Molecular characterization of Enterocytozoon bieneusi genotypes in wild Altai marmot (Marmota baibacina) in Xinjiang, China: host specificity and adaptation.

Enterocytozoon bieneusi is responsible for opportunistic infections leading to gastrointestinal diseases in humans and animals worldwide. A total of 334 fresh fecal samples were collected from wild Altai marmots (Marmota baibacina) in Xinjiang, China, and E. bieneusi was screened via PCR amplification of the internal transcribed spacer (ITS) region of the small submit ribosomal RNA (SSU rRNA). The results indicated that 22.8% (76/334) of the wild Altai marmot fecal samples were positive for E. bieneusi, and the highest positive rate was detected in Akqi (51.9%, 27/52), with a significant difference from other sampling sites (p < 0.01). Four known genotypes (BEB6, CHG3, GX2, and YAK1) and three novel genotypes (XJHT2 to XJHT4) were identified in the present study. Genotype XJHT3 was dominant and detected in 48 fecal samples. In the phylogenetic analysis, the novel genotypes XJHT2 and XJHT3 were clustered in Group 1 together with the known genotype YAK1, while genotypes CHG3 and BEB6 were clustered in Group 2. The novel genotype XJHT4 was clustered together with other rodent-derived genotypes and generated a novel Group 14. These data confirmed the host specificity and adaptation of E. bieneusi in rodents. These findings enrich our understanding of the prevalence and genetic diversity of E. bieneusi in wild Altai marmots in Xinjiang, China.

Prevalence and sequence diversity of Balantioides coli in pigs in Xinjiang, China.

Balantioides coli is a common intestinal parasitic protozoan in pigs. In the present study, 801 fecal samples of pigs from seven farms in Xinjiang were analyzed based on the ITS1-5.8S rRNA-ITS2 gene. The prevalence of B. coli was 4.2% (34/801), with the highest prevalence of 18.9% (18/95) occurring in Alaer, Xinjiang. B. coli was detected in all age groups (pre-weaned pigs, post-weaned pigs, fattening pigs and sows), with the highest rate in fatteners (6.9%, 9/129) and the lowest (1.2%, 2/169) in pre-weaned pigs. Significant differences (P = 0.000) were found among sampling sites but not among age groups (P = 0.084). Sequence analysis indicated that 34 sequence variants, including sequence type A (n = 11) and sequence type B (n = 23), occurred in all age groups. In this study, the existence of sequence type A suggested that B. coli poses a potential threat to human health. More studies are needed to better understand the distributions and public health significance of B. coli in China.

Molecular characterization of Blastocystis spp. in Hotan Black chickens in southern Xinjiang.

To determine the infection status and assess the zoonotic potential of Blastocystis spp. in Hotan Black chickens in southern Xinjiang, China, fecal samples were collected from 617 chickens on 18 large-scale farms. The presence of Blastocystis spp. was determined using polymerase chain reaction based on the small subunit rRNA (SSU rRNA) locus. The results revealed an overall infection rate of 26.3% (162/617). Samples from Farm 1 in Luopu County showed the highest infection rate (76.3%, 29/38). The highest and lowest infection rates were detected in the <30-day (34.4%, 43/125) and > 90-day age groups (12.4%, 11/89), respectively. The infection rate decreased with increasing age. Statistical analysis showed significant differences in the infection rates of Blastocystis spp. among the different sampling sites (p < 0.05) and age groups (p < 0.05). Four Blastocystis spp. subtypes (ST6, ST7, ST10, and ST23) were identified. The infection rates of the zoonotic subtypes, ST6 and ST7, were 3.2% (20/617) and 22.2% (137/617), respectively. The presence of Blastocystis spp. and zoonotic subtypes provided evidence for the potential transmission of this pathogen between Hotan Black chickens and humans, especially in animal handlers in this area.

Occurrence and genetic characteristics of Giardia duodenalis in donkeys in Xinjiang, China.

