Prevalence and genotype/subtype distribution of Enterocytozoon bieneusi and Blastocystis in donkeys in Shanxi Province, north China.

Enterocytozoon bieneusi and Blastocystis may cause diarrhea in humans and various animals. However, little information is available regarding the prevalence and genetic diversity of E. bieneusi and Blastocystis in donkeys. To fill this gap, we molecularly assessed E. bieneusi and Blastocystis in fecal samples from donkeys (n = 815) in Shanxi Province, north China. The overall prevalence of E. bieneusi and Blastocystis in donkeys was 8.1% and 0.2%, respectively. Region and age were risk factors associated with E. bieneusi infection in donkeys. Three internal transcribed spacer (ITS) genotypes of E. bieneusi were identified in the current study, including two previously described genotypes (D and Henan-IV) and one novel genotype (named SXD1). Of which, genotype D was found to be the most prevalent. Phylogenetic analysis demonstrated that the three genotypes belonged to group 1, implying a potential of zoonotic transmission. Multilocus sequence typing showed that 19, 15, 13, and 22 types were identified at the loci MS1, MS3, MS4, and MS7, respectively, forming six multilocus genotypes (MLGs) distributed in the genotype D. One Blastocystis subtype (ST33) was identified, which has previously been reported only in horses. This is the first molecular-based description of E. bieneusi and Blastocystis infections in donkeys in Shanxi Province, north China, contributing to a better understanding of transmission dynamics and molecular epidemiological characteristics of the two intestinal protozoa.

Prevalence and Multilocus Genotyping of Giardia duodenalis in Donkeys in Shanxi Province, North China.

Giardia duodenalis is a ubiquitous flagellated protozoan, causing significant economic losses to animal husbandry and posing threats to public health. China ranks the world's sixth largest major producer of donkeys, rearing approximately 2.6 million donkeys in 2019, but limited investigation of G. duodenalis prevalence has been conducted in the past, and it is yet to be known whether donkeys in Shanxi Province are infected with G. duodenalis. In the present study, a total of 815 fecal samples collected from donkeys in representative regions of Shanxi Province, North China, were examined for G. duodenalis using nested PCR. Then, the assemblages and multilocus genotypes (MLGs) were examined based on three established loci: namely, ß-giardin (bg), triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh). The overall prevalence of G. duodenalis in donkeys in Shanxi Province was 16.81% (137/815). The region was identified as the main risk factor for the observed difference in G. duodenalis prevalence in donkeys among the three study areas (χ2 = 21.611, p < 0.001). Assemblages A, E, and B were identified, with the latter as the predominant assemblage. Three MLGs (MLG-novel-1 to 3) were formed based on sequence variation among the three loci. The present study reveals the presence of G. duodenalis in donkeys in Shanxi Province, North China, for the first time, which not only enriches the data on the distribution of G. duodenalis in donkeys in China but also provides useful baseline data for planning control strategies against G. duodenalis infection in the sampled areas.

iTRAQ-Based Phosphoproteomic Analysis Exposes Molecular Changes in the Small Intestinal Epithelia of Cats after Toxoplasma gondii Infection.

Toxoplasma gondii, an obligate intracellular parasite, has the ability to invade and proliferate within most nucleated cells. The invasion and destruction of host cells by T. gondii lead to significant changes in the cellular signal transduction network. One important post-translational modification (PTM) of proteins is phosphorylation/dephosphorylation, which plays a crucial role in cell signal transmission. In this study, we aimed to investigate how T. gondii regulates signal transduction in definitive host cells. We employed titanium dioxide (TiO2) affinity chromatography to enrich phosphopeptides in the small intestinal epithelia of cats at 10 days post-infection with the T. gondii Prugniuad (Pru) strain and quantified them using iTRAQ technology. A total of 4998 phosphopeptides, 3497 phosphorylation sites, and 1805 phosphoproteins were identified. Among the 705 differentially expressed phosphoproteins (DEPs), 68 were down-regulated and 637 were up-regulated. The bioinformatics analysis revealed that the DE phosphoproteins were involved in various cellular processes, including actin cytoskeleton reorganization, cell necroptosis, and MHC immune processes. Our findings confirm that T. gondii infection leads to extensive changes in the phosphorylation of proteins in the cat intestinal epithelial cells. The results of this study provide a theoretical foundation for understanding the interaction between T. gondii and its definitive host.

