Duration: 10/01/1999 to 09/30/2004

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

STATEMENT OF THE PROBLEM:

The Western regional research project W-102 is the only regional project dedicated to the understanding and control of livestock parasites. The objective of this revised project is to study the biology, pathogenesis and treatment of protozoan and helminth diseases of livestock. Development of control programs combining traditional chemotherapy with novel techniques of parasite control is anticipated. The reduced dependence on antiparasitic drug use will follow development of alternative parasite control measures and will be more appropriate for new, more sustainable systems of contemporary livestock production.


JUSTIFICATION:

Importance in agriculture, rural life and consumer concerns

Agriculture continues to be the dominant industry in rural communities; however, it is becoming more and more evident that stewardship of our natural resource base is not only the responsibility of farmers, but of all citizens. Consequently, society is demanding that agriculture implement environmentally sound sustainable systems of production that have low chemical usage, reduced movement of sediment and nutrients from the land, and have minimal or no off-site impacts. One component of these production systems must be parasite control. Parasites continue to be a common limiting factor in livestock production systems, decreasing the quality and quantity of livestock products and requiring periodic treatment with parasiticides. However, a thorough understanding of the pathogenesis and transmission of parasitic disease must be developed before integrated programs of parasite control can be formulated.


Extent of the problem

Parasites continue to decrease animal productivity in all livestock species and their importance is recognized by producers. The antiparasitic market is the fastest growing segment of the veterinary pharmaceutical industry (Conder, personal communication). In 1997 more equine operations, regardless of size, gave dewormers to horses than vaccinated at least one horse (CAHM, 1997). In the sheep industry, more producers indicated moderate to high levels of concern about gastrointestinal worms than any other health condition (CAHM, 1997), and in a recent veterinary services survey of cattle producers (USDA/APHIS, 1994), internal parasites were listed as the second most common cause of significant economic impact in the past 12 months.

Over the past 30 years, there has been a dramatic increase in the use of chemotherapeutic methods as a means of endoparasite control. Many of the newest generation of parasiticides offer longer residual times and increased ease of administration for the producer. While it continues to be important to evaluate the activity of new drugs against major livestock parasites it has become clear that the real utility of modern chemotherapeutic agents lies in their role as a basic component of an integrated parasite control program.

The need to look beyond conventional chemotherapy is immediate in the case of those parasitic infections where no effective treatments exist. Cryptosporidium parvum and Neospora caninum are coccidial protozoan parasites whose full economic impact is still under study. Neospora caninum has recently been recognized as a cause of abortion and neonatal paralysis in cattle. It has been estimated that abortion caused by the parasite costs the diary industry $35 million a year in California alone (Dubey and Lindsay, 1996). Cattle can be infected by two routes of transmission: 1) congenitally infected heifers may mature, become pregnant, and the organism may then cross the placenta to infect the next generation of offspring; 2) previously uninfected cows may consume N.caninum and then bear infected offspring or aborted fetuses. The relative importance of these two methods of transmission in causing reproductive losses has not been determined. Both a better understanding of the transmission of this parasite and new approaches to control are important in limiting losses to the dairy industry. Since treatment to eliminate the parasite and prevent abortion is unavailable, development of a vaccine against the parasite probably offers the most effective means of controlling cattle infections in the future. Successful vaccine production will require a thorough understanding of the host immune response to N. caninum.

Crytosporidium parvum has become increasingly important to US agriculture because of its ability to infect domestic and wild animals and humans. Outbreaks of human gastroenteritis caused by this parasite are commonly blamed on run-off from farming operations, most notably dairy operations. It is frequently assumed that young calves are the source of the parasites in the water supply. In 1997, the Science and Technology Committee of the National Beef Cattlemans Association listed C. parvum ninth in a list of production issues. Since there are no effective treatments for C. parvum at this time, future agricultural practices must be targeted to reducing the potential of farm operations for the release of C parvum and other potential human pathogens. The development of such programs depends on an accurate knowledge of the epidemiology of the parasite, and upon the development of sensitive and specific detection tools. (Fayer, 1997). Additionally, efforts to discover effective chemotherapeutic agents for C. parvum and better understand the pathogenesis of cryptosporidiosis must continue. Other coccidian parasites are of similar importance to US producers. Eimeria infections in ruminants annually produce significant losses and in poultry, treatment for this genus is universal in broiler production. This practice has led to serious levels of resistance to many of the available poultry coccidiostats.

The limitations of chemotherapy are also evident in the dramatic increase in anthelmintic resistance seen in the trichostrongylid nematode Haemonchus contortus in the south central and south eastern United States. H. contortus is a blood sucking parasite and the most important of the GI worms of small ruminants. The mild climate of the southeastern US is particularly well suited to the life cycle of this group of nematodes. Development from the egg to the infective stage occurs on pasture and is dependent on warm weather and adequate mositure. To prevent both morbidity and mortality of animals, small ruminant producers are often forced to deworm their animals repeatedly during the grazing season. These frequent treatments can rapidly select for anthelmintic resistance (Conder and Cambell, 1995). While there are ample anecdotal reports of resistance to 1 or more drugs, the extent of resistance in the US has not been well documented. In the absence of practical alternatives, the efficacy of available anthelmintics needs to be preserved. Once the prevalence and distribution of the resistance problem are established, rational strategies of drug use can be developed and tested for efficacy in areas where resistance threatens the ability of producers to control parasite infections. Resistance to anthelmintics has also now been documented in the strongylids of horses and swine but the extent of the problem in these species is also unknown.

The increasing levels of anthelmintic resistance and efforts to reduce our dependence on traditional chemotherapeutic approaches to parasite control, provide impetus for development of novel approaches to the management of parasitism. Vaccination against gastrointestinal helminths offers an alternative approach to control in an integrated parasite control program (Smith, 1999). However, effective vaccines for nematode parasites have been very difficult to develop (at this time there is only one availablethe European bovine lungworm vaccine). One of the primary reasons for this difficulty is the complexity of the immune response to gastrointestinal nematodes. To date a large number of bovine immune responses have been measured including serum antibody, serum eosinophils, cellular reactivity, and the induction of mRNA's of 6 different cytokines. None have correlated with protective immunity, although work with the economically important bovine abomasal parasite, Ostertagia ostertagi, and other animal nematodes has demonstrated the central role of cytokines in mediating immune and other responses to parasitism (Finkelman, Shea-donohue eta al, 1997; Gasbarre, 1997). Additional analysis of antihelminth immunity is required in conjunction with efforts to identify effective antigens for vaccine use.

