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.
This report is the February edition of the Swine Disease Eradication Center (SDEC) research update. Not sure what the SDEC is? Check out this quick read about our research group collaborating with industry partners to solve problems faced by the swine industry.
Dr. Vilalta collaborated with Minnesota-based producers, the Veterinary Diagnostic Laboratory and the MycoLab to investigate processing fluids as a sample type for the detection of Mycoplasma hyopneumoniae.
This new publication in Veterinary Microbiology describes the best methodology to monitor 3-day-old piglets for PRRS, using both serum and processing fluid samples. The first author of the publication is Dr. Carles Vilalta, member of the Morrison Swine Health Monitoring Program (MSHMP) team.
Processing fluids (PF) constitute a useful sample to detect PRRSV infections at processing.
PRRSV can circulate in the farm at a low prevalence, increasing the chances of a re-break.
Young parity female litters should be targeted for PRRSV detection.
Current practice to bleed 30 pigs could be underestimating PRRSV prevalence in the herd.
The decrease in sensitivity at the litter level can be compensated by sampling more litters to detect PRRSV at the herd level.
The study was conducted in a 6,000 sow farm with a PRRS stable status. Every 3 weeks, serum samples and processing fluids were collected from all piglets in 10 randomly chosen litters. This process was then repeated 8 times, meaning that the farm was monitored for a total of 24 weeks. All samples were tested via PCR. 3 samples with the lowest Ct value were tested by virus isolation and sequencing of the ORF5 gene was performed.
10.6% of the piglets tested positive for PRRSv via serum PCR, representing 29.8% of the litters. The same number of litters tested positive via processing fluid PCR testing.
The percentage of processing fluid positive samples was significantly higher is parity 1 and 2 sows compared to parity 3 and older sows. Additionally, a significant association between parity and probability of detecting a positive pig was observed.
A significant higher proportion of positive serum samples was observed in males compared to females. A similar trend was obtained when comparing positive Ct values by gender with values from males being lower (i.e., higher viral load) than those from females.
Using a Ct value of 37, processing fluid samples had a Se and Sp of 87% (95% CI: 66%–97%) and 94% (95% CI: 85%–99%), respectively when compared with litter RT-PCR results obtained from individual serum samples. The total agreement between both tests was 92.2% and the positive and negative predictive values were 87% (95% CI: 66%–97%) and 94% (95% CI: 85%–99%), respectively. False negative processing fluids were identified in litters having 2 or less PRRSV positive piglets
The agreement between the PF and serum results was kappa = 0.81 (95% CI: 0.59–1.00). The difference in the proportion of positive samples between both types of sample was not statistically significant (McNemar test, p = 1).
Collection of serum samples of pigs at weaning to monitor for porcine reproductive and respiratory syndrome virus (PRRSV) has become a common practice to determine PRRSV herd infection status. Diagnostic sensitivity of this practice is low in herds undergoing PRRSV elimination once prevalence of infection is near zero. Thus, the goal of this study was to characterize the dynamics of PRRSV infection in 3 day-old pigs overtime using serum and serosanguineous fluids obtained as part of castration and tail docking practices (processing fluids (PF)). Secondary goal was to estimate sensitivity and specificity of PF in the 3 day old population. A 6000 breed-to-wean sow herd was monitored every three weeks for 23 weeks after a PRRSV outbreak by collecting both PF and individual serum samples from all pigs in the selected litters. Out of the 77 litters tested, 23 (29.8%) were identified as positive using the PF and the serum samples, with a Cohen’s kappa statistic of 0.81 (95% CI: 0.59–1) between the results obtained in each sample type. The sensitivity and specificity of the PF relative to the results in serum was 87% (95% CI: 66%–97%) and 94% (95% CI: 85%–99%) respectively. The percentage of PRRSV positive litters decreased over time and litters from gilts were more likely to test positive than those from older sows. Overall, the study demonstrates that PF can be a convenient and reliable specimen to monitor PRRSV infection in breeding herds.