Progress 01/19/11 to 01/18/16
Outputs
Progress Report Objectives (from AD-416): 1) Determine what genetic and genomic features distinguish Trichinella spiralis from Trichinella murrelli. 2) Utilize genomics to determine if microsatellite loci can be used to trace zoonotic outbreaks of Trichinella spiralis. 3) Determine the genetic features that account for the epidemic spread of certain strains of Toxoplasma gondii. Approach (from AD-416): Investigations will be conducted to clarify how infections in wildlife influence the safety of pastured pork. Accordingly, first identify heritable differences between two related species of Trichinella, only one of which (T. spiralis) severely compromises pork safety by evading swine immunity. The other, Trichinella murrelli, predominates in North American wildlife but fails to thrive in swine. By comparing the genomes of these two parasites, the intent is to establish a basis for exploring what makes pigs so especially vulnerable to T. spiralis. Secondly, develop the means to trace chains of transmission of Trichinella spp. Using markers which have already established the long- term dispersal history of T. spiralis (to the Americas in the pigs and rats brought by European colonists), researchers will attempt to discriminate instances of persistent on-farm transmission from sporadic introductions of T. spiralis to swine herds. Finally, the genetics of T. gondii reproduction will be characterized. Both sexual and asexual reproduction can occur in T. gondii, and available data provide conflicting evidence as to the relative importance of each reproductive mode. These incongruous data leave in doubt whether this parasite evolves as an assemblage of distinct lineages, or whether it more closely resembles a coherent, interbreeding species. Additional data are needed to better resolve how T. gondii propagates and evolves. These results will help determine whether particular strains pose elevated food safety risk will help anticipate this parasite's evolutionary response to preventative interventions. This is the final report for the project 8042-42000-016-00D terminated in May, 2016. A series of studies were completed that advance, beyond what had been initially proposed, knowledge in the comparative genetics and genomics of species of Trichinella. In particular, we used high- throughput, long-read sequences and high quality short-read sequences to obtain a complete genome assembly of Trichinella murreli, and we have used that assembly to improve the assembly of Trichinella spiralis ten- fold. We completed a study documenting the consequences of reproductive hybridization between two types of Trichinella, and we reported in another study the unexpected occurrence of natural hybrids between Trichinella spiralis (associated with swine) and Trichinella britovi (prevalent in wildlife) in Europe. Under Objective 2 we evaluated a class of variable genetic markers (termed microsatellites) for their usefulness in tracing zoonotic outbreaks of Trichinella spiralis and found them to be of limited value in locales where the parasite is highly inbred; when such analyses are restricted to fewer than a dozen loci, as has been typical, these genetic features offer limited traceback power in the Americas and in Europe; but this limitation has been largely overcome by our taking advantatage of advances in genome sequencing technology. The broader goal (outbreak tracing) was furthered by better, cheaper, and more precise and reproducible alternatives. Those opportunities are being more fully elaborated in the successor project. The goal of determining genetic features that account for the epidemic spread of certain strains of Toxoplasma gondii was satisfied, under Objective 3, by a landmark study, published in Nature Communications, identifying classes of secreted pathogenesis determinants whose elaboration and diversification has driven diversification within this important zoonotic species. These accomplishments cap a five-year period in which a great deal was accomplished towards each of the three objectives specified at the outset. Genetic and genomic features distiguishing Trichinella spiralis from Trichinella murrelli (Objective 1) have been identified, and that approach has been extended to other parasitic species in this genus, as well. Genetic markers to trace outbreaks of Trichinella (Objective 2) have been evaluated, leading to more promising approaches to addressing this difficult but important goal. Finally, genetic features accounting for the epidemic spread of certain strains of Toxoplasma gondii (Objective 3) were achieved beyond what could have been imagined at the outset, owing to effective collaboration and aided by substantial advances in genome sequencing technologies. Accomplishments 01 Foodborne parasites have evolved to influence host defenses. In a landmark study published in Nature Communications, ARS researchers in Beltsville, Maryland, collaborated with an international consortium to determine what is unique to the zoonotic parasite Toxoplasma gondii and to determine which features of the parasite�s genome differ to the greatest extent from one isolate to another. Dozens of isolates were sequenced in their entirety, and compared to one another and to their closest relatives that do not burden human health. Several classes of genes were found to be unique to the agent of toxoplasmosis, or were found to be especially diverse in this disease agent. Protein which exert powerful effects on the physiology of host cells, and which help evade host immunity, were found to have evolved in this parasite to an especially great extent. The resulting database and analysis will serve as a major reference point for future work seeking to prevent and/ or treat animals and people contracting this disease. 02 Interbreeding is limited between two lineages of freeze-resistant Trichinella parasites. Interbreeding may homogenize otherwise distinct lineages and result in the spread of traits from one type to another; alternatively, lineages may avoid or be penalized by the occurrence of such interbreeding, resulting in the maintenance of stable differences. Trichinella nativa and Trichinella T6 are zoonotic nematode parasites which can be distinguished genetically despite occasional hybridization. ARS researchers in Beltsville, Maryland, determined that two types of Trichinella were able to mate, but that mating preferences, genetic incompatibilities, and/or differences in the fitness of offspring minimized the resulting genetic mixing. These barriers likely limit gene flow between these two lineages in natural settings, serving as a barrier to their homogenization and promoting their persistence as distinct and separate entities. As a result, epidemiologists, geneticists, and infectious disease authorities have a clearer understanding of the processes that keep related parasite taxa distinct. 