Source: AGRICULTURAL RESEARCH SERVICE submitted to NRP
MANAGING THE EMERGING RISK OF TRICHINELLOSIS IN ORGANIC AND FREE RANGE PORK
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0225009
Grant No.
2011-67005-30348
Cumulative Award Amt.
$266,226.00
Proposal No.
2010-04461
Multistate No.
(N/A)
Project Start Date
Apr 1, 2011
Project End Date
Mar 31, 2015
Grant Year
2011
Program Code
[A4141]- Food Safety: Addressing Critical and Emerging Food Safety Issues
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
RM 331, BLDG 003, BARC-W
BELTSVILLE,MD 20705-2351
Performing Department
Agricultural Research Service
Non Technical Summary
This project is needed to evaluate whether or not practices promoted by USDA's Organic Production standards indeed compromise the safety of pork by elevating exposure to the zoonotic parasite Trichinella spiralis, as has been recently debated. Determining whether outdoor swine husbandry increases exposure to zoonotic parasites will enable producers, regulators, and consumers to make more informed choices. In order to do so, we will develop and then apply diagnostic reagents that discriminate between two, related parasite species. One of these (Trichinella spiralis) multiplies and persists in swine hosts (Trichinella spiralis), posing appreciable risk to the health of anyone consuming such pork. The other parasite (Trichinella murrelli) generally fails to thrive or persist in swine, as it is effectively controlled by swine immunity. At present, serological assays do not discriminate between the two. The ability to make such distinctions will enable: Greater precision of the actual health risk posed by a given animal. Valid characterization of a herd's exposure risks and zoonotic potential. This importance of this work is augmented by recent findings that transient exposure to T. murrelli may render an animal immune to subsequent infection with T. spiralis. Thus, the presence of T. murrelli in the American environment may decrease the risks associated with outdoor swine husbandry. Success in developing and applying discriminatory diagnoses will enable the testing of this hypothesis.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3132210111010%
3133310111025%
3133410111025%
3133510111010%
3133729111010%
3133799111010%
3133810111010%
Knowledge Area
313 - Internal Parasites in Animals;

Subject Of Investigation
3310 - Beef cattle, live animal; 3510 - Swine, live animal; 2210 - Chemurgic crops; 3729 - Other cultured shellfish; 3410 - Dairy cattle, live animal; 3799 - Cultured aquatic animals, general/other; 3810 - Horses, ponies, and mules;

Field Of Science
1110 - Parasitology;
Goals / Objectives
Overall objective of this project is to develop serological assays that differentiate swine chronically infected with zoonotic T. spiralis from swine only transiently exposed to T. murrelli.
Project Methods
ARS will use what is known about antigenic gene families to identify prospects, among these candidates, that deserve to be prioritized for experimental validation. A complementary attack will use a differential screening strategy to identify T. murrelli specific antigens from a cDNA library that are uniquely recognized by the host immune response during a T. murrelli infection and that can be used to delineate T. murrelli infected animals from T. spiralis infected animals. To this end, muscle larvae (ML) from T. murrelli will be propagated in mice. After 30 days, the mice will be euthanized and the ML isolated using an experimental digestion fluid (1% pepsin:1 percent HCL at 40 C for 3 h). The liberated ML will be used to isolate total RNA by homogenization and purification in Trizol (TM). Total RNA will be converted to cDNA then dscDNA using the Clontech SMART (TM) technology, then cloned into a suitable lambda expression vector (lambda gt11 -derived) for subsequent immunological screening. The integrity of the library (minimum 1 x 10x6 plaques) will be validated by picking at least 100 plaques and subjecting them to plaque PCR using vector primers to determine the occurrence and length of inserts. Cloning efficiency will also be evaluated by blue/white screening on agar plates using the vector-derived Beta-galactosidase gene and the ability of plaques to convert X-gal substrate. To start, a minimum of 100,000 plaques will be plated onto agar (approximately 7K plaques per plate) and transferred to Nytran filters impregnated with IPTG to induce protein expression from the cloned sequences. These filters will be screened with pooled sera from 3 pigs experimentally-exposed to T. murrelli (provided by D. Hill). Clones that react positively will be handpicked and suspended in SM storage medium, replated, and screened secondarily with pooled sera from 3 T. spiralis-infected pigs. Clones reacting to T. murrelli infection serum but not with T. spiralis infection serum will be sequenced and subcloned into a suitable bacterial expression vector (pSUMO) to allow affinity purification of the cloned antigens. The cloned antigen(s) will again be validated by Western blot and ELISA as T. murrelli. The genotypic and serological reagents will then be employed to assess the prevalence and diversity of each parasite in pastured swine and in sympatric wildlife, enabling unprecedented precision in defining the extent and direction of parasite transmission into and out of such swine herds. Equivalent studies will focus on the parasites in feral swine, which may serve both as reservoirs and vectors for zoonotic trichinellosis. By sampling domesticated and wild hosts on and around several farms (representing various regional environmental conditions and varying degrees of biosecurity) our aim will be to assess which, if either species of trichinella, pastured swine typically encounter.

