[Evaluation of Toll-Like Receptor Gene Expressions in Encephalitozoon intestinalis Infection]. / Encephalitozoon intestinalis Enfeksiyonunda Toll-Like Reseptör Gen Ekspresyonlarinin Degerlendirilmesi.

Microsporidia are obligate intracellular pathogens that can infect many vertebrate and invertebrate hosts. While the Microsporidia phylum was defined as protozoa until the 1990s, it has been associated with fungi in line with the data obtained as a result of phylogenetic and molecular analyzes in recent years. Although approximately 200 genera and 1400 Microsporidia species related to these genera have been reported to date, only 14 species are known to cause infection in humans. Encephalitozoon intestinalis is one of the most frequently detected species in humans and causes serious clinical conditions in immunosuppressed individuals. Little information is available about the immunology of this infection. This study was aimed to investigate the changes in Toll-Like receptor (TLR) gene expressions in Madin-Darby canine kidney (MDCK) cells treated with E.intestinalis spores. Three groups were formed in the study. In the first group, only the medium prepared for E.intestinalis was added to the MDCK cells. In the second group, 108 live spores waiting at +4 °C were added. In the third group, 108 heat-inactivated spores were added. All three groups were incubated at 37ºC with 5% CO2 . RNA isolation and cDNA synthesis were performed from samples taken from these groups at the 1st, 3rd, 6th, 12th and 24th hours. Expression of TLR1-10 genes from the obtained cDNAs was evaluated by real-time polymerase chain reaction (Rt-PCR). GAPDH and ACTB genes were used as housekeeping genes in the study. Target genes were normalized by taking the average of these two genes and statistical analysis was performed by applying the 2-ΔΔCt formula. Genes detected above the threshold value (threshold 1) were considered to have increased expression. Genes detected below the threshold value were considered to have decreased expression. The growth of the live and inactive spores were followed simultaneously with the experimental groups. Approximately two weeks after the start of the culture, it was observed that E.intestinalis grew in the culture with live spore, but did not grow in the culture with inactivated spores. No statistically significant change was observed in gene expressions in the inactivated spore group. In the live spore group, a significant increase was seen in the expression of only two genes. These genes were TLR3 and TLR4. It was observed that there was a significant increase in TLR3 gene expression at the first hour (1.6-fold of control group) but the expression level started to decrease at the third hour (1.4-fold of control group) and returned to the control level at the sixth hour. It was observed that TLR4 gene expression continued parallel to the control until the 24th hour and increased significantly (2.1-fold of control group) at the 24th hour. In conclusion, this study is the f irst report in which the changes in ten different TLR gene expressions were evaluated at different times in MDCK cells stimulated with E.intestinalis and the change in TLR3 gene expression.

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.

The role of microsporidian polar tube protein 4 (PTP4) in host cell infection.

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.

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.

Encephalitozoon intestinalis Inhibits Dendritic Cell Differentiation through an IL-6-Dependent Mechanism.

Microsporidia are a group of intracellular pathogens causing self-limited and severe diseases in immunocompetent and immunocompromised individuals, respectively. A cellular type 1 adaptive response, mediated by IL-12, IFNγ, CD4+, and CD8+ T cells has been shown to be essential for host resistance, and dendritic cells (DC) play a key role at eliciting anti-microsporidial immunity. We investigated the in vitro response of DC and DC precursors/progenitors to infection with Encephalitozoon intestinalis (Ei), a common agent of human microsporidosis. Ei-exposed DC cultures up-regulated the surface expression of MHC class II and the costimulatory molecules CD86 and CD40, only when high loads of spores were used. A vigorous secretion of IL-6 but not of IL-1ß or IL-12p70 was also observed in these cultures. Ei-exposed DC cultures consisted of immature infected and mature bystander DC, as assessed by MHC class II and costimulatory molecules expression, suggesting that intracellular Ei spores deliver inhibitory signals in DC. Moreover, Ei selectively inhibited the secretion of IL-12p70 in LPS-stimulated DC. Whereas Ei-exposed DC promoted allogeneic naïve T cell proliferation and IL-2 and IFNγ secretion in DC-CD4+ T cell co-cultures, separated co-cultures with bystander or infected DCs showed stimulation or inhibition of IFNγ secretion, respectively. When DC precursors/progenitors were exposed to Ei spores, a significant inhibition of DC differentiation was observed without shifting the development toward cells phenotypically or functionally compatible with myeloid-derived suppressor cells. Neutralization experiments demonstrated that this inhibitory effect is IL-6-dependent. Altogether this investigation reveals a novel potential mechanism of immune escape of microsporidian parasites through the modulation of DC differentiation and maturation.

