Bieneusi Genotypes Research Articles

Wastewater-based intestinal protozoa monitoring in Shanghai, China.

Intestinal protozoa Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi have been implicated in serious waterborne outbreaks worldwide. Wastewater-based epidemiology (WBE) is a promising approach for evaluating the disease prevalence in a catchment population in that it monitors the contamination level of the intestinal pathogens in wastewater. We collected 48 urban wastewater samples (24 from influents and 24 from effluents) from the Yangpu Wastewater Treatment Plant (YPWTP) in Shanghai, China. We identified Cryptosporidium spp., G. duodenalis, and E. bieneusi by nested polymerase chain reaction (PCR) amplification. Cryptosporidium hominis and subtype IdA14 were identified in two samples by analyzing the sequences of small subunit ribosomal RNA (SSU rRNA) and 60-kDa glycoprotein (gp60) genes, respectively. The G. duodenalis sub-assemblage AII (n = 8) and assemblage C (n = 4) in 12 samples were determined by analyzing triosephosphate isomerase (tpi) gene sequences. The E. bieneusi genotype A was identified in one sample by analyzing the sequence of the internal transcribed spacer (ITS) region of the rRNA gene. These findings suggest that improving wastewater treatment and monitoring the virility of pathogens in effluents is critical. We observed similar prevalence and genotypes/subtypes of the three intestinal protozoa in our wastewater samples as those reported in previous studies, providing evidence that WBE can be used as an effective epidemic management tool.IMPORTANCECryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common intestinal protozoa causing diarrhea. The infective oocysts, cysts, and spores released in feces can survive in different environments, including multiple types of water bodies. Humans can acquire these intestinal protozoan infections via the fecal-oral route as in waterborne transmission. Wastewater-based epidemiology can rapidly and reliably detect and monitor the emergence and spread of waterborne diseases. We detected Cryptosporidium spp., G. duodenalis, and E. bieneusi in a wastewater treatment plant in Shanghai, China, reflecting the occurrence and genetic characterizations of the three intestinal pathogens from community members served by the wastewater treatment plant.

Pet chinchillas (Chinchilla lanigera): Source of zoonotic Giardia intestinalis, Cryptosporidium ubiquitum and microsporidia of the genera Encephalitozoon and Enterocytozoon

The domestic chinchilla (Chinchilla lanigera) is kept as a pet and previous studies suggest that it may play an important role as a source of zoonotic parasites, including Giardia intestinalis, Cryptosporidium spp. and microsporidia. In this study, we examined the occurrence and genetic diversity of above mentioned parasites in pet chinchillas in the Czech Republic by PCR/sequencing of the 18S rRNA, TPI, and ITS genes. Of 149 chinchillas from 24 breeders, 91.3 % were positive for G. intestinalis, 8.1 % for Cryptosporidium spp., 2.0 % for Encephalitozoon spp., and 5.4 % for E. bieneusi. Molecular analyses revealed presence of G. intestinalis assemblage B, C. ubiquitum (XIIa family), E. bieneusi genotypes D, SCF2, and, CHN-F1, and E. intestinalis. The infection intensity of G. intestinalis determined by qRT-PCR reached up to 53,978 CPG, C. ubiquitum up to 1409 OPG, E. intestinalis up to 1124 SPG, and E. bieneusi up to 1373 SPG. Only two chinchillas with C. ubiquitum and five with G. intestinalis had diarrhoea at the time of the screening. Three chinchillas in the long-term study were consistently positive for G. intestinalis, with intermittent excretion of C. ubiquitum, E. intestinalis, and E. bieneusi over 25 weeks. The findings indicate that chinchillas are frequently infected with zoonotic parasitic protists, but that these infections rarely show clinical signs. The lack of visible signs could reduce the vigilance of pet owners when handling their chinchillas, increasing the risk of transmission within breeding groups and possibly to humans.

