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.

Characterization of WRSs2 and WRSs3, new second-generation virG(icsA)-based Shigella sonnei vaccine candidates with the potential for reduced reactogenicity.

Live, attenuated Shigella vaccine candidates, such as Shigella sonnei strain WRSS1, Shigella flexneri 2a strain SC602, and Shigella dysenteriae 1 strain WRSd1, are attenuated principally by the loss of the VirG(IcsA) protein. These candidates have proven to be safe and immunogenic in volunteer trials and in one study, efficacious against shigellosis. One drawback of these candidate vaccines has been the reactogenic symptoms of fever and diarrhea experienced by the volunteers, that increased in a dose-dependent manner. New, second-generation virG(icsA)-based S. sonnei vaccine candidates, WRSs2 and WRSs3, are expected to be less reactogenic while retaining the ability to generate protective levels of immunogenicity seen with WRSS1. Besides the loss of VirG(IcsA), WRSs2 and WRSs3 also lack plasmid-encoded enterotoxin ShET2-1 and its paralog ShET2-2. WRSs3 further lacks MsbB2 that reduces the endotoxicity of the lipid A portion of the bacterial LPS. Studies in cell cultures and in gnotobiotic piglets demonstrate that WRSs2 and WRSs3 have the potential to cause less diarrhea due to loss of ShET2-1 and ShET2-2 as well as alleviate febrile symptoms by loss of MsbB2. In guinea pigs, WRSs2 and WRSs3 were as safe, immunogenic and efficacious as WRSS1.

Microsporidiosis in South Africa: PCR detection in stool samples of HIV-positive and HIV-negative individuals and school children in Vhembe district, Limpopo Province.

Microsporidia were initially recognized as pathogens of insects and fish but have recently emerged as an important group of human pathogens, especially in immune-compromised individuals, such as those with HIV infection. In this study, we used a PCR-RFLP assay confirmed by quantitative real-time PCR and trichrome staining to determine the prevalence of microsporidian infections among hospital patients and school children in Vhembe region. Enterocytozoon bieneusi was the only microsporidian species detected in these stool samples. It was found in 33 (12.9%) of 255 samples from the hospitals and in 3 (4.5%) of 67 samples from primary school children and was significantly associated (P=0.039) with diarrhea in HIV-positive patients (21.6%) compared to HIV-negative individuals (9%). However, microsporidian infections were not associated with intestinal inflammation as indicated by the lactoferrin test. These results suggest that microsporidia might be a cause of secretory diarrhea in HIV-positive patients. To our knowledge, this is the first report of E. bieneusi in the Vhembe region of South Africa. Further investigations are needed in order to clarify the pathogenesis of E. bieneusi in HIV-positive patients.

Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices.

AIMS: The aims of this study were to validate a portable continuous flow centrifuge (PCFC) as an alternative concentration step of US-EPA Method 1623 and to demonstrate it's efficacy for recovery of low numbers of protozoa from large volumes of various water matrices. METHODS AND RESULTS: Recoveries of Cryptosporidium parvum oocysts, Giardia intestinalis cysts and Encephalitozoon intestinalis spores spiked into 10-1000 l volumes of various water matrices were evaluated during in-house and collaborative trials. Spiked protozoa were either approved standards or diluted stock samples enumerated according to USEPA Method 1623. Cryptosporidium recoveries exceeded method 1623 criteria and substantially high recoveries were observed for Giardia and E. intestinalis. CONCLUSIONS: Portable continuous flow centrifuge methodology exceeded method 1623 acceptance criteria for Cryptosporidium and could be easily adopted for other protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCFC could be adopted as an alternative user-friendly concentration method for Cryptosporidium and for monitoring of large volumes of source and tap water for accidental or deliberate contamination with protozoa and potentially with other enteric pathogens. It is anticipated that PCFC would also be equal or superior to filtration for protozoa monitoring in wastewater and effluents.

Characterization of a human monoclonal antibody against Shiga toxin 2 expressed in Chinese hamster ovary cells.

Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 microg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.

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.

Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species.