03 Genetic evidence of natural interspecies genetic mixing among the foodborne parasites Trichinella spiralis and Trichinella britovi. Over a period of decades, ARS researchers in Beltsville, Maryland have played a leading role in recognizing distinctions between several distinct kinds of Trichinella, some of which can threaten food safety by consumption of infected meat. Working with collaborators in Europe, they discovered parasites of mixed ancestry, descended from a species common in European wildlife and also one more typical in domesticated swine lacking adequate biosecurity. This discovery raises the possibility that traits could spread from one species to another. Scientists and those managing the spread of animal and zoonotic diseases will benefit by understanding that connections between parasites that were previously considered entirely distinct, a circumstance with the potential to undermine efforts to improve health and trade relationships. 04 Likely origins, in fish, of a widespread and consequential group of single-celled parasites. Fish are the oldest and most diverse group of vertebrates; it therefore stands to reason that fish may have been the original hosts for many types of extant vertebrate parasites. ARS researchers in Beltsville, Maryland, working with colleagues at the Hungary Veterinary Medical Research Institute found remarkable diversity among these parasites of fish, and found evidence that each of the major lineages of coccidian parasites in mammals, birds, reptiles, and amphibians most likely originated as parasites of fish, first. These include the ancestors of Toxoplasma gondii and other parasites that form tissue cysts in intermediate hosts, a finding not previously appreciated. These results revise our view of the history of life on the planet, and direct renewed attention to the impact that such parasites may have on veterinary and human health. Parasitologists, evolutionary biologists, epidemiologists, and the public health community will benefit from more fully understanding the diversity and origins of this parasitic group, and diagnosticians will benefit from the wealth of genetic data made publicly available through this effort. 05 Defining another source of potential human exposure to Toxoplasma gondii. Horses are valued recreational and work animals in the United States, but in certain European countries they are also an important source of food. Consequently, it is important to understand the risks associated with consuming horse meat, especially when eaten raw (as is customary in some European countries). ARS scientists in Beltsville, Maryland, assisted a team of scientists at the Veterinary College in Cluj-Napoca, Romania in order to better characterize the risk posed to human health by the zoonotic parasite Toxoplasma gondii in horses. More than one-third of the Romanian horses surveyed were found to be infected. This indicates a significant risk to human health that had not been previously appreciated, a fact that will be important to those seeking to understand the epidemiology of this infection and limit the impact of human infections.
Impacts
(N/A)
Publications
- Lorenzi, H., Khan, A., Behnke, M.S., Liu, L., Namasivayam, S., Seshadri, S. , Hadjithomas, M., Karamycheva, S., Pinney, D., Brunk, B., Ajioka, J., Azjenberg, D., Boothroyd, J.C., Boyle, J., Darde, M.L., Dubey, J.P., Grigg, M., Howe, D., Kim, K., Rosenthal, B.M., Saeij, J., Su, C.L., White, M., Zhu, X.Q., Parkinson, J., Kissinger, J.C., Roos, D.S., Sibley, L.D. 2016. Comparative sequence analysis of Toxoplasma gondii reveals local genomic admixture drives concerted expansion and diversification of secreted pathogenesis determinants. Nature Communications. doi: 10.1038/ncomms10147.
- Hecht, L., Thompson, P., Zarlenga, D.S., Lavin, E., Rosenthal, B.M. Hybridization is limited between two lineages of freeze-resistant Trichinella during coinfection in a mouse model. Infection, Genetics and Evolution. 38:146-51.
- Franssen, F., Bilska-Zajac, E., Deksne, G., Sprong, H., Pozio, E., Rozycki, M., Rosenthal, B.M., Van Der Giessen, J. 2015. Genetic evidence of interspecies introgression of mitochondrial genomes between Trichinella spiralis and Trichinella britovi under natural conditions. Infection, Genetics and Evolution. 36:323-332.
- Rosenthal, B.M., Dunams, D.B., Ostoros, G., Molnar, K. 2016. Coccidian parasites of fish encompass profound phylogenetic diversity and gave rise to each of the major parasitic groups in terrestrial vertebrates. Infection, Genetics and Evolution. 40L219-227.
- Pastiu, A.I., Gyorke, A., Kalmar, Z., Bolfa, P., Rosenthal, B.M., Oltean, M., Villena, I., Spinu, M., Cozma, V. 2015. Toxoplasma gondii in horse meat intended for human consumption in Romania. Veterinary Parasitology. 15:393-395.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 1) Determine what genetic and genomic features distinguish Trichinella spiralis from Trichinella murrelli. 2) Utilize genomics to determine if microsatellite loci can be used to trace zoonotic outbreaks of Trichinella spiralis. 3) Determine the genetic features that account for the epidemic spread of certain strains of Toxoplasma gondii. Approach (from AD-416): Investigations will be conducted to clarify how infections in wildlife influence the safety of pastured pork. Accordingly, first identify heritable differences between two related species of Trichinella, only one of which (T. spiralis) severely compromises pork safety by evading swine immunity. The other, Trichinella murrelli, predominates in North American wildlife but fails to thrive in swine. By comparing the genomes of these two parasites, the intent is to establish a basis for exploring what makes pigs so especially vulnerable to T. spiralis. Secondly, develop the means to trace chains of transmission of Trichinella spp. Using markers which have already established the long- term dispersal history of T. spiralis (to the Americas in the pigs and rats brought by European colonists), researchers will attempt to discriminate instances of persistent on-farm transmission from sporadic introductions of T. spiralis to swine herds. Finally, the genetics of T. gondii reproduction will be characterized. Both sexual and asexual reproduction can occur in T. gondii, and available data provide conflicting evidence as to the relative importance of each reproductive mode. These incongruous data leave in doubt whether this parasite evolves as an assemblage of distinct lineages, or whether it more closely resembles a coherent, interbreeding species. Additional