The nematode intestine has provided a valuable source of antigens that are capable of inducing protective immunity against Haemonchus contortus and potentially other gastrointestinal nematode parasites Whole intestinal preparations, intestinal antigen complexes and individual intestinal proteins (Munn, Greenwood, et al. 1987; Jasmer, McGuire 1991; Smith 1993; Smith, Munn, et al. 1993; Jasmer, Perryman, et al. 1993) have all induced significant levels of protective immunity against H. contortus challenge infections in sheep and goats. Genes encoding some of these individual antigens have been cloned and genes encoding related proteins have been identified from other parasitic nematodes (Redmond, Knox, et al. 1997; Longbottom, Redmond, et al. 1997; Rehman, Jasmer 1998). Therefore, observations made with H. contortus in small ruminants may apply to other nematode infections. Vaccination against the equine nematode, Strongylus vulgaris, is also being studied (Klei, 1997). There are numerous questions that need to be addressed regarding vaccines based on intestinal and other antigens and their significance in control of gastrointestinal nematodes. One relates to the ability of these antigens to protect against field challenge, since most immunization trials have utilized single laboratory infections for challenge. Another is identification of recombinant proteins and presentation protocols able to induce protective immune responses that approximate those of corresponding antigens isolated from worms. A third relates to host mechanisms responsible for the protective immunity, which may provide guidance for development of antigen presentation protocols.

Successful vaccination is based on manipulation of the individual host immune response to parasites, but there is also considerable evidence that part of the natural variation seen in resistance to helminth infection in ruminants is under genetic control (Albers and Gray, 1986; Barger, 1989; Stear et al.,1990b; Gogolin-Ewens et al., 1992; Gray and Gill, 1993; Ruvuna and Taylor, 1994; Stear and Murray, 1994; Miller and Gray, 1996). It has been shown that some breeds of sheep are more resistant than others to effects of nematode infection (Altaif and Dargie, 1978; Preston and Allonby, 1978, 1979a; Baker etal., 1993, 1994; Gruner et al., 1986; Radhakrishnan et al., 1972; Courtney et al., 1985; Bahirathan et al., 1996; Miller et al., 1998). Evaluating breed resistance under field conditions and understanding the genetic mechanisms of resistance are 2 important components of this approach to parasite control. Moreover, identification of some of the genes involved in regulating resistance to helminth infection will allow early selection of genetically superior animals and may increase the rate of selection for resistance. Attempts to associate genetic markers with nematode resistance has been under investigation for a number of years (Beh and Maddox, 1996), without much success. Because nematode resistance is probably a function of multiple loci and a quantitative trait, this and investigations by others (ILRI, Kenya; AgResearch, New Zealand; and CSIRO, Australia, Roslin Institute, Scotland) are addressing a total genomic approach to finding and mapping resistance QTL. Numerous microsatellite markers have been and are continuing to be identified for gene mapping purposes. At present, an autosomal genetic linkage map of the sheep genome with over 550 markers has been published (Crawford et al., 1995) and updated (Maddox et al., 1996).

In addition to alternative techniques of parasite control based on host responses, attention is also being directed to elimination of parasite stages in the environment. It has long been recognized that chemical control, although convenient and fast acting, is only a partial and short term control procedure. In contrast, there are implications that biological control, although slow acting, may be a persistent and enduring control methodology. Biological control is highly dependent on a thorough knowledge of the control agents. For example, the nematophagous fungi (e.g. Duddingtonia flagrans) are environmentally safe and would decrease pasture contamination by trapping and killing infective nematode parasite larvae (Sterling, 1991), reducing the need for drug treatment and slowing the rate of development of drug resistance. Additionally, reduction in anthelmintic treatments will decrease drug residues in meat and meat products and also reduce chemical contamination of the environment.

Strategic management of pastures has also been shown to affect levels of infective parasitic nematode larvae (Michel, 1976). Several techniques have been utilized to reduce pasture parasite burdens including treatment of the host animal with anthelmintics at the time of peak egg shedding and then allowing the remaining larvae to die off during periods when the pastures were not grazed (Brunsdon, 1980; Donald, 1974; and Williams et al., 1986). The practice of moving cattle to crop residues following the harvesting of a non-grazed crop has been shown to offer relatively safe pastures (Michel, 1976). There are many factors influencing development of safe pastures that have not been investigated. This is particularly true of production systems that involve rotation of crop and livestock production. Little is known about the minimal time for removal of all helminth species from a particular land area or how soil and crop management practices influence the removal of certain species. Further, little is known about how long safe pastures can be maintained as safe pastures or what contamination prevention techniques are practical for maintenance of such pastures. These issues must be examined regionally because of the enormous variation in forage and crop biology and management across the U.S.



Needs and Advantages of a cooperative approach

While chemotherapy will remain an important part of parasite control, vaccines, use of genetic variation and biocontrol offer exciting alternatives to reliance on antiparasitics alone. However, the value of any parasite control technique cannot be fully evaluated in the laboratory. Any technique of parasite control must be examined in the field within the context of the total management system. While the ultimate goal of this regional project is the development of parasite control programs in sustainable agricultural systems, there is still considerable knowledge to be gathered about the individual components of these programs and how they interact. The problem is of such magnitude that it can be addressed effectively only by a multidisciplinary approach involving scientists working on many diverse specific aspects of this problem. Progress in controlling parasitic disease of livestock can best be achieved by a comprehensive approach to research utilizing regional cooperation in the maintenance of parasite strains, exchange of samples, pooling of expertise, information and equipment and exchange of research data, while permitting individual laboratories to specialize in aspects of the master project which they are best equipped to perform. No individual station or investigator is likely to develop an effective control program. Additionally, field testing of any parasite programs must be replicated in different regions to account for the wide variation in climatic and environmental conditions in the U.S. We feel that the objectives of this project are more likely to be accomplished by pooling our efforts in the multifaceted approach proposed.

Related, Current and Previous Work

The participants in regional research project W-102, Integrated Methods of Parasite control for Improved Livestock Production, have published 192 peer-reviewed articles and book chapters (10 in press). In addition, 53 technical or popular articles, 10 theses/dissertations and 135 abstracts were published. This effort clearly demonstrates the continued progress shown by the cooperative efforts of the technical committee.

