Frequent occurrence of mixed Enterocytozoon bieneusi infections in humans.

Enterocytozoon bieneusi (phylum Microsporidia) is a human pathogen with a broad host range. Following the sequencing of 3.8 Mb of the estimated 6-Mb E. bieneusi genome, simple sequence repeats (micro- and minisatellites) were identified. Sequencing of four such repeats from various human and animal E. bieneusi isolates identified extensive sequence polymorphism and enabled the development of a multilocus genotyping method to study the epidemiology of this pathogen. We genotyped E. bieneusi DNA extracted from 197 fecal samples originating from children with diarrhea who were residing in Kampala, Uganda. Three newly identified microsatellite markers and the internal transcribed spacer were PCR amplified, and multiple cloned amplicons for each marker were sequenced from each individual. Most microsatellite sequences were unique to the Ugandan population. Significantly, polymorphism not only was present among isolates but was also found within isolates. This observation suggests that infections with heterogeneous E. bieneusi populations are common in this region. However, the data do not exclude that some of the polymorphism originates from divergent paralogs within the genome. The frequent occurrence of multiple sequences within an isolate precluded the identification of multilocus genotypes. This observation raises the possibility that in a region in which the prevalence of E. bieneusi is high, sequencing of uncloned PCR products may not be adequate for multilocus genotyping.

Cryptosporidium meleagridis: infectivity in healthy adult volunteers.

Most Cryptosporidium infections in humans are caused by C. parvum or C. hominis. However, genotyping techniques have identified infections caused by unusual Cryptosporidium species. Cryptosporidium meleagridis has been identified in ≤ 1% of persons with diarrhea, although prevalence is higher in developing nations. We examined the infectivity of C. meleagridis in healthy adults. Five volunteers were challenged with 10(5) C. meleagridis oocysts and monitored six weeks for fecal oocysts and clinical manifestations. Four volunteers had diarrhea; three had detectable fecal oocysts; and one infected volunteer remained asymptomatic. Fecal DNA from two volunteers was amplified by using a polymerase chain reaction specific for the Cryptosporidium small subunit ribosomal RNA gene. Nucleotide sequence of these amplicons was diagnostic for C. meleagridis. All infections were self-limited; oocysts were cleared within ≤ 12 days of challenge. These studies establish that healthy adults can be infected and become ill from ingestion of C. meleagridis oocysts.

Noninvasive saliva collection techniques for free-ranging mountain gorillas and captive eastern gorillas.

This study was designed to develop a simple, noninvasive method for saliva collection: a first step toward developing new diagnostic tests to survey gorillas for infectious diseases. The subjects included free-ranging mountain gorillas (Gorilla beringei beringei) in the Parc National des Volcans, Rwanda, and a group of orphan mountain and Grauer's gorillas (Gorilla heringei graueri) housed nearby in a temporary holding facility. Three collection methods were used to recover saliva from discarded forest food: swabbing, soaking, and washing. Saliva was also collected from orphan gorillas maintained in a captive setting by using dental ropes inside mesh bags. The presence of gorilla saliva in each sample was confirmed by using a salivary s-amylase assay and forensic press test paper. The recovery of gorilla DNA was verified by polymerase chain reaction by using primers specific to mountain and Grauer's gorillas. Of the three collection techniques used to recover saliva from forest food, directly swabbing plant bite marks was the most effective. Wild celery (Peucedanum linderi) provided for the most consistent saliva recovery and is eaten year round by mountain gorillas in Rwanda. This study shows that gorilla saliva can be recovered easily and noninvasively from known individual free-ranging gorillas by collecting pieces of wild celery discarded as the gorillas forage and from captive gorillas by offering them juice-soaked dental ropes inside mesh bags. Both methods can be used to recover gorilla DNA for genetic studies. Saliva collected from free-ranging and captive gorillas may prove to be a useful biologic sample for the development of new diagnostic tests and hormonal analysis.

