Enterocytozoon bieneusi Infection after Hematopoietic Stem Cell Transplant in Child, Argentina.

We report a case of Enterocytozoon bieneusi infection in a pediatric hematopoietic stem cell transplant recipient in Argentina. Spores were visualized in feces using Calcofluor White and modified trichrome stainings. PCR and sequencing identified E. bieneusi genotype D in fecal samples and liver samples, confirming extraintestinal dissemination of the parasite.

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.

First report of Enterocytozoon bieneusi in Erinaceus europaeus and Hemiechinus auritus hedgehogs from recovery centers of Portugal.

Enterocytozoon bieneusi microsporidia are emerging pathogens infecting a wide range of vertebrate and invertebrate hosts, known to have zoonotic features since they infect both wild and domestic animals, and humans. Despite their significance, there is very limited epidemiological data on microsporidia in hedgehogs, especially European hedgehogs (Erinaceus europaeus) and long-eared hedgehogs (Hemiechinus auritus), the former known as synantropic hedgehogs, and the latter suited as pets. As such, the present study aimed to assess the presence of E. bieneusi in hedgehogs from Portugal. For this purpose, fecal samples from 110 hedgehogs of three species-E. europaeus (n = 106), H. auritus (n = 1), and Atelerix albiventris (n = 3)-were collected and tested for E. bieneusi by PCR targeting the internal transcribed spacer region and the flanking small and large subunits of the rRNA. We found an overall occurrence of 22.7% (25/110; 95% confidence interval [CI]: 15.28-31.70), with 22.6% (24/106; 95% [CI]: 15.08-31.79) in E. europaeus, 100% (1/1) in H. auritus, and 0% in A. albiventris. Interestingly, three novel genotypes were identified, all belonging to the potentially zoonotic Group 1. Our findings highlight the importance of hedgehogs as potential reservoirs for E. bieneusi and emphasize the need for further research to understand their role in transmission dynamics and assess the associated risks to public and veterinary health.

Synanthropic hedgehogs were tested for Enterocytozoon bieneusi, the main cause of human microsporidiosis. Results showed 22.7% of hedgehogs were shedding E. bieneusi spores, with three new genotypes from the zoonotic Group 1. Hedgehogs may transmit to humans/animals, warranting more research.

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

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

Transcriptomic analysis provides insights into the mechanisms underlying the resistance of Penaeus vannamei to Enterocytozoon hepatopenaei (EHP).

The Penaeus vannamei aquaculture industry is facing a significant challenge in the form of hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP), resulting in substantial economic losses. However, the extent of knowledge regarding the mechanisms by which shrimp resist EHP is limited. We screened resistant and susceptible shrimp and found that resistant shrimp had lower EHP load and less tissue damage. To gain insight into the molecular mechanisms underlying the EHP resistance of shrimp, a comparison was conducted at the transcriptional level between the resistant and susceptible families. Transcriptomic analysis of shrimp hepatopancreas revealed significant differences between the resistant and susceptible families. Compared to the susceptible family, the immune system of the resistant family was activated. The resistant family showed up-regulation in the expression of cathepsin L, C-type lectin, penaeidin, chitinase genes, and metabolism of xenobiotics by cytochrome P450-related genes. Additionally, the resistant shrimp exhibited a higher capacity for amino acid uptake. The observed differences in the resistant and susceptible family transcriptome may contribute to the shrimp's resistance to EHP.

White feces syndrome in shrimp: Comprehensive understanding of immune system responses.

White feces syndrome (WFS) is a multifactorial disease that affects global shrimp production. The diagnostic approach to identify WFS involves traditional and molecular scientific methods by examining histopathology, bioassays, PCR (polymerase chain reaction), and calorimetric estimation. The pathogenesis of WFS is closely associated with Vibrio spp., intestinal microbiota (IM) dysbiosis, and Enterocytozoon hepatopenaei (EHP). It also has caused over 10-15 % loss in the aquaculture industry and is also known to cause retardation, lethargy and slowly leading to high mortality in shrimp farms. Therefore, it is necessary to understand the molecular mechanisms processed under the association of IM dysbiosis, Vibrio spp., and EHP to analyze the impact of disease on the innate immune system of shrimp. However, only very few reviews have described the molecular pathways involved in WFS. Hence, this review aims to elucidate an in-depth analysis of molecular pathways involved in the innate immune system of shrimp and their response to pathogens. The analysis and understanding of the impact of shrimp's innate immune system on WFS would help in developing treatments to prevent the spread of disease, thereby improving the economic condition of shrimp farms worldwide.

