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.

The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model.

The eaeA gene of enteropathogenic Escherichia coli (EPEC) is necessary for intimate attachment to epithelial cells in vitro. Enterohemorrhagic E. coli (EHEC) strains also possess an eae gene and are capable of intimate attachment and microvillus effacement in vitro and in animal models. To assess the role of the EHEC eae gene in intimate attachment, we constructed an eae deletion/insertion mutation in wild-type EHEC O157:H7 strain 86-24 by using linear electroporation of a recombinant allele. The mutant obtained was deficient in inducing f-actin accumulation in HEp-2 cells and was incapable of attaching intimately to colonic epithelial cells in a newborn piglet model of infection. Intimate attachment in vivo was restored when the EHEC eae gene or the eaeA gene of EPEC was introduced into the mutant on a plasmid. These results indicate that the eae gene is necessary for intimate attachment of EHEC in vivo. In addition, the complementation achieved by the EPEC locus indicates that the eae gene of EHEC and the eaeA gene of EPEC are functionally homologous.

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.

Treatment with bovine hyperimmune colostrum of cryptosporidial diarrhea in AIDS patients.

Cryptosporidium parvum may cause severe, debilitating diarrhea in patients with AIDS. Recent anecdotal reports have suggested that hyperimmune bovine colostrum may be effective. We conducted a double-blind, controlled pilot study of hyperimmune bovine colostrum for diarrhea due to cryptosporidiosis in five AIDS patients. The patients were randomized to receive either hyperimmune or control colostrum by continuous nasogastric infusion for 10 days. All stools were collected, graded, and weighed, and the concentration of oocysts excreted was determined daily. One of the three patients treated with hyperimmune colostrum had a reduction in diarrhea and in the concentration of oocysts excreted. A second treated patient had a modest decrease in the concentration of oocysts excreted. Two patients who received control colostrum also had decreases in the volume of diarrhea but no change in the concentration of oocysts excreted. We conclude that hyperimmune colostrum with high titers of specific anti-Cryptosporidium antibody could be effective in treating patients with cryptosporidiosis. However, more studies of cow colostral immunoglobulin need to be performed so that the efficacy of this treatment can be assessed more thoroughly.

Intestinal parasites in patients with diarrhea and human immunodeficiency virus infection in Zimbabwe.

OBJECTIVES: To determine the prevalence of intestinal parasites and risk factors for infection associated with diarrhea in HIV-infected patients in Harare, Zimbabwe. DESIGN: Prospective observational study. METHODS: Single stool samples were collected from 88 HIV-infected individuals presenting with diarrhea of greater than 1 week duration. Stools were examined for intestinal parasites using modified acid fast stain, fluorescence- labeled monoclonal antibody for Cryptosporidium parvum, as well as a modified trichrome stain and a PCR-based protocol for Enterocytozoon bieneusi. RESULTS: C. parvum was detected in 9% (seven out of 82) of samples evaluated, but no Cyclospora was detected. E. bieneusi was detected in 18% (10 out of 55) of stool by trichrome staining and in 51% (28 out of 55) of stool examined by PCR. Risk factors for E. bieneusi infection were: living in rural areas, consumption of nonpiped water, contact with cow dung and household contact with an individual with diarrhea. CONCLUSION: E. bieneusi infection was common in HIV-infected patients with diarrhea in Zimbabwe and may be acquired through person-to-person and fecal-oral transmission.

Cryptosporidiosis in perspective.

