Tartrolon E, a secondary metabolite of a marine symbiotic bacterium, is a potent inhibitor of asexual and sexual Plasmodium falciparum.

Due to the spread of resistance to front-line artemisinin derivatives worldwide, there is a need for new antimalarials. Tartrolon E (TrtE), a secondary metabolite of a symbiotic bacterium of marine bivalve mollusks, is a promising antimalarial because it inhibits the growth of sexual and asexual blood stages of Plasmodium falciparum at sub-nanomolar levels. The potency of TrtE warrants further investigation into its mechanism of action, cytotoxicity, and ease with which parasites may evolve resistance to it.

The Marine Compound Tartrolon E Targets the Asexual and Early Sexual Stages of Cryptosporidium parvum.

New therapeutic agents for cryptosporidiosis are a critical medical need. The marine organic compound, tartrolon E (trtE), is highly effective against multiple apicomplexan parasites, including Cryptosporidium. Understanding the mechanism of action of trtE is required to advance in the drug development pipeline. Here, we validate using Nluc C. parvum parasites for the study of trtE and pinpoint the life stage targeted by trtE. Results show that trtE kills Nluc and wild type C. parvum with equal efficiency, confirming the use of the Nluc C. parvum to study this compound. Results revealed that trtE kills the parasite within an hour of treatment and while the compound has no effect on viability of sporozoites, trtE does inhibit establishment of infection. Targeting treatment at particular life cycle stages demonstrated that trtE is effective against asexual of the parasite but has reduced efficacy against mature sexual stages. Gene expression analysis shows that trtE inhibits the early sexual stage of the parasite. Results from these studies will aid the development of trtE as a therapeutic for cryptosporidiosis.

High-Throughput Screening of a Marine Compound Library Identifies Anti-Cryptosporidium Activity of Leiodolide A.

Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds.

Dual Transcriptomics To Determine Gamma Interferon-Independent Host Response to Intestinal Cryptosporidium parvum Infection.

Animals with a chronic infection of the parasite Toxoplasma gondii are protected against lethal secondary infection with other pathogens. Our group previously determined that soluble T. gondii antigens (STAg) can mimic this protection and be used as a treatment against several lethal pathogens. Because treatments are limited for the parasite Cryptosporidium parvum, we tested STAg as a C. parvum therapeutic. We determined that STAg treatment reduced C. parvum Iowa II oocyst shedding in gamma interferon knockout (IFN-γ-KO) mice. Murine intestinal sections were then sequenced to define the IFN-γ-independent transcriptomic response to C. parvum infection. Gene Ontology and transcript abundance comparisons showed host immune response and metabolism changes. Transcripts for type I interferon-responsive genes were more abundant in C. parvum-infected mice treated with STAg. Comparisons between phosphate-buffered saline (PBS) and STAg treatments showed no significant differences in C. parvum gene expression. C. parvum transcript abundance was highest in the ileum and mucin-like glycoproteins and the GDP-fucose transporter were among the most abundant. These results will assist the field in determining both host- and parasite-directed future therapeutic targets.

Boromycin Has Potent Anti-Toxoplasma and Anti-Cryptosporidium Activity.

Toxoplasma gondii and Cryptosporidium parvum, members of the phylum Apicomplexa, are significant pathogens of both humans and animals worldwide for which new and effective therapeutics are needed. Here, we describe the activity of the antibiotic boromycin against Toxoplasma and Cryptosporidium Boromycin potently inhibited intracellular proliferation of both T. gondii and C. parvum at half-maximal effective concentrations (EC50) of 2.27 nM and 4.99 nM, respectively. Treatment of extracellular T. gondii tachyzoites with 25 nM boromycin for 30 min suppressed 84% of parasite growth, but T. gondii tachyzoite invasion into host cells was not affected by boromycin. Immunofluorescence of boromycin-treated T. gondii showed loss of morphologically intact parasites with randomly distributed surface antigens inside the parasitophorous vacuoles. Boromycin exhibited a high selectivity for the parasites over their host cells. These results suggest that boromycin is a promising new drug candidate for treating toxoplasmosis and cryptosporidiosis.

Cryptosporidium: host and parasite transcriptome in infection.

Cryptosporidium is a waterborne gastrointestinal parasite that causes outbreaks of diarrheal disease worldwide. Despite the impact of this parasite on human health there are no effective drugs or vaccines. Transcriptomic data can provide insights into host-parasite interactions that lead to identification of targets for therapeutic interventions. However, for Cryptosporidium, interpreting transcriptomes has been challenging, in part due to the presence of multiple life cycle stages, the lack of appropriate host cells and the inability to culture the parasite through its complete life cycle. The recent improvements in cell culture and the ability to tag and isolate specific life cycle stages will radically improve transcriptomic data and advance our understanding of Cryptosporidium host-parasite interactions.