Giardia duodenalis is a common enteric parasite in humans and animals. To examine the occurrence and genetic characteristics of Giardia in donkeys in Xinjiang, China, 758 fecal samples from donkeys were collected, and Giardia was screened via PCR at the SSU rRNA gene. A total of 17.0% (129/758) of samples tested positive for Giardia, with the infection rate in large-scale farm and domestic donkeys being 21.4% (124/580) and 2.8% (5/178), respectively; the infection rates in <1-year-old and ≥1-year-old donkeys were 19.3% (72/374) and 12.7% (41/323), respectively. Three Giardia assemblages were identified, with assemblage B (n = 102) as the prevalent assemblage, followed by assemblage A (n = 23) and assemblage E (n = 4). Of the 129 Giardia-positive isolates, 40, 34 and 59 sequences were obtained at the bg, gdh and tpi genes, respectively. Twenty-one isolates successfully allowed multilocus genotyping (MLG), with four novel assemblage A MLGs, named MLG-AI-1 (n = 1), MLG-AI-2 (n = 1), MLG-AI-3 (n = 1), and MLG-AI-4 (n = 1) and three novel assemblage B MLGs, named MLG-B1 (n = 1), MLG-B2 (n = 14), and MLG-B3 (n = 1). Moreover, two isolates formed two MLG-mixed sequences. The results suggest that donkeys are commonly infected with Giardia in Xinjiang, and there is genetic diversity and host adaptability among the isolates.

Title: Présence et caractéristiques génétiques de Giardia duodenalis chez les ânes du Xinjiang, Chine. Abstract: Giardia duodenalis est un parasite entérique courant chez les humains et les animaux. Pour étudier la présence et les caractéristiques génétiques de Giardia chez les ânes du Xinjiang, en Chine, 758 échantillons fécaux d'ânes ont été collectés et Giardia a été criblé par PCR du gène de l'ARNr SSU. Au total, 17,0 % (129/758) des échantillons ont été testés positifs pour Giardia. Le taux d'infection, respectivement chez les ânes des élevages à grande échelle et domestiques, étaient de 21,4 % (124/580) et 2,8 % (5/178). Les taux chez les ânes de < 1 an et ≥ 1 an étaient respectivement de 19,3 % (72/374) et 12,7 % (41/323). Trois assemblages de Giardia ont été identifiés, l'assemblage B (n = 102) étant l'assemblage prédominant, suivi de l'assemblage A (n = 23) et de l'assemblage E (n = 4). Sur les 129 isolats positifs pour Giardia, 40, 34 et 59 séquences ont été obtenues respectivement au niveau des gènes bg, gdh et tpi. Vingt et un isolats ont permis du génotypage multilocus (MLG), avec quatre nouveaux MLG de l'assemblage A, nommés MLG-AI-1 (n = 1), MLG-AI-2 (n = 1), MLG-AI-3 (n = 1) et MLG-AI-4 (n = 1) et trois nouveaux MLG de l'assemblage B, nommés MLG-B1 (n = 1), MLG-B2 (n = 14) et MLG-B3 (n = 1). De plus, deux isolats formaient deux séquences MLG mélangés. Les résultats suggèrent que les ânes sont couramment infectés par Giardia au Xinjiang, et qu'il existe une diversité génétique et une adaptabilité à l'hôte parmi les isolats.

Molecular evaluation of Cryptosporidium spp. in sheep in southern Xinjiang, China.

Cryptosporidium spp. are diarrheagenic intestinal parasites with multiple hosts worldwide. A total of 1252 fresh fecal samples of sheep were collected from 10 large-scale farms in southern Xinjiang. Based on the small subunit ribosomal (SSU rRNA) gene of Cryptosporidium, 100 Cryptosporidium-positive samples (8.0%, 100/1252) were detected by PCR. Nine out of 10 farms were positive for Cryptosporidium, with the highest infection rate being 18.4% (23/125) on farm 9 in Qira. The infection rates of Cryptosporidium in pre-weaned lambs, weaned lambs, fattening sheep, and adult sheep were 20.3% (61/301), 10.3% (34/329), 0.9% (3/327), and 0.7% (2/295), respectively. Three Cryptosporidium species were identified, namely, C. xiaoi (n = 61), C. parvum (n = 22), and C. ubiquitum (n = 17). Of them, C. xiaoi was detected on all positive farms and in different age groups of sheep. The subtypes of C. parvum and C. ubiquitum were identified by PCR at the 60 kDa glycoprotein (gp60) gene. Two C. parvum subtypes were identified: IIdA19G1 (n = 21) and IIdA15G1 (n = 1). One C. ubiquitum subtype was identified with XIIa (n = 17). These results indicated the common transmission and genetic diversity of Cryptosporidium in sheep in southern Xinjiang, and further investigations are needed on the zoonotic potential of C. parvum and C. ubiquitum in this region.

