USE OF WATER SAMPLES FROM DRINKING TROUGHS FOR EARLY IDENTIFICATION OF INFECTED ANIMALS; PROOF OF CONCEPT USING ORF AS A MODEL FOR FOOT-AND-MOUTH DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1002610
• Project Start Date: Mar 18, 2014
• Project End Date: Jan 5, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Population Health & Reproduction

Non Technical Summary
The use of water samples from drinking troughs for identification of infected animals has the potential to revolutionize disease surveillance in dairy and non-dairy livestock species. For example, foot-and-mouth disease (FMD) research to date has focused on use of bulk tank milk samples to monitor the spread of infection and early detection of infected premises. The use of milk is an obvious choice for active FMD surveillance on dairies during an epidemic. However, the rationale for identifying an alternative or complementary sample is that milk excludes non-dairy animals such as non-lactating dairy cattle, beef cattle, sheep, meat goats, swine and camelids. The objective of this study is to test whether water samples from drinking troughs can be used for surveillance of food animals with orally transmitted infectious diseases.

Animal Health Component

100%

Research Effort Categories

Basic

0%

Applied

100%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3699	1170	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3699 - Sheep and wool, general/other;

Field Of Science
1170 - Epidemiology;

Keywords

water samples

foreign animal disease

orf

foot-and-mouth

Goals / Objectives
In the event of an infectious disease epidemic, early identification of cases through active surveillance for infected premises is crucial to limit the disease spread and impact. For example, foot-and-mouth disease (FMD) research to date has focused on use of bulk tank milk samples to monitor the spread of infection and early detection of infected premises. The use of milk is an obvious choice for active FMD surveillance on dairies during an epidemic. However, the rationale for identifying an alternative or complementary sample is that milk excludes non-dairy animals such as non-lactating dairy cattle, beef cattle, sheep, meat goats, swine and camelids. Water samples have been tested for FMD during an outbreak in one of Egypt's governorates however no information was provided on the condition of the water sampled (Ghoneim et al., 2010). Furthermore, little is known about the conditions that may affect the success of such a sample in early surveillance of infectious diseases. The objective of this study is to test whether water samples from drinking troughs can be used for surveillance of food animals with orally transmitted infectious diseases. A disease that can act as a model for orally transmitted infectious diseases such as FMD in food animals is Contagious Ecthema or Orf. Orf is a viral disease that mainly affects small ruminants and is endemic in California. The disease is characterized by a distinct oral lesion that can also occur on the feet. We propose to investigate the use of water samples from drinking troughs as a tool for identification of infected animals with clinical signs. Biologically, drinking water trough samples may be more sensitive than bulk tank milk samples since the former represents fewer animals (animals in a pen versus an entire herd or flock) and thus may provide a sample with higher virus concentration. The proposed research is a proof-of-concept study that will provide preliminary data for an NSF disease Ecology grant.

