Investigating the transcriptomic variances in two phases Ecytonucleospora hepatopenaei (EHP) in Litopenaeus vannamei.

This study explores the transcriptomic differences in two distinct phases of Ecytonucleospora hepatopenaei (EHP) in Litopenaeus vannamei, a crucial aspect in shrimp health management. We employed high-throughput sequencing to categorize samples into two phases, 'Phase A' and 'Phase B', defined by the differential expression of PTP2 and TPS1 genes. Our analysis identified 2057 genes, with 78 exhibiting significant variances, including 62 upregulated and 16 downregulated genes. Enrichment analyses via GO and KEGG pathways highlighted these genes' roles in cellular metabolism, signal transduction, and immune responses. Notably, genes like IQGAP2, Rhob, Pim1, and PCM1 emerged as potentially crucial in EHP's infection process and lifecycle. We hypothesize that these genes may influence trehalose metabolism and glucose provision, impacting the biological activities within EHP during different phases. Interestingly, a lower transcript count in 'Phase A' EHP suggests a reduction in biological activities, likely preparing for host cell invasion. This research provides a foundational understanding of EHP infection mechanisms, offering vital insights for future studies and therapeutic interventions.

Interactions between the gut bacterial community of Exopalaemon carinicauda and infection by Enterocytozoon hepatopenaei.

To explore the relationship between the intestinal flora of Exopalaemon Carinicauda and infection by Enterocytozoo Hepatopenaei (EHP), we analyzed the species and richness of gut microbiota in infected individuals in different EHP load groups [i.e., control (C), high load (H), and low load (L)] using gene sequencing after infection. The results showed that the abundance of intestinal flora in the high-load EHP group was significantly lower than that in the healthy group. Based on the UPGMA cluster tree and PCoA analysis, with comparisons to healthy shrimp, the gut microbiota of the EHP high load and low load groups were clustered into one branch, which indicated that EHP infection changed the composition of the gut microbiota of infected shrimps. The heat map analysis of species abundance clustering revealed that the dominant bacteria in the low EHP load group and the control group were beneficial genera such as Lactococcus, Ligilactobacillius, and Bifidobacterium, but the dominant bacteria in the high EHP load group were harmful genera such as Pseudomonas, Photobacterium, and Candidatus hepatincola. The functions of the intestinal flora predicted that most genes related to metabolism were more abundant in healthy shrimp, most genes related to metabolism and the organisms' system were more abundant in the low EHP load group, and most genes related to diseases and environmental information processing were more abundant in the high EHP load group. After separation and purification, the dominant bacteria (Bifidobacterium animalis in healthy shrimp and Lactococcus garvieae in the low EHP load group) and the non-dominant bacteria (Macrococus caseolyticus in the low EHP load group) were obtained. Each of these isolated strains were used together with EHP to infect E. carinicauda, and the results showed that Bifidobacterium animali and Lactococcus garvieae significantly reduced the EHP load in EHP-infected individuals. At the same time, the morphology and structure of the hepatopancreas and intestinal tissue of EHP-infected E. carinicauda were improved. No improvement was seen in tissue that was infected with Macrococus caseolyticus.

White feces syndrome in Penaeus vannamei is potentially an Enterocytozoon hepatopenaei (EHP) associated pathobiome origin of Vibrio spp.

White feces syndrome (WFS) is a commercially important disease in Penaeus vannamei (whiteleg shrimp) farming. The aetiology beyond the white or golden white midgut with mediocre growth performance producing a floating mass of white fecal strings in WFS-affected shrimp farms remains uncharted. To give WFS a perception of pathobiome, healthy P. vannamei shrimps were subjected to an enteric microsporidian Enterocytozoon hepatopenaei (EHP) infection along with Vibrio harveyi and V. alginolyticus in different combinations. Immune responses in haemolymph (total haemocyte count (THC), prophenoloxidase activity (proPO), respiratory burst activity (RBA), superoxide dismutase activity (SOD) and catalase activity (CAT)), plasma biochemical changes (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)) and digestive enzymes activity (alpha-amylase (AMY), lipase (LIP) and protease (PRO)) were assessed in the challenged shrimps at 5, 10 and 15 days post-infection (dpi). The microbial interactions between the EHP and Vibrio spp. have led to the formation of WFS in the challenged shrimps. The histological sections of the hepatopancreas revealed the presence of EHP along with colonized bacterial masses, leading to the formation of aggregated transformed microvilli (ATM) structures and increased sloughing of lipid vacuoles into the tubule lumen. A significantly decreased THC and increased proPO levels, dysregulated antioxidant system, prominent hepatic damage, reduced energy metabolism and higher lipid production were the key records supporting that EHP-associated WFS in P. vannamei is due to the pathobiome.