Dominant infection of Cryptosporidium baileyi in broiler chickens in Zhejiang Province, China.

Cryptosporidium is a common enteric parasite in chickens. A total of 812 fecal specimens were collected from 11 broiler farms in Zhejiang Province, China, and analyzed by nested PCR amplification based on the small subunit ribosomal RNA (SSU rRNA) gene. The overall infection rate of Cryptosporidium was 6.3% (51/812), and five of 11 farms were Cryptosporidium positive. Broilers aged > 90 days accounted for the highest infection rate of 16.1% (6/56), followed by those aged 30-60 days (10.6%, 38/358) and 60-90 days (4/378, 1.1%). Two Cryptosporidium species were identified by sequence analysis, with the predominant species being C. baileyi (96.1%, 49/51) and the minor infection being C. meleagridis (3.9%, 2/51). Based on the 60-kDa glycoprotein (gp60) gene, two C. meleagridis-positive isolates were identified as one known subtype, IIIbA24G1R1. This study indicated the common occurrence of C. baileyi in broiler chickens in this region and low zoonotic transmission potential of Cryptosporidium to humans.

Glyphosate induces autophagy in hepatic L8824 cell line through NO-mediated activation of RAS/RAF/MEK/ERK signaling pathway and energy metabolism disorders.

Glyphosate is an herbicide commonly used worldwide, and its substantial use causes widespread pollution with runoff. However, research on glyphosate toxicity has mostly remained at the embryonic level and existing studies are limited. In the present study, we investigated whether glyphosate can induce autophagy in hepatic L8824 cells by regulating energy metabolism and rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) signaling by activating nitric oxide (NO). First, we selected 0, 50, 200, and 500 µg/mL as the challenge doses, according to the inhibitory concentration of 50% (IC50) of glyphosate. The results showed that glyphosate exposure increased the enzyme activity of inducible nitric oxide synthase (iNOS), which in turn increased the NO content. The activity and expression of enzymes related to energy metabolism, such as hexokinase (HK)1, HK2, phosphofructokinase (PFK), phosphokinase (PK), succinate dehydrogenase (SDH), and nicotinamide adenine dinucleotide with hydrogen (NADH), were inhibited, and the RAS/RAF/MEK/ERK signaling pathway was activated. This led to the negative expression of mammalian target of rapamycin (mTOR) and P62 in hepatic L8824 cells and the activation of the autophagy marker genes microtubule-associated proteins light chain 3 (LC3) and Beclin1 to induce autophagy. The above results were dependent on glyphosate concentration. To verify whether autophagy can be excited by the RAS/RAF/MEK/ERK signaling pathway, we treated L8824 cells with the ERK inhibitor U0126 and found that the autophagy gene LC3 was reduced due to the inhibition of ERK, thus demonstrating the reliability of the results. In conclusion, our results demonstrate that glyphosate can induce autophagy in hepatic L8824 cells by activating NO, thus regulating energy metabolism and the RAS/RAF/MEK/ERK signaling pathway.

