Progress report for ONE20-372
Toxoplasmosis is the zoonotic disease caused by Toxoplasma gondii. Symptoms only become easily noticeable once an animal is immunocompromised (ill or pregnant). Majority of the symptoms occur in females with abortion, fetal mummification, reabsorption, weak kids, and in severe cases, blindness and brain damage. In order to effectively report the incidence and stop the cycle of toxoplasmosis on farms, it is imperative animals with the parasite are identified and treated or culled and preventative measure are taken. Over 40 years ago, molecular detection identified surface antigen 1 (SAG-1) as a major surface antigen with specificity to T. gondii. The SAG-1 gene has been utilized by many to detect T. gondii livestock across the world. Therefore, this project seeks to characterize the level and understand the impact of Toxoplasma gondiiinfection on small ruminant farms in Delaware. To complete this project, fifteen small ruminant farms (at least 10 animals) in Delaware will be selected and blood samples taken from their animals. Utilizing molecular techniques with SAG-1 specific primers, polymerase chain reaction (PCR) will be conducted and the product fractionated to identify SAG-1 gene. The results from this study will be used to inform producers of actual status of T. gondii presence and impact it has on the reproduction efficiency on their farms. This study will generate data that will update the information on anthelmintic resistance in Delaware. Results will be distributed to animal scientists, extension agents, and producers through presentations at scientific meetings, workshops, extension meetings and publications.
This project seeks to characterize the level of and understand the impact of Toxoplasma gondii infection on small ruminant farms in Delaware. Molecular detection method (polymerize chain reaction; PCR) will be used to determine the levels of T. gondii parasite in small ruminants in Delaware and surveys will be created to gain information from farmers to understand the impact this parasite had on the reproductive efficiency on their farm. The results from this objective will be used to educate producers of the popularity of T. gondii within Delaware and to help them come up with ways to prevent or limit the impacts of this parasite.
Agriculture is one of the largest enterprises in Delaware with animal-based agriculture the largest and most profitable. Due to the increased immigrant population in the United States (U.S.; specifically, on the East Coast) and the desire for healthier diets, there is an increased demand for small ruminant meat (Ibrahim et al., 2017). The increasing need to meet this demand provides an attractive, affordable, and profitable alternative enterprise for small and beginning farmers on the eastern seaboard including those in Delaware. Although this demand provides an opportunity for many producers, growth of the goat industry is impacted by the prevalence of internal parasite infections that lead to losses in production and increased mortality in many herds/flocks. One parasite that can significantly reduce small ruminant production is the opportunistic zoonotic apicomplexan protozoan parasite, Toxoplasma gondii, that causes the disease toxoplasmosis.
Toxoplasmosis is a disease that is common among animals and humans with up to one-third of the world’s population chronically infected (Halonen and Weiss, 2014). It is transmitted from a definitive host (feral and domestic cats) to an intermediate host (human, sheep, goat, cattle, etc.) or between different intermediate hosts (Dubey and Lindsay, 2006). Sheep and goats generally become infected by eating contaminated feed or drinking contaminated water while the leading cause of infection for human is the consumption of under-cooked/raw meat or unpasteurized goat milk (Dubey and Lindsay, 2006). In the U.S., small ruminants are primarily raised on pastures which are frequently visited by stray cats resulting in high infestation by T. gondii. Toxoplasma gondii infection in sheep and goats is very detrimental to production as it causes abortion, mummification, weak kids, stillbirth, embryonic and fetal death, and neonatal death (Dubey and Lindsay, 2006). Additionally, once this parasite enters the bloodstream it is able to spread to other tissues within the animal and in some cases causing diminished vision and brain damage. Currently, there are limited information in the U.S. on the prevalence of this parasite in small ruminants. However, in a study conduct on lambs in 22 states it was found that 58.8% of the farms tested on the east coast had animals positive for T. gondii infection (APHIS, 2014). Additionally, study conducted at Delaware State University (DSU) after a series of abortions found 18 out of 36 meat-goat does infected with T. gondii (unpublished, 2018).
With the potential of this parasite to be detrimental to animal production and having the ability to cause severe infection in humans, identifying the prevalence and impacts of this parasite within Delaware is of greave importance. Therefore, this research will address a critical area in small ruminant production and human health in Delaware and will enlighten producers on how widespread T. gondii is and how to best limit the effects on their farm. This will lead to different management decisions (such as adding anti-protozoal treatment to feed, limit feral and domestic cat interaction with sheep and goats) and health precautions that could prevent further spread of T. gondii parasites both to animals and humans.
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Materials and Methods
In order to conduct this project, Institutional Animal Care and Use Committee (IACUC) and Institutional Review Board (IRB) approvals were acquired to ensure safe use of animals and corporation from humans. Samples were collected from 15 small ruminant farms (goat = 10; sheep = 4; sheep and goat = 1) having a minimum of 10 animals each. Blood sampling were done in duplicated collection from each animal and used for peripheral blood mononuclear cell (PBMC) extraction. After which, genomic deoxyribonucleic acid (DNA) was isolated and used to conduct conventional polymerase chain reaction (PCR). The PCR product was then separated by gel electrophoresis and imaged in order to tell if the animals were positive or negative for Toxoplasma gondii. In order to evaluate farmer knowledge of the parasite and its potential impact a survey was given to all participating farmer and 14 was complete and returned.