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.

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.

Prevalence and genotype/subtype distribution of Enterocytozoon bieneusi and Blastocystis in donkeys in Shanxi Province, north China.

Enterocytozoon bieneusi and Blastocystis may cause diarrhea in humans and various animals. However, little information is available regarding the prevalence and genetic diversity of E. bieneusi and Blastocystis in donkeys. To fill this gap, we molecularly assessed E. bieneusi and Blastocystis in fecal samples from donkeys (n = 815) in Shanxi Province, north China. The overall prevalence of E. bieneusi and Blastocystis in donkeys was 8.1% and 0.2%, respectively. Region and age were risk factors associated with E. bieneusi infection in donkeys. Three internal transcribed spacer (ITS) genotypes of E. bieneusi were identified in the current study, including two previously described genotypes (D and Henan-IV) and one novel genotype (named SXD1). Of which, genotype D was found to be the most prevalent. Phylogenetic analysis demonstrated that the three genotypes belonged to group 1, implying a potential of zoonotic transmission. Multilocus sequence typing showed that 19, 15, 13, and 22 types were identified at the loci MS1, MS3, MS4, and MS7, respectively, forming six multilocus genotypes (MLGs) distributed in the genotype D. One Blastocystis subtype (ST33) was identified, which has previously been reported only in horses. This is the first molecular-based description of E. bieneusi and Blastocystis infections in donkeys in Shanxi Province, north China, contributing to a better understanding of transmission dynamics and molecular epidemiological characteristics of the two intestinal protozoa.

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.

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.

First report on occurrence and genotypes of Enterocytozoon bieneusi, Cryptosporidium spp. and Cyclospora cayetanensis from diarrheal outpatients in Ningbo, Southeast China.

Enterocytozoon bieneusi, Cryptosporidium spp. and Cyclospora cayetanensis are three important zoonotic pathogens which were a major cause of foodborne or waterborne intestinal diseases in humans and animals. However, very little data about occurrence and genotypes of the three parasites in Ningbo in the south wing of the Yangtze River Delta, China, which is important for a tourist city. In the present study, molecular characterization of E. bieneusi, C. cayetanensis and Cryptosporidium spp. in fecal samples from 489 diarrheal outpatients were carried out. As a result, a total of 35 (7.16%, 35/489) and three (0.61%, 3/489) samples were positive for E. bieneusi and C. cayetanensis respectively. No Cryptosporidium-positive sample or mixed-infections were detected. Four known E. bieneusi genotypes (Type IV, D, I and CHN4) and 8 novel genotypes (NBH1-NBH8) were identified with type IV was the dominant genotype (n = 14), followed by genotypes D (n = 5), NBH8 (n = 5) and NBH7 (n = 3). The remaining genotypes were found in one sample each, and these genotypes were belonged to the previously described high-potential zoonotic group 1. One novel sequence named NBC315, and the other two sequences (NBC30 and NBC370) identical with the reported sequence were detected. Therefore, the existence and importance of zoonotic potential of E. bieneusi and C. cayetanensis in diarrheal outpatients in Ningbo indicates the public health threats, and more investigations should be carried out in human populations, animals and other environmental sources from the One Health perspective.

A comparative transcriptome analysis of how shrimp endure and adapt to long-term symbiosis with Enterocytozoon hepatopenaei infection.

