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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #431978

Research Project: Immune, Molecular, and Ecological Approaches for Attenuating GI Nematode Infections of Ruminants

Location: Animal Parasitic Diseases Laboratory

2018 Annual Report

Objective 1. Identify and characterize parasitic immune modulators and local immune cell responses associated with GI nematodes of livestock. There is a pressing need for alternative control measures, such as vaccines, to complement/reduce antiparasite drug usage. Parasites evade host immunity by down-regulating or manipulating immune responses in favor of their own survival. Regulatory immune (T and B) cells that are up-regulated during infection may actually control an otherwise hostile environment and, in so doing, limit the host protective response. We propose to characterize parasite-provoked regulatory T/B cells. Further, parasitic immune modulators (PIMs) will be identified and characterized for their ability to induce host regulatory cells. Those PIMs responsible for cross-regulation will be selected as vaccine candidates. Objective 2. Identify proteomic and molecular markers for defining anthelmintic resistance among GI nematodes of livestock. Identifying genetic markers that differentiate the resistant and susceptible parasites will assist in herd management and long-term control. Our approach will be to utilize proteomic analyses and high-throughput transcript sequencing to discern genotypic differences that occur when resistant parasites are placed under drug selection. This will involve comparing drug treated vs. non-drug treated parasites that are resistant to macrocylic lactone class of drugs. This approach will minimize our risk of pursuing coincidentally-associated markers, and provide targets for new therapies. Putative new markers will be confirmed from environmentally-derived samples and will assist in reducing treatments with ineffective drugs. Objective 3. Explore the effects of accelerating climate change and ecological perturbation on managed and wild systems with emphasis on complex host-parasite relationships. Drug resistance cannot be viewed only as a problem of domestic livestock, but must include an evaluation of wild ungulates and the effects that environmental change can have on parasite transmission. With environmental change will come the movement of hosts and therefore exotic parasites into more temperate climates. Comprehensive definitions of parasite faunal diversity serve as the basis for exploring the impact of environmental change on complex systems, and are dependent on accurate baselines for host and parasite distributions. To discern the effects of accelerating climate change, information on parasite diversity will be obtained and summarized based on available georeferenced data from the literature, and on suitable biological collections. Basic studies in taxonomy, systematics and phylogeny of specific nematode groups will enable us to define species diversity in complex faunas among wild and domesticated North American ruminants. Parasite distributions will be mapped using geographic information systems (GIS) and Species Distribution Models (SDM) to assess the effects of global change on how invasion, colonization, and climate change influence the dissemination and persistence of drug resistance genes in the GI nematodes of ruminants.

Objective 1. Hypothesis: Parasite infection-elicited host regulatory cells are directly or indirectly induced by the parasitic immune modulators (PIMs). Rationale: Our recent report indicates that B cells and T cells with regulatory phenotypes are expanded in abomasa and the draining lymph nodes (dLN) in cattle raised on pastures. The proposed studies will investigate if single-infection by O. ostertagi, C. oncophora, or H. contortus, and mixed infections thereof induce a similar change in immune cell phenotypes. The ability of PIMs from ES products to enrich host regulatory cells will be investigated. Objective 2: Hypothesis: Drug treatment uniquely alters the genetic and proteomic profiles in resistant worms, enabling the identification of parasite targets associated with the resistance phenotype. Rationale: It is anticipated that the mechanism of resistance to macrocyclic lactones (ML) will be conserved among this broad group of parasites. We intend to focus on C. punctata, given that resistance is well documented and we possess two strains resistant to Ivermectin or Dectomax. Also, once putative markers have been identified, these can be tested against drug resistant forms of Trichostrongylus and Haemonchus, which are also available at our facility. Given the high level of genetic variation in and between nematode populations, resistant and sensitive isolates will also differ genetically in ways unrelated to resistance. To overcome this problem, we will compare proteomic and genomic data from a given, resistant isolate in the presence or absence of ML, hypothesizing that drug treatment will alter proteomic and gene expression profiles. This approach will focus on those genes and gene products regulated by drug treatment and which may differ from those isolates that are not drug resistant. Preliminary data will be generated using Ivermectin and the data will be validated using Dectomax. Objective 3: Hypothesis: Goal: Characterize diversity among species of Haemonchus in North American ruminants. Rationale: Surveys continue to broaden our understanding of parasite diversity, and establish baselines to assess changing patterns of distribution. Several key taxa remain poorly known in North America, and important biogeographic zones have been poorly documented. Borderland areas between managed and natural systems remain a concern given the poor understanding of species diversity for nematodes and their exchange between free-ranging and domestic hosts. Haemonchus nematodes are present in the Nearctic by recent anthropogenic introduction. Thus, radiation in tropical environments suggests this fauna is currently constrained in distribution by patterns of temperature and humidity. Global distributions are attributable to human-related translocation with domestic stock and would be anticipated to respond to accelerating climate warming and environmental change. To better assess change and distributions, we will collect and characterize isolates of Haemonchus and other parasites both morphologically and genetically, then use GIS and SDM’s to model parasite distributions based applications and mapping for ruminant helminth faunas.

