Impacts of a highly pathogenic ovine Eimeria ovinoidalis on the growth of Hu lambs.

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.

Molecular characterization and zoonotic risk assessment of Cryptosporidium spp. in children and calves in Bangladesh.

Cryptosporidium is a gastro-intestinal protozoan parasite that has been found to infect both humans and livestock. This study investigated the parasite in 998 fecal samples from Bangladeshi children (n = 299) and calves (n = 699) to determine its prevalence, genetic variation, and zoonotic importance. The nested PCR and sequencing of the SSU rRNA gene in the samples showed a Cryptosporidium infection rate of 2.3% (7/299) in children and 15.7% (110/699) in calves. Statistical analysis revealed insignificant variations in Cryptosporidium infections among children across age, gender, and study area, while in calves, the infection rate significantly differed based on location and breed. Genotyping of seven human isolates of Cryptosporidium confirmed C. hominis (n = 5) and C. parvum (n = 2). After characterizing 110 Cryptosporidium isolates from calves, C. andersoni (n = 55), C. ryanae (n = 29), C. bovis (n = 14), C. parvum (n = 10), C. ubiquitum (n = 1), and C. occultus (n = 1) were identified. Cryptosporidium hominis and C. parvum-positive samples were further subjected to nested PCR and sequencing of the glycoprotein 60 (gp60) gene for subtyping. Four C. hominis subtypes (IaA19R3, IaA23R3, IbA9G3, and IdA15G1) and one C. parvum subtype (IIdA15G1) were observed. In conclusion, Cryptosporidium was prevalent in calves but less common in children in the study locations, and the presence of zoonotic Cryptosporidium species and subtypes in calves raises concerns regarding zoonotic transmission to humans.

Occurrence and genotyping of Enterocytozoon bieneusi in flying squirrels (Trogopterus xanthipes) from China.

Enterocytozoon bieneusi is an obligate intracellular microsporidian parasite with a worldwide distribution. As a zoonotic pathogen, E. bieneusi can infect a wide range of wildlife hosts through the fecal-oral route. Although the feces of flying squirrels (Trogopterus xanthipes) are considered a traditional Chinese medicine (as "faeces trogopterori"), no literature is available on E. bieneusi infection in flying squirrels to date. In this study, a total of 340 fresh flying squirrel fecal specimens from two captive populations were collected in Pingdingshan city, China, to detect the prevalence of E. bieneusi and assess their zoonotic potential. By nested PCR amplification of the ITS gene, six specimens tested positive, with positive samples from each farm, with an overall low infection rate of 1.8%. The ITS sequences revealed three genotypes, including known genotype D and two novel genotypes, HNFS01 and HNFS02. Genotype HNFS01 was the most prevalent (4/6, 66.7%). Phylogenetic analysis showed that all genotypes clustered into zoonotic Group 1, with the novel genotypes clustering into different subgroups. To our knowledge, this is the first report of E. bieneusi infection in flying squirrels, suggesting that flying squirrels could act as a potential reservoir and zoonotic threat for E. bieneusi transmission to humans in China.