Early responses in Penaeus vannamei during experimental infection with Enterocytozoon hepatopenaei (EHP) spores by injection and oral routes.

Hepatopancreatic microsporidiosis caused by Enterocytozoon hepatopenaei (EHP) is associated with severe production losses in Penaeus vannamei farming. Early responses in P. vannamei experimentally infected with EHP was assessed in this study by feeding infected hepatopancreatic tissue and by injecting purified EHP spores (∼1 × 105 Spores/shrimp). Immune responses to EHP infection were assessed in the haemolymph by analysing the total haemocyte count (THC), superoxide dismutase (SOD) activity, prophenoloxidase activity (proPO), respiratory burst activity (RBA), catalase activity (CAT), lysozyme activity (LYS) and Toll gene expression in hepatopancreas at 0, 6, 12, 24, 36, 48, 60 and 72 h post-infection (hpi). Experimental infection with EHP resulted in a significant (p < 0.05) reduction in the immune parameters such THC, CAT and LYS at 6, 24 and 24 hpi respectively while there was a significant increase (p < 0.05) in the levels of SOD, proPO and RBA at 6 hpi. The expression of the Toll gene was significantly upregulated (p < 0.05) after experimental infection with EHP from 6 hpi. These findings on immune responses in P. vannamei during EHP infection will assist in the development of suitable management measures to reduce the negative impacts of EHP in P. vannamei farming. This is the first report on early responses in P. vannamei during EHP infection.

The effect of salinity on enterocytozoon hepatopenaei infection in Penaeus vannamei under experimental conditions.

BACKGROUND: Enterocytozoon hepatopenaei (EHP) is an enteric pathogen that affects Penaeus vannamei and Penaeus monodon shrimp in many SE Asian countries. In the western hemisphere, EHP was reported for the first time in 2016 in farmed P. vannamei in Venezuela. Anecdotal evidence suggests that EHP is more prevalent in grow-out ponds where the salinity is high (> 15 parts per thousand (ppt)) compared to grow-out ponds with low salinities (< 5 ppt). Considering that P. vannamei is an euryhaline species, we were interested in knowing if EHP can propagate in P. vannamei in low salinities. RESULTS: In this study, we described an experimental infection using fecal strings as a source inoculum. Specific Pathogen Free (SPF) P. vannamei were maintained at three different salinities (2 ppt, 15 ppt, and 30 ppt) while continuously challenged using feces from known EHP-infected P. vannamei over a period of 3 weeks. The fecal strings, used as a source of EHP inocula in the challenges, was sufficient to elicit an infection in shrimp maintained at the three salinities. The infectivity of EHP in shrimp reared at 2 ppt, 15 ppt, and 30 ppt salinities was confirmed by PCR and histopathology. The prevalence and the severity of the EHP infection was higher at 30 ppt than at 2 ppt and 15 ppt. CONCLUSION: The data suggests that fecal strings are a reliable source of EHP inoculum to conduct experimental challenges via the fecal-oral route. An EHP infection can occur at a salinity as low as 2 ppt, however, the prevalence and the severity of the EHP infection is higher at a salinity of 30 ppt.

The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control.