Di(2-ethylhexyl) phthalate and microplastics cause necroptosis and apoptosis in hepatocytes of mice by inducing oxidative stress.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer and an endocrine disruptor. Microplastics (MPs) are pathogenic small plastic particles and abundant in the aqueous environment. The problem of residual hazards of plastic products is worthy of study, especially the joint exposure of a variety of plastic-related products to the toxic effect. We used 200 mg/kg DEHP and 10 mg/L MPs to establish exposure model in vivo and 2 mM DEHP and 200 µg/L MPs to establish AML12 cell exposure model in vitro. In vivo study results showed that compared with the control group (NC) group, DEHP and MPs significantly increased the contents of malondialdehyde and hydrogen peroxide, and significantly decreased the contents of glutathione and the activity of superoxide dismutase, total antioxidant capacity, catalase and glutathione peroxidase. The level of oxidative stress was further aggravated after combined exposure. The reactive oxygen species level of AML12 exposed to DEHP and MPs in vitro was significantly higher than NC group, and the combined exposure was significantly higher than the single exposure. The in vivo and in vitro also confirmed that DEHP and MPs could significantly increase the mRNA and protein levels of apoptosis markers and necroptosis markers and there was an additive effect. After N-acetylcysteine treatment in vitro, the above-mentioned oxidative stress level and cell damage decreased significantly. This study provided a reference for advocating the reduction of the mixed use of plastic products, and provided a basis for preventing the harm of plastic products residues.

Tannin alleviates glyphosate exposure-induced apoptosis, necrosis and immune dysfunction in hepatic L8824 cell line by inhibiting ROS/PTEN/PI3K/AKT pathway.

Glyphosate can cause tissue damage such as liver and kidney in mammals. Tannin has anti-inflammatory, antibacterial and anti-inflammatory properties. However, the effect of glyphosate on the growth of L8824 cell line and the effect of tannin on antagonism of glyphosate through the ROS/PTEN/PI3K/AKT axis are unclear. In this study, L8824 cells were treated with glyphosate (50 µg/mL) and/or tannin (4.5 µM) for 24 h to establish a model. The results showed that glyphosate exposure increased ROS and MDA levels, decreased CAT and SOD activities. PTEN was activated and the PI3K/AKT signaling pathway was inhibited. The P53/Bcl-2/Bax/CytC/Caspase3 and RIPK1/RIPK3/MLKL pathways were also activated. In addition, the cytokines and antimicrobial peptides LEAP-2, TNF-α and IL-1ß were increased while ß-defensin, Hepcidin, IL-2 and IFN-γ were decreased. The use of tannin reduced the adverse effects of glyphosate exposure on L8824 cells significantly. In conclusion, tannin can trigger oxidative stress via PTEN/PI3K/AKT pathway to cause apoptosis, necroptosis and immune dysfunction of L8824 cells.

Identification and genetic characterization of Blastocystis subtypes in Père David's deer (Elaphurus davidianus) from Shishou, China.

Blastocystis is a protozoan parasite frequently reported in the gastrointestinal tract of humans and animals, with a global distribution. No information on the infections of Blastocystis in Père David's deer (Elaphurus davidianus, PDD) is available. In this study, a total of 128 fecal samples collected in the National Nature Reserve of Shishou, Hubei Province of China, were used to determine the occurrence and subtypes of Blastocystis in PDD. Amplification of the SSU rDNA gene confirmed the common presence of Blastocystis infection, with an observed prevalence of 56.3% (72/128). Through nucleotide sequencing and phylogenetic analysis, five known subtypes, which consisted of one zoonotic subtype (ST10) and four ruminant-specifical subtypes (ST21, ST23, ST25, and ST26), were identified in this study. This represents a high degree genetic diversity of parasites within the host. Of the 72 PCR-positive samples, ruminant-specific subtypes were the most frequently observed, with ST21 being the most prevalent subtype (56.9.7%, 41/72). This is the first report characterizing the molecular subtypes of Blastocystis in PDD. The findings demonstrate a broad diversity and zoonotic potential of Blastocystis in PDD. Further study of the transmission potential between wildlife and humans or domestic animals in the region is required.

Molecular detection and genetic characteristics of Cryptosporidium spp. in Chinese racehorses.

