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Summary: 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. [ABSTRACT FROM AUTHOR]

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To find out whether and how the prevalence and genetic diversity of Cryptosporidium in neonatal calves vary with the season, 380 fecal samples from neonatal calves on two large‐scale farms in Xinjiang (Alar and Wensu) were studied using molecular biology techniques. Cryptosporidium was detected in 48.7% (185/380) of the samples and was most frequent in summer (56.8%), followed by spring (50.0%), winter (46.8%), and autumn (41.7%; p > 0.05). Calves with diarrhea seem to be more likely infected by Cryptosporidium than those without diarrhea (p < 0.01). We also found that C. parvum (n = 173), C. bovis (n = 7), and C. ryanae (n = 3) were the Cryptosporidium species detected in this study, and co‐infections of these three species (n = 2) were also identified. Two subtypes (IIdA14G1 and IIdA15G1) of C. parvum were identified, and both can infect human. These results also show that neonatal calves commonly suffer diarrhea caused by C. parvum throughout the year. [ABSTRACT FROM AUTHOR]

Cryptosporidium spp., Enterocytozoonbieneusi, and Giardiaduodenalis are common enteric pathogens that are capable of infecting humans and animals. Total of 1,005 fecal samples from captive pet birds were collected from seven locations in Henan Province, China. The results demonstrated that 9.9% (99/1,005) of the captive birds were infected with one of these three pathogens. Enterocytozoonbieneusi was the most prevalent species among the birds (45/1,005, 4.5%) followed by G. duodenalis (33/1,005, 3.3%) and Cryptosporidium spp. (21/1,005, 2.1%). Five Cryptosporidium species were identified, namely, C. baileyi (10), C. galli (5), C. meleagridis (4), C. andersoni (1), and C. parvum (1). Two known E. bieneusi genotypes were identified: Peru 6 (44) was identified in pigeons (34) and European turtle doves (10); whereas, the genotype PtEb I (1) was only identified in a pigeon. Only G. duodenalis assemblage E (33) was identified in some pet birds. To the best of our knowledge, this study is the first to undertake the molecular identification of G. duodenalis in birds in China. The identification of potentially zoonotic species/genotypes of the pathogens suggests that exposure to the excreta of these birds, either directly or via food and water, may pose a threat to human health. [ABSTRACT FROM AUTHOR]

To determine the occurrence and genotypes of Enterocytozoon bieneusi in captive mammals at Bangladesh National Zoo and to assess their zoonotic significance, 200 fecal samples from 32 mammalian species were examined using a nested PCR and sequencing of internal transcribed spacer (ITS) gene. Enterocytozoon bieneusi was detected in 16.5% (33/200) of the samples. Seven different ITS genotypes were identified, including two known genotypes (D and J) and five new ones (BAN4 to BAN8). Genotype D was the most common genotype being observed in 19 isolates. In phylogenetic analysis, four genotypes (D, BAN4, BAN5, and BAN6), detected in 30 isolates (90.9%), belonged to Group 1 having zoonotic potential. The sequence of genotype J found in a Malayan pangolin was clustered in so‐called ruminant‐specific Group 2. The other two genotypes BAN7 and BAN8 were clustered in primate‐specific Group 5. To our knowledge, this is the first report of molecular characterization of E. bieneusi in Bangladesh, particularly in captive‐bred wildlife in this country. The potentially zoonotic genotypes of E. bieneusi are maintained in zoo mammals that may transmit among these animals and to the humans through environmental contamination or contact. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi is an important opportunistic pathogen widely distributed in humans and animals that causes diarrhea or fatal diarrhea in immunocompromised hosts. To examine the infection status and molecular characteristics of E. bieneusi in pigs, 725 fecal samples were collected from pigs in six areas of Fujian Province. The E. bieneusi genotypes were identified based on the internal transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene by nested PCR, and its population genetics were analyzed by multilocus sequence typing (MLST). The results showed that the infection rate of E. bieneusi was 24.4% (177/725), and 11 known genotypes (EbpC, EbpA, CHN‐RR2, KIN‐1, CHG7, CHS5, CM11, CHG23, G, PigEBITS, and D) and 2 novel genotypes (FJF and FJS) were identified. All the genotypes were found to be clustered into zoonotic Group 1. Moreover, 52 positive samples were successfully amplified at minisatellite and microsatellite loci and formed 48 distinct multilocus genotypes (MLGs). Further population structure analyses showed strong genetic linkage disequilibrium (LD) and several recombination events (Rm), indicating that E. bieneusi has a clonal population structure. This study is the first to investigate the prevalence and molecular characteristics of E. bieneusi in Fujian Province and could provide baseline data to control E. bieneusi infection in pigs and humans and deepen our understanding of the zoonotic risk of E. bieneusi and its distribution in China. [ABSTRACT FROM AUTHOR]