Serological screening of selected microsporidia in HPV-positive women.

Microsporidiosis is considered to be emerging opportunistic infection in immunocompromised individuals worldwide. The aim of this study was to identify the specific serum antibodies to intestinal microsporidia Encephalitozoon cuniculi and Encephalitozoon intestinalis in women with Human Papillomavirus HPV and without HPV by the indirect immunofluorescence (IFA). From total number of 669 examined women, 225 were HPV positive and 444 women HPV negative. Overall the study comprised of 10.8% women with positive result for presence of E. cuniculi antibodies. In group 1 (HPV-positive women) it was more than 28% and in group 2 (HPV-negative women) it was less than 2% (p<0.001). E. intestinalis infection was found in total of 4.48% women, in group 1 it was present in less than 6% and in group 2 in less than 4% of women.

Microsporidia and its relation to Crohn's disease. A retrospective study.

BACKGROUND: The cause of Crohn's Disease (CD) remains unknown. Recently a decrease in the global lymphocyte population in the peripheral blood of CD patients has been reported. This decrease was more evident in γδ T lymphocytes, especially γδ CD8+T subsets. Furthermore, a decrease of IL-7 was also observed in these patients. We propose the hypothesis that microsporidia, an obligate intracellular opportunistic parasite recently related to fungi, in CD patients can take advantage of the lymphocytes and IL-7 deficits to proliferate and to contribute to the pathophysiology of this disease. METHODS AND FINDINGS: In this case-control study, serum samples were collected from 36 CD patients and from 36 healthy individuals (controls), IgE and IgG anti-Encephalitozoon antibodies were determined by ELISA; and forty-four intestinal tissue samples were analyzed through real time Polymerase Chain Reaction (PCR), twenty CD patients, nine with others diseases and 15 healthy subjects. We observed that IgE anti-Encephalitozoon levels were significantly higher in patients with CD: 0.386(±0.256) vs control group, 0.201(±0.147), P<0.001. However, IgG anti-Encephalitozoon values were significantly lower in CD patients: 0.361(±0.256) vs control group, 0.876(±0.380), P<0.001. In the group of CD patients, 6/20 (30%) were positive by real time PCR for microsporidia and, all the patients of the control group were negative by real time PCR. CONCLUSIONS: These results suggest that CD patients are a group at risk for microsporidiasis and, moreover that microsporidia may be involved as a possible etiologic factor of CD.

Immune response to Encephalitozoon infection review.

The microsporidia are emerging agents of infectious disease in both immunocompromised and immunocompetent mammals. Recently, there has been an increased interest in studying the immunobiology of microsporidiosis. This paper discusses the humoral and cell-mediated immune responses to Encephalitozoon spp. The T-cell-mediated responses appear to be most important in conferring resistance. This has become evident by the lethal effects of microsporidiosis in T-cell-deficient hosts. However, much still needs to be learned about the immunobiology of microsporidiosis regarding the specific T-cell responses and the cytokines that provide protective immunity and facilitate the macrophage-mediated killing of microsporidia. Such information will become important in developing immunotherapeutic strategies to control microsporidiosis in the future.

Latent microsporidial infection in immunocompetent individuals - a longitudinal study.