Blood Sampling and Peripheral Blood Mononuclear Cells
Blood samples were collected at each participating producer farm (n = 15) by jugular venipuncture from individual animals (n = 250) and deposited into blood collection tubes with ethylenediaminetetraacetic acid (EDTA; to prevent clotting). Samples were placed on ice and then transported to Delaware State University’s parasitology laboratory for white blood cell isolation. Individual blood samples (n = 250) were separated into serum, red blood cells and PBMC using Histopaque-1077 (Sigma-Aldrich) following the manufacturer’s instructions. Peripheral blood mononuclear cell were aspirated and washed twice with phosphate buffered saline (PBS) to eradicate red blood cell, histopaque, and plasma residues. The pellets were then transferred to 2 ml centrifuge tubes and stored at -80 °C prior to DNA extraction for T. gondii identification.
Deoxyribonucleic Acid (DNA) Extraction and Conventional Polymerase Chain Reaction (PCR)
In order to identify T. gondii from blood samples, genomic DNA were isolated from individual animal's (n = 250) PBMC and PCR conducted to amplify T. gondii specific surface antigen 1 (SAG-1). Briefly, genomic DNA were isolated by first incubating PBMC in a lysis buffer containing acetic acid, tris base, sodium dodecyl sulfate (SDS) and proteinase K for one hour. After which, a conventional phenol/chloroform extraction method was performed and the isolated DNA pellet resuspended in 20 μL nuclease free water and stored at -20°C for PCR amplification.
The amplification of SAG-1 full coding sequence, was then conducted by conventional PCR. Polymerase chain reaction was conducted utilizing forward and reverse specific oligonucleotide primers designed based on the coding sequences described by Selseleh et al., 2012 and ordered from Integrated DNA Technologies, Coralville, Iowa. After PCR, the amplicons were fractionated on agarose/ethidium bromide (EtBr) gel in 1X Tris-acetate-EDTA (TAE) Buffer for DNA separation based on size and visualized by using a Gel Doc Imager. Once a band was identified on the gel, this allowed researchers to know the animals that were positive for T. gondii and the percent infected on each farm was calculated.
A 22-question survey was designed and approved by the institutional review board (IRB) in order to acquired information on the knowledge and awareness of producers about T. gondii and its impact on small ruminants. Farm demographic data, current disease management practices, presence or absence of cats, handling of cat litter, and abortion history were collected from each farm in order to help understand if there was a spread or impact of toxoplasmosis on these farms. This survey was given to all 15 farms where samples were collected.
To gain knowledge on the prevalence and the level of understanding producers have about Toxoplasma gondii infection, producers were given a 22-question IRB approved survey that covered farm demographics, rate of abortion, and knowledge of toxoplasmosis. Based on the survey data, 78.6% of the farmers had cats on their farms and 91.0% of the farms that had cats allowed their cats to have access to feed room and feed products. Additionally, it was found that 35.7% of the farmers had no knowledge about T. gondii while the other 64.3% of farmers had some knowledge about T. gondii.
In order to identify T. gondii on small ruminant farms, blood samples were collected individually from a total of 165 goats and 85 sheep from fifteen producer farms throughout Delaware. White blood cells were isolated from each animal's blood sample and used for genomic deoxyribonuclease acid (DNA) isolation. The isolated DNA was then used in a polymerase chain reaction (PCR) to amplify surface antigen-1 (SAG-1) that is specific to T. gondii. Gel electrophoresis and imaging of the PCR product, indicated that T. gondii was present in 15.2% (38/250) of animals tested and on 40.0% (6/15) of farms tested. Additionally, the data indicated that 33.3% of the farms that had positive animals belonged to sheep producers while 66.7% of the positive animals belonged to goat producers.
It can be concluded that T. gondii was present on several farms in Delaware. However, not all the animals on these farms were positive for T. gondii.
Education & Outreach Activities and Participation Summary
The results of the proposed research project will be presented at the American Society of Animal Science (ASAS) sectional meeting, producer workshops and student research meetings. The data collected from the proposed project will be used to produce at least one factsheet that will be available at meetings and posted online for download. The information will be presented to the general public through agricultural experiment station progress reports, field day presentations, Delaware Ag Week Small Ruminant Sessions, DSU’s Research Day and web-based publications. Therefore, the data will be presented in appropriate formats to industry personnel, teachers, researchers, producers and students. Farmer partners on this project will be included via producer seminars detailing their experiences and knowledge gain on T. gondii at the Delaware State University’s Profiting from a Few Acres conference held annually. After the factsheet has been written, it will be shared on the DSU small ruminant facebook page so that producers outside of Delaware can view this information and realize the significance of knowing about T. gondii infection and potential impacts on their farm.
The data from this project will be presented at approximately five different meetings with an expected total of 200 participants (farmers, students, researchers and extension agents). The publication of factsheet electronically will have the possibility of reaching more than 10,000 people as it will be shared on the DSU website, DSU small ruminant Facebook page and shared by other extension specialists’/agents’ websites.