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Advancing Diagnostics for Hunter Food Safety: Comparing the Efficacy of PCR and Serology Tests for Trichinella in Game Meat

Investigators
Ernest, Holly
Institutions
University of California - Davis
Start date
2011
End date
2015
Objective
Our overall goal is to decrease the incidence of foodborne disease in hunters and their families through the advancement of diagnostic tools necessary to monitor and control this potentially lethal parasite in consumable wildlife species by addressing the following objectives: 1) comparing the sensitivity and specificity of an indirect serological test and a novel direct real-time PCR (rt-PCR) to the gold standard test, tissue digestion. 2) performing a cost-benefit analysis for each diagnostic technique in order to make recommendations to wildlife and food safety officials. 3) extending project findings and Trichinella awareness through workshops and publications to elevate knowledge amongst hunters and wildlife specialists.

This proposal aims to advance diagnostic tools for the detection of Trichinella in wildlife meat by comparing an accepted indirect serology test with a novel, highly sensitive and specific, rt-PCR. Our long term goals include reducing foodborne illness for under-served segments of the public, developing food handling guidance to minimize microbial risks, and disseminating the results via workshops and in lay and scientific publications to increase awareness of the potential risk of trichinellosis for wildlife meat consumers.

More information
Non-Technical Summary:
Trichinellosis is a serious disease caused by one of the most widely distributed parasite groups in the world, Trichinella. Infection and illness in humans occurs following the consumption of undercooked meat containing Trichinella worms. Many cases of trichinellosis are misdiagnosed, but common symptoms include nausea, diarrhea, vomiting, fatigue, and in severe cases, death. Despite the danger this parasite poses to the public, particularly those who consume wildlife meat, it has dropped off the radar of many healthcare professionals and food safety officials. Diagnostic testing is essential for understanding the burden of disease in populations, yet little advancement in diagnostic testing for Trichinella spp. has occurred in the last decade, thus precluding the establishment of effective monitoring programs and creating the potential for a reemergence of this dangerous disease. Current diagnostic tests for Trichinella spp. were primarily developed for use in domestic animals, such as pigs and horses. However, recent data shows that wildlife meat has become the main cause of trichinellosis in the United States, and yet no diagnostic tests have been validated for use in wildlife. Artificial digestion is considered the best test, however it cannot detect all species of Trichinella, low level infections or infections in samples that have been frozen. In wildlife, there is typically a lower level infection and samples are almost always frozen before testing, therefore digestion is not a suitable option for this purpose. Testing blood serum, or serology, for the detection of antibodies to Trichinella is a widely used diagnostic test due to its ease of use and rapid generation of results. Serology can be used for the detection of Trichinella in wildlife species, however, significant drawbacks, such as the inability to detect long term infections and false positives (due to cross reactions with other parasites) may limit the interpretation and applicability of diagnostic results. A more novel technology, real-time-PCR (rt-PCR) has recently been developed for detection of Trichinella in wildlife. It is highly specific, rapid and robust and is capable of detecting and identifying all species of Trichinella infection with as few as 1 larva per 100 g of tissue. Once validated, this technique could be integral to the advancement of Trichinella diagnostics in wildlife species. The United States Department of Agriculture, California Department of Food and Agriculture (CDFA), California Department of Fish and Game (CDFG), and the general public have repeatedly expressed concern over the lack of data related to the occurrence of Trichinella in California wildlife, and the risk of human infection from consumption of wildlife meat. Therefore, we believe it is important to validate the diagnostic tests used to estimate the burden of Trichinella in wildlife, and ultimately assess the foodborne risk this parasite poses to the public. This study would responsibly fill the needs of an under-served hunting population by developing the diagnostic tools necessary for wildlife and health officials to estimate Trichinella burdens in California wildlife meat.

Approach:
Naturally occurring positive and negative controls (determined by artificial digestion) will be obtained from reference collections at the USDA, Alaska Department of Fish and Game and International Trichinella Reference Center. We will assemble 25 positive and 25 negative black bear tissue samples as well as 25 positive and 25 negative wild pig tissue samples from these sources. Therefore, we aim to test 100 samples in total. This proposal will serve as the preliminary step of an overall comparative Trichinella diagnostic tool evaluation, as we aim to increase our sample size to 100 positive and 100 negative controls for each species in subsequent proposals. Serology will be performed using tissue fluids. This alternative source of antibodies is preferable to sera due to its ease of collection in the field, allowing for greater consistency for the purpose of this study. Tissue samples will be approximately 10 g in size and originate from known predilection sites including the tongue and diaphragm. The base of the tongue and the diaphragm are the most suitable sites for detecting the parasite in wildlife due to high accessibility and preferential accumulation of larvae. Samples will stored and frozen for transfer to UC Davis. All samples will be compiled and processed at Dr. Ernest's laboratory at UC Davis. Each 10 g muscle sample will be thawed and homogenized. Nobuto filter strips will be used to collect tissue fluid from the homogenized mixture, then dried and stored for later used. The remaining homogenate will be digested with Proteinase K buffer for 1 hour. DNA will be extracted from 1.5 g subsamples using a Qiagen Dneasy extraction kit. DNA samples will be analyzed by rt-PCR, an assay developed to target the 5S rRNA region that allows for the simultaneous detection of the 10 species and 3 genotypes of Trichinella. Serology will be performed using the tissue fluid collected on the Nobuto strips and a commercially available ELISA kit for the detection of IgG antibodies to excretory/secretory (E/S) Trichinella antigens. We anticipate that the rt-PCR test will be easy to perform, highly sensitive and specific (99% or greater), and more cost-effective than serological analysis for the wildlife species under consideration. Published values of sensitivity and specificity in domestic species for serology are 94% and 94% respectively, however we expect a lower value in our wildlife samples due to the issues discussed previously. Sensitivity and specificity (and 95% confidence intervals) will be estimated by comparing the results of each test with the "gold-standard" of tissue digestion in a 2x2 table. A cost-benefit analysis will be performed by comparing the varying costs of the diagnostic tests with respect to accuracy of their results, and also the test feasibility, to indicate circumstances in which use of a test becomes inefficient.

Progress:
2012/01 TO 2012/12
OUTPUTS: Networks of state and federal agency collaborators were put in place and samples acquired and/or identified. Training and mentoring of the PhD student and lab personnel in quantitative PCR (attended training workshop) and other laboratory techniques was accomplished. Agency personnel and members of the public were taught about zoonotic disease from wildlife and preventive measures. Large amount of planned activities will be conducted summer 2013 therefore following that period, outputs will be generated.
PARTICIPANTS: PhD student Jamie Sherman received workshop training in quantitative PCR and other laboratory techniques. She received wildlife training through the California Department of Fish and Wildlife, and developed collaborative networks with state and federal agencies. Jamie secured additional collaborations for this project and made plans for sample collection and laboratory analysis that will occur summer 2013. Holly Ernest (PI) mentored Ms. Sherman regularly for the project, including guiding additional grant proposal development, report-writing, etc. Partner Organizations include the USDA, California Department of Fish and Wildlife, and California Department of Food and Agriculture.
TARGET AUDIENCES: Target audiences for the products and efforts of this project include state and federal agency biologists, veterinarians, and the public. Specific segments of the public that will benefit from this project include hunters of wildlife in California.
PROJECT MODIFICATIONS: Not relevant to this project.

IMPACT: The project is in progress and much of the work will be conducted summer 2013. Therefore we do not have results and an outcome yet.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
CALV-AH-321
Accession number
229199
Categories
Bacterial Pathogens
Natural Toxins
Commodities
Meat, Poultry, Game