Immune signaling of Litopenaeus vannamei c-type lysozyme and its role during microsporidian Enterocytozoon hepatopenaei (EHP) infection.

The microsporidian Enterocytozoon hepatopenaei (EHP) is a fungi-related, spore-forming parasite. EHP infection causes growth retardation and size variation in shrimp, resulting in severe economic losses. Studies on shrimp immune response have shown that several antimicrobial peptides (AMPs) were upregulated upon EHP infection. Among those highly upregulated AMPs is c-type lysozyme (LvLyz-c). However, the immune signaling pathway responsible for LvLyz-c production in shrimp as well as its function against the EHP infection are still poorly understood. Here, we characterized major shrimp immune signaling pathways and found that Toll and JAK/STAT pathways were up-regulated upon EHP infection. Knocking down of a Domeless (DOME) receptor in the JAK/STAT pathways resulted in a significant reduction of the LvLyz-c and the elevation of EHP copy number. We further elucidated the function of LvLyz-c by heterologously expressing a recombinant LvLyz-c (rLvLyz-c) in an Escherichia coli. rLvLyz-c exhibited antibacterial activity against several bacteria such as Bacillus subtilis and Vibrio parahaemolyticus. Interestingly, we found an antifungal activity of rLvLyz-c against Candida albican, which led us to further investigate the effects of rLvLyz-c on EHP spores. Incubation of the EHP spores with rLvLyz-c followed by a chitin staining showed that the signals were dramatically decreased in a dose-dependent manner, suggesting that rLvLyz-c possibly digest a chitin coat on the EHP spores. Transmission electron microscopy analysis revealed that an endospore layer, which is composed mainly of chitin, was digested by rLvLyz-c. Lastly, we observed that EHP spores that were treated with rLvLyz-c showed a significant reduction of the spore germination rate. We hypothesize that thinning of the endospore of EHP would result in altered permeability, hence affecting spore germination. This work provides insights into shrimp immune signaling pathways responsible for LvLyz-c production and its anti-EHP property. This knowledge will serve as important foundations for developing EHP control strategies.

High-throughput phenotyping of infection by diverse microsporidia species reveals a wild C. elegans strain with opposing resistance and susceptibility traits.

Animals are under constant selective pressure from a myriad of diverse pathogens. Microsporidia are ubiquitous animal parasites, but the influence they exert on shaping animal genomes is mostly unknown. Using multiplexed competition assays, we measured the impact of four different species of microsporidia on 22 wild isolates of Caenorhabditis elegans. This resulted in the identification and confirmation of 13 strains with significantly altered population fitness profiles under infection conditions. One of these identified strains, JU1400, is sensitive to an epidermal-infecting species by lacking tolerance to infection. JU1400 is also resistant to an intestinal-infecting species and can specifically recognize and destroy this pathogen. Genetic mapping of JU1400 demonstrates that these two opposing phenotypes are caused by separate loci. Transcriptional analysis reveals the JU1400 sensitivity to epidermal microsporidia infection results in a response pattern that shares similarity to toxin-induced responses. In contrast, we do not observe JU1400 intestinal resistance being regulated at the transcriptional level. The transcriptional response to these four microsporidia species is conserved, with C. elegans strain-specific differences in potential immune genes. Together, our results show that phenotypic differences to microsporidia infection amongst C. elegans are common and that animals can evolve species-specific genetic interactions.

Molecular prevalence and genotypes of Enterocytozoon bieneusi in cancer patients under chemotherapy in Aegean region of Türkiye.

