GENETIC COMPARISON OF BOVINE AND FELINE ISOLATES OF TRITRICHOMONAS FOETUS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0230160
• Project Start Date: Jun 1, 2012
• Project End Date: Jun 1, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
College of Veterinary Medicine

Non Technical Summary
Tritrichomonas foetus is a protozoan parasite found in the reproductive tract of cattle and intestine of cats. In cows, pyometra can occur, leading to mid- and late-term abortions, while bulls are asymptomatic carriers. It is estimated that the beef industry in the United States loses millions each year due to infections of this parasite within their herd. This includes both calf and bull losses. Infections are predominantly found in the western United States due to large, free-roaming herds that are allowed to mate freely, however has also been found in Florida herds. In cats, disease results in chronic diarrhea. It is usually found in young cats, less than two years of age. The exact means of transmission is unknown, however it is most likely due to fecal contamination. There are no approved treatments for T. foetus in either cattle or cats. Research has shown that there are genetic differences, as well as differences in pathogenicity during experimental cross-infection studies, however they are morphologically indistinct. Research has shown that a feline isolate of T. foetus can experimentally infect cows, however there is significantly less pathogenicity, or none at all. This holds true for cats experimentally infected with a bovine isolate of T. foetus, infection occurred but there was no disease caused by the infection. Due to this finding, we believe that the feline isolates may be used as a potential vaccine for the cow. Our ultimate goal with this pilot project is to gather enough genetic data to support our hypothesis that these isolates are different. Bulls will be sampled by preputial wash and cows by vaginal mucus aspiration. Feces will be collected from suspect cats. Samples will be transferred to Diamond's TYM culture media and examined for the presence of trichomonads using light microscopy. Verification of T. foetus will be via polymerase chain reaction (PCR). Once isolates have been collected, DNA will be isolated from cultured organisms. PCR will be used to amplify various genes throughout the organism genome. These will be submitted for sequencing of PCR products. In addition, genomic DNA will be submitted for a pilot pyrosequencing experiment from both strains. The genetic information we receive could enable us to potentially develop a more efficacious vaccine for Bovine Trichomoniasis.

Animal Health Component

25%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
313	3310	1110	25%
313	3310	1090	50%
313	3310	1040	25%

Knowledge Area
313 - Internal Parasites in Animals;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1110 - Parasitology;

Keywords

tritrichomonas foetus

parasite

pyometra

Goals / Objectives
To determine genetic differences and similarities between bovine and feline isolates of Tritrichomonas foetus.

Project Methods
Cattle herds in Florida will be identified for sampling. T. foetus has been found in several large herds in south Florida, and an attempt to resample those herds will be made. Additional herds will be identified via veterinarians and cattlemen who suspect infection. Bulls will be sampled by preputial wash using a sterile insemination pipette connected by plastic or rubber tube to a 20 ml sterile disposable syringe. Cows will be sampled by vaginal mucus aspiration. Vaginal mucus samples will be collected using a sterile 52.5-cm infusion pipette with a flex adaptor and 20-ml syringe. Samples will be transferred to2mL of Diamond's trypticase-yeast-maltose (TYM) media without agar and returned to the laboratory and maintained TYM media at 37 degreesC for 5 days. Samples will be examined at 24hrs, 48 hrs and 120hrs for the presence of trichomonads using standard light microscopy at x100 magnification. Isolates from laboratories currently conducting research on feline trichomoniasis will be collected. In addition, veterinarians who have previously diagnosed T. foetus in client cats will be contacted to request additional samples. Feces will be collected from suspect cats. Immediately upon arrival at the University of Florida College of Veterinary Medicine a sample of the contents of the feces were examined via direct smear by light microscopy at 100X magnification and remaining feces will be subjected to culture in trypticase-yeast-maltose (TYM) media without agar at 37 degrees C (Diamond, 1983). Samples will be designated as positive (+) or negative (-) based on the presence of motile T. foetus trophozoites. Positive cultures were verified by polymerase chain reaction (PCR) using DNA extracted from each sample (Billeter et al., 2007, Grahn et al., 2005). Primers TFR-3 and TFR-4 were used to probe 5.8S rRNA gene sequences and internal transcribed spacer (ITS) regions for confirmation of T. foetus-specific sequences (Felleisen et al., 1998; Grahn et al., 2005). Organisms will be stored in liquid nitrogen in freezing medium made under sterile conditions using fetal calf serum, dimethylsulfoxide, and TYM at 3:2:5, respectively. Organisms will remain frozen until all isolates have been collected. Organisms will then be thawed quickly in a warm water bath, suspended in 10mL TYM media and maintained at 37 degrees C. Organisms will be passaged every 2-3 days in fresh TYM media

Progress 06/01/12 to 06/01/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Libraries were prepared from bovine and feline isolates of Tritrichomonas. After one round of Illumina-based sequencing, the following were obtained: Bovine: 58,420,890 sequences totalling 16,352,808,948 nucleotides. Feline: 49,190,316 sequences totalling 14,050,558,750 nucleotides. More than half of all sequences are greater than 250bp and have average PHRED quality values greater than 35, indicating high-quality sequences. De novo assembly of these sequences has produced 12,314 contigs for the feline isolate, ranging from 146bp to 334,485bp and 26,594 contigs for the bovine isolate, ranging from 123 bp to 1,932,831bp. Analysis of these sequences is still ongoing. Initial analysis suggests there is some contamination of the bovine culture with bacteria, suggesting another sequencing run may be necessary to generate more sequences for comparison. However, there appear to be differences in several genes that may prove significant for disease pathogenesis, including an analogue of heat shock protein 70 (HSP70); however, given the early stage of these analyses, the significance of these changes remains unknown.

Publications

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Libraries were prepared from bovine and feline isolates of Tritrichomonas. After one round of Illumina-based sequencing, the following were obtained: Bovine: 58,420,890 sequences totalling 16,352,808,948 nucleotides. Feline: 49,190,316 sequences totalling 14,050,558,750 nucleotides. More than half of all sequences are greater than 250bp and have average PHRED quality values greater than 35, indicating high-quality sequences. De novo assembly of these sequences has produced 12,314 contigs for the feline isolate, ranging from 146bp to 334,485bp and 26,594 contigs for the bovine isolate, ranging from 123 bp to 1,932,831bp. Analysis of these sequences is still ongoing. Initial analysis suggests there is some contamination of the bovine culture with bacteria, suggesting another sequencing run may be necessary to generate more sequences for comparison.

Publications

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: We have collected over 30 bovine Tritrichomonas foetus samples and over 10 feline Tritrichomonas foetus samples. All samples have been cultured and DNA has been collected for analysis. We have identified the gene targets and are currently validating the PCR protocols required for each genetic analysis. Once the PCR is validated and standardized, we will begin submitting the PCR products for genetic sequencing. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have just completed the collection phase of this project, and have just started the genetic analysis. We have not compiled a data set of genetic sequencing information yet.

Publications

• No publications reported this period