In this review I have examined the vast literature which has accumulated on Cryptosporidium, particularly in the past 3 years, in an attempt to highlight areas in which progress has been made in relation to the organism and the disease, and to indicate areas in which knowledge is still lacking. Since 1982, a global effort by scientists and clinicians has been directed towards determining the nature of the disease in humans and the relative contribution of cryptosporidiosis to gastroenteritis. From published data, the incidence of diarrhoea is 1-5% in most developed countries, and 4-7% in less developed countries, when measured throughout the year and in all age groups. The frequency of cryptosporidiosis is highest in children aged between 6 months and 3 years, and in particular locations (e.g., day-care centres) and at particular times of the year. Although susceptibility to infection is life-long, one suspects that the lower prevalence among older children and adults is due to immunity acquired from frequent exposure. Other important factors contributing to higher prevalence are the season--it is more frequent in a wet, warm climate--association with travel to particular destinations, poor hygiene, intimate contact with certain animals, and congregation of large numbers of young previously unexposed children in day-care centres. The association between cryptosporidiosis and giardiasis presumably results from the existence of a common source of infection. The immune status of the host appears to be a major determinant of whether the infection is self-limiting or persistent. It is clear that both branches of the immune system are required for complete recovery, since T-lymphocyte dysfunction or hypogammaglobulinaemia can both lead to persistent illness. Chronic diarrhoea and malabsorption attributed to cryptosporidiosis also occur in the absence of evidence of immune defect. The importance of respiratory tract infection in humans, other than in the terminal stages of chronic illness, requires investigation. The infection has now been identified in all classes of vertebrates; it has been observed in all domestic animals including pets, and a wide range of wildlife including birds. Cryptosporidiosis seems to cause diarrhoea in young ruminants, less frequently in pets. In birds the parasite has been observed in the gastrointestinal tract, without ill effect, and in the respiratory tract, in which clinical symptoms of variable severity have been described. The mucosal response of the gastrointestinal tract to infection appears to vary among mammals and may be the key to the variable clinical manifestations observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Cryptosporidiosis: laboratory investigations and chemotherapy.

Much progress has been achieved in the last decade in terms of development of laboratory techniques, reagents and in vivo models. They have undoubtedly contributed to better and more accurate investigations. Despite a concerted effort by many investigators, however, breakthroughs have been minimal. The development of adequate in vitro and in vivo techniques for drug screening, and the intensified and systematic screening, has so far not resulted in the discovery of an effective therapy. The reason for the failure may well be due to the unique biological niche the parasite occupies (discussed at length in the first chapter in this volume). Its location beneath the cell membrane, but outside the cell cytoplasm, may prove a crucial element that needs to be considered when designing new therapeutic approaches. Laboratory investigations on two drugs currently used against chronic Cryptosporidium parvum in acquired immune deficiency syndrome (AIDS) are discussed. This chapter also provides information and the rationale for work in progress in our laboratory that relates to the development of novel approaches for control of the disease. This includes the identification of molecular targets of parasite origin for drug design, and studies on the structure-activity relationships of partially effective drugs with a view to synthesize more effective derivatives. Other investigations attempt to establish the role of secretory antibody, and the merit of repeated mucosal immunizations as a means of providing protection to individuals with AIDS who are at risk of developing chronic C. parvum infection.

Natural history and biology of Cryptosporidium parvum.

The taxonomy of the genus Cryptosporidium remains ambiguous, because the current criteria for speciation are insufficient to validate the 6-8 named species. Cross-transmission experiments have shown varying and conflicting results, and the limited genetic data available do not necessarily support currently proposed species designations. The reasons for this ambiguity lie with the ubiquitous nature of Cryptosporidium, probably infecting all vertebrates and variety of tissues therein, and the absence of reference strains with defined virulence attributes that can be linked to genetic markers for comparative analysis. The inability to classify oocysts or confidently to identify their origin, implicate oocysts from all sources as hazardous to humans. Another major issue is the unusual degree of resistance that Cryptosporidium has shown to antiprotozoan and antimicrobial agents. The intracellular but extracytoplasmic domain the parasite occupies is in itself a significant barrier to drug entry. In support of this we outline how the intracellular niche of this parasite differs from the related Apicomplexans, Plasmodium and Toxoplasma, and delineate why the feeder organelle membrane, rather than, or in addition to, the parasitophorous membrane, is the major portal of nutrient entry for Cryptosporidium. The broad conclusion is that anticryptosporidial agents will have to enter the parasite via the multiple apical membranes that camouflage the parasite, or via the host cell, possibly transported by vesicles to the feeder organelle membrane. This may have major implications for rational drug discovery and design.

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.

Cerebral infection with Escherichia coli O157:H7 in humans and gnotobiotic piglets.