Project Methods
RESEARCH HYPOTHESES: Our hypothesis is that the concentration of Orf viral DNA (as measured in CT) in water from drinking troughs used by Orf cases is highly correlated with the concentration of Orf viral DNA (as measured in CT) in saliva and oral mucosa swabs collected during clinical signs. OBJECTIVES: To validate the use of drinking trough water samples as a surveillance tool for detection of infected animals showing clinical signs of orally-transmitted infectious diseases. Using the Orf-causing parapoxvirus in sheep and goats as a model, we will estimate the correlation between Orf viral DNA concentration in saliva of cases naturally infected with the Orf causing parapoxvirus and in water samples from the animals' drinking water troughs. Materials and Methods: Suspect Orf cases (although more common in lambs and kids, other ages will be pursued) during the lambing and kidding season of 2014 will be identified through a call made to the sheep and goat industry in California through producer meetings, dairy herd improvement associations and personal communication. The Wool Growers Association and several small ruminant associations will also be contacted and producers requested to inform us of any suspect cases within 24 hours of appearance of clinical signs. A total of 20 cases will be identified for oral lesion and saliva swabs. In addition, water samples from each case's drinking water trough will be sampled. Samples will be collected twice a day until disappearance of signs or a maximum of 4 days. A standardized swabbing technique of the oral lesion and saliva using Dacron swabs passed 6 times along the lesion will be used to reduce variability due to swabbing technique. The swab will then be placed into a sterile red top tube. The swab's shaft will be broken off and the tube sealed and transported the same day to CAHFS Laboratory on wet ice. At the laboratory, viral transport media will be added and DNA extracted using a high-throughput magnetic bead approach. Real-time PCR testing will be performed on a ABI 7500 fast machine. Ten ml of water will be collected from the drinking trough of each case using a disposable plastic Pasteur pipette and placed in a 15 ml sterile polypropylene tube stored on ice and transported the same day to the laboratory. Information on each case will be collected including history, age, breed and severity and approximate age of lesions. Information on the water samples will be collected including, presence of an automatic refill system, number of animals with access to the trough, indoor or outdoor location, exposure to direct sunlight, as well as the condition of the water including pH (using litmus paper), temperature, mold growth, transparency and presence of dirt/bedding. In addition, to characterize the field water as a matrix, standard plate counts and Coliform counts from appropriate agar plates will be determined and used to rank the water samples by degree of contamination. The Orf status of the naturally infected cases will be verified using qPCR and the virus concentration in saliva will be quantified in cycles-to-threshold (CT). The mean CT results of oral and salivary swabs will be correlated with that of water samples over time using the Intra-class Correlation Coefficient (Aly et al., 2009). References Ghoneim, N. H., A. K. Abdel-Karim, L. El-Shehawy, and K. A. Abdel-Moein. 2010. Foot and mouth disease in animals in Sharkia governorate - Egypt. Transbound Emerg Dis 57(1-2):19-21. Aly, S. S., R. J. Anderson, R. H. Whitlock, T. L. Fyock, S. McAdams, J. M. Adaska, J. Jiang, and I. A. Gardner. 2009. Reliability of environmental sampling to quantify Mycobacterium avium subspecies paratuberculosis on California free-stall dairies. J Dairy Sci 92(8):3634-3642.

Progress 03/18/14 to 01/05/18

Outputs
Target Audience:Extension specialists performing outreach education to food animal producers were the initial target audience. In addition, results of this study specifically concern the sheep industry since Orf virus affects newborn lambs every year causing loss in production (growth). Changes/Problems:Among the factors that may have contributed to the negative PCR results for water samples from drinking troughs of positive animals is dilution of the virus shed in the water and hence concentrating techniques and more robust preparation of sample should be explored, similarly, producers tended to clean water troughs before visiting them in preparation for water sample collection which included refilling water troughs, use of clorox to clean the containers, all these may have contributed to the negative PCR results. What opportunities for training and professional development has the project provided?Undergraduate and graduate students were able to participate in the sample collection and processing in the field, data entry and reporting back to producers. This provided a training opportunity on animal handling, sample collection and submission, data form preparation and entry. How have the results been disseminated to communities of interest?Results were shared back with the producers. Animals with orf clinical signs were indeed positive for Orf virus PCR. Only a single water sample from a drinking water trough was positive for Orf virus. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Several announcements to the sheep industry through producer newsletters, small ruminant practitioners association communications to invite producers to participate in the study. Several outbreaks have been identified and producers contacted and enrolled in the study. Simultaneous water trough and animal swab samples were collected and submitted to the California Animal Health and Food Safety laboratory.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Extension specialists performing outreach education to food animal producers were the initial target audience. In addition, results of this study specifically concern the sheep industry since Orf virus affects newborn lambs every year causing loss in production (growth). Changes/Problems:Among the factors that may have contributed to the negative PCR results for water samples from drinking troughs of positive animals is dilution of the virus shed in the water and hence concentrating techniques and more robust preparation of sample should be explored, similarly, producers tended to clean water troughs before visiting them in preparation for water sample collection which included refilling water troughs, use of clorox to clean the containers, all these may have contributed to the negative PCR results. What opportunities for training and professional development has the project provided?Undergraduate and graduate students were able to participate in the sample collection and processing in the field, data entry and reporting back to producers. This provided a training opportunity on animal handling, sample collection and submission, data form preparation and entry. How have the results been disseminated to communities of interest?Results were shared back with the producers. Animals with orf clinical signs were indeed positive for Orf virus PCR. Only a single water sample from a drinking water trough was positive for Orf virus. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Several announcements to the sheep industry through producer newsletters, small ruminant practitioners association communications to invite producers to participate in the study. Several outbreaks have been identified and producers contacted and enrolled in the study. Simultaneous water trough and animal swab samples were collected and submitted tothe California Animal Health and Food Safety laboratory.