BACKGROUND: Enterocytozoon bieneusi is the most common species found in humans. Although E. bieneusi has been investigated in humans, genotype profile of E. bieneusi is not known in Türkiye. METHODS: In this study, we screened E. bieneusi in patients (n = 94) with different types of malignant solid tumors by Real Time PCR and then sequenced E. bieneusi positive samples. All cancer patients were undergoing chemotherapy and had diarrhea. Moreover, as control groups, we also screened E. bieneusi in patients with diarrhea (n = 50) and without diarrhea (n = 50). RESULTS: Among all patients analyzed, 33 (17%) were found to be E. bieneusi-positive. As the patients were categorized, the molecular prevalence of E. bieneusi increased to 25.5% among cancer patients with diarrhea. However, the molecular prevalence of E. bieneusi was found to be lower in patients with presenting only diarrhea (8%) and patients without diarrhea (10%). The high molecular prevalence value detected among cancer patients with diarrhea was also statistically significant compared to other patient groups (P = 0.00112 and P = 0.0269). Among the 33 Real Time PCR positive samples, 10 of them were amplified by nested PCR and among these 10 samples, 6 of them were successfully genotyped. The phylogenetic tree showed the presence of D and Type IV which were also identified in stray cats living in Izmir in our previous study. CONCLUSIONS: High molecular prevalence value indicates the importance of screening stool samples of cancer patients with diarrhea for E. bieneusi and genotyping results indicate that D and Type IV are circulating between humans and cats.

Molecular detection of Enterocytozoon bieneusi in bats from Portugal.

Enterocytozoon bieneusi is a microsporidia commonly found in the gastrointestinal tract of humans and a wide range of other animals, constituting a major cause of microsporidiosis in humans. Although E. bieneusi has been detected in humans, domestic, and wild animals in Portugal, and its presence in bats has been linked to zoonotic characteristics, its occurrence in bats within the country has not been reported. In this study, we investigated the presence of E. bieneusi in 380 bat fecal samples collected in mainland Portugal through a nested PCR assay targeting the internal transcribed spacer region and the flanking small and large subunits of the ribosomal RNA. Enterocytozoon bieneusi was detected in one bat sample (i.e., 0.26%; Pipistrellus pipistrellus). Additionally, another sample tested positive for Enterocytozoon sp. Phylogenetic analysis of the obtained ITS sequence of E. bieneusi revealed clustering within the potentially zoonotic Group 1. This study represents the first report of E. bieneusi in a bat from Europe. Findings presented here contribute to an enhanced understanding of E. bieneusi epidemiology.

Enterocytozoon bieneusi is the most frequent cause of microsporidiosis in humans. In this study, E. bieneusi, belonging to a potentially zoonotic Group, was detected in 0.26% bat samples from Portugal, highlighting bats' potential role in transmitting this microsporidia to humans and other animals.

Differential molecular responses of hemolymph and hepatopancreas of swimming crab, Portunus trituberculatus, infected with Ameson portunus (Microsporidia).

Ameson portunus (Microsporidia) has caused serious economic losses to the aquaculture industry of swimming crab, Portunus trituberculatus. The hemolymph and hepatopancreas are the main immune organs of P. trituberculatus, and the main sites of A. portunus infection. Elucidating the response characteristics of hemolymph and hepatopancreas to microsporidian infection facilitates the development of microsporidiosis prevention and control strategy. This study performed comparative transcriptomic analysis of hemolymph (PTX/PTXA) and hepatopancreas (PTG/PTGA) of P. trituberculatus uninfected and infected with A. portunus. The results showed that there were 223 and 1309 differentially expressed genes (DEGs) in PTX/PTXA and PTG/PTGA, respectively. The lysosome pathway was significantly enriched after the invasion of the hemolymph by A. portunus. Also, immune-related genes were all significantly up-regulated in the hemolymph and hepatopancreas, suggesting that the invasion by A. portunus may activate host immune responses. Unlike hemolymph, antioxidant and detoxification-related genes were also significantly up-regulated in the hepatopancreas. Moreover, metabolism-related genes were significantly down-regulated in the hepatopancreas, suggesting that energy synthesis, resistance to pathogens, and regulation of oxidative stress were suppressed in the hepatopancreas. Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. The differential genes and pathways identified in this study can provide references for the prevention and control of microsporidiosis.