Prevalence and Genetic Characterization of Blastocystis in Sheep and Pigs in Shanxi Province, North China: From a Public Health Perspective.

Blastocystis is a common zoonotic intestinal protozoan and causes a series of gastrointestinal symptoms in humans and animals via the fecal-oral route, causing economic losses and posing public health problems. At present, the prevalence and genetic structure of Blastocystis in sheep and pigs in Shanxi province remains unknown. Thus, the present study collected 492 sheep fecal samples and 362 pig fecal samples from three representative counties in northern, central and southern Shanxi province for the detection of Blastocystis based on its SSU rRNA gene. The results showed that the overall prevalence of Blastocystis in the examined sheep and pigs were 16.26% and 14.09%, respectively. Sequences analyses showed that four known subtypes (ST5, ST10, ST14 and ST30) in sheep and two subtypes (ST1 and ST5) in pigs were detected in this study, with ST5 being the predominate subtype among the study areas. Phylogenetic analysis showed that the same subtypes were clustered into the same branch. This study reveals that sheep and pigs in Shanxi province are hosts for multiple Blastocystis subtypes, including the zoonotic subtypes (ST1 and ST5), posing a risk to public health. Baseline epidemiological data are provided that help in improving our understanding of the role of zoonotic subtypes in Blastocystis transmission.

Molecular Identification and Genotyping of Cryptosporidium spp. and Blastocystis sp. in Cattle in Representative Areas of Shanxi Province, North China.

Both Cryptosporidium spp. and Blastocystis sp. are common intestinal protozoa, which can cause zoonotic diseases and economic losses to livestock industry. To evaluate the prevalence and genetic population structure of Cryptosporidium spp. and Blastocystis sp. in beef and dairy cattle in Shanxi Province, north China, a total of 795 fecal samples were collected from beef and dairy cattle in three representative counties in Shanxi Province, and these fecal samples were examined using molecular approaches based on 18S small-subunit ribosomal RNA (SSU rRNA) of Cryptosporidium spp. and Blastocystis sp., respectively. Among 795 cattle fecal samples, 23 were detected as Cryptosporidium-positive and 103 were detected as Blastocystis-positive, and the overall prevalence of Cryptosporidium spp. and Blastocystis sp. in cattle in Shanxi Province was 2.9% and 13.0%, respectively. For Cryptosporidium spp., DNA sequence analysis indicated that all 23 positive samples were identified as C. andersoni. Furthermore, five known subtypes (ST1, ST10, ST14, ST21 and ST26) and three unknown subtypes of Blastocystis sp. were detected among 103 positive samples using DNA sequence analysis. This study reported the occurrence and prevalence of Cryptosporidium spp. and Blastocystis sp. in cattle in Shanxi Province for the first time, which extends the geographical distribution of these two zoonotic parasites and provides baseline data for the prevention and control of these two important zoonotic parasites in cattle in Shanxi Province.

Dynamic RNA profiles in the small intestinal epithelia of cats after Toxoplasma gondii infection.