A conserved coccidian gene is involved in Toxoplasma sensitivity to the anti-apicomplexan compound, tartrolon E.

New treatments for the diseases caused by apicomplexans are needed. Recently, we determined that tartrolon E (trtE), a secondary metabolite derived from a shipworm symbiotic bacterium, has broad-spectrum anti-apicomplexan parasite activity. TrtE inhibits apicomplexans at nM concentrations in vitro, including Cryptosporidium parvum, Toxoplasma gondii, Sarcocystis neurona, Plasmodium falciparum, Babesia spp. and Theileria equi. To investigate the mechanism of action of trtE against apicomplexan parasites, we examined changes in the transcriptome of trtE-treated T. gondii parasites. RNA-Seq data revealed that the gene, TGGT1_272370, which is broadly conserved in the coccidia, is significantly upregulated within 4 h of treatment. Using bioinformatics and proteome data available on ToxoDB, we determined that the protein product of this tartrolon E responsive gene (trg) has multiple transmembrane domains, a phosphorylation site, and localizes to the plasma membrane. Deletion of trg in a luciferase-expressing T. gondii strain by CRISPR/Cas9 resulted in a 68% increase in parasite resistance to trtE treatment, supporting a role for the trg protein product in the response of T. gondii to trtE treatment. Trg is conserved in the coccidia, but not in more distantly related apicomplexans, indicating that this response to trtE may be unique to the coccidians, and other mechanisms may be operating in other trtE-sensitive apicomplexans. Uncovering the mechanisms by which trtE inhibits apicomplexans may identify shared pathways critical to apicomplexan parasite survival and advance the search for new treatments.

A symbiotic bacterium of shipworms produces a compound with broad spectrum anti-apicomplexan activity.

Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics.

Production and Purification of Functional Cryptosporidium Glycoproteins by Heterologous Expression in Toxoplasma gondii.

Development of an effective vaccine against cryptosporidiosis is a medical and veterinary priority. However, many putative Cryptosporidium vaccine candidates such as surface and apical complex antigens are posttranslationally modified with O- and N-linked glycans. This presents a significant challenge to understanding the functions of these antigens and the immune responses to them. Isolation of large amounts of native antigen from Cryptosporidium oocysts is expensive and is only feasible for C. parvum antigens. Here, we describe a method of producing recombinant, functional Cryptosporidium glycoprotein antigens in Toxoplasma gondii. These functional recombinant proteins can be used to investigate the role of glycotopes in Cryptosporidium immune responses and parasite-host cell interactions.

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection.

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an in vitro culture system that recapitulates the conditions in the host is needed. Organoids, which closely resemble the tissues of their origin, are ideal for studying host-parasite interactions. Organoids are three-dimensional (3D) tissue-derived structures which are derived from adult stem cells and grow in culture for extended periods of time without undergoing any genetic aberration or transformation. They have well defined polarity with both apical and basolateral surfaces. Organoids have various applications in drug testing, bio banking, and disease modeling and host-microbe interaction studies. Here we present a step-by-step protocol of how to prepare the oocysts and sporozoites of Cryptosporidium for infecting human intestinal and airway organoids. We then demonstrate how microinjection can be used to inject the microbes into the organoid lumen. There are three major methods by which organoids can be used for host-microbe interaction studies-microinjection, mechanical shearing and plating, and by making monolayers. Microinjection enables maintenance of the 3D structure and allows for precise control of parasite volumes and direct apical side contact for the microbes. We provide details for optimal growth of organoids for either imaging or oocyst production. Finally, we also demonstrate how the newly generated oocysts can be isolated from the organoid for further downstream processing and analysis.

Up-regulated expression of spherical body protein 2 truncated copy 11 in Babesia bovis is associated with reduced cytoadhesion to vascular endothelial cells.

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.

Identification of a putative methyltransferase gene of Babesia bigemina as a novel molecular biomarker uniquely expressed in parasite tick stages.