Progress 04/01/11 to 03/31/15

Outputs
Target Audience:Results of the project have been reported at the International Conference on Trichinellosis, sponosored by the International Commission on Trichiniellosis (the principal body that promotes scientific exchange and advises governemental and inter-governmental bodies on matters related to trichinella infection). A manuscript is also being prepared that will target scientists interested in the biology, genetics, and genomics of parasitic infection. Changes/Problems:Our major shift was to a comparative genomics approach, when the comparative immunological approach revealed the two species to be indistinguishable. What opportunities for training and professional development has the project provided?We trained a post-doctoral student, a visiting PhD student from China, and a bioinformatics trainee who is a candidate for a Masters degree. An undergraduate also co-authored a study resulting from this work. How have the results been disseminated to communities of interest?Through conference presentations, poster days, and several peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The antigenic profiles of the two species were found to be virtually entirely identical. The parasites differed in the degree of expression of particular antigens, but the peptide sequence of candidate to distinguish immune responses proved elusive, owing to the conservation that became evident through our studies.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Hybridization is limited between two lineages of freeze-resistant Trichinella during coinfection in a mouse model. Hecht LB, Thompson PC, Lavin ES, Zarlenga DS, Rosenthal BM. Infect Genet Evol. 2016 Mar;38:146-51. doi: 10.1016/j.meegid.2015.12.016. Epub 2015 Dec 23
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Outbreak of Trichinella spiralis infections associated with a wild boar hunted at a game farm in Iowa. Holzbauer SM, Agger WA, Hall RL, Johnson GM, Schmitt D, Garvey A, Bishop HS, Rivera H, de Almeida ME, Hill D, Stromberg BE, Lynfield R, Smith KE. Clin Infect Dis. 2014 Dec 15;59(12):1750-6. doi: 10.1093/cid/ciu713. Epub 2014 Sep 11.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Surveillance of feral swine for Trichinella spp. and Toxoplasma gondii in the USA and host-related factors associated with infection. Hill DE, Dubey JP, Baroch JA, Swafford SR, Fournet VF, Hawkins-Cooper D, Pyburn DG, Schmit BS, Gamble HR, Pedersen K, Ferreira LR, Verma SK, Ying Y, Kwok OC, Feidas H, Theodoropoulos G. Vet Parasitol. 2014 Oct 15;205(3-4):653-65. doi: 10.1016/j.vetpar.2014.07.026. Epub 2014 Aug 12.
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Screening of early antigen genes of adult-stage Trichinella spiralis using pig serum from different stages of early infection. Liu P, Wu XP, Bai X, Wang XL, Yu L, Rosenthal B, Blaga R, Lacour S, Vallee I, Boireau P, Gherman C, Oltean M, Zhou XN, Wang F, Zhao Y, Liu MY. Vet Parasitol. 2013 May 20;194(2-4):222-5. doi: 10.1016/j.vetpar.2013.02.001. Epub 2013 Feb 9.
  • Type: Journal Articles Status: Accepted Year Published: 2012 Citation: Discernible but limited introgression has occurred where Trichinella nativa and the T6 genotype occur in sympatry. Dunams-Morel DB, Reichard MV, Torretti L, Zarlenga DS, Rosenthal BM. Infect Genet Evol. 2012 Apr;12(3):530-8. doi: 10.1016/j.meegid.2012.01.004. Epub 2012 Jan 15. PMID: 22266240