Nathan Winkelman, DVM, president and co-owner of Swine Services Unlimited, Inc. (SSUI), is the 2019 recipient of the Allen D. Leman Science in Practice Award, which is presented at the annual Allen D. Leman Swine Conference in Saint Paul, MN. Dr. Winkelman caught the science bug early in his career and, thankfully, never found a cure for his orientation toward discovery.
In an effort to examine the role of ingredients, especially vitamins, in feed biosecurity, the Swine Health Information Center (SHIC) and the University of Minnesota organized a vitamin manufacturing sector-wide workshop. Representatives from pork industry organizations including National Pork Board, National Pork Producers Council, American Association of Swine Veterinarians, vitamin manufacturers and blenders, and feed industry associations joined SHIC and the University of Minnesota for the workshop in late April in St. Paul, Minnesota. Participants focused on vitamins and the processes involved prior to delivery to a producer’s farm, with special focus on African swine fever transport and transmission risk.
The use of processing fluids (PF) to detect and monitor PRRSvand other pathogens is increasing among producers and veterinarians. Preliminary data from our research team identified Mycoplasma hyopneumoniae in PF at the litter level, using a speciesͲspecific realͲtime PCR, in a M. hyopneumoniae endemically infected farm.
To investigate the detection of M. hyopneumoniae in non-respiratory tissues and fluids collected from suckling pigs at processing age.
To develop an in situ hybridization (ISH) assay to further identify M. hyopneumoniae in non-respiratory tissues.
Material and methods
Freshly farrowed litters were sampled at two sow farms with previous detection of M. hyopneumoniae in PF. The following samples were obtained from:
Dams: Whole blood, serum,colostrum, whole placenta and vaginal swab.
Stillborn: Individually bagged and submitted for full diagnostics M. hyopneumoniae workup at the UMN-VDL. Whole blood was also collected during sampling.
Viable piglets: New born piglets were processed prior to suckling. Tails and testicles were collected individually per piglet and gender was recorded. Whole blood and laryngeal swabs were collected for all piglets. (PPE and sampling supplies were changed or disinfected between collection for each piglet)
Daily aggregated PF were collected at a sow farmover a 10-week period. A novel RNA-based ISH was developed using hybridization-coupled signal amplification system in histological tissue sections. To aid visualization of transcriptionally active bacterial organism expressing ribosomal and adhesin proteins.
Mycoplasma hyopneumoniae detection in non-respiratory tissues or fluids
All dams tested negative for M. hyopneumoniae by RT-PCR in blood, serum, colostrum, placenta, and vaginal swabs. Fifty percent of dams were seropositive by Oxoid™ Mycoplasma hyopneumoniae ELISA. All blood samples from stillborn and piglets resulted negative to M. hyopneumoniae by RT-PCR. Mycoplasma hyopneumoniae was detected in 2/54 individual fluid samples (tails and testicles). M. hyopneumoniae was detected (Ct<40) over the 10-week period by RT-PCR (Figure 1). PF and their associated testicles were collected individually at the litter level. All PF were tested by M. hyopneumoniae by RT-PCR. Samples were fixed in formalin to perform ISH on positive samples.
Development of an In situ hybridization assay
The ISH-RNA technique established the distribution of M. hyopneumoniae in affected tissues in association with histological lesions, characterized by lymphoplasmocytic peribronchiolitis and/or hyperplasia of the broncho-associated lymphoid tissues. In M. hyopneumoniae positive lungs, hybridization signals were observed in the apical membrane of the respiratory epithelium of bronchi and bronchioles. Positive signals were also observed in inflammatory cells and degenerative epithelial cells within the bronchial and bronchiolar lumen. The ISH-RNA technique provided molecular detection of M. hyopneumoniae cells expressing mRNA of proteins and elucidated the localization patterns by visualization in tissue.
Conclusions and Implications
Mycoplasma hyopneumoniae was detected intermittently in aggregated PF. In this investigation, M. hyopneumoniae was not detected in piglet tissues or samples, regardless of M. hyopneumoniae detection in aggregated PF. Regardless of the fact that environmental contamination can not be ruled out, aggregated PF could be a good indicator of M. hyopneumoniae a farm level. A specific In situ hybridization assay for M. hyopneumoniae was developed, which will be applied to nonͲrespiratory piglet tissue samples.