BACKGROUND: Cryptosporidium is a protozoan parasite causing diarrhoea in humans and animals. Although Cryptosporidium has been found in domestic horses (farmed or kept at pasture), there has been only one published study of Cryptosporidium infections in Chinese racehorses, which was restricted to a very small geographical area. OBJECTIVES: To investigate the presence of Cryptosporidium spp. in the faeces of racehorses in China and to perform molecular characterisation of the parasite. STUDY DESIGN: Cross-sectional. METHODS: A total of 621 fresh faecal samples were collected for DNA extraction from racehorses at 17 equestrian clubs from 12 provinces of China from December 2016 to May 2018. All the DNA were analysed for the presence of Cryptosporidium species/genotypes and subtypes by PCR amplification of the small subunit ribosomal RNA and 60 kDa glycoprotein genes respectively. RESULTS: PCR analysis revealed that 11 samples (1.8%) were positive for Cryptosporidium spp. Among them seven samples were identified as C. parvum and four were C. hominis. The C. parvum isolates were identified as subtype IIdA14G1 (n = 4) and IIdA15G1 (n = 3), while all C. hominis isolates were identified as subtype IkA18G1 (n = 4). MAIN LIMITATIONS: A single faecal sample from each horse was used instead of multiple samples that could improve the detection rates of the parasite. CONCLUSIONS: Although Cryptosporidium infection rate was relatively low in the investigated racehorses, the presence of zoonotic subtypes IIdA14G and 1IIdA15G1 of C. parvum and IkA18G1 of C. hominis, suggesting that these animals are a potential source of Cryptosporidium in humans.

Prevalence and genetic characterization of Theileria equi and Babesia caballi in grazing horses in Xinjiang, northwestern China.

Piroplasmosis is a disease that negatively affects equine health worldwide. Hence, 324 blood samples were collected from grazing horses in ten sites in Xinjiang and testing them for the presence of Theileria equi and Babesia caballi by PCR of the EMA-1 gene and BC48 gene, respectively. Of the 324 blood samples, 161 (49.7%) were positive for equine piroplasms. The prevalence of T. equi was 38.9% (126/324), while that of B. caballi was 30.2% (98/324). The T. equi and B. caballi co-infection rate was 19.4% (63/324). From the 126 EMA-1 gene sequences and 98 BC48 gene sequences we obtained, 21 and 27 genotypes were identified, respectively. The EMA-1 sequences together with the GenBank reference sequences grouped into four clusters, with those from the present study forming two distinct clusters. In contrast, the BC48 sequences formed eight clusters with the GenBank reference sequences, while those obtained in the present study formed five distinct clusters. Our results highlight the widespread distribution and abundant gene polymorphism of T. equi and B. caballi in grazing horses from Xinjiang.

Genetic characterization of Cryptosporidium spp. in Hotan Black Chickens in China reveals two novel subtypes of Cryptosporidium meleagridis.

A total of 617 fecal specimens were collected on 18 Hotan Black chicken farms in Southern Xinjiang, China, and tested for the presence of Cryptosporidium spp. by PCR of the small subunit ribosomal RNA (SSU rRNA) gene. The overall infection rate by Cryptosporidium spp. was 11.5% (71/617), and ten of the 18 farms were positive. The infection rate by Cryptosporidium spp. was 14.5% (48/331) in the 30-60 d group, higher than chickens in the <30 d (12.0%, 15/125), 60-90 d (6.9%, 5/72), and >90 d (3.4%, 3/89) groups. Cryptosporidium meleagridis (n = 38) and C. baileyi (n = 33) were confirmed by sequencing analysis. A total of 25 of the 38 C. meleagridis-positive specimens were subtyped successfully at the gp60 gene, including one known subtype (IIIbA23G1R1, n = 1) and two novel subtypes, named IIIbA25G1R1 (n = 20) and IIIbA31G1R1 (n = 4). The results showed that infection by Cryptosporidium spp. in Hotan Black Chickens was common in this area and the distribution of C. meleagridis subtypes had regional characteristics.