BACKGROUND: Felids are the only definitive hosts of Toxoplasma gondii. However, the biological features of the feline small intestine following T. gondii infection are poorly understood. We investigated the changes in the expression of RNAs (including mRNAs, long non-coding RNAs and circular RNAs) in the small intestinal epithelia of cats following T. gondii infection to improve our understanding of the life cycle of T. gondii and cat responses to T. gondii infection. METHODS: Fifteen cats were randomly assigned to five groups, and the infection groups were inoculated with 600 tissue cysts of the T. gondii Pru strain by gavage. The small intestinal epithelia of cats were collected at 6, 10, 14, and 30 days post infection (DPI). Using high-throughput RNA sequencing (RNA-seq), we investigated the changes in RNA expression. The expression levels of differentially expressed (DE) genes and non-coding RNAs (ncRNAs) identified by RNA-seq were validated by quantitative reverse transcription PCR (qRT-PCR). Differential expression was determined using the DESeq R package. RESULTS: In total, 207 annotated lncRNAs, 20,552 novel lncRNAs, 3342 novel circRNAs and 19,409 mRNAs were identified. Among these, 70 to 344 DE mRNAs, lncRNAs and circRNAs were detected, and the post-cleavage binding sites between 725 ncRNAs and 2082 miRNAs were predicted. Using the co-location method, we predicted that a total of 235 lncRNAs target 1044 protein-coding genes, while the results of co-expression analysis revealed that 174 lncRNAs target 2097 mRNAs. Pathway enrichment analyses of the genes targeted by ncRNAs suggested that most ncRNAs were significantly enriched in immune or diseases-related pathways. NcRNA regulatory networks revealed that a single ncRNA could be directly or indirectly regulated by multiple genes or ncRNAs that could influence the immune response of cats. Co-expression analysis showed that 242 circRNAs, mainly involved in immune responses, were significantly associated with T. gondii infection. In contrast, 1352 protein coding RNAs, mainly involved in nucleic acid process/repair pathways or oocyte development pathways, were negatively associated with T. gondii infection. CONCLUSIONS: This study is the first to reveal the expression profiles of circRNAs, lncRNAs and mRNAs in the cat small intestine following T. gondii infection and will facilitate the elucidation of the role of ncRNAs in the pathogenesis of T. gondii infection in its definitive host, thereby facilitating the development of novel intervention strategies against T. gondii infection in humans and animals.

A combined miRNA-piRNA signature in the serum and urine of rabbits infected with Toxoplasma gondii oocysts.

BACKGROUND: Increasing evidence has shown that non-coding RNA (ncRNA) molecules play fundamental roles in cells, and many are stable in body fluids as circulating RNAs. Study on these ncRNAs will provide insights into toxoplasmosis pathophysiology and/or help reveal diagnostic biomarkers. METHODS: We performed a high-throughput RNA-Seq study to comprehensively profile the microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) in rabbit serum and urine after infection with Toxoplasma gondii oocysts during the whole infection process. RESULTS: Total RNA extracted from serum and urine samples of acutely infected [8 days post-infection (DPI)], chronically infected (70 DPI) and uninfected rabbits were subjected to genome-wide small RNA sequencing. We identified 2089 miRNAs and 2224 novel piRNAs from the rabbit sera associated with T. gondii infection. Meanwhile, a total of 518 miRNAs and 4182 novel piRNAs were identified in the rabbit urine associated with T. gondii infection. Of these identified small ncRNAs, 1178 and 1317 serum miRNAs and 311 and 294 urine miRNAs were identified as differentially expressed (DE) miRNAs in the acute and chronic stages of infections, respectively. A total of 1748 and 1814 serum piRNAs and 597 and 708 urine piRNAs were found in the acute and chronic infection stages, respectively. Of these dysregulated ncRNAs, a total of 88 common DE miRNAs and 120 DE novel piRNAs were found in both serum and urine samples of infected rabbits. CONCLUSIONS: These findings provide valuable data for revealing the physiology of herbivore toxoplasmosis caused by oocyst infection. Circulating ncRNAs identified in this study are potential novel diagnostic biomarkers for the detection/diagnosis of toxoplasmosis in herbivorous animals.

Lung Lipidomic Alterations in Beagle Dogs Infected with Toxocara canis.

