Performing Department
Veterinary Sciences
Non Technical Summary
The State of Wyoming has approximately 365,000 sheep based on the 2011 National Animal Health Monitoring System (NAHMS). This is the 3rd highest in the nation, behind Texas and California. This same study identified 817 Wyoming farms raising sheep, indicating a mean flock size of over 440 sheep. For ranches in Wyoming, approximately 70% of the household income comes from ranching or farming from this size of flock (2012, University of Wyoming Extension). For Wyoming sheep producers, maintaining a healthy herd is important not only for animal welfare but also for their own livelihood.In Wyoming, lamb production accounts for 35% of gross annual agricultural sales (2012, University of Wyoming Extension).Thus, any factors that may affect lamb production are considered a priority. This includes nutrition, genetics, and disease prevention. One of the diseases that directly affects reproduction in a flock is Brucella ovis (B. ovis). This bacteria may cause epidymitis in rams, but the greater implications for the flock include: ram infertility, decreased ewe conception rates, more abortions in pregnant ewes, and higher numbers of premature lambs. B. ovis has direct negative effects on lamb production and profit for the producer. Due to the negative consequences of this disease being introduced into a herd, most producers with larger flocks (greater than 50 ewes) perform ram breeding soundness exams which includes a blood test to detect whether there has been exposure to B. ovis bacteria. This is particularly important when it comes to new introductions into the herd. For sheep producers, maintaining a disease free herd with strong genetics is invaluable. If a ram has been exposed to B. ovis, he will most likely be infected for life and it is necessary to cull him from the herd.It only takes one producer to decide that the reproductive losses within a flock are acceptable, and deciding that a flock is "healthy" when in fact it is not. A recent example is the B. ovis infection that affected a previously uninfected flock of approximately 8000 Wyoming sheep. The affected producer routinely tests his newly introduced rams, and the rams he maintains always test negative. When the flock began having an unusually high number of abortions in the spring of 2014, the rams were tested again and most of them were now positive to B. ovis. It was determined that the source of infection came from another flock of sheep that had mingled with the Wyoming producer's flocks. The 2nd producer, did not routinely test his rams for B. ovis, and now there were major consequences for the Wyoming producer. His unexpected financial loss included culling 145 rams ($700/ram was average cost and investment = $101,500), veterinary fees for two different blood samples ($6915), B. ovis testing fees ($3455), additional brand inspections ($145), an extra ton hay for those segregated from the rest of the flock ($160/ton). Other losses included the potential loss in profit from the aborted lambs, and loss of invaluable genetics in the rams they were forced to cull from the flock. While B. ovis is not a federal regulated disease, there is discussion among the producers and at the state level of regulation on what the testing requirements should be. As shown in the above example, this becomes particularly important when there is "mingling" of flocks. And while Wyoming has a lot of range land, when there are large flocks, it is hard NOT to mingle on occasion.For sheep producers, it is imperative that good management decisions are made in order to protect and maintain the health of the animals under their watch. While $5 per test may not seem expensive, when you are looking at testing 300 animals for B. ovis, $1500 (300 X $15) becomes expensive. It is important to maintain healthy herds, with confidence that the flock is disease free, and testing is minimized to the new introductions, in order to protect the profit margin. The money spent for Brucella ovis diagnostic testing, is typically money well spent. However, no diagnostic tests are perfect (100% sensitivity and 100% specificity). The test currently used in the United States to detect exposure to B. ovis, is an ELISA (Enzyme-Linked Immunosorbant Assay). However, this test is not pre-manufactured, rather each laboratory buys the necessary components (i.e. antigen, controls, etc) and following specific instructions "creates" the test. This unfortunately causes some variation in results between laboratories, and frustration for the producer. For example, if there is a POSITIVE result by one lab and NEGATIVE result by another, what is the actual status of the animal? As a producer, do you cull that animal or do you keep it? In the proposed project, one objective is to compare the current ELISA to another one manufactured and marketed in Europe (IDEXX B.ovis Antibody Test) on the same samples and determine which one is better at detecting positive animals (higher sensitivity) that are truly positive (higher specificity). This ultimately will assist the sheep producer by providing the best test available for managing B. ovis in their sheep flocks. This overall goal of this study is to determine the seroprevalence of B. ovis in