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Molecular epidemiology of Mycoplasma hyopneumoniae from outbreaks of enzootic pneumonia in domestic pig and the role of wild boar
Veterinary Microbiology 174 (2014) 261–266
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Molecular epidemiology of Mycoplasma hyopneumoniae from outbreaks of enzootic pneumonia in domestic pig and the role of wild boar Peter Kuhnert *, Gudrun Overesch Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 May 2014 Received in revised form 15 August 2014 Accepted 22 August 2014
Mycoplasma hyopneumoniae is the major cause of enzootic pneumonia (EP) in domestic pigs, a disease with low mortality but high morbidity, having a great economic impact for producers. In Switzerland EP has been successfully eradicated, however, sporadic outbreaks are observed with no obvious source. Besides the possibility of recurrent outbreaks due to persisting M. hyopneumoniae strains within the pig population, there is suspicion that wild boars might introduce M. hyopneumoniae into swine herds. To elucidate possible links between domestic pig and wild boar, epidemiological investigations of recent EP outbreaks were initiated and lung samples of pig and wild boar were tested for the presence of specific genotypes by multilocus sequence typing (MLST). Despite generally different genotypes in wild boar, outbreak strains could be found in geographically linked wild boar lungs after, but so far not before the outbreak. Recurrent outbreaks in a farm were due to the same strain, indicating unsuccessful sanitation rather than reintroduction by wild boar. In another case outbreaks in six different farms were caused by the same strain never found in wild boar, confirming spread between farms due to hypothesized animal transport. Results indicate the presence of identical lineages of wild boar and domestic pig strains, and possible transmission of M. hyopneumoniae between wild boar and pig. However, the role of wild boar might be rather one as a recipient than a transmitter. More important than contact to wild boar for sporadic outbreaks in Switzerland is apparently persistence of M. hyopneumoniae within a farm as well as transmission between farms. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Swine Disease MLST Epidemiology Transmission Genotyping
1. Introduction Enzootic pneumonia (EP) of swine, also called mycoplasmal pneumonia, is caused by Mycoplasma hyopneumoniae and is one of the most important causes of diseaseassociated losses in swine production (Sibila et al., 2009;
* Corresponding author at: Institute of Veterinary Bacteriology, University of Bern, Laenggassstr. 122, 3001 Bern, Switzerland. Tel.: +41 31 6312485; fax: +41 31 6312634. E-mail address: [email protected] (P. Kuhnert). http://dx.doi.org/10.1016/j.vetmic.2014.08.022 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
Simionatto et al., 2013). Infection with M. hyopneumoniae is characterized by a sporadic, dry and non-productive cough, retarded growth rate and inefficient utilization of feed. The disease has almost no mortality but a high morbidity. Infection occurs generally through direct contact with respiratory secretions from carrier animals. However, an airborne transmission over several kilometers is also reported (Stark et al., 1998; Otake et al., 2010). Despite the success in combating the disease in Switzerland, sporadic outbreaks are observed with no obvious source of infection (Stark et al., 2007). Recurrent outbreaks could be due to insufficient sanitation since
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M. hyopneumoniae can persist in the organs of pigs even after antibiotic treatment (Le Carrou et al., 2006). Besides, there is an increasing suspicion that wild boar transmit M. hyopneumoniae to the swine population, especially since outdoor pig production has become more popular, allowing contacts with wild animals. Nevertheless, the role of wild boar as a reservoir for M. hyopneumoniae has been poorly investigated so far and mainly focused on serological analyses (Vengust et al., 2006; Sibila et al., 2010; Baker et al., 2011). Thereby seroprevalences of 21% in Slovenia and Spain as well as 32% in parts of the US were reported. Genotyping as a molecular tool for epidemiological investigations has proved useful for EP outbreak investigations in pig and can be directly applied on clinical material (Mayor et al., 2007, 2008). For Switzerland a high variability of M. hyopneumoniae genotypes from various outbreaks was shown. However, a single strain was found responsible for an individual outbreak on a farm and farms in close geographical or organizational contacts had identical strains. The genotyping approach was also shown applicable to wild boar with some limitations (Kuhnert et al., 2011). Even though prevalence is rather high (>40%), wild boar harbor fewer amounts of M. hyopneumoniae than domestic pig, reducing the number of typeable wild boar samples. Moreover, the lungs show less macroscopic lesions and therefore deciding on the precise sampling spot is difficult. Finally, in contrast to pig more than one strain might be present in wild boar. In order to assess the potential role of wild boar as a reservoir and a transmitter of M. hyopneumoniae, genotypes derived from lung swab samples from EP outbreak cases and wild boar were investigated. Resulting genotypes were compared including pre- and post-outbreak samples from wild boar in the vicinity. Identical genotypes were found in pig and wild boar, indicating no difference in lineages and therefore possible transmission of M. hyopneumoniae between these two hosts. 