Experimental reproduction of White Feces Syndrome in whiteleg shrimp, Penaeus vannamei.

White Feces Syndrome (WFS) is an emergent disease of penaeid shrimp (Penaeus monodon and P. vannamei) that is identified by the presence of floating white fecal strings on pond water in grow-out ponds. Although the clinical manifestations of WFS are well defined, the underling etiology remains obscure. WFS has been associated with several enteric pathogens, including Enterocytozoon hepatopenaei (EHP). The association is based on studies that found areas where WFS has been reported, the prevalence and severity of EHP infection are high. In this study, we describe an experimental reproduction of WFS in P. vannamei pre-infected with EHP and challenged with a unique isolate of Vibrio parahaemolyticus isolated from the gastrointestinal tract of a shrimp displaying WFS. Upon laboratory challenge, shrimp displaying white fecal strings and white discoloration of the gastrointestinal tract were analyzed by histopathology, in-situ hybridization and quantitative PCR. Histological analysis confirmed the lesions of EHP and septic hepatopancreatic necrosis in the hepatopancreas of shrimp exposed to both pathogens. Quantitative PCR showed shrimp infected with both EHP and V. parahaemolyticus had a significantly higher load of EHP compared to shrimp infected with EHP alone. This is the first demonstration of experimental reproduction of WFS under laboratory conditions when animals are infected with EHP and V. parahaemolyticus concurrently. The data revealed a synergistic relation between EHP and V. parahaemolyticus isolate that led to the manifestation of WFS. We propose the gross signs of WFS can be used as an indicator of the presence of EHP infection in association with a particular strain of an enteric Vibrio spp. in countries where EHP is endemic.

Simultaneous visual diagnosis of acute hepatopancreatic necrosis disease and Enterocytozoon hepatopenaei infection in shrimp with duplex recombinase polymerase amplification.

Shrimp is a globally popular seafood. Shrimp farming has been challenged by various infectious diseases that lead to significant economic losses. The prevention of two important shrimp infectious diseases, the acute hepatopancreatic necrosis disease (AHPND) and the Enterocytozoon hepatopenaei (EHP) infection, is highly dependent on early and accurate diagnostic. On-site monitoring of the two diseases in shrimp farming facilities demands point-of-care-testing (POCT) type of diagnostic assays. This study established a duplex recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) combined assay that could simultaneously diagnose the two diseases. The optimized RPA-LFD assay could finish the diagnostic in 35 min with good specificity, and the sensitivity reached 101 and 102 gene copies per reaction for EHP and AHPND, respectively, which were at the same level as the currently available molecular diagnostic assays. Test results of clinical samples showed 100% agreement of this assay with the industrial standard nested polymerase chain reaction (PCR) assays, and samples with both diseases were simultaneously identified. Because of the isothermal 37℃ amplification and the visual reading of the signal on dipsticks, the dependence on equipment is minimal. This duplex RPA-LFD assay is well suited for simultaneous POCT diagnostic of the two important shrimp infectious diseases. Moreover, the principle can be applied to multiplex POCT diagnostic of other infectious diseases in aquaculture.

Molecular characterization of the waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp. in wastewater and sewage in Guangzhou, China.

