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.
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.
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.

Microsporidia are obligate intracellular pathogens identified ∼150 years ago as the cause of pébrine, an economically important infection in silkworms. There are about 220 genera and 1,700 species of microsporidia, which are classified based on their ultrastructural features, developmental cycle, host-parasite relationship, and molecular analysis. Phylogenetic analysis suggests that microsporidia are related to the fungi, being grouped with the Cryptomycota as a basal branch or sister group to the fungi. Microsporidia can be transmitted by food and water and are likely zoonotic, as they parasitize a wide range of invertebrate and vertebrate hosts. Infection in humans occurs in both immunocompetent and immunodeficient hosts, e.g., in patients with organ transplantation, patients with advanced human immunodeficiency virus (HIV) infection, and patients receiving immune modulatory therapy such as anti-tumor necrosis factor alpha antibody. Clusters of infections due to latent infection in transplanted organs have also been demonstrated. Gastrointestinal infection is the most common manifestation; however, microsporidia can infect virtually any organ system, and infection has resulted in keratitis, myositis, cholecystitis, sinusitis, and encephalitis. Both albendazole and fumagillin have efficacy for the treatment of various species of microsporidia; however, albendazole has limited efficacy for the treatment of Enterocytozoon bieneusi. In addition, immune restoration can lead to resolution of infection. While the prevalence rate of microsporidiosis in patients with AIDS has fallen in the United States, due to the widespread use of combination antiretroviral therapy (cART), infection continues to occur throughout the world and is still seen in the United States in the setting of cART if a low CD4 count persists.

Microsporidia are a group of spore-forming, fungus-related pathogens that can infect both invertebrates and vertebrates including humans. The primary infection site is usually digestive tract, but systemic infections occur as well and cause damages to organs such as lung, brain, and liver. The systemic spread of microsporidia may be intravascular, requiring attachment and colonization in the presence of shear stress. Von Willebrand Factor (VWF) is a large multimeric intravascular protein and the key attachment sites for platelets and coagulation factors. Here in this study, we investigated the interactions between VWF and microsporidia Encephalitozoon hellem (E. hellem), and the modulating effects on E. hellem after VWF binding. Microfluidic assays showed that E. hellem binds to ultra-large VWF strings under shear stress. In vitro germination assay and infection assay proved that E. hellem significantly increased the rates of germination and infection, and these effects would be reversed by VWF blocking antibody. Mass spectrometry analysis further revealed that VWF-incubation altered various aspects of E. hellem including metabolic activity, levels of structural molecules, and protein maturation. Our findings demonstrated that VWF can bind microsporidia in circulation, and modulate its pathogenicity, including promoting germination and infection rate. VWF facilitates microsporidia intravascular spreading and systemic infection.

BACKGROUND: Microsporidia are common opportunistic parasites in humans and animals, including rabbits. However, only limited epidemiology data concern about the prevalence and molecular characterization of Enterocytozoon bieneusi and Encephalitozoon spp. in rabbits. This study is the first detection and genotyping of Microsporidia in pet rabbits in China.
RESULTS: A total of 584 faecal specimens were collected from rabbits in pet shops from four cities in Sichuan province, China. The overall prevalence of microsporidia infection was 24.8% by nested PCR targeting the internal transcribed spacer (ITS) region of E. bieneusi and Encephalitozoon spp. respectively. E. bieneusi was the most common species (n = 90, 15.4%), followed by Encephalitozoon cuniculi (n = 34, 5.8%) and Encephalitozoon intestinalis (n = 16, 2.7%). Mixed infections (E. bieneusi and E. cuniculi) were detected in five another rabbits (0.9%). Statistically significant differences in the prevalence of microsporidia were observed among different cities (χ2 = 38.376, df = 3, P < 0.01) and the rabbits older than 1 year were more likely to harbour microsporidia infections (χ2 = 9.018, df = 2, P < 0.05). Eleven distinct genotypes of E. bieneusi were obtained, including five known (SC02, I, N, J, CHY1) and six novel genotypes (SCR01, SCR02, SCR04 to SCR07). SC02 was the most prevalent genotype in all tested cities (43.3%, 39/90). Phylogenetic analysis showed that these genotypes were clustered into group 1-3 and group 10. Meanwhile, two genotypes (I and II) were identified by sequence analysis of the ITS region of E. cuniculi.
CONCLUSIONS: To the best of our knowledge, this is the first report of microsporidia infection in pet rabbits in China. Genotype SC02 and four novel genotypes were classified into potential zoonotic group 1, suggesting that pet rabbits may cause microsporidiosis in humans through zoonotic transmissions. These findings provide preliminary reference data for monitoring microsporidia infections in pet rabbits and humans.