The reduced genome of the parasitic microsporidian Enterocytozoon bieneusi lacks genes for core carbon metabolism.

Reduction of various biological processes is a hallmark of the parasitic lifestyle. Generally, the more intimate the association between parasites and hosts the stronger the parasite relies on its host's physiology for survival and reproduction. However, some systems have been held to be indispensable, for example, the core pathways of carbon metabolism that produce energy from sugars. Even the most hardened anaerobes that lack oxidative phosphorylation and the tricarboxylic acid cycle have retained glycolysis and some downstream means to generate ATP. Here we describe the deep-coverage genome resequencing of the pathogenic microsporidiian, Enterocytozoon bieneusi, which shows that this parasite has crossed this line and abandoned complete pathways for the most basic carbon metabolism. Comparing two genome sequence surveys of E. bieneusi to genomic data from four other microsporidia reveals a normal complement of 353 genes representing 30 functional pathways in E. bieneusi, except that only 2 out of 21 genes collectively involved in glycolysis, pentose phosphate, and trehalose metabolism are present. Similarly, no genes encoding proteins involved in the processing of spliceosomal introns were found. Altogether, E. bieneusi appears to have no fully functional pathway to generate ATP from glucose. Therefore, this intracellular parasite relies on transporters to import ATP from its host.

Evaluation of Fab and F(ab')2 fragments and isotype variants of a recombinant human monoclonal antibody against Shiga toxin 2.

5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin's ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 microg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab')(2)] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab')(2) fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.

Host-specific segregation of ribosomal nucleotide sequence diversity in the microsporidian Enterocytozoon bieneusi.

Enterocytozoon bieneusi is a unicellular enteric fungal pathogen and the most common cause of human microsporidiosis. The frequent detection of this organism in animals, including companion animals, livestock and wildlife, has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen. A partial sequence of the ribosomal internal transcribed spacer (ITS) has been widely used as a genetic marker for studying the molecular epidemiology of E. bieneusi. With the aim of comparing E. bieneusi ITS genotypes originating from different host species, and assess the potential for zoonotic transmission, E. bieneusi ITS sequences retrieved from GenBank were analyzed using two metrics of diversity, rarefaction and phylogenetic distance. In spite of the human ITS sample being geographically more diverse, ITS sequence diversity in animals exceeded that of humans. In both host groups much of the ITS diversity remains to be sampled. Using quantitative phylogenetic tests we found evidence for a partial but significant segregation of E. bieneusi ITS sequences according to host species. Host-specific segregation was confirmed by hierarchical analysis of molecular variation. To improve our understanding of the epidemiology of human microsporidiosis and strengthen the study of E. bieneusi populations, efforts to genotype additional E. bieneusi isolates from wildlife and companion animals should be prioritized and the geographic and species diversify of animal samples should be increased. Due to the possibility of genetic recombination in this species, additional unlinked genetic markers need to be developed and included in future studies.

OpuC--an ABC transporter that is associated with Staphylococcus aureus pathogenesis.

RIP is a novel antibiotic against staphylococci. It acts at least in part by competing with RNAIII activating protein (RAP) by downregulating TRAP histidine phosphorylation, and by downregulating the expression of the acessory gene regulator (agr). While much is known about the function of the agr as a quorum sensing system that regulates virulence, not much is known about TRAP. TRAP is a 167-kDa protein that is highly conserved among staphylococci and is involved in DNA protection from stress. TRAP is membrane-associated but does not have a transmembrane domain, and thus it may be bound to the membrane through other proteins. To search for these proteins, protein-protein interaction studies were carried out using a bacterial two-hybrid system, and OpuCA was discovered as a TRAP-binding protein. OpuCA is an ATP binding-cytoplasmic (ABC) domain of an OpuC ABC transporter. S. aureus OpuC- mutant strain was constructed and shown to be less tolerant to salt stress, and was defective in choline uptake. OpuC- cells were less pathogenic and showed reduced TRAP phosphorylation and agr activity, did not respond to RAP, and were defective in biofilm formation in vitro and in vivo. These results suggest that OpuC acts as a transporter and also plays a role in S. aureus pathogenesis.

Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470.

Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin and its analogues have been demonstrated to have activity invitro and in animal models of microsporidiosis and human infections due to E. bieneusi. Fumagillin and its analogues inhibit methionine aminopeptidase type 2. Encephalitozoon cuniculi MetAP2 (EcMetAP2) was cloned and expressed as an active enzyme using a baculovirus system. The crystal structure of EcMetAP2 was determined with and without the bound inhibitors fumagillin and TNP-470. This structure classifies EcMetAP2 as a member of the MetAP2c family. The EcMetAP2 structure was used to generate a homology model of the E. bieneusi MetAP2. Comparison of microsporidian MetAP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporidian MetAP2.

Genomic survey of the non-cultivatable opportunistic human pathogen, Enterocytozoon bieneusi.

Enterocytozoon bieneusi is the most common microsporidian associated with human disease, particularly in the immunocompromised population. In the setting of HIV infection, it is associated with diarrhea and wasting syndrome. Like all microsporidia, E. bieneusi is an obligate, intracellular parasite, but unlike others, it is in direct contact with the host cell cytoplasm. Studies of E. bieneusi have been greatly limited due to the absence of genomic data and lack of a robust cultivation system. Here, we present the first large-scale genomic dataset for E. bieneusi. Approximately 3.86 Mb of unique sequence was generated by paired end Sanger sequencing, representing about 64% of the estimated 6 Mb genome. A total of 3,804 genes were identified in E. bieneusi, of which 1,702 encode proteins with assigned functions. Of these, 653 are homologs of Encephalitozoon cuniculi proteins. Only one E. bieneusi protein with assigned function had no E. cuniculi homolog. The shared proteins were, in general, evenly distributed among the functional categories, with the exception of a dearth of genes encoding proteins associated with pathways for fatty acid and core carbon metabolism. Short intergenic regions, high gene density, and shortened protein-coding sequences were observed in the E. bieneusi genome, all traits consistent with genomic compaction. Our findings suggest that E. bieneusi is a likely model for extreme genome reduction and host dependence.

Inferences about the global population structures of Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium parvum and Cryptosporidium hominis are two related species of apicomplexan protozoa responsible for the majority of human cases of cryptosporidiosis. In spite of their considerable public health impact, little is known about the population structures of these species. In this study, a battery of C. parvum and C. hominis isolates from seven countries was genotyped using a nine-locus DNA subtyping scheme. To assess the existence of geographical partitions, the multilocus genotype data were mined using a cluster analysis based on the nearest-neighbor principle. Within each country, the population genetic structures were explored by combining diversity statistical tests, linkage disequilibrium, and eBURST analysis. For both parasite species, a quasi-complete phylogenetic segregation was observed among the countries. Cluster analysis accurately identified recently introduced isolates. Rather than conforming to a strict paradigm of either a clonal or a panmictic population structure, data are consistent with a flexible reproductive strategy characterized by the cooccurrence of both propagation patterns. The relative contribution of each pattern appears to vary between the regions, perhaps dependent on the prevailing ecological determinants of transmission.

Patterns of genome evolution among the microsporidian parasites Encephalitozoon cuniculi, Antonospora locustae and Enterocytozoon bieneusi.