BACKGROUND: The waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Cyclospora cayetanensis can cause intestinal diseases in humans. An understanding of their occurrence and transport in the environment is essential for accurate quantitative microbial risk assessment. METHODS: A total of 238 influent samples were collected from four wastewater treatment plants (WWTPs) and 88 samples from eight sewer locations in Guangzhou, China. PCR-based tools were used to detect and genetically characterize Cryptosporidium spp., G. duodenalis and E. bieneusi. Eimeria spp. and Cyclospora spp. were also analyzed to assess the sources of Cryptosporidium spp., G. duodenalis and E. bieneusi in wastewater. RESULTS: The overall occurrence rates in the WWTP and sewer samples were 14.3% (34/238) and 13.6% (12/88) for Cryptosporidium spp., 55.5% (132/238) and 33.0% (29/88) for G. duodenalis, 56.3% (134/238) and 26.1% (23/88) for E. bieneusi and 45.4% (108/238) and 47.7% (42/88) for Eimeria spp., respectively. Altogether, 11 Cryptosporidium species and genotypes, six G. duodenalis genotypes, 11 E. bieneusi genotypes and four C. cayetanensis were found, together with the presence of nine Eimeria species. The common occurrence of Cryptosporidium rat genotype IV, C. muris and Eimeria papillata and E. nieschulzi suggested that rodents were significant sources of the enteric pathogens detected in the wastewater samples. CONCLUSIONS: While the dominant Cryptosporidium spp. detected in the raw wastewater sampled in this study are not pathogenic to humans, the widely detected G. duodenalis assemblage A and E. bieneusi genotypes D and Type IV are well-known zoonotic pathogens. Further studies are needed to monitor the occurrence of these waterborne pathogens in WWTPs to better understand their transmission and environmental transport in China.

Enterocytozoon bieneusi of animals-With an 'Australian twist'.

Enterocytozoon bieneusi is a microsporidian microorganism that causes intestinal disease in animals including humans. E. bieneusi is an obligate intracellular pathogen, typically causing severe or chronic diarrhoea, malabsorption and/or wasting. Currently, E. bieneusi is recognised as a fungus, although its exact classification remains contentious. The transmission of E. bieneusi can occur from person to person and/or animals to people. Transmission is usually via the faecal-oral route through E. bieneusi spore-contaminated water, environment or food, or direct contact with infected individuals. Enterocytozoon bieneusi genotypes are usually identified and classified by PCR-based sequencing of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA. To date, ~600 distinct genotypes of E. bieneusi have been recorded in ~170 species of animals, including various orders of mammals and reptiles as well as insects in >40 countries. Moreover, E. bieneusi has also been found in recreational water, irrigation water, and treated raw- and waste-waters. Although many studies have been conducted on the epidemiology of E. bieneusi, prevalence surveys of animals and humans are scant in some countries, such as Australia, and transmission routes of individual genotypes and related risk factors are poorly understood. This article/chapter reviews aspects of the taxonomy, biology and epidemiology of E. bieneusi; the diagnosis, treatment and prevention of microsporidiosis; critically appraises the naming system for E. bieneusi genotypes as well as the phylogenetic relationships of these genotypes; provides new insights into the prevalence and genetic composition of E. bieneusi populations in animals in parts of Australia using molecular epidemiological tools; and proposes some areas for future research in the E. bieneusi/microsporidiosis field.

Comparison of infectious agents detected from hatchery and wild juvenile Coho salmon in British Columbia, 2008-2018.

Infectious diseases are potential contributors to decline in Coho salmon (Oncorhynchus kisutch) populations. Although pathogens are theoretically considered to pose higher risk in high-density rearing environments like hatcheries, there is no direct evidence that hatchery-origin Coho salmon increase the transmission of infectious agents to sympatric wild populations. This study was undertaken to compare prevalence, burden, and diversity of infectious agents between hatchery-reared and wild juvenile Coho salmon in British Columbia (BC), Canada. In total, 2,655 juvenile Coho salmon were collected between 2008 and 2018 from four regions of freshwater and saltwater in BC. High-throughput microfluidics qPCR was employed for simultaneous detection of 36 infectious agents from mixed-tissue samples (gill, brain, heart, liver, and kidney). Thirty-one agents were detected at least once, including ten with prevalence >5%. Candidatus Brachiomonas cysticola, Paraneuclospora theridion, and Parvicapsula pseudobranchiocola were the most prevalent agents. Diversity and burden of infectious agents were substantially higher in marine environment than in freshwater. In Mainland BC, infectious burden and diversity were significantly lower in hatchery smolts than in wild counterparts, whereas in other regions, there were no significant differences. Observed differences in freshwater were predominantly driven by three parasites, Loma salmonae, Myxobolus arcticus, and Parvicapsula kabatai. In saltwater, there were no consistent differences in agent prevalence between hatchery and wild fish shared among the west and east coasts of Vancouver Island. Although some agents showed differential infectious patterns between regions, annual variations likely contributed to this signal. Our findings do not support the hypothesis that hatchery smolts carry higher burdens of infectious agents than conspecific wild fish, reducing the potential risk of transfer to wild smolts at this life stage. Moreover, we provide a baseline of infectious agents in juvenile Coho salmon that will be used in future research and modeling potential correlations between infectious profiles and marine survival.