data are needed to better resolve how T. gondii propagates and evolves. These results will help determine whether particular strains pose elevated food safety risk will help anticipate this parasite's evolutionary response to preventative interventions. Good progress was made on each of the project�s objectives. Differences between species of Trichinella were characterized using genome sequencing of T. murrelli and improvements to the genome assemblies of it and T. spiralis. A manuscript describing important differences is in preparation. The work on traceback analysis adapted to improvements in technology. As opposed to relying on microsatellites (as envisioned in the project plan five years ago) we have instead elected to use genome sequencing as a more comprehensive and more powerful means to identify attributes that uniquely characterize particular outbreak strains. Data are still being collected for this revised effort, and analyses are pending. The third milestone was met by completing analyses of natural isolates of Toxoplasma gondii, in which we found striking new evidence for genetic and phenotypic transformation that occasioned its exploitation of domesticated animals. For some years, we have noted that strains of the parasite isolated from people and from domesticated livestock harbor notably little genetic diversity. Our recent work contrasts that homogeneity with far greater diversity in parasites derived from wildlife hosts. Moreover, we recently found the first evidence that parasites from certain wild hosts are ill-suited for transmission through domesticated cats; this was a remarkable and unexpected finding, since cats are so extraordinarily susceptible to most strains that have heretofore been encountered to date. Having used house cats as a means to research toxoplasmosis, we now must consider that those cats have selected for particular strains of parasites and encouraged their proliferation on farms, suburbs, and cities. These studies have also more clearly resolved patterns of recombination in this ubiquitous and costly parasite. Accomplishments 01 Discovered that domesticated forms of the agent of toxoplasmosis may be especially well-adapted for transmission through house cats. Toxoplasmosis remains an important source of foodborne illness and can result in severe congenital infections. Agricultural Research Service (ARS) researchers in Beltsville, Maryland, working with an international team of academic collaborators, compared parasites derived from domestic settings to those derived from the jungle interior of French Guiana. Although the sampling sites were not very distant, striking differences were observed among the parasites derived from each. To the great surprise of the team, these differences were not merely reflected in the genetics profiles of the parasites (far more diverse than those from wildlife), but also in the capacity of the parasites to be propagated by domestic cats. This finding indicates that those parasites commonly infecting people and food animals may have specifically adapted to transmission opportunities characteristic of domestic and agricultural landscapes. Finding the basis for efficient transmission through domestic cats could provide an important means to limit future human exposure to this important and pervasive parasite. (306669, 309231, 310714) 02 Characterized a new infectious zoonosis. ARS scientists in Beltsville, Maryland collaborated with Center for Disease Control (CDC) epidemiologists and an international team of pathologists and field biologists in order to characterize the agent of an emerging infectious disease, the symptoms of which derive from reactions to tissue cysts of parasite previously only known in non-human primates. Travelers to Malaysia were frequent victims. Based on evolutionary analyses conducted by ARS scientists, the source of infections was suspected to be a predatory snake or reptile. That prediction proved key to tracing the infection to its source, and prompted ongoing public health interventions to protect water supplies from the offending, newly discovered pathogen. 03 Determined that traditional goat husbandry may substantially contribute to toxoplasmosis exposure. Raising goats in settings that are highly contaminated with oocysts of Toxoplasma gondii may contribute significantly to human exposure to this zoonotic parasite. Increasing consumption of meat from goats raised in backyards that are also homes to the definitive host of this parasite (cats) elevates such concern. Working with veterinary researchers in Romania, ARS researchers in Beltsville, Maryland, estimated the prevalence of T. gondii goat-kids raised there for human consumption found one-third of them to be infected. This finding has alerted veterinary and health officials to a potentially important source of human infection. (Log 306689)
Impacts
(N/A)
Publications
- Khan, A., Shaik, J.S., Behnke, M., Wang, Q., Dubey, J.P., Lorenzi, H.A., Ajioka, J.W., Rosenthal, B.M., Sibley, L.D. 2014. NextGen sequencing reveals short double crossovers contribute disproportionately to genetic diversity in Toxoplasma gondii. Biomed Central (BMC) Genomics. 15:1168.
- Pastiu, A., Ajzenberg, D., Gyorke, A., Suteu, O., Balea, A., Domsa, C., Rosenthal, B.M., Cozma, V. 2014. Traditional goat husbandry may substantially contribute to human toxoplasmosis exposure. Journal of Parasitology. DOI:10.1645/13-483.1.
- Esposito, D.H., Stich, A., Epelboin, L., Malvy, D., Han, P., Bottieau, E., Da Silva, A., Zanger, P., Slesak, G., Van Genderen, P.J., Rosenthal, B.M., Fayer, R. 2014. Acute Muscular Sarcocystosis: an international investigation among ill travelers returning from Tioman Island, Malaysia, 2011 and 2012. Clinical Infectious Diseases. DOI:10.1093/cid/ciu622.
- Zarlenga, D.S., Hoberg, E.P., Rosenthal, B.M., Mattiucci, S., Nascetti, G. 2014. Anthropogenics: Human influence on global and genetic homogenization of parasite populations. Journal of Parasitology. 100(6):756-772. doi: 10. 1645/14-622.1.
- Khan, A., Ajzenberg, D., Mercier, A., Demar, M., Simon, S., Darde, M., Rosenthal, B.M., Verma, S., Dubey, J.P., Sibley, L. 2014. Geographic separation of domestic and wild strains of T. gondii in French Guiana correlates with a monomorphic version of chromosome 1a and enhanced transmission in the domestic cat. PLOS Neglected Tropical Diseases. DOI: 10.1371/journal.pntd.0003182.
- Calero-Bernal, R., Verma, S., Oliveira, S., Yang, Y., Rosenthal, B.M., Dubey, J.P. 2015. In the United States, negligible rates of zoonotic sarcocystosis occur in feral swine that, by contrast, frequently harbor infections with Sarcocystis meischeriana, a related parasite contracted from dogs. Parasitology. 142:549-556.