A search for related work was conducted in the Current Research Information System (CRIS) database. The search retrieved 102 resumes of current (termination dates of 1998 or later) Hatch, State, Industry, Animal Health and USDA Special Grant projects within the scope of the work conducted by the W-102 participants. Forty-seven (46%) were directed by W-102 participants and collaborators (6 were W-102 projects; the remainder were special grants, Hatch, etc., projects under the leadership of W-102 participants). The remaining related projects (55 or 54%) were from 23 states (including 10 of the W-102 states) and included research on parasitism in wildlife (3), general protozoal infections in domestic animals (34) and general helminth infections in domestic animals (10). Three projects involved molecular and immunological research on protozoan and helminthic disease in non-domestic animals. These latter projects represent efforts of specific individuals and/or laboratories and are not coordinated as are the W-102 efforts.

In addition to the above-mentioned projects, W-102 shares interests with other regional projects:

NC-62 Prevention and Control of enteric Diseases of Swine

NC-107 Bovine Respiratory Diseases: Risk Factors, Pathogens, Diagnosis and Management

NE-60 Genetic Bases for resistance and Immunity to Avian Diseases

S-145 Nutrition and Management of Swine for Increased Reproductive Efficiency

S-221 Development of Profitable Beef-Forage Production Systems for the Southern Region

W-112 Reproductive Performance in Domestic Ruminants

NRSP-8 National Animal Genome Research Program

These shared interests are largely evident in the types of technologies used to study, understand, and control diseases and are not manifest in the specific type of pathogen-host relationship. There is no overlap of W-102 with any of these regional projects. However, control methodologies generated by W-102 research will contribute to the missions of the related projects. The reader is referred to The extent of the problem under the Justification section for a review of the science related to this project.

Objectives

1. Control of parasitic diseases using biological and chemical agents and physical methods
   
2. Define the roles of pathogenesis, immunomodulation, vaccination and genetic manipulations in parasite control
   
3. Integration of parasite control practices into livestock production systems
   
4. 
   