Genotypes and public health potential of Enterocytozoon bieneusi and Giardia duodenalis in crab-eating macaques.

BACKGROUND: Enterocytozoon bieneusi and Giardia duodenalis are common human and animal pathogens. Studies have increasingly shown that non-human primates (NHPs) are common hosts of these two zoonotic parasites. However, few studies have explored the genetic diversity and public health potential of these pathogens in laboratory monkeys. In this study, we examined the genetic diversity of the two pathogens in crab-eating macaques (Macaca fascicularis) in a commercial facility in Hainan, China. RESULTS: Enterocytozoon bieneusi and G. duodenalis were detected by PCR analysis in 461/1452 (31.7%) and 469/1452 (32.3%) fecal specimens from the animals, respectively. Significantly higher detection rates of E. bieneusi were detected in males (36.5%, 258/706) than in females (26.7%, 160/599; χ2 = 14.391, P = 0.0001), in animals with loose stools (41.4%, 151/365) than those with normal stool (28.5%, 310/1087; χ2 = 20.83, P < 0.0001), and in animals of over 3 years of age (38.6%, 135/350) than those of 1-3 years (29.6%, 326/1,102; χ2 = 9.90, P = 0.0016). For G. duodenalis, the detection rate in males (33.4%, 236/706) was higher than in females but not statistically significant (30.2%, 181/599; χ2 = 1.54, P = 0.2152), in monkeys with loose stools (41.1%, 150/365) than those with normal stools (29.3%, 319/1087; χ2 = 17.25, P < 0.0001), and in monkeys of 1-3 years of age (36.6%, 403/1102) than those over 3 years (18.9%, 66/350; χ2 = 38.11, P < 0.0001). Nine E. bieneusi genotypes were detected in this study by DNA sequence analysis of the internal transcribed spacer of the rRNA gene, namely Type IV (236/461), Peru8 (42/461), Pongo2 (27/461), Peru11 (12/461), D (4/461) and PigEbITS7 (1/461) previously seen in NHPs as well as humans, and CM1 (119/461), CM2 (17/461) and CM3 (3/461) that had been only detected in NHPs. DNA sequence analyses of the tpi, gdh and bg loci identified all G. duodenalis specimens as having assemblage B. Altogether, eight (4 known and 4 new), seven (6 known and 1 new) and seven (4 known and 3 new) subtypes were seen at the tpi, gdh and bg loci, leading to the detection of 53 multi-locus genotypes (MLG-B-hn01 to MLG-B-hn53). Most of them were genetically related to those previously seen in common Old-World monkeys. CONCLUSIONS: Data from this study indicate a common occurrence of zoonotic genotypes of E. bieneusi and assemblage B of G. duodenalis in farmed crab-eating macaques in Hainan, China.

The prevalence of microsporidia in China : A systematic review and meta-analysis.