Cryptosporidium parvum TU502, a genotype 1 isolate of human origin, was passaged through three different mammalian hosts, including humans, pigs, and calves. It was confirmed to be genotype 1 by PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene, direct sequencing of PCR fragments of the small subunit rRNA and beta-tubulin genes, and microsatellite analysis. This isolate was shown to be genetically stable when passaged through the three mammalian species, with no evidence of the emergence of new subpopulations as observed by a genotype-specific PCR assay. TU502 oocysts from different sources failed to infect gamma interferon knockout mice, a characteristic of genotype 1 isolates. The genotypic and phenotypic characterization of TU502 is significant since it is the isolate selected to sequence the genome of C. parvum genotype 1 and is currently used in several research projects including human volunteer studies.

Variability among Cryptosporidium parvum genotype 1 and 2 immunodominant surface glycoproteins.

Published genomic differences between Cryptosporidium parvum genotype 1 (human-derived) and genotype 2 (animal and human-derived) isolates suggest that these may belong to two distinct species. This is of significant interest since genotype 1 isolates are associated with sporadic cases of human cryptosporidiosis in 30-40 % of cases in contrast to 60-70 % of cases caused by genotype 2. The lower genetic sequence similarity between genotype 1 and 2 surface glycoproteins (gp40/15) suggests that antigenic differences should also occur, a feature that was investigated in this study. Using immune and convalescent serum samples from gnotobiotic piglets previously inoculated with genotype 1 and 2 isolates, we demonstrated that C. parvum gp15 was immunodominant for both genotype 1 and 2 isolates. Lower genetic sequence similarity between genotype 1 and 2 Cpgp40/15 did correspond to gp15 protein differences as detected by Western blot. Moreover, we confirmed that gp15 contains epitopes that are also immunodominant. Deglycosylation of C. parvum proteins resulted in decreased ability of gp15, gp23 and gp900 to react with homologous polyclonal antibodies, suggesting that these proteins also express carbohydrate epitopes. Taken together, our data suggest that there is a high phenotypic variability between C. parvum genotype 1 and 2 isolates at the level of gp15. We contemplate that gp15 surface glycoprotein plays an important role in the biology of C. parvum as a potent inducer of immune response and a possible virulence factor.

The utilization of sodium taurocholate in excystation of Cryptosporidium parvum and infection of tissue culture.

The present work deals with optimization of excystation of Cryptosporidium parvum oocysts and the infection process of tissue culture cells by the parasite. It was shown that presence of the bile salt sodium taurocholate in the incubation medium expedited excystation of the tested GCH1 isolate and enhanced it, as compared with bleaching of the oocysts. This bile salt had no effect on the viability of tissue culture cell lines MDBK and HCT-8 at the tested concentration of 0.375% for up to 2 hr of coincubation. Infection studies conducted on tissue culture cells showed higher infection rates in the presence of sodium taurocholate than with bleached oocysts in the absence of this bile salt. It may be concluded that, at least as regards the GCH1 strain of C. parvum, the whole infection process can be performed in the presence of sodium taurocholate, and does not require separation and cleaning of the excysted sporozoites before their application to tissue culture cells.

Hyperimmune bovine colostrum in the treatment of shigellosis in children: a double-blind, randomized, controlled trial.

UNLABELLED: Immunological approaches have been considered as an alternative therapeutic option for the treatment of enteric infections over the past few years. Hyperimmune bovine colostrum (HBC) is a potentially innovative immunological option in the management of shigellosis together with traditional antibiotic therapy. Children aged 1-12 y with a history of bloody mucoid diarrhoea of less than 5 d duration were enrolled after their stool specimen was found to be positive for Shigella dysenteriae type I antigen by a rapid diagnostic fluorescent antibody staining test. They were randomized to receive either HBC containing very high titres of antibody against S. dysenteriae type I antigen or bovine colostrum (BC) without any antibody. The study group received 100 ml of HBC three times a day orally for 3 d and control group received BC. Children also received pivmecillinam in a dose of 50 mg kg(-1) d(-1) in four divided doses orally for 5 d. Admission characteristics of the 34 children in the HBC group and 35 in the BC group were comparable. No significant differences were observed in duration of diarrhoea, fever, anorexia, abdominal pain, tenesmus, stool frequency or visible blood in the stool between the groups. Two (6%) children in the study and five (14%) in the control group remained stool culture positive for S. dysenteriae type 1, even after 5 d of sensitive antimicrobial therapy. CONCLUSION: The results indicate that HBC as an adjuvant is unable to show any beneficial effect in reducing the stool frequency, duration or severity of childhood shigellosis due to S. dysenteriae type I infection.