BACKGROUND: Microsporidia (Fungi) have been repeatedly identified as the cause of opportunistic infections predominantly in immunodeficient individuals such as AIDS patients. However, the global epidemiology of human microsporidiosis is poorly understood and the ability of microsporidia to survive and multiply in immunocompetent hosts remains unsolved. AIMS: To determine the presence of latent microsporidia infections in apparently healthy humans in the Czech Republic, the authors tested sera, urine and stool originating from fifteen persons within a three month period examined on a weekly basis. METHODS: Sera, stool and urine samples originating from fifteen HIV-negative people at risk with occupational exposure to animals, aged 22-56 years, living in the Czech Republic were tested by indirect immunofluorescence assay (IFA) for the presence of specific anti-microsporidial antibodies, standard Calcofluor M2R staining for the detection of microsporidian spores in all urine sediments and stool smears and molecular methods for the microsporidial species determination. RESULTS: Specific anti-microsporidial antibodies were detected in fourteen individuals, asymptomatic Encephalitozoon spp. infection was found in thirteen and E. bieneusi infection was detected in seven of those examined. While E. hellem 1A and E. cuniculi II were the major causative agents identified, seven different genotypes of E. bieneusi were recorded. CONCLUSIONS: These findings clearly show that exposure to microsporidia is common and chronic microsporidiosis is not linked to any clinical manifestation in healthy population. Moreover, our results indicate much higher incidence of microsporidial infections among an apparently healthy population than previously reported. These results open the question about the potential risk of reactivation of latent microsporidiosis in cases of immunosupression causing life-threatening disease.

Encephalitozoonosis in New Zealand rabbits and potential transmission risk.

Encephalitozoon cuniculi is a small protozoan parasite in the phylum Microspora. It has been shown to naturally infect several host species, including humans. Encephalitozoonosis is routinely diagnosed in vivo by serological examination or post mortem by histopathology. In a conventional rabbit colony, two animals suddenly showed clinical signs (torticollis and asthenia of limbs). Serum samples of these rabbits were seropositive for E. cuniculi after definitive diagnosis (Toxoplasma gondii and Listeria monocytogenes). The animals in the same breeding facility were also clinical examined, and the present study evaluated the prevalence of specific anti-E. cuniculi antibodies using serological testing, both in animals and in people working with animals, after two clinical cases. The rabbits showed no clinical symptoms of the disease. Blood samples were taken for E. cuniculi infection from 50 clinically healthy rabbits. Anti-E. cuniculi antibodies were found in two asymptomatic and two clinically affected animals belonging to the same rabbit colony. Finally, the present study found that the 7.7% (4/52) prevalence of CIA, test positive in rabbits. E. cuniculi spores were detected in the urine of one clinically affected rabbit, and one seropositive animal caretaker after staining with the modified trichrome stain. In conclusion, the presence of seropositive, but apparently healthy rabbits indicates the need for screening examinations to detect the anti-E. cuniculi antibody in rabbits, especially considering the potential zoonotic risk. Therefore, persons should avoid contact with the urine of infected or healthy animals, and always use good personal hygiene when handling animals.

Evaluation of the co-agglutination test in diagnosis of experimental microsporidiosis.

Microsporidiosis is an emerging and opportunistic infection associated with wide range of clinical syndromes in humans. Confirmation of the presence of microsporidia in different samples is laborious, costly and often difficult. The present study was designed to evaluate the utility of the Co-agglutination test (Co-A test) for detection of urinary, fecal and circulating microsporidial antigens in experimentally infected mice. One hundred and twenty male Swiss albino mice were divided into non infected control and infected experimental groups which were further subdivided into two equal subgroups; immunosuppressed and immunocompetent. Microsporidial spores were isolated from human stools and identified to be Encephalitozoon intestinalis by the molecular methods. They were used to infect each subgroup of mice, then their urine, stools and sera were collected at the 1st, 3rd, 5th, 7th and 9th days post-infection (PI). Co-A test, using prepared hyperimmune serum, was used to detect antigens in all samples collected. The cross reactivity of microsporidial hyperimmune sera with antigens of Cyclospora cyatenensis and Cryptosporidium parvum was investigated by Co-A test. The results showed that Co-A test was effective in detecting microsporidial antigen in stool of immunosuppressed infected mice from the 1st day PI, and in urine and serum from the 3rd day PI till the end of the study. In the immunocompetent subgroup, Co-A test detected microsporidial antigens in stool, serum and urine of mice from the 1st day, 3rd day and the 5th day PI, respectively till the end of the study, without cross reactivity with C. cyatenensis or C. parvum in both subgroups. Co-A test proved to be simple and suitable tool for detecting microsporidial antigen in different specimens and did not need sophisticated equipment. It is very practical under field or rural conditions and in poorly equipped clinical laboratories.