Heat shock protein 70 (Hsp70), a highly conserved protein family, is widely distributed in organisms and plays fundamental roles in biotic and abiotic stress responses. However, reports on Hsp70 genes are scarce in microsporidia, a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. In this study, we identified 37 Hsp70 proteins from eight microsporidian genomes and classified them into four subfamilies (A-D). The number of Hsp70 genes in these microsporidia was significantly fewer than in Rozella allomycis and yeast. All microsporidian species contained genes from each subfamily and similar subcellular locations (mitochondria, endoplasmic reticulum, cytosol, and cytosol and/or nucleus), indicating that each Hsp70 member may fulfil distinct functions. The conserved structures and motifs of the Hsp70 proteins in the same subfamily were highly similar. Expression analysis indicated that the subfamily C cytosol (cyto)-associated Hsp70s is functional during microsporidia development. Immunofluorescence assays revealed that Cyto-NbHsp70 was cytoplasmically located in the proliferation-stage of Nosema bombycis. Cyto-NbHsp70 antiserum also labeled Encephalitozoon hellem within infected cells, suggesting that this antiserum is a potential molecular marker for labeling the proliferative phases of different microsporidian species. The propagation of N. bombycis was significantly inhibited following RNAi of Cyto-NbHsp70, indicating that Cyto-NbHsp70 is important for pathogen proliferation. Our phylogenetic data suggest that Hsp70 proteins evolved during microsporidia adaption to intracellular parasitism, and they play important roles in pathogen development.

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

The sub-3 Mbp genomes from microsporidian species of the Encephalitozoon genus are the smallest known among eukaryotes and paragons of genomic reduction and compaction in parasites. However, their diminutive stature is not characteristic of all Microsporidia, whose genome sizes vary by an order of magnitude. This large variability suggests that different evolutionary forces are applied on the group as a whole. In this study, we have compared the codon usage bias (CUB) between eight taxonomically distinct microsporidian genomes: Encephalitozoon intestinalis, Encephalitozoon cuniculi, Spraguea lophii, Trachipleistophora hominis, Enterocytozoon bieneusi, Nematocida parisii, Nosema bombycis and Nosema ceranae. While the CUB was found to be weak in all eight Microsporidia, nearly all (98%) of the optimal codons in S. lophii, T. hominis, E. bieneusi, N. parisii, N. bombycis and N. ceranae are fond of A/U in third position whereas most (64.6%) optimal codons in the Encephalitozoon species E. intestinalis and E. cuniculi are biased towards G/C. Although nucleotide composition biases are likely the main factor driving the CUB in Microsporidia according to correlation analyses, directed mutational pressure also likely affects the CUB as suggested by ENc-plots, correspondence and neutrality analyses. Overall, the Encephalitozoon genomes were found to be markedly different from the other microsporidians and, despite being the first sequenced representatives of this lineage, are uncharacteristic of the group as a whole. The disparities observed cannot be attributed solely to differences in host specificity and we hypothesize that other forces are at play in the lineage leading to Encephalitozoon species.

We characterized a complete Sec61 complex in Nosema bombycis, which has been shown to consist of Sec61alpha, Sec61beta, and Sec61gamma genes. Comparing the genomic regions that harbor the respective subunit genes between N. bombycis, Encephalitozoon cuniculi, and Antonospora locustae, we found that microsporidian genomes have high degree of synteny in short genomic fragment, and that this gene synteny in general might exist throughout microsporidian genomes.