Gilt acclimation has become a common practice in the United States swine industry to control Mycoplasma hyopneumoniae infections. The goal of gilt acclimation is to achieve uniform exposure of gilts prior to entry to the breeding herd and to create a “Day 0” for disease elimination purposes. Utilization of M. hyopneumoniae-positive lung homogenate (LH) for gilt exposure has shown to be efficient. However, the characterization, preparation, handling and storage of the LH have not been tested experimentally, which poses a risk of failure of successful exposure. Therefore, the purpose of this study was to establish methods of LH preparation and storage to optimize the process and result in a successful exposure of gilts during acclimation.
Three aims were completed in this study, including:
- To evaluate the M. hyopneumoniae concentration in different anatomical lung sections of naturally infected pigs,
- To evaluate the effect of the LH homogeneity on M. hyopneumoniae detection by real-time PCR,
- To evaluate the viability of M. hyopneumoniae in the LH under various storage and dilution conditions.
For aim one, lung donor pigs were selected, and categorized into low, medium and high Ct value groups based on deep tracheal catheter (DTC) samples. After humane euthanasia, lung lesion scores were recorded and bronchial swabs (BS) were collected from each lung lobe. For each lobe, tissue was blended at a 70:30 proportion of tissue and Friis medium to create lung lobe-specific homogenate samples. Real-time PCR for M. hyopneumoniae detection was performed on BS and lung lobe homogenates. For aim two, lungs were collected from four donor pigs and blended whole at a 70 tissue:30 Friis ratio. The resulting LH was used to obtain 50:50 and 30:70 dilutions. All three LH dilutions were further diluted at a 1:10 ratio and treated as follows: i) No filtration, ii) Gross filtration using a nylon stocking and iii) Fine filtration (100 µm). Filtered and diluted products were tested for M. hyopneumoniae via real-time PCR. For aim three, whole lungs were obtained from two M. hyopneumoniae-positive donors and blended separately. For each donor lung, two preparations were created including a 70:30 tissue:Friis medium dilution and a 70:30 tissue:saline dilution. Lung homogenate samples were then stored in different conditions: 1) Fresh at 25ºC, 2) 1 week frozen at -20ºC, 3) 1 month frozen at -20ºC and 4) 6 months frozen at -20ºC. There were four challenge groups based on the length of storage. For each challenge group, two gilts were randomly assigned a treatment of either LH prepared with saline or medium from donor pig one or two resulting in four gilts for both saline and medium. For all challenge groups, DTC and sera were collected at the start of the trial and four weeks post-inoculation. At necropsy, lung lesions were evaluated, and a BS was collected.
Gilts with low DTC Ct values had a relatively uniform distribution of M. hyopneumoniae throughout the whole lung, as well as a high bacterial load, compared to pigs with medium or high DTC Ct values. In addition, lung lesions were not a reliable indicator of the presence of M. hyopneumoniae in the lesioned lobe as M. hyopneumoniae was detected in lobes without any lesions and vice versa. Second, dilution and filtration method of the lung homogenate generated conflicting results, without a clear indication of their effect on M. hyopneumoniae detection by real-time PCR. Regarding the third aim of the investigation, both Friis medium and saline were adequate for dilution of LH, even when LH was frozen for up to six months at -20ºC. However, the capability of LH to induce disease in gilts appeared to be slightly lower for LH that was frozen for six months, compared to LH frozen for one month or less.