Microsporidia are a diverse parasite phylum infecting host from all major taxa in all global biomes. This research was conducted to conclude the prevalence of microsporidia in China. All published articles up to February 16, 2018 were considered, including descriptive, cross-sectional, case-control and epidemiology studies. A total of 1052 articles were separated after literature search. After a strict selection according to our criteria, 82 articles were included in qualitative synthesis and ultimately 52 studies were included in quantitative synthesis. Three species of microsporidia were confirmed to exist in China, including Enterocytozoon bieneusi (E. bieneusi), Nosema and Encephalitozoon cuniculi (E. cuniculi). The highest overall estimated prevalence of E. bieneusi in humans was 8.1%, which was observed in acquired immunodeficiency syndrome patients (AIDS). Moreover, the prevalence of E. bieneusi in animals including the cattle, dogs, pigs, deer, sheep and goats were analyszed in this study. The overall estimated prevalence of E. bieneusi acquired by using the random effects model in meta-analysis in cattle, dogs, pigs, sheep and goats and deer was 20.0% (95% confidence intervals: 0.133-0.266, I2 = 98.031%, p < 0.0001), 7.8% (95% CI: 0.050-0.106, I2 = 60.822%, p = 0.0537), 45.1% (95% CI: 0.227-0.674, I2 = 98.183%, p < 0.0001), 28.1% (95% CI: 0.146-0.415, I2 = 98.716%, p < 0.0001) and 19.3% (95% CI: 0.084-0.303, I2 = 96.995%, p < 0.0001) respectively. The overall detection rate of E. bieneusi in water acquired by using the random effects model in meta-analysis was 64.5% (95% CI: 0.433-0.857, I2 = 98.486%, p < 0.0001). Currently, 221 genotypes of E. bieneusi, 1 genotype of E. cuniculi and 6 Nosema were detected in China. The most prevalent genotype of E. bieneusi was genotype D, followed by BEB6 and EbpC.

Occurrence, Molecular Characterization, and Assessment of Zoonotic Risk of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Pigs in Henan, Central China.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common gastrointestinal pathogens in humans and animals. Little is known about them and the range of species/assemblages/genotypes occurring in domestic pigs in China. Here, we present data on the occurrence and molecular diversity of these pathogens detected in the feces from farms in Henan, central China. Of 897 fecal samples tested, 28 (3.1%), 15 (1.7%), and 408 (45.5%) samples were positive for Cryptosporidium, G. duodenalis, and E. bieneusi, respectively. Cryptosporidium and G. duodenalis were most frequently detected in piglets, while E. bieneusi was markedly more prevalent in fattening pigs. Sequence analysis of SSU rRNA gene revealed that positive Cryptosporidium strains belonged to C. suis (n = 18) and C. scrofarum (n = 10). Giardia duodenalis assemblages E (n = 9), assemblages A (n = 3), and assemblages C (n = 3) were characterized based on the sequence analysis of tpi gene. Thirteen E. bieneusi genotypes comprising four novel (pigHN-I to pigHN-IV) and nine known (EbpC, EbpA, pigEbITS5, LW1, H, CM8, G, CHG19, and CHS5) genotypes were identified by ITS sequence analysis of a large proportion (n = 200) of E. bieneusi-positive samples. EbpC was the most frequent genotype, detected in 60 specimens. All 13 genotypes identified in this study clustered in zoonotic Group 1. The findings indicate that the presence of zoonotic species/assemblages/genotypes of these pathogens poses a threat to public health, suggesting that pigs in Henan province could be a significant source of human infection and water pollution.

Molecular characterization and new genotypes of Enterocytozoon bieneusi in pet chipmunks (Eutamias asiaticus) in Sichuan province, China.