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Infectious disease epidemiologists and foreign animal disease prevention agencies. Changes/Problems:However, detection of Orf virus in water using traditional techniques have been challenging and we have encountered several cases with animal swabs that test positive however their respective water trough samples tested negative. These false negative results is could be due to inhibition of PCR assay on water samples due to contamination (bacteria, algae etc.) in the water. Further research using water concentrating techniques is warranted. What opportunities for training and professional development has the project provided?Several residents and veterinary students at the Veterinary Medicine Teaching Hospital at UC Davis were trained on the infectious disease Orf, animal handling and zoonotic risks. 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?Finalize our findings and report them in a manuscript.

Impacts
What was accomplished under these goals? Sample collection and testing was completed from over 4 farms with Orf outbreaks.

Publications

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Researchers working on surveillance of domestic ruminants for foreign animal diseases or endemic diseases with etiological pathogen shed through the oral route, examples include Foot-and-Mouth-Disease (FMD), Rift Valley Fever, Orf (Contagious Ecthema). Private practitioners and ruminant production facilities' owners including (sheep, goats - meet or dairy breeds, beef, dairy cattle, swine). Changes/Problems:Mainly low incidence or Orf and when it occurs lack of information channels allowing us to know about these cases. As a result we changed our approach for contacting producers and veterinarians, we are now relying on personal contact more, yet we are still letting producers and vets know that we need cases that are potentially Orf by mail and internet. 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?Attempt to complete the data collection,sample testing, data analysis and write up,

Impacts
What was accomplished under these goals? After the first year, we revisited our plan for identifying facilities and now we have expanded our search by contacting farm advisers, 4H advisers, small ruminant practitioners and lists both in person (as much as possible) and by mail or phone. We hope that the upcoming Orf season (winter through spring) we will be called more often to outbreaks that seem to be Orf in sheep.

Publications

Progress 03/18/14 to 09/30/14

Outputs
Target Audience: Findings from this study would be of interest to veterinary epidemiologists at federal and state government in charge of devising prevention and control measures for infectious diseases due to viral diseases transmitted by oral fluids from infected animals such as Foot-and-mouth disease. Changes/Problems: Case load reporting has been low, we will work with private practitioners too to help us identify more cases. What opportunities for training and professional development has the project provided? Dr. Alyssa Louie a masters student in the Masters in Preventive Veterinary Medicine and Dr. Gaby Meyer a resident at the Livestock reproduction and herd health residency at the teaching hospital at SVM UCDavis were trained on the study protocol and helped collect samples. How have the results been disseminated to communities of interest? Study still on going and results in preliminary stage, we are not ready yet to share our findings at this stage without a completed study and analysis. What do you plan to do during the next reporting period to accomplish the goals? Compelte enrollment of cases and sample collection

Impacts
What was accomplished under these goals? A detailed protocol for collecting water samples from water troughs and swabing lesions from cases. IACUC protocol for animal use was applied for and approved. We designed a complete protocol for a farm visit to collect required samples and data on husbandry and management conditions. Call made to several industry partners and organizations about the importance of the study and the search for cases consistent with Contagious ecthyma in sheep for enrollment and participation in our study. Two different farms reported cases and were sampled. Given late dispersal of fundings we requested extension 1 year so we may be able to complete the required sample size

Publications