Methods

Control of parasitic diseases using biological and chemical agents and physical methods. Coordinator: Healey (UT) 1A.Evaluate the anthelmintic activity of naturally occurring fungi in laboratory and field trials. 1.Efficacy of biological control measures on parasite transmission will be studied. The ability of fungi such as Verticillium, Rhopalomyces, and Catenaria to attack nematode, cestode, and trematode eggs under laboratory conditions will be determined. Selection of effective fungal species and strains will be implemented as well as improved cultivation technology to produce infective propagules for field studies. Field studies conducted by CA-Riverside will involve small pasture plots and will be done in collaboration with MD and LA. After development of successful small field tests, more extensive field testing with other members of the technical committee will be sought. 2.Another study using biological control is planned to determine how the nematode-trapping fungus Duddingtonia flagrans might be used in combination with anthelmintic treatment protocols in sheep grazing pastures predominantly contaminated with Haemonchus contortus. During a 2 to 3 year period, one group of sheep will be supplemented with feed containing fungal spores during the haemonchosis season (April through September). Another group of sheep will receive the same supplemental feed without fungal spores. Parasite infection levels will be monitored using fecal nematode egg per gram (EPG) counts and blood packed cell volumes (PCV). The pasture contamination level will be discerned either by grazing tracer lambs or enumerating infective larvae recovered from pasture herbage samples. Intervention with anthelmintic treatments will be deemed necessary at times by comparative EPG counts and PCV. It is expected that the fungal spore-fed group of sheep will have cleaner pastures and reduced reliance on anthelmintic treatment. This research will be conducted in LA and in collaboration with the Danish Center for Experimental Parasitology. IB.Evaluation of the efficacy of the latest generation of endecto-parasiciticides and novel anthelmintic agents. 1.The latest generations of endectoparasiticide and other novel anthelmintic drugs will be screened in cattle, sheep, goats, and horses to determine better efficacy, broader spectrum, safety, zero or minimal withdrawal times, and most appropriate programming time for administration. Drugs will include the avermectins eprinomectin, moxidectin, doramectin, and ivermectin and any other new classes of anthelmintic drugs that become available. Formulations will include solutions for injection as well as topical pour-ons. Both internal and external parasites will be targeted, including nematodes of the gastrointestinal and respiratory tracts, migrating arthropods and nematodes in the extraintestinal viscera, lice, fleas, grubs, and maggots of horn and house flies. The primary intent will be to determine effectiveness of drug activity, especially in beef cattle, on fecal egg count reduction of natural occurring gastrointestinal nematodes. In addition, body weights per animal and means per group will be analyzed. Speciation of gastrointestinal nematodes will be determined by coproculture. In addition to MO, 3 geographically separated regions (MN, LS, and MS) will cooperate to provide data. 1C.Determine the prevalence of anthelmintic resistance in the US and characterize resistant parasites 1.Anthelmintic resistance in the trichostrongylid parasite Haemonchus contortus has become an urgent problem in small ruminant production throughout the world. In the United States, resistance has not been widely documented. The extent of anthelmintic resistance in H. contortus and other trichostrongyles of small ruminants will be established using both fecal egg counting techniques and a larval development assay. Fecal samples will be collected from llamas to assist with the evaluation of the extent of anthelmintic resistance that occurs in trichostrongylid parasites of llamas in the United States. Llamas are often frequently dewormed to control meningeal worm infections and anthelmintic resistance is likely to occur in their gastrointestinal parasites. Since many of the trichostrongyle parasites of llamas are shared with small ruminants, strains from llamas may infect small ruminants and increase the prevalence of resistance in those animals. This research will be conducted in VA in collaboration with MO, LA, and MS. 2.In addition to llamas, captive sylvatic ruminants and exotic bovids will be examined for anthelmintic resistance to gastrointestinal nematode populations. The larval inhibition assay (DrenchRite., Horizon Technology, Australia) will be used to detect resistant trichostrongylid nematodes. This in vitro assay is designed to detect resistance to avermectin/milbemycin, benzimidazoles, levamisole and benzimidazole/levamisole combinations in the major gastrointestinal nematodes of sheep. The prevalence of resistance to specific target compounds in these animals will be discerned using samples anaerobically shipped to collaborating laboratories in order to avoid problems with egg development prior to the assay. Evaluation of new and existing anthelmintic compounds is necessary in order to integrate their use into strategic parasite control programs so that producers can be advised as to development of anthelmintic resistance. Documentation of the presence and extent of anthelmintic resistance in traditional livestock, alternative livestock, captive ruminants, and exotic bovids will advance our understanding of how anthelmintic resistance develops and is maintained. This research will represent a collaborative effort between MS, KS, TX, and VA. Moreover, the larval inhibition assay will be used to test these anthelmintics in small ruminants and cattle in cooperation with extension veterinarians and animal scientists in KS, MO, MS, MN, and TX. 1D.Development of an animal model for cryptosporidiosis 1.Research will be conducted to determine the feasibility of using the non-neonatal pig (1 week to 6 months of age) as an animal model for cryptosporidiosis. Initially, experiments will be designed to provide preliminary data on various immunosuppressive regimens when administered to pigs of different age groups. Later, the non-neonatal pig model will be fully developed by conducting experiments designed to define the optimum immunosuppressive regimen, ages most susceptible to infection, minimum dosage of oocysts that will produce patent infections, and isolates of C. parvum capable of infecting the pigs. Demonstrating efficacy against C. parvum following the administration of paromomycin to infected pigs will be used to validate the model. This model can then be used for testing of novel therapies for cryptosporidiosis. This research will be conducted primarily in UT, with possible collaborations with MS and MD. Objective 2 Define the roles of pathogenesis, immunomodulation, vaccination and genetic manipulations in parasite control. Coordinator: Jasmer (WA) 2A.Define immune mechanisms that lead to host immunity or pathology in parasitic infections. 1.Studies in Maryland will continue to define immune responses elicited by gastrointestinal nematodes of cattle. Using the assay systems developed in cattle infected with Ostertagia ostertagi, future studies will compare responses of resistant and susceptible cattle to infection by the parasites in an effort to identify responses that are correlated with protective immunity. These studies will involve genetically selected cattle and cattle that have been immunized in a such a manner that they reduce the number of parasites developing after challenge infection. Similar studies will be performed to define protective immune responses elicited by other parasite species, such as Haemonchus placei which occupies the same organ as Ostertagia but elicits strongly protective immune responses. These studies will focus on cytokine responses in the local tissues, and also on effector cell populations at the site of infection. A number of new parameters will also be evaluated including levels of pro?inflammatory cytokines, levels of colony stimulating cytokines, and numbers of effector cell populations in the abomasal mucosa. These studies should provide more accurate markers for functional immunity in the bovine host, and will aid mapping of genes involved in immunity to GI nematodes. Through collaborative efforts to produce reagents specific for bovine cytokines efforts will be made to determine if cytokine gene expression correlates with biologically active products. The purpose of all studies in this area will be to identify protective immune mechanisms or immune responses that directly correlate with protective immunity. The identification of immune responses directly responsible for protection will result in further studies aimed at enhancing the generation of these responses, while the identification of responses that are correlates of protective immunity would allow for a more accurate identification of resistant animals and thus facilitate the search for the protective responses. 2.Products of Ostertagia ostertagi that have immunoregulatory activity will be identified. Recent studies have shown that products of O ostertagi inhibit in vitro growth of T lymphocytes. Preliminary work implies that this activity may reside in a TGF-beta like moiety. It is very intriguing that a molecule of this class is also involved in the regulation of Dauer larvae formation in C. elegans. Future studies will attempt to purify the inhibitory moiety and confirm or dismiss the idea that it is a TGF-beta-like substance. Once identified the inhibitory molecule will be characterized, and the gene(s) encoding the substance will be cloned and expressed for further studies of the mechanism of immunosuppression. Collaborative studies (LA, MD, MN) will continue to determine if Ostertagia infections also result in functional immunosuppression of their host. A growing body of evidence indicates that immune responses to certain microorganisms result in the suppression of immunity to other infections that require different immune effector mechanisms. Studies will be performed in which parasite infected and parasite free cattle will be subjected to immunization which unrelated antigens. The ability of the cattle to respond to these immunological insults will be assessed to ascertain the effect of the existing nematode infections. 