Molecular characterization and zoonotic potential of Entamoeba spp., Enterocytozoon bieneusi and Blastocystis from captive wild animals in northwest China

BackgroundParasites Entamoeba spp., Enterocytozoon bieneusi and Blastocystis are prevalent pathogens causing gastrointestinal illnesses in animals and humans. Consequently, researches on their occurrence, distribution and hosts are crucial for the well-being of both animals and humans. Due to the confined spaces and frequent interaction between animals and humans, animal sanctuaries have emerged as potential reservoirs for these parasites. In this study, the wildlife sanctuary near the Huang Gorge of the Qinling Mountains in northwest China is chosen as an ideal site for parasite distribution research, considering its expansive stocking area and high biodiversity.ResultsWe collected 191 fecal specimens from 37 distinct wildlife species and extracted genomic DNA. We identified these three parasites by amplifying specific gene regions and analyzed their characteristics and evolutionary relationships. All the parasites exhibited a high overall infection rate, reaching 90.05%. Among them, seven Entamoeba species were identified, accounting for a prevalence of 54.97%, with the highest infection observed in Entamoeba bovis. In total, 11 Enterocytozoon bieneusi genotypes were discovered, representing a prevalence of 35.08%, including three genotypes of human-pathogenic Group 1 and two novel genotypes (SXWZ and SXLG). Additionally, 13 Blastocystis subtypes were detected, showing a prevalence of 74.87% and encompassing eight zoonotic subtypes. All of the above suggests significant possibilities of parasite transmission between animals and humans.ConclusionsThis study investigated the occurrence and prevalence of three intestinal parasites, enhancing our understanding of their genetic diversity and host ranges in northwest China. Furthermore, the distribution of these parasites implies significant potential of zoonotic transmission, underscoring the imperative for ongoing surveillance and implementation of control measures. These efforts are essential to mitigate the risk of zoonotic disease outbreaks originating from wildlife sanctuary.

Host specificity and zoonotic Enterocytozoon bieneusi genotypes in wild rodents from the Inner Mongolian Autonomous Region and Liaoning Province of China.

Wild rodents can serve as reservoirs or carriers of E. bieneusi, thereby enabling parasite transmission to domestic animals and humans. This study aimed to investigate the prevalence of E. bieneusi in wild rodents from the Inner Mongolian Autonomous Region and Liaoning Province of China. Moreover, to evaluate the potential for zoonotic transmission at the genotype level, a genetic analysis of the isolates was performed. A total of 486 wild rodents were captured from two provinces in China. Polymerase chain reaction (PCR) was performed to amplify the vertebrate cytochrome b (cytb) gene in the fecal DNA of the rodents to detect their species. The genotype of E. bieneusi was determined via PCR amplification of the internal transcribed spacer (ITS) region of rDNA. The examination of genetic characteristics and zoonotic potential requires the application of similarity and phylogenetic analysis. The infection rates of E. bieneusi in the four identified rodent species were 5.2% for Apodemus agrarius (n = 89), 4.5% for Cricetulus barabensis (n = 96), 11.3% for Mus musculus (n = 106), and 38.5% for Rattus norvegicus (n = 195). Infection was detected at an average rate of 17.4% among 486 rodents. Of the 11 identified genotypes, nine were known: SHR1 (detected in 32 samples), D (30 samples), EbpA (9 samples), PigEbITS7 (8 samples), HNR-IV (6 samples), Type IV (5 samples), HNR-VII (2 samples), HNH7 (1 sample), and HNPL-V (1 sample). Two novel genotypes were also discovered, NMR-I and NMR-II, each comprising one sample. The genotypes were classified into group 1 and group 13 via phylogenetic analysis. Based on the initial report, E. bieneusi is highly prevalent and genetically diverse in wild rodents residing in the respective province and region. This indicates that these animals are crucial for the dissemination of E.bieneusi. Zoonotic E. bieneusi-carrying animals present a significant hazard to local inhabitants. Therefore, it is necessary to increase awareness regarding the dangers presented by these rodents and reduce their population to prevent environmental contamination.

Identification of common human infectious and potentially zoonotic novel genotypes of Enterocytozoon bieneusi in cavernicolous bats in Thailand.