- Oltean, M., Kalm�r, Z., Kiss, B.J., Marinov, M., Vasile, A., S�ndor, A.D., Domsa, C., Gherman, C.M., Boireau, P., Rosenthal, B.M., Cozma, V., Mihalca, A.D. 2014. European mustelids occupying pristine wetlands in the Danube delta harbor infections with trichinella likely derived from domesticated swine. Journal of Wildlife Diseases. 50(4)972-975.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): 1) Determine what genetic and genomic features distinguish Trichinella spiralis from Trichinella murrelli. 2) Utilize genomics to determine if microsatellite loci can be used to trace zoonotic outbreaks of Trichinella spiralis. 3) Determine the genetic features that account for the epidemic spread of certain strains of Toxoplasma gondii. Approach (from AD-416): Investigations will be conducted to clarify how infections in wildlife influence the safety of pastured pork. Accordingly, first identify heritable differences between two related species of Trichinella, only one of which (T. spiralis) severely compromises pork safety by evading swine immunity. The other, Trichinella murrelli, predominates in North American wildlife but fails to thrive in swine. By comparing the genomes of these two parasites, the intent is to establish a basis for exploring what makes pigs so especially vulnerable to T. spiralis. Secondly, develop the means to trace chains of transmission of Trichinella spp. Using markers which have already established the long- term dispersal history of T. spiralis (to the Americas in the pigs and rats brought by European colonists), researchers will attempt to discriminate instances of persistent on-farm transmission from sporadic introductions of T. spiralis to swine herds. Finally, the genetics of T. gondii reproduction will be characterized. Both sexual and asexual reproduction can occur in T. gondii, and available data provide conflicting evidence as to the relative importance of each reproductive mode. These incongruous data leave in doubt whether this parasite evolves as an assemblage of distinct lineages, or whether it more closely resembles a coherent, interbreeding species. Additional data are needed to better resolve how T. gondii propagates and evolves. These results will help determine whether particular strains pose elevated food safety risk will help anticipate this parasite's evolutionary response to preventative interventions. Progress was made on all three of the project�s goals, which support NP108 Food Safety Component 1: Food-borne Contaminants. To determine features that distinguish Trichinella spiralis from Trichinella murrelli, expression libraries of each parasite were screened with serum from experiementally infected animals. The draft genome sequence of T. spiralis was used as a scaffold assembly for short reads derived from T. murrelli, and a comparative genomic analysis initiated. This was used as the basis for a novel gap closure campaign, premised on the (often validated) belief that the overall structure of the two genomes has been maintained over evolutionary time. To enhance efforts to trace outbreaks of trichinellosis, a combination of genetic markers was applied to additional field isolates to better understand the degree of individuality associated with given foci of transmission. In addition, further data were collected on an experimental cohort of parasites given the opportunity to interbreed from distinct parental lineages. The culmination of this study will define, with unprecedented precision, the limits of gene flow among two freeze-resistant forms of the parasite. Finally, the process of strain formation and dissemination in the agent of toxoplasmosis was explored as part of a consortium that has sequenced the genomes of tens of strains. The consortium of experts is finalizing a definitive report on genome variation in this important zoonotic parasite. In addition, the evolutionary dynamics of a second protozoan species (important to oyster health) was defined in a pair of notable papers, identifying patterns that resemble, to a striking degree, those characteristic of Toxoplasma gondii, providing a basis to surmise general trends for these and related parasites. Significant Activities that Support Special Target Populations: ARS scientists are working with three historically black colleges and universities (University of Maryland Eastern Shore, State University, and Tuskegee University) as well as minority students at area High Schools to enhance capacity and foster interest in research in poultry health and food safety. Accomplishments 01 Where goats are raised in poor hygiene, very substantial risks of toxoplasmosis ensue. Working with a Romanian study team, ARS scientists at the Beltsville Agricultural Research Center determined rates of infection with the agent of toxoplasmosis in goats raised in backyards. Because backyards are frequently also inhabited by cats which serve as a reservoir of infection, they suspected that goats raised there may experience elevated rates of infection. Indeed, one- third of the sampled goats was found to harbor infection, underscoring food safety concern as meat from these goats was intended for human consumption. 02 Hybridization among distinct parasite lineages can engender ones with new, epidemiologically important properties. It remains uncertain how continuously parasitic protists exchange genetic information, or whether they persist for long evolutionary intervals as reproductively independent strains. ARS researchers working at the Beltsville Agricultural Research Center have contributed to a growing awareness that certain foodborne parasites persist for long intervals as clonal types, punctuated by occasional but consequential episodes of genetic change. Turning their attention to Perkinsus marinus, the agent of Dermo disease that constitutes a major disease of oysters, they determined that current parasite populations harbor the vestiges of two lineages that have been evolving in isolation for an interval likely enduring several million years. New properties may emerge when long- diverged lineages are brought into contact, and it is possible that movements of oysters, whether owing to changing water temperature patterns or through intentional translocation of oyster seed, may have accelerated the pace of this form of genetic change. 03 Certain forms of the agent of Dermo disease in oysters have undergone markedly widespread dissemination. Dermo disease causes major economic losses for the oyster industry. Perkinsus marinus, like the agent of foodborne toxoplasmosis, is a protozoan parasite in the phylum Apicomplexa. Its population genetics were studied by ARS scientists at the Beltsville Agricultural Research Center owing to its economic impact and to develop comparative data on the nature of such veterinary parasites. Fine-scale genotyping of an array of populations sampled from throughout the Eastern Seaboard and the Gulf of Mexico showed that whereas certain genotypes were endemic to particular regions, others have become widely established throughout the range. Moreover, certain important estuaries, such as Delaware and Chesapeake Bays, are home to an especially diverse array of such parasites, making them likely incubators for new genetic forms. These data provide a new lens through which to trace the movement of oysters and their parasites, and underscore the potential risks associated with intentional movement of oysters between regions. 04 Evolutionary responses of innate and adaptive immunity. Immunity in human beings and our livestock depends both on innate pathogen recognition molecules as well �adaptive immunity,� a system that adjusts to changing microbial challenges throughout the lifecourse. Adaptive immunity was refined in vertebrates, but invertebrates continue to rely entirely on inate pathogen recognition. An ARS researcher at the Beltsville Agricultural Research Center, working with a student at the University of Maryland, considered how the advent of �adaptive immunity� may have promoted diversification in pathogens and altered the role and evolution of innate pathogen recognition receptors. The diversity of innate receptors in organisms lacking adaptive immunity far exceeds that of organisms, such as humans, that have adaptive immunity. Although innate immunity remains important to vertebrates, these components have become entrained in complex signaling pathways, constraining their freedom to diversify. By contrast, invertebrates continue to elaborate novelty in their innate receptors, necessitated by ever-changing microbial and parasitic threats.
