Background: A dedicated program to monitor for freedom of several economically important diseases is present within most of the breeding companies that currently deliver high health breeding animals to their customers. Serology is therefore the preferential approach in order to screen for most of these diseases, including Mycoplasma hyopneumoniae (M. hyopneumoniae). However, in case of positive serology, further decisions on farm health status and the related consequences should be based on additional confirmation tests. Case presentation: The current case report demonstrates that tracheo-bronchial swab (TBS) sampling is a suitable alternative to confirm a suspect M. hyopneumoniae-seropositive situation. A Central-European SPF herd was shown positive (90% positive, 10% suspect; n = 10) for M. hyopneumoniae using the conventional ELISA serology (Idexx HerdChek Mhyo ELISA) and a second ELISA test (IDEIA™ Mycoplasma hyopneumoniae EIA kit) did not exclude potential M. hyopneumoniae infection (10% positive, 70% suspect; n = 10). Further follow-up remained inconclusive on both tests. Throughout the entire monitoring period of 6 months, no coughing, necropsy lesions or lesions at slaughter could be detected which could confirm the M. hyopneumoniae health status. TBS sampling was used to confirm the health status for M. hyopneumoniae. In total, 162 samples were collected at different ages (n = 18 per age category): piglets at 3–6–9-12 and 15 wks of age, rearing gilts at 18–21-24 and 27 weeks of age. Collected TBS samples were negative for M. hyopneumoniae until 15 wks of age, but rearing gilts were highly M. hyopneumoniae- positive from 18 wks onwards with 87–100% M. hyopneumoniae-positive animals and PCR Ct-values between 25 and 33. Conclusions: This case report shows that collection of TBS samples to confirm the M. hyopneumoniae infection status of a breeding herd was able to provide additional information to serology in order to make crucial decisions concerning health management and eradication strategies within the breeding herd. Background Porcine Respiratory Disease Complex (PRDC) through in- Mycoplasma hyopneumoniae (M. hyopneumoniae),the teractions with several other respiratory pathogens. primary pathogen of enzootic pneumonia, occurs world- Piglets can become infected with M. hyopneumoniae wide and causes major economic losses to the pig indus- during the suckling period and many studies have shown try. The pathogen adheres to and damages the ciliated M. hyopneumoniae-positive animals from weaning on- epithelium of the respiratory tract. Affected pigs usually wards [2–7]. Moreover, once infected with M. hyopneu- show chronic coughing, are more susceptible to other re- moniae, animals can excrete the pathogen over a long spiratory infections and have a reduced performance ). period of time, with total clearance lasting till 254 days Moreover, M. hyopneumoniae plays a key role in the post-infection . This implies that infected gilts could carry M. hyopneumoniae well across their first preg- nancy into their first lactation cycle, infecting their off- spring with M. hyopneumoniae in early life. * Correspondence: firstname.lastname@example.org Elanco Animal Health Benelux, Benelux, Plantijn en Moretuslei 1 – 3rd floor, Therefore, dedicated programs to monitor for freedom 2018 Antwerpen, Belgium of M. hyopneumoniae have been developed within Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vangroenweghe et al. Porcine Health Management (2018) 4:12 Page 2 of 6 breeding companies that currently deliver high health hyopneumoniae, the farm was also negative for Pasteur- breeding animals to their customers. Serology using ella multocida DNT+, Sarcoptes scabiei var. suis, ELISA is the preferential approach in order to screen for Brachyspira hyodysenteriae, Porcine Reproductive and M. hyopneumoniae [9–14]. In case of positive serology, Respiratory Syndrome Virus (PRRSV) and Actinobacillus further decisions on farm health status and the related pleuropneumoniae. consequences should be based on additional confirm- External biosecurity is at the highest level, with no en- ation tests. Clinical diagnosis of enzootic pneumonia can trance to visitors and strict shower protocols for all farm be verified by serological analysis . However, in SPF personnel upon entrance of the farm. Internal biosecur- programmes, the herd prevalence of M. hyopneumoniae ity is also well established with boot hygiene (washing infections is often low and the positive herd predict- and