Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Veterinary Population Medicine
Non Technical Summary
Pig welfare is a critical aspect of the ethics of animal husbandry and also a key for the advancement and public perception of the swine industry. Welfare can be greatly compromised in the presence of joint infections and lesions, which can have severe clinical presentation with lameness. The easily perceived economic losses involved in the lameness process are directly related to treatment costs, secondary conditions, and reduced growth rates. Mycoplasma hyosynoviae has been considered a re-emerging and important bacterial cause of infectious arthritis in growing pigs and, consequently, lameness in the US swine industry. However, little is known about the epidemiology and pathogenesis of this bacterium in pig lameness.Thus, we propose this study with the objective of performing an epidemiological investigation to determine the prevalence of detection of the bacterium and lameness at the farm level by combining sampling with oral fluids and lameness scoring. Also we plan to perform an in-depth investigation to identify bacterial agents present in the joint fluid of affected pigs, as the potential for co-factors or co-infections should not be discarded. Moreover, it is our objective to analyze the metabolic profile of the joint fluid in order to identify the biomarkers and better understand likely processes responsible for lameness in growing pigs. This proposal represents our efforts to give a comprehensive evaluation of the potential development of disease associated with M. hyosynoviae infections in growing pigs.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
With this investigation we propose to gain understanding on Mycoplasma hyosynoviae lameness in growing pigs, its presentation at the farm level, diagnostics, and pathogenesis associated with it. This will be accomplished by determining the level of detection of M. hyosynoviae in the field and by selecting clinically diseased animals and performing an in-depth study of the bacterial agents and metabolites present in the joint fluid. With this study, information will be generated regarding the extent of the post-weaning infectious arthritis and lameness, answering questions such as if M. hyosynoviae is actually a re-emerging concern, or if the combination of several factors and infectious agents are needed for disease presentation. In addition, this research will lay a foundation for the development of an infection model for pig lameness, which is largely missed.
Project Methods
Ten (10) wean-to-finish farms will be enrolled in this investigation. The farms will be selected based on the following criteria:- Willingness to participate and allow for one farm visit, pig sampling, lameness scoring, and euthanasia of three affected pigs.- Recent history of lameness in the flow (at least in the last year).- Housing pigs from an origin different that the other farms in this study.- A minimum site size of 1000 pigs.Farms will be visited at one time point and 4 oral fluid samples will be collected from pigs of each of the following ages: 4, 10, 16, 18, and 22 weeks for a total of 20 oral fluid samples from each farm.Field samplingOral fluids will be collected by pen, as described by Prickett and Zimmerman (2010). Briefly, a 70-cm length of 3-strand twisted rope will be untwisted and suspended at shoulder height from a metal bar in the pens. The rope will be left in the pens so that the pigs could chew on it for 20-30 min.Mycoplasma hyosynoviae detection by real time PCR in oral fluidsOral fluid samples will be processed for DNA extraction using MagMAX™-96 Viral RNA isolation kit and MagMAX™ Express-96 Magnetic Particle Processor (Life Technologies, Grand Island, NY, USA) and prepared to be assayed for detection of genetic material specific for M. hyosynoviae using an in-house real-time PCR (UMN Veterinary Diagnostic laboratory). PCR results will be used to calculate the prevalence of M. hyosynoviae at the farm level, along with the clinical presentation of lameness.Clinical assessment of lamenessClinical evaluation of the gait of the pigs will be performed and recorded prior to sample collection, preferably at the same time of the day, and before 11:00am. A pen lameness assessment will be performed in each pen of the visited barn. A total prevalence by barn will be obtained after analyzing and summarizing the prevalence by pen. An instrument (record sheet) will be designed by the student to record data regarding clinical assessment at the visit.The gait and leg position of sampled pigs will be assessed (as described by Nielsen et al., 2001) and recorded. Briefly, the pig's movement pattern will be from 0 to 4: Score 0. Pig gets up immediately from a lying position and moves freely in the pen with balanced weight on all four limbs. Score 1. Pig rises immediately but a reluctant movement is observed, with short steps and uneven distribution of body weight. Score 2. Pig moved slowly in the pen with short steps and reduced weight in the sore limb, or pig rises slowly and the affected limb was not weight bearing. Score 3. Pig reluctant to rise, with muscle shivering when standing