Host-specific Cryptosporidium, Giardia and Enterocytozoon bieneusi in shelter dogs from central Europe.

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

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.

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

BACKGROUND: Parasites 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. RESULTS: We 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. CONCLUSIONS: This 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.

A novel neurotropic microsporidium from the swamp guppy Micropoecilia picta from Grenada, West Indies.

A novel microsporidium was observed in wild swamp guppies Micropoecilia picta from Levera Pond within Levera National Park Grenada, West Indies. Initial observations indicated similarity with Pseudoloma neurophilia, an important pathogen in zebrafish Danio rerio. P. neurophilia exhibit broad host specifity, including members of the family Poecillidae, and both parasites infect the central nervous system. However, spore morphology and molecular phylogeny based on rDNA showed that the swamp guppy microsporidium (SGM) is distinct from P. neurophilia and related microsporidia (Microsporidium cerebralis and M. luceopercae). Spores of the SGM were smaller than others in the clade (3.6 µm long). Differences were also noted in histology; the SGM formed large aggregates of spores within neural tissues along with a high incidence of numerous smaller aggregates and single spores within the surface tissue along the ventricular spaces that extended submeninx, whereas P. neurophilia and M. cerebralis infect deep into the neuropile and cause associated lesions. Analysis of small subunit ribosomal DNA sequences showed that the SGM was <93% similar to these related microsporidia. Nevertheless, one of 2 commonly used PCR tests for P. neurophilia cross reacted with tissues infected with SGM. These data suggest that there could be other related microsporidia capable of infecting zebrafish and other laboratory fishes that are not being detected by these highly specific assays. Consequently, exclusive use of these PCR tests may not accurately diagnose other related microsporidia infecting animals in laboratory and ornamental fish facilities.

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), 60 kDa 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.

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.

The global epidemiology of Microsporidia infection in birds: A systematic review and meta-analysis.

This study aimed to assess the global status and genetic diversity of Microsporidia infection in different birds. An online search was conducted in international databases from 1 January 1990 to 30 June 2022. A total of 34 articles (including 37 datasets) were included for the final meta-analysis. The pooled global prevalence of Microsporidia infection in birds was 14.6% (95% CI: 11.6-18.1). The highest prevalence of Microsporidia was found in wild waterfowl which was 54.5% (28.1-78.6). In terms of detection methods, the pooled prevalence was estimated to be 21.2% (95% CI: 12.1-34.4) and 13.4% (95% CI: 10.3-17.3) for using microscopic and molecular detection methods, respectively. Enterocytozoon bieneusi was the most common pathogen (24/31; 77.42% of the studies) according to PCR-based methods, and genotype D was the highest reported genotype (nine studies). In conclusion, designing strategies for the control and prevention of Microsporidia infection in birds should be recommended.

Non-coding RNAs identification and regulatory networks in pathogen-host interaction in the microsporidia congenital infection.