Title: Occurrence et génotypage d'Enterocytozoon bieneusi chez les écureuils volants (Trogopterus xanthipes) de Chine. Abstract: Enterocytozoon bieneusi est un parasite microsporidien intracellulaire obligatoire présent dans le monde entier. En tant qu'agent pathogène zoonotique, E. bieneusi peut infecter un large éventail d'hôtes sauvages par la voie fécale-orale. Bien que les excréments d'écureuils volants (Trogopterus xanthipes) soient considérés comme un ingrédient de médecine traditionnelle chinoise (comme « faeces trogopterori ¼), aucune littérature n'est disponible à ce jour sur l'infection par E. bieneusi chez les écureuils volants. Dans cette étude, un total de 340 spécimens fécaux frais d'écureuils volants provenant de deux populations captives ont été collectés dans la ville de Pingdingshan, en Chine, pour détecter la prévalence d'E. bieneusi et évaluer leur potentiel zoonotique. Par amplification PCR nichée du gène ITS, six échantillons se sont révélés positifs, avec des échantillons positifs dans chaque ferme, et un taux d'infection global faible, à 1,8 %. Les séquences ITS ont révélé trois génotypes, dont le génotype D connu et deux nouveaux génotypes, HNFS01 et HNFS02. Le génotype HNFS01 était le plus répandu (4/6, 66,7 %). L'analyse phylogénétique a montré que tous les génotypes se regroupaient dans le groupe zoonotique 1, les nouveaux génotypes se regroupant en différents sous-groupes. À notre connaissance, il s'agit du premier rapport d'infection par E. bieneusi chez des écureuils volants, ce qui suggère que les écureuils volants pourraient agir comme un réservoir potentiel et une menace zoonotique pour la transmission d'E. bieneusi aux humains en Chine.

miR-181d targets BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to Cryptosporidium parvum infection via the intrinsic apoptosis pathway.

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.

Predominance of the Blastocystis subtype ST5 among free-living sympatric rodents within pig farms in China suggests a novel transmission route from farms.

Blastocystis is a parasitic protist that can infect humans and various domestic and wild animals. However, there is limited research on the prevalence of this parasite among rodents, particularly those living in pig farm settings. Therefore, to investigate the occurrence, molecular characterization, and zoonotic potential of Blastocystis among rodents within pig farm environments, we conducted an investigation of 227 rodents and shrews from 34 pig farms located in Henan, Shaanxi, and Shanxi provinces of China using nested PCR of the SSU rRNA gene of Blastocystis. The potential transmission and public health implications were also assessed from a One Health perspective. Blastocystis was detected in 86 (37.9%) fecal samples. The highest infection rate was observed among Ruttus norvegicus (73.7%, 42/58), followed by Ruttus tanezumi (30.1%, 41/136), and Mus musculus (12.0%, 3/25). However, it was not detected among individuals with Apodemus agrarius (n = 1) and Crocidura shantungensis (n = 7). Five known zoonotic Blastocystis subtypes (ST1-ST5) were identified, with ST4 (51.2%, 44/86) and ST5 (40.7%, 35/86) being the predominant ones, followed by ST1 (3.5%, 3/86), ST3 (3.5%, 3/86), and ST2 (1.2%, 1/86). ST4 was prevalent among R. norvegicus (83.3%, 35/42), while ST5 dominated R. tanezumi (70.7%, 29/41). Furthermore, ST5 exhibited the widest distribution at pig farm level, accounting for 65.0% (13/20) of Blastocystis-positive pig farms. This investigation presents the first documented Blastocystis infection in R. tanezumi and M. musculus, highlighting the predominant presence of the zoonotic ST5 subtype in rodents for the first time. The results demonstrate that sympatric rodents can serve as natural reservoirs for Blastocystis and play a role in its transmission. These findings provide information on the dynamics of rodent transmission and emphasize the potential public health threat posed by zoonotic Blastocystis subtypes spillover from pig farms.

The global prevalence of Cyclospora cayetanensis infection: A systematic review, meta-analysis, and meta-regression.