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.

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals.

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p<0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p<0.0075).

Innate immune responses to Encephalitozoon species infections.

Microsporidia are obligate intracellular, eukaryotic fungi, which have gained recognition as opportunistic parasites in immunocompromised patients. Resistance to lethal microsporidia infections requires a Th1 immune response; how this protection is initiated against Encephalitozoon species is the focus of this review article.

Interferon-gamma-mediated activation of enterocytes in immunological control of Encephalitozoon intestinalis infection.

The microsporidian Encephalitozoon intestinalis develops within intestinal epithelial cells (enterocytes) and is an important opportunistic diarrhoeal pathogen associated with AIDS. Little is known about the protective immune response against the parasite although in mice IFN-gamma is involved and is required to prevent dissemination of the infection to other organs. The present study was designed to establish a suitable short-term in vitro culture technique for E. intestinalis that would enable studies of the role of cytokines such as IFN-gamma in the effector phase of immunity. Encephalitozoon intestinalis reproduced considerably better in the murine enterocyte cell line CMT-93 than in the three human enterocyte cell lines Caco-2, HT29 and HCT-8. Treatment of CMT-93 cells with IFN-gamma significantly reduced parasite reproduction in a dose- and time-dependent manner. IFN-gamma also inhibited development of the parasite in Caco-2 cells. Neither production of NO nor Fe deprivation appeared to be involved in IFN-gamma-mediated parasite killing. However studies suggested that tryptophan catabolism by indoleamine 2,3-dioxygenase played an important part in inactivation of E. intestinalis.

Toll-like receptor 2 recognition of the microsporidia Encephalitozoon spp. induces nuclear translocation of NF-kappaB and subsequent inflammatory responses.

Microsporidia are obligate intracellular parasites that are ubiquitous in nature and have been recognized as causing an important emerging disease among immunocompromised individuals. Limited knowledge exists about the immune response against these organisms, and virtually nothing is known about the receptors involved in host recognition. Toll-like receptors (TLR) are pattern recognition receptors that bind to specific molecules found on pathogens and signal a variety of inflammatory responses. In this study, we show that both Encephalitozoon cuniculi and Encephalitozoon intestinalis are preferentially recognized by TLR2 and not by TLR4 in primary human macrophages. This is the first demonstration of host receptor recognition of any microsporidian species. TLR2 ligation is known to activate NF-kappaB, resulting in inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8). We found that the infection of primary human macrophages leads to the nuclear translocation of NF-kappaB in as early as 1 h and the subsequent production of TNF-alpha and IL-8. To verify the direct role of TLR2 parasite recognition in the production of these cytokines, the receptor was knocked down in primary human macrophages using small interfering RNA. This knockdown resulted in decreases in both the nuclear translocation of NF-kappaB and the levels of TNF-alpha and IL-8 after challenge with spores. Taken together, these experiments directly link the initial inflammatory response induced by Encephalitozoon spp. to TLR2 stimulation in human macrophages.

Encephalitozoonosis in household pet Nederland Dwarf rabbits (Oryctolagus cuniculus).

The paper presents the results of examination of 32 domestically bred rabbits, the breed Nederland Dwarf of Oryctolagus cuniculus, for the presence of Encephalitozoon cuniculi microsporidian species. The results of serological tests for E. cuniculi in 32 rabbits are reviewed along with other follow-up studies of clinical cases. Blood samples were taken from 7 asymptomatic rabbits and 25 rabbits showing neurological and ocular signs suggestive of encephalitozoonosis. In the asymptomatic group, 5 out of 7 rabbits were seropositive (71%). 16 rabbits with clinical diseases showed neurological sings, including torticollis, circus-like movements, loss of weight; 6 of them also showed ataxia, anorexia, asthenia of hind-limbs and 3 showed ocular signs. All 25 rabbits were seropositive. The spores of E. cuniculi were isolated from the faecal samples or kidneys and brain of an animal and subsequently were used for DNA isolation and PCR analysis.

Kinetics of Encephalitozoon spp. infection of human macrophages.