Enterocytozoon bieneusi is a common cause of human microsporidiosis and can infect a variety of animal hosts worldwide. In Thailand, previous studies have shown that this parasite is common in domestic animals. However, information on the prevalence and genotypes of this parasite in other synanthropic wildlife, including bats, remains limited. Several pathogens have been previously detected in bats, suggesting that bats may serve as a reservoir for this parasite. In this study, a total of 105 bat guano samples were collected from six different sites throughout Thailand. Of these, 16 from Chonburi (eastern), Ratchaburi (western), and Chiang Rai (northern) provinces tested positive for E. bieneusi, representing an overall prevalence of 15.2%. Based on ITS1 sequence analysis, 12 genotypes were identified, including two known genotypes (D and type IV) frequently detected in humans and ten novel potentially zoonotic genotypes (TBAT01-TBAT10), all belonging to zoonotic group 1. Lyle's flying fox (Pteropus lylei), commonly found in Southeast Asia, was identified as the host in one sample that was also positive for E. bieneusi. Network analysis of E. bieneusi sequences detected in this study and those previously reported in Thailand also revealed intraspecific divergence and recent population expansion, possibly due to adaptive evolution associated with host range expansion. Our data revealed, for the first time, multiple E. bieneusi genotypes of zoonotic significance circulating in Thai bats and demonstrated that bat guano fertilizer may be a vehicle for disease transmission.

Prevalence and Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Cattle in Heilongjiang Province, Northeast China.

Crytosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are important diarrheal pathogens with a global distribution that threatens the health of humans and animals. Despite cattle being potential transmission hosts of these protozoans, the associated risks to public health have been neglected. In the present study, a total of 1155 cattle fecal samples were collected from 13 administrative regions of Heilongjiang Province. The prevalence of Cryptosporidium spp., G. duodenalis, and E. bieneusi were 5.5% (64/1155; 95% CI: 4.2-6.9), 3.8% (44/1155; 95% CI: 2.7-4.9), and 6.5% (75/1155; 95% CI: 5.1-7.9), respectively. Among these positive fecal samples, five Cryptosporidium species (C. andersoni, C. bovis, C. ryanae, C. parvum, and C. occultus), two G. duodenalis assemblages (E and A), and eight E. bieneusi genotypes (BEB4, BEB6, BEB8, J, I, CHS7, CHS8, and COS-I) were identified. Phylogenetic analysis showed that all eight genotypes of E. bieneusi identified in the present study belonged to group 2. It is worth noting that some species/genotypes of these intestinal protozoans are zoonotic, suggesting a risk of zoonotic disease transmission in endemic areas. The findings expanded our understanding of the genetic composition and zoonotic potential of Cryptosporidium spp., G. duodenalis, and E. bieneusi in cattle in Heilongjiang Province.

Molecular characterization of Cryptosporidium spp., Giardia spp. and Enterocytozoon bieneusi in eleven wild rodent species in China: Common distribution, extensive genetic diversity and high zoonotic potential

Cryptosporidium spp., Giardia spp. and Enterocytozoon bieneusi are common zoonotic pathogens in humans and animals. Although rodents are important parts of the ecosystem and common hosts for these pathogens, little is known of the distribution, genetic diversity and zoonotic potential of these pathogens in wild rodents. A total of 442 fecal samples were collected from eleven wild rodent species in three provinces of China, and analyzed for these pathogens by PCR and DNA sequencing. The infection rates of Cryptosporidium spp., Giardia spp. and E. bieneusi were 19.9% (88/442), 19.8% (75/378) and 12.2% (54/442), respectively. Altogether, 23 known Cryptosporidium species/genotypes were identified and their distribution varied among different sampling locations or rodent species. Subtyping of the zoonotic Cryptosporidium species identified two novel subtype families XVe and XVf in C. viatorum, the subtype family XIIh and a novel subtype family XIIj in C. ubiquitum, and the subtype family IId in C. parvum. Three Giardia species were identified, including G. microti (n = 57), G. muris (n = 15) and G. duodenalis (n = 3), with G. duodenalis assemblages A and G identified in brown rats in urban areas of Guangdong. In addition, 13 E. bieneusi genotypes including eight known and five novel ones were identified, belonging to Groups 1, 2, 10, 14 and 15. Within nine genotypes in the zoonotic Group 1, common human-pathogenic genotypes D, Type IV, PigEbITS7 and Peru8 were detected only in brown rats and Lesser rice-field rats in urban areas of Guangdong. Apparent host adaptation and geographical differences were observed among Cryptosporidium spp., Giardia spp. and E. bieneusi genotypes in wild rodents in the present study. Furthermore, the zoonotic Cryptosporidium species and E. bieneusi genotypes commonly found here suggest a high zoonotic potential of these pathogens in wild rodents, especially in brown rats in urban areas. Hygiene and One Health measures should be implemented in urban streets and food stores to reduce the possible direct and indirect transmission of these rodent-related pathogens.

