Protozoan Parasites Affecting Food Animals, Food Safety, and Public Health

APPROACH: 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.

<P>

PROGRESS: 2006/10 TO 2007/09<BR>
Progress Report 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. <BR>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. <BR>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. <BR>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. 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. Significant Activities that Support Special Target Populations 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 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. 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. <BR>Technology Transfer Number of New CRADAS and MTAS: 1 Number of Active CRADAS and MTAS: 7 Number of Patent Applications filed: 2 <BR>Number of Non-Peer Reviewed Presentations and Proceedings: 12

<P>

PROGRESS: 2005/10/01 TO 2006/09/30<BR>
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aligned with National Program 108. This project replaces 1265-32000-073-00D. The protozoan parasites Cryptosporidium and Giardia are clearly recognized as food- and water-borne pathogens worldwide. Microsporidia have been designated by the CDC as an emerging pathogen of concern. However, little is known of their prevalence, distribution, or epidemiology. These three groups of agents have been responsible for numerous outbreaks of severe diarrheal disease in thousands to hundreds of thousands of persons in the United States. The economic impact of giardiasis and cryptosporidiosis, stemming from lost productivity, is enormous, while the impact from microsporidial illnesses remains largely unknown. Cryptosporidium parasites have now been reported from 154 species of mammals, mostly by microscopic identification. It is now recognized that this method is unable to distinguish one species of Cryptosporidium from another. Cryptosporidium parvum, a zoonotic species, is the most prevalent species in preweaned dairy and beef calves, causing morbidity and mortality with associated economic losses. A single infected calf can excrete tens of billions of infectious oocysts into the environment. Healthy persons can acquire infection from the ingestion of less than 100 such oocysts in contaminated water or food items, resulting in several days of diarrhea and other flu-like symptoms. Infants, the elderly, and immune-compromised persons are at high risk because pharmaceuticals are not available to prevent infection and only one drug has been approved by the FDA- for treatment of children. Despite numerous reports of Cryptosporidium in cattle, their role in environmental contamination with species infectious for humans is not clear. The ARS is investigating the prevalence and species of Cryptosporidium in livestock of different ages and attempting to determine the relationship of the animal isolates to the organisms that cause disease in humans and in the animals themselves. Giardia is the most commonly diagnosed intestinal parasite in public health laboratories in the United States and has been the most frequently identified agent associated with drinking water outbreaks in the U.S. for many years. There has been increasing evidence of Giardia infection in livestock, raising the question of how agriculture may contribute to environmental contamination. The ARS is investigating the prevalence of Giardia in livestock species of different ages and attempting to determine the relationship of isolates from livestock to the organisms that cause disease in humans. Thirteen species of Microsporidia have been identified in fulminating infections in AIDS patients. Little is known about the actual sources of human infection with Microsporidia but reports from Europe indicate that cattle and pigs are potential sources. ARS scientists are conducting surveys of cattle to detect Microsporidia, to determine the prevalence in cattle of different ages, and to compare the species found in cattle with those infecting humans. Cryptosporidium, Giardia and Microsporidia have been identified as waterborne pathogens of concern by the U.S. Environmental Protection Agency and the World Health Organization. Because clean drinking water starts with clean source water, protection of both surface and ground water against contamination with manure from animal feeding operations and discharge from waste water treatment plants have become issues of national importance. These issues are affecting decisions by such agencies as the US/EPA and state environmental agencies by restricting animal farming practices in proximity to water. In addition, the impact of cryptosporidiosis on the animals themselves can be devastating, resulting in morbidity, poor growth, and even mortality, with associated economic losses. The impact of the work will be to provide accurate information on the prevalence and species (or genotypes) of