disinfection) between production groups (sows, pig- ive value of a serological result decreases progres- lets, rearing gilts), clean disinfection baths at entrance of sively with the decreasing herd prevalence . each individual compartment and no movement of other Moreover, ELISA testing of sera from naturally in- materials (cleaning equipment, pig handling materials, fected pigs does not detect early-stage infection prior etc.) between production groups. to seroconversion [16, 17], and infection and vaccin- The sow farm is run on a 3-week batch management ation responses are indistinguishable. Under field con- system with 7 groups of 32 sows each. Productive sows, ditions, the mean time to onset of coughing following weaned piglets and rearing gilts are housed in separate an M. hyopneumoniae infection was 13 days, whereas the buildings on the premises. Successive batches of weaned mean time between onset of coughing and seroconversion piglets from 4 weeks until 16 weeks of age are housed in as measured by ELISA was 9 days . Recent research separate nursery compartments with strict all-in all-out has shown that currently used ELISA tests only start (AI/AO) strategies. From 16 weeks of age onwards, rear- showing a seroconversion from 21 days post-infection on- ing gilts are housed in a larger barn that is not managed wards . The percentage of animals seroconverting in according to AI/AO strategies. the early stages of M. hyopneumoniae infection using one of the commercially available M. hyopneumoniae ELISAs Standard serological monitoring for M. hyopneumoniae remains relatively low (16–22% at 21 days and 35–45% at At gilt delivery (27 weeks of age), regular serological sam- 28 days post-infection) . This implies that a large pling to assess M. hyopneumoniae status was performed number of samples is needed to reliably detect the pres- throughout the last decade, repeatedly confirming the M. ence of M. hyopneumoniae within the monitored herd. In hyopneumoniae-negative status. The farm first tested posi- the Danish SPF program, the final verification of herd in- tive for M. hyopneumoniae using the first ELISA serology fection with M. hyopneumoniae is consequently per- (Idexx HerdChek Mhyo ELISA, indirect ELISA; Idexx formed by demonstration of the agent . A recent Laboratories) in March 2017. Additional monitoring one comparative study on diagnostic sampling approach for month later (Idexx) confirmed the M. hyopneumoniae M. hyopneumoniae detection showed that laryngeal swabs positivity and therefore, a second ELISA test (IDEIA™ were a reliable option to establish early detection of M. Mycoplasma hyopneumoniae EIA kit; Oxoid – Thermo hyopneumoniae, followed by brocho-alveolar lavage fluids Scientific) was performed, demonstrating a clear evolution and nasal swabs . Other innovative sampling tech- towards lower M. hyopneumoniae-positivity in both niques, such as tracheo-bronchial swab (TBS) sampling ELISA tests used. This decreasing trend did however not [6, 7, 19] have been introduced in combination with PCR persist in the fourth sampling showing again a gradual in- detection of M. hyopneumoniae to reliably detect the crease in M. hyopneumoniae-titers. The number of posi- pathogen of infected animals. The objective of the current tive samples obtained with both ELISA tests on each case report is to show that TBS sampling is a suitable sampling date are given in Table 1. method to confirm a suspect M. hyopneumoniae-seroposi- tive situation. Monitoring of clinical signs and lung lesions Moreover, throughout the entire monitoring period, no Case presentation coughing, lung lesions at necropsy or at slaughter could Case description be detected. A high health breeding farm in Central Eastern Europe (220-sow herd) had been negative for M. hyopneumoniae Epidemiological information: M. hyopneumoniae in for more than 20 years using a standard serological neighboring farms and wind direction monitoring schedule (3×/year; 25 samples per time Several other swine farms, some belonging to the point) with a commercially available M. hyopneumoniae same production group, are located within a range of ELISA test (Idexx HerdChek Mhyo ELISA, indirect 2–3kmfromthe describedSPF farm.FarmVS, afat- ELISA; Idexx Laboratories). Besides freedom for M. tening unit directly related to the SPF source farm, is Vangroenweghe et al. Porcine Health Management (2018) 4:12 Page 3 of 6 Table 1 Mycoplasma hyopneumoniae standard ELISA (Idexx HerdChek Mhyo ELISA, indirect ELISA; Idexx