Impacts
(N/A)
Publications
- Ward, A.E., Rosenthal, B.M. 2014. Evolutionary responses of innate Immunity to adaptive immunity. Infection, Genetics and Evolution. 21:492- 496.
- Thompson, P.C., Rosenthal, B.M., Hare, M.P. 2014. Microsatellite genotypes reveal some long distance gene flow in Perkinsus marinus, a major pathogen of eastern oysters. Journal of Shellfish Research. 33(1):195-206.
- Thompson, P.C., Hare, M.P., Rosenthal, B.M. 2014. Hybridization between previously isolated ancestors may explain the persistence of exactly two ancient lineages in the genome of the oyster parasite Perkinsus marinus. Infection, Genetics and Evolution. 24:167-176.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): 1) Determine what genetic and genomic features distinguish Trichinella spiralis from Trichinella murrelli. 2) Utilize genomics to determine if microsatellite loci can be used to trace zoonotic outbreaks of Trichinella spiralis. 3) Determine the genetic features that account for the epidemic spread of certain strains of Toxoplasma gondii. Approach (from AD-416): Investigations will be conducted to clarify how infections in wildlife influence the safety of pastured pork. Accordingly, first identify heritable differences between two related species of Trichinella, only one of which (T. spiralis) severely compromises pork safety by evading swine immunity. The other, Trichinella murrelli, predominates in North American wildlife but fails to thrive in swine. By comparing the genomes of these two parasites, the intent is to establish a basis for exploring what makes pigs so especially vulnerable to T. spiralis. Secondly, develop the means to trace chains of transmission of Trichinella spp. Using markers which have already established the long- term dispersal history of T. spiralis (to the Americas in the pigs and rats brought by European colonists), researchers will attempt to discriminate instances of persistent on-farm transmission from sporadic introductions of T. spiralis to swine herds. Finally, the genetics of T. gondii reproduction will be characterized. Both sexual and asexual reproduction can occur in T. gondii, and available data provide conflicting evidence as to the relative importance of each reproductive mode. These incongruous data leave in doubt whether this parasite evolves as an assemblage of distinct lineages, or whether it more closely resembles a coherent, interbreeding species. Additional data are needed to better resolve how T. gondii propagates and evolves. These results will help determine whether particular strains pose elevated food safety risk will help anticipate this parasite's evolutionary response to preventative interventions. Progress was made on all three of the project�s goals, which support NP108 Food Safety Component 1: Food-borne Contaminants. To determine features that distinguish Trichinella spiralis from Trichinella murrelli, a series of experiments established differences in protein expression by these two parasites at various stages of their development. Proteins expressed exclusively, or especially abundantly, by one of these two species were sequenced and characterized. Immunological assays were designed in order to establish which of these might offer the most potential to diagnose animals exposed to one or the other of these parasites, only one of which persists in swine to an extent that gravely threatens pork safety. To enhance efforts to trace outbreaks of trichinellosis, isolates known to share a transmission history were compared using variable genetic markers. Greater resolution will be sought using additional markers. Finally, the process of strain formation and dissemination in the agent of toxoplasmosis was explored as part of a consortium that has sequenced the genomes of tens of strains. When completed, we will have our clearest view yet of the extent of diversity in this ubiquitous foodborne parasite and the methods it employs to adapt and disseminate. Significant Activities that Support Special Target Populations: ARS scientists are working with three Historically Black Colleges and Universities (University of Maryland Eastern Shore, State University, and Tuskegee University) as well as minority students at area High Schools to foster collaborative research in poultry health and food safety. Accomplishments 01 Proteins show important variation among species of Trichinella. Of late, increasing concern has been raised about the possibility that trichinellosis (once nearly completely eliminated from the American pork supply) may resurge as a foodborne parasitic threat, given the growing practice of raising swine outdoors where they may more frequently encounter infections. If that were to occur, the health of Americans could be compromised and very significant costs to production and testing might ensue. ARS researchers at the Beltsville Agricultural Research Center were therefore interested in comparing a parasite capable of thriving and persisting in swine with a closely related parasite that is instead adapted to wildlife hosts. In spite of the fact that the parasites are far more similar than they are different, proteins were discovered that specifically characterize one or the other of these parasites. Ongoing work will seek to understand the biological significance of those differences, and will seek to exploit those differences as a basis to judge whether or not a given pig harbors a parasite that endangers human health. These findings will interest veterinarians, public health officials, producers, and consumers of pork and may influence policies governing risk reduction in free-range and organic animal husbandry. 02 Backyard pigs are more dangerous than wild boar as a potential source of human trichinellosis. Americans rarely experience trichinellosis, and most cases can be attributed to consuming poorly cooked wild game. Conventionally raised pork no longer conveys appreciable risk, owing to progress in sanitation and control over the swine diet. In Romania, human outbreaks routinely persist, owing to either the hunting of wild boar or the slaughter of domesticated pigs raised by families for their own consumption. Working with a team of Romanian veterinarians, ARS researchers in Beltsville, MD established that pigs raised in for familial consumption actually are twice as likely to be infected as are wild boar. More encouragingly, the team confirmed that swine raised in Romania using modern confinement procedures were entirely free of infection. These data underscore that risk varies not so much as a function of geography, but rather as a function of the type of animal husbandry practiced. Risk would appear especially elevated where swine are raised near people, but without proper attention to rodent control or to the quality of swine feed. These data will interest veterinarians, epidemiologists, and public health officials concerned to reduce human exposure to illness. They may also bear on policies governing agricultural trade with Europe. 