2. Materials and methods 2.1. Samples and sample preparation Lungs from three pigs of a slaughter batch were received from the abattoirs for routine diagnosis in case of typical EP lesions. They resulted from 10 farm outbreaks during 2010–2013 (farms A–J). In two farms (farms A and C) recurrent outbreaks were observed after sanitation. Wild boar lungs were organized in coordination with the cantonal veterinarians and received from game wards as soon as possible after shooting of the animals. A few lungs were obtained in the framework of a preliminary study on the prevalence of M. hyopneumoniae in wild boar. A total of 47 individual wild boar lung samples were included in the genotyping study. All samples are indicated in Fig. 1 and their geographic distribution is shown in Fig. 2. Lysates were prepared from bronchial swabs from pig and wild boar lungs as previously described, tested by realtime PCR for presence of M. hyopneumoniae and positive samples were stored at 20 8C until further analysis by genotyping (Dubosson et al., 2004; Kuhnert et al., 2011).
2.2. Genotyping Multilocus sequence typing (MLST) was done according to Mayor et al. (2008). For this purpose the three housekeeping genes adk, rpoB and tpiA were amplified by PCR and subsequently sequenced. Sequences were edited using Sequencher (GeneCodes, Ann Arbor, MI, USA) and proofread sequences were entered into Bionumerics v7.1 (Applied Maths, Sint-Martens-Latem, Belgium). Cluster analyses were performed in Bionumerics by UPGMA using individual similarity matrices from the three genes. 3. Results 3.1. Genotypes Successful genotyping was generally achieved with all three individual animal lung samples from real-time PCR positive pig farms. Genotypes from all three animals from a single farm were always identical. With wild boar samples the rate of successful genotyping was much lower and the Ct-values from the real-time PCR were much higher than with positive pig samples (data not shown). About half of the PCR positive wild boar samples could be effectively genotyped. Given an observed prevalence in our sample set of about 50% in wild boar based on testing bronchial swabs, roughly one-quarter of all investigated wild boar lungs resulted in a genotype. A cluster analysis of EP outbreak genotypes and all observed wild boar genotypes generated in the study is given in Fig. 1. In total 18 clusters were observed, five of them containing pig and 16 containing wild boar samples. Two clusters were only formed by pig genotypes whereas 13 only by wild boar genotypes. The wild boar clusters formed by multiple entries were generally formed by samples that were also geographically clustered (Fig. 2). 3.2. Outbreak analysis The geographic location of outbreak and wild boar samples analyzed are shown in Fig. 2. From all outbreak scenarios wild boar samples were available. In the Geneva region (GE) no outbreak occurred during the analyzed time period but wild boar samples were available from this part. Looking in more detail at the EP outbreaks there was one segregated case (farm B). This was a sentinel farm in the Jura right at the border to France and the outbreak did not show any connection with other cases nor with any wild boar samples. Furthermore, the genotype retrieved in farm B was not previously observed in Switzerland (Mayor et al., 2008). It therefore remained a completely independent outbreak in contrast to the others. In another outbreak scenario in the Eastern part the identical genotype was found in six different farms (farms D–I). Three of these farms were in close proximity (D, F, and G), two of them actually neighbors. The others were in some distance southwards from these, with farm E 20 km and farms H and I more than 40 km away. A similar but still divergent genotype to this cluster (no. 48) was found in a wild boar from the region of the probable start of the outbreak. Another farm 40 km northwards (farm J) showed a clearly different genotype not related to the one found in the previous six farms. This outbreak continued for at least
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Fig. 1. Samples analyzed and genotypes determined in the study. The sample number, date of sampling, origin, ZIP code and the state are indicated. Samples from pig are indicated by farm the ones from wild boar by WB. Cluster analysis was done in Bionumerics v7.1 based on partial sequences of adk, rpoB and tpiA using UPGMA.