Cyclospora cayetanensis (C. cayetanensis) is a significant pathogen that causes diarrheal illness and causes large foodborne diarrhea outbreaks in the USA and Canada. However, there is currently a lack of published meta-analysis on the prevalence of C. cayetanensis infection in the global population. A real estimation of a disease prevalence should always be done on the basis of studies designed for that purpose. We conducted a comprehensive search of various databases for articles pertaining to the prevalence of C. cayetanensis infection in humans, spanning from the inception of these databases to March 10, 2023. Utilizing a random effects model, we estimated the prevalence of C. cayetanensis infection in humans. Our analysis included a total of 150 datasets sourced from 42 different countries, which were ultimately selected for the final quantitative assessment. The prevalence of C. cayetanensis infection in humans worldwide was estimated to be 3.4 % (5636/166,611). Notably, Africa exhibited the highest prevalence rate at 5.9 % (606/11,068). Further subgroup analysis revealed a significantly higher infection rate in humans residing in low-income countries (7.6 %, 83/921) compared to those in lower-middle-income countries (4.8 %, 3280/48,852), upper-middle-income countries (2.9 %, 2194/99,419), and high-income countries (0.4 %, 79/17,419). The results indicate that the global prevalence of C. cayetanensis infection in humans is relatively low, despite its extensive geographical distribution and children were found to be more susceptible to C. cayetanensis infection compared to those adults. Sensitivity analysis revealed that one study significantly affects the prevalence of C. cayetanensis, which was adjusted to 2.9 % (4017/160,049; 95 % CI: 2.7-3.1 %) by excluding this study. The findings highlight the relatively high prevalence of C. cayetanensis infection in low-income countries and among humans with diarrhea, particularly in Africa. Consequently, routine surveillance for intestinal protozoa is crucial in these regions.

Identification of host protein ENO1 (alpha-enolase) interacting with Cryptosporidium parvum sporozoite surface protein, Cpgp40.

BACKGROUND: Cryptosporidium parvum is an apicomplexan zoonotic parasite causing the diarrheal illness cryptosporidiosis in humans and animals. To invade the host intestinal epithelial cells, parasitic proteins expressed on the surface of sporozoites interact with host cells to facilitate the formation of parasitophorous vacuole for the parasite to reside and develop. The gp40 of C. parvum, named Cpgp40 and located on the surface of sporozoites, was proven to participate in the process of host cell invasion. METHODS: We utilized the purified Cpgp40 as a bait to obtain host cell proteins interacting with Cpgp40 through the glutathione S-transferase (GST) pull-down method. In vitro analysis, through bimolecular fluorescence complementation assay (BiFC) and coimmunoprecipitation (Co-IP), confirmed the solid interaction between Cpgp40 and ENO1. In addition, by using protein mutation and parasite infection rate analysis, it was demonstrated that ENO1 plays an important role in the C. parvum invasion of HCT-8 cells. RESULTS: To illustrate the functional activity of Cpgp40 interacting with host cells, we identified the alpha-enolase protein (ENO1) from HCT-8 cells, which showed direct interaction with Cpgp40. The mRNA level of ENO1 gene was significantly decreased at 3 and 24 h after C. parvum infection. Antibodies and siRNA specific to ENO1 showed the ability to neutralize C. parvum infection in vitro, which indicated the participation of ENO1 during the parasite invasion of HCT-8 cells. In addition, we further demonstrated that ENO1 protein was involved in the regulation of cytoplasmic matrix of HCT-8 cells during C. parvum invasion. Functional study of the protein mutation illustrated that ENO1 was also required for the endogenous development of C. parvum. CONCLUSIONS: In this study, we utilized the purified Cpgp40 as a bait to obtain host cell proteins ENO1 interacting with Cpgp40. Functional studies illustrated that the host cell protein ENO1 was involved in the regulation of tight junction and adherent junction proteins during C. parvum invasion and was required for endogenous development of C. parvum.

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in pet cats in Henan Province, central China.

