Prevalence and genotypic identification of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in pre-weaned dairy calves in Guangdong, China.

BACKGROUND: Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi are common enteric pathogens in humans and animals. Data on the transmission of these pathogens are scarce from Guangdong, China, which has a subtropical monsoon climate and is the epicenter for many emerging infectious diseases. This study was conducted to better understand the prevalence and identity of the three pathogens in pre-weaned dairy calves in Guangdong. METHODS: The occurrence and genetic identity of three pathogens were analyzed by polymerase chain reaction. PCR-positive products were sequenced to determine the species and genotypes. A Chi-square test was used to compare the prevalence of pathogens among sampling dates, age groups, or clinical signs. RESULTS: The detection rates of Cryptosporidium spp., G. duodenalis and E. bieneusi were 24.0% (93/388), 74.2% (288/388) and 15.7% (61/388), respectively. Three Cryptosporidium species were detected, including C. bovis (n = 73), C. parvum (n = 12) and C. ryanae (n = 7); one animal had concurrence of C. bovis and C. parvum. C. parvum was the dominant species during the first two weeks of life, whereas C. bovis and C. ryanae were mostly seen at 3-9 weeks of age. Sequence analysis identified the C. parvum as subtype IIdA19G1. Assemblage E (n = 282), assemblage A (n = 1), and concurrence of A and E (n = 5) were identified among G. duodenalis-positive animals using multilocus genotyping (MLG). Altogether, 15, 10 and 17 subtypes of assemblage E were observed at the bg, gdh and tpi loci, respectively, forming 49 assemblage E MLGs. The highest detection rate of G. duodenalis was found in winter. Sequence analysis identified genotypes J (n = 57), D (n = 3) and one concurrence of J and D among E. bieneusi-positive animals. The detection rate of E. bieneusi was significantly higher in spring (38.0%; 41/108) than in summer (7.2%; 8/111) and winter (7.1%; 12/169). CONCLUSIONS: These results indicate a common occurrence of C. parvum subtype IIdA19G1, G. duodenalis assemblage E, and E. bieneusi genotype J in pre-weaned dairy calves in Guangdong. More studies are needed to understand the unique genetic characteristics and zoonotic potential of the three enteric pathogens in the province.

Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages.

BACKGROUND: Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques. RESULTS: Seven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target. CONCLUSIONS: The IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy.

Evaluation of Immunoflourescence antibody assay for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis.

Detection of microsporidia at the species level is important for therapeutic purpose. The available techniques, modified trichrome (MT) staining cannot differentiate between species, while polymerase chain reaction (PCR) requires a reference laboratory and skilled technical staff. Immunoflourescence antibody (IFA) assay is another technique, which can differentiate among commonest species of microsporidia. However, there are very limited studies on its efficacy worldwide. Therefore, we aimed to evaluate IFA assay for the detection of microsporidia and differentiation among commonest species, Enterocytozoon bieneusi (E. bieneusi) and Encephalitozoon intestinalis infecting immunocompromised patients. Stool samples from 200 immunocompromised patients (19 with microsporidia and 181 without microsporidia using MT staining) were tested for species identification by PCR-RFLP and IFA assay. Sensitivity, specificity, diagnostic accuracy, and positive and negative predictive values were calculated as per standard formulae. Kappa statistics was used to assess the agreement between three tests. Of 200 immunocompromised patients, 21 and 20 patients had microsporidia using PCR and IFA assay, respectively. IFA assay and PCR identified E. bieneusi in all patients infected with microsporidia. Considering MT stain as gold standard, sensitivity and specificity of IFA assay was 100 and 99.4 %, respectively. Upon considering PCR as gold standard, sensitivity and specificity of IFA assay was 95.2 and 100 %, respectively. Diagnostic accuracy of IFA assay was 99.5 % along with its high test agreement with MT staining and PCR (K = 0.915, p = 0.049; K = 0.973, p = 0.027). IFA assay is highly sensitive and specific technique for detecting and identifying species of microsporidia among immunocompromised patients. E. bieneusi was the commonest species identified.