03 Detected early infections with Trichinella in swine may reduce public health risks. Parasitic infection with species of Trichinella is only rarely reported among American consumers of conventionally raised pork, but remains a concern for consumers of wild game and has been raised as an emerging concern for organic and free-range pork. Although most species of Trichinella excrete substances to which both pigs and people raise strong immune responses, the earliest stages of infection can go undetected (because immature parasites fail to make their presence known to the immune system) and certain parasites never develop the structures most irritating to our immune systems. ARS researchers at the Beltsville Agricultural Research Center, working in collaboration with colleagues from China and France, identified antigens expressed at particular stages of parasite development in order to overcome such limitations. This accomplishment provides a means to reduce the risk of �false-negative� tests for this parasite, which could further help ensure the safety of pork to American consumers. 04 Parasitic infections provide clues to fighting cancer. Although eliminating animal-borne parasites from the food supply unquestionably benefits food safety, experiments in laboratory animals has demonstrated properties of such parasites that could benefit human health. For example, mice prone to certain cancers are protected by infection with parasites of Trichinella. Other mice prone to inflammatory bowel disease can have their symptoms reduced by exposure to parasitic infection. Working with colleagues from China, ARS researchers at the Beltsville Agricultural Research Center explored the basis of such tumor and inflammatory suppression. By identifying how parasites reprogram cells in ways that prevent their cancerous growth, and modify the signals that otherwise induce inflammation, these findings may promote new avenues for fighting cancer and autoimmune disease. These results will be of interest to clinicians, veterinarians, parasitologists, immunologists, and cancer biologists. 05 Newly discovered variants of Toxoplasma are especially dangerous. Infection with Toxoplasma gondii has variable clinical outcomes, ranging from asymptomatic lifelong infection to serious neurological disease, blindness, and congenital defects. Preventing the most serious outcomes depends, in part, on reducing the exposure of women to infection while they are pregnant. But additional variation in risk evidently derives from the genotype of the parasite. Working with an international team, ARS scientists at the Beltsville Agricultural Research Center identified new parasite variants that provoke especially marked veterinary disease. Clinicians and epidemiologists will need to be on guard for the possibility that these genotypes may convey elevated risk to human health. 06 Ancient diversity among parasites found in fish. Parasites belonging to the Coccidia include members that threaten veterinary and human health. Although the best-studied of these organisms infect mammals and birds, the diversity of this parasitic group is vast and their ultimate origins heretofore unknown. In a groundbreaking study, ARS researchers at the Beltsville Agricultural Research Center working with Hungarian colleagues established that the coccidia infecting fish harbor especially ancient evolutionary diversity, and may well be the ultimate source of infections that plague people and our livestock. This information will be of interest to evolutionary biologists as a step forward in our understanding of the history of life, but may well have practical application as it points to the possibility of foodborne infection with uncharacterized parasites via the consumption of uncooked fish. 07 Food-borne bacteria have similar genomes despite very different origins. Listeria monocytogenes is an important foodborne infection, and it is important to identify the major routes of human exposure as well as the efficacy of measures taken to render food products free of contamination. Here, ARS scientists in Beltsville, MD worked with a team from Denmark to characterize and compare the genomes of two isolates derived from separate fish processing plants. In spite of their distinct origins, and in spite of a several year interval between isolations, these two isolates proved to be almost identical to each other, more so than to any other isolate presently recorded. Particular attributes of this strain of Listeria.
Impacts
(N/A)
Publications
- Turner, M., Lenhart, S., Rosenthal, B.M., Sullivan, A., Zhao, X. 2013. Modeling effective transmission strategies and control of the world�s most successful parasite. Theoretical Population Biology. 86:50-61.
- Zhao, Y., Liu, M.Y., Wang, X.L., Liu, X.L., Yang, Y., Zou, H.B., Sun, S.M., Yu, L., Rosenthal, B.M., Shi, H.N., Boireau, P., Wu, X.P. 2013. Modulation of inflammatory bowel disease in a mouse model following infection with Trichinella spiralis. Veterinary Parasitology. 194:211-216.
- Rosenthal, B.M., Ajioka, J.W. 2012. Population genetics, diversity and spread of virulence in Toxoplasma gondii. In: David, L.S., Howlett, B.J., Heitman, J., editors. Evolution of Virulence in Eukaryotic Microbes. Boston, MA: Wiley-Blackwell. p. 231-245.
- Holch, A., Webb, K., Lukjancenko, O., Ussery, D., Rosenthal, B.M., Gram, L. 2013. Genome sequencing identifies two nearly unchanged strains of persistent Listeria monocytogenes isolated in two different fish processing plants sampled six years apart. International Journal of Food Microbiology. 79:2944-2951.
- Wang, X.L., Liu, M.Y., Sun, S.M., Liu, Y., Wang, X.R., Chu, L.X., Rosenthal, B.M., Shi, H.N., Boireau, P., Wang, F., Zhao, Y., Wu, X.P. 2013. An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells. Veterinary Parasitology. 194:186-188.
- Wu, X.P., Liu, X.L., Wang, W.L., Blaga, R., Fu, B.Q., Liu, P., Bai, X., Wang, Z.J., Rosenthal, B.M., Shi, H.N., Sandrine, L., Vallee, I., Boireau, P., Wang, F., Zhou, X.N., Zhao, Y., Liu, M.Y. 2013. Unique antigenic gene expression at different developmental stages of Trichinella pseudospiralis. Veterinary Parasitology. 194:198-201.
- Pastiu, A.I., Gyorke, A., Blaga, R., Mircean, V., Rosenthal, B.M., Cozma, V. 2013. In Romania, exposure to Toxoplasma gondii occurs twice as often in swine raised for familial consumption as in hunted wild boar, but occurs rarely if ever among fattening pigs raised in confinement. Parasitology Research. 112(6):2403-2407.