Host cell apoptosis impairs Cryptosporidium parvum development in vitro.

The absence of a self-sustaining in vitro propagation method for Cryptosporidium parvum is a major obstacle for research on this parasite. Conventional cell monolayers are unsuitable for long-term parasite propagation because the level of infection decreases over time and few oocysts, if any, are produced. The interaction between parasite and host cell was studied to identify factors limiting parasite development in vitro. Loss of substrate adherence and death of parasitized host cells was observed in 2 epithelial cell lines. Nuclear morphology, DNA laddering, annexin V binding, and terminal deoxytransferase-mediated dUTP nick end labeling indicated that host cell death occurred by apoptosis. At 6 hr postinfection, only a minority of infected cells remained in the monolayer, and few survived the initial phase of parasite development without losing adherence. Treatment of infected monolayers with caspase inhibitors drastically reduced cell detachment but failed to increase the number of parasites in monolayers. In contrast, cell cultures grown on laminin-coated plates showed a higher proportion of infected cells. These observations indicate that cell detachment and apoptosis in C. parvum-infected cell culture negatively affect parasite survival in vitro.

Mediation of Cryptosporidium parvum infection in vitro by mucin-like glycoproteins defined by a neutralizing monoclonal antibody.

The protozoan parasite Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. Attachment to and invasion of host intestinal epithelial cells by C. parvum sporozoites are crucial steps in the pathogenesis of cryptosporidiosis. The molecular basis of these initial interactions is unknown. In order to identify putative C. parvum adhesion- and invasion-specific proteins, we raised monoclonal antibodies (MAbs) to sporozoites and evaluated them for inhibition of attachment and invasion in vitro. Using this approach, we identified two glycoproteins recognized by 4E9, a MAb which neutralized C. parvum infection and inhibited sporozoite attachment to intestinal epithelial cells in vitro. 4E9 recognized a 40-kDa glycoprotein named gp40 and a second, >220-kDa protein which was identified as GP900, a previously described mucin-like glycoprotein. Glycoproteins recognized by 4E9 are localized to the surface and apical region of invasive stages and are shed in trails from the parasite during gliding motility. The epitope recognized by 4E9 contains alpha-N-acetylgalactosamine residues, which are present in a mucin-type O-glycosidic linkage. Lectins specific for these glycans bind to the surface and apical region of sporozoites and block attachment to host cells. The surface and apical localization of these glycoproteins and the neutralizing effect of the MAb and alpha-N-acetylgalactosamine-specific lectins strongly implicate these proteins and their glycotopes as playing a role in C. parvum-host cell interactions.

Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis.

Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.

Intestinal lesions associated with disseminated candidiasis in an experimental animal model.

In human patients, disseminated candidiasis, a life-threatening disease for immunocompromised patients, is often associated with intestinal lesions. In this study, we demonstrate that immunosuppressed gnotobiotic (IGB) piglets orally inoculated with wild-type Candida albicans developed extensive intestinal lesions and disseminated infection. Severe ulceration of the ileal mucosa was observed overlying regions of colonization and necrosis of the gut-associated lymphoid tissue. Despite the high susceptibility of IGB piglets to many microbial pathogens, an avirulent mutant strain of C. albicans failed to produce intestinal lesions and exhibited poor dissemination, demonstrating that these effects required virulent organisms. It is likely that in IGB piglets, as in human patients, intestinal lesions provide the mechanism for escape of C. albicans from the gastrointestinal tract. Multinucleated giant cells containing fungal organisms were observed within lymph nodes and lymphatic vessels, and as with other pathogens, such cells could provide a mechanism for dissemination of C. albicans.

Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15.