Wyoming sheep flocks. By utilizing samples collected from previous animal health screenings, we will have a good cross sectional representation of the Wyoming flocks. While the bacteria is typically considered a disease of rams, some ewes can maintain the infection for more than one estrus cycle and then become a reservoir for infection for other rams in subsequent breedings. Additionally, some rams do not generate antibodies and will test negative by the blood test even if they are currently infected. Thus testing the ewes in addition to the rams is a good way to assess the overall health of the flock. By determining if Wyoming flocks are infected, we can then look at other factors to assist producers in management of the disease. Ultimately this information will be useful for regulation decisions and testing recommendations for all the sheep producers in Wyoming.The results of this study will have direct implications on a national B. ovis seroprevalence project. The National Sheep Industry is interested in determining the extent to which domestic sheep flocks are exposed to this bacteria, as there is a direct effect on production profit when flocks are infected. This Wyoming study would be instrumental in providing the data to justify a national study.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
Brucella ovis has direct negative effects on lamb production, and is of major concern for Wyoming producers as lamb production accounts for 35% of gross agricultural sales (2012, University of Wyoming Extension). Brucella ovis (B. ovis) is a, gram negative bacterial pathogen that is present in most major sheep-producing regions of the world. Infection is introduced into a flock through an infected ram, and historically is associated with epididymitis. However, less than half of infected rams have palpable clinical abnormalities of the epididymis. The implications of a B.ovis infection for the flock include: ram infertility, decreased ewe conception rates, more abortions in pregnant ewes, and higher numbers of premature lambs. The effect of B.ovis infection is not only economic, as valuable genetics are also lost when infected rams are culled from the flock. Infection spreads throughout a flock of sheep by direct contact between rams, but can also be transmitted through the ewe when multiple rams mate with the same ewe during the breeding season. Serology can be used to detect exposure to B.ovis, and for Wyoming producers with larger flocks (>50 ewes) it is used as part of the breeding soundness exam. While ewes are not typically tested, there is evidence that they can harbor the bacteria for multiple estrus cycles and be a source of ram re-infection. Additionally, some infected rams do not develop antibodies, and by testing ewes an infected but sero-negative ram would be identified. The enzyme-linked immunosorbent assay (ELISA) is utilized by most veterinary diagnostic labs that test for this bacteria. There are multiple reasons to use this assay; consistent results, easily train personnel, fast turnarounds, and low cost. There are also a few drawbacks such as laboratory to laboratory variability, and the classification of samples as Indeterminate (not quite positive and not quite negative). This is extremely frustrating from the producers' perspective. How do they determine the actual infection status when one laboratory indicates the animal is Positive and another indicates Negative? And what does a producer do with an "Indeterminate" result? Is it worth the risk to the flock to introduce an "Indeterminate" ram into the flock?The seroprevalence of B. ovis has not been well documented in the United States, and a basic study concerning seroprevalence would be useful to better understand factors associated with its prevalence.The objectives of this study are: 1) To determine the historical seroprevalence of B. ovis in Wyoming sheep flocks2) Comparatively evaluate the currently available serologic tests for B. ovis3) Determine the current seroprevalence of B. bovis in Wyoming sheep flocks, and compare it to the historical seroprevalenceUtilizing samples collected from the National Animal Health Monitory System (NAHMS) study, we will have information about each sample regarding gender, location, age of animal, breed, and flock size. Due to the negative effect of B.ovis on sheep production, and the recent infection of an 8000 head flock in Wyoming resulting in an estimated loss of over $100,000, a study to determine the seroprevalence of B.ovis in Wyoming flocks is of interest to sheep producers, the Livestock Board and the Woolgrower's Association. Due to the inter-laboratory variability of the NVSL-ELISA, it is of interest to the Wyoming State Diagnostic Laboratory to determine if there is a better ELISA to utilize. The overall objective of this proposal is to determine the seroprevalence of Brucella ovis in Wyoming sheep flocks. By determining the seroprevalence of B.ovis, we can inform producers with data to assist them in making management decisions to improve the reproductive health, increase the economic return, and prevent huge economic losses due to the infection in their flocks. Future studies would include determining B.ovis seroprevalence across other regions in the United States.
