Cryptosporidium Research 2001: Workshop Summary

The proceedings of the Cryptosporidium papers presented at the International Workshops on Opportunistic Infections in Cincinnati, June 13–16, 2001 are briefly summarized below. The current direction of Cryptosporidium research was represented by a total of 25 papers focusing on chemotherapy, host/parasite interactions, epidemiology, biochemistry, immunology, genotyping, and molecular biology. This overview represents most of the papers that were presented in the oral and poster presentations during the meeting.

CHEMOTHERAPY

Papers were presented on therapeutics that demonstrated promising anticryptosporidial activities. Studies were reported by Yirlett et al. on the potential of polyamine analogs as anticryptosporidial agents. It was found that conformationally unrestricted bis-ethyl analogs were competitive inhibitors of spermine:spermidine N-acetyltransferase (SSAT), an enzyme involved in the polyamine biosynthetic pathway. Restricted analogs were found to be effective in an ap T cell knockout mouse model when mice were treated for 5–6 days. Riggs et al. reported on the immunotherapeutic efficacy of the monoclonal antibody (3E2). This monoclonal antibody was generated against the apical complex glycoprotein (GSL), a glycan ligand that functions in the infection process and is expressed on sporozoites and merozoites. Treatment with this monoclonal antibody in mice resulted in a 85–92% reduction in parasite load in C.B.17 SCID/anti-gamma interferon treated mice. Further characterization of this glycan ligand showed it is mainly composed of beta glucose.

BIOCHEMISTRY AND CELL BIOLOGY

Characterization of the putative mitochondrion of C. parvum was presented by J. Keithly and colleagues. Data showed that C. parvum appears to present an acristae, ribosome-studded mitochondrion posterior to the nucleus. It exhibited a cyanide and oligomycin-sensitive membrane potential. Four nucleus-encoded mitochondrion specific genes were identified, including iron-sulfur proteins, chaperonins, adenylate kinase and valyl t-RNA synthase. It was proposed that FeS assembly takes place in the mitochondria and that C. parvum may have a unique type of energy metabolism that could provide insight into the development of specific drugs. Zhu et al. described how overlapping subunits of a multifunctional type 1 fatty acid synthase were cloned in pMAL expression vectors to be used for enzymatic analysis and to elucidate biochemical function of the CpFASl gene. Clearly targeted drug development will be greatly facilitated by the elucidation of biochemical pathways and especially the identification of key parasite organelles and enzymes.

HOST PARASITE INTERACTIONS

Little is yet known about the parasite's interaction with the host cell or changes that occur to either the host cell or the parasite as a result of invasion. Chen et al. presented a paper on the mechanisms by which C. parvum interacts with epithelial cells. Some of the findings that were described included the observation that invasion is associated with actin and that apoptosis is inhibited in infected cells through activation of the NF-kB survival pathway. The response of human epithelial cells to infection by C. parvum was also studied by detection of changes in mRNA using microarray expression analysis. Deng et al. showed that many genes of epithelial cells are up regulated or down regulated in response to infection.

GENOTYPING

Phylogenetic analysis of C. parvum isolates show that the organism can be differentiated into 2 different genotypes: human (genotype 1) and cattle/animal (genotype 2). Several different methods for genotyping and identifying sub genotypes of isolates were described. Alves et al. described multilocus genotyping using four genetic loci: COWP, TRAP-C1, DHRF and SSU-rRNA genes. They found that HrV-seropositive patients were infected with both genotype 1 and 2 but also with isolates with loci matching C. felis, and C. meleagridis. Similar results were observed by Guyot et al., who used the 18S rRNA gene to identify different genotypes in 57 patients. Multilocus genotyping was also used to distinguish differences in C. meleagridis (Glaberman et al.). The glycoprotein 60 kDa, heat shock 70 kDa, and SSU rRNA were used to divide C. meleagridis in to 6 genotypes and the human and bovine genotypes into 8 and 3 sub genotypes, respectively.

EPIDEMIOLOGY AND ENVIROMENTAL METHODS

Several studies focused on prevalence and characterization of C. parvum isolates and genotypes in specific populations. C. parvum was detected in 23.3% of the cattle sample in the Center and South regions of Portugal. All isolates were shown to be the cattle genotype. The parasite was detected in 55% of the animal water supplies, suggesting that cattle can be important in the transmission of infections for humans as well as animals. In a separate study (Learmonth et al.), fecal specimens from 618 cattle and 47 humans from New Zealand were examined. Thirty-six of the dairy cattle and 47 humans were positive for C. parvum. Forty-six of the 47 human isolates were genotype 2 (bovine), demonstrating the zoonotic transmission of the parasite. A study by Guyot et al. was performed to determine the species and genotypes in HIV-infected patients in Haiti. Both genotype 1 and genotype 2 were found in patients. Children were infected most often with genotype 1. Two patients were infected with isolates matching C. felis at target loci.

Models were described for environmental studies. Follet-Dumoulin et al. described the use of maggots and contaminated meat to study the dissemination of C. parvum from insects. The use of the steroid methylprednisolone acetate was used to uniformly deliver the steroid and increase the number of oocysts per mouse (Miller and Schaefer).

In another study (Phelps et al.) hydrogen peroxide was shown to inactivate C. parvum oocysts at a concentration of 0.025%. It was suggested that hydrogen peroxide could be used as an alternative to pasteurization in fruit juices.

IMMUNOLOGY

Susceptibility to infection was studied through the use of cytokine knockout mice. Four different knockout mice (IL-4, IL-10, IL-12 and CCR5) were infected C. parvum. IL-12 knockout mice were very susceptible to infection but recovered 3 weeks post infection. No infection was detected in IL-4 and IL-10 knockout mice. Modest infections were observed in CCR5 knockout mice with peak infections by day 8. The chemokine receptor, CCR5, is responsible for chemotaxis and lymphocyte activation in the gut. In another study, it was shown that C. parvum oocysts could be inactivated by different enterobacterial strains found in the feces of normal animal feces, indicating a role for bacterial flora in resistance to infection.

MOLECULAR BIOLOGY

Characterization of cryptosporidial genes and gene sequences has markedly increased in recent years and has the potential to identify new chemotherapeutic targets. The characterization of three thrombospondin-related adhesive proteins were described. This group of proteins has been shown to play a role in motility in other apicomplexans. O'Connor et al. presented a paper on the characterization of the Cpgp40/15 gene: a single copy gene with multiple transcripts resulting from alternate polyadenylation (purportedly responsible for message stabilization and regulation of gp40/15).

While advances in molecular biology and genotyping continue to be made, more basic research is needed in areas difficult to explore, such as biochemistry, physiology and immune response. Hopefully, insights into these basic areas of study will aid in the development of more efficacious therapeutics.