Title: La caractérisation génétique de Cryptosporidium spp. chez les poulets noirs du Hotan en Chine révèle deux nouveaux sous-types de Cryptosporidium meleagridis. Abstract: Un total de 617 échantillons fécaux ont été prélevés dans 18 élevages de poulets noirs du Hotan dans le sud du Xinjiang, en Chine, et testés pour la présence de Cryptosporidium spp. par PCR du gène de la petite sous-unité de l'ARN ribosomique (ARNr SSU). Le taux d'infection global par Cryptosporidium spp. était de 11,5 % (71/617) et dix des 18 élevages étaient positifs. Le taux d'infection de Cryptosporidium spp. était de 14,5 % (48/331) dans le groupe 30­60 jours, supérieur à celui des poulets dans les groupes <30 jours (12,0 %, 15/125), 60­90 jours (6,9 %, 5/72) et >90 jours (3,4 %, 3/89). Cryptosporidium meleagridis (n = 38) et C. baileyi (n = 33) ont été confirmés par analyse de séquençage. Vingt-cinq des 38 spécimens positifs pour C. meleagridis ont été sous-typés avec succès au niveau du gène gp60, dont un sous-type connu (IIIbA23G1R1, n = 1) et deux nouveaux sous-types, nommés IIIbA25G1R1 (n = 20) et IIIbA31G1R1 (n = 4). Les résultats ont montré que l'infection par Cryptosporidium spp. chez les poulets noirs du Hotan était commune dans cette zone et que la distribution des sous-types de C. meleagridis avait des caractéristiques régionales.

First detection of coccidia species in blue peafowl (Pavo cristatus) in China via both microscopic and molecular methods.

Coccidia are protozoan parasites in the class Conoidasida. To determine the prevalence and molecular characteristics of coccidia species in blue peafowl (Pavo cristatus) in Henan, China, 240 fecal specimens were collected and screened for the presence of Eimeria spp. and Isospora spp. The overall prevalence was 65.0% (156/240), and seven different coccidia species were identified: E. pavonis (51.3%, 123/240), E. arabic (40.0%, 96/240), E. riyadhae (37.1%, 89/240), E. mandali (22.9%, 55/240), E. mayurai (14.2%, 34/240), I. mayuir (10.9%, 26/240), and I. lacazei (8.5%, 21/240). E. arabic and E. riyadhae were detected for the first time in China. Additionally, we provide molecular data of the seven different coccidia species at the 18S and 28S ribosomal RNA and the COI loci. Sequence homology percentages among the five species of Eimeria at the 18S rRNA, 28S rRNA, and COI loci were 96.0%-98.6%, 90.7%-98.2%, and 85.0%-94.9%, respectively, whereas for two species of Isospora the sequence homology percentages were 98.8%, 99.1%, and 95.4% at three corresponding loci. This is the first report of the molecular data of the seven coccidia species in blue peafowl in China.

Cineole regulates Wnt/ß-catenin pathway through Nrf2/keap1/ROS to inhibit bisphenol A-induced apoptosis, autophagy inhibition and immunosuppression of grass carp hepatocytes.

Bisphenol A (BPA), an environmental pollutant, can cause multiple organ tissue damage by inducing oxidative stress. Cineole (CIN) is a terpene oxide existing in a variety of plant essential oils, which has anti-inflammatory, analgesic, and antioxidant effects. This study examined the effects of 200 nM BPA and 20 µM CIN on apoptosis, autophagy, and immunology in grass carp hepatocytes (L8824). The treatments were categorized as NC, CIN, BPA + CIN, and BPA. The findings demonstrated that BPA exposure could increase ROS levels and oxidative stress-related indicators, decrease the expression of the Nrf2/keap1 pathway and the Wnt/ß-catenin pathway, increase the expression of genes involved in the apoptotic pathway (Bax and Caspase3), and decrease the expression of the anti-apoptotic gene Bcl-2 by lowering mitochondrial membrane potential. BPA also reduced the expression of genes linked to autophagy (ATG5, Beclin1, LC3). Changes in immunological function after BPA exposure were also shown by changes in the amounts of antimicrobial peptides (HEPC, ß-defensin, LEAP2) and cytokines (INF-γ, IL-1ß, IL-2, and TNF-α). After the co-treatment of CIN and BPA, CIN can inhibit BPA-induced apoptosis and recover from autophagy and immune function to a certain extent by binding to keap1 to exert an anti-oxidative regulatory effect of Nrf2 incorporation into the nucleus. Molecular docking provides strong evidence for the interaction of CIN ligands with keap1 receptors. Therefore, these results indicated that CIN could inhibit BPA-induced apoptosis, autophagy inhibition and immunosuppression in grass carp hepatocytes by regulating the Wnt/ß-catenin pathway with Nrf2/keap1/ROS. This study provided further information to the risk assessment of the neuroendocrine disruptor BPA on aquatic organisms and offered suggestions and resources for further research into the function of natural extracts in the body's detoxification process.