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein in C. parvum attachment to and invasion of host cells. We have cloned and sequenced a gene designated Cpgp40/15 that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated in C. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bp Cpgp40/15 revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predicted O-glycosylation sites, a single potential N-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy of Cpgp40/15 in the C. parvum genome, and this gene does not contain introns. Our data indicate that the two Cpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis.

Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum.

Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.

Escherichia coli O157:H7 strains that express Shiga toxin (Stx) 2 alone are more neurotropic for gnotobiotic piglets than are isotypes producing only Stx1 or both Stx1 and Stx2.

Infection with Escherichia coli O157:H7 can lead to hemolytic uremic syndrome (HUS) in some children. Epidemiologic data suggest that Shiga toxin (Stx) 2-producing strains are more frequently associated with HUS than are Stx1-producing strains. Less clear is whether strains that express Stx2 alone are more frequently associated with HUS than strains that express Stx1 and Stx2. Isogenic mutants 933stx1- and 933stx2- were produced from strain 933 (Stx1 and Stx2 producer), and 86-24stx2- was produced from strain 86-24 (Stx2 producer). Neurologic lesions or symptoms developed in 18 (90%) of 20 gnotobiotic piglets orally infected with strain 86-24, in 15 (85%) of 18 infected with mutant 933stx1-, in 9 (31%) of 29 infected with strain 933, in 0 of 5 infected with mutant 86-24stx2-, and in 0 of 6 infected with mutant 933stx2-. It was concluded that strains expressing Stx2 alone are more neurotropic for piglets when fed orally than are those strains expressing Stx1 and 2, whereas Stx1-producing strains induce only diarrhea. It is also conceivable that strains that produce Stx2 may constitute a significant predictive risk factor for HUS in humans.

Enterocytozoon bieneusi infection in patients without evidence of immunosuppression: two cases from Zimbabwe found to have positive stools by PCR.

Patients infected with human immunodeficiency virus (HIV) often also have intestinal infections with Enterocytozoon bieneusi. Recently, infection with this microsporidian has been described in immunocompetent subjects, mainly from Europe. When the stools of six HIV-negative patients who presented with diarrhoea in Zimbabwe were investigated, using a recently described protocol based on PCR, two patients were found to have E. bieneusi infections. These two individuals presented with a self limited diarrhoea, abdominal cramping and nausea. These data indicate that E. bieneusi may be a more common cause of diarrhoea in Zimbabwe than previously thought. Larger, prospective studies are needed.

Tacrolimus-FK506 induced immunosuppression in gnotobiotic piglets.

Tacrolimus (FK506), an inhibitor of calcineurin, is an immunosuppressive agent used in clinical trials of transplant patients. Although FK506 targets Ca(2+)-mediated T-cell signaling, phenotype(s) of the specific target cells and the corresponding cytokine pathways are not well known. In this study, the impact of FK506 on number and characteristic of T-cells in selected lymphoid tissues of gnotobiotic (GB) piglets was determined. FK506-treated GB piglets were compared with untreated GB and conventional piglets. The T-helper, cytotoxic, natural killer, double-positive, and activated T-cell populations were analyzed in suspensions of mononuclear cells isolated from thymus, mesenteric lymph nodes and peripheral blood. In vitro secretion of interleukin-8 and interferon-gamma in concanavalin A-stimulated lymphoid cell-cultures was measured by ELISA. Daily intramuscular treatment of GB piglets with 1mg/kg of FK506 from birth for 4 weeks resulted in lowered (P<0.05) in vitro secretion of interferon-gamma and interleukin-8. Moreover, depletions of MNC in systemic and mucosa-associated lymphoid tissues were observed in piglets treated with FK506. The depletions of mononuclear cells and low levels of interferon-gamma and interleukin-8 in piglets treated with FK506 were accompanied by lower proportion of CD3+, CD2+CD4+ and CD2+CD8+ T-cell phenotypes in peripheral blood but not in thymus and mesenteric lymph nodes. These results indicate that FK506-treatment causes immunosuppression in GB piglet, and this effect could be exploited further to study opportunistic pathogens in pig model.