In the present study, fecal samples from a total of 620 Tibetan sheep and 260 Tibetan goats from six counties in Tibet were examined by nested PCR. The results showed that the overall infection rates of Giardia duodenalis and Enterocytozoon bieneusi were 0.8% (5/620) and 15% (93/620), respectively, in Tibetan sheep, and 0% (0/260) and 9.6% (25/260), respectively, in Tibetan goats. Based on sequence analysis of the SSU rRNA, tpi, bg, and gdh genes of G. duodenalis, only assemblage E was identified. Based on sequence analysis of the ribosomal internal transcriptional spacer (ITS) region of E. bieneusi, a total of 12 genotypes (three novel and nine known) were detected, and these clustered into two separate phylogenetic groups. Genotypes CHG19, EbpA, EbpC, H, PigEBITS5, and CTS3 clustered into Group 1 with high zoonotic potential, while genotypes BEB6, CHC8, CHG1, I, CTS1, and CTS2 fell within the host‐specific Group 2. Ten genotypes were detected in Tibetan sheep, and two genotypes were found in Tibetan goats. The current study indicated that E. bieneusi infections are widespread among these livestock, and Tibetan goats may play an important role as a reservoir of zoonotic E. bieneusi genotypes. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi is one of the most frequently diagnosed Microsporidia of humans and most animals. However, there is no information on E. bieneusi infection of pigs in Tibet and Henan, China. In this study, 1,190 fecal samples were collected from pigs in Tibet and Henan and screened for the presence of E. bieneusi. The overall prevalence of E. bieneusi infection was 54.2% (645/1,190), with differences in prevalence observed among geographical areas, ages, and pig breeds. Moreover, 10 E. bieneusi genotypes were identified based on internal transcribed spacer region genotyping, including eight known genotypes (EbpC, EbpA, CHG19, CHC5, Henan‐III, I, D, and H) and two novel genotypes (XZP‐I and XZP‐II). Multilocus sequence typing revealed 18, 7, 17, and 13 genotypes at minisatellite/microsatellite loci MS1, MS3, MS4, and MS7, respectively. Strong linkage disequilibrium (LD) and few numbers of recombination events, suggest a clonal structure of the E. bieneusi population examined in this study. The low pairwise genetic distance (FST) and gene flow (Nm) values indicated limited gene flow in the E. bieneusi population from different hosts, with phylogenetic, structure, and median‐joining network analyses all indicating the existence of host and geographical isolation. The identification of isolates belonging to nine human‐pathogenic genotypes indicates that pigs play an important role in the dissemination of E. bieneusi, improving our present understanding of E. bieneusi epidemiology in the studied region. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi causes microsporidiosis, a condition with complex epidemiology involving both direct and indirect transmission routes. To assess the potential role of synanthropic rodents and flies in the transmission of this pathogen, a total of 277 cattle fecal samples, 199 synanthropic rodents, and 50 batches of 20 flies were collected from a cattle farm. These samples were screened for the presence of E. bieneusi by PCR and sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. The positive rates of cattle, synanthropic rodents, and flies were 11.9% (33/277), 4.0% (8/199) and 12.0% (6/50), respectively. Nineteen genotypes were identified, including 11 known genotypes (BEB6, I, COS‐I, EbpC, D, J, CHS5, CHG1 to CHG3 and CHG14) and eight novel genotypes (named CHC9 to CHC16). The dominant genotype detected in the present study, BEB6, was found in all three categories of hosts. Moreover, human pathogenic genotypes D and EbpC were also observed in both synanthropic rodents and flies. These results demonstrate that synanthropic rodents and flies may act as biological disseminator or mechanical vector in the transmission of microsporidiosis to humans. Efforts should be made to minimize threats from these commensal animals to public health. [ABSTRACT FROM AUTHOR]