BACKGROUND: Microsporidia are intracellular parasites that are highly-derived relatives of fungi. They have compacted genomes and, despite a high rate of sequence evolution, distantly related species can share high levels of gene order conservation. To date, only two species have been analysed in detail, and data from one of these largely consists of short genomic fragments. It is therefore difficult to determine how conservation has been maintained through microsporidian evolution, and impossible to identify whether certain regions are more prone to genomic stasis. PRINCIPAL FINDINGS: Here, we analyse three large fragments of the Enterocytozoon bieneusi genome (in total 429 kbp), a species of medical significance. A total of 296 ORFs were identified, annotated and their context compared with Encephalitozoon cuniculi and Antonospora locustae. Overall, a high degree of conservation was found between all three species, and interestingly the level of conservation was similar in all three pairwise comparisons, despite the fact that A. locustae is more distantly related to E. cuniculi and E. bieneusi than either are to each other. CONCLUSIONS/SIGNIFICANCE: Any two genes that are found together in any pair of genomes are more likely to be conserved in the third genome as well, suggesting that a core of genes tends to be conserved across the entire group. The mechanisms of rearrangments identified among microsporidian genomes were consistent with a very slow evolution of their architecture, as opposed to the very rapid sequence evolution reported for these parasites.

Characterization of subtilase protease in Cryptosporidium parvum and C. hominis.

Cryptosporidium spp., enteropathogens of humans and other animals, are members of the Apicomplexa. In parasites belonging to this phylum, proteases have been shown to play a key role in the invasion of host cells, organelle biogenesis, and intracellular survival. The subtilases constitute a family of serine proteases present in prokaryotes, eukaryotes, and viruses. The C. parvum subtilase gene, CpSUB1, encodes a transcript of 3972 base pairs (bp) and 1324 amino acids. Using homologous polymerase chain reaction primers, a similar gene, ChSUB1, which has 98% (4007 bp/4050 bp) identity to CpSUB1, was found in C. hominis. The alignment of the CpSUB1 and ChSUB1 nucleotide sequences identified primarily silent substitutions, consistent with the absence of diversifying selection. The catalytic domain of CpSUB1 is very similar to that of other Apicomplexa (> 38% amino acid identity and >57% similarity) and to the bacterial subtilisin BPN from B. subtilis (36 and 47%). Transcriptional upregulation during merozoite development was observed in cell culture, and a predicted 76-bp intron located near the 3' end of the open reading frame was confirmed experimentally. Cryptosporidium parvum infection in cell culture was significantly inhibited by subtilisin inhibitor III and other serine protease inhibitors, emphasizing the importance of the parasite's subtilase for intracellular development and the enzyme's potential as a drug target.

Analysis of the beta-tubulin genes from Enterocytozoon bieneusi isolates from a human and rhesus macaque.

Enterocytozoon bieneusi is the most common and clinically significant microsporidium associated with chronic diarrhea and wasting in immunocompromised humans. Albendazole, which is effective against several helminths, protozoa, and microsporidia, is relatively ineffective against infections due to E. bieneusi. A likely explanation for the observed clinical resistance to albendazole was discovered from sequence analysis of the E. bieneusibeta-tubulin from isolates from an infected human and a naturally infected rhesus macaque. The beta-tubulin of E. bieneusi has a substitution at Glu(198), which is one of six amino acids reported to be associated with benzimidazole sensitivity.

Subtype analysis of Cryptosporidium isolates from children in Uganda.

Cryptosporidium hominis and Cryptosporidium parvum isolates from children in Uganda were characterized by DNA sequence analysis of the GP60 gene. Eight alleles were identified, 4 C. hominis and 4 C. parvum, of which 3 represent new C. parvum families. The data show that it is highly likely that the route of transmission is anthroponotic.

Cryptosporidium hominis: experimental challenge of healthy adults.

Cryptosporidium hominis causes diarrhea in humans and has been associated with community outbreaks. This study describes the infectivity, illness, and serologic response after experimental challenge of 21 healthy adult volunteers with 10-500 C. hominis (TU502) oocysts. Sixteen subjects (76.2%) had evidence of infection; the 50% infectious dose (ID(50)) was estimated to be 10-83 oocysts using clinical and microbiologic definitions of infection, respectively. Diarrhea occurred in 40% of subjects receiving 10 oocysts with a stepwise increase to 75% in those receiving 500 oocysts. A serum IgG response was seen in those receiving more than 30 oocysts. Greatest responses were seen in volunteers with diarrhea and oocyst shedding. Volunteers with no evidence of infection had indeterminant or negative IgG responses. Cryptosporidium hominis 10 oocysts) and is clinically is infectious for healthy adults (ID(50) = similar to C. parvum-induced illness. In contrast to C. parvum, C. hominis elicted a serum IgG response in most infected persons.