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Fig. 2. Geographic map of outbreak farms and wild boar samples analyzed in the study. Numbered white circles represent wild boar samples, black dots indicate domestic pig outbreak farms. Panel A: overview of localization of samples including the most Western Geneva region (GE). Panel B: samples from the Bern (BE) and Jura (JU/BL) region. Panel C: samples from Eastern Switzerland (SG/TG/SH).
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two months and the same genotype was later found in the wild boar population in the same region (SH), including all seven wild boar samples that were typed from this region (nos. 51–57). Finally, in two of the outbreaks recurrent infection was observed (farms A and C). In the case of farm A (BE), three wild boar samples taken after the first outbreak showed the identical genotype as the outbreak strain (nos. 2, 5 and 6). The most abundant dataset was obtained in the context of the outbreak in farm C (JU). In this scenario wild boar samples from the region were available before and after the outbreak. Whereas the outbreak genotype was not observed in any wild boar sample before, it was frequently present in the wild boar population in this region after the outbreak (nos. 36–39). 4. Discussion This is the first epidemiological study comparing M. hyopneumoniae genotypes from EP outbreaks with genotypes found in temporally and geographically related wild boar samples. About half of the investigated wild boar lungs were positive in the M. hyopneumoniae specific realtime PCR, indicating a relatively high prevalence around 50% in the investigated set of wild boar samples. In some cases not only indirect detection, but also actually isolation of M. hyopneumoniae from wild boar lungs was possible (e.g. no. 14), providing final proof that this organism is in fact present and can colonize wild animals. Moreover, the genotype of the isolate corresponded to the one obtained by direct analysis of clinical material, what confirms the validity of the direct genotyping approach also for wild boar lung samples. These isolates available will allow for more detailed analyses including whole genome sequencing and comparative genomics with pig strains. Genotyping directly from wild boar lung swabs was less successful than from pig, as has been previously observed (Kuhnert et al., 2011). The relative amount of M. hyopneumoniae DNA in wild boar was lower than in affected pig and Ct-values below 30 in the REP assays were necessary to provide enough genetic material for successful genotyping. Sibila et al. (2010) showed, that wild boar harboring M. hyopneumoniae did not show macroscopic lesions and supposed, that wild boars are only subclinically infected by this swine pathogen. This is in agreement with our observation that wild boars are affected by fewer amounts of M. hyopneumoniae than diseased pig and could therefore be just carriers of the organism. The fact that less mycoplasmas are detected in wild boar than in diseased swine also means that there is less discharge of organisms given, compared to affected pigs. The environmental contamination by M. hyopneumoniae is therefore much higher in the surrounding of an outbreak farm than it is around an infected individual wild animal or even a positive wild boar horde. Whereas similar genotypes as in pigs were observed previously in wild boar (Kuhnert et al., 2011), this is the first time that identical genotypes in pig and wild boar could be repeatedly identified. This allowed for more detailed outbreak investigations. A total of 10 EP outbreaks in individual farms were investigated, with two farms having recurrent outbreaks after 8 (farm A) and 14 months (farm C). In both these cases
265