BACKGROUND: Bovine babesiosis is caused by apicomplexan pathogens of the genus Babesia such as B. bigemina and B. bovis. These tick-borne pathogens have a complex life-cycle involving asexual multiplication in vertebrate hosts and sexual reproduction in invertebrate vectors. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. Understanding the mechanisms that underlie formation of extracellular Babesia tick stages is an important step towards developing control strategies for preventing tick infection and subsequent parasite transmission. RESULTS: We induced B. bigemina sexual stages in vitro by exposing parasites to Tris 2-carboxyethyl phosphine (TCEP). Subsequently, we identified a novel putative methyltransferase gene (BBBOND_0204030) that is expressed uniquely in all B. bigemina tick stages but not in blood stages. In vitro TCEP-exposed B. bigemina presented diverse morphology including parasites with long projections, round forms and clusters of round forms indicative of sexual stage induction. We confirmed the development of sexual stages by detecting upregulation of previously defined B. bigemina sexual stage marker genes, ccp2 and 3, and their respective protein expression in TCEP-induced B. bigemina cultures. Next, transcription analysis of in vitro TCEP-induced B. bigemina culture based on an in silico derived list of homologs of Plasmodium falciparum gamete-specific genes demonstrated differential expression of the gene BBBOND_0204030 in induced cells. Further examination of ex vivo infected ticks demonstrated that BBBOND_0204030 is transcribed by multiple stages of B. bigemina during parasite development in tick midgut, ovary and hemolymph. Interestingly, ex vivo results confirmed our in vitro observation that blood stages of B. bigemina do not express BBBOND_0204030 and validated the in vitro system of inducing sexual stages. CONCLUSIONS: Herein we describe the identification of a B. bigemina gene transcribed exclusively by parasites infecting ticks using a novel method of inducing B. bigemina sexual stages in vitro. We propose that this gene can be used as a marker for parasite development within the tick vector. Together, these tools will facilitate our understanding of parasite-tick interactions, the identification of novel vaccine targets and, consequently, the development of additional strategies to control bovine babesiosis.

Modelling Cryptosporidium infection in human small intestinal and lung organoids.

Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.

High-throughput screen of drug repurposing library identifies inhibitors of Sarcocystis neurona growth.

The apicomplexan parasite Sarcocystis neurona is the primary etiologic agent of equine protozoal myeloencephalitis (EPM), a serious neurologic disease of horses. Many horses in the U.S. are at risk of developing EPM; approximately 50% of all horses in the U.S. have been exposed to S. neurona and treatments for EPM are 60-70% effective. Advancement of treatment requires new technology to identify new drugs for EPM. To address this critical need, we developed, validated, and implemented a high-throughput screen to test 725 FDA-approved compounds from the NIH clinical collections library for anti-S. neurona activity. Our screen identified 18 compounds with confirmed inhibitory activity against S. neurona growth, including compounds active in the nM concentration range. Many identified inhibitory compounds have well-defined mechanisms of action, making them useful tools to study parasite biology in addition to being potential therapeutic agents. In comparing the activity of inhibitory compounds identified by our screen to that of other screens against other apicomplexan parasites, we found that most compounds (15/18; 83%) have activity against one or more related apicomplexans. Interestingly, nearly half (44%; 8/18) of the inhibitory compounds have reported activity against dopamine receptors. We also found that dantrolene, a compound already formulated for horses with a peak plasma concentration of 37.8 ±â€¯12.8 ng/ml after 500 mg dose, inhibits S. neurona parasites at low concentrations (0.065 µM [0.036-0.12; 95% CI] or 21.9 ng/ml [12.1-40.3; 95% CI]). These studies demonstrate the use of a new tool for discovering new chemotherapeutic agents for EPM and potentially providing new reagents to elucidate biologic pathways required for successful S. neurona infection.

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk.

Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.

Cryptosporidiosis in HIV/AIDS patients in Kenya: clinical features, epidemiology, molecular characterization and antibody responses.

We investigated the epidemiological and clinical features of cryptosporidiosis, the molecular characteristics of infecting species and serum antibody responses to three Cryptosporidium-specific antigens in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients in Kenya. Cryptosporidium was the most prevalent enteric pathogen and was identified in 56 of 164 (34%) of HIV/AIDS patients, including 25 of 70 (36%) with diarrhea and 31 of 94 (33%) without diarrhea. Diarrhea in patients exclusively infected with Cryptosporidium was significantly associated with the number of children per household, contact with animals, and water treatment. Cryptosporidium hominis was the most prevalent species and the most prevalent subtype family was Ib. Patients without diarrhea had significantly higher serum IgG levels to Chgp15, Chgp40 and Cp23, and higher fecal IgA levels to Chgp15 and Chgp40 than those with diarrhea suggesting that antibody responses to these antigens may be associated with protection from diarrhea and supporting further investigation of these antigens as vaccine candidates.

Biochemical and functional characterization of CpMuc4, a Cryptosporidium surface antigen that binds to host epithelial cells.