Toxocariasis, mainly caused by Toxocara canis, and to a lesser extent, Toxocara cati, is a neglected parasitic zoonosis. The mechanisms that underlie the changes in lipid metabolism of T. canis infection in Beagle dogs' lungs remain unclear. Lipidomics is a rapidly emerging approach that enables the global profiling of lipid composition by mass spectrometry. In this study, we performed a non-targeted lipidomic analysis of the lungs of Beagle dogs infected with the roundworm T. canis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1197 lipid species were identified, of which 63, 88, and 157 lipid species were significantly altered at 24 h post-infection (hpi), 96 hpi, and 36 days post-infection (dpi), respectively. This global lipidomic profiling identified infection-specific lipid signatures for lung toxocariasis, and represented a comprehensive comparison between the lipid composition of dogs' lungs in the presence and absence of T. canis infection. The potential roles of the identified lipid species in the pathogenesis of T. canis are discussed, which has important implications for better understanding the interaction mechanism between T. canis and the host lung.

Molecular Detection and Genotyping of Enterocytozoon bieneusi in Beef Cattle in Shanxi Province, North China.

Enterocytozoon bieneusi is an intracellular pathogen that can parasitize humans and a variety of animals. The infection of E. bieneusi in most hosts is asymptomatic, but in immunocompromised individuals, it can lead to serious complications such as acute diarrhea, dehydration, and even death. However, no data on the prevalence and genotyping of E. bieneusi in beef cattle in Shanxi province are currently available. In this study, a total of 401 fecal samples were collected from beef cattle in farms from two representative counties­Qi county and Jishan county­in Shanxi province, north China. Nested PCR was applied to determine the prevalence and genotypes of E. bieneusi by amplifying and sequencing the internal transcribed spacer (ITS) regions of the rRNA gene. A total of 90 out of 401 samples were detected as E. bieneusi-positive, with 22.44% overall prevalence of E. bieneusi in beef cattle in Shanxi province. The highest prevalence of E. bieneusi was detected in calves (28.67%, 41/143) and male beef cattle (28.13%, 54/192). Statistical analysis revealed that the prevalence of E. bieneusi was significantly associated with gender and age factors (p < 0.05), but without any statistical difference among regions. Moreover, six known E. bieneusi genotypes (BEB4, BEB6, BEB8, J, I, and PigSpEb2) and two novel genotypes (designated CSC1 and CSC2) were identified by analysis of ITS sequences, and genotype I was the predominant genotype in these two counties. Phylogenetic analysis showed that five known genotypes and two novel genotypes were clustered into Group 2, but PigSpEb2 belonged to Group 1. To our knowledge, the present study demonstrated the presence and identified genotypes of E. bieneusi in beef cattle in Shanxi province for the first time, extending the data on prevalence and genotypes of E. bieneusi in beef cattle and providing baseline data for executing intervention measures to control it in the study regions.

Differential Spleen miRNA Expression Profile of Beagle Dogs Infected with Toxocara canis.

Toxocara canis is an unnoticed zoonotic helminth that causes severe disease in animals and humans. The spleen has a wide range of immunological functions in protecting the host against infection by many pathogens, but the function of the spleen in T. canis infection is still to be clarified, especially for the role of spleen microRNAs (miRNAs). In this study, deep sequencing of spleen RNA samples of 18 Beagle puppies was conducted to uncover the miRNAs expression profiling at 24 h post-infection (hpi), 96 hpi, and 36 days post infection (dpi). A total of 20, 34, and 19 differentially expressed miRNAs (DEmiRNAs) were identified at 24 hpi, 96 hpi, and 36 dpi, respectively. These DEmiRNAs (e.g., cfa-miR-206, cfa-miR-331, and cfa-miR-339) could play critical roles in Beagle puppies against T. canis infection, such as influencing inflammatory and immune-related cells and cytokines, by regulating target genes that are tightly associated with host immune function and enriched in immune response and immune pathways based on GO annotation and KEGG enrichment analysis. The current study discovered marked alterations of spleen miRNAs after T. canis infection, with potential effects on the pathogenesis of toxocariasis.