Disease is a major limiting factor in the global production of cultivated shrimp. The microsporidian parasite Enterocytozoon hepatopenaei (EHP) was formally characterized in 2009 as a rare infection of the black tiger shrimp Penaeus monodon. It remained relatively unstudied until mid-2010, after which infection with EHP became increasingly common in the Pacific whiteleg shrimp Penaeus vannamei, by then the most common shrimp species farmed in Asia. EHP infects the hepatopancreas of its host, causing hepatopancreatic microsporidiosis (HPM), a condition that has been associated with slow growth of the host in aquaculture settings. Unlike other infectious disease agents that have caused economic losses in global shrimp aquaculture, EHP has proven more challenging because too little is still known about its environmental reservoirs and modes of transmission during the industrial shrimp production process. This review summarizes our current knowledge of the EHP life cycle and the molecular strategies that it employs as an obligate intracellular parasite. It also provides an analysis of available and new methodologies for diagnosis since most of the current literature on EHP focuses on that topic. We summarize current knowledge of EHP infection and transmission dynamics and currently recommended, practical control measures that are being applied to limit its negative impact on shrimp cultivation. We also point out the major gaps in knowledge that urgently need to be bridged in order to improve control measures.

Transcriptome analysis of pacific white shrimp (Penaeus vannamei) intestines and hepatopancreas in response to Enterocytozoon hepatopenaei (EHP) infection.

Penaeus vannamei is the most economically important species of shrimp cultured worldwide. Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that severely affects the growth and development of shrimps. In this study, the transcriptome differences between EHP-infected and uninfected shrimp were investigated through next-generation sequencing. The unigenes were assembled with the reads from all the four libraries. The differentially expressed genes (DEGs) of intestines and hepatopancreas were analyzed. There were 2,884 DEGs in the intestines and 2,096 DEGs in the hepatopancreas. The GO and KEGG enrichment analysis indicated that DEGs were significantly enriched in signaling pathways associated with nutritional energy metabolism and mobilizing autoimmunity. Moreover, the results suggested the downregulation of key genes in energy synthesis pathways contributed greatly to shrimp growth retardation; the upregulation of immune-related genes enhanced the resistance of shrimp against EHP infection. This study provided identified genes and pathways associated with EHP infection revealing the molecular mechanisms of growth retardation.

Molecular Characterization of Giardia duodenalis and Enterocytozoon bieneusi Isolated from Tibetan Sheep and Tibetan Goats Under Natural Grazing Conditions in Tibet.

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

A comparative study of Enterocytozoon hepatopenaei (EHP) challenge methods in Penaeus vannamei.

The microsporidium Enterocytozoon hepatopenaei (EHP) is considered as an emerging pathogen threating the shrimp industry worldwide. It is an intracellular parasite that has been associated with retarded growth syndrome and white feces syndrome in shrimp. Although the impact of EHP to the shrimp industry is well known, many aspects of host-pathogen interactions are not well understood. A major limitation in the study of EHP is the lack of a reliable method to produce large quantities of inoculum rapidly and reproducibly. The present study was designed to compare different challenge methods including intramuscular injection, oral administration, co-habitation, hepatopancreas (HP) injection and reverse gavage. The results showed that the HP injection and the reverse gavage are two promising methods to infect shrimp rapidly and generate inoculum in a reproducible manner starting with a limited amount of inoculum. Therefore, the HP injection and reverse gavage were chosen for a scale-up study. Histopathology results showed that EHP proliferated in the epithelial cells of the HP in shrimp challenged via direct injection of inoculum into HP and reverse gavage treatments. In accordance with the histopathology results, the qPCR data showed that EHP loads in the challenged shrimp increased significantly with the HP injection and reverse gavage methods. Furthermore, the histopathological and quantification results indicate that HP injection and reverse gavage are two novel methods that can be used in EHP-challenge studies and for rapidly generating viable EHP inoculum.

Zoonotic potential of Enterocytozoon bieneusi and Giardia duodenalis in horses and donkeys in northern China.