Escherichia coli O157:H7 was isolated from a fatal case of haemorrhagic colitis with haemolytic uraemic syndrome and neurological symptoms. This strain induced diarrhoea and neurological symptoms including incoordination, ataxia, and convulsions in piglets after oral inoculation. Similar neurological signs were seen in piglets inoculated intraperitoneally with bacterial extracts containing a shiga-like toxin that is elaborated by the bacteria. Histological examination of the brains from these piglets showed vascular damage and small infarcts confined to the cerebellum. Comparable lesions were also seen in the brain of the child from whom E coli O157:H7 was isolated. We suggest that the cerebral changes in the piglets and in the patient were caused by the shiga-like toxin elaborated by E coli O157:H7. The shiga-like toxin is thought to cause neurological abnormalities by damage to cerebral blood vessels rather than by a direct effect on the neurones.

Cryptosporidiosis in hospital patients with gastroenteritis.

Among 884 hospital patients with gastroenteritis, 36 (4.1%) were excreting Cryptosporidium oocysts in their stools; only 5 of the 36 patients were also excreting other enteropathogens, while none of 320 hospital patients without gastroenteritis were excreting Cryptosporidium oocysts. Children were more commonly infected with Cryptosporidium (4.8%) than were adults (1.6%). The prevalence of infection was higher (7%) during the summer period of February-May 1981 than in the remainder of the observation period to the beginning of June 1982 (1.9%). The most common clinical manifestation of gastroenteritis in Cryptosporidium-infected patients was diarrhea, lasting from 3 to over 14 days, accompanied by vomiting, anorexia, and abdominal pain. The results show that a small proportion of patients with gastroenteritis are infected with Cryptosporidium, and the importance of the infection needs to be examined.

Simultaneous infection with multiple serotypes of Shigellae in a patient.

We isolated three different serotypes of Shigella on admission from a patient with dysentery as well as a Shigella-like organism and Campylobacter jejuni upon follow-up. The patient produced serum antibodies to all three serotypes of shigellae.

The association of haemagglutination and adhesion with lipopolysaccharide of Shigella dysenteriae serotype 1.

In this study the ability of strains of Shigella dysenteriae serotype 1 to agglutinate mammalian erythrocytes is attributed to the polysaccharide fraction of bacterial-cell lipopolysaccharide (LPS). LPS obtained from a rough, mutant strain of S. dysenteriae serotype 1, lacking the O-antigen polysaccharide side-chain, did not agglutinate erythrocytes, clearly demonstrating a link between O-antigen polysaccharides and haemagglutinating activity (HA). Strains of S. dysenteriae serotype 1 adhered well to cultured Henle Intestinal 407 cells, whereas rough strains adhered poorly. Pre-treatment of bacteria with LPS-specific antisera inhibited both HA and binding to cultured human-intestinal cells. The contribution of the polysaccharide side-chain and its associated HA--which appear to facilitate binding to cultured cells--to bacterial attachment to colonocytes and to the pathogenesis of shigellosis in vivo needs to be confirmed in animal studies.

The pathogenesis of Yersinia enterocolitica infection in gnotobiotic piglets.

Yersinia enterocolitica is an important cause of enteritis and mesenteric adenitis in many countries. However the pathogenesis of the disease caused by this organism has not been fully elucidated. Most isolates from clinical material possess two independent properties associated with virulence whose relative contribution to the development of disease is not known. These are the ability to penetrate the intestinal wall, which is thought to be controlled by a plasmid gene, and the production of heat-stable enterotoxin, which is controlled by a chromosomal gene. In this study, we infected neonatal gnotobiotic piglets with strains of Y. enterocolitica expressing these two properties in various combinations. The suitability of the piglet model was shown in experiments in which piglets fed virulent Y. enterocolitica serogroup O3 developed a clinical illness related to the size of the inoculum, which was accompanied by intestinal lesions similar to those reported in naturally and experimentally infected people and animals. The results confirmed the key role of a 47 X 10(6)-mol. wt plasmid in the pathogenicity of Y. enterocolitica, but suggested that penetration of the intestinal wall may be governed by chromosomal rather than plasmid-borne genes. No role for enterotoxin in the pathogenesis of yersiniosis was shown, although there was evidence that enterotoxin may promote intra-intestinal proliferation of Y. enterocolitica, thus favouring increased shedding of bacteria and encouraging their spread between hosts.