Progress Report
Proteins secreted by parasitic nematodes that control inflammation and blood coagulation are potential vaccine candidates. Haemonchus contortus is a parasitic nematode that infects small (sheep and goats) and large (cattle) ruminants. Haemonchus can be devastating because it is the most prevalent blood feeding nematode in temperate regions of the world that raise livestock. Infections with Haemonchus cause severe anemia in moderately to heavily-infected animals because the worms require blood meals beginning as early as the fourth stage and continuing throughout the adult stage. Once embedded in the mucosal lining of the host’s stomach (abomasum), the worm not only controls the inflammatory response which is necessary for the host to abate the infection, but it also controls platelet activation which is necessary for blood coagulation and would otherwise prevent the worm from feeding. In so doing, the parasite creates an environment within the host that is conducive to longevity. Unfortunately, this parasite has become refractory to all classes of drugs typically used to treat nematode infections. Thus, alternative approaches to control are required. We have identified a class of secreted enzymes in nematodes i.e., apyrases, that degrade ATP and ADP which are important in regulating inflammation and blood coagulation. Controlling the parasite’s ability to degrade ATP and ADP may result in blood coagulation, interfere with the parasite’s feeding and therefore protect the host against infection. Therefore, we cloned, expressed, purified and tested the activity of this enzyme. Preliminary results showed nearly 100% protection against infection to challenge in cattle. Additional trials are ongoing in sheep, the primary and most natural host for Haemonchus. There are 1.6 billion sheep and another 0.5 billion goats worldwide. Of these, 800 million sheep and 50 million goats are raised in countries where control of haemonchosis is financially and logistically feasible and required. If successful, this will be the first vaccine of its kind to prevent haemonchosis. We sought to better understand how parasitic worms control local inflammation in the bovine gut by characterizing the immune cells that proliferate in lymph nodes proximate and distal to worm infections and by examining tissue-specific expression of genes essential to important immune responses. We have found that early in the infection process, immune B cells increased while immune T cells decreased in the draining lymph nodes, but that tissues proximate to the infection site undergo immunosuppressive effects suggestive of local suppression of inflammation leading to maintenance of the infection. These mixed and unfocused responses help explain why cattle generally fail to develop protective immunity to re-infection with O. ostertagi. Having a better understanding of how host immune responses are manipulated by the parasite will better facilitate developing vaccine candidates, and have prompted development of new recombinant proteins that may overcome such local immune suppression. The biggest deterrent to controlling nematodes of large and small ruminants is the escalation of drug resistance among the populations of worms most prevalent on North American pastures (Objective 3). Management is multifaceted; first, by locally controlling the worms that exist on commercial farms and second by ascertaining and controlling transmission to local wildlife that can act as a reservoir to reintroduce pathogens back onto production facilities. To date, no information has been generated that describes the prevalence of worms within wild ruminants that are also common in domestic livestock. We have contacted State Wildlife Departments from all 50 states and received responses from 25. From these, we requested and received approximately 600 fecal samples which were subsequently cultured for parasites and assayed by molecular methods. Among the 600 samples, 180 were deemed positive for nematodes. We have collaborated with scientists in the Department of Veterinary Medicine at the University of Calgary to sequence these DNAs and identify the species content and proportions. These results will be the first of their kind to ascertain the level of putatively drug resistant parasites within wild ruminants that also frequent U.S. production facilities. Trichostrongylus colubriformis is a parasitic nematode most frequently found in sheep and goats but can also infect cattle along with the cattle species Trichostrongylus axei. In attempts to collate data from the drug resistant forms of Haemonchus, Cooperia and Teladorsagia, we have embarked on a project to sequence the genome and transcriptome of Trichostrongylus colubriformis for comparative studies (Objective 2). To date, the genome has been sequenced and is now being assembled. Transcriptomic and proteomic approaches for identifying genes and proteins that are regulated in drug–resistant parasites will require that RNA from the various stages of development be generated. RNA from the infective L3 has been produced and sequencing will ensue once tissue from parasitic L3, L4 and adult worms has been isolated. Within this population of genes and proteins, we hypothesize using this information to validate genetic markers for differentiating drug-resistant from drug-sensitive worm populations. This will provide the means to identify and manage the early stages of drug-resstant parasites within U.S. beef herd production systems. Haemonchus contortus is a gastro-intestinal (GI), blood-feeding parasite of livestock that causes major production losses globally. Current control measures rely heavily on anthelmintics which are no longer effective given the rise in drug resistance. Thus, alternative control strategies are in demand. Recent advances in genomics have revealed key factors in pathogenesis, development and host-parasite relationships. Among these, parasite-derived proteases and protease inhibitors have been implicated in controlling host responses to the parasite, host evasion and digestion. We used database analyses and comparative genomics to identify Haemonchus peptidases and peptidase inhibitors that are conserved among blood feeding nematodes and arthropods. A collection of 335 proteins was identified and characterized where active site sequence predictions revealed conserved cleavage sites among similar proteins found in blood feeding pathogens, even in those that are not parasitic. Since this collection of proteins is likely involved in host adaptation and parasitism, it may provide new vaccine targets for controlling haemonchosis (Objective 1). Assessment of Haemonchus specimens (Objective 3) and insuring we have pure stocks of nematodes (Objective 2) demand accurate, sensitive and specific methods of identification stages which are morphologically indistinguishable i.e., eggs. Diagnosis of GI nematodes in production facilities is critical for ascertaining if drug intervention is necessary and if drug resistance is present on the farm and in the surrounding environment. Work has been advanced to develop a test to differentiate and quantify mixed infections of GI nematodes using fluorescently-labeled polymerase-chain reaction (PCR) products and a capillary based sequencer. Tests on monospecific infections, experimentally-mixed infections and environmentally-derived samples have now shown the test to meet all criteria as an antemortem assay for nematode infections. The assay can identify at the genus level Haemonchus, Ostertagia, Cooperia, Trichostrongylus, Oesophagostomum and Nematodirus parasites. All these parasite groups infect cattle and small ruminants, and drug resistance has been identified in most of them. Species of Haemonchus introduced into North America are H. contortus, H. placei, and H. similis. For many years, these were considered the dominant pathogens of this genus that infect cattle, sheep and goats. Recently, specimens of Haemonchus collected from Pronghorn antelope in Texas, although like H. contortus, exhibit morphological characters not typically found in of any North American species. Extensive comparative morphological assessments of these specimens are being conducted to determine; 1) if morphological boundaries defined for the Haemonchus contortus are applicable to these specimens from Pronghorn; 2) if the current morphological descriptions require revision for this species, or; 3) if the isolates from Pronghorn represent a new, previously unrecognized species of Haemonchus (Objective 3). This work is important because Pronghorn may act as a reservoir for introducing this new phenotype into domestic livestock. If drug resistant, it poses yet another threat to the large and small ruminant production systems in North America.

