Xin Yang

Escherichia coli, Clostridium perfringens and Cryptosporidium are common diarrhea-related pathogens in dairy calves, posing considerable economic losses to animal husbandry and threatening public health. A previous study in our lab found the frequent occurrence of Escherichia coli virulence genes (eaeA, stx1 and stx2), Clostridium perfringens cpa and Cryptosporidium in diarrheic dairy calves in Ningxia Hui Autonomous Region, China. The present study aimed to develop a multiplex PCR for simultaneous detection of these virulence genes and Cryptosporidium in diarrheic dairy calves. The multiplex PCR demonstrated sensitivities of 2060 copies, 18200 copies, 1300 copies, 1990 copies and 974 copies for stx1, stx2, eaeA, cpa and 18S rRNA, respectively. Moreover, the method showed no cross-reactivity with Giardia duodenalis, Enterocytozoon bieneusi, Eimeria, Haemonchus contortus, Oesophagostomum, Moniezia, Salmonella, Proteus mirabilis and Staphylococcus aureus. Further application of the multiplex PCR in 20 clinical faecal samples from diarrheic dairy calves found that the positive rates of the multiplex PCR assay were 55% (11/20), 50% (10/20), 60% (12/20), 45% (9/20) and 25% (5/20) for stx1, stx2, eaeA, cpa and 18S rRNA, respectively, which were not significantly lower than those of the conventional PCR targeting stx1 (60%, 12/20) and eaeA (65%, 13/20), but higher than those of the reported PCR targeting stx2 (45%, 9/20) and cpa (40%, 8/20), and were consistent with those of the reported nested PCR targeting 18S rRNA (25%, 5/20). Taken together, the present study preliminarily developed a multiplex PCR assay for the rapid detection of selected E. coli virulence genes, C. perfringens cpa and Cryptosporidium 18S rRNA in dairy calves, which could provide basic data and technical support for the diagnosis and prevention of calf diarrhea. However, more samples from divergent clinical settings are needed to validate the assay in the diagnosis of selected E. coli virulence genes, C. perfringens cpa and Cryptosporidium 18S rRNA in future studies.

Giardia duodenalis, Enterocytozoon bieneusi, and Cryptosporidium spp. are common zoonotic intestinal protozoa in dairy goats, capable of cross-species transmission. These pathogens impair the growth and development of dairy goats and present a significant threat to public health. Conventional detection methods are time-consuming and lack sensitivity, requiring a rapid and efficient technique for simultaneous detection. A TaqMan probe-based multiplex real-time qPCR assay for the detection of the three protozoa was established in this study using specific primers and probes. Specificity validation demonstrated no cross-reactivity with other common intestinal pathogens of dairy goats. Sensitivity testing revealed minimum detection limits of 29.83, 39.33, and 33.15 copies/μL for the three protozoa, with excellent repeatability. Detection of clinical fecal samples and comparison with PCR confirmed the assay’s superior performance, which was capable of identifying positive samples missed by PCR. In conclusion, this rapid, specific, and sensitive assay provides technical support for large-scale epidemiological surveys and real-time monitoring of related protozoan diseases in dairy goats, thereby laying a scientific foundation for prevention and control and safeguarding both the dairy goat industry and public health.

Cryptosporidium spp. are important zoonotic pathogens causing diarrhea in humans and various animals. To date, there are still no effective drugs and vaccines for the prevention and treatment of cryptosporidiosis, mainly due to the absence of knowledge on the interaction mechanisms between hosts and Cryptosporidium . Increasing evidence has indicated that long non-coding RNAs (lncRNAs) play significant roles in various physiological and pathological processes. Our previous study showed that Cryptosporidium parvum infection induced significant differential changes in lncRNA expression profiles in HCT-8 cells. BACE1-AS was a significantly downregulated lncRNA in HCT-8 cells during C. parvum infection, but its roles were still unknown. Results of the present study indicated that C. parvum infection significantly downregulated the expression of BACE1-AS probably through regulating the nuclear factor kappa-B (NF-κB) signaling pathway, and the expression of BACE1-AS was negatively related to the propagation of C. parvum in HCT-8 cells. BACE1-AS was mainly localized outside of the nucleus of HCT-8 cells and promoted the expression of IRF3 by sponging miR-6805-5p during C. parvum infection. Further studies indicated that the BACE1-AS/miR-6805-5p/IRF3 axis delayed in vitro propagation of C. parvum through regulating BCL2-mediated apoptosis of HCT-8 cells. The present study enriches our knowledge on the function of host lncRNAs during Cryptosporidium infection and contributes to our understanding of the interaction between hosts and Cryptosporidium . IMPORTANCE: Recent studies indicate that Cryptosporidium can regulate cell apoptosis to promote its development in host cells, but the mechanism is still unclear. This study identified a significantly downregulated host lncRNA BACE1-AS during C. parvum infection, which could regulate cell apoptosis to affect the propagation of C. parvum in vitro by targeting the miR-6805-5p/IRF3 axis. The present study contributes to our understanding of the pathogenesis of Cryptosporidium and provides potential targets for the development of drugs and vaccines against cryptosporidiosis.