Longitudinal surveillance of Cryptosporidium spp. in broiler chickens in Xinjiang, northwest China: genetic diversity of Cryptosporidium meleagridis subtypes.

Cryptosporidium spp. are common enteric parasites in humans and animals. Herein, 175 faecal specimens were collected from a broiler farm in Xinjiang, China, including seven repeated samplings at 10-day intervals of broilers aged 10 to 70 days. Cryptosporidium was detected and identified by PCR-RFLP analysis. The overall infection rate of Cryptosporidium in broilers was 23.4% (41/175), with the highest infection rate of 48.0% (12/25) at 40 days of age, and no infection was detected at 10 days of age. Two Cryptosporidium species were confirmed, namely, C. baileyi (3.4%, 6/175) and C. meleagridis (20%, 35/175). In total, 21 of 35 C. meleagridis isolates were successfully subtyped based on the gp60 gene, and one known subtype, IIIgA22G3R1 (n = 1), and three novel subtypes, IIIbA25G1R1 (n = 10), IIIgA24G3R1 (n = 9) and IIIgA25G2R1 (n = 1), were identified. Our findings highlight the genetic diversity of C. meleagridis in Xinjiang and the potential endemic characteristics of the subtypes.

Genotyping of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi from sheep and goats in China.

BACKGROUND: Few studies have molecularly characterized the potential zoonotic protozoa, Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in sheep and goats in China, therefore total 472 fecal samples were collected from eight provinces and infection rates of three protozoa were determined by PCR analysis of corresponding loci. All PCR positive samples were sequenced to identify the genotype. RESULTS: The overall infection rates for Cryptosporidium, G. duodenalis, and E. bieneusi were 1.9% (9/472), 20.6% (97/472), and 44.5% (210/472), respectively. C. xiaoi (n = 5), C. ubiquitum (n = 3), and C. anderson (n = 1) were identified in goats. 97 G. duodenalis strains were successfully detected, and assembly E (n = 96) and assembly A (n = 1) were identified. Two novel G. duodenalis multilocus genotype (MLGs) were identified, with one belonging to subgroup AI and the other to subgroup E5. Nine known genotype (BEB6, CD6, CHC8, CHG3, CHG5, Peru6, CHG1, CHG2, and COS-I) and four new genotype (CHG26, CHG27, CHG28, and CHS18) were identified in E. bieneusi, with CHG3 dominant in this group. CONCLUSIONS: The present results highlight the role of sheep and goats as reservoir hosts for this three gastrointestinal pathogens. In summary, we provided a platform for more detailed research on genotyping or subtyping intestinal pathogens to better understand their risks and modes of transmission.

Molecular detection and characterization of Enterocytozoon bieneusi in captive alpine musk deer (Moschus chrysogaster) in Central and Eastern of Gansu Province, China.

The most ubiquitously diagnosed microsporidian species in animals and humans is Enterocytozoon bieneusi (E. bieneusi). In this case, this work aimed to probe the occurrence and genotypes of this pathogen in captive alpine musk deer (Moschus chrysogaster) in Gansu Province, China. After fecal sample collection (n = 201) from three farms in Gansu, the internal transcribed spacer (ITS) region of the rRNA gene was probed by PCR for detection and genotyping of E. bieneusi. The infection rate of E. bieneusi in alpine musk deer was 6.0% (12/201), with 0% (0/36), 7.7% (5/65) and 7.0% (7/100) in farms 1 to 3, respectively. The infection rate of E. bieneusi in young alpine musk deer (3.2%; 1/31) is lower than that of adults (6.5%; 11/170), with no evident significant differences between age groups (P > 0.05). Three known genotypes D (n = 8), EbpA (n = 3) and BEB6 (n = 1) were identified by sequence analysis. This is the first such scrutiny of E. bieneusi infection in alpine musk deer in China as per our knowledge. Genotypes D and EbpA were common in humans and animals that is suggestive of the plausible zoonotic role in E. bieneusi transmission by alpine musk deer.