Enterocytozoon bieneusi and Cryptosporidium bat genotype XXI and bat genotype XXII in fruit bats (Rousettus leschenaultii) inhabiting a tropical park in Hainan Province, China

Bats stand as one of the most diverse groups in the animal kingdom and are key players in the global transmission of emerging pathogens. However, their role in transmitting Enterocytozoon bieneusi and Cryptosporidium spp. remains unclear. This study aimed to evaluate the occurrence and genetic diversity of the two pathogens in fruit bats (Rousettus leschenaultii) in Hainan, China. Ten fresh fecal specimens of fruit bats were collected from Wanlvyuan Gardens, Haikou, China. The fecal samples were tested for E. bieneusi and Cryptosporidium spp. using Polymerase Chain Reaction (PCR) analysis and sequencing the internal transcribed spacer (ITS) region and partial small subunit of ribosomal RNA (SSU rRNA) gene, respectively. Genetic heterogeneity across Cryptosporidium spp. isolates was assessed by sequencing 4 microsatellite/minisatellite loci (MS1, MS2, MS3, and MS16). The findings showed that out of the ten specimens analyzed, 2 (20 %) and seven (70.0 %) were tested positive for E. bieneusi and Cryptosporidium spp., respectively. DNA sequence analysis revealed the presence of two novel Cryptosporidium genotypes with 94.4 to 98.6 % sequence similarity to C. andersoni, named as Cryptosporidium bat-genotype-XXI and bat-genotype-XXII. Three novel sequences of MS1, MS2 and MS16 loci identified here had 95.4 to 96.9 % similarity to the known sequences, which were deposited in the GenBank. Two genotypes of E. bieneusi were identified, including a novel genotype named HNB-I and a zoonotic genotype PigEbITS7. The discovery of these novel sequences provides meaningful data for epidemiological studies of the both pathogens. Meanwhile our results are also presented that the fruit bats infected with E. bieneusi, but not with Cryptosporidium, should be considered potential public health threats.

Detection and genotyping of zoonotic microsporidia in the endangered Iberian lynx (Lynx pardinus).

Microsporidia is a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most frequently reported species in humans. Limited information is available about the presence and molecular diversity of microsporidian species in the endangered Iberian lynx (Lynx pardinus). Presence of Enterocytozoon bieneusi and Encephalitozoon spp. was investigated by molecular methods in wild and captive Iberian lynxes from Spain. Overall, E. bieneusi was detected in 3.2% (8/251) of the animals examined. None of the samples tested were positive for Encephalitozoon spp. Four known (D, EbfelA, PigEBITS7, and Type IV) and a novel (named as LynxSpEb1) E. bieneusi genotypes were identified. All the genotypes found belonged to the zoonotic Group 1 of E. bieneusi. This study provides the first genotyping data of E. bieneusi in Iberian lynx in Spain. Our result indicate that the Iberian lynx does not seem to play a relevant role in the epidemiology of Encephalitozoon spp., and that this endangered felid is likely acting as spillover host rather than a true reservoir of E. bieneusi. Additional studies should be conducted to assess the impact of this parasite in the health status of the endangered Iberian lynx.

Genetic diversity of Cryptosporidium spp., Encephalitozoon spp. and Enterocytozoon bieneusi in feral and captive pigeons in Central Europe.

Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon spp. are the most common protistan parasites of vertebrates. The results show that pigeon populations in Central Europe are parasitised by different species of Cryptosporidium and genotypes of microsporidia of the genera Enterocytozoon and Encephalitozoon. A total of 634 and 306 faecal samples of captive and feral pigeons (Columba livia f. domestica) from 44 locations in the Czech Republic, Slovakia and Poland were analysed for the presence of parasites by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (18S rDNA), 60kDa glycoprotein (gp60) and internal transcribed spacer (ITS) of SSU rDNA. Phylogenetic analyses revealed the presence of C. meleagridis, C. baileyi, C. parvum, C. andersoni, C. muris, C. galli and C. ornithophilus, E. hellem genotype 1A and 2B, E. cuniculi genotype I and II and E. bieneusi genotype Peru 6, CHN-F1, D, Peru 8, Type IV, ZY37, E, CHN4, SCF2 and WR4. Captive pigeons were significantly more frequently parasitised with screened parasite than feral pigeons. Cryptosporidium meleagridis IIIa and a new subtype IIIl have been described, the oocysts of which are not infectious to immunodeficient mice, whereas chickens are susceptible. This investigation demonstrates that pigeons can be hosts to numerous species, genotypes and subtypes of the studied parasites. Consequently, they represent a potential source of infection for both livestock and humans.

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

BackgroundEnterocytozoon 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.ResultA 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.ConclusionsThese 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 genetic characterization of Enterocytozoon bieneusi in pet dogs in Yunnan Province, China.

Enterocytozoon bieneusi is the most common microsporidian species in humans and can affect over 200 animal species. Considering possible increasing risk of human E. bieneusi infection due to close contact with pet dogs and identification of zoonotic E. bieneusi genotypes, 589 fresh fecal specimens of pet dogs were collected from Yunnan Province, China to determine the occurrence of E. bieneusi, characterize dog-derived E. bieneusi isolates, and assess their zoonotic potential at the genotype level. Enterocytozoon bieneusi was identified and genotyped by PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. Twenty-nine specimens (4.9%) were positive. A statistical difference was observed in occurrence rates of E. bieneusi in pet dogs among 11 sampling sites by Fisher's exact test. Fifteen genotypes were identified and all of them phylogenetically belonged to zoonotic group 1, including four known genotypes (EbpC, D, Peru 8, and Henan-III) and 11 novel genotypes. Genotype Henan-III was reported in dogs for the first time. The finding of known genotypes found previously in humans and novel genotypes falling into zoonotic group 1 indicates that dogs may play a role in the transmission of E. bieneusi to humans in the investigated areas.

Wild sympatric rodents inhabiting pig farm environments may facilitate the spillover of Enterocytozoon bieneusi from pig farms.

Enterocytozoon bieneusi is a zoonotic pathogen prevalent in mammalian and avian hosts across the globe. Wild small mammals, being abundant worldwide, serve as important sources of zoonotic disease transmission to humans. Here, 227 fecal samples were collected from five rodent and shrew species on 34 pig farms in China to investigate the prevalence and molecular characterization of E. bieneusi. The overall prevalence of E. bieneusi was 17.18% (39/227), with a distribution of 23.53% (32/136) in Rattus tanezumi, 8.62% (5/58) in Rattus norvegicus, and 8.00% (2/25) in Mus musculus. Eight E. bieneusi genotypes were identified, comprising four known genotypes: D (n=8), EbpC (n=8), PigEBITS7 (n=9), and EbpA (n=2), and four novel genotypes: CHPR1 (n=7), CHPR2 (n=1), CHPR3 (n=2), and CHPR4 (n=2). This study is the first to report E. bieneusi in rodents from pig farms in Henan, Shaanxi, and Shanxi Provinces in China. The host range of genotype EbpC was expanded with its first detection in M. musculus and R. tanezumi. All identified E. bieneusi genotypes belong to group 1, raising concerns about these sympatric rodents being reservoirs of zoonotic transmission. Moreover, the widespread distribution of genotype EbpC suggests potential cross-species transmission between sympatric rodents and domestic pigs. Our findings highlight the potential role of sympatric rodents in facilitating the spillover of E. bieneusi from pig farms, which could pose a potential public health threat.

Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Escherichia coli in Dairy Goat Kids with Diarrhea in Partial Regions of Shaanxi Province, China.

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.

Genotyping of Enterocytozoon bieneusi isolates detected in stray cats of İzmir, Türkiye.