3.Previous studies have demonstrated that ponies immunized with irradiated L3 of the nematode S.vulgaris are protected from challenge infections whereas those immunized with worm extracts in RIBI adjuvant show signs of increased pathology as compared to uninfected controls (Monahan et al., 1994). These observations suggest that different immune responses are induced by these two different immunization regimes and that these two responses are associated with either protection or pathology. Cytokine gene expression will be measured in ponies immunized by these two methods during a challenge infection and these profiles compared to uninfected controls. Cytokine gene expression in cells collected from blood and cecal lymph nodes will be measured using an RT-QPCR method previously described (Swiderski et al.,1998). Cells will be collected from ponies at day -4, 4, 9 and 14 following challenge infections of 1000 L3. Surgical methods previously described will be used to collect cecal lymph nodes (Swiderski et al., 1998). Lymphocytes will also be cultured with parasite antigen, separated into CD4+ and CD8+ cells by magnetic bead separation and cytokine gene measured. Cytokine gene expression to be measured includes, IL-2,IL-4, IFN gamma, IL-10 and IL-5 (LA). 4.Recent studies demonstrate that resistance to the gastrointestinal worms N. brasiliensis, Heligmosomoides polygyrus, Strongyloidies venezuelensis, and Trichinella spiralis are regulated by activation of the intracellular enzyme, the STAT-6 molecule, that binds to the common alpha chain of the IL-4/IL-13 receptor. This indicates a common mechanism of control of phylogenetically and physiologically different nematodes that infect the mammalian gut. Elucidation of the mechanism of IL-4 versus IL-13-induced immune responses to gastrointestinal parasites, and their relationship to the interleukin receptor-activated STAT-6 molecule that regulates IL-4 and IL-13 gene activation will be a key event in the understanding of resistance to gastrointestinal parasites. Parasite infection models provide insight into the design of more complicated and costly studies of responses of swine to parasitic infections. Human and mouse agonists and antagonists of the IL-4/IL-13 receptor will be evaluated on pig lymphoid and epithelial cells isolated from the intestines in order to determine their application to control of infection. Development of cytokine measurement techniques in swine infected with Ascaris suum and Trichuris suis will be evaluated to provide recommendations for control of these important economic and the emerging zoonotic aspects of these infections. Recent data indicates that these infections may have a broader role in foodborne infections. It is now clear that prior infection with Trichuris suis enables Campylobacter pylori to establish more vigorously in the intestine with resultant effects on animal health and on foodborne transmission of this human pathogen. 5.Studies will be undertaken to clone, express and evaluate cryptic gut-associated proteins derived from Ascaris suum that can be used to attenuate host infection. Attention will be directed toward proteins that may have more universal applicability in reducing parasitosis from other common nematodes parasites of swine. IL-4 will be cloned and expressed to develop quantities of the biologically active molecule to test its therapeutic and adjuvant activities against gastrointestinal nematodes. Combinational treatment with common nematode antigens and IL-4/IL-13 receptor agonists will be evaluated for its potential to activate protective mechanism common to the STAT-6-dependent resistance that exists in murine models of resistance to infection. Swine IL-12, and other important swine cytokines will also be cloned and expressed to test for use as immune modulators to enhance resistance to economically important protozoan parasites and to evaluate their use as activators of innate immunity. Potentially these compounds could be used to reduce early weaning and transportation-induced losses in production costs in neonatal swine. 6.Immunity to the protozoan parasite, Neospora caninum, will be examined in several systems. The cell mediated immune response to N. caninum in dogs will be determined with lymphocyte proliferation assays using Alamar blue (Zhi-Jun et al., 1998). Flow cytometry will be used to examine specific T and B cell populations in infected dogs. Humoral responses will be determined using an Immunofluorescent Antibody (IFA) test. Immunodominant antigens will be identified by western immunoblotting techniques. Sheep will be used as a model for N. caninum induced abortions. Pregnant ewes will be infected at various times in gestation and the fetal outcome evaluated (VA). 7.Studies have been initiated to define immune responses in naove and immunized cattle after infection with Eimeria (MD, MN) or Neospora (MD, VA). Initial studies are focused on the identification of the recognition sites of parasite antigens by the host immune system, and upon the characterization of lymphoid populations stimulated. Competitive PCR techniques for at least 13 different bovine cytokines are available, and these assays are being used to discern important shifts in cytokine gene expression. Once important cell populations and/or cytokines responses are identified, attempts will be made to employ a number of immunomodulators, including recombinant cytokines, to enhance desirable immune responses. 8. Studies will continue to define the immune responses to Cryptosporidium infections in neonatal calves. Susceptible and resistant calves will be challenged with the parasite to assess immune responses such as cytokine profiles, cellular reactivity, changes in lymphocyte subpopulations, and effector cell numbers in the mucosa. In addition the effect of exogenous cytokines and gamma-Interferon inducing agents on immunity to the parasite will be determined 9.Current efforts to produce colostrum for immunotherapy of cryptosporidiosis in young calves will be expanded. Research plans will build on recent experiments wherein gene gun immunization of periparturient cows with plasmid DNA encoding C. parvum antigens elicited anti-C. parvum antibodies in colostrum. Studies will include using other plasmid expression vectors, genes for other C. parvum antigens, and use of liposomes or cytokines to enhance immune responses. The colostrum will be tested in an adult mouse model and in calves against C. parvum infection. Some calves will be tested for response to immunization by oral inoculation with irradiated C. parvum oocysts. 10.Efforts will continue to identify protective antigens of Eimeria species in chickens and to develop a comprehensive understanding of host protective immunity. Molecular probes, anti-cytokine antibodies, and sensitive molecular techniques to evaluate cytokine production in chickens with coccidiosis will be developed and used in conjunction with strategies to express avian cytokines. Develop an effective mucosal immunization strategy against coccidiosis using viral and bacterial vectors as well as naked DNA. 2B.Identify and test parasite antigens that can serve as targets for control of parasitic infections. 1.Monoclonal Antibodies (Mabs) against the sporozoite and oocyst stages of Cryptosporidium parvum have been produced. To identify antigens from other stages in the host, a panel of Mabs against the intermediate stages (meronts) of this parasite will be generated (UT). Briefly, neonatal BALB/c mice (1 week of age) will be experimentally infected with C. parvum via oral inoculation with infective oocysts. Intestinal infections will be resolved as the mice become older. At approximately 2 to 3 months of age, mice will be challenged by intraperitoneal injections with antigens prepared from intermediate stages (meronts) of the parasite. Meronts will be produced by infecting 1-day-old white Leghorn chickens. At 3 or 4 days of age, the chickens will be killed and meronts collected from the terminal ilea. Mice will be subsequently challenged at 2 to 3 month intervals using antigens prepared from sonicated oocysts. Five to 7 days prior to killing, mice will be challenged a final time by intraperitoneal injection of either chicken-derived meront antigens or sonicated oocysts. A standard fusion protocol will then be followed. Resultant hybridomas will be screened with the ELISA and an indirect immunofluorescence assay (IFA) for production of Mabs against C. parvum meronts. The IFA will be used to observe Mabs specific for meronts growing in bovine fallopian tube epithelial (BFTE) cell culture. These monoclonal antibodies will be used to monitor in vitro cultures for screening of antiparasite inhibitors and may identify antigens that have potential value for immune protection against C. parvum infections. (UT). 2.Due to the initial success of field trials using a gel formulation of irradiated Eimeria oocysts to protect against coccidiosis, we plan to expand our vaccination trialsusing lower doses of oocysts to achieve protection. Recombinant Eimeria antigens associated with metabolic stages of intracellular parasites will be produced based on reverse transcriptase and PCR reaction of mRNA to identify targets for protective immunity. Cooperative agreements will be developed to test metabolic proteins as targets for drug treatment to prevent coccidiosis. 