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PCR detection of Mycoplasma hyopneumoniae in piglet processing fluids in the event of a clinical respiratory disease outbreak
  1. Carles Vilalta,
  2. Beatriz Garcia-Morante,
  3. Juan Manuel Sanhueza,
  4. Mark Schwartz and
  5. Maria Pieters
  1. Veterinary Population Medicine, University of Minnesota, St Paul, Minnesota, USA
  1. Correspondence to Dr Maria Pieters; piet0094{at}


Diagnosis of early infection with Mycoplasma hyopneumoniae in breeding herds remains challenging. M hyopneumoniae has been recently detected in processing fluids (PFs), an emerging sample suitable for porcine reproductive and respiratory syndrome virus monitoring in pig breeding farms. This clinical report describes the unusual detection of M hyopneumoniae in PF at the same time that a clinical respiratory disease outbreak occurred in a previously M hyopneumoniae-negative sow farm. These results provide new insights into the value that testing PF to detect M hyopneumoniae may have in breeding herds.

  • <i>Mycoplasma hyopneumoniae</i>
  • outbreak
  • processing fluids
  • pigs

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Mycoplasma hyopneumoniae is the aetiological agent of porcine enzootic pneumonia and one of the major pathogens involved in the porcine respiratory disease complex (PRDC).1 Piglets are thought to be free from M hyopneumoniae at birth, but colonisation may occur as early as the first week of life.2–4 Notwithstanding, colonisation prevalence at weaning is highly variable, ranging from 0 to over 50 per cent in farms endemically infected with M hyopneumoniae.2–7

Sampling procedures that better approximate to the lower respiratory airways are considered the most sensitive for detection of this bacterium in live pigs.8–11 However, these sample types (eg, tracheobronchial swabs) are invasive and, therefore, less conveniently obtained. This, together with the high piglet colonisation variability, makes it extremely challenging to monitor early stages of infection with M hyopneumoniae in breeding farms. Processing fluids (PF), which consist of serosanguineous exudates from tissues obtained during newborn piglet processing activities, have arisen as an appropriate sample for monitoring porcine reproductive and respiratory syndrome virus (PRRSV) presence in pig breeding herds.12 13 Moreover, a recently published study highlighted the feasibility of detecting M. hyopneumoniae in PF.14

The present case report describes the detection of M hyopneumoniae in PF by means of real-time PCR in the event of a clinical respiratory disease outbreak that took place in a previously M hyopneumoniae-negative sow farm. Results provide new insights for the potential use that PF may have to detect M hyopneumoniae in breeding farms.

Case presentation

Case herd

The sow farm was located in the US Midwest and housed approximately 5450 sows and gilts divided in six barns: four for breeding and gestation, and two for farrowing. The farm had a 50 to 55 per cent average annual replacement rate and received replacement gilts from an on-site gilt development unit (GDU) of approximately 1600–1800 gilts managed by continuous flow. Gilts were obtained at weaning age from a single high health status multiplier. A PRRSV and M hyopneumoniae eradication herd closure was conducted at the sow farm early in 2015. However, the herd experienced a porcine reproductive and respiratory syndrome outbreak in October 2017. A modified live PRRSV vaccine was then applied to the breeding herd population. For the purpose of monitoring PRRSV, PF were collected weekly starting in March 2018. By then, the farm was deemed as clinically stable for PRRSV and negative for M hyopneumoniae. The farm was regarded as seronegative for M hyopneumoniae since no vaccination was applied. In addition, periodical laboratory tests from the wean-to-finish flow sourced from the sow farm were negative to M hyopneumoniae during the period 2016–2018.

Outbreak description

An outbreak of respiratory disease in the breeding herd was detected during the first week of August 2018. The outbreak was initially characterised by sudden coughing in the farrowing and gestation units. No cough was reported in the on-site GDU. To diagnose the ongoing respiratory disease, blood and nasal swab samples were immediately collected across clinically affected sows in the farrowing units. As the outbreak progressed, sow mortality and abortion rate increased in gestation barns. By mid-September, lungs from sow mortalities were submitted for further analysis and additional blood samples were obtained from gestating sows.