BACKGROUND: The interaction networks between coding and non-coding RNAs (ncRNAs) including long non-coding RNA (lncRNA), covalently closed circular RNA (circRNA) and miRNA are significant to elucidate molecular processes of biological activities and interactions between host and pathogen. Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-feeding industry. However, little is known about ncRNAs that take place in the microsporidia congenital infection. Here we conducted whole-transcriptome RNA-Seq analyses to identify ncRNAs and regulatory networks for both N. bombycis and host including silkworm embryos and larvae during the microsporidia congenital infection. RESULTS: A total of 4,171 mRNAs, 403 lncRNA, 62 circRNAs, and 284 miRNAs encoded by N. bombycis were identified, among which some differentially expressed genes formed cross-talk and are involved in N. bombycis proliferation and infection. For instance, a lncRNA/circRNA competing endogenous RNA (ceRNA) network including 18 lncRNAs, one circRNA, and 20 miRNAs was constructed to describe 14 key parasites genes regulation, such as polar tube protein 3 (PTP3), ricin-B-lectin, spore wall protein 4 (SWP4), and heat shock protein 90 (HSP90). Regarding host silkworm upon N. bombycis congenital infection, a total of 14,889 mRNAs, 3,038 lncRNAs, 19,039 circRNAs, and 3,413 miRNAs were predicted based on silkworm genome with many differentially expressed coding and non-coding genes during distinct developmental stages. Different species of RNAs form interacting network to modulate silkworm biological processes, such as growth, metamorphosis and immune responses. Furthermore, a lncRNA/circRNA ceRNA network consisting of 140 lncRNAs, five circRNA, and seven miRNAs are constructed hypothetically to describe eight key host genes regulation, such as Toll-6, Serpin-6, inducible nitric oxide synthase (iNOS) and Caspase-8. Notably, cross-species analyses indicate that parasite and host miRNAs play a vital role in pathogen-host interaction in the microsporidia congenital infection. CONCLUSION: This is the first comprehensive pan-transcriptome study inclusive of both N. bombycis and its host silkworm with a specific focus on the microsporidia congenital infection, and show that ncRNA-mediated regulation plays a vital role in the microsporidia congenital infection, which provides a new insight into understanding the basic biology of microsporidia and pathogen-host interaction.

Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera.

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee's ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

First identification and coinfection detection of Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp. and Giardia duodenalis in diarrheic pigs in Southwest China.

BACKGROUND: Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp., and Giardia duodenalis (G. intestinalis) are enteric pathogens that cause diarrhea in pigs. This study aimed to determine the prevalence of these enteric parasites and their coinfection with E. bieneusi in diarrheic pigs in Southwest China (Chongqing and Sichuan) using nested polymerase chain reaction (nPCR) based methods. RESULTS: A total of 514 fecal samples were collected from diarrheic pigs from 14 pig farms in Chongqing (five farms) and Sichuan (nine farms) Provinces. The prevalence of Encephalitozoon spp., Cryptosporidium spp. and G. duodenalis was 16.14% (83/514), 0% (0/514), and 8.95% (46/514), respectively. Nested PCR revealed 305 mono-infections of E. bieneusi, six of E. cuniculi, two of E. hellem, and nine of G. duodenalis and 106 concurrent infections of E. bieneusi with the other enteric pathogens. No infections of E. intestinalis and Cryptosporidium species were detected. The highest coinfection was detected between E. bieneusi and E. cuniculi (10.5%, 54/514), followed by E. bieneusi and G. duodenalis (5.8%, 30/514) and E. bieneusi and E. hellem (2.9%, 15/514). E. bieneusi was the most frequently detected enteric pathogen, followed by E. cuniculi, G. duodenalis and E. hellem. There was a significant age-related difference in the prevalence of E. cuniculi in fattening pigs (χ2 = 15.266, df = 3, P = 0.002) and G. duodenalis in suckling pigs (χ2 = 11.92, df = 3, P = 0.008) compared with the other age groups. Sequence analysis of the ITS region of Encephalitozoon species showed two genotypes (II and III) for E. cuniculi and one (TURK1B) for E. hellem. Only G. duodenalis assemblage A was identified in all nested PCR-positive samples. E. bieneusi was found more often than other enteric pathogens. CONCLUSIONS: This study showed that E. bieneusi, Encephalitozoon spp. [E. cuniculi and E. hellem] and G. duodenalis were common enteric parasites in diarrheic pigs in Chongqing and Sichuan Provinces. In case of both mono-infection and coinfection, E. bieneusi was the most common enteric pathogen in diarrheic pigs. Thus, it may be a significant cause of diarrhea in pigs. Precautions should be taken to prevent the spread of these enteric parasites.

Occurrence and molecular characterization of Enterocytozoon bieneusi in wild and domestic animal species in Portugal.