Enterocytozoon hepatopenaei (EHP) is a prevalent microsporidian pathogen responsible for hepatopancreatic microsporidiosis (HPM) in Litopenaeus vannamei. This infection not only leads to slowed growth in shrimp abut aslo inflicts substantial economic losses in the global aquaculture industry. However, the molecular mechanisms by which EHP influences the host during various infection stages remain unclear. This study employed comparative transcriptomics to examine the effects of EHP infection on Litopenaeus vannamei between early and late stage of infection groups. Utilizing transcriptomic approaches, we identified differentially expressed genes (DEGs) with notable biological significance through the COG, GO, KEGG, GSEA, and Mufzz time-series methodologies. The results reveal that EHP infection considerably influences host gene expression, with marked differences between early and late infection across distinct timeframes. Key processes such as detoxification, cell apoptosis, and lipid metabolism are pivotal during host-parasite interactions. Hexokinase and phosphatidic acid phosphatase emerge as key factors enabling invasion and sustained effects. Cytochrome P450 and glucose-6-phosphate dehydrogenase could facilitate infection progression. EHP significantly impacts growth, especially through ecdysteroids and 17ß-estradiol dehydrogenase. By delineating stage-specific effects, we gain insights into interaction between EHP and Litopenaeus vannamei, showing how intracellular pathogens reprogram host defenses into mechanisms enabling long-term persistence. This study provides a deeper understanding of host-pathogen dynamics, emphasizing the interplay between detoxification, metabolism, immunity, apoptosis and growth regulation over the course of long-term symbiosis.

Analysis of differentially expressed proteins after EHP-infection and characterization of caspase 3 protein in the whiteleg shrimp (Litopenaeus vannamei).

Whiteleg shrimp (Litopenaeus vannamei) is the most important species of shrimp farmed worldwide in terms of its economic value. Enterocytozoon hepatopenaei (EHP) infects the hepatopancreas, resulting in the hepatopancreatic microsporidiosis (HPM) of the host, which causes slow growth of the shrimp and poses a threat to the farming industry. In this study, differentially expressed proteins (DEPs) between EHP-infected and uninfected shrimp were investigated through proteomics sequencing. A total of 9908 peptides and 2092 proteins were identified. A total of 69 DEPs were identified in the hepatopancreas (HP), of which, 28 were upregulated and 41 were downregulated. Our results showed that the differences among the level of multiple proteins involved in the apoptosis were significant after the EHP infection, which indicated that the apoptosis pathway was activated in whiteleg shrimp. In addition, expression leve of caspase 3 gene were identified related to the EHP infection. Furthermore, predictions of spatial structure, analysis of phylogeny and chromosome-level linearity of the caspase 3 protein were performed as well. In conclusion, a relatively complete proteomic data set of hepatopancreas tissues in whiteleg shrimp were established in this study. Findings about genes involved in the apoptosis here will provide a further understanding of the molecular mechanism of EHP infection in the internal immunity of whiteleg shrimp.

Transcriptome analysis of the hepatopancreas in Penaeus vannamei under experimental infection with Enterocytozoon hepatopenaei (EHP).

Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasite that mainly resides in the hepatopancreas of shrimp, causing significant growth retardation in shrimp. In this study, Penaeus vannamei was infected with EHP through an artificial challenge experiment, and the different genes and pathways in the hepatopancreas between EHP-infected and healthy shrimp were analyzed by transcriptome sequencing. The results showed that a total of 240 significantly differentially expressed genes were obtained, including 99 up-regulated genes and 141 down-regulated genes. Immune-related genes such as Astakine, lysozyme, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), and macrophage mannose receptor 1-like (MMR) were up-regulated, and the expression levels of lipid metabolism-related genes pancreatic lipase-related protein 2 (PLRP2), lysosomal acid lipase (LIPA), and adiponectin receptor protein (AdipoR) were also increased. However, several genes were down-regulated in carbohydrate and protein metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), trypsin-1, and delta-1-pyrroline-5-carboxylate synthase (ALDH18A1). The results suggested that EHP infection of shrimps could significantly activate the immune system, but the energy and material metabolism processes were disturbed. This study identified a substantial number of genes and pathways associated with EHP infection, providing a valuable resource for revealing the molecular mechanism of growth retardation in shrimp.

Role of hemocytin from Litopenaeus vannamei in immune response against microsporidian, Enterocytozoon hepatopenaei.