Cryptosporidium spp. and G. duodenalis often infect humans, cats, and other mammals, causing diarrhea and being responsible for numerous outbreaks of waterborne and foodborne infections worldwide. The rapid increase in the number of pet cats poses a substantial public health risk. However, there were few reports about the infection of Cryptosporidium spp. and G. duodenalis infections in pet cats in Henan Province, central China. Thus, to understand the prevalence and genetic distribution of Cryptosporidium spp. and G. duodenalis in pet cats, and to evaluate the zoonotic potential, possible transmission routes and public health implications of isolates, fecal samples (n = 898) were randomly collected from pet cats in 11 cities in Henan Province, central China. Nested PCR based on the SSU rRNA gene and bg gene was used to the prevalence of Cryptosporidium spp. and G. duodenalis, respectively. The prevalence was 0.8 % (7/898) and 2.0 % (18/898) for Cryptosporidium spp. and G. duodenalis respectively. Additionally, the Cryptosporidium spp. positive isolates were identified as C. parvum subtype IIdA19G1 by gp60 gene. In the present study, the IIdA19G1 subtype was discovered in pet cats for the first time in China, enriching the information on the host type and geographical distribution of Cryptosporidium spp. in China. For G. duodenalis, a total of 18 G. duodenalis positive samples were identified, belonging to four assemblages: a zoonotic assemblage A1 (4/898), three host-specific assemblages C (8/898), D (5/898), and F (1/898). Interestingly, we found that pet cats infected with Cryptosporidium spp. and G. duodenalis are more likely to experience emaciation symptoms compared to the negative group. More importantly, the prevalence of Cryptosporidium spp. and G. duodenalis detected in the present study were low, but the subtype IIdA19G1 of Cryptosporidium spp. and the assemblages A1, C, D, and F of G. duodenalis have the potential for zoonotic transmission. Thus, we should focus on preventing and controlling the risk of cross-species transmission that may occur in pet cats in Henan Province.

Host specific Eimeria genus diagnosis and qPCR development in Ovis aries and Capra hircus.

The objective of the present study was to develop a real-time PCR (qPCR) technique for the diagnosis of Eimeria spp. in Ovis aries and Capra hircus. The qPCR technique was developed using SYBR Green, resulting in a PCR with high sensitivity, specificity, and reproducibility.

Widespread distribution of human-infective Enterocytozoon bieneusi genotypes in small rodents in northeast China and phylogeny and zoonotic implications revisited.

Enterocytozoon bieneusi features high genetic diversity among host species and environmental sources and over 500 genotypes in 11 phylogenetic groups have been defined. Here we investigated 291 small rodents in Heilongjiang province, northeast China, for the presence of E. bieneusi by PCR of the ribosomal internal transcribed spacer (ITS). Nine of 60 (15.0 %) gray squirrels from a park in Harbin, 120 of 201 (59.7 %) guinea pigs from a pet shop in Harbin, and two of 30 (6.7 %) peridomestic rats from a pasture in Qiqihar were positive for the parasite. Six known genotypes (EbpB, SCC-1, SCC-2, D, S7 and HLJ-CP1) and two novel genotypes (NESQ1 and NEGP1) were identified by sequence analysis of the ITS, with EbpB, SCC-1, SCC-2 and NESQ1 found in squirrels, D, S7 and NEGP1 in guinea pigs, and EbpB and HLJ-CP1 in rats. Widespread distribution of human-infective Group 10 genotype S7 and Group 1 genotype D in guinea pigs raised our concerns about the importance of pet animals as zoonotic reservoirs of microsporidiosis. Co-occurrence of Group 1 genotypes D and HLJ-CP1 in cancer patients and rodents in Heilongjiang indicated a possibility of zoonotic transmission. The host range of Group 1 genotype EbpB previously considered pig-adapted was extended. A potential variant of genotype S7, namely NESQ1, went into the existing Group 10 in phylogenetic analysis. The other new genotype, NEGP1, was clustered in an undefined clade we proposed as Group 15. With the emerging epidemiologic evidence, the host specificity of existing E. bieneusi genotypes is now being challenged.

Molecular epidemiology of Enterocytozoon bieneusi from foxes and raccoon dogs in the Henan and Hebei provinces in China.