Laboratories; S/P-ratio, sample to positive ratio) monitoring results from March 2017 onwards. Results of the second ELISA test (IDEIA™ Mycoplasma hyopneumoniae; Oxoid – Thermo Scientific; PI, percentage of inhibition) are also given. Table demonstrates total number of samples and number of samples with negative (Idexx, S/P < 0.30; IDEIA™,PI ≥ 65%), suspect (Idexx, 0.30 ≤ S/P ≤ 0.40; IDEIA™,50% ≥ PI > 65%) or positive (Idexx, S/P > 0.40; IDEIA™,PI < 50%) M. hyopneumoniae ELISA results. Mean titers (± SEM) for Idexx ELISA (expressed as S/P-ratio) and IDEIA™ ELISA (expressed as PI) are given per sampling timepoint Date Analytical Analytical result Results (mean ± SEM) Total test sample Negative Suspect Positive number 20.3.2017 Idexx 0 1 9 0.86 ± 0.15 10 IDEIA™ 2 7 1 59.7 ± 7.7% 10 21.4.2017 Idexx 11 7 7 0.43 ± 0.08 25 IDEIA™ 17 4 4 65.4 ± 14.3% 25 25.4.2017 Idexx 18 0 4 0.17 ± 0.04 22 IDEIA™ 4 0 0 90.3 ± 15.0% 4 9.8.2017 Idexx 11 1 8 0.36 ± 0.06 20 IDEIA™ 2 0 2 58.0 ± 11.8% 4 located at 1.35 km air distance in western direction and based on magnetic bead technology. One millilitre of TBS Farm VH, an unrelated 35-sow herd, is located at 2.25 km was centrifuged for 5 min at 16,000 g, the pellet suspended air distance in south-south-eastern direction. Epidemio- in 400 μL lysis buffer, and 400 μL of the suspension was logical information on disease state, including M. hyop- used as the sample. If no pellet was observable, 300 μLof neumoniae, is actively exchanged among these different the TBS was used as the sample. Bead mix and lysis/bind- farms. Under the local conditions, wind direction is most ing solution were added and the mix transferred onto a often from a western direction with high wind speed 96-well plate in the processor. Nucleic acid isolation was (source: http://oze.tzb-info.cz/vetrna-energie/9800-vetrne- performed according to the manufacturer’s instructions. podminky-v-ceske-republice-ve-vysce-10-m-nad- The PCR results were reported as negative (Ct ≥ 37) or povrchem-ii). During the last quarter of 2017, both farms positive (Ct < 37) for the presence of M. hyopneumoniae (VS, fattening unit and VH, sow unit) were detected M. based on a Ct-threshold value. The detection limit range hyopneumoniae-positive on serological monitoring using for M. hyopneumoniae reported  was from 10 ng/μL the conventional ELISA test (Idexx HerdChek Mhyo to 2.5 fg/μL. The detection limit for the PCR was validated ELISA, indirect ELISA; Idexx Laboratories). for TBS spiked with dilutions of M. hyopneumoniae strain J (ATCC 25934) of at least 5 fg/μL. Diagnostic approach with TBS Tracheo-bronchial sampling was performed as previ- Sampling for determination of freedom of disease ously described [6, 7]. Briefly, TBS samples were ob- The minimal number of samples needed to show ‘free- tained following restraint of the piglets with a nose dom of disease’ in the farm was calculated (http://epi- snare, and subsequent use of a mouth opener. The aspir- tools.ausvet.com.au), using FreeCalc – sample size ation tube used (CH12 × 50 cm; Medinorm) was inserted calculation for freedom testing with imperfect tests through the mouth and glottis down to the trachea- using the modified hypergeometric distribution for exact bronchial bifurcation where mucus was collected hypothesis testing, with the assumption that maximum 2. through gentle swab movement. The tip of the swab was 5% of the animals (n = 6000) present on the farm were collected in a sterile 10 mL polystyrene tube (MLS), positive for M. hyopneumoniae, assuming test sensitivity mixed with 1 mL sterile saline and kept at 3–5 °C until and specificity of the TBS/qPCR combination of 75 and analysis within 48 h of sampling. 100%, respectively [19, 20]. Based on this calculation, a total of 162 samples were collected at different ages (n =18 Analysis of tracheo-bronchial swabs per age category): piglets at 3–6–9-12 and 15 weeks of age The material collected by TBS was processed in a M. and rearing gilts at 18–21-24 and 27 weeks of age. hyopneumoniae p183 real-time-PCR . Nucleic acid was extracted from TBS using an RNA/DNA isolation Statistical analysis for TBS positivity and Ct value kit (MagMAX Pathogen RNA/DNA Kit; Life Technolo- For the assessment of the overall effect of sampling date gies) and an automated nucleic acid isolation processor on the probability of a positive qPCR result, a Pearson (MagMAX Express 96 processor; Life Technologies) chi-squared test was used. Pairwise comparisons Vangroenweghe et al. Porcine Health Management (2018) 4:12 Page 4 of 6 between sampling dates was also performed with subsequently increased up to 30 and 33 in animals of 24 Pearson chi-squared tests, with p-values adjusted with and 27 weeks of age, respectively (Fig. 1). Ct values were the Holm procedure. All per-comparison p-values were significantly different (P < 0.05) from 37, the cut-off computed by referring to the permutation null distribu- value for PCR test positivity. tion of the test statistics. The latter were approximated based on 2000 random permutations. The overall effect Discussion of age (n = 9 age groups) on the average Ct values was The current case report demonstrated that diagnosis of assessed by means of an F-test in a one-way ANOVA. an M. hyopneumoniae infection can be difficult using Post-hoc multiple comparisons of means were per- the standard monitoring tools, such as clinical observa- formed with Tukey’s method. The overall effect of age tion, serology  and slaughterhouse checks for typical (n = 9 age groups) on the probability of a positive qPCR lung lesions . It was also shown that PCR testing on result was assessed by means of the Pearson chi-squared TBS samples confirmed the M. hyopneumoniae health test. Multiple pairwise comparisons were also done with status of a breeding herd. This allowed the farmer to Pearson chi-squared tests, with p-values adjusted with make decisions concerning health management within Holm’s procedure. the breeding company. All overall tests were performed at the 5% level of sig- Interestingly, the collected TBS samples only showed nificance. Multiple comparison tests were performed M. hyopneumoniae-positive results from 18 weeks simultaneously at the 5% familywise error rate (FWER) onwards, which is the age category that enters the gilt level. All data analyses were performed with the R statis- rearing barn. Until now, it is unclear how the M. hyop- tical software version 3.4.3. . neumoniae infection has entered the farm. Generally, the major source of M. hyopneumoniae introduction into Results a farm is considered newly arrived subclinically M. hyop- M. hyopneumoniae detection using TBS and subsequent neumoniae-infected gilts, especially since M. hyopneu- PCR moniae-infected animals can excrete the pathogen for During lactation and nursery phase (from 3 until up to 254 days following initial infection . However, 15 weeks of age), no M. hyopneumoniae could be de- this nucleus herd has not received any live breeding ani- tected using PCR testing. However, once the animals en- mals for many years. tered the rearing barn located at the same site, M. A second hypothesis is that the M. hyopneumoniae en- hyopneumoniae was clearly present. Among the gilts tered the farm by airborne transmission from infected from 18 until 27 weeks of age, the percentage of positive neighboring farms. Under favorable climatic conditions, animals significantly increased (P < 0.05) to 87–100% in aerogenous spread of M. hyopneumoniae between neigh- the oldest age group (Fig. 1). PCR Ct values initially de- boring herds over varying distances has been demon- creased with age, starting at 29 in animals of 18 weeks strated [22–24]. Indeed, the farm in the reported case of age, to 25 in animals of 21 weeks of age; and had neighboring swine herds within a 1.5 to 3-km range. Fig. 1 Tracheo-bronchial swab (TBS) sampling results of different age categories (n = 18 animals per age category) at the Central-European SPF herd. Piglets of 3–6–9-12 and 15 weeks and rearing gilt of 18–21-24 and 27 weeks of age were sampled. The qPCR M. hyopneumoniae-positive animals per age category were expressed as percentage (red line). The Ct values of those qPCR M. hyopneumoniae-positive TBS samples (Ct < 37) were expressed as means (± SEM; blue bars) Vangroenweghe et al. Porcine Health Management (2018) 4:12 Page 5 of 6 Moreover, epidemiological information shared among monitoring, a specified eradication plan will be designed these neighboring farms clearly indicated that a break in to result in a renewed M. hyopneumoniae-negative gilt M. hyopneumoniae health status had also happened dur- outflow to the end customers. ing this year based on the results of their regular sero- logical monitoring. Unfortunately, we do not have Conclusions details on which of these swine farms first broke with its The present case report showed that for several months, M. hyopneumoniae health status, although based on pre- M. hyopneumoniae infection in an SPF-herd may occur vailing wind direction, farm VS