Serial propagation of the microsporidian Enterocytozoon bieneusi of human origin in immunocompromised rodents.

Enterocytozoon bieneusi, a microsporidian, is clinically one of the most significant opportunistic causes of diarrhea and wasting associated with profound human immunodeficiencies. The lack of an animal model for E. bieneusi hinders serious investigations and limits the availability of spores to individuals with severe human immunodeficiency virus/AIDS disease who are infected with E. bieneusi. The development of procedures for purification and concentration of spores from stools of infected humans has led to the production of immune reagents and provided a source of spores to conduct research, including attempts to develop and serially propagate E. bieneusi in rodent models. We have evaluated and successfully infected six different immunodeficient and/or immunosuppressed rodent models and have demonstrated persistent infections lasting at least 18 weeks in SCID mice and in nude rats. To enhance the intensity and duration of infection in these two models, animals were given anti-gamma interferon monoclonal antibody injections at regular intervals. Of the six models evaluated, nude rats and gerbils immunosuppressed with dexamethasone excreted the highest number of spores and for longer time periods. Four different E. bieneusi isolates were equally infectious, and one of them was serially propagated in nude rats six times over a period of 10 months. Typically, rats challenged orally with 10(4) spores yielded 2 x 10(7) to 6.3 x 10(7) spores per single fecal sample when the level of spores was measured 2 weeks later. Rodent models and a nonhuman source of fresh spores will considerably enhance future investigations on this important opportunistic pathogen, including the screening and evaluation of urgently needed chemotherapeutic agents.

Failure to propagate Cryptosporidium spp. in cell-free culture.

The successful propagation of Cryptosporidium parvum in cell-free culture medium was recently reported. To investigate whether this phenomenon could be broadened to include other C. parvum isolates, as well as Cryptosporidium hominis, we attempted to propagate 3 isolates in cell-free medium under reported culture conditions. Cryptosporidium oocysts from C. parvum strains Moredun (MD) or IOWA or C. hominis strain TU502 were added to media containing coagulated newborn calf serum. The cultures were sampled at various times throughout a 45 (IOWA) or 78 (MD, TU502)-day period and were microscopically examined for various life stages of Cryptosporidium. Cell-free cultures harvested on days 45 and 68 postinoculation were tested for in vitro infectivity on Madrin-Darby bovine kidney cells. In vivo infectivity testing was performed using either infant or 2-wk-old immunosuppressed C57BL mice with cell-free cultures harvested on days 52 and 78. Fecal and gut samples collected from mice were examined by modified acid-fast staining. Data from wet mounts, electron microscopy, and in vitro and in vivo infectivity testing showed that the original oocysts did not complete their life cycle and produce new, viable, infectious oocysts in cell-free culture. Thus, we conclude that this is not a universal phenomenon or readily accomplished.

Cryptosporidiosis and microsporidiosis in ugandan children with persistent diarrhea with and without concurrent infection with the human immunodeficiency virus.