BACKGROUND: Enterocytozoon bieneusi is a zoonotic pathogen widely distributed in animals and humans. It can cause diarrhea and even death in immunocompromised hosts. Approximately 800 internal transcribed spacer (ITS) genotypes have been identified in E. bieneusi. Farmed foxes and raccoon dogs are closely associated to humans and might be the reservoir of E. bieneusi which is known to have zoonotic potential. However, there are only a few studies about E. bieneusi genotype identification and epidemiological survey in foxes and raccoon dogs in Henan and Hebei province. Thus, the present study investigated the infection rates and genotypes of E. bieneusi in farmed foxes and raccoon dogs in the Henan and Hebei provinces. RESULT: A total of 704 and 884 fecal specimens were collected from foxes and raccoon dogs, respectively. Nested PCR was conducted based on ITS of ribosomal RNA (rRNA), and then multilocus sequence typing (MLST) was conducted to analyze the genotypes. The result showed that infection rates of E. bieneusi in foxes and raccoon dogs were 18.32% and 5.54%, respectively. Ten E. bieneusi genotypes with zoonotic potential (NCF2, NCF3, D, EbpC, CHN-DC1, SCF2, CHN-F1, Type IV, BEB4, and BEB6) were identified in foxes and raccoon dogs. Totally 178 ITS-positive DNA specimens were identified from foxes and raccoon dogs and these specimens were then subjected to MLST analysis. In the MLST analysis, 12, 2, 7 and 8 genotypes were identified in at the mini-/ micro-satellite loci MS1, MS3, MS4 and MS7, respectively. A total of 14 multilocus genotypes were generated using ClustalX 2.1 software. Overall, the present study evaluated the infection of E. bieneusi in foxes and raccoon dogs in the Henan and Hebei province, and investigated the zoonotic potential of the E. bieneusi in foxes and raccoon dogs. CONCLUSIONS: These findings expand the geographic distribution information of E. bieneusi' host in China and was helpful in preventing against the infection of E. bieneusi with zoonotic potential in foxes and raccoon dogs.

Occurrence and genotypic identification of Blastocystis sp., Enterocytozoon bieneusi, and Giardia duodenalis in dairy cattle in Heilongjiang Province, China.

Blastocystis sp., Enterocytozoon bieneusi, and Giardia duodenalis are three common zoonotic intestinal parasites, and cattle are important hosts of these three intestinal protozoa. In this study, 1632 fecal samples were collected from dairy farms in Heilongjiang Province, China, and screened for Blastocystis sp., E. bieneusi, and G. duodenalis using polymerase chain reaction. Of these, 149 (9.13%) were positive for three zoonotic pathogens, including 104 (6.40%), 22 (1.35%), and 23 (1.41%) for Blastocystis sp., E. bieneusi, and G. duodenalis, respectively. Based on partial SSU rRNA gene sequencing analysis, 104 positive samples of Blastocystis sp. were found, and a total of nine known subtypes were identified, including ST10 (61), ST3 (18), ST14 (6), ST26 (7), ST24 (3), ST25 (2), ST1 (2), ST5 (2), and ST21 (1). Among these, three subtypes (ST1, ST3, and ST5) were recognized as zoonotic subtypes, and two subtypes (ST10 and ST14) were specific to animals. All 23 Giardia duodenalis-positive samples belonged to assemblage E (n = 23) based on sequenced beta-giardin (bg) and triosephosphate isomerase (tpi) genes. Three known genotypes of E. bieneusi, namely J (n = 9), I (n = 6), and BEB4 (n = 7), were identified by sequence analysis of the internal transcriptional spacer region gene. Our study provides basic data for prevention and control in Heilongjiang Province; however, further research is required to better understand the prevalence and public health significance of these pathogens in the Heilongjiang region.

Study on genetic characteristics of Cryptosporidium isolates and first report of C. parvum IIdA24G2 subtype in dairy cattle in China.