Diagnostic test results and control measures

Table 1 summarises test results obtained from samples collected from sows during the outbreak. M hyopneumoniae-like pneumonia lesions consisting of acute bronchopneumonia with lymphoplasmacytic alveolitis and perivasculitis were observed in lungs submitted for histopathology. Bacteriological examination of lung samples also revealed implication of Pasteurella multocida and Streptococcus suis. Altogether, findings pointed out that the respiratory problems were associated with an outbreak of PRDC as other bacterial agents and porcine circovirus type 2 (PCV2) were coinfections, along with M hyopneumoniae. Lymphoid tissues were not examined. Thus, the full extent of PCV2 infection was uncertain. At the first suspicion of M hyopneumoniae infection, feed containing 200 parts per million tilmicosin phosphate (Pulmotil 200 Premix, Elanco Animal Health) was pulsed to the breeding herd for a period of 3 weeks starting on August 24 2018.

Table 1

Proportion (per cent) of positive samples to a battery of laboratory tests performed for differential diagnosis of the clinical respiratory disease in the sow farm

PF sampling and testing procedures

PF collection consisted in gathering all tails and testicles from 15 farrowing gilts, 15 second parity sows, and 15 third parity sows and above, on a weekly basis. Tails and testicles were kept frozen for 1 or 2 weeks at the farm prior to submission to the University of Minnesota. At arrival, processing tissues were thawed at room temperature and PFs were aggregated by sow parity in a sterile tube. As a result, three PF samples were obtained on a given processing week: PF1 from primiparous sows’ litters, PF2 from second parity sows’ litters and PF3+ from third or more parity sows’ litters. In addition, individual litter samples from one of the processing weeks per month were also stored. All collected PF were frozen (−80°C) until tested. Once the PRDC outbreak was confirmed, a retrospective investigation was performed. PF previously collected and stored between March 19 and October 8 2018, were tested for M hyopneumoniae using real-time PCR. DNA extraction with a viral RNA isolation kit and a particle processor (Life Technologies) was performed as previously described.15 Following extraction, the nucleic acid templates were prepared for TaqMan-based real-time PCR with VetMAX qPCR Master Mix and VetMAX M hyopneumoniae Reagent Kit (Life Technologies),16 according to manufacturer’s instructions. In addition, VetMAX M hyopneumoniae control synthetic DNA (Life Technologies) at a concentration of 1000 copies/µl was used for relative quantification in suspect or positive PF samples. Briefly, 10-fold serial dilutions of the control DNA were prepared in nuclease-free water and were run in duplicate together with the corresponding PF. Thus, DNA quantity in PF was expressed relative to control DNA copies.

Laboratory findings

A total of 90 PF were tested by real-time PCR for M hyopneumoniae. All PF tested negative, except for three samples collected on August 13, 20 and 27, which showed cycle threshold (Ct) values of >37 (figure 1). The first two suspect PF were obtained from parity 3+ sows, whereas the last one was obtained from parity 2 sows. Remarkably, all three suspect PF were collected while the clinical respiratory disease outbreak was taking place, starting from the first week of August and ending by the second week of September. Individual litter PF gathered from the positive PF2 (week of August 27 2018) were available for further testing. PF2 was composed of PF from 12 litters that were individually tested by real-time PCR. All litter PF were detected negative, except for one litter with a Ct value of 32.21. The above-mentioned PF were retested by real-time PCR and their DNA were relatively quantified by a standard curve method. The number of DNA copies per millilitre of PF were 2.2, 1.4 and 3.5×103 for the samples from August 13, 20 and 27 August, respectively. The individual litter PF had 350×103 copies/ml. Results are depicted in figure 2.

Figure 1

Ct values detected in PF tested for Mycoplasma hyopneumoniae by real-time PCR from March 19 to October 8 2018. The shadowed graphical area represents an estimation of the duration of the clinical respiratory disease outbreak. The arrow points out the day when the antibiotic treatment was started. Ct, cycle threshold; PF, processing fluid; PF1, primiparous sows’ litters; PF2, second parity sows’ litters; PF3+, third or more parity sows’ litters.