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.

Lipidomic changes in the liver of beagle dogs associated with Toxocara canis infection.

A global lipidomic analysis using liquid chromatography-tandem mass spectrometry was performed on the liver of beagle dogs infected with Toxocara canis to profile hepatic lipid species at 12 h post-infection (hpi), 24 hpi, and 36 days post-infection (dpi). This analysis identified six categories and 42 subclasses of lipids, including 173, 64, and 116 differentially abundant lipid species at 12 hpi, 24 hpi, and 36 dpi, respectively. Many of the identified lysophospholipids, such as lysophosphatidylglycerol, lysophosphatidylserine, and lysophosphatidylcholine, may contribute to the migration and development of T. canis during the early infection stage. Pathway analysis revealed significant alterations of several immune-inflammatory pathways, such as the B-cell receptor signaling pathway, the NF-kappa B signaling pathway, and the C-type lectin receptor signaling pathway at 12 and 24 hpi. These findings demonstrate the value of lipidomic profiling in revealing the extent of changes in the composition and abundance of hepatic lipidome caused by T. canis infection and their relevance to the pathophysiology of toxocariasis in beagle dogs.

Research Note: Transcriptomic analysis of LMH cells in response to the overexpression of a hypothetical protein identified in Eimeria tenella SD-01 strain.

Though genome sequencing of Eimeria tenella predicts more than 8,000 genes, the molecular functions of many proteins remain unknown. In this study, the coding region corresponding to the mature peptide of a hypothetical protein of E. tenella (ETH_00023950) was amplified and expressed in a bacterial system. Following preparation of polyclonal antibody that recognizes ETH_00023950, the expression of ETH_00023950 in merozoites was examined. Meanwhile, we determined the transcriptomic responses of the leghorn male hepatoma (LMH) cells to its expression. Sequencing analysis showed that one single nucleotide polymorphism and one indel of ETH_00023950 of E. tenella SD-01 strain were found compared with that of the UK reference Houghton strain, leading to a frame shift and a premature stop codon. The expression of ETH_00023950 in E. tenella merozoites was confirmed by indirect immunofluorescence and Western blot analysis. Transcriptomic analysis showed that ETH_00023950 altered the expression of 2,680 genes (321 downregulated genes and 2,359 upregulated genes) in LMH cells. The RNA-sequencing data were consistent with the results of the quantitative real-time polymerase chain reaction (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that differentially expressed transcripts were significantly related to 8 pathways, including oxidative phosphorylation and TGF-beta signaling pathway. These findings contribute to understanding host-pathogen interaction and secondary bacterial infections related to E. tenella.

Molecular Detection and Genotyping of Enterocytozoon bieneusi in Pigs in Shanxi Province, North China.

Enterocytozoon bieneusi is a common opportunistic intestinal pathogen that can cause acute diarrhea in immunosuppressed humans and animals. Though E. bieneusi has been widely detected in pigs around the world, little is known of its prevalence and genotype distribution in pigs in Shanxi province, north China. In this study, a total of 362 fecal samples were collected from pigs in three representative counties in north, south, and central Shanxi province, China. The prevalence and genotypes of E. bieneusi were investigated by nested PCR amplification of the ribosomal internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. Overall, the prevalence of E. bieneusi in pigs in Shanxi province was 54.70% (198/362). Statistical analysis showed the difference in prevalence was statistically significant between regions (χ2 = 41.94, df = 2, P < 0.001) and ages (χ2 = 80.37, df = 1, P < 0.001). In addition, 16 genotypes of E. bieneusi were identified in this study by sequence analysis of the ITS region, including 15 known genotypes (EbpC, EbpA, EbpB, pigEb4, PigEBITS5, I, Henan-I, G, WildBoar 7, SH10, EbpD, CHC5, PigSpEb1, PigSpEb2, and CHG19) and one novel genotype (designated as PigSX-1). Phylogenetic analysis revealed that 14 known genotypes and the novel genotype were clustered into Group 1, whereas genotype I belonged to Group 2. To the best of our knowledge, this is the first report on the prevalence and genotypes of E. bieneusi in pigs in Shanxi province. These findings enrich the genetic diversity of E. bieneusi and provide the baseline data for the prevention and control of E. bieneusi in pigs in the study regions.