which is located west without clinical symptoms and typical lung lesions. In from the SPF nucleus herd could be a good candidate. addition, serological testing may be difficult to interpret, Another source of potential introduction might be and PCR testing on TBS may be needed to establish a through an M. hyopneumoniae-infected transport ve- conclusive diagnosis. hicle. Especially under winter conditions, cleaning and Abbreviations disinfection of transport vehicles is not always easy to AI/AO: All-in/all-out; ANOVA: Analysis of variance; Ct value: threshold value; perform. Recently, M. hyopneumoniae survival on stain- DNT: Dermo-necrotic toxin; ELISA: Enzyme-linked immunosorbent assay; less steel at 4 °C was demonstrated for at least 2 days M.hyopneumoniae: Mycoplasma hyopneumoniae; PCR: Polymerase chain reaction; PRRSV: Porcine Reproductive and Respiratory Syndrome Virus; qPCR: quantitative (max. 8 days for some strains) . This would imply PCR; SPF: Specific pathogen free; TBS: Tracheo-bronchial swab that the regular procedure for SPF pig-free downtime of 48 h for transport vehicles cannot always guarantee a Acknowledgements 100% M. hyopneumoniae-free vehicle. Analysis of the M. The authors greatly acknowledges all the swine herd personnel for assistance during sampling. The authors acknowledge the background hyopneumoniae infection kinetics on the farm revealed support by dr. Katerina Nechvatalova in arranging the farm visit and that the M. hyopneumoniae infection was first detected providing the details on weather data. within the gilt rearing barn. This is also the location from which gilts are selected and loaded for external Funding The study was funded by Elanco Animal Health, which facilitated the transport. Therefore, the probability of a biosecurity sampling and analysis of TBS samples. breach at the gilt loading point seems possible, since ex- ternal biosecurity at all other levels has always been at Availability of data and materials the very high level. Moreover, once the M. hyopneumo- The datasets analysed during the current study are available from the corresponding author on reasonable request. niae infection entered this section of the farm, M. hyop- neumoniae could easily further spread within the age Authors’ contributions groups from 18 until 27 weeks, which is not managed FV coordinated the entire study from study design to data collection and writing of the manuscript. EV and JM provided the herd for this case report under strict AI/AO conditions. The reason why no and JM supported the sampling of the 162 animals at the herd. OT coughing nor typical lung lesions were present is not performed data analysis and DM advised in study design and supported the clear. It may be due to the rather good housing condi- manuscript writing. All authors read and approved the final manuscript. tions, e.g. air quality and level of dust [26, 27] and/or the Authors’ information fact that a low-virulent strain was circulating . FV is currently a Sr. Technical Consultant Swine for Benelux within Elanco Although a break in biosecurity seems unlikely, it Animal Health. He holds a DVM and a PhD in Veterinary Sciences, has a should be considered as another option for M. hyopneu- specific specialization in TBS sampling in pigs from 2 weeks of age until slaughter, including replacement gilts and productive sows. moniae introduction into the farm. A 4-year research demonstrated that a 1-night downtime period is suffi- Ethics approval and consent to participate cient to prevent mechanical spread of both PRRSV and Not applicable M. hyopneumoniae by personnel and fomites . More- over, that study concluded that basic sanitation proce- Competing interests The authors declare that they have no competing interests. dures, such as hand hygiene and the use of both boots and coveralls should be enough to prevent mechanical spread and therefore, the implementation of a shower Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in protocol is even not necessary . In the present breed- published maps and institutional affiliations. ing farm, besides regular personnel, showering in on every entry, no visitors were admitted, except for the Author details Elanco Animal Health Benelux, Benelux, Plantijn en Moretuslei 1 – 3rd floor, TBS sampling needed to confirm the M. hyopneumoniae 2018 Antwerpen, Belgium. Topigs-Norsvin International, Vught, The infection status. 3 4 Netherlands. Topigs Norsvin CZ, Brno, Czech Republic. 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