Assessment of nebulization technology for gilt exposure to Mycoplasma hyopneumoniae as an acclimation strategy

Mycoplasma hyopneumoniae is the etiologic agent of mycoplasmal pneumonia, a chronic bronchopneumonia which impacts swine health worldwide.¹ Infection with M hyopneumoniae predisposes pigs to infections with other respiratory bacteria and viruses, playing an important role in more clinically and economically relevant diseases known as enzootic pneumonia (EP) and the porcine respiratory disease complex (PRDC).¹ Despite all efforts implemented to reduce the economic impact attributed to M hyopneumoniae, EP and the PRDC are still associated with important economic losses to the swine industry.

Although indirect contact has importance in the infection dynamics of M hyopneumoniae, direct nose-to-nose contact between infected and susceptible pigs is considered the main route of transmission.² First exposure to M hyopneumoniae occurs during lactation when piglets may become infected in the farrowing unit through shedding sows.^3-5 Several studies have demonstrated that piglets may be colonized during the lactation period and are then positive with this bacterium when weaned.^6-9 Moreover, it has been shown that disease severity in growing pigs could be correlated with M hyopneumoniae piglet prevalence at weaning.^9,10

Circulation of M hyopneumoniae is thought to occur among existing sows and be transmitted to incoming gilts.³ An inverse relationship between parity number and M hyopneumoniae shedding has been described, thus, gilts and low parity sows infected for the first time are considered the main source of the bacteria to suckling piglets.^2,11,12 In addition, the existence of negative subpopulations that can reach 20% of the gilt population have been described in positive herds.¹³ This, together with M hyopneumoniae shedding that can persist up to 214 days post infection,¹⁴ make the implementation of an early and proper gilt acclimation process against M hyopneumoniae of paramount importance. Therefore, an adequate gilt acclimation pursues the elimination of bacterial shedding at first farrowing to minimize piglet colonization and later problems in the growing phase.^15,16

Vaccination is the main strategy used for replacement gilt acclimation procedures against M hyopneumoniae in both Europe and North America.¹⁶ Vaccination protects the gilts although it does not stop them from being infected and shedding the organism.^17,18 Another frequently used acclimation strategy is direct contact with pigs that are suspected to be infected.^19-21 In this strategy, a uniform infection with M hyopneumoniae is difficult to achieve as transmission is known to be very slow.^22,23 To increase the success of infection, the use of lung tissue homogenate containing M hyopneumoniae to deliberately infect replacement gilts has been recently reported in the United States and Mexico.^20,21,24,25

Controlled exposure of naïve gilts to M hyopneumoniae infective material might be a complementary method for gilt acclimation that deserves further investigation. The intratracheal (IT) method is the most widely used in M hyopneumoniae experimental inoculation procedures.²⁶ However, due to the difficulty that this method represents at a large scale in the swine industry, this study assessed the efficacy of M hyopneumoniae exposure using nebulization (NEB), also referred to as fogging, under field conditions. Efficacy of exposure was determined by real-time polymerase chain reaction (qPCR) testing of pooled tracheobronchial swabs (TBS) collected at 2 and 4 weeks post exposure to confirm M hyopneumoniae infection.

Materials and methods

Animals and housing

The study was conducted from July 2017 to June 2018 in a 2200-head gilt development unit (GDU) located in Aragón, Spain. This farm consists of two barns, each 854 m²; each barn had two 420 m² units. The gilts (Landrace × Large White crossbred) in the study were housed in 9 m² pens in groups of 10 within the same unit. The GDU worked in batches depending on sow farm demand and used an all-in/all-out management system. Feed and water were available ad libitum in stainless steel feeders and through water nipples, respectively. Gilts were raised in facilities with fully slatted floors and fed a diet to meet or exceed their nutritional needs. All animals were under veterinary oversight and care with a veterinarian-client-patient relationship and a Welfare Quality based certification in place (animal welfare certification by Asociación Española de Normalización y Certificación). Gilts weighed approximately 20 kg upon entry to the GDU (approximately 7 weeks of age) and approximately 100 kg upon departure from the GDU (approximately 28 weeks of age). All gilts were obtained from a unique nucleus and multiplier pig farm known to be negative for M hyopneumoniae, wild type porcine reproductive and respiratory syndrome virus (PRRSV), and influenza A virus. The routine vaccination program included immunization against PRRSV, Aujeszky’s disease virus, influenza A virus, porcine parvovirus, Erysipelothrix rhusiopathiae, Actinobacillus pleuropneumoniae, porcine circovirus type 2, and M hyopneumoniae as a growing pig (starting at 10-12 weeks of age) followed by a booster immunization 3 to 4 weeks apart. Prior to study initiation, no signs of any major pig diseases were observed. No antimicrobials were administrated to gilts under study.