RNA sequencing reveals dynamic expression of spleen lncRNAs and mRNAs in Beagle dogs infected by Toxocara canis.

BACKGROUND: Toxocara canis is a cosmopolitan parasite with a significant adverse impact on the health of humans and animals. The spleen is a major immune organ that plays essential roles in protecting the host against various infections. However, its role in T. canis infection has not received much attention. METHODS: We performed sequencing-based transcriptome profiling of long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression in the spleen of Beagle puppies at 24 h post-infection (hpi), 96 hpi and 36 days post-infection (dpi). Deep sequencing of RNAs isolated from the spleen of six puppies (three infected and three control) at each time point after infection was conducted. RESULTS: Our analysis revealed 614 differentially expressed (DE) lncRNAs and 262 DEmRNAs at 24 hpi; 726 DElncRNAs and 878 DEmRNAs at 96 hpi; and 686 DElncRNAs and 504 DEmRNAs at 36 dpi. Of those, 35 DElncRNA transcripts and 11 DEmRNAs were detected at all three time points post-infection. Many DE genes were enriched in immune response, such as ifit1, ifit2 and rorc. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that some genes (e.g. prkx and tnfrsf11a) were involved in the T cell receptor signaling pathway, calcium signaling pathway, Ras signaling pathway and NF-κB signaling pathway. CONCLUSIONS: The findings of this study show marked alterations in the expression profiles of spleen lncRNAs and mRNAs, with possible implications in the pathophysiology of toxocariasis.

Transcriptional changes in LMH cells induced by Eimeria tenella rhoptry kinase family protein 17.

Though a number of Eimeria tenella rhoptry kinase family proteins have been identified, little is known about their molecular functions. In the present study, the gene fragment encoding the matured peptide of E. tenella rhoptry kinase family protein 17 (EtROP17) was used to construct a recombinant vector, followed by transfection into leghorn male hepatoma (LMH) cells. Then, the transcriptional changes in the transfected cells were determined by RNA-seq. The expression of EtROP17 in LMH cells was validated by both Western blot and indirect immunofluorescence analysis. Our analysis showed that EtROP17 altered the expression of 309 genes (114 downregulated genes and 195 upregulated genes) in LMH cells. The quantitative real-time polymerase chain reaction (qRT-PCR) results of the selected differentially expressed genes (DEGs) were consistent with the RNA-seq data. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in nine pathways, such as toll-like receptor signaling pathway, ECM-receptor interaction, intestinal immune network for IgA production and focal adhesion. These findings reveal several potential roles of EtROP17, which contribute to understanding the molecular mechanisms underlying the host-parasite interplay.

Metabolomics study of cat small intestine during the early stage of Toxoplasma gondii oocyst formation identifies potential biomarkers.