The phylum Microsporidia encompasses a diverse group of obligate, intracellular, and spore-forming organisms able to infect a wide range of animal hosts. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and animals. Little is known about the presence and epidemiology of E. bieneusi in wildlife. We investigated E. bieneusi occurrence and genetic diversity in wild and domestic mammals, through molecular-detection methods, from different regions across Portugal. A total of 756 samples were collected from 288, 242, and 226 wild carnivores, wild ungulates, and domestic animals, respectively. Overall, eight specimens were E. bieneusi-positive (1.1%, 8/756) obtained from five wild (Iberian lynx, Iberian wolf, red fox, stone marten, and wild boar) and one domestic (sheep) host. Nucleotide sequence analysis identified four genotypes of E. bieneusi, Type IV, Wildboar3, BEB6, and PtEbIX. Three of those genotypes belong to Groups 1 (Type IV and Wildboar3) and 2 (BEB6), which are known to contain genotypes capable of infecting a variety of hosts, including humans, highlighting their public health importance. PtEbIX belongs to the dog-specific Group 11. This study represents the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal in wild and domestic animals to date and the first worldwide identification of E. bieneusi in wolf species. Our study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals.

The identification of human-pathogenic genotypes of fungi-related Enterocytozoon bieneusi in wild carnivores and ungulates in Portugal suggests cross-species infection events and overlapping of the sylvatic and domestic transmission cycles, demonstrating a potential transmission risk to humans.

Enterocytozoon bieneusi and Encephalitozoon intestinalis (microsporidia) in HIV-positive patients in central Spain.

Microsporidia are fungi-related eukaryotic intracellular parasites that opportunistically infect immunocompromised individuals such as those infected by the human immunodeficiency virus (HIV). Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most clinically relevant species. We investigated the occurrence and genetic diversity of microsporidial and protist infections in mostly immunocompetent HIV-positive patients in Madrid, Spain. A structured questionnaire was used to retrieve data on factors potentially associated with an increased risk of infection, including sexual attitudes and sex-risk behaviour. Faecal samples (n = 96) from 81 HIV-positive patients were collected and analysed by molecular (PCR and Sanger sequencing) methods. Two microsporidial pathogens were detected: Ent. bieneusi (2.5%, 95% CI: 0.3-8.6) and Enc.intestinalis (4.9%, 95% CI: 1.4-12.2). The two Ent. bieneusi isolates were identified as zoonotic genotype A. Among protists, Entamoeba dispar was the species most prevalently found (33.3%, 95% CI: 23.2-44.7), followed by Blastocystis spp. (19.8%, 95% CI: 11.7-30.1), Giardia duodenalis (13.6%, 95% CI: 7.0-23.0), and Cryptosporidium spp. and Entamoeba histolytica (2.5%, 95% CI: 0.3-8.6 each). Cyclospora cayetanensis and Cystoisospora belli were not detected. Subtypes ST1 (70.6%, 12/17) and ST3 (29.4%, 5/17) were identified within Blastocystis sp., sub-assemblages AII and BIII (50%, 1/2 each) within G. duodenalis, and Cry. parvum and canine-adapted Cry. canis (50%, 1/2 each) within Cryptosporidium spp. Microsporidial and protist parasites were frequent in well-controlled, mostly immunocompetent HIV-positive patients and should be included in diagnostic algorithms when diarrhoea is present.

Opportunistic microsporidial and protist intestinal infections were relatively common in well-controlled HIV-positive patients in Madrid, Spain. These agents should be suspected and appropriately diagnosed in HIV-positive patients presenting with diarrhoea regardless of their immunological status.

Microsporidial keratoconjunctivitis - first outbreak in Japan.

