence analysis of the internal transcribed spacer (ITS) gene identified six known E. bieneusi genotypes, I (n=87), J (n=83), BEB4 (n=18), BEB6 (n=3), N (n=1) and Ebpc (n=1), along with three new genotypes, CHC6 (n=1), CHC7 (n=1) and CHC8 (n=7). Phylogenetic analysis showed that Ebpc belonged to zoonotic group 1, whereas the other E. bieneusi genotypes clustered within group 2. More studies are needed to better understand the species distributions and public health significance of these pathogens in the study areas., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enterocytozoon bieneusi is the most common microsporidia species in humans and has a variety of animal hosts. To assess the prevalence and molecular characteristics of E. bieneusi in dairy calves in the Xinjiang Uyghur Autonomous Region of China, 514 fecal samples were collected from 15 farms and examined by polymerase chain reaction based on the internal transcribed spacer of the ribosomal RNA gene of E. bieneusi. The overall prevalence of E. bieneusi in calves was 16.5% (85/514). No significant difference in prevalence was observed between pre- and post-weaned calves. Sequence analysis of ITS nucleotide sequences identified six known genotypes (BEB4, CC4, D, I, J, and EbpC), five of these previously detected in humans. Genotype J was the most prevalent genotype (57/85) and was identified on 11 farms. The high prevalence of zoonotic E. bieneusi genotypes in dairy calves suggests they are a potential source of zoonotic infection in humans., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In recent years, tick-borne diseases like anaplasmosis have become widespread worldwide threatening the health of both human and animals. Dogs play an important role in the epidemiology of several zoonotic tick-borne pathogens by acting as reservoirs. In this study, the status of Anaplasma phagocytophilum, A. platys, A. bovis and A. ovis infection were assessed in dogs in Henan, China, with PCR and phylogenetic analyses. Nested PCRs on 243 blood samples collected from dogs from different sampling sites revealed that thirty-three (13.6%) dogs were positive for one or more pathogens. The prevalence of Anaplasma spp. in stray dogs was 40.7% (24/59), which was much higher than that of pet dogs (4.0%, 7/175). The prevalence for A. ovis, A. bovis and A. phagocytophilum was 6.2%, 4.1% and 0.4%, respectively and mixed-infection of these three pathogens was found in only one stray dog (prevalence, 0.4%). None of the dogs was positive for A. platys. Phylogenetic analyses classified A. phagocytophilum into two distinct groups (East Asia and south Africa group, Europe and America group), whereas A. ovis and A. bovis showed a general classification into two groups (cluster 1 and cluster 2), respectively. The isolate (KX190783) of A. ovis from a stray dog fell in a clade with a human isolate from Cyprus (FJ460443) and shared 99.8% similarity with it. To the best of our knowledge, this study is the first report to identify A. bovis and A. ovis DNA in dogs in China and the mixed-infection of the three Anaplasma spp. (A. phagocytophilum, A. bovis and A. ovis) in dogs., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Organisms of the genera Anaplasma and Theileria are important intracellular bacteria and parasites that cause various tick-borne diseases, threatening the health of numerous animals as well as human beings. In the present study, a 12-month-old male wild South African giraffe (Giraffa camelopardalis giraffa) originating from South Africa, and living in Zhengzhou Zoo (located in the urban district of Zhengzhou in the provincial capital of Henan), suddenly developed an unknown fatal disease and died 1day after the onset of the clinical signs. By microscopic examination of Giemsa-stained blood smears combined with nested PCR and DNA sequence analysis, Anaplasma phagocytophilum, Anaplasma bovis and a novel Theileria spp. were found in the blood of this giraffe. The six other Cervidae animals in the zoo and three ruminants living in the same colony house with them were found to be negative for both Anaplasma and Theileria in their blood specimens. We report on the first case of an A. phagocytophilum infection and the occurrence of a novel Theileria spp. in the blood of a giraffe. This is the first reported case of a multi-infection of A. bovis, A. phagocytophilum and Theileria spp. in a giraffe, as revealed by microscopic examination of blood smears and the results of nested PCR and DNA sequencing., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