Experimental design and sample collection

This research consisted of two phases: phase I had a total of 448 gilts from 4 batches (1-4) and phase II had 1160 gilts from 3 batches (5-7). The experimental design is presented in Table 1. In all cases, batches were formed with a varying number of gilts and exposure to M hyopneumoniae occurred at 10 to 13 weeks of age (study day 0). In phase I, batches 1 and 2 were used as positive controls and were inoculated via IT, while batches 3 and 4 were exposed to the pathogen by NEB. In phase II, all batches (5-7) were exposed to M hyopneumoniae via NEB at 10 to 13 weeks of age. In each batch, a subgroup of 30 gilts was randomly selected and monitored for infection confirmation during the acute phase of infection. For this purpose, TBS were collected at 2 and 4 weeks post exposure (D14 and D28, respectively) in phase I, and at D14 in phase II.

M hyopneumoniae infectious material

The infectious material for batches 1 and 2 was obtained from a commercial farrow-to-wean herd with EP problems in replacement gilts at entry and in offspring at finishing stages. The herd received gilts from the GDU used in this study. Ten, clinically affected, 24-week old gilts were selected and subjected to TBS sampling. The seed material donor was identified based on the minimum presence of other swine respiratory pathogens and the lowest cycle threshold (Ct) to M hyopneumoniae as determined by qPCR and described by Robbins et al.²⁷ Subsequently, the selected donor was euthanized, necropsied, and the lung tissue was used to prepare the seed tissue homogenate. For batches 3 and 4, seven gilts from the GDU were artificially inoculated with M hyopneumoniae (batch 1), euthanized, and necropsied. Seed material donors were selected by testing lung homogenate and using qPCR and the same criteria previously mentioned. In this case, lungs from 3 gilts were selected to proceed with the seed tissue homogenate preparation. Lastly, lungs from 10 gilts belonging to previous GDU batches exposed to M hyopneumoniae were used for the seed lung homogenate preparation for batches 5, 6, and 7. In all cases, seed tissue homogenates were prepared roughly as a ratio of 6 g of lung tissue for every 4 mL of homemade Friis medium. Thereafter, the homogenates were confirmed to be positive for M hyopneumoniae and stored in 30 mL aliquots at -80°C until used. Presence of M hyopneumoniae and other pathogens in the three seed lung homogenates are shown in Table 2.

M hyopneumoniae inoculation

Gilts from batches 1 and 2 (phase I) were inoculated once via IT with 10 mL of inoculum. The inoculum was prepared with the seed lung homogenate at a dilution of 1:50 in Friis medium and at a final concentration of 4.6 × 10⁶ genome copies/mL, as determined by qPCR. The inoculation technique was performed as previously described by Pieters et al,¹⁴ but without the use of anesthesia. Briefly, a post cervical insemination catheter (Magaplus; Magapor) was used for lung homogenate delivery into the trachea, and a laryngoscope and a mouth gag used for visualization. An electric portable aerosol applicator (Hurricane Ultra; Curtis Dyna-Fog Ltd) was used to expose gilts in batches 3 to 7 (phase I and II) to M hyopneumoniae via NEB. In this case, inoculum was prepared with the seed lung homogenate at a dilution of 1:50 in Friis medium and grossly filtered to discard tissue debris to avoid equipment malfunction. In phase I, inocula final concentrations of M hyopneumoniae were 6.6 × 10⁶ and 8.9 × 10⁶ genome copies/mL in batch 3 and 4, respectively. In phase II, inocula final concentrations were 1.1 × 10⁴, 2.1 × 10⁵, and 1.6 × 10⁷ genome copies/mL in batch 5, 6, and 7, respectively. Infectious material was administered with a total output rate of approximately 236 mL/min at 220 volts over 2 minutes in each pen housing 10 gilts. The fogger was manually focused toward the gilts’ snouts and a left-right movement made to ensure that all animals inhaled the aerosol. The particle sizes generated ranged from 7 to 30 µm of volume mean diameter depending on the flow rate and viscosity of the inoculum. During the NEB procedure, all barn windows were closed to avoid air flows that could interfere with the exposure of the gilts to the infectious material. Additionally, all personnel that could be putatively exposed to the aerosol used personal protection equipment including a respirator (3M 4279 Reusable Half Face Masks; 3M) and goggles (3M GoggleGear 500 Series GG501SGAF; 3M).