Limited data are available on infection rates and genetic identity of Enterocytozoon bieneusi and Giardia duodenalis in horses and donkeys. In this study, 865 fecal specimens were collected from donkeys (n = 540) and horses (n = 325) in three provinces and autonomous regions in northern China during 2015-2019. Enterocytozoon bieneusi was detected and genotyped by PCR and sequence analyses of the ribosomal internal transcribed spacer (ITS) and G. duodenalis was detected and genotyped by PCR and sequence analyses of the ß-giardin, glutamate dehydrogenase, and triosephosphate isomerase genes. The overall infection rates of E. bieneusi and G. duodenalis were 21.9% (118/540) and 11.5% (62/540) in donkeys, and 7.4% (24/325) and 2.8% (9/325) in horses, respectively. These differences in infection rates of E. bieneusi and G. duodenalis between donkeys and horses were significant (χ2 = 30.9, df = 1, P < 0.0001; χ2 = 20.4, df = 1, P < 0.0001, respectively). By age, the 28.9% infection rate of E. bieneusi in donkeys under 6 months was significantly higher than that in animals over 6 months (6.0%; χ2 = 35.2, df = 1, P < 0.0001). In contrast, donkeys of 6-12 months had higher infection rate (35.9%) of G. duodenalis than donkeys under 6 months (9.9%; χ2 = 22.1, df = 1, P < 0.0001) and over 12 months (8.7%; χ2 = 17.3, df = 1, P < 0.0001). In horses, animals of > 12 months had significantly higher infection rate (31.1%) of E. bieneusi than horses under 6 months (3.4%; χ2 = 29.4, df = 1, P < 0.0001) and 6-12 months (3.8%; χ2 = 26.1, df = 1, P < 0.0001). Twenty genotypes of E. bieneusi were detected, including six known ones and 14 new genotypes. Among them, nine genotypes in 45% E. bieneusi-positive specimens belonged to the zoonotic group 1. Similarly, three G. duodenalis assemblages were detected, including A (in 2 horses and 30 donkeys), B (in 6 horses and 29 donkeys), and E (in 1 horse); three donkeys had coinfections of assemblages A and B. The assemblage A isolates identified all belong to the sub-assemblage AI. These results indicate that unlike in other farm animals, there is a common occurrence of zoonotic E. bieneusi and G. duodenalis genotypes in horses and donkeys.

Enterocytozoon bieneusi genotypes in cats and dogs in Victoria, Australia.

BACKGROUND: Enterocytozoon bieneusi is one of the commonest microsporidians contributing to human microsporidiosis, and is frequently found in animals in various countries. However, there is limited epidemiological information on this microorganism in Australia. Here, we undertook the first molecular epidemiological study of E. bieneusi in cats and dogs in Victoria. RESULTS: Genomic DNAs were extracted from 514 individual faecal deposits from cats (n = 172) and dogs (n = 342) and then tested using PCR-based sequencing of the internal transcribed spacer (ITS) of nuclear ribosomal DNA. Four distinct genotypes (designated D, PtEb IX, VIC_cat1 and VIC_dog1) of E. bieneusi were identified in 20 of the 514 faecal samples (3.9%). Genotype D is known to have a broad host range (humans and other animals) and has a wide geographical distribution around the world. The identification of this genotype here suggests that companion animals might represent reservoir hosts that are able to transmit E. bieneusi infection to humans in Australia. A phylogenetic analysis of ITS sequence data revealed that the novel genotype VIC_cat1 is related to the known genotype type IV within Group 1, and the new genotype VIC_dog1 is linked to a contentious "Group 3", which includes genotypes reported previously in the published literature to represent Group 2 or 3. CONCLUSIONS: A future, large-scale phylogenetic study of all known E. bieneusi genotypes, including VIC_dog1, should aid in clarifying their relationships and assignment to Groups, and in the identification of new genotypes, thus assisting epidemiological investigations.

Multilocus Typing of Enterocytozoon bieneusi in Pig Reveals the High Prevalence, Zoonotic Potential, Host Adaptation and Geographical Segregation in China.

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

The Potential Role of Synanthropic Rodents and Flies in the Transmission of Enterocytozoon bieneusi on a Dairy Cattle farm in China.

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

Proteomic and metabolomic responses in hepatopancreas of whiteleg shrimp Litopenaeus vannamei infected by microsporidian Enterocytozoon hepatopenaei.

Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. HPM is not normally associated with shrimp mortality, but is associated with significant growth retardation. In this study, the responses induced by EHP were investigated in hepatopancreas of shrimp Litopenaeus vannamei using proteomics and metabolomics. Among differential proteins identified, several (e.g., peritrophin-44-like protein, alpha2 macroglobulin isoform 2, prophenoloxidase-activating enzymes, ferritin, Rab11A and cathepsin C) were related to pathogen infection and host immunity. Other proteomic biomarkers (i.e., farnesoic acid o-methyltransferase, juvenile hormone esterase-like carboxylesterase 1 and ecdysteroid-regulated protein) resulted in a growth hormone disorder that prevented the shrimp from molting. Both proteomic KEGG pathway (e.g., "Glycolysis/gluconeogenesis" and "Glyoxylate and dicarboxylate metabolism") and metabolomic KEGG pathway (e.g., "Galactose metabolism" and "Biosynthesis of unsaturated fatty acids") data indicated that energy metabolism pathway was down-regulated in the hepatopancreas when infected by EHP. More importantly, the changes of hormone regulation and energy metabolism could provide much-needed insight into the underlying mechanisms of stunted growth in shrimp after EHP infection. Altogether, this study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of microsporidial infection in the shrimp L. vannamei.

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.

Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP).

BACKGROUND: Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. It is probably endemic in Australasia and was first characterized and named from the giant or black tiger shrimp Penaeus monodon from Thailand in 2009. Later, it was also found to infect exotic Penaeus vannamei imported for cultivation in Asia. HPM is not normally associated with shrimp mortality, but information from shrimp farmers indicates that it is associated with significant growth retardation that is not clearly noticeable until 2-3 months of cultivation. In order to study modes of HPM transmission and to test possible control measures, a laboratory challenge model was needed that would mimic the mode of infection in shrimp ponds. RESULTS: We describe successful transmission in a cohabitation model with natural E. hepatopenaei (EHP)-infected shrimp in closed, perforated plastic containers placed in aquaria together with free-swimming, uninfected shrimp. After a period of 14 days all the free-swimming shrimp tested positive by PCR (approximately 60% with heavy infections evident by 1-step PCR positive test results) and gave positive histological and in situ hybridization results for E. hepatopenaei (EHP) in the hepatopancreas. CONCLUSIONS: A laboratory cohabitation model for studying E. hepatopenaei (EHP) has been developed and used to confirm that E. hepatopenaei (EHP) can be directly transmitted horizontally among shrimp via water. The model will facilitate studies on methods to prevent the E. hepatopenaei (EHP) transmission.

Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP-infected whiteleg shrimp, Litopenaeus vannamei (Boone, 1931), in India.

Stunted growth in pond-reared Litopenaeus vannamei was observed in different farms located in Tamil Nadu and Andhra Pradesh, India. No mortality was associated with stunted growth. PCR assay on these samples revealed the presence of Enterocytozoon hepatopenaei (EHP) in stunted shrimp. Tissue distribution of EHP in naturally and experimentally infected shrimp was studied by PCR and histology. Histological examination revealed the presence of EHP in hepatopancreas and gut, but not in other organs. The PCR assay revealed the presence of EHP in all the organs tested in both naturally and experimentally infected shrimp. Healthy shrimp were challenged with E. hepatopenaei by intramuscular injection and oral route, and no mortality was observed in both routes after 30 days post-challenge. Different developmental stages of the microsporidian parasite were observed in the hepatopancreatic epithelial cells. Biochemical parameters such as total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase were measured in the haemolymph of naturally and experimentally EHP-infected shrimp. All biochemical parameters mentioned were found to be significantly higher in EHP-infected shrimp when compared to normal shrimp. This is the first report relating AST and ALT levels to EHP infection in naturally and experimentally infected shrimp.

High prevalence of Enterocytozoon hepatopenaei in shrimps Penaeus monodon and Litopenaeus vannamei sampled from slow growth ponds in India.

Hepatopancreatic microsporidiosis in cultivated Litopenaeus vannamei and Penaeus monodon is caused by the newly emerged pathogen Enterocytozoon hepatopenaei (EHP). It has been detected in shrimp cultured in China, Vietnam and Thailand and is suspected to have occurred in Malaysia and Indonesia and to be associated with severely retarded growth. Due to retarded shrimp growth being reported at farms in the major grow-out states of Tamilnadu, Andhra Pradesh and Odisha in India, shrimp were sampled from a total of 235 affected ponds between March 2014 and April 2015 to identify the presence of EHP. PCR and histology detected a high prevalence of EHP in both P. monodon and L. vannamei, and infection was confirmed by in situ hybridization using an EHP-specific DNA probe. Histology revealed basophilic inclusions in hepatopancreas tubule epithelial cells in which EHP was observed at various developmental stages ranging from plasmodia to mature spores. The sequence of a region of the small subunit rDNA gene amplified by PCR was found to be identical to EHP sequences deposited in GenBank. Bioassays confirmed that EHP infection could be transmitted orally to healthy shrimp. Histology also identified bacterial co-infections in EHP-infected shrimp sampled from slow-growth ponds with low-level mortality. The data confirm that hepatopancreatic microsporidiosis caused by EHP is prevalent in shrimp being cultivated in India. EHP infection control measures thus need to be implemented urgently to limit impacts of slowed shrimp growth.

