1a.Objectives (from AD-416)
The objectives are to improve food safety and reduce contamination of drinking water by improving detection, determining sources, and reducing transmission of protozoan parasites infecting humans. Objective 1 a) Improve speed and accuracy of methods to detect Cryptosporidium, Giardia, and Microsporidia in selected environmental specimens and in specimens from food animals, other farm animals, wildlife, and transport hosts that might harbor multiple species or genotypes. b) Develop monoclonal antibodies specifically to identify zoonotic species of Cryptosporidium.

Objective 2 a) Determine the prevalence Blastocystis spp. in 1000 pre- and post-weaned dairy cattle from farms in eastern states utilizing DNA from our immediate past project; determine the prevalence of Microsporidia, Blastocystis, Giardia, and Cryptosporidium in 150 sheep and 500 pigs from birth to market age from multiple farms and states, and from 1000 feedlot beef cattle in Nebraska. b) Determine the presence of these same organisms in environmental specimens provided by NOAA collaborators from waters impacted by agricultural runoff. c) Assess the potential infectivity, duration of infection, and numbers of parasites excreted throughout a period of infection, by experimentally infecting parasite-free cattle, sheep, pigs, chickens, turkeys, and laboratory rodents with any unique genetic isolates found in the field studies described above.

Objective 3 a) Test for protective immunity of HBC fed to neonatal calves experimentally challenged with C. parvum oocysts by observing the severity and duration of infection. b) Conduct biochemical and molecular studies that might serve as a basis for future treatment strategies to interfere with transmission of parasites. c) Test anti-viral drugs associated with reduction of cryptosporidiosis in AIDS patients and in vitro will be tested for efficacy against zoonotic Giardia and Cryptosporidium, both of which have been shown to contain RNA viruses.

1b.Approach (from AD-416)
Studies will identify Giardia, Cryptosporidium, and Microsporidia of livestock and wildlife by developing multiplex PCR techniques and examining new gene sequences to provide improved characterization of these organisms. Viruses have been found within Giardia and Cryptosporidium, and studies will determine if differences in the quality or quantity of such viruses using newly developed reagents can facilitate detection and differentiate pathogenic and non-pathogenic strains. The prevalence of Giardia, Cryptosporidium, Microsporidia, and Blastocystis in sheep and pigs, and feedlot cattle will be determined. The prevalence of Blastocystis also will be determined in dairy cattle. Unique genotypes of these pathogens from field isolates will be tested in transmission studies to determine their potential to infect other animal hosts. The presence of zoonotic protozoan pathogens in environmental specimens in areas impacted by runoff from agricultural animals will be assessed. Studies will identify methods to provide protective immunity against Cryptosporidium. Cows will be immunized with recombinant proteins and immune stimulators to produce colostrum with high levels of anti-Cryptosporidium antibody for passive immunization of calves. Biochemical and molecular techniques will be used to study encystation/excystation in Giardia and Cryptosporidium to identify proteins that can be targeted to disrupt transmission. Anti-viral and anti-protozoal drugs will be tested against Cryptosporidium and Giardia using cell culture and animal infectivity models.

3.Progress Report
A multiplex PCR test was developed to detect and differentiate the major species of Cryptosporidium that infect humans and cattle from minor or noninfectious species that infect cattle. A manuscript is in progress. Monoclonal antibodies were produced against virus-like particles (CPV) found within the fecal stage of Cryptosporidium (the oocyst stage). These antibodies were found to be capable of detecting oocysts by dot blot analysis. Using this test and reverse transcriptase polymerase chain reaction the concentration of virus-like particles was found to differ amongst isolates suggesting a link with pathogenesis of the isolates. After developing laboratory methods to improve recovery and detection of Cryptosporidium oocysts and Giardia cysts from sediment, surface water and sediment from sewage outfalls at 3 sites entering the Chesapeake Bay were analyzed in collaboration with scientists from NOAA. These methods are being transferred to NOAA technicians to continue monitoring these sites. Cross species testing of Giardia from cattle to rodents, sheep, and pigs, in addition to gene sequencing of specimens, has demonstrated a new species unique to cattle. Data analysis is proceeding and a manuscript will follow. Evaluating hyperimmune colostrum for protecting calves against cryptosporidiosis has concluded. Although protective antibodies have been demonstrated and severity of infection was reduced in some calves, infection was not prevented and protection was not universal. cDNA libraries enriched for sequences specific for Giardia cysts and trophozoites and another cDNA library enriched for specific sequences for Cryptosporidium oocysts were developed. Sequences of activated genes called “Expressed Sequence Tags (EST’s) from the Giardia DNA libraries are being analyzed and will be deposited into GenBank. A method called subtractive hybridization will be used to determine how these parasites form protective cysts that enable them to survive in the environment and how they are released from oocysts after entering the gastrointestinal tract. These studies are ongoing. SYs and technicians meet weekly on Monday morning to discuss findings from the previous week and to make plans for the coming week.

4.Accomplishments
Virus-like particles in Cryptosporidium may control severity of disease. Cryptosporidiosis is a severe gastrointestinal disease with no treatment for infected food animals and only one drug (with limited and often ineffective application) for treatment of humans. Differences were detected in the concentration of (CPV40) within the (oocyst) environmental stage of two strains (genotypes) of Cryptosporidium infecting calves. One strain, containing a higher concentration of virus-like particles, produced greater numbers of oocysts per infection, and resulted in more severe diarrhea than the strain with a lower concentration of virus-like particles. These differences suggest that the virus-like particles play a role in the infectivity and pathology of these isolates. Furthermore, this finding offers a possible means of treatment of cryptosporidiosis by targeting drugs or biologicals against viral proteins. Addresses Program 108 objectives specifically related to action plan component 1.1.1: sampling, isolation, identification and quantification of pathogens in animal fluids and tissues, manure; and the environment, including feed, water, and wild animals.

Sheep should be considered as a potential source of human infectious Cryptosporidium and Giardia. A study was undertaken to investigate the presence of Cryptosporidium and Giardia in sheep on a farm in Maryland. This was the first molecular based studies in North America to confirm the importance of sheep with regard to these human pathogens. Two assemblages of Giardia duodenalis were detected, Assemblage E, a type that is not infectious for humans, and Assemblage A, a known human pathogen. Three species/genotypes of Cryptosporidium were identified: C. parvum, Cryptosporidium cervine genotype, and a novel Cryptosporidium genotype (bovis-like genotype). Cryptosporidium parvum as well as the cervine genotype have been reported worldwide infecting humans. Cryptosporidium cervine genotype could possibly emerge as an important human pathogen because current evidences suggestes that this genotype has a wide host range and zoonotic potential. Because there is confusion in the taxonomy of the cervine genotype we characterized 30 of those isolates obtained from sheep using three different genes. These data were compared with information from all previous reports to facilitate a right identification when trying to detect this parasite. Addresses Program 108 objectives specifically related to action plan component 1.1.1: sampling, isolation, identification and quantification of pathogens in animal fluids and tissues, manure; and the environment, including feed, water, and wild animals.

5.Significant Activities that Support Special Target Populations
None

6.Technology Transfer

Number of new CRADAs and MTAs	1
Number of active CRADAs and MTAs	7
Number of patent granted	2
Number of non-peer reviewed presentations and proceedings	12

Review Publications
Fayer, R., Santin, M., Trout, J.M., Destefano, S., Koenen, K., Kaur, T. 2006. Prevalence of Microsporidia, Cryptosporidium, and Giardia in beavers (Castor canadensis) in Massachusetts. Journal of Zoo and Wildlife Medicine. 37:492-497.