Sample collection, processing, and testing

Thirty gilts within each exposed batch were randomly selected for TBS sampling at D14 and D28, which were obtained as previously described by Fablet et al.²⁸ A gilt was restrained with a nose snare and a mouth gag and laryngoscope were used for visualization. A post cervical insemination catheter was used to reach the trachea-bronchial bifurcation where mucus was collected through gentle catheter movement. The tip of the catheter (2-cm diameter) was placed in a 5 mL BD Serum Vacutainer tube (Becton Dickinson and Company), mixed with 2 mL sterile saline, and refrigerated until testing. Individual TBS were tested in pools of 5. Each sample or sample pool was sent to EXOPOL S.L.U. (Zaragoza, Spain) and analyzed using an M hyopneumoniae specific qPCR (EXOone M hyopneumoniae oneMIX qPCR; EXOPOL S.L.U.), which has been validated using a DNA purification kit (UltraClean Tissue & Cells DNA Isolation Kit; MOBIO Lab, Inc) for DNA extraction. The qPCR kit contains an endogenous control to avoid false negative results and ensure that the entire process has been correctly performed. A sample was considered positive for M hyopneumoniae when the Ct value was ≤ 38.

Data analysis

Statistical analyses and data summaries were performed using Graph Pad Prism 8 software. All data were summarized descriptively based on the type of variable and analyzed assuming a completely random design structure. An analysis of variance through the ordinary one-way ANOVA was applied for mean comparison of qPCR Ct values among gilt batches at D14 and D28. The Chi square test was used to evaluate the proportion of positive qPCR samples between groups at different sampling points. Tests on differences were designed as 2-sided tests at α = .05, with differences considered significant if P ≤ .05.

Results

Phase I: Batches exposed via IT vs NEB

The Ct values of the positive TBS pooled samples in the acute phase of infection (D14 and D28) from batches 1 to 4 are shown in Figure 1. Overall, no statistically significant differences were observed in proportion of positive TBS pooled samples or in potential bacterial load (mean Ct value) between batches at any time point. At D14, all samples were positive for M hyopneumoniae in all batches, regardless of the exposure method (ie, IT or NEB). At D28, 4 of 6 (66.7%) TBS pooled samples were positive for M hyopneumoniae in batch 1, 5 of 6 (83.3%) samples were positive in batches 2 and 3, and 6 of 6 (100%) samples were positive in batch 4.

Phase II: Batches exposed via NEB

The Ct values of the positive TBS pools in the acute phase of infection (D14) from batches 5 to 7 are shown in Figure 2. No statistically significant differences were observed in proportion of positive TBS pools or in indicative bacterial load (mean Ct value) between batches at D14. In parallel to phase I, all pooled samples were positive for M hyopneumoniae in all batches at D14.

Discussion

Because gilts might be the major source of M hyopneumoniae to newborn pigs,^2,3 a suitable gilt acclimation focused on reducing the bacterial shedding at first farrowing has been suggested.¹⁵ Information on gilt acclimation strategies for M hyopneumoniae is limited; a recent review has pointed out that vaccination is the main strategy used in Europe, Mexico, and the United States.¹⁶ Vaccination of gilts for M hyopneumoniae at acclimation may be effective to decrease shedding and infectious pressure,²⁹ however, studies under experimental¹⁸ and field conditions^7,30 showed that vaccination did not prevent infection and transmission of the pathogen. To control the time of infection with M hyopneumoniae and minimize the likelihood of bacterial shedding at the first farrowing, early controlled exposure has been attempted by administering lung tissue homogenate containing M hyopneumoniae to replacement gilts.^20,21,24,27 Inoculation with lung homogenate prepared from infected pigs might have the potential to introduce adventitious agents and aggravate the inflammatory response due to the administration of foreign antigens.³¹ While the use of M hyopneumoniae pure culture would avoid these concerns, this bacterium is notoriously fastidious, and bacterial culture remains challenging and time consuming.³² Moreover, potential legal aspects could arise from the use of bacterial isolates at farm level.