A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh.

We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea. E. coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence. With 1136 isolates from 387 patients, there was general agreement between the two assay methods. When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay. In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively. Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe-positive isolates gave positive results in the bioassays. Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe. Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results. None of the other probe-positive isolates hybridised with any of the cloning vectors used. The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation.

Intestinal changes associated with rotavirus and enterotoxigenic Escherichia coli infection in calves.

Newborn calves inoculated with rotavirus, enterotoxigenic Escherichia coli (ETEC) serotype 020:K' x 106':K99:HNM, either alone or in combination, became depressed, anorectic, diarrhoeic and dehydrated. ETEC did not adhere to the intestine although there was extensive proliferation in the lumen. Only slight mucosal changes were induced by ETEC and the activity of membrane bound lactase remained normal. More severe mucosal damage and a decrease in lactase activity were found in newborn calves inoculated with either rotavirus or rotavirus and ETEC in combination. The most severe clinical illness was found in calves inoculated with both rotavirus and ETEC. Calves inoculated at 1 week of age with either rotavirus or ETEC remained clinically normal. Rotavirus infection produced slight mucosal changes and a reduction of lactase activity. In contrast, colostrum-fed or suckling calves up to 2 weeks old inoculated with both rotavirus and ETEC became clinically affected, showed severe mucosal damage and decreased lactase activity. There was no bacterial adhesion to the intestinal mucosa as observed by immunofluorescent labelling and light microscopy.

Screening of pig intestines for K88 non-adhesive phenotype by enzyme immunoassay.

An adhesion test for binding of porcine brush border membranes to Escherichia coli cells that possess the K88 antigen (K88+) has been developed using enzyme immunoassay procedures. K88 pilus protein or K88+ E. coli cells were immobilized in the wells of polystyrene microtitre plates. These plates were incubated in the presence of material obtained by scraping the villous surface of pig small intestines. Adhesion of membrane material to immobilized K88 was detected by adding rabbit anti-brush border IgG followed by urease-labelled sheep anti-rabbit IgG conjugate. Action of bound enzyme on urea/bromo-cresol purple substrate solution (pH 4.8) produced an intense colour change from yellow to purple, enabling the test to be read visually. This test enables simple, rapid testing of large numbers of intestial samples and gives results that agree well with the more cumbersome microscopic adhesion test for adhesion of K88+ E. coli to purified brush border membranes.

Attachment of E. coli-bearing K88 antigen to equine brush-border membranes.

Equine small intestinal brush-border membranes, from 40 adult horses were tested in vitro for the presence of receptors for the Escherichia coli adhesive antigens K88ab, K88ac and K99. Only K88-positive strains of E. coli adhered strongly to horse brush-border membranes. In contrast, a K88-negative mutant strain J2, 2 K99-positive strains and 3 E. coli strains isolated from foals failed to adhere to horse brush-border membranes. Purified K88ac pili when reacted with equine brush-border membranes inhibited to a great extent the adhesion of K88-positive E. coli. Similarly, K88-positive E. coli previously reacted with K88 antibody, did not attach to equine brush-border membranes. Oral inoculation of 4 newborn foals with strains of K88-positive enterotoxigenic E. coli, producing either heat-stable or heat-labile enterotoxin, caused diarrhoea in 1 animal.

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.

Enterocolitis in piglets caused by Cryptosporidium sp. purified from calf faeces.

Twelve gnotobiotic piglets were dosed with a bacteria-free calf faecal homogenate which contained Cryptosporidium oocysts. The infection induced severe enterocolitis in piglets when inoculated at 1 day of age, moderate diarrhoea at 7 days of age and a subclinical infection at 15 days of age. In piglets aged 3 days or less, the entire intestine was extensively infected with Cryptosporidium and the mucosa was severely damaged. In piglets 7 days of age or older, the upper small intestine was sparsely infected with the organisms, but the ileum and the large bowel were heavily infected with associated mucosal damage.