Hemocytin, a multidomain hemostasis-related protein, is a homologous protein of hemolectin in Drosophila melanogaster and von Willebrand factor (vWF) in humans. The vWF type D (VWD) domain in hemocytin is thought to be a major mediator of hemocyte aggregation and the prophenoloxidase (proPO) activation system. Here, we report for the first time the role of hemocytin from Litopenaeus vannamei (LvHCT) against Enterocytozoon hepatopenaei (EHP), the pathogenic microsporidian causing hepatopancreatic microsporidiosis in Pacific white shrimp (L. vannamei). The LvHCT gene contains 58,366 base pairs consisting of 84 exons encoding for 4267 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that LvHCT was clustered with crustacean hemocytins. Gene expression analysis by quantitative real-time RT-PCR showed that LvHCT in hemocytes was significantly upregulated at 9 and 11 days post-EHP cohabitation, which was consistent with EHP copy numbers in the infected shrimp. To further investigate the biological function of LvHCT in EHP infection, a recombinant protein containing an LvHCT-specific VWD domain (rLvVWD) was expressed in Escherichia coli. In vitro agglutination assays showed that rLvVWD was functionally representative of LvHCT and induced aggregation of pathogens, including Gram-negative and -positive bacteria, fungi, and EHP spore. LvHCT suppression resulted in higher EHP copy numbers and proliferation due to the lack of hemocytin-mediated EHP spore aggregation in LvHCT-silenced shrimp. Moreover, immune-related genes in the proPO-activating cascade and Toll, IMD and JAK/STAT signaling pathways were upregulated to eliminate the over-controlled EHP in LvHCT-silenced shrimp. Furthermore, the impaired phenoloxidase activity due to LvLGBP suppression was recovered after rLvVWD injection, suggesting that LvHCT may be directly involved in phenoloxidase activation. In conclusion, a novel LvHCT is involved in shrimp immunity against EHP via EHP spore aggregation and possible activation of the proPO-activating cascade.

Molecular characterization of turtle-like protein in whiteleg shrimp (Litopenaeus vannamei) and its role in Enterocytozoon hepatopenaei infection.

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects shrimp hepatopancreas, causing growth retardation and disease susceptibility. Knowledge of the host-pathogen molecular mechanisms is essential to understanding the microsporidian pathogenesis. Turtle-like protein (TLP) is part of the immunoglobulin superfamily of proteins, which is widely distributed in the animal kingdom. TLP has multiple functions, such as cell surface receptors and cell adhesion molecules. The spore wall proteins (SWPs) of microsporidia are involved in the infection mechanisms. Some SWPs are responsible for spore adherence, which is part of the activation and host cell invasion processes. Previous studies showed that TLP from silkworms (Bombyx mori) interacted with SWP26, contributing to the infectivity of Nosema bombycis to its host. In this study, we identified and characterized for the first time, the Litopenaeus vannamei TLP gene (LvTLP), which encodes an 827-aa protein (92.4 kDa) composed of five immunoglobulin domains, two fibronectin type III domains, and a transmembrane region. The LvTLP transcript was expressed in all tested tissues and upregulated in the hepatopancreas at 1 and 7 days post-cohabitation (dpc) and at 9 dpc in hemocytes. To identify the LvTLP binding counterpart, recombinant (r)LvTLP and recombinant (r)EhSWP1 were produced in Escherichia coli. Coimmunoprecipitation and enzyme-linked immunosorbent assays demonstrated that rLvTLP interacted with rEhSWP with high affinity (KD = 1.20 × 10-7 M). In EHP-infected hepatopancreases, LvTLP was clustered and co-localized with some of the developing EHP plasmodia. Furthermore, LvTLP gene silencing reduced the EHP copy numbers compared with those of the control group, suggesting the critical role of LvTLP in EHP infection. These results provide insight into the molecular mechanisms of the host-pathogen interactions during EHP infection.

Comparative transcriptome analysis of non-germinated and germinated spores of Enterocytozoon hepatopenaei (EHP) in vitro.

Enterocytozoon hepatopenaei (EHP), an obligate intracellular parasite classified as microsporidia, is an emerging pathogen with a significant impact on the global shrimp aquaculture industry. The understanding of how microsporidia germinate has been a key factor in exploring its infection process. However, the germination process of EHP was rarely reported. To gain insight into the germination process, we conducted a high-throughput sequencing analysis of purified EHP spores that had undergone in vitro germination treatment. This analysis revealed 137 differentially expressed genes, with 84 up-regulated and 53 down-regulated genes. While the functions of some of the genes remain unknown, this study provides important data on the transcriptomic changes before and after EHP germination, which can aid in further studies on the EHP infection mechanism.