Cryptosporidium spp. and Enterocytozoon bieneusi are enteric pathogens that have emerged as significant causes of persistent diarrhea (PD) in immunologically compromised individuals particularly in association with HIV/AIDS. We conducted a cross-sectional study on the clinical epidemiology of E. bieneusi and Cryptosporidium in children with PD, with and without HIV/AIDS, attending Uganda's Mulago National Referral Hospital. Two hundred forty-three children aged < 60 months, admitted between November 2002 and May 2003 with PD (> 14 days), were analyzed for HIV status and CD4 lymphocyte counts, and stools were screened for the presence of E. bieneusi and Cryptosporidium by microscopy and positive samples genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Eighty (32.9%) of the children were excreting E. bieneusi, and 76 (31.3%) were excreting Cryptosporidium. Ninety-one of the 243 children had HIV, of who 70 (76.9%) had E. bieneusi, versus 10 (6.6%) of the 152 without (odds ratio = 47.33; 95% CI = 19.88 to 115.97), while 67 (73.6%) had Cryptosporidium, versus 9 (5.9%) without (odds ratio = 44.36; 95% CI = 18.39 to 110.40). Children with counts < 25% CD4 cells were more likely to have either E. bieneusi (odds ratio = 7.42; 95% CI = 3.77 to 14.69) or Cryptosporidium (odds ratio = 6.45; 95% CI = 3.28 to 12.76) than those with higher CD4 percentages. However, only HIV status was independently associated with either Cryptosporidium or E. bieneusi. Among the 243 children with PD, 67 (27.8%) were infected with both enteric pathogens, with HIV being the only independent predictor of coinfection. Finally, some 81% of HIV-infected children with PD excreted one or both organisms, compared with only 10% of children with PD testing negative for HIV. Seventy-four percent of isolates were C. hominis, the anthroponotic species, 17% were C. parvum, the zoonotic species, and 8% were a mixture of the two or others.

Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves.

Genotypic analyses of Cryptosporidium parvum oocysts have divided the species into two genotypes, referred to as type 1 and type 2. Although humans are susceptible to both types, mixed type 1/type 2 infections have rarely been identified. The paucity of mixed infections could be explained by the predominance of one type over the other in mixed infections, or by the poor sensitivity of restriction fragment length polymorphism (RFLP) analyses for detecting subpopulations. Using a type-specific real-time PCR assay capable of detecting type 1 or type 2 constituting as little as 0.01% of the population, archived and new isolates of human, bovine, and mouse origin were genotyped. Mixed type 1/type 2 infections were identified in humans and calves, including in samples previously found to be homogeneous by RFLP. Isopycnic fractionation of mixed isolates revealed that type 1 and type 2 oocysts differ in their sedimentation properties. The detection of a type 1 subpopulation in serially-propagated bovine isolates indicates that type 1 and type 2 are stably maintained during long-term passage. Together with recently reported experimental bovine and ovine type 1 infections, the persistence of type 1 subpopulation in experimentally infected animals suggests that animals may play a previously unrecognized role in the maintenance of C. parvum type 1.

Rapid displacement of Cryptosporidium parvum type 1 by type 2 in mixed infections in piglets.

Genotypes 1 and 2 of Cryptosporidium parvum are the primary types associated with infections in humans, with type 1 being by far the predominant genotype. The frequency of mixed infection with both genotypes in humans is relatively rare, while type 1, which experimentally infects other mammals, has been found to naturally infect almost exclusively humans. One possible explanation for the absence of type 1 in other mammals and the low frequency of mixed infections in humans is the inability of type 1 to compete with type 2 in nature when both occur simultaneously. To investigate this, we challenged gnotobiotic piglets with equal number of oocysts of type 1 and type 2, given either simultaneously or with type 2 given 24 or 48 h after type 1. The genotype of the oocysts excreted in feces and the relative distribution of each of the genotypes throughout the gut at necropsy were determined. Regardless of the time interval between challenges with the two genotypes, type 2 invariably displaced type 1. The rate of displacement was rapid when both genotypes were given simultaneously, after which no traces of type 1 were detected in the feces or in gut sections by PCR. Infection with type 1 24 or 48 h before challenge with type 2, while permitting type 1 to become established, was still rapidly eliminated within 3 days after challenge with type 2. These observations have major implications regarding the relative perpetuation and survival of these two genotypes in mammals.