Project Methods
Objective 1: Determine historical B. ovis seroprevalence from samples collected from domestic sheep in Wyoming in 2001 and 2011.The overall goal for this project is to determine the seroprevalence of Brucella ovis in Wyoming sheep flocks. We will begin the project utilizing previously collected sheep serum from the National Health Animal Monitoring System in 2001 and 2011. The first diagnostic assay that we will perform will be the NVSL-ELISA, which is currently the only ELISA utilized in the United States. The ELISA (Enzyme Linked Immuno-Absorbant Assay) detects antibodies to B. ovis in sheep serum samples. This assay is routinely performed at the Wyoming State Diagnostic Laboratory (WSVL), and Molly Elderbrook is currently being trained on practice samples to become proficient. The reagents including the B. ovis antigen, and controls (high positive, low positive and negative) will be purchased from the National Veterinary Services Laboratory (NVSL). The assay will be performed according to the Standard Operating Procedure (SOP) at WSVL, with samples run in duplicate. The ELISA will be read at a wavelength of 620nm on a spectrophotometer, to obtain an optical density (OD) reading for each sample. The average OD will be calculated for each sample and the controls. Calculations for each sample will be performed using the following formula: [ODsample/ODlowpos = S/P ratio]. For the test to be valid, the ODlowpos must be >0.350 and <0.650; the ODnegative must be <0.150; and the ODlowpos - ODnegative must be >0.150; and the ODhighpos must be >0.900. The sample is then classified at Positive, Negative or Indeterminate based on the cut-offs supplied by the reagent data sheet accompanying the specific lot number of the low positive control. Any samples testing "Indeterminate" will be identified and retested using the Complement Fixation (CF) assay. The CF assay is reported to be more specific than the ELISA, and can potentially resolve the "Indeterminate" status of samples. While this test is technically more challenging to perform, there are technicians at WSVL who have previously performed this assay and are willing to provide training. The reagents and SOP for this assay will also be obtained from NVSL.Objective 2: Compare B. ovis seroprevalence between two different ELISA tests (IDEXX vs NVSL), using the complement fixation test to resolve any "indeterminate" results.The goal for this objective is to utilize the same serum samples tested by the NVSL-ELISA in objective (1), and compare it to the IDEXX-ELISA. This B. ovis Antibody ELISA is manufactured and marketed in Europe by IDEXX. Each testing kit contains all the reagents and controls for two ELISA plates. The plates are already manufactured, so there is the potential for less variability in results due to fewer steps in the assay. The underlying principle of this assay is the same as the NVSL-ELISA, to detect the presence of antibodies to B. ovis in sheep serum samples. Samples will be classified as Positive or Negative following this calculation: [(ODsample-ODneg) / (ODpos- ODneg) = Value]The calculated value >0.800 is considered positive. The NVSL-ELISA and the IDEXX-ELISA results can then be compared. If there are sample discrepancies (i.e. positive by NVSL-ELISA, negative by IDEXX-ELISA or vice versa), then those samples will also be tested by the Complement Fixation (CF) assay. Base on the OIE recommendations, the CF is still the gold standard. An additional subset of 2001 samples will be run by CF in order to compare the results from the NVSL-ELISA and IDEXX-ELISA to the gold standard. A kappa statistic will be performed on all 2001 samples from both ELISAs to determine their agreement. At the end of this objective we will be able to determine if one ELISA out-performs the other for future diagnostic testing recommendations.Objective 3: Determine current B. ovis seroprevalence from samples collected from domestic sheep in 2015-2016, and compare it to historical seroprevalence.The goal for this objective is to obtain current B. ovis seroprevalence data from Wyoming sheep flocks. Sampling will be statistically determined, to obtain a representative cross-section that includes different regions, flock sizes, gender, age, and breed of sheep. Blood sampling will be coordinated with the producers and occur during their processing times in order to minimize stress on the sheep and make efficient use of available volunteers/workers in handling the sheep. The Animal Health Monitoring Service had previously performed statistics for their study, and collected approximately 1700 samples across Wyoming. This number may fluctuate after we perform our own analysis. Once the samples are collected they will be analyzed by ELISA. Finally, the seroprevalence for the 2015-2016 samples will be compared to that of 2001 and 2011 to determine if there is fluctuation in B.ovis infection in Wyoming sheep flocks.