Two cases of opportunistic parasite infections in patients receiving alemtuzumab.

Two cases are reported of rare digestive opportunistic parasites in patients being treated with alemtuzumab for lymphoid haematological malignancies. In both patients, classical biological examinations were insufficient to reach the diagnosis. Only specific parasitological techniques enabled diagnoses of cryptosporidiosis and microsporidiosis, respectively. In both cases, cellular immune reconstitution was sufficient to eradicate these opportunistic infections. In this context, parasitological diagnosis is often underestimated by medical practitioners, so immunologists and oncohaematologists need to be aware of this kind of opportunistic pathogen.

MyD88-dependent pathway is essential for the innate immunity to Enterocytozoon bieneusi.

Enterocytozoon bieneusi is clinically the most significant microsporidian parasite associated with persistent diarrhoea, wasting and cholangitis in 30-50% of individuals with HIV/AIDS, as well as in malnutritional children and in the recipients of immunosuppressive therapy. However, the host immune responses to E. bieneusi have not been investigated until recently because of lack of sources of spores, cell culture system and animal models. In this study, we purified spores from heavily infected human or monkey faeces by serial salt-Percoll-sucrose-iodixanol centrifugation, and the purity of spores was confirmed by FACS and scanning electron microscopy. Exposure of dendritic cells to E. bieneusi spores induced the upregulation of the surface markers and production of pro-inflammatory cytokines. The cytokine production was independent of toll-like receptor 4, but MyD88 dependent, because dendritic cells from MyD88 knockout mice failed to secrete these pro-inflammatory cytokines, whereas dendritic cells from C3H/HeJ (a toll-like receptor 4 mutant) were activated by E. bieneusi and secreted these cytokines. Furthermore, MyD88-deficient mice were susceptible to E. bieneusi infection, in contrast to wild-type mice that resisted the infection. Collectively, the data demonstrate innate recognition of E. bieneusi by dendritic cells and the importance of MyD88-dependent signalling in resisting infection in a murine challenge model.

Microsporidiosis and Cryptosporidiosis in HIV/AIDS Patients in St. Petersburg, Russia: Serological identification of microsporidia and Cryptosporidium parvum in sera samples from HIV/AIDS patients.

To determine seroprevalence of the opportunistic organisms Cryptosporidium parvum and microsporidia (Encephalitozoon cuniculi, E. intestinalis, E. hellem, and Enterocytozoon bieneusi) in Russian HIV/AIDS patients, we evaluated 46 sera from HIV/AIDS patients from the S.P. Botkin Clinical Infectious Diseases Hospital, St. Petersburg, Russia. Five (10.9%) sera were seropositive for E. cuniculi and 19 (41.3%) were positive for C. parvum by ELISA. By IFAT, 6 (13.0%) sera were seropositive for E. bieneusi, 4 (8.7%) for E. intestinalis, and 9 (19.6%) for E. hellem. This study is the first report to estimate the prevalence of infection with Cryptosporidium and microsporidia among Russian HIV/AIDS patients.

Monoclonal antibodies against Enterocytozoon bieneusi of human origin.

Enterocytozoon bieneusi is clinically the most significant among the microsporidia infecting humans, causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. The lack of immune reagents is largely due to the absence of methods for laboratory propagation of E. bieneusi. We recently described a procedure for the concentration and purification of spores from diarrheic stool of infected humans. Purified spores were used to immunize mice for production and screening of monoclonal antibodies (MAbs) against E. bieneusi. The eight immunoglobulin M MAbs generated and fully characterized did not cross-react with other human microsporidia or with other microorganisms normally present in stool. One of the MAbs, 2G4, reacted with E. bieneusi spores in stools from monkeys and humans, without background fluorescence, which makes it an ideal diagnostic reagent. It also recognizes intracellular stages of the parasite and will be suitable for determining tissue distribution of E. bieneusi in infected hosts. At least two immunodominant antigens of E. bieneusi of 33,000 and 35,000 Da exist, which were recognized by rabbit and mouse antisera. The availability of MAbs against E. bieneusi will simplify considerably the diagnosis of this infection in humans and will provide tools for epidemiologic investigations regarding the true prevalence of the infection in various human and mammalian populations and the environmental sources of infection.