BACKGROUND: Enterocytozoon bieneusi, the most commonly identified microsporidian species in humans, is also identified in livestock, birds, rodents, reptiles, companion animals, even wastewater. However, there is no information available on occurrence of E. bieneusi in pet chipmunks. The aim of the present study was to determine the genotypes, molecular characterization of E. bieneusi in pet chipmunks, and assess the zoonotic potential. RESULTS: A total of 279 fecal specimens were collected from chipmunks from seven pet shops and one breeding facility in Sichuan province, China. The prevalence for E. bieneusi was 17.6% (49/279) based on nested PCR targeting the internal transcribed spacer (ITS) region. The prevalence of E. bieneusi in chipmunks < 90 days of age was significantly higher than that in older chipmunks; however, differences among different sources and between genders were not significant. Eight genotypes of E. bieneusi were identified, including four known genotypes (D, Nig7, CHG9, and CHY1) and four novel genotypes (SCC-1 to 4). Phylogenetic analysis classified these genotypes into four distinct groups as follows: genotypes D and CHG9 clustered into group 1 of zoonotic potential; genotypes Nig7 and CHY1 clustered into group 6 and a new group, respectively; the four novel genotypes (SCC-1 to 4) formed a separate group named group 10. CONCLUSIONS: To the best of our knowledge, this is the first study reporting the prevalence and genotypes of E. bieneusi in pet chipmunks in China. Genotypes D and Nig7, found in chipmunks in this study, have also been previously identified in humans, which suggests that chipmunks might play a role in the transmission of this pathogen to humans.

Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP).

BACKGROUND: The microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp. RESULTS: We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein. CONCLUSIONS: Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.

First report of Giardia duodenalis and Enterocytozoon bieneusi in forest musk deer (Moschus berezovskii) in China.

BACKGROUND: Giardia duodenalis and Enterocytozoon bieneusi are widespread pathogens that can infect humans and various animal species. Thus far, there are only a few reports of G. duodenalis and E. bieneusi infections in ruminant wildlife. Thus, the objective of this study was to examine the prevalence of G. duodenalis and E. bieneusi in forest musk deer in Sichuan, China, as well as identifying their genotypes. RESULTS: In total, we collected 223 faecal samples from musk deer at the Sichuan Institute of Musk Deer Breeding in Dujiangyan (n = 80) and the Maerkang Breeding Institute (n = 143). Five (2.24%) faecal samples were positive for G. duodenalis; three belonged to assemblage E, and two belonged to assemblage A based on the sequence analysis of the ß-giardin (bg) gene. One sample each was found to be positive based on the glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) gene, respectively. Thirty-eight (17.04%) faecal samples were found to be E. bieneusi-positive based on the internal transcribed spacer (ITS) sequence, and only SC03 genotype was identified, which belonged to the zoonotic group 1 according to the phylogenic analysis. The infection rates were significantly different among the different geographical areas and age groups but had no apparent association with gender or clinical symptoms. CONCLUSIONS: To our knowledge, this was the first molecular characterisation of G. duodenalis and E. bieneusi in musk deer. Identification of the zoonotic genotypes indicated a potential public health threat, and our study suggested that the forest musk deer is an important carrier of these parasites.

Enterocytozoon bieneusi Microsporidiosis in Stem Cell Transplant Recipients Treated with Fumagillin

Enterocytozoon bieneusi microsporidiosis is an emerging disease in immunocompromised patients. We report 2 cases of this disease in allogeneic hematopoietic stem cell transplant patients successfully treated with fumagillin. Thrombocytopenia occurred but without major adverse events. Modifications of immunosuppression could be avoided when E. bieneusi is rapidly identified and fumagillin therapy is started promptly.

Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia.

Glycolysis and oxidative phosphorylation are the fundamental pathways of ATP generation in eukaryotes. Yet in microsporidia, endoparasitic fungi living at the limits of cellular streamlining, oxidative phosphorylation has been lost: energy is obtained directly from the host or, during the dispersive spore stage, via glycolysis. It was therefore surprising when the first sequenced genome from the Enterocytozoonidae - a major family of human and animal-infecting microsporidians - appeared to have lost genes for glycolysis. Here, we sequence and analyse genomes from additional members of this family, shedding new light on their unusual biology. Our survey includes the genome of Enterocytozoon hepatopenaei, a major aquacultural parasite currently causing substantial economic losses in shrimp farming, and Enterospora canceri, a pathogen that lives exclusively inside epithelial cell nuclei of its crab host. Our analysis of gene content across the clade suggests that Ent. canceri's adaptation to intranuclear life is underpinned by the expansion of transporter families. We demonstrate that this entire lineage of pathogens has lost glycolysis and, uniquely amongst eukaryotes, lacks any obvious intrinsic means of generating energy. Our study provides an important resource for the investigation of host-pathogen interactions and reductive evolution in one of the most medically and economically important microsporidian lineages.