Toxoplasma gondii (T. gondii) is a zoonotic intracellular protozoan parasite that can invade, replicate and survive in almost all cells of warm-blooded animals. T. gondii infection threatens the life of the fetus or can cause morbidity in the infant. As the only definitive host of T. gondii, felids spread the pathogen mainly by forming oocysts in the small intestines and discharging the oocysts into the ambient environment, consequently polluting water, vegetables, and meat products. In this study, we used untargeted metabolomics technology to study the changes in metabolites that occurred during the early stage of oocyst formation in the cat small intestine following T. gondii infection and attempted to identify metabolic biomarkers that could potentially be used as diagnostic molecular markers in the future. Domestic cats (Felis catus) were infected with T. gondii Pru tissue cysts, and samples of their small intestinal epithelium were collected at 2 and 4 days post-infection (DPI) for metabolic analysis. LC-MS/MS and multivariate statistical analysis were employed to detect metabolomic signatures that discriminated between the infected and control groups. A total of 1673 ions and 1201 ions were obtained in the positive and negative modes, respectively. Of these ions, 175 were up-regulated and 127 were down-regulated in the positive ion mode; whereas, 123 were up-regulated and 81 were down-regulated in the negative ion mode. Three commonly altered ions (0.74_313.0414 m/z, 8.82_615.2621 m/z and 8.16_325.2362 m/z) were determined to have potential research value. Seventy common metabolic pathways were enriched at two time points, with arginine biosynthesis, pyrimidine metabolism, pantothenate and CoA biosynthesis being the three most significant pathways related to T. gondii. The area under the curve (AUC) of differential metabolites combined with relevant literature analysis showed that N-Methylpelletierine and 3,3-Difluoro-17-methyl-5alpha-androstan-17beta-ol have higher predictability and better potential application value than other metabolites. Our analysis of metabolic markers during the early stage of T. gondii oocyst formation in the small intestine of the definitive host (cat) provided novel insight for understanding oocyst development and a theoretical basis for the application of potential biomarkers.

Toxocara canis Infection Alters mRNA Expression Profiles of Peripheral Blood Mononuclear Cells in Beagle Dogs at the Lung Infection Period.

Toxocara canis is a neglected zoonotic roundworm distributed all over the world, causing toxocariasis in humans and animals. However, so far, the immune mechanism of T. canis infection in definitive hosts remains to be clarified. In this study, the transcriptional alterations of Beagle dogs' peripheral blood mononuclear cells (PBMCs) induced by T. canis infection during the lung infection period were analyzed using RNA-seq technology. A total of 2142 differentially expressed genes were identified, with 1066 upregulated genes and 1076 downregulated genes. Many differentially expressed genes participated in the biological process of intracellular signal transduction, as well as the immune- or inflammation-related KEGG signaling pathway, such as the Notch signaling pathway, Toll-like receptor signaling pathway, and NF-kappa B signaling pathway, through KEGG enrichment analysis. This study indicated that T. canis infection could suppress the biological function of Beagle dogs' PMBCs and provided basic data to further clarify the interaction mechanism between T. canis and host immune cells.

Molecular Identification and Genotyping of Enterocytozoon bieneusi in Sheep in Shanxi Province, North China.

Enterocytozoon bieneusi is a fungus-like protist that can cause malabsorption and diarrhea in sheep, other animals, and humans, threatening the development of animal husbandry and public health. To date, there are no data about the prevalence and genotypes of E. bieneusi in sheep in Shanxi Province, North China. In this study, 492 fecal samples were collected from sheep in three representative counties in northern, central, and southern Shanxi Province. Nested PCR amplification was performed to detect the prevalence and identify the genotypes of E. bieneusi based on the internal transcribed spacer (ITS) region of the rRNA gene. Overall, 168 of 492 examined samples were E. bieneusi-positive, with a prevalence of 34.2% (168/492). Significant differences in the prevalence of E. bieneusi were observed among the three sampled regions (χ2 = 95.859, df = 2, p < 0.001), but the differences in E. bieneusi prevalence were not statistically significant between different genders and age groups (p > 0.05). Sequence analysis showed that four known genotypes (BEB6, COS-I, CHS7, and CHC8) and one novel genotype (named SY-1) were identified. BEB6 was the prevalent genotype found within the three counties. Phylogenetic analysis revealed that the five genotypes observed in this study belong to Group 2. The present study reported the presence and genotypes of E. bieneusi infection in sheep in Shanxi Province for the first time, which enriches the knowledge of the genetic diversity of E. bieneusi and provides baseline data for the prevention and control of E. bieneusi infection in animals and humans.

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.