Cryptosporidium spp. and Giardia intestinalis are important zoonotic protozoa that are closely related to diarrhea and cause considerable economic losses in the livestock breeding industry. Ganzi is one of the main production areas for yaks in China, but there have been few reports on the occurrence of Cryptosporidium spp. and G. intestinalis in yaks. This study used PCR-based sequencing techniques to survey the prevalence and species/genotypes of Cryptosporidium spp. and G. intestinalis in faecal samples from 223 yaks in Ganzi, Sichuan Province. The positive rate of Cryptosporidium spp. was 7.2% (16/223), with the highest positive rate of yaks found in Yajiang (37%, 10/27), which was significantly higher than that in Litang (3.1%, 4/130) and Seda (3%, 2/66). The positive rate of Cryptosporidium spp. in young animals aged 24 months (3.3%, 3/90). Three Cryptosporidium species were found by sequence analysis of 18S rRNA locus, namely C. andersoni, C. ryanae, and C. bovis. The total positive rate of G. intestinalis was 15.7% (35/223), with significant differences identified between Yajiang (40.7%, 11/27), Litang (17.7%, 23/130), and Seda (1.5%, 1/66). One genotype (assemblage E) was found by analyzing the sequence of gdh, bg, and tpi loci. Meanwhile, co-infection of Cryptosporidium spp. and Giardia intestinalis was identified in five samples. The present study explores the infection of Cryptosporidium spp. and G. intestinalis from yaks in Ganzi, aiming to enrich our understanding of the occurrence of these protozoa in livestock.

Enterocytozoon bieneusi is a common pathogen in humans and various animals, threatening the breeding industry and public health. However, there is limited information on the molecular characteristics of E. bieneusi in yaks, an economically important animal mainly domesticated in the Qinghai Tibet Plateau in China. In the present study, nested PCR targeting the ITS gene region was applied to investigate the positive rates and genetic diversity of E. bieneusi in 223 faecal samples of yaks from three locations in Ganzi Tibetan Autonomous Prefecture, Sichuan Province. The total positive rate of E. bieneusi was 23.8% (53/223). Significant differences in positive rates were identified among yaks from three locations (χ² = 8.535, p = 0.014) and four age groups (χ² = 17.259, p = 0.001), with the highest positive rates in yaks from Yajiang and aged < 6 months, respectively. Sequence analysis identified seven known (EbpC, LW1, LQ10, PigEBITS5, ESH-01, J and BEB4) and five novel (Ganzi1–5) ITS genotypes. Phylogenetic analysis showed eight genotypes (EbpC, LW1, LQ10, PigEBITS5, ESH-01, Ganzi1, Ganzi2 and Ganzi4) in group 1 and three genotypes (J, BEB4 and Ganzi3) in group 2, indicating high genotype diversity and zoonotic potential of E. bieneusi in yaks from Ganzi. Considering the increasing zoonotic genotypes in yaks in the present study compared with previous findings, interventions should be developed to reduce the potential transmission of E. bieneusi between humans and animals.

Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli are important diarrheal pathogens threatening the health of humans and various animals. Goats, especially pre-weaned goat kids, that carry these pathogens are important reservoirs related to human infection. In the present study, PCR-based sequencing techniques were applied to characterize Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in 202 fecal samples of diarrheal kids for Guanzhong dairy goats from five locations in Shaanxi Province. The positive rates of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli were 37.6% (76/202), 16.3% (33/202), 55.4% (112/202) and 78.7% (159/202) in these goat kids, respectively. Co-infection of two to four pathogens was found in 114 of 202 fecal samples. Significant differences (p < 0.001) in the positive rates of Cryptosporidium spp. and G. duodenalis were found among locations and age groups. Furthermore, two Cryptosporidium species (C. parvum and C. xiaoi), two G. duodenalis assemblages (E and A), nine E. bieneusi genotypes (CHG3, CHG1, BEB6, CHG5, CHG2, SX1, CHG28, COS-II and CD6) and two E. coli pathotypes (EPEC and EHEC) were identified. As for Cryptosporidium, two (IIdA19G1 and IIdA19G2) and two (XXIIIa and XXIIIg) subtypes were recognized in samples positive for C. parvum and C. xiaoi, respectively. A phylogenetic analysis based on the ITS locus of E. bieneusi indicated that all nine genotypes of E. bieneusi identified in this study belonged to the group 2. Four virulence factors (ehxA, eae, stx2 and stx1) of EPEC and EHEC were found in E. coli strains. Collectively, this study explored the colonization frequency of Cryptosporidium spp., G. duodenalis, E. bieneusi and E. coli in diarrheal kids of Guanzhong dairy goats in Shaanxi Province and expanded our understanding of the genetic composition and zoonotic potential of these pathogens in goats.