- Liu, P., Wu, X.P., Bai, X., Wang, X.L., Yu, L., Rosenthal, B.M., Blaga, R., Lacour, S., Vallee, I., Boireau, P., Gherman, C., Oltean, M., Wang, F., Zhao, Y., Liu, M.Y. 2013. Screening of early antigen genes of adult-stage Trichinella spiralis using pig serum from different stages of early infection. Veterinary Parasitology. 194:222-225.
- Molnar, K., Ostoros, G., Dunams, D.B., Rosenthal, B.M. 2012. Eimeria that infect fish are diverse and are related to, but distinct from, those that infect terrestrial vertebrates. Infection, Genetics and Evolution. 12(8) :1810-1815.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): 1) Determine what genetic and genomic features distinguish Trichinella spiralis from Trichinella murrelli. 2) Utilize genomics to determine if microsatellite loci can be used to trace zoonotic outbreaks of Trichinella spiralis. 3) Determine the genetic features that account for the epidemic spread of certain strains of Toxoplasma gondii. Approach (from AD-416): Investigations will be conducted to clarify how infections in wildlife influence the safety of pastured pork. Accordingly, first identify heritable differences between two related species of Trichinella, only one of which (T. spiralis) severely compromises pork safety by evading swine immunity. The other, Trichinella murrelli, predominates in North American wildlife but fails to thrive in swine. By comparing the genomes of these two parasites, the intent is to establish a basis for exploring what makes pigs so especially vulnerable to T. spiralis. Secondly, develop the means to trace chains of transmission of Trichinella spp. Using markers which have already established the long- term dispersal history of T. spiralis (to the Americas in the pigs and rats brought by European colonists), researchers will attempt to discriminate instances of persistent on-farm transmission from sporadic introductions of T. spiralis to swine herds. Finally, the genetics of T. gondii reproduction will be characterized. Both sexual and asexual reproduction can occur in T. gondii, and available data provide conflicting evidence as to the relative importance of each reproductive mode. These incongruous data leave in doubt whether this parasite evolves as an assemblage of distinct lineages, or whether it more closely resembles a coherent, interbreeding species. Additional data are needed to better resolve how T. gondii propagates and evolves. These results will help determine whether particular strains pose elevated food safety risk will help anticipate this parasite's evolutionary response to preventative interventions. Progress was made on all three of this project's objectives, which support NP108 Food Safety Component 1: Food-borne Contaminants. To commence assembly of the Trichinella murrelli genome, short sequence reads were successfully organized by scaffold assembly to homologous regions of the Trichinella spiralis genome as a proof of principle and to guide ongoing assembly and annotation efforts. To improve the ability to trace outbreaks of Trichinella spiralis, additional variable genetic markers (containing microsatellite repeats) were validated and shown to discriminate among field isolates. Analyzing these markers also demonstrated that in nature, animals can become infected more than once, but that they seldom ever do. This finding is relevant for considering the possible efficacy of vaccines intended to safeguard veterinary and public health. Finally, new insights were gained on the global diversity of Toxoplasma gondii, including a comprehensive description of parasite diversity using all extant systems for genetic characterization. This achievement provides researchers, the world over, a common set of reference strains, overcoming a history of distinct genotyping systems in different laboratories. The result should hasten communication and progress in the field of toxoplasmosis epidemiology. In addition, progress was made in understanding the identities, distributions, and risks associated with several other food-borne parasitic agents including those in the genera Sarcocystis and Besnoitia. Significant Activities that Support Special Target Populations: Toxoplasmosis poses particular health risks to pregnant women, newborns, and persons with HIV-AIDS. Scientists have participated in outreach activities including presentations to the "Minorities in Agriculture, Natural Resources, and Related Sciences" annual meeting and mentoring a summer intern attending a Tribal College. Accomplishments 01 Genetics of Trichinella infections. Strong immune responses may strictl limit the opportunities for unrelated parasites, such as foodborne speci of Trichinella, to mate. ARS researchers in Beltsville, Maryland examin the diversity of Trichinella larvae in naturally infected animals. Almo without exception, they found that animals had been successfully infecte by only a single pair of parasites, as judged by the fact that larvae (their progeny) were full siblings. Therefore, immunity may typically preclude establishment of subsequent infections, a finding that further substantiates vaccination as a plausible strategy for future public heal interventions. 02 Gene flow among dissimilar types of Trichinella. The spread of biologic traits relevant to public health may be constrained by geographical, physical, and/or physiological barriers to reproduction among diverse forms of Trichinella. ARS biologists working in Beltsville, Maryland genotyped isolates of these food-borne parasites at loci (located in the nucleus) inherited from each parent, as well as at a locus (in the mitochondrion) inherited solely from the maternal line. Where each of t parasite lineages was known to infect wildlife, intermediate forms of th parasite (containing ancestry from both lines) were documented. In particular, one maternal lineage was found where two had been expected, substantiating the notion that introgression has resulted in the displacement, locally, of one by the other. These findings are among th first to explore the capacity of genes to flow from one lineage of Trichinella to another. Understanding whether traits can be shared amon lineages is important, because a prevalent method to ensure the safety o meat (freezing) would be undermined if it certain traits (i.e. freeze resistance) were capable of being shared. 03 An unexpectedly widespread chromosomal variant in Toxoplasma gondii. Toxoplasma gondii, is an important and prevalent parasite that can be contracted either by eating contaminated meat or by ingesting contaminat produce or water. In spite of the fact that the parasite can undergo sexual recombination, producing myriad variants in certain locales, othe strains are temporally stable and geographically widespread. The basis for persistence and dissemination of such strains was studied by ARS researchers in Beltsville, Maryland in collaboration with an internation team of academic scientists. They found that a particular variant of on of the parasite�s chromosomes occurs in myriad, otherwise unrelated stra This finding focuses attention on the genes encoded by this chromosome as it may offer functional explanation for the basis of this parasite�s dispersal capacity, and new means to intervene in its transmission to fo animals and to people.