To intentionally infect pigs with M hyopneumoniae, the IT inoculation route has been extensively used under experimental settings.²⁶ The IT route is expected to apply a greater inoculum volume to the pig’s lower respiratory tract, achieving greater infectious doses in shorter times and promoting an earlier M hyopneumoniae colonization.³³ Nevertheless, IT application is labor intensive, time consuming, and invasive. Practically, these factors present a great challenge to implementation of this method on a large scale in the swine industry. Another option would be the controlled exposure of naïve gilts to knowingly shedding animals (seeders). Roos et al²³ concluded that 6 seeders infected via IT were required in a group of 10 gilts for successful exposure to M hyopneumoniae in a 4-week exposure period. Due to the high ratio of infected animals needed and the unfeasibility of the IT methodology under field conditions, practical alternatives to ensure infection with M hyopneumoniae are needed. While NEB is supposed to closely mimic the natural conditions of M hyopneumoniae infection, it may also pose some constraints such as biosecurity and biocontainment issues, or lower dosage accuracy. In earlier work, pigs inoculated via IT displayed a significantly earlier upper respiratory tract colonization of M hyopneumoniae compared to those inoculated through NEB using individual exposure via an inhalation mask.³³ This finding could suggest a certain time lag in M hyopneumoniae infection dynamics partly due to the method of pathogen exposure. The present study assessed the validity of NEB to purposely expose naïve gilts to M hyopneumoniae in a field context. In this framework, efficacy of NEB to infect gilts was evaluated by collecting TBS after exposure (D14 and D28) and testing pooled TBS by qPCR.

Tracheobronchial swab sampling in combination with qPCR testing for M hyopneumoniae has been introduced as an innovative technique to consistently detect the pathogen in different contexts.^28,34-38 It has been suggested that TBS is the most sensitive antemortem sample available to assess M hyopneumoniae prevalence in a pig population.^35,38 In our study, 30 TBS were collected per batch and time point. This sample size is widely used in the field to detect at least 1 positive from a population of 1000 pigs, assuming a 10% prevalence and 95% CI.³⁹ Applying this to the present research, at least one negative exposed pig per batch would have been detected by using this sample size. However, to significantly reduce the number of tests and associated costs, samples were submitted for testing in pools of 5. While this dilution step has been proposed as the preferential approach for field studies collecting TBS,^24,35,40 pooling more than one animal for each sample minimizes the effects of biological variation between individuals. In this context where high prevalence and low Ct values are expected, there is a risk of not detecting negative animals in a pool. This can certainly be understood as a limitation of the study as it can give false confidence about the data significance. Nevertheless, there is evidence in the literature that prove it unlikely that the pooled positive samples from this study were mainly composed of negative individual TBS. According to Sponheim et al,³⁸ the cumulative incidence of M hyopneumoniae infection, as detected by individual TBS, was 100% at about 14 weeks post infection in pigs inoculated via IT. A previous work using a mechanical fogger to expose gilts to M hyopneumoniae reported 100% positive individual TBS between 8 and 11weeks post fogging.²⁵ More recently, 100% of pooled TBS were PCR positive to M hyopneumoniae at 14 days post infection in pigs also exposed by NEB.²⁴ In this latter case, all samples were tested individually and every sample was confirmed PCR positive.