Production and characterization of monoclonal antibodies against Enterocytozoon bieneusi purified from rhesus macaques.

Enterocytozoon bieneusi spores derived from rhesus macaque feces were purified by serial salt-Percoll-sucrose-iodixanol centrifugation, resulting in two bands with different specific densities of 95.6% and 99.5% purity and with a recovery efficiency of 10.8%. An ultrastructural examination revealed typical E. bieneusi spores. Twenty-six stable hybridomas were derived from BALB/c mice immunized with spores and were cloned twice by limiting dilution or growth on semisolid medium. Four monoclonal antibodies (MAbs), reacting exclusively with spores, were further characterized. These MAbs specifically reacted with spores present in stools of humans and macaques, as visualized by immunofluorescence, and with spore walls, as visualized by immunoelectron microscopy. A blocking enzyme-linked immunosorbent assay and Western blotting revealed that the epitope recognized by 8E2 was different from those recognized by 7G2, 7H2, and 12G8, which identified the same 40-kDa protein. These MAbs will be valuable tools for diagnostics, for epidemiological investigations, for host-pathogen interaction studies, and for comparative genomics and proteomics.

Purification of Enterocytozoon bieneusi from stools and production of specific antibodies.

Enterocytozoon bieneusi is clinically the most significant of the microsporidia in humans, causing chronic diarrhea wasting and cholangitis in individuals with human immunodeficiency virus infection and AIDS. Little progress on this infection has been made because of the inability to propagate E. bieneusi in vitro and in vivo, which limits the source of parasite spores to the stools of infected human patients. Given the size and shape of the E. bieneusi spores (1.1 to 1.6 by 0.7 to 1.0 microm) and the lack of specific immune reagents, the identification and purification of large quantities of spores from feces are technically challenging. Consequently, diagnosis relies entirely on PCR, a labor-intensive approach that requires highly skilled personnel. We describe a method for the purification of E. bieneusi spores from human stools and the production of rabbit-specific antisera. Spores were purified by a combination of isopycnic Percoll gradient centrifugation and continuous sucrose gradient centrifugation. Specific polyclonal antibodies raised in mice and rabbits reacted by indirect immunofluorescence with E. bieneusi but not with Encephalitozoon spp., Candida albicans, Staphylococcus aureus, Escherichia coli, or other forms present in human stools.

Quantitative evaluation of Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected rhesus monkeys.

The association of the microsporidia Enterocytozoon bieneusi with chronic diarrhea and wasting in individuals with acquired immunodeficiency syndrome (AIDS) has been demonstrated. The disease caused by E. bieneusi has been linked to decreased levels of circulating CD4+ T lymphocytes. In this study, we investigated the relationship between the extent of excretion of E. bieneusi in feces of simian immunodeficiency virus (SIV)-infected juvenile macaques and the CD4+ T lymphocyte counts in the peripheral blood. Twelve juvenile rhesus monkeys (Macaca mulatta) were intravenously inoculated with the pathogenic molecular clone SIVmac239. Numbers of CD4+ T lymphocytes were assessed by three-color flow cytometry. The presence of E. bieneusi DNA in feces was assessed by nested PCR. In addition, selected samples of feces were examined by competitive quantitative PCR to assess the level of E. bieneusi infection. Low (n = 5) to undetectable (n = 7) quantities of E. bieneusi were present in feces of the twelve animals in prior to inoculation with SIV. After SIV inoculation the number of animals shedding E. bieneusi increased (n = 10) as did the quantity of E. bieneusi shedding in the feces. Of the twelve juvenile animals, five animals died within 8 months post-SIV inoculation with symptoms of AIDS. Four of the five deceased animals showed shedding of E. bieneusi DNA in feces (> or =100 spores/g) for at least three consecutive months. Increased number of E. bieneusi in feces was accompanied by decreased counts of circulating CD4+ T lymphocytes and increased SIV plasma viral load.