Cryptosporidium spp. are intracellular apicomplexan parasites that cause outbreaks of waterborne diarrheal disease worldwide. Previous studies had identified a Cryptosporidium parvum sporozoite antigen, CpMuc4, that appeared to be involved in attachment and invasion of the parasite into intestinal epithelial cells. CpMuc4 is predicted to be O- and N-glycosylated and the antigen exhibits an apparent molecular weight 10kDa larger than the antigen expressed in Escherichia coli, indicative of post-translational modifications. However, lectin blotting and enzymatic and chemical deglycosylation did not identify any glycans on the native antigen. Expression of CpMuc4 in Toxoplasma gondii produced a recombinant protein of a similar molecular weight to the native antigen. Both purified native CpMuc4 and T. gondii recombinant CpMuc4, but not CpMuc4 expressed in E. coli, bind to fixed Caco-2A cells in a dose dependent and saturable manner, suggesting that this antigen bears epitopes that bind to a host cell receptor, and that the T. gondii recombinant CpMuc4 functionally mimics the native antigen. Binding of native CpMuc4 to Caco2A cells could not be inhibited with excess CpMuc4 peptide, or an excess of E. coli recombinant CpMuc4. These data suggest that CpMuc4 interacts directly with a host cell receptor and that post-translational modifications are necessary for the antigen to bind to the host cell receptor. T. gondii recombinant CpMuc4 may mimic the native antigen well enough to serve as a useful tool for identifying the host cell receptor and determining the role of native CpMuc4 in host cell invasion.

Infectious diarrhoea in antiretroviral therapy-naive HIV/AIDS patients in Kenya.

BACKGROUND: Diarrhoea is a significant cause of morbidity and mortality in immunocompromised patients. The objectives of this study were to investigate the aetiological agents, risk factors and clinical features associated with diarrhoea in HIV/AIDS patients in Kenya. METHODS: Sociodemographic, epidemiological and clinical data were obtained for 164 HIV/AIDS patients (70 with and 94 without diarrhoea) recruited from Kenyatta National Hospital, Kenya. Stool samples were examined for enteric pathogens by microscopy and bacteriology. RESULTS: Intestinal protozoa and fungi were identified in 70% of patients, more frequently in those with diarrhoea (p<0.001). Helminths were detected in 25.6% of patients overall, and bacterial pathogens were identified in 51% of patients with diarrhoea. Polyparasitism was more common in patients with diarrhoea than those without (p<0.0001). Higher CD4(+) T-cell count (OR = 0.995, 95% CI 0.992-0.998) and water treatment (OR = 0.231, 95% CI 0.126-0.830) were associated with a lower risk of diarrhoea, while close contact with cows (OR = 3.200, 95% CI 1.26-8.13) or pigs (OR = 11.176, 95% CI 3.76-43.56) were associated with a higher risk of diarrhoea. CONCLUSIONS: Multiple enteric pathogens that are causative agents of diarrhoea were isolated from stools of antiretroviral therapy-naïve HIV/AIDS patients, indicating a need for surveillance, treatment and promotion of hygienic practices.

Microbial distribution and abundance in the digestive system of five shipworm species (Bivalvia: Teredinidae).

Marine bivalves of the family Teredinidae (shipworms) are voracious consumers of wood in marine environments. In several shipworm species, dense communities of intracellular bacterial endosymbionts have been observed within specialized cells (bacteriocytes) of the gills (ctenidia). These bacteria are proposed to contribute to digestion of wood by the host. While the microbes of shipworm gills have been studied extensively in several species, the abundance and distribution of microbes in the digestive system have not been adequately addressed. Here we use Fluorescence In-Situ Hybridization (FISH) and laser scanning confocal microscopy with 16S rRNA directed oligonucleotide probes targeting all domains, domains Bacteria and Archaea, and other taxonomic groups to examine the digestive microbiota of 17 specimens from 5 shipworm species (Bankia setacea, Lyrodus pedicellatus, Lyrodus massa, Lyrodus sp. and Teredo aff. triangularis). These data reveal that the caecum, a large sac-like appendage of the stomach that typically contains large quantities of wood particles and is considered the primary site of wood digestion, harbors only very sparse microbial populations. However, a significant number of bacterial cells were observed in fecal pellets within the intestines. These results suggest that due to low abundance, bacteria in the caecum may contribute little to lignocellulose degradation. In contrast, the comparatively high population density of bacteria in the intestine suggests a possible role for intestinal bacteria in the degradation of lignocellulose.

Serum antibody responses to polymorphic Cryptosporidium mucin antigen in Bangladeshi children with cryptosporidiosis.

Cryptosporidium is a significant cause of diarrheal disease in children in developing countries. The sporozoite antigen Muc4 is important for infection of host cells, and could be a candidate vaccine antigen. However, this antigen is polymorphic between Cryptosporidium hominis and C. parvum. We investigated antibody responses to C. hominis Muc4 and C. parvum Muc4 antigen in children in Bangladesh infected with C. hominis. Antibody responses were compared between children with cryptosporidial diarrhea (cases) and uninfected children with diarrhea (controls). There was a significant IgM response to Muc4 from both species in cases compared with controls, which increased over time, and was higher in children with persistent diarrhea. Despite sequence polymorphisms, antibody responses to C. hominis Muc4 and C. parvum Muc4 were significantly correlated. These results suggest that the human antibody response to Muc4 is cross-reactive between species, but in young children does not mature to an IgG response within the period observed in this study.