Zoonotic potential of Enterocytozoon genotypes in humans and pigs in Thailand.

Enterocytozoon bieneusi is an opportunistic intestinal pathogen infecting humans and a variety of animals. Its mode of transmission and zoonotic potential are not completely understood. E. bieneusi has been frequently identified in pigs. The objective of our study was to investigate E. bieneusi in pigs and humans in Western and Central Thailand to determine its presence, genetic diversity, and zoonotic potential. A total of 277 human and 210 pig faecal samples were collected and analysed. E. bieneusi was found in 5.4% and 28.1% of human and pig samples, respectively, by nested PCR. Genotyping based on the internal transcribed spacer regions of the small subunit ribosomal RNA demonstrated three known genotypes (D, H, PigEb10) and eight novel genotypes (TMH1-8) in humans, and five known genotypes (D, EbpA, EbpC, H, O) and 11 novel genotypes (TMP1-11) in pigs. All known genotypes identified in humans and pigs had zoonotic potential. Further studies are needed to evaluate zoonotic risk of novel genotypes, as pigs may play an important role in the transmission of E. bieneusi.

Enterocytozoon bieneusi Genotypes in Grazing Horses in China and their Zoonotic Transmission Potential.

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.

Enterocytozoon bieneusi in sika deer (Cervus nippon) and red deer (Cervus elaphus): deer specificity and zoonotic potential of ITS genotypes.

As the most common cause of the human microsporidiosis, Enterocytozoon bieneusi has been found in a wide variety of animal hosts. Deers are the ruminant mammals living in a variety of biomes, and the distribution of deer species differ by geography. To understand the prevalence of natural infection of E. bieneusi in deer and to assess their epidemiological role in the transmission of microsporidiosis caused by E. bieneusi, 91 fecal specimens were collected from 86 sika deers and five red deers in the northeast of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an average infection rate of 31.9% (29/91) was observed in deer, with 32.6% (28/86) for sika deer, and 20% (1/5) for red deer. Six ITS genotypes were identified: one known genotype BEB6 (n = 20) and five novel genotypes HLJD-I to HLJD-IV (one each) and HLJD-V (n = 5). A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that genotypes HLJD-II and HLJD-III fell into group 1 of zoonotic potential, while the other genotypes (BEB6, HLJD-I, HLJD-IV, HLJD-V) were clustered into so-called bovine-specific group 2. This is the first report of E. bieneusi in deer in China. The observation of genotype BEB6 in humans previously and in deer here and also the findings of the two novel genotypes (HLJD-II to HLJ-III) belonging to potential zoonotic group 1 suggested the possibility of deer in the transmission of E. bieneusi to humans.

The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei.

BACKGROUND: The microsporidian Enterocytozoon hepatopenaei was first described from Thailand in 2009 in farmed, indigenous giant tiger shrimp Penaeus (Penaeus) monodon. The natural reservoir for the parasite is still unknown. More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS). Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS. RESULTS: Generic primers used to amplify a fragment of the small subunit ribosomal RNA (ssu rRNA) gene for cloning and sequencing revealed that the new parasite from WFS ponds had 99% sequence identity to that of E. hepatopenaei, suggesting it was conspecific. Normal histological analysis using tissue sections stained with hematoxylin and eosin (H&E) revealed that relatively few tubule epithelial cells exhibited spores, suggesting that the infections were light. However, the H&E results were deceptive since nested PCR and in situ hybridization analysis based on the cloned ssu rRNA gene fragment revealed very heavy infections in tubule epithelial cells in the central region of the hepatopancreas in the absence of spores. Despite these results, high prevalence of E. hepatopenaei in shrimp from ponds not exhibiting WFS and a pond that had recovered from WFS indicated no direct causal association between these infections and WFS. This was supported by laboratory oral challenge trials that revealed direct horizontal transmission to uninfected shrimp but no signs of WFS. CONCLUSIONS: The microsporidian newly found in P. vannamei is conspecific with previously described E. hepatopenaei and it is not causally associated with WFS. However, the deceptive severity of infections (much greater than previously reported in P. monodon) would undoubtedly have a negative effect on whiteleg shrimp growth and production efficiency and this could be exacerbated by the possibility of horizontal transmission revealed by laboratory challenge tests. Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.