Overall, data obtained in the present study support the idea that NEB may be a convenient and effective methodology to infect gilts with M hyopneumoniae for acclimation purposes. Tracheobronchial swab pooled samples revealed no statistically significant differences in proportion of positives or in mean bacterial loads between batches early after exposure via IT and NEB. Although real prevalence of negative animals to M hyopneumoniae after exposure could not be addressed, the only pooled TBS samples that tested negative were collected on D28 from gilts exposed by both the IT and NEB routes. Due to the high analytical sensitivity yield by qPCR, all individuals within the negative pools are generally considered negative. However, and using PRRSV as an example, about 6% of the samples that would be detected by reverse-transcriptase PCR on individual serum would be missed if they were run in pools of 5.⁴¹ In processing fluids, samples with initial Ct values of 35 would fall above the suspect threshold if further diluted.⁴² However, pools that test positive indicate that at least one individual within each pool is positive, and individual retesting of each specimen is needed to discern between positives and negatives. Unfortunately, retesting of individual TBS samples could not be performed in this study as prepooled samples were not available. Moreover, to the knowledge of the authors, there is no literature assessing the changes in Ct values of M hyopneumoniae positive TBS pools due to the presence of negative samples. In consequence, the existence of negative subpopulations after exposure to M hyopneumoniae by the NEB technology cannot be discarded. Whether such negative subpopulations shortly after exposure influence the efficacy of acclimation strategies in reducing M hyopneumoniae shedding and prevalence of disease in downstream flow is unexplored and needs to be addressed in future work. Another scenario would be a herd undergoing an M hyopneumoniae elimination protocol. In this case, the presence of susceptible subpopulations represents a major risk for program failure,⁴³ which emphasizes the need to develop accurate diagnostic protocols to determine the success of M hyopneumoniae exposure. In summary, tailored diagnostic protocols are needed to reach the objective pursued with each acclimation strategy, which can be either control (low prevalence) or eradication of the infection.

Besides the inoculation methodology, successful exposure to M hyopneumoniae was also observed irrespective of the inoculum bacterial concentration. Thus, different titrations (expressed as genome copies per mL) of the final lung tissue homogenates were obtained, but no significant differences in proportion of positivity or in bacterial loads from TBS pooled samples were detected between any of the batches. While many other factors are probably involved, qPCR is not indicative of the bacterium viability in the inoculum as DNA fragments have been reported to be present in culture for long periods even when M hyopneumoniae cells are no longer viable.⁴⁴

Mycoplasma hyopneumoniae-infected pigs via IT can potentially excrete the bacterium for up to 214 days following initial infection,¹⁴ though the duration of shedding could vary in naturally infected gilts under field conditions.²⁹ In the present study, no excretion data was obtained late post exposure. This information for NEB also is lacking in the literature, thus, whether the excretion pattern is different in pigs exposed to M hyopneumoniae by NEB remains unknown and should be the subject of further investigation. The age of exposure has major importance when the goal is to obtain nonshedding gilts by the time of first farrow. Gilts from the present study entered and left the GDU at approximately 7 and 28 weeks of age, respectively, and the age at first mating was about 35 weeks. Considering a shedding duration of 214 days (approximately 31 weeks) and that the acclimation process started around 10 to 13 weeks of age, the protocol used in this study would likely have ensured the elimination of M hyopneumoniae shedding at first farrowing, as suggested by Pieters and Fano.¹⁵ Regrettably, M hyopneumoniae status of gilts at first farrowing was not checked, therefore, it remains unknown whether this acclimation protocol was effective in reducing bacterial shedding at that critical time. Also, there are numerous factors that could impact the duration of M hyopneumoniae shedding, for instance, the immunological status of the infected animals. The gilts enrolled in this study were vaccinated against M hyopneumoniae before their entrance to the GDU. In naturally infected gilts under field conditions, a lower duration of M hyopneumoniae shedding has been suggested in vaccinated gilts when compared to their nonvaccinated counterparts.²⁹ The latter, however, should be corroborated in experimentally inoculated animals where the exact time of exposure to M hyopneumoniae is known. Still, protocols including exposure of vaccinated gilts could be advantageous for reducing acclimation timings.

Implications

• Gilt acclimation to M hyopneumoniae is key for sustainable EP and PRDC control.
• Controlled exposure to M hyopneumoniae may be a complementary acclimation method.
• Nebulization could be used consistently to expose gilts to M hyopneumoniae.

Acknowledgments

We gratefully acknowledge the employees from Piensos Costa that allowed and facilitated sample collection and submissions and follow up on this case. We would also like to thank EXOPOL S.L.U., especially to Gema Chacon, for their excellent technical assistance. This study was funded by Boehringer Ingelheim Animal Health.

Conflict of interest

None reported.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.

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