Zoonotic and Potentially Host-Adapted Enterocytozoon bieneusi Genotypes in Sheep and Cattle in Northeast China and an Increasing Concern about the Zoonotic Importance of Previously Considered Ruminant-Adapted Genotypes.

This study investigated fecal specimens from 489 sheep and 537 cattle in multiple cities in northeast China for the prevalence and genetic characteristics of Enterocytozoon bieneusi by PCR and sequencing of the ribosomal internal transcribed spacer. Sixty-eight sheep specimens (13.9%) and 32 cattle specimens (6.0%) were positive for E. bieneusi. Sequence polymorphisms enabled the identification of 9 known genotypes (BEB4, BEB6, CM7, CS-4, EbpC, G, I, J, and OEB1) and 11 new genotypes (NESH1 to NESH6 and NECA1 to NECA5). The genotypes formed two genetic clusters in a phylogenetic analysis, with CS-4, EbpC, G, NESH1 to NESH3, and NECA1 to NECA5 distributed in zoonotic group 1 and BEB4, BEB6, CM7, EbpI, J, OEB1, and NESH4 to NESH6 distributed in potentially host-adapted group 2. Nearly 70% of cases of E. bieneusi infections in sheep were contributed by human-pathogenic genotypes BEB6, CS-4, and EbpC, and over 80% of those in cattle were by genotypes BEB4, CS-4, EbpC, I, and J. The cooccurrence of genotypes BEB4, CS-4, EbpC, I, and J in domestic ruminants and children in northeast China and the identification of BEB6 and EbpC in humans and water in central China imply the possibility of zoonotic transmission. This study also summarizes E. bieneusi genotypes obtained from ruminants worldwide and displays their host ranges, geographical distributions, and phylogenetic relationships. The data suggest a host range expansion in some group 2 genotypes (notably BEB4, BEB6, I, and J) that were previously considered to be adapted to ruminants. We should be concerned about the increasing zoonotic importance of group 2 genotypes with low host specificity.

Host specificity and source of Enterocytozoon bieneusi genotypes in a drinking source watershed.

To assess the host specificity of Enterocytozoon bieneusi and to track the sources of E. bieneusi contamination, we genotyped E. bieneusi in wildlife and stormwater from the watershed of New York City's source water, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 255 specimens from 23 species of wild mammals and 67 samples from stormwater were analyzed. Seventy-four (29.0%) of the wildlife specimens and 39 (58.2%) of the stormwater samples from streams were PCR positive. Altogether, 20 E. bieneusi genotypes were found, including 8 known genotypes and 12 new ones. Sixteen and five of the genotypes were seen in animals and stormwater from the watershed, respectively, with WL4 being the most common genotype in both animals (35 samples) and stormwater (23 samples). The 20 E. bieneusi genotypes belonged to five genogroups (groups 1, 3, 4, and 7 and an outlier), with only 23/113 (20.4%) E. bieneusi-positive samples belonging to zoonotic genogroup 1 and 3/20 genotypes ever being detected in humans. The two genogroups previously considered host specific, groups 3 and 4, were both detected in multiple groups of mammals. Thus, with the exception of the type IV, Peru11, and D genotypes, which were detected in only 7, 5, and 2 animals, respectively, most E. bieneusi strains in most wildlife samples and all stormwater samples in the watershed had no known public health significance, as these types have not previously been detected in humans. The role of different species of wild mammals in the contribution of E. bieneusi contamination in stormwater was supported by determinations of host-adapted Cryptosporidium species/genotypes in the same water samples. Data from this study indicate that the host specificity of E. bieneusi group 3 is broader than originally thought, and wildlife is the main source of E. bieneusi in stormwater in the watershed.