Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
ANIMAL SCIENCE
Non Technical Summary
The overall goal of this exploratory proposal is to determine the prevalence of herds positive to Mycoplasma wenyonii and C. Mycoplasma hemobos in selected regions of Wisconsin and Michigan and to generate preliminary data about potential risk factors associated with herd-level prevalence of these organisms. In this study we will be visiting dairy farms in Michigan and Wisconsin to determine if a blood parasite is causing infections in the cattle. To acheive this aim, we will be selecting farms in counties that contain a large number of dairy farms and then visting those farms to collect blood samples from a representative propostion of the cows. The blood samples will be tested to determine if the animals have been infected with these blood parasites. During our farm visits, we will also collect data about farm management practices to identify potential management practices that may contribute to increased risk of infection. Results of this exploratory study will be used to determine if hemotrophic mycoplasma represent an emerging pathogen of dairy cows.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
M. Wenyonii and a closely associated organism named Candidatus Mycoplasma hemobos are part of a group of host-specific animal pathogens collectively referred to as hemoplasmas. Cattle infected with hemoplasmas have exhibited immune mediated anemia, edema of the mammary gland and rear legs, pyrexia, lymphadenopathy, reduced milk yield, weight loss and infertility. The organisms have also been identified in blood of apparently healthy cattle and researchers have hypothesized that infection with hemotrophic mycoplasmas may enhance expression of other diseases. Transmission of hemoplasmas is thought to be primarily through contact with blood and possible routes of transmission include biting insects or vectors such as use of common needles, rectal palpation sleeves or other veterinary instruments. Recent testing of calves and heifers on farms in both Michigan and Wisconsin has revealed widespread apparent prevalence of PCR positive dairy cattle in herds that show the aforementioned clinical signs of disease. The prevalence of infection with these organisms is unknown. We are concerned that these organisms represent an unrecognized newly emerging disease and hypothesize that transmission may be facilitated by widespread use of shared needles in timed-breeding programs. The overall goal of this exploratory project is to determine the distribution of herds positive to Mycoplasma wenyonii and C. Mycoplasma hemobos in dairy intensive regions of Wisconsin and Michigan and to generate preliminary data about potential risk factors associated with herd-level prevalence of these organisms. The following aims will be performed to achieve this aim: Aim 1. Estimate herd-level prevalence of PCR positive herds for M wenyonii and C.M. hemobos in selected dairy intensive regions of Wisconsin and Michigan. Aim 2. Identify potential risk factors for disease (including prevalence of cows positive for Bovine Leukemia virus) associated with with-in herd prevalence of hemotrophic mycoplasmas in dairy herds in selected regions of Michigan and Wisconsin.
Project Methods
This study is designed as a prospective cross-sectional study to estimate prevalence and identify potential risk factors for infection with these organisms.Methods Used for Aim 1. The experimental unit is herd and the study is designed to estimate the proportion of herds that contain at least 10% of the cows that are positive for M. wenyonii, C. M. hemobos or both hemoplasma. Blood samples will be collected from cows (n = 30 per farm) on 100 dairy farms located in Michigan (n = 25) and Wisconsin (n = 75). The number of herds sampled per state is roughly equivalent to the proportion of herds located in the 2 states. Herds are eligible for the study if they are located in the top 10 (WI) or top 5 (MI) dairy counties (based on number of dairy cows). Selection of herds will be stratified by herd size to reflect milk production with 60% of enrolled herds containing >200 cows (larger herds) and 40% of herds containing <200 cows (small herds). Herds that contain a minimum of 50 lactating cows will be eligible for selection.Each farm will be visited once for sample collection and to administer the survey. All lactating and dry cows (including sick cows and cows designated to be culled) will be eligible. Upon arriving at the farm, research personnel will collect a list of all mature cows (parity 1+) and select 40 cows that will be eligible for blood sampling. Five ml of blood will be collected into an EDTA tube from the first 30 eligible cows using venipuncture of the coccygeal vein. An additional 12 cc of blood will be collected and used to separate serum for BLV testing. Blood samples will be immediately cooled for transport to the laboratory.Lab Procedures. Whole blood harvested into evacuated tubes containing EDTA will be used for DNA extraction. Briefly, 200 µl of blood will be processed following instructions suppled with a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, Valencia, CA). The kit uses a silica-based DNA purification method and the purified DNA will be eluted with 50 µl of molecular biology grade water. The eluted DNA will be used in PCR assays that target the 16S ribosomal RNA gene of Mycoplasma wenyonii and Candidatus Mycoplasma haemobos. The PCR primers used amplify DNA products that allow differentiation of the source mycoplasmas by number of base pairs in the product. The reagent mix for the PCR reactions will be AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City, CA). The PCR reaction conditions will be one cycle at 95° C for 5 minutes followed by 40 cycles of 95° C for 30 sec, 56° C for 30 sec, and 72° C for 30 sec. A final extension step of 72° C for 5 min completes the reaction. Detection of amplified product will be done using an agarose based electrophoresis system and ethidium bromide staining. Representative amplicons will be cut from gels, purified, and submitted to the Research Technology Support Facility at Michigan State University for Sanger sequencing to verify identity of the PCR product.A commercially available ELISA kit (VMRD, Inc., Pullman, WA) will be used for detection of antibody against the viral glycoprotein 51 (gp51) molecule. This assay is done routinely in the diagnostic serology laboratory. The directions for performance of the ELISA supplied by the manufacturer will be followed. The directions supplied by the manufacturer of the kit for operation of an automated ELSA plate washer also will be followed.Statistical analysis & Sample Size Calculation. The experimental unit is herd and prevalence will be calculated at the herd-level. Statistical analysis will be performed using SAS v9.4 (SAS Institute, Cary, NC). Herds with one or more cattle positive for M. wenyonii and/or C.M. hemobos by PCR will be considered positive for hemotrophic mycoplasma. Herd-level prevalence will be defined as the proportion of positive herds, and will reported as a point estimate with 95% CI using the exact method. Samples sizes were calculated to determine the number of cows/herd that need to be sampled to detect hemoplasma, given a hypothesized within-herd prevalence (Table 1). Based on the budget, we are proposing to sample 30 cows/herd and will be able to determine if tested herds have >10% prevalence of infected cows.Methods used for Aim 2. A survey instrument about herd- and animal-level putative risk factors will be adapted from a survey instrument previously used to identify risk factors for BLV. After development of the instrument, it will be pre-tested on several farms and adapted based on input from local veterinarians and producers. Before using the survey, all researchers will be cross trained to ensure consistency. During the data collection period, to ensure consistency among researchers, weekly conference calls will be conducted among research team members. During each farm visit, researchers will administer the survey to the farm owner or an appropriate farm manager. Data will be entered into an on-line database that will be accessible to all researchers.Statistical Analysis. Within-herd prevalence will be determined as a weighted average of positive cows in 1st, 2nd, 3rd and 4th+ parities. Individual risk factors associated with herd-level or within-herd prevalence at P ≤ 0.25 will be used to build multivariable models of risk factors associated with herd-level and within-herd prevalence using logistic and linear or ordinal regression models, respectively.Sample Size and Power Calculation. Assuming a 1:1 ratio of exposed/unexposed for a potential risk factors and 30% of unexposed herds being positive for hemoplasma, using 48 exposed and 48 unexposed herds, we will be able to detect an odds ratio of 3.3 with confidence and power of 95% and 80%, respectively.