BACKGROUND: Most cases of microsporidial keratoconjunctivitis are found in the Southern hemisphere. Our purpose was to investigate the first outbreak of microsporidial keratoconjunctivitis in Japan among healthy, immunocompetent soccer players from the same team during a 1-month period. CASE PRESENTATION: This study is an observational case series. The medical records were analyzed for five cases with microsporidial keratoconjunctivitis who presented within September 2022. All five cases were males between 28 and 36 years old. These previously healthy individuals belonged to the same football team. Their eyes were considered susceptible to contaminated water or dirt from the turf at game and practice sites. All cases involved unilateral conjunctivitis, with scattered round white lesions that showed positive fluorescein staining in the corneal epithelium. All cases experienced diminution of vision in the affected eye. In three cases, direct smears showed spores of approximately 2-3 µm in diameter. Polymerase chain reaction (PCR) analysis of corneal scrapes revealed partial amplification of microsporidial 18 S ribosomal RNA gene in four cases. Sequences of PCR products from all four cases showed 100% identity with strains of Vittaforma corneae previously reported from an outbreak in Singapore. All cases were treated with topical therapy, including voriconazole, fluorometholone, and levofloxacin. Four eyes underwent corneal scraping. After treatment, all eyes healed without residual opacities. CONCLUSIONS: Only a few sporadic case reports of this disease have previously been reported in Japan. We detected V. corneae in our case series, representing what appears to be the first outbreak of microsporidial keratoconjunctivitis in Japan. Exposure to contaminated water or soil, in addition to inadequate sanitary facilities, represents a potential source of infection. Further investigations to clarify the characteristics of microsporidia seem warranted.

Global prevalence and genotype distribution of Microsporidia spp. in various consumables: a systematic review and meta-analysis.

Water and food sources play a major role in the distribution and transfer of microsporidia infection to animals and humans. So, this systematic review and meta-analysis aimed to assess the status and genetic diversity of microsporidia infection in water, vegetables, fruits, milk, cheese, and meat. The standard protocol of Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines was followed. Scopus, PubMed, Web of Science, and Google Scholar were searched from 1 January 2000 and 1 February 2023. The point estimates and 95% confidence intervals (CIs) were calculated using a random-effects model. Of the 1,308 retrieved studies, 35 articles were included in the final meta-analysis. The pooled prevalence of microsporidia infection in mixed water, mixed fruits, mixed vegetables, and milk was 43.3% (95% CI, 33-54.2%; I2, 94.86%), 35.8% (95% CI, 5.3-84.8%; I2, 0), 12% (95% CI, 4.9-26.6%; I2, 96.43%), and 5.8% (95% CI, 2.7-12%; I2, 83.72%), respectively. Considering the genotypes, microsporidia with genotype D in water sources and genotype CD6 in vegetables/fruits were the highest reported genotypes. Given the relatively high prevalence of microsporidiosis (especially in water sources), designing strategies for control, and prevention of microsporidia infection in these sources should be recommended.

Characterization of Enterocytozoon hepatopenaei causing hepatopancreatic microsporidiosis in L. vannamei and a new molecular method for its detection in shrimps, and other environmental samples.

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is a disease of utmost concern in almost all shrimp growing countries. The pathogen was characterized by ultramicrography, histopathology and phylogenetic analysis of 18srDNA. A total of 183 biological samples were collected from all major shrimp growing states of the country.The histology technique could be used very well in identifying the site of infection and can aid in diagnosis of EHP. Wet mount and Ultramicrography were employed to observe the structure of spores. A single step PCR based method was developed for detecting the pathogen from variety of DNA samples including shrimp and non-shrimp sources.The developed PCR assay proved to be a robust and reliable technique to detect EHP in shrimps and environmental samples and for assessing the distribution of pathogen within geographical zones, thus aid in mitigating the disease. The PCR primers was also used to generate DIG labelled probe which was successful in binding to the EHP infected cells in HP of shrimp. The presence of pathogen was confirmed from many non-shrimp environmental samples suggests that they could act as reservoirs for recurrent infection in shrimp ponds. Proper control of these reservoirs will be the first step in recovering an EHP affected pond back to normal.