The phylum Microsporidia includes obligate intracellular parasites that can infect humans and various animals. To date, 17 different species within the phylum have been reported to infect humans. Among them, Enterocytozoon bieneusi (E. bieneusi) is one of the most frequently detected species in humans. Identification of E. bieneusi as well as its genotypes in humans and animals is important to reveal their role in transmission to each other. Cats are blamed as the source of E. bieneusi transmission to humans. In this study, we aimed to genotype 170 E. bieneusi positive samples isolated from stool of stray cats living in İzmir province of Türkiye. According to the results, 47 samples were amplified by nested PCR protocol targeting ITS region and successfully sequenced. The phylogenetic analysis showed the presence of zoonotic genotype D and type IV in stray cats, which are also frequently detected in humans. Among the E. bieneusi genotypes detected, the prevalence of type IV (93.6%; 44/47) was very high compared to genotype D. Overall, the identification of zoonotic genotypes of E. bieneusi supports that stray cats can play an important role in the transmission of E. bieneusi to humans in İzmir.

The Frequency of Intestinal Parasitic Infections in COVID-19 Patients: A Case-Control Study in Tehran, Capital of Iran.

The present study was done to evaluate the prevalence of intestinal parasitic infections (IPIs) in patients with COVID-19 in health care centers (Imam Reza and Golestan hospitals), Tehran, capital of Iran. By designing a matched case-control study, 200 fecal samples were collected for each of the COVID-19 patients and healthy individuals. Nasopharyngeal/oropharyngeal swab samples were collected from all participants for the diagnosis of COVID-19. RNA extraction was performed, and then real time reverse-transcription polymerase chain reaction (rRT-PCR) assay was applied to detect viral RNA. Considering the lung complications, 25%> lung complications was detected in 49 patients, 25-49% in 42 patients, and 50%≤ in 109 patients. Fecal samples were examined using different parasitological techniques. After nested-PCR, sequencing was applied to identify Cryptosporidium spp. and microsporidia spp. A relatively lower prevalence of IPIs was detected among control group (7.5%), than in COVID-19 patients (13%), though not significant (P=0.13). The most prevalent parasite among patients was Blastocystis sp. (6%). Also, 13.76% of IPIs were detected in inpatients with more than 50% lung complication. As well, a remarkably significant difference in IPIs was observed among diarrheic COVID-19 patients, in comparison with nondiarrheic patients (P < 0.00001). Moreover, the isolated sequences in the present study belonged to C. parvum subtype IIa and Enterocytozoon bieneusi genotypes D and Peru 8. In conclusion, more epidemiological and clinical research studies are needed to better understand the status and interaction of IPI in COVID-19 in Iran and other countries.

Microscopic and Molecular Investigation of Intestinal Microsporidia in HIV + /AIDS and Cancer Patients Undergoing Chemotherapy in Mazandaran Province, North of Iran.

The aim of this study was to identify Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples of HIV + /AIDS and cancer patients undergoing chemotherapy, and comparing the results to healthy individuals in Mazandaran province, north of Iran. Stool samples were collected from 50 HIV + /AIDS patients, 50 cancer patients, and 50 healthy samples referred to medical centers in north of Iran. Stool samples were kept in 2.5% potassium dichromate at 4°C, and stained by modified trichrome for light microscopy examination. The multiplex/nested-PCR targeted the small subunit ribosomal RNA (SSU rRNA) gene. To characterize genotypes, the nested PCR products sequencedby Bioneer Companyand was subjected to phylogenetic analyses. Ten of 50 samples (20%) of HIV + /AIDS patients, 5of50 samples (10%) of cancer patients, and 1 of healthy individuals (2%) were microscopically positive. From 50 HIV + / AIDS patients, E. bieneusi and Encephalitozoon spp. were detected in 10 (20%) and 6 (12%) cases, respectively. Furthermore, among cancer patients, 7 (14%) and 2 (4%) cases were E. bieneusi and Encephalitozoon spp., respectively. Out of 50 samples of healthy individuals, only 3 (6%)cases of E. bieneusi were observed. The genotypes D and M were detected among positive samples of E. bieneusi. E. bieneusi and then Encephalitozoon spp. are common intestinal microsporidia in HIV + /AIDS patients and cancer patients undergoing chemotherapy in Mazandaran province. E. bieneusi genotype D seems to be the predominant genotype in Mazandaran province.Due to the considerable prevalence of intestinal microsporidia, physicians are advised to pay more attention to this opportunistic infection in high-risk groups.