3.Studies on vaccination with recombinant Eimeria antigens using direct DNA injection of plasmid DNA will be expanded by comparing different expression plasmids and the use of liposomes and cytokines to enhance responses. Through an understanding of the mechanism of tissue gene expression, testing of live delivery vectors that may replicate antigen processing and expression will be attempted. 4.The importance of cytokines in response to vaccination and pathology will be examined, particularly against foodborne parasites like Toxoplasma gondii. Expression of recombinant IL-12 will be evaluated for its effect on protection and its potential as an adjuvant in combination with vaccination against toxoplasmosis. Detailed studies to define immunity to T. gondii will focus on effective vaccines; efforts will be aimed at identifying unique antigens that stimulate protective anti-parasite responses. Parallel efforts will be focused on use of DNA vaccination to stimulate protective immune responses against this foodborne parasite. 5.Collaborative efforts will identify antigens of Haemonchus contortus for use in vaccine trials and the geographic distribution of antigens Multiple genes encoding intestinal antigens from H. contortus have been cloned. These include GA1, cysteine protease and metallopeptidase proteins. Recombinant proteins encoded by these antigens will be expressed in bacteria as glutathione transferase (GST) fusion proteins, isolated by conventional methods and tested in immunization trials (WA). Moreover, antibody, nucleic acid probes and PCR primers exist to determine geographic conservation of gut antigens that are vaccine candidates of this parasite. Field isolates of H. contortus from geographically disparate worm populations will be evaluated for conservation of these antigens (LA, TX, VA. WA). 6.Whole intestine dissected from adult worms (WA), or H11-HgalGP (Moredun Research Institute) will be used to immunize kids or lambs, respectively. Immunized animals will be field challenged (kids in MO; lambs in LA) on pasture during known periods of high parasite transmission. Immunized and challenged animals will be compared with respective control groups to determine whether immunization methods and antigens, shown to induce protection against laboratory infections, will have efficacy against natural challenge infections. Recent work has indicated that adjuvants other than those currently being used should be evaluated for sustaining the antigenic activity of the antigens. These studies will be conducted in MO, LA and WA with additional collaboration with the Moredun Research Institute in Scotland(D Smith). 2C.Identify genetic differences in responses to gastrointestinal nematodes both within and between breeds. 1. Microsatellite genomic markers for sheep will be used to identify alleles that may account for differences in resistance to H. contortus infections among sheep breeds (LA,TX,UT). Gulf coast sheep (Native sheep) are significantly more resistant to H. contortus than Suffolk sheep. Approximately 400 F2 lambs (from large half-sib families) will be phenotyped for H. contortus infection level based on fecal nematode egg count data. DNA from lambs classified as resistant (20% lowest infection level) and susceptible (20% highest infection level) will be compared and screened across a panel of 150 microsatellite markers that are spaced 20-30 cM apart on the ovine gene map. We aim to map Quantitative Trait Loci (QTL) fixed for alternative alleles in the two breed groups which explain the breed difference. We will also test the hypothesis that major QTLs are not fixed, but are segregating within one or the other breed. Analyses will be performed by regression based interval mapping methods. In addition, a similar, but smaller resource population consisting of Native and Rambouillet sheep at Texas A&M University (TX) will be similarly assessed. Other collaborations on this study are with Utah State University, the University of Georgia (A McGraw), and the Roslin Institute in Scotland (C Haley). 2. The feasibility of selection for resistance to H. contortus infection as a management tool in profitable wool breeds will be investigated by crossbreeding experiments using resistant St. Croix sheep. In addition, individual variation will be assessed in a University flock as a prelude to an intrabreed selection program aimed at increasing parasite resistance (VA). Positive results on these projects will provide resources for mechanistic investigations in collaboration with LA. Experiments will also be conducted to characterize parasite resistance among goat breeds using fecal egg count analysis and hematologic parameters. 3.Cooperative studies (MD, UT) have been initiated to begin linkage mapping of genes involved in resistance to GI nematodes in cattle. Phenotypic data (currently third generation) will be combined with linkage mapping to identify genomic areas associated with enhanced or diminished resistance to the parasites. A number of polymorphic probes which give moderately good coverage of the genome have been identified, and the statistical model for data analyses based on the population structure has been developed. Offspring of selected breedings of cattle will continue to be tested for their parasite resistance phenotype. Past work has indicated that several commonly used measures of parasite burdens are inaccurate, or are applicable to the less pathogenic genera infecting cattle. New parameters are being continually tested for their accuracy and precision in both detecting parasite numbers in the host, and for measuring host immunity to the parasites. 4.Immune based mechanisms that contribute to differences in resistance demonstrated by different sheep breeds will be evaluated by several approaches. Dexamethasone will be used in attempts to abrogate resistance (LA). CD4+ T lymphocytes will also be depleted in resistant lambs in attempts to abrogate resistance (LA, WA). Humoral and T lymphocyte responses, including mucosal and lymph node responses from the abomasum, will be compared between suppressed/depleted and control lambs. Samples of abomasum will be evaluated by quantitative histological analysis of mast cells, globule leukocytes, B cells and intracellular cytokines (LA, VA). 5.Determination of the genes which control T. gondii resistance will require testing many more SLA inbred pigs, as well as testing outbred swine. As candidate genes involved in resistance are identified, studies will be needed to determine their applicability in both inbred and outbred populations of pigs. Currently most pigs which are resistant have fewer parasites in their tissues. For foodborne protozoan parasite infections it remains to be determined whether genetic resistance must be complete, i.e., whether no parasite can be left in the tissue or whether significant decreases in parasite burden are effective. An alternate approach to controlling this infection could involve combination of genetic control with vaccination. Because the swine industry has been so effective in controlling parasitic infections in their modern facilities research in MD has expanded into detailed analyses of the developing immune system of the neonatal pig. In collaboration with PIG USA studies are underway to assess mucosal immunity in neonatal pigs. These data are being collected on pigs from defined genetic lines so that future studies can be performed to correlate parameters associated with overall disease resistance with the genetic alleles that regulate mucosal immunity and the related cytokine responses. 6.Due to increasing drug?resistance of Eimeria parasites, development of alternative control strategies toward coccidiosis control is critically needed. Although the nature of host genes which are involved in the control of coccidiosis is not known, many chicken DNA markers which are available now enable us to explore a marker?assisted genetic improvement strategy for poultry disease control. Researchers at the Avian Disease and Oncology Laboratory, East Lansing, MI, are collaborating on an effort to use chicken genome markers to map different commercial lines of poultry. A new program will be initiated to identify microsatellite markers which are associated with coccidiosis disease resistance in commercial broiler chickens. Lines of poultry are simultaneously being evaluated for their genotypes, using the microsatellite markers, and for their resistance to several important poultry diseases, including Marek's disease and coccidiosis. These studies should lead to the identification of DNA markers associated with disease resistance and should enable producers to establish a marker?assisted genetic improvement strategy for their core breeding lines. These DNA marker?based gene improvements in chickens will lessen the production losses due to parasites both by developing parasite resistant stock and by avoiding costs associated with use of drugs to control these infections. Overall, consumer confidence will be enhanced. 2D.Identify new or improved methods of diagnosis of parasitic infections 1.Although easy to perform, current diagnostic tests for cryptosporidiosis are based on positive binding to parasite oocysts by antibodies which are non-specific. Future work will build on the identification of C. parvum specific antigens. Preparation of specific sera and confirmation of reactivity with recombinant proteins will facilitate the development of C. parvum?specific monoclonal antibodies for detection of C. parvum oocysts in environmental samples. 2.Experimentation will attempt to develop novel nucleic?acid based molecular diagnostic assays for the identification of Cryptosporidium species, genotypes of C. parvum, and other emerging agriculture?mediated pathogens (MD). The value of LDS isozymes of C. parvum as targets for therapeutic intervention or for detection of viable oocysts in a simple dip stick test will be examined. These assays will be evaluated using portable, analytical thermal cycler platforms. The feasibility of multilabeled, fluorogenic PCR probes for simultaneous one?tube, real?time identification of several pathogenic microorganisms will also be evaluated. Both inexpensive, high?throughput sample preparation methods and automated sample processing platforms will be tested for use in providing nucleic acids suitable for molecular diagnostic assays. Testing will be conducted to determine if these techniques are applicable to the detection of parasitic organisms in the environment. Studies will also be conducted to examine the utility and cost?effectiveness of these assays for use by various agricultural, food safety, and environmental?quality agencies and laboratories. 