Impacts
(N/A)
Publications
- Khan, A., Miller, N., Roos, D., Dubey, J.P., Ajzenburg, D., Darde, M., Ajioka, J.W., Rosenthal, B.M., Sibley, L. 2011. A monomorphic halotype of chromosome Ia is associated with widespread success in clonal and nonclonal populations of Toxoplasma gondii. mBio. 2:e00228-11.
- La Rosa, G., Marucci, G., Rosenthal, B.M., Pozio, E. 2012. Development of a single larva microsatellite analysis to investigate the population structure of Trichinella spiralis. Infection, Genetics and Evolution. 12(2) :369-376.
- Dunams, D.B., Reichard, M.V., Torretti, L., Zarlenga, D.S., Rosenthal, B.M. 2012. Discernible but limited introgression has occurred where Trichinella nativa and the T6 genotype occur in sympatry. Infection, Genetics and Evolution. 12:530-538.
- Chen, X., He, Y., Yonghua, L., Olias, P., Rosenthal, B.M., Cui, L., Zuo, Y. , Yang, Z. 2012. Infections with Sarcococystis wenzeli are prevalent in the chickens of Yunnan Province, China, but rare or absent from the flocks of domesticated pigeons and ducks. Experimental Parasitology. 131(1):31-34.
- Tian, M., Chen, Y., Rosenthal, B.M., Liu, X., He, Y., Dunams, D.B., Cui, L. , Yang, Z. 2012. Phylogenetic analysis of of Sarcocystis nesbitti (Coccidia: Sarcocystidae) suggests a snake as its probable definitive host. Veterinary Parasitology. 183:373-376.
- Madubata, C., Dunams, D.B., Elkin, B., Oksanen, A., Rosenthal, B.M. 2012. Evidence for a population bottleneck in an Apicomplexan parasite of caribou and reindeer, Besnoitia tarandi. Infection, Genetics and Evolution. DOI: 10.1016/j.meegid.2012.06.007.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) 1) Determine what genetic and genomic features distinguish Trichinella spiralis from Trichinella murrelli. 2) Utilize genomics to determine if microsatellite loci can be used to trace zoonotic outbreaks of Trichinella spiralis. 3) Determine the genetic features that account for the epidemic spread of certain strains of Toxoplasma gondii. Approach (from AD-416) Investigations will be conducted to clarify how infections in wildlife influence the safety of pastured pork. Accordingly, first identify heritable differences between two related species of Trichinella, only one of which (T. spiralis) severely compromises pork safety by evading swine immunity. The other, Trichinella murrelli, predominates in North American wildlife but fails to thrive in swine. By comparing the genomes of these two parasites, the intent is to establish a basis for exploring what makes pigs so especially vulnerable to T. spiralis. Secondly, develop the means to trace chains of transmission of Trichinella spp. Using markers which have already established the long- term dispersal history of T. spiralis (to the Americas in the pigs and rats brought by European colonists), researchers will attempt to discriminate instances of persistent on-farm transmission from sporadic introductions of T. spiralis to swine herds. Finally, the genetics of T. gondii reproduction will be characterized. Both sexual and asexual reproduction can occur in T. gondii, and available data provide conflicting evidence as to the relative importance of each reproductive mode. These incongruous data leave in doubt whether this parasite evolves as an assemblage of distinct lineages, or whether it more closely resembles a coherent, interbreeding species. Additional data are needed to better resolve how T. gondii propagates and evolves. These results will help determine whether particular strains pose elevated food safety risk will help anticipate this parasite's evolutionary response to preventative interventions. Progress has been made on all three objectives of this project (implemented in January of 2011), and significant new support for related objectives was procured in the form of a grant from NIFA with the objective to distinguish the genetic and genomic features that differentiate Trichinella spiralis from Trichinella murrelli, we sequenced certain housekeeping genes, introns, and non-coding sequences. These data will provide an estimate of genomic differences between the two, and will be used as a basis to determine if the Trichinella spiralis genome assembly can be used as a scaffold to interpret short sequence reads derived from Trichinella murrelli. In addition, we published a comprehensive comparison of the mitochondrial genomes of these two zoonotic parasite species. Finally, a proteomics approach was initiated to identify antigens in Trichinella murrelli that differ from homologues in Trichinella spiralis, with the intention that these may serve as a basis for differential serological diagnosis in swine. To determine if microsatellites can be used to trace zoonotic outbreaks of Trichinella spiralis, we assessed the capacity to detect mixed infections and applied these assays in an experimental evaluation of the vulnerability of swine to repeated infection (heterologous challenge). To determine the genetic features that account for the epidemic spread of certain strains of Toxoplasma gondii, we established (and published) new criteria for recognizing a fourth major lineage of this parasite in North American wildlife. Significant Activities that Support Special Target Populations The work on toxoplasmosis is especially important to the health of pregnant women, fetal and newborn health, and the health of persons afflicted by HIV-AIDS. Accomplishments 01 Used novel genome sequencing methods to comprehensively describe differences between the mitochondrial genome of this important parasite swine and a related parasite found in wildlife hosts. Understanding the differences will allow clearer understanding of how frequently food animals may be exposed to either of these two, otherwise indistinguishab parasite species (which however may pose very different levels of risk t human health). This work will lay a foundation for future work to asses risks posed by either parasite to the safety of pasture-raised pork.
Impacts
(N/A)
Publications
- Dubey, J.P., Rosenthal, B.M. 2010. Morphologic and molecular characterization of the sarcocysts of Sarcocystis rileyi (Apicomplexa: sarcocystidae) from the mallard duck (Anas platyrhynchos). Journal of Parasitology. 96:765-770.
- Xiang, Z., He, Y., Rosenthal, B.M., Li, X., Zuo, Y., Feng, G., Cui, L., Yang, Z. 2011. Comparative studies confirm natural infections of buffaloes by Sarcocystis cruzi. Parasitology International. 127(2):460-466.
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