1. Synthesizing the study of insect pests and animal parasites. Parasite–host and insect–plant researchers make notably little use of one another’s contributions to understanding the forces that drive host-switching, despite obvious similarities in the systems they study and the agricultural processes they seek to safeguard. For example, parasitologists traditionally have assumed parasites to have co-speciated with their hosts, resulting in a high degree of specialization and a poor ability to exploit new kinds of hosts, whereas those studying insect–plant associations have placed greater attention on host shifts and the absence of extreme specialization. ARS scientists, working with an international team of collaborators, synthesized and interrogated these contrasting scientific traditions and developed a synthetic framework for understanding the primary forces governing the likelihood, pace, and character of new host colonization via a process termed “ecological fitting.” A common research program, drawing on the breadth of available data, better prepares us for the challenges posed by introduced species and global change.

2. Quantifying harms imposed by parasites in wild ungulates. Domestic livestock are managed in ways intended to promote animal health, but little is known about the consequences of parasitic infection to the survival and reproduction odds (fitness) of wild ungulates. This question deserves attention, because parasitic infection does not always induce clinical illness, and illness may not always harm fitness, perhaps explaining the mix of resistance and tolerance traits that have evolved in these wild hosts. ARS scientists in Beltsville, Maryland, working with an international team of collaborators, evaluated the harms imposed by each of nine parasitic agents prevalent in one wild ungulate (Dall’s sheep), evaluating associations between prevalence, host age, and host body condition. Doing so allowed them to document harms induced by one of these, the abomasal nematode Marshallagia marshalli, and underscoring the resilience that these wild hosts exhibit in the face of many other parasitic threats. The basis of that resilience may be useful in enhancing the health of domestic livestock, bread for production characteristics but often more susceptible to infection-induced harms.

Review Publications
Aleuy, O., Ruckstuhl, K., Hoberg, E.P., Veitch, A., Simmons, N., Kutz, S. 2018. Diversity of gastrointestinal helminths in Dall's sheep and the negative association of the abomasal nematode, Marshallagia marshalli, with fitness indicators. International Journal for Parasitology. 13:3.
Nylin, S., Agosta, S., Bensch, S., Boeger, W.A., Braga, M., Brooks, D.R., Forister, M.L., Hamback, P.A., Hoberg, E.P., Nyman, T., Schapers, A., Stigall, A. 2018. Embracing colonizations: A new paradigm for species association dynamics. Trends in Ecology and Evolution. 33(1):4-14.