Cryptosporidium is an important gastrointestinal parasite that can cause mild to severe diarrhea in various vertebrates, including humans and domestic animals. Infection is prevalent in dairy cattle, particularly calves, resulting in diarrhea and increased mortality with significant production losses. However, the prevalence and identity of Cryptosporidium spp. in cattle in Heilongjiang Province is still poorly known. Our study aimed to investigate the prevalence and species and subtype distribution of Cryptosporidium in cattle in the region. In addition, we evaluated the zoonotic potential of Cryptosporidium isolates and assessed possible transmission routes and health effects of this organism. We collected 909 fecal samples from five different farms in Heilongjiang Province between August and September 2022. The samples underwent Cryptosporidium detection by nested PCR and small subunit (SSU) rRNA gene sequence analysis. Four Cryptosporidium species were identified, including C. parvum, C. bovis, C. ryanae, and C. andersoni, with an overall prevalence of 4.4% (40/909). Based on sequence analysis of the 60 kDa glycoprotein gene of C. parvum and C. bovis, three subtypes of C. parvum were identified, namely two previously known subtypes (IIdA19G1 and IIdA20G1), and one novel subtype (IIdA24G2). Two distinct subtype families were identified in C. bovis (XXVId and XXVIe). The high diversity of Cryptosporidium in dairy cattle and the emergence of a novel subtype of C. parvum in Heilongjiang Province suggest that dairy cattle may serve as a significant source of zoonotic cryptosporidiosis infection in this region.

A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice.

The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin-neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 µg of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 µg completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.

Morphological and molecular biology identification of Cystoisospora sp. in the blue fox, Alopex lagopus (Linnaeus, 1758).

To investigate the prevalence and molecular characteristics of Cystoisospora sp. in blue fox (Alopex lagopus), Sheather's sugar floatation method was conducted to detect coccidia in 423 fresh fecal samples randomly collected from blue fox farms from three cities in China. The overall prevalence of coccidia was 1.4% (6/423), and three Cystoisospora sp. (Cystoisospora fennechi, Cystoisospora sp. I and Cystoisospora vulpina) were identified by their morphological characteristics. The 18S ribosomal RNA (rRNA) and cytochrome c oxidase subunit I (COI) locus sequences were sequenced for molecular biological identification, homology comparison, and phylogenetic analysis of Cystoisospora sp. by single-oocyst selection technology and multi-locus-nested PCR amplification. At the 18S rRNA and COI loci, C. vulpina had 99.48% and 99.59% homology, respectively, with Cystoisospora canis and Cystoisospora ohioensis from canines. Phylogenetic analysis indicated that C. vulpina was clustered in a clade with Cystoisospora sp. from Canidae, which the relatives are consistent with the hosts. To our knowledge, this is the first report on molecular identification and evolutionary analysis of C. vulpina at two different loci.

Assessment of potential zoonotic transmission of Giardia duodenalis from dogs and cats.

Giardia duodenalis is one of the major causes of diarrhea among humans, especially in young children. Statistical analysis revealed that the pooled prevalence of G. duodenalis in humans, dogs, and cats was 9.72% (10,921/112383), 15.60% (7510/48140), and 14.53% (1125/7740), respectively. Unquestionably, the canine-specific assemblages C and D and the feline-specific assemblage F were the dominant genotypes in dogs and cats, respectively. Additionally, the prevalence of zoonotic G. duodenalis assemblages (A and B) in dogs and cats was 23.07% (875/3792) and 41.42% (169/408), respectively, implying that the potential transmission of G. duodenalis from dogs and cats to human infection cannot be ignored. The highest frequency of potentially zoonotic assemblages was found among working dogs (3.55%, 25/705) and the 1-5 age group (22.92%, 11/48). In summary, dogs and cats have a significant role in the zoonotic transmission of G. duodenalis due to their close contact with humans and the higher frequency presence of zoonotic assemblages. Further studies are necessary to explore the presence of G. duodenalis among humans and animals and in environmental samples. Researchers should adopt a one-health approach to gain a deeper understanding of G. duodenalis in dogs and cats and potential transmission routes to humans.

Metagenomic analysis of gut microbiome and resistome of Whooper and Black Swans: a one health perspective.