The reduced genome of the parasitic microsporidian Enterocytozoon bieneusi lacks genes for core carbon metabolism.

Reduction of various biological processes is a hallmark of the parasitic lifestyle. Generally, the more intimate the association between parasites and hosts the stronger the parasite relies on its host's physiology for survival and reproduction. However, some systems have been held to be indispensable, for example, the core pathways of carbon metabolism that produce energy from sugars. Even the most hardened anaerobes that lack oxidative phosphorylation and the tricarboxylic acid cycle have retained glycolysis and some downstream means to generate ATP. Here we describe the deep-coverage genome resequencing of the pathogenic microsporidiian, Enterocytozoon bieneusi, which shows that this parasite has crossed this line and abandoned complete pathways for the most basic carbon metabolism. Comparing two genome sequence surveys of E. bieneusi to genomic data from four other microsporidia reveals a normal complement of 353 genes representing 30 functional pathways in E. bieneusi, except that only 2 out of 21 genes collectively involved in glycolysis, pentose phosphate, and trehalose metabolism are present. Similarly, no genes encoding proteins involved in the processing of spliceosomal introns were found. Altogether, E. bieneusi appears to have no fully functional pathway to generate ATP from glucose. Therefore, this intracellular parasite relies on transporters to import ATP from its host.

Microsporidiosis and malnutrition in children with persistent diarrhea, Uganda.

We show that the microsporidian fungus Enterocytozoon bieneusi is associated with lower rates of weight gain in children in Uganda with persistent diarrhea. This relationship remained after controlling for HIV and concurrent cryptosporidiosis. Children with microsporidiosis were predicted to weigh 1.3 kg less than children without microsporidiosis at 5 years of age.

Outbreak of microsporidiosis caused by Enterocytozoon bieneusi in falcons.

Four falcons from a private collection of 137 falcons in Abu Dhabi (UAE) died suddenly in summer 2005. In order to screen for a possible disease among the remaining falcons in the aviary, all other birds were caught, examined and treated if necessary. Most of the falcons suffered from massive lice infestation and 74 falcons additionally from a heavy Caryospora sp. burden. Endoscopy revealed yellowish plaques on intestines, livers or kidneys in 70 birds (51.1% morbidity). Proliferative serositis was seen in 17 out of 24 necropsied birds with plaques on intestines, livers or kidneys, which did not resemble any known disease in falcons. However, apart from 20 falcons, which died within a 6-week period after the initial examinations due to advanced disease stages, all other falcons responded well to the treatment with dimetridazole (Emtryl), indicating protozoal disease. Immunohistochemistry confirmed the presence of microsporidial antigen. The final diagnosis of Enterocytozoon (E.) bieneusi genotype D was confirmed with materials from 6 birds by PCR and sequencing. To our knowledge this is the first report of microsporidiosis caused by E. bieneusi in raptors in general and in falcons in particular. However, it is still unclear for how long E. bieneusi was present in the falcon flock, and which role it played in the development of the disease. Predisposing factors such as high temperature and overcrowding in the aviary induced immune suppression causing massive lice infestation as well as coccidiosis, thus paving the way for invasion with microsporidial spores.