Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
Biomedical & Diagnostic Sciences
Non Technical Summary
Trichomonas gallinae is considered the most important disease in passerines and columbids, and several large epidemics have been reported. Previous research strongly suggests that birds are infected with T. gallinae isolates via ingestion of contaminated food or water. We have previously found that T. gallinaecan persist in water with organic material for at least 4 hrs; however, the factors associated with persistence are unknown. Similarly the efficacy of various decontaminants that can be used to control trichomonosis outbreaks is unknown. The purpose of our study is to determine the persistence of up to 10 different T. gallinae isolates in water and feed and determine the efficacy of three decontaminants (UV light, bleach, and ammonia) against the trichomonad isolates. In addition, we will perform molecular testing to determine the T. gallinae genotypes associated with a large passerine mortality event that occurred in the eastern United States in 2009. National Center for Veterinary Parasitology; 1/1/14 - 12/31/14; $7,000
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Determine the epidemiology of the 2009 T. gallinae-associated outbreakDetermine the persistence of up to 10 isolates in food in waterExamine decontamination procedures to mitigate future outbreaks.
Project Methods
PCR testing of 2009 samples: Oral cavity or liver tissue samples from passerine cases submitted to several wildlife disease and veterinary diagnostic labs have been collected in our laboratory and will be tested by PCR using Trichomonidida-family wide primers ITS1 (5'- TGCTTCACTTCAGCGGGTCTTCC-3') and ITS2 (5'-CGGTAGGTGAACCTGCCGTTGG-3'). These primers amplify the internal transcribed spacer (ITS) regions of the ribosomal RNA. Additionally, we will examine the iron hydrogenase gene using primers TrichhydF (5'- GTTTGGGATGGCCTCAGAAT-3) and TrichhydR (5'-AGCCGAAGATGTTGTCGAAT-3). The iron hydrogenase and ITS regions will provide the fine scale separation to determine the various genotypes associated with the 2009 outbreak. To date, approximately 200 samples from 2009 have been collected and from a small subset of the samples (N=10), T. gallinae was detected in three birds by PCR in our laboratory. We will conduct PCR testing on the remaining samples and resultant amplicons will be sequenced to determine which genotypes are present. This genotype information is useful in investigating the epidemiology of trichomonosis outbreaks, since these various genotypes are associated with particular avian hosts (i.e. rock pigeon, Eurasian collared-dove, white-winged dove).Persistence trials: The persistence of T. gallinae in water and feed will be examined in efforts to determine the transmission dynamics of T. gallinae associated with artificial feeders and waters. We will inoculate 500 ml of distilled or chlorinated water and various bird feeds with 1X106 trichomonads from at least 10 different T. gallinae isolates cryopreserved in our laboratory. These 10 isolates will be comprised of known virulent and avirulent isolates from various genotypes. Aliquots of the inoculated water or feed kept at various ambient temperatures (10 C, 20 C, 30 C), and containing various levels of organic matter will be inoculated into in flasks containing complete Diamond's media at various time points post inoculation and examined for live trichomonads by microscopy for five consecutive days. These studies will simulate the persistence of T. gallinae at different seasonal temperatures and in progressively deteriorating sanitation levels (as can be found in backyard feeders). Similarly, 1X106 trichomonads from at least 10 different T. gallinae isolates will be exposed at various concentrations or durations of decontaminates including UV light (1 min, 10 min, 20 min, and 30 min) and bleach and ammonia (10%, 20%, and 30%) for various durations (1 min, 10 min, 20 min, 30 min, and 60 min) to determine the most efficacious concentration and duration for the various decontaminates.Each treatment will be replicated at least three times to produce adequate data for meaningful statistical analysis. The resultant information will be immediately useful to parasitologists, biologists, avian disease professionals, and citizens and allow for citable information on feeder disease mitigation procedures. Furthermore for the water samples, we will collect approximately 10% of the total volume and centrifuge the sample at 750 x g for 20 min. The remaining pellet will be resuspended and live trichomonads counted using a hemocytometer to determine the relative concentration of trichomonads persisting at the respective time points for the various treatments.Data Analysis: Positive amplicons from the PCR testing of the 2009 outbreak will be bi-directionally sequenced. Sequences will be aligned and a phylogenetic analysis performed with the sequences generated from this study as well as closely related sequences from GenBank. For the persistence studies, we will analyze the length of persistence for the various treatments using descriptive analysis as performed previously. For studies determining the concentration of viable trichomonads in water, we will compare the trichomonad concentrations for the various treatments using an ANOVA.