To infer population genetics of Enterocytozoon bieneusi in nonhuman primates (NHPs), 126 positive specimens in 839 fecal specimens from 23 NHP species in China based on ITS locus were used, belonging to genotypes Type IV, D, Peru8, Henan V, Peru11, PigEBITS7 and 3 novel ones (CM1, CM2 and CM3). Multilocus sequence typing employing four micro and minisatellites (MS1, MS3, MS4 and MS7) and ITS were used to analyze population structure of 85 isolates successfully amplified at all five loci, which yielded 59 multilocus genotypes. Linkage disequilibrium (LD) was measured using both multilocus sequences and allelic profile data. The observation of strong and significant LD with limited recombination in multilocus sequence analysis indicated the presence of overall clonal population structure of E. bieneusi, which was supported by allelic profile data analysis. Fu's selective neutrality test demonstrated the absence of neutral mutations and molecular selection. The population structure of common ITS genotypes (CM1, Type IV and D) was compared. Strong LD in multilocus sequence analysis versus insignificant LD and/or LE in allelic profile data analysis implied epidemic population in common ITS genotypes. No significant genetic isolation was evidenced by either phylogenetic or substructural analyses. The population genetics was also compared among the sub-population 1 (contained mainly genotype Type IV), sub-population 2 (contained mainly genotypes CM1 and D), sub-population 3 (contained mixed genotypes) and sub-population 4 (contained genotype Henan V). The presence of strong LD in multilocus data analysis with insignificant LD and/or LE in allele profile data analysis suggested the epidemic population in sub-populations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In the present study, chick embryo tracheal organ (TOCs) was used to cultivate oocysts or sporozoites of Cryptosporidium baileyi. Approximately 5 × 10(4) sporozoites and oocysts mixture for group I; 5 × 10(5), 1 × 10(6), 2 × 10(6) purified sporozoites for group II, group III and group IV, respectively, were inoculated into respective chick embryo tracheal rings maintained in RPMI 1640 supplemented with 5% heat-inactivated FBS, and cultivated in each well of the 24-well culture plate at 40°C and 5% CO(2). The tracheal rings in four experimental groups (I-IV) were successfully infected with C. baileyi, and different stages of parasites were also observed under light and electron microscopy. Parasite infection and cytological alterations were noted as early as PI 72 h. The Cryptosporidium were seen attached to the edge of the tracheal epithelium, with more number of parasites in group I than that in group II, group III and group IV. The moderate nuclear swelling and chromatin margination were also detected, and the normal vertical orientation and basilar location of the nuclei of the epithelial cells were almost lost. C. baileyi that has been passed by TOCs exhibited similar immunity and molecular features with parasites before intratracheal inoculation. These results suggest that chick embryo tracheal organ is a new and effective in vitro culture model for C. baileyi and other respiratory pathogens., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Dairy industry plays an important role in the agricultural economy of China. To estimate the prevalence and public health significance of cryptosporidiosis in post-weaned and adult dairy cattle in China, during four consecutive years (from 2006 to 2009), a total of 1315 fecal samples from 22 dairy cattle farms in ten prefectures in Henan Province were examined for the presence of Cryptosporidium oocysts. The overall prevalence of Cryptosporidium was 7.9%, with the highest infection rate (11.3%) in 3 to 11-month-old calves and the lowest infection rate (1.0%) in >2-year-old cows (p<0.01). Cryptosporidium-positive samples (n=104) were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene, and 25 representative samples were further analyzed by DNA sequencing of the PCR products. Cryptosporidium bovis and Cryptosporidium andersoni were identified. C. andersoni (84/104) was the predominant species and was found in all age groups, whereas C. bovis (20/104) was only detected in 3 to 11-month-old calves. Thus, C. andersoni appears to be the dominant species in weaned dairy calves and heifers in China, in contrast with its common occurrence in adult cattle in other parts of the world., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

A total of 469 fecal samples were collected from American minks (Mustela vison) on a farm in Hebei Province in China and examined for Cryptosporidium by Sheather's sugar flotation technique and 8 Cryptosporidim isolates were obtained. The partial 18S rRNA, 70kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes of six isolates were sequenced. Sequence data were analyzed together with known Cryptosporidium spp. and genotypes. Results of this multi-locus genetic characterization indicated that the six Cryptosporidium isolates in this study shared the same sequences of the genes studied and were different from known Cryptosporidium species and genotypes. The closest relative was Cryptosporidium ferret genotype with 7, 22, 2 and 2 nucleotide differences in the 18S rRNA, HSP70, COWP and actin genes, respectively. The homology to ferret genotype at the 18S rRNA locus was 99.1%, which is comparable to that between C. parvum and C. hominis (99.2%), or between C. muris and C. andersoni (99.4%). Therefore, the Cryptosporidium in minks in this study is considered a new genotype, the Cryptosporidium mink genotype.