Cryptosporidium parvum is an important zoonotic protozoon that threatens the health of humans and animals, but the interaction mechanisms between C. parvum and hosts are poorly understood. Our previous study indicated that the expression levels of C3a and C3aR were up-regulated in mice during C. parvum infection, but the mechanisms of C3a/C3aR signaling during C. parvum infection have not been elucidated. In the present study, an optimized BALB/c suckling mouse model infected with C. parvum was used to explore the function of C3a/C3aR signaling during C. parvum infection. The expression levels of C3aR in the ileum tissues of mice infected with C. parvum were analyzed using real-time PCR, Western blot and immunohistochemistry. The mRNA levels of the Cryptosporidium 18S rRNA gene, tight junction proteins (zo-1, claudin 3, and occludin), intestinal stem cell marker lgr5, cell proliferation marker ki67, Th1 cell-related cytokine ifn-γ, and Treg cell-related cytokine tgf-β in mouse ileum tissues were analyzed by real-time PCR. The pathological injury of ileal mucosa was examined by histopathology analysis. The mRNA expression levels of Cryptosporidium 18S rRNA gene were significantly up-regulated in the ileum tissues of C3aR-inhibited mice during C. parvum infection. Meanwhile, histopathology analysis of ileal mucosa in mice showed that inhibition of C3aR significantly aggravated the changes in villus length, villus diameter, mucosal thickness and the ratio of villus length to crypt depth during C. parvum infection. Further studies found inhibition of C3aR aggravated the down-regulation of occludin at most time points during C. parvum infection. The mRNA levels of ki67 and lgr5 in the ileum tissues of mice infected with C. parvum were significantly down-regulated. Inhibition of C3aR significantly down-regulated the mRNA expression levels of lgr5 at most time points, but significantly up-regulated the mRNA expression levels of ki67 at most time points. The mRNA expression levels of ifn-γ and tgf-β were significantly up-regulated and down-regulated in the ileum tissues of mice infected with C. parvum, respectively. However, inhibition of C3aR significantly increased the mRNA expression levels of ifn-γ and tgf-β in the ileum tissues of mice infected with C. parvum. Taken together, C3a/C3aR signaling could possibly affect the propagation of C. parvum in mouse ileum tissues by regulating the gut barrier, cell proliferation and CD4⁺ T cell main effectors, which would contribute to our understanding of the interaction between Cryptosporidium and hosts.

Background: The effective transmission mode of Neospora caninum, with infection leading to reproductive failure in ruminants, is vertical transmission. The uterus is an important reproductive organ that forms the maternal–fetal interface. Neospora caninum can successfully invade and proliferate in the uterus, but the molecular mechanisms underlying epithelial-pathogen interactions remain unclear. Accumulating evidence suggests that host long noncoding RNAs (lncRNAs) play important roles in cellular molecular regulatory networks, with reports that these RNA molecules are closely related to the pathogenesis of apicomplexan parasites. However, the expression profiles of host lncRNAs during N. caninum infection has not been reported. Methods: RNA sequencing (RNA-seq) analysis was used to investigate the expression profiles of messenger RNAs (mRNAs) and lncRNAs in caprine endometrial epithelial cells (EECs) infected with N. caninum for 24 h (TZ_24h) and 48 h (TZ_48 h), and the potential functions of differentially expressed (DE) lncRNAs were predicted by using Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of their mRNA targets. Results: RNA-seq analysis identified 1280.15 M clean reads in 12 RNA samples, including six samples infected with N. caninum for 24 h (TZ1_24h-TZ3_24h) and 48 h (TZ1_48h-TZ3_48h), and six corresponding control samples (C1_24h-C3_24h and C1_48h-C3_48h). Within the categories TZ_24h-vs-C_24h, TZ_48h-vs-C_48h and TZ_48h-vs-TZ_24h, there were 934 (665 upregulated and 269 downregulated), 1238 (785 upregulated and 453 downregulated) and 489 (252 upregulated and 237 downregulated) DEmRNAs, respectively. GO enrichment and KEGG analysis revealed that these DEmRNAs were mainly involved in the regulation of host immune response (e.g. TNF signaling pathway, MAPK signaling pathway, transforming growth factor beta signaling pathway, AMPK signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway), signaling molecules and interaction (e.g. cytokine-cytokine receptor interaction, cell adhesion molecules and ECM-receptor interaction). A total of 88 (59 upregulated and 29 downregulated), 129 (80 upregulated and 49 downregulated) and 32 (20 upregulated and 12 downregulated) DElncRNAs were found within the categories TZ_24h-vs-C_24h, TZ_48h-vs-C_48h and TZ_48h-vs-TZ_24h, respectively. Functional prediction indicated that these DElncRNAs would be involved in signal transduction (e.g. MAPK signaling pathway, PPAR signaling pathway, ErbB signaling pathway, calcium signaling pathway), neural transmission (e.g. GABAergic synapse, serotonergic synapse, cholinergic synapse), metabolism processes (e.g. glycosphingolipid biosynthesis-lacto and neolacto series, glycosaminoglycan biosynthesis-heparan sulfate/heparin) and signaling molecules and interaction (e.g. cytokine-cytokine receptor interaction, cell adhesion molecules and ECM-receptor interaction). Conclusions: This is the first investigation of global gene expression profiles of lncRNAs during N. caninum infection. The results provide valuable information for further studies of the roles of lncRNAs during N. caninum infection. Graphical