Figure 2

Real-time PCR standard curve. The real-time PCR standard curve is graphically represented as a semilog regression line plot of Ct value versus log of input DNA. Blue dots represent the 10-fold serial dilutions of the control DNA, whereas the green squares represent the retested PF samples. Y-intercept=43.102; slope=−3.635; R2=0.999. Ct, cycle threshold; PF, processing fluid.


PF have been postulated as an appropriate sample for monitoring PRRSV in pig herds,12 13 and recently published data showed detection of M hyopneumoniae in this sample type.14 This latter finding is difficult to explain as M hyopneumoniae is regarded as an extracellular pathogen that resides uniquely in the respiratory tract of pigs by attaching to the cilia that lines its epithelium.17 Nonetheless, M hyopneumoniae has been cultured from tissues outside the respiratory tract, such as the liver, spleen or kidneys of experimentally infected pigs.18–20 Additionally, recent evidence suggested that this bacterium could persist intracellularly and traffic to extrapulmonary sites.21 Although the potential mechanisms or routes by which M hyopneumoniae is detected in PF remain largely unknown, the role that this sample could play in detection of early stages of M hyopneumoniae infection deserves to be further evaluated.

The present work reports the detection of M hyopneumoniae on a breeding farm that collected PF on a weekly basis for PRRSV monitoring. In the event of a PRDC outbreak, PF were retrospectively tested for M hyopneumoniae by real-time PCR. Remarkably, the unique three samples that turned to be suspect were collected in the period in which clinical signs of respiratory disease took place. In a previous study, M hyopneumoniae was consistently detected by real-time PCR in daily PF over a 2-month period.14 The report indicated the sow farm to be subclinically infected, as no clinical signs suggestive of M hyopneumoniae were observed, but were occasionally detected in the progeny, in the finishing pig population. A different clinical picture was seen in the present farm, where M hyopneumoniae was detected only in PF during a clinical respiratory disease outbreak in a previously M hyopneumoniae-negative farm. Most probably, the antibiotic feed grade pulsed to the breeding herd in response to the outbreak mitigated shedding, preventing further detections of M hyopneumoniae. In any case, PFs have been shown to be useful for detecting M hyopneumoniae in two different clinical scenarios.

The origin of the genetic material detected in PF remains uncertain and environmental contamination cannot be ruled out, especially since M hyopneumoniae has been shown to survive outside the host for up to 8 days.22 In this investigation, M hyopneumoniae was detected with Ct values of >37. PF with Ct values between 37.01 and 40.0 have been regarded as doubtful in previous work using the same real-time PCR.14 Fortunately, PF from each of the litters gathered in one of the suspect samples could be individually tested. One out of 12 of the gathered PF tested positive with a Ct value of 32.21. Additionally, all suspects could be relatively quantified using a standard curve method by real-time PCR, which confirmed the presence of M hyopneumoniae genetic material in PF. Since PFs are an aggregated sample, the sensitivity to detect a positive pig or litter, if present in the sample, may decrease as more negative pigs or litters are collected. For PRRSV, however, it has already been estimated that aggregation of up to 40 litters does not hinder detection by real-time PCR when a pig with a Ct value of ~33 is present in the sample.23 Here,~15 litters were aggregated. Thus, even Ct values above 33 should have been detected. Besides possible environmental contamination, the high Ct values observed in the present work might be due to a very low proportion of M hyopneumoniae-positive litters and the dilution factor inherent to aggregation and pooling in PF.

The fact that M hyopneumoniae has been consistently detected in PF from two different epidemiological contexts has raised questions about the current understanding of the pathogenesis and epidemiology of this pathogen. While with the current data it is not possible to draw definite conclusions, more efforts are needed to elucidate the origin of the genetic material from M hyopneumoniae detected in PF. Even in the case of environmental contamination, the value of using this accessible sample type to detect M hyopneumoniae in breeding herds deserves further investigation.

Learning points

  • Genetic material of Mycoplasma hyopneumoniae from processing fluid (PF) was quantified by real-time PCR.

  • The origin of M hyopneumoniae genetic material detected in PF needs to be clarified.

  • PF are a potential sample to detect M hyopneumoniae in breeding herds.


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  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available upon request.

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