Neospora caninum inhibits tumor development by activating the immune response and destroying tumor cells in a B16F10 melanoma model.

BACKGROUND: Melanoma is a malignant tumor with a high mortality rate. Some microorganisms have been shown to activate the immune system and limit cancer progression. The objective of this study is to evaluate the anti-melanoma effect of Neospora caninum, a livestock pathogen with no pathogenic activity in humans. METHODS: Neospora caninum tachyzoites were inoculated into a C57BL/6 mouse melanoma model by intratumoral and distal subcutaneous injections. Tumor volumes were measured, and cell death areas were visualized by hematoxylin and eosin staining and quantified. Apoptosis in cell cultures and whole tumors was detected by propidium iodide (PI) and TUNEL staining, respectively. Cytokine and tumor-associated factor levels in tumors and spleens were detected by real-time quantitative polymerase chain reaction. Infiltration of macrophages and CD8+ T cells in the tumor microenvironment (TME) were detected by immunohistochemistry with anti-CD68 and anti-CD8 antibodies, respectively. Finally, 16S rRNA sequencing of mice cecal contents was performed to evaluate the effect of N. caninum on gut microbial diversity. RESULTS: Intratumoral and distal subcutaneous injections of N. caninum resulted in significant inhibition of tumor growth (P < 0.001), and more than 50% of tumor cells were dead without signs of apoptosis. Neospora caninum treatment significantly increased the mRNA expression levels of IL-12, IFN-γ, IL-2, IL-10, TNF-α, and PD-L1 in the TME, and IL-12 and IFN-γ in the spleen of tumor-bearing mice (P < 0.05). An increase in the infiltration of CD8+ T cells and macrophages in the TME was observed with these cytokine changes. Neospora caninum also restored the abundance of gut microbiota Lactobacillus, Lachnospiraceae, Adlercreutzia, and Prevotellaceae associated with tumor growth, but the changes were not significant. CONCLUSION: Neospora caninum inhibits B16F10 melanoma by activating potent immune responses and directly destroying the cancer cells. The stable, non-toxic, and efficacious properties of N. caninum demonstrate the potential for its use as a cancer treatment.

Molecular detection and genetic characterization of Cryptosporidium in kindergarten children in Southern Xinjiang, China.

Cryptosporidium is a common cause of diarrhea in children globally. However, there is limited information on the prevalence and genetic characteristics of Cryptosporidium in children in Xinjiang, China. This study aimed to assess the genetic characteristics and epidemiological status of Cryptosporidium in kindergarten children in Southern Xinjiang, China. A total of 609 fecal samples were collected from kindergartners aged 2-6 years from 11 counties in Southern Xinjiang, China. We used nested PCR amplification of the partial SSU rDNA gene to screen samples for Cryptosporidium spp. Isolates containing Cryptosporidium parvum and C. hominis were further subtyped for a gene encoding a 60-kDa glycoprotein (gp60). We used MEGA7 to construct a phylogenetic tree to study the genetic relationship between the gp60 subtypes of these two species via the Maximum Likelihood method based on the Tamura-Nei model. Only 1.3% (8/609) of asymptomatic children were confirmed to be infected with Cryptosporidium, with a 2.0% (6/299) infection rate in boys and 0.6% (2/310) infection rate in girls. Three Cryptosporidium species were identified including C. felis (37.5%; 3/8), C. hominis (37.5%; 3/8), and C. parvum (25.0%; 2/8). Three C. hominis subtypes (IbA9G3, IdA14, and IfA12G1) and two C. parvum subtypes (IIdA14G1 and IIdA15G1) were also found. This study is the first to identify the presence of Cryptosporidium in kindergarten children in Southern Xinjiang, China. The presence of zoonotic C. parvum subtypes IIdA14G1 and IIdA15G1 indicates the possible cross-species transmission of Cryptosporidium between children and animals.