these three groups of organisms so that action agencies can make decisions based on appropriate and reliable scientific data. <BR><BR>2. List by year the currently approved milestones (indicators of research progress) <BR>OBJECTIVE 1: Detection (2006-2007) Exp Design 1a - Develop multiplex PCR test 2006-2007 Exp Design 1b Monoclonal antibody detection 2006-2007 <BR>OBJECTIVE 2: Epidemiology (2006-2010) Exp Design 2a - 2006-2010 1. Dairy cattle: Blastocystis 2. Sheep: all zoonotics 3. Pigs: all zoonotics 4. Feedlot cattle: all zoonotics Exp Design 2b - Environ contam from agric runoff 2006-2010 Exp Design 2c - Cross species infectivity 2006-2010 <BR>OBJECTIVE 3: Treatment/control (2006-2010) Exp Design 3a - Colostrum production/testing 2006-2007 Exp Design 3b Subobjective 3b1 - Cryptosporidium excystation 2008-2010 Exp Design 3b Subobjective 3b2 - Giardia encystation 2006-2008 Exp Design 3c - Nucleoside drug testing 2009-2010 4a List the single most significant research accomplishment during FY 2006. ARS scientists conducted cross species infectivity studies with Giardia isolated from cattle. The most prevalent Giardia found in cattle in a multistate study (genotype E) was not infectious for other animal species tested. The less often found genotype (genotype A),which has been reported to infect humans and other animals, was isolated from cattle and found to infect gerbils and sheep. This finding demonstrated that cattle can be a source of a Giardia genotype reported in human infections but the predominant genotype of Giardia found in cattle does not appear to be zoonotic. This finding could influence state and federal regulations regarding farm management, manure management, and proximity of livestock to surface water. <BR><BR>National Program Component 108. 4b List other significant research accomplishment(s), if any. <BR>1. A new genotype of Cryptosporidium was identified in sheep, distinguishing them from the pathogenic C. parvum providing epidemiologists with a new parasite to differentiate from pathogenic species found in drinking water or food specimens. <BR>2. Identified potentially zoonotic Giardia in sheep.<BR> 3. Developed monoclonal antibodies against a virus found within Cryptosporidium parvum. <BR>5. Describe the major accomplishments to date and their predicted or actual impact. <BR><BR>Data are still being collected early in this first year of the project. Molecular, biological, and immunological techniques that were developed in the previous project will aid in the rapid detection of the Cryptosporidium parasite in surface water, ground water, and drinking water. A recombinant protein that was produced by genetic engineering that is similar to a constituent on the surface of Cryptosporidium and monoclonal antibodies were developed that detect the parasite. ARS scientists developed a multiplex molecular test for detection of Cryptosporidium oocysts in water samples and are now analyzing the data. This assay speeds up the analysis time for identifying water samples that contain Cryptosporidium parasites because, once a specimen has been prepared, multiple species can be analyzed in a single test in a short time. ARS scientists also refined methods for recovery and molecular testing of Cryptosporidium oocysts recovered from the environmental waters and sediments. This research is essential in identifying sources of the parasite in the environment and could ultimately be used to manage run-off from agricultural and urban areas. The most in-depth study of Giardia in any animal species has been completed in cattle over the past four years and has demonstrated the persistance of relatively high levels of infection with a livestock specific strain and the sporadic presence of a strain infectious for humans. In the new project this type of study is being applied to other livestock species- sheep and swine. Enterocytozoon bienuesi a human pathogen was identified in cattle and wildlife for the first time in North America, and the most extensive and widespread documentation of its presence in animals was conducted in the past project. In the new project this type of study is being applied to other livestock species- sheep and swine. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Three gene sequences were patented and licensed by ARS to biotechnology/ pharmaceutical industries from the immediate past project. Potential new products have not yet been developed in the new project. As participants in a collaborative project with scientists from the National Oceanic and Atmospheric Administration (NOAA) and the Centers for Disease Control (CDC) ARS scientists are developing technologies for detecting contamination of environmental waters with fecal-borne pathogens potentially from humans and livestock species. Submitted seventeen (17) new gene sequences to GenBank for Cryptosporidium, Giardia and Microsporidia from coyotes, beavers and cats.