First report on the molecular detection of Enterocytozoon bieneusi in livestock and wildlife around Qinghai Lake in the Qinghai-Tibetan Plateau area, China.

Enterocytozoon bieneusi is considered to be a microsporidial species of humans and animals in the worldwide. Limited data have been reported on the prevalence and genotypes of E. bieneusi in livestock and wild animals around Qinghai Lake in the Qinghai-Tibetan Plateau area, which shares water sources, grasslands, and harsh climate with high altitudes. In this study, fecal samples from 110 Tibetan sheep, 128 yaks, 227 wild birds, 96 blue sheep (Pseudois nayaur) and 268 Przewalski's gazelle (Procapra przewalskii) around Qinghai Lake were collected, and then tested for E. bieneusi by PCR and sequencing analysis based on the ribosomal internal transcribed spacer. Among them, ten (9.09%) samples from Tibetan sheep, five (3.91%) from yaks, five (2.20%) from wild birds, one (1.04%) from wild blue sheep and two (0.75%) from Przewalski's gazelle were positive for E. bieneusi. Among sheep, there were nine E. bieneusi genotypes, including two known genotypes (BEB6 and J), and seven novel genotypes (named CHS18-CHS24). From yaks, four genotypes were identified, including two known ones (BEB4 and J) and two novel genotypes (named CHN15 and CHN16). While in wild animals, eight genotypes were found, including five different genotypes from wild bids, with three known genotypes (EbpC, J and NCF2), two novel genotypes (named CHWB1 and CHS24), and two genotypes from Przewalski's gazelle, with one known genotype J and one novel genotype CHWPG1, and one novel genotype CHWBS1 from blue sheep. According to the phylogenetic analysis, five isolates belonged to group 1, and the others were clustered into group 2. This study provides unique data on the epidemiological reports and potential risk factors for E. bieneusi in both domesticated livestock and wild animals around Qinghai Lake in the Qinghai-Tibetan Plateau area; it is important to better understand the molecular epidemiology and zoonotic potential of E. bieneusi in the Qinghai-Tibetan Plateau area.

Prevalence and Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Diarrheic and Non-Diarrheic Calves from Ningxia, Northwestern China.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are significant parasitic gastrointestinal pathogens with global distribution in humans and domestic animals, including calves. The main symptoms of calf infection are severe diarrhea, dehydration, growth retardation, and sometimes even death. To date, there has been limited information on the prevalence of Cryptosporidium spp., G. duodenalis, and E. bieneusi infections in calves in Ningxia, China, especially between diarrheic and non-diarrheic calves. A total of 438 fecal samples were collected from diarrheic (201) and non-diarrheic (237) calves in Ningxia. PCR and DNA sequencing were used to find the prevalence of Cryptosporidium spp. at 46.8% (205/438), G. duodenalis at 16.9% (74/438), and E. bieneusi at 10.0% (44/438). The prevalence of Cryptosporidium spp. infection in diarrheic and non-diarrheic calves was 54.0% (128/237) and 38.3% (77/201), respectively, and statistical analysis showed a positive correlation between the prevalence of Cryptosporidium spp. infection and calf diarrhea (p < 0.01). However, in this study, there was no statistical correlation between the prevalence of G. duodenalis infection as well as E. bieneusi infection and calf diarrhea (p > 0.05). Furthermore, four known Cryptosporidium species were successfully identified by comparing them with SSU rRNA gene sequences, including C. parvum, C. bovis, C. ryanae, and C. andersoni. In addition, all 74 G. duodenalis-positive samples were identified as assemblage E by comparative analysis of bg gene sequences. Among the 44 E. bieneusi-positive samples sequenced in the present study, 4 distinct E. bieneusi genotypes were successfully identified by comparative analysis of ITS sequences, including 3 known genotypes (J, BEB4, and N) and 1 novel genotype, the latter of which was identified and designated as NX1. These findings indicated that the high genetic diversity and complex population structures of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Ningxia diarrhea calves and non-diarrhea calves, which provide new data for understanding the epidemiological status of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Ningxia calves.