3.Ostertagia ostertagi is the most economically important parasite of cattle in the US. Because of the particular biology of this host-parasite system, there are no effective means to adequately estimate the number of Ostertagia ostertagi without killing the host. Recently studies in MD demonstrated that Ostertagia differ from all other trichostrongyle nematodes by the addition of a repeat sequence in a highly conserved region of ribosomal DNA. Using PCR technology it is possible to identify DNA from Ostertagia eggs, and to develop a semi-quantitative assay that detects Ostertagia eggs in the feces. This assay will be used to determine if the number of Ostertagia eggs in feces is representative of the number of adult Ostertagia in cattle (MD). As a corollary, attempts will be made to identify the number of eggs that correlate with economic loss. This latter aspect is very complex, and will involve the input of a number of the co-operating institutions (GA, KS, LA, MD, MO, MN, TX, VA). OBJECTIVE 3: Integration of parasite control practices into livestock production systems. Coordinator: Stromberg (MN) 3A.Examine the effect of soil and crop management practices on parasite transmission. 1.These studies will determine the impact of various soil and crop management strategies on the cleansing of parasitic larvae from contaminated pastures. A long-term experiment has been underway to determine the rate of soil carbon restoration on highly degraded, previously cultivated land using coastal bermuda grass pasture, hayed and unharvested management systems. The area was free of parasitic larvae prior to grass establishment and an effort has been made to prevent contamination of the area by judicious pre-grazing anthelmintic treatment of cattle. Parasites present will be identified and quantified in an effort to associate grazing intensity, pasture fertilizer source (poultry litter and inorganic), with the pasture parasite burden. Pasture parasite burdens will be determined using parasite-free tracer calves. The next phase of this experiment will include overseeding the bermuda grass with tall fescue in order to move toward almost year-around grazing. Characterization of the pasture parasite burden will continue in the second phase. Another experiment is being planned to investigate the impact of endophyte-infected tall fescue on subsequent cropping systems. As a part of this study, which will be executed on parasite-contaminated pastures, the impact of various soil and crop management systems (including conventional and minimum tillage, with and without cover cropping) on pasture larval survival will be evaluated (ARS-GA, GA). 3B.Evaluate efficacy of integrated anthelmintic/pasture management programs in parasite control. 1.Another group of studies will evaluate how combinations of chemotherapeutic treatment and animal/pasture management strategies impact species shifts and pasture larval contamination. These studies will determine the impact of managed intensive rotational grazing and the use of anthelmintics on the productivity of stocker beef cattle, cows and their calves, dairy replacement heifers and lactating dairy cows as well as the maintenance of grass (forage) availability and gastrointestinal parasitism. Most if these will be multi year studies using replicates and comparison to conventional methods. These studies will be conducted at multiple sites (ARS-GA, ARS-MD, MO, MN, TX) 2.Studies will continue on a Pennsylvania dairy farm that has been practicing intensive rotational grazing for the past 13 years. Previous work had demonstrated that the parasite control procedures initiated at the onset of the grazing program were insufficient to control economic losses due to GI nematodes. Using an intensive strategic anthelmintic program, the producers pastures were returned to an infection level where little economic impact due to the parasites could be demonstrated (LA, MD). Future studies will use this well-characterized working dairy to determine the effect of different management and drug programs on the control of GI nematode parasites. Because this producer is an active member of the Northeast Grazing and Research Center, the Northeast Pasture Consortium, and a number of grazing discussion groups, the results of these studies will be directly conveyed to producers through the auspices of these organizations (LA, MD, MN). 3C.Evaluate the impact of management systems on the development of anthelmintic resistance. 1.Sheep and goat farms using a variety of anthelmintic parasite control programs will be tested for anthelmintic resistance in LA, MN, MS, TX using a larval development assay. The prevalence of resistance and its association with management practices in both sheep and goat flocks will be evaluated and resistant parasite genera will be identified. 2.Strain differences in O. dentatum in swine will be compared experimentally by allowing development to the infective stage under four different environmental conditions. These infective L3 will be used to infect pigs and eggs will be harvested as the infections become patent and then weeks later into the patent period. This will be repeated using the early and late patency eggs. The determination of the pre-patent period for all 8 strains as well as the original will be compared. Preliminary data suggests that different prepatent periods will result. Morphological and DNA characteristics will be used in evaluating these differences. These strains will then be used in studies to determine anthelmintic resistance in LA. 3D.Examine the impact of anthelmintic treatment and nematode trapping fungi on transmission of equine strongyles 1.The efficacy of daily feeding of Duddingtonia flagrans following anthelmintic treatment on the reduction of strongyle parasite numbers in ponies maintained on pasture in LA. Mixed breed ponies 1 to 3 years of age, with naturally acquired infections of strongyles will be used. These will be housed on six pastures of approximately 1 acre each. Each pasture will house 3 ponies. Three pastures will be included in each treatment group. Ponies in one treatment group will receive fungal treatment and the control will not. Prior to turnout (day 0) all ponies will be treated with ivermectin at the recommended dose of 0.2 mg/kg of body weight. Fungal treatment will begin five weeks after treatment (day 35) a time prior to the reappearance of strongyle eggs in the feces. This will allow time for ponies to become accustomed to being fed fungus on a daily basis. It will also insure that the pastures are not contaminated with strongyle L3. Necropsies and worm counts will be conducted on ponies 10 weeks after the initiation of the fungal feeding (day 105) as previously described (Monahan et al., 1998). This period will allow for eggs to reappear, contaminate the pasture and provide for a significant exposure of ponies to pasture L3 during the peak transmission season. 3E.Impact of management practices on transmission of protozoan parasites 1.The efficacy of on-farm treatment technologies for destroying C parvum oocysts will be examined as well as the role of environmental factors affecting transport of oocysts to surface waters, and the use of grass buffer strips as a mechanism to minimize/eliminate oocyst transport. On-farm composting of manure to temperatures over 350C may be an effective method of reducing or eliminating oocyst viability. Pilot composting experiments in resulted in a 50% reduction in oocyst viability. Additional experiments are needed to optimize the time-temperature relationship and the effect of bulking agents on oocyst mortality It is frequently postulated that contamination of surface waters is due to surface runoff of oocysts from land-applied manure. However, environmental factors affecting the rates/extent of oocyst runoff are poorly understood. Major factors likely to control runoff are soil texture, soil slope, soil moisture, vegetation cover, and rainfall duration/intensity. The effects of these variables will be systematically investigated in order to formulate manure management strategies which eliminate or minimize the potential for oocyst transport. Grass buffer strips have been proposed as a means for reducing runoff of sediments, nutrients, and protozoan parasites/bacterial pathogens. Previous research has demonstrated the efficacy of buffer strips in removing sediments from runoff water. There is conflicting data regarding the efficacy of buffer strips in removing bacterial pathogens; there is no data with respect to protozoan parasites. To the extent that buffer strips may be implemented as a mechanism for reducing sediments and nutrients in runoff, it is important to determine their efficacy with respect to removal of pathogens/parasites from runoff waters (MD). 2. A herd of breeding cows that previously suffered an outbreak of abortion due to neosporosis will be studied over the course of 2 or more years. Pregnancy and calving rates will be determined and compared to serologic status of animals. Heifers born to seropositive cows will be included in the study, until they have calved themselves. The results of avidity ELISA testing, performed in Dr. Camilla Bjvrkman's laboratory in Uppsala, Sweden, will be compared over time to help refine test parameters used to determine duration of infection. Standard production parameters will be compared between seropositive and seronegative animals. These data should allow development of management and culling strategies based upon economic impact (IL).