In present study, 262 fecal specimens were collected from 12 groups of grazing horses in the Xinjiang Uyghur Autonomous Region of China. The specimens were subjected to PCR and sequencing analyses of the ribosomal internal transcribed spacer ( ITS). The overall prevalence of E. bieneusi in horses was 30.9% (81/262). No significant differences in prevalence were observed between horses of different ages or sexes. Nineteen genotypes were identified: 15 known genotypes ( BEB6, CHG19, CM6, CM7, CM8, CS-1, CS-4, D, EpbA, EbpC, G, horse1, horse2, O, and Peru8) and four new genotypes ( XJH1- XJH4). Six of these genotypes were previously detected in humans: BEB6, D, EbpA, EbpC, O, and Peru8. Genotype EbpC was the most prevalent (21/81), followed by EpbA (20/81), BEB6 (9/81), CM6 (4/81), horse1 (4/81), O (4/81), G (3/81), CHG19 (2/81), CM7 (2/81), horse2 (2/81), and XJH1 (2/81), whereas the remaining eight genotypes were seen in one specimen each. In a phylogenetic analysis, 14 genotypes (65/81, 80.2%), excluding genotypes BEB6, CM7, horse2, XJH1, and XJH4, belonged to group 1, which have zoonotic potential. The high diversity in the E. bieneusi genotypes and their zoonotic potential suggest that grazing horses are a potential source of zoonotic infection in humans. [ABSTRACT FROM AUTHOR]

Giardia duodenalis is an important zoonotic pathogen, causes diarrhea in humans and animals worldwide. To date, few data are available on the prevalence of G. duodenalis in rabbits in China. In total, 955 fecal samples were collected from rabbits during 2008-2011 in Henan Province, Central China. The overall prevalence of G. duodenalis was 8.4% (80/955) on microscopic analysis, with the highest infection rate (11.3%) in rabbits aged 91-200 d. All G. duodenalis-positive isolates were characterized at the small subunit ribosomal RNA, β-giardin ( bg), glutamate dehydrogenase ( gdh), and triosephosphate isomerase genes. Two assemblages and a mixed assemblage were detected in the rabbits: assemblage B ( n = 26), assemblage E ( n = 2), and a mixed assemblage of B and E ( n = 4). Assemblage B isolates showed variability at the nucleotide sequences belonging to the so-called subtype BIV, based on analysis of multiple genes. This is the first report of G. duodenalis assemblage E in rabbits, and one novel subtype of assemblage E was identified through sequence analysis of gdh and bg genes, respectively. Our data suggest that rabbits may be reservoirs of G. duodenalis cysts potentially infectious to humans. [ABSTRACT FROM AUTHOR]

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common gastrointestinal protists in humans and animals. Two hundred and three fecal specimens from 80 wildlife species were collected in Zhengzhou Zoo and their genomic DNA extracted. Three intestinal pathogens were characterized with a DNA sequence analysis of different loci. Cryptosporidium felis, C. baileyi, and avian genotype III were identified in three specimens (1.5%), the manul, red-crowned crane, and cockatiel, respectively. Giardia duodenalis was also found in five specimens (2.5%) firstly: assemblage B in a white-cheeked gibbon and beaver, and assemblage F in a Chinese leopard and two Siberian tigers, respectively. Thirteen genotypes of E. bieneusi (seven previously reported genotypes and six new genotypes) were detected in 32 specimens (15.8%), of which most were reported for the first time. A phylogenetic analysis of E. bieneusi showed that five genotypes (three known and two new) clustered in group 1; three known genotypes clustered in group 2; one known genotype clustered in group 4; and the remaining four genotypes clustered in a new group. In conclusion, zoonotic Cryptosporidium spp., G. duodenalis, and E. bieneusi are maintained in wildlife and transmitted between them. Zoonotic disease outbreaks of these infectious agents possibly originate in wildlife reservoirs. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans. It has been found in a wide range of animals and is considered an important zoonotic pathogen. To date, little information is available on the role that cattle play in the epidemiology of human microsporidiosis caused by E. bieneusi in China. In this study, 133 fecal specimens from dairy cattle were collected in Heilongjiang Province, China. Enterocytozoon bieneusi was identified and genotyped by nested PCR analysis of the internal transcribed spacer ( ITS) region of the rRNA gene, with 30.1% positive. Nine ITS genotypes were identified: six known genotypes-O ( n = 26), EbpA ( n = 2), I ( n = 2), J ( n = 2), D ( n = 1) and BEB4 ( n = 1)-and three novel genotypes named as CC-I to CC- III (two each). Genotype O was identified in cattle for the first time. The observation of all the six known genotypes here reported previously in humans, and also the fact of all the three novel genotypes ( CHN- DC1 to CHN- DC3) falling into zoonotic group 1, indicate the possibility of cattle in the transmission of E. bieneusi to humans. [ABSTRACT FROM AUTHOR]