BACKGROUND: With the promotion of "One Health," the health of animals and their impact on the environment have become major concerns recently. Widely distributed in China, the whooper swans (Cygnus cygnus) and black swans (Cygnus atratus) are not only important to the ecological environment, but they may also potentially influence public health security. The metagenomic approach was adopted to uncover the impacts of the gut microbiota of swans on host and public health. RESULTS: In this study, the intestinal microbiome and resistome of migratory whooper swans and captive-bred black swans were identified. The results revealed similar gut microbes and functional compositions in whooper and black swans. Interestingly, different bacteria and probiotics were enriched by overwintering whooper swans. We also found that Acinetobacter and Escherichia were significantly enriched in early wintering period swans and that clinically important pathogens were more abundant in black swans. Whooper swans and black swans are potential reservoirs of antibiotic resistance genes (ARGs) and novel ARGs, and the abundance of novel ARGs in whooper swans was significantly higher than that in black swans. Metagenomic assembly-based host tracking revealed that most ARG-carrying contigs originated from Proteobacteria (mainly Gammaproteobacteria). CONCLUSIONS: The results revealed spatiotemporal changes in microbiome and resistome in swans, providing a reference for safeguarding public health security and preventing animal epidemics.

Functional characterization of CpADF, an actin depolymerizing factor protein in Cryptosporidium parvum.

Cryptosporidium is a highly pathogenic water and food-borne zoonotic parasitic protozoan that causes severe diarrhea in humans and animals. Apicomplexan parasites invade host cells via a unique motility process called gliding, which relies on the parasite's microfilaments. Actin depolymerizing factor (ADF) is a fibrous-actin (F-actin) and globular actin (G-actin) binding protein essential for regulating the turnover of microfilaments. However, the role of ADF in Cryptosporidium parvum (C. parvum) remains unknown. In this study, we preliminarily characterized the biological functions of ADF in C. parvum (CpADF). The CpADF was a 135-aa protein encoded by cgd5_2800 gene containing an ADF-H domain. The expression of cgd5_2800 gene peaked at 12 h post-infection, and the CpADF was located in the cytoplasm of oocysts, middle region of sporozoites, and cytoplasm of merozoites. Neutralization efficiency of anti-CpADF serum was approximately 41.30%. Actin sedimentation assay revealed that CpADF depolymerized but did not undergo cosedimentation with F-actin and its ability of F-actin depolymerization was pH independent. These results provide a basis for further investigation of the roles of CpADF in the invasion of C. parvum.

Micro-RNA expression profile of BALB/c mouse glandular stomach in the early phase of Cryptosporidium muris infection.

Cryptosporidiosis is a zoonotic disease in humans and animals that is caused by infection with the oocysts of Cryptosporidium. MicroRNAs (miRNAs) are important players in regulating the innate immune response against parasitic infection. Public miRNAs data for studying pathogenic mechanisms of cryptosporidiosis, particularly in natural hosts, are scarce. Here, we compared miRNA profiles of the glandular stomach of C. muris-infected and uninfected BALB/c mice using microarray sequencing. A total of 10 miRNAs (including 3 upregulated and 7 downregulated miRNAs) with significant differential expression (|FC| ≥ 2 and P value < 0.05) were identified in the glandular stomach of BALB/c mice 8 h after infection with C. muris. MiRWalk and miRDB online bioinformatics tools were used to predict the target genes of differentially expressed miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to annotate the target genes. GO analysis indicate that gene transcription-related and ion transport-related GO terms were significantly enriched. In addition, the KEGG analyses showed that the target genes were strongly related to diverse types of tumor disease progression and anti-pathogen immunity pathways. In the current study, we firstly report changes in miRNA expression profiles in the glandular stomach of BALB/c mice at the early phase of C. muris invasion. This dysregulation in miRNA expression may contribute to our understanding of cryptosporidiosis pathology. This study provides a new perspective on the miRNA regulatory mechanisms of cryptosporidiosis, which may help in the development of effective control strategies against this pathogen.

Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.