Measurement of Progress and Results

Outputs

• Completion of the objectives described in this proposal will not only increase our understanding of host parasite relationships but bring us closer to applying this knowledge to both traditional and innovative methods of parasite control. Knowledge of the extent of anthelmintic resistance and evaluation of new products will allow cooperating members of this proposal to recommend the most efficient and economic use of antiparasitics in diverse regions of the U.S. This information will be presented to producer and practitioner groups around the country by committee members. Members of the regional project have been actively involved in outreach or continuing education programs in the past and the committee is now evaluating the feasibility of establishing a site on the world wide web (WWW) that would provide interested individuals and groups with state of the art information on parasite control. We believe that this form of technology transfer should reach our target audience in an effective manner. Although it is unlikely that vaccines for helminth and protozoan parasites will be commercially available in the next 5 years, continuing the collaborative research begun in the previous project will substantially increase the probability that important commercial vaccines will be developed in the near, and not the distant, future. Similarly, other innovative strategies like the use of nematophagous fungi will receive important testing during the 5 years covered by the next project and the relevance of these techniques to modern animal management will be evaluated under field conditions.

Outcomes or Projected Impacts

• It is important that the American livestock industries look toward development of integrated pest management systems for control of parasite pathogens. The development of these systems requires a thorough knowledge of the biology of the infecting organisms, the delineation of host responses that reduce parasite transmission and parasite induced pathology, the identification of potential agents for biological control, along with a better understanding of anthelmintic usage. A more complete knowledge in these areas will permit the development of sustainable agricultural systems that utilize host resistance and strategic drug treatment to reduce parasite transmission and pathology. Such integrated management systems will improve the efficiency of livestock operations by reducing costs of parasite control, lessening the potential for drug resistance in parasite populations and reducing the amount of drug usage in the food supply. Unfortunately, transmission of the parasites is intimately correlated with local environmental conditions and local management practices. So, while general tenets of parasite-host relationships will be applicable among producers throughout the U.S., specific recommendations aimed at parasite control will have to be tailored for different geographical regions. This need to refine information to fit local conditions mandates that attempts to develop integrated pest management systems for parasite control involve researchers from diverse geographical areas, and include experts in parasite epidemiology, immunology, genetics and molecular biology.

Milestones

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

Regional Technical Committee: The Technical Committee shall consist of the Administrative Advisor (non-voting), CSRS representative (non-voting), a technical representative from each participating SAES appointed by the director, and a technical representative of each cooperating USDA research laboratory named by the appropriate administrator. The responsibility of the Technical Committee shall be to coordinate research activities of the participants and to carry out such other functions as outlined in the Manual for Cooperative Regional Research SEA-CR/OD-1082.

Officers: These shall consist of a Chair, a Secretary, and a Member-at-Large. The Secretary will assume the office of the Chair and the Member-at-Large will assume the office of secretary.

Executive Committee: this subcommittee, consisting of the Chair, Secretary, Member-at-Large, and the Administrative Advisor will act as directed by, and for, the Technical Committee between meetings.

The time and place of the annual meetings will be decided by vote of the members after consultation with the Administrative Advisor or by the Executive Committee when so directed.

Literature Cited

Attachments

Land Grant Participating States/Institutions

AZ, CA, GA, IL, KS, MN, MS, NY, TX, VA

Non Land Grant Participating States/Institutions