and lifts the sore limb from the floor, or pig refuses to walk or walks on three limbs only. Score 4. Pig only rises when forced. Pig stand with marked signs of pain.At earlier stages of the design of this proposal, the investigators considered the potential of using a more objective measurement of the clinical presentation by way of a portable force plate platform in order to quantify pig lameness. While the idea of using the force plate was attractive, certain aspects detracted enthusiasm for the use of such method. The use of the force plate would have needed to be on site, at each farm, thus pigs could walk on it to be evaluated. This approach results difficult to apply for this investigation due to reasons associated with biosecurity. In other words, using the force plate at the farm would have implied to bring in the pieces of equipment to the farm and to leave them on-site, without the possibility of using the same equipment in all ten farms. And a cost of ~$8,500/force plate platform, the use of different equipment at each farm would have resulted extremely high cost and sub-use of the equipment, which would be unjustified. Thus, it was preferred to use a scoring system, that although subjective, maybe one of the best instruments for the screening of large number of animals in several operations (Nielsen et al., 2001).Selection of clinically affected pigs for histopathology, metagenomics and metabolomicsA total of three pigs will be identified at each one of the ten visited farms, for a total of 30 pigs in the investigation. The pigs will be selected in order to identify one apparently healthy pig (score 0) and two pigs clinically affected by lameness (score 3 or 4). Pig selection will be performed by the same investigator in all farms. Swine producers will be compensated for the value of the pig selected for euthanasia, and the cost of euthanasia. A total of three legs will be selected from each one of the euthanized pigs and will be brought to the VDL at the University of Minnesota. At the VDL the main joints of each leg will be aseptically sampled in order to collect synovial fluid and a sample of synovial membrane tissue will be fixed for histopathology. The three legs to be sampled would have been selected at the farm of origin including affected and non-affected limbs in the same pig. However, the identification and clinical characteristics of each limb will be blinded to the pathologist and to personnel collecting synovial fluid.Synovial membrane tissue will be fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 microns and stained with hematoxylin/eosin for evaluation by light microscopy. The presence, type, and relative number of inflammatory cells present in the sample will be recorded and the association with the identification of certain bacteria and metabolites will be investigated.The genomics analysis of the potential microorganisms present in the synovial fluid of pigs will be performed by 16s next generation sequencing. This new methodology has been of crucial importance to understanding the critical role microorganisms play in many biological processes, as well as the biological diversity of a microbial population in complex samples. Thus, we propose the use of 16s next generation sequencing in order to identify potential bacterial species that may participate or be present in the joint fluid of lame pigs. For example, species like M. hyosynoviae, M. hyorhinis and H. parasuis can cause articular disease in pigs. In the past we have approached disease investigations by targeted testing for each bacterial species. Instead, in this investigation we will be able to identify the main bacterial species causing lameness, and we will also have the potential of identifying other bacterial species not previously described, in the case they were to be present.Untargeted metabolomics analysis will be conducted to determine the metabolite composition of synovial fluid. Samples will be processed by solvent extraction, protein precipitation, and chemical derivatization to become compatible with LC-MS analysis. Respective LC-MS methods will be utilized to analyze organic acids, amines, lipids, and general metabolites (Chen and Kim, 2013). A coupled ultra-performance liquid chromatography (UPLC) and quadrupole-time-of-flight (QTOF) MS system will be used for LC-MS-based metabolomic analysis. The potential for identification of antibiotics or their metabolites in the synovial fluid is considered an important aspect of this sample analysis and will allow better understanding the effect of antibacterial drugs at the joint level.Data analysisPairwise t-test comparison with unequal variances with the Bonferroni-Holm correction for multiple comparisons will be used to compare the prevalence among age groups. Terms with a p-value < 0.05 will be considered significant. The correlation of results for M. hyosynoviae prevalence in oral fluids and lameness score will be evaluated using the Pearson's analysis. Histopathology and metagenomics data will be analyzed in a descriptive manner. LC-MS data will be processed by multivariate data analysis for identifying the metabolites associated with bacterial infection and lameness.