Microsporidia are obligate intracellular, eukaryotic parasites that are known to infect a variety of invertebrate and vertebrate species and have been reported to include a broad range of host specificities for various cell types. Although it is clear that some species of microsporidia have the ability to disseminate, causing multiorgan infections, it is not understood how dissemination occurs. One hypothesis suggests that mononuclear phagocytes engulf the pathogen and migrate to various organs while the parasite persists and proliferates. This implies that microsporidia have developed methods by which to escape intracellular degradation and can, instead, use the host as a source of nourishment and a vehicle for dissemination. In our study, we investigated the infection kinetics of 2 Encephalitozoon spp. known to cause disseminated disease in humans. Using fluorescence and scanning electron microscopy, it was determined that spore adherence to the host was rapid (3-6 hr), as was the uptake and organization of internal parasitophorous vacuoles (24 hr). Furthermore, replication was shown to occur within macrophages at 72 hr, as measured by the bromodeoxyuridine proliferation assay, and the production of mature spores occurred in host cells at 120 hr. Parasitic replication could be reduced by pretreatment of macrophages with interferon-gamma and bacterial lipopolysaccharide.

Immune responsiveness associated with experimental Encephalitozoon intestinalis infection in immunocompetent rats.

PURPOSE: Microsporidial infections have been recognized as an increasingly important infection in immunocompromized patients, particularly those infected with HIV/AIDS. This study was designed to study immune responses associated with experimental Encephalitozoon intestinalis infection in immunecompetent rats. MATERIALS AND METHODS: Thirty-four rats in 3 groups, A (Control), B (Intraperitoneal) and C (Oral) were given injections of 0.5 ml of 2 x 10(6) of purified spores of Encephalitotozoon intestinalis spores and were observed for serum specific IgG for 21 days using both Direct and Indirect ELISA. RESULTS: In indirect ELISA, specific lgG were detected on days 7, 14 and 21 for the group B rats and on day 21 for group C and in direct ELISA method, specific lgG were detected in-group B rats on days 7 and 21, for group C rats on day 21 only, while in the control rats, specific lgG were not detected. There was no significant difference between the direct and indirect methods (df=1, X(2), P>0.05). E. intestinalis was observed in stool samples of rats in 1/12 (08.33%) on days 14 and 21 in group B and in 4/10 (33.33%), 3/10 (25.00%) and 2/10 (16.67%) on days 7, 14 and 21 respectively in group C. In-group, A which is the control rats, no microsporidia were observed on days 0, 7, 14 and 21. CONCLUSIONS: There were no changes in the T-lymphocyte counts of rats prior to and after inoculation with spores. Extensive lesions were observed along the intestinal walls especially on the middle and lower sections of group C rats only.

Induction of host chemotactic response by Encephalitozoon spp.

Microsporidians are a group of emerging pathogens typically associated with chronic diarrhea in immunocompromised individuals. The number of reports of infections with these organisms and the disseminated pathology is growing as diagnostic tools become more readily available. However, little is known about the innate immune response induced by and generated against these parasites. Using a coculture chemotaxis system, primary human macrophages were infected with Encephalitozoon cuniculi or Encephalitozoon intestinalis, and the recruitment of naïve monocytes was monitored. Encephalitozoon spp. induced an average threefold increase in migration of naïve cells 48 h postinfection, which corresponded to optimal infection of monocyte-derived-macrophages. A limited microarray analysis of infected macrophages revealed several chemokines involved in the inflammatory responses whose expression was upregulated, including CCL1, CCL2, CCL3, CCL4, CCL7, CCL15, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8. The levels of 6 of 11 chemokines also present in the microarray were confirmed to be elevated by protein profiling. Kinetic studies confirmed that secreted CCL2, CCL3, and CCL4 were expressed as early as 6 h postinfection, with peak expression at 12 to 24 h and expression remaining until 48 h postinfection. Neutralization of these chemokines, specifically CCL4, significantly reduced the number of migrating cells in vitro, indicating their role in the induction of monocyte migration. This mechanism of recruitment not only supports the evidence that in vivo cellular infiltration occurs but also provides new hosts for the parasites, which escape macrophages by rupturing the host cell. To our knowledge, this is the first documentation that chemokine production is induced by microsporidian infections in human macrophages.