as well as in the outbreak in farm J, identical pig and wild boar genotypes were found as shown by cluster analysis (Fig. 1). Whereas for farms A and J no wild boar samples were available from the region before the outbreak, several such samples were obtained from the same region (perimeter of <20 km) before the outbreak in farm C. In this case, all wild boar samples taken before showed a clearly different genotype to the outbreak strain of farm C. The observations in these three farms indicate that identical genotypes of M. hyopneumoniae can be found in pig and wild boar and transmission between the two certainly occurs. Data for farm C, showing that the outbreak genotype is only observed in wild boar after the outbreak but never before is an indication that M. hyopneumoniae is transmitted from an affected pig farm to the environment. It remains an open question if a transmission from wild boar to pig is equally possible. Free ranging or at least outdoor access in pig production became very popular if not legally demanded. Under these circumstances, interactions of pigs with wild animals and especially boar are observed and a transmission of the pathogen cannot be excluded. However, in our experience the mycoplasmal load in wild boar is much less than the one in affected pigs. Moreover, an affected and coughing pig herd sheds much more of the pathogen than an individual wild boar in the environment. Therefore, while for a transmission from wild boar to pig a direct contact might be necessary, the pathogen can easily be transmitted at high loads from an affected pig farm to the environment even in a closed building by mechanical ventilation. There was a clear geographic clustering of wild boar genotypes identical to outbreak genotypes around the corresponding pig farms. Moreover, the outbreak genotype was frequently found in wild boar and in the case of farm C never before in the numerous samples available from the region. This is another hint, that M. hyopneumoniae is probably released in high amounts from affected pig farms and transmitted to several wild boar animals in the vicinity. Farm B served as a sentinel farm located at the very border to France only 20 km East to farm C. However, the genotype responsible for this outbreak was clearly different from the one in farm C as well as from any wild boar genotype and this outbreak remained a single observation. Unfortunately, no wild boar or any pig samples from the French part of the Jura were available to check for this specific genotype. There were six farms from two different cantons affected by EP in the Eastern part of Switzerland. Genotyping revealed a single type being responsible for all six farm outbreaks. There was a geographic clustering of these farms, however, with 50 km between the most distant farms. Inquiries at the Cantonal Veterinary Service revealed that these outbreaks might have been related to animal transport what was supported by genotyping results and all six farms must be regarded as a single outbreak. This case illustrates the potential of genotyping to elucidate such epidemiological connections. Finally, the study confirmed previous findings, with geographic clustering of genotypes from wild boar indicating that a certain genotype circulates within a wild boar horde (Kuhnert et al., 2011).
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In conclusion the study showed that identical genotypes of M. hyopneumoniae can be found in wild boar and pig, indicating that there are no different lineages between the two. Furthermore, it shows that transmission from one to the other is possible with transmission from affected pig farms to wild boar being more probable than the other way. Transmission from farm to farm by (illegal) animal transport as well as incomplete sanitation has still to be considered as the major source and main reason for EP outbreaks observed in Switzerland. Genotyping allows tracing of such inter-farm outbreaks. In addition it would be the appropriate tool to more precisely elucidate the role of wild boar in sporadic cases by consequent monitoring of M. hyopneumoniae genotypes in the wild boar population before EP outbreaks occur. Acknowledgements We are grateful for the technical help of Amandine Ruffieux and Romie Jonas. We thank the cantonal veterinarians Dr. Urs-Peter Brunner and Peter Uehlinger (Canton of Schaffhausen), Dr. Anne Ceppi (Canton of Jura) and Dr. Paul Witzig (Canton of Thurgau) for organizing wild boar samples and/or providing information on EP outbreaks. This work was funded by the Federal Veterinary Office (grant 1.11.16). References Baker, S.R., O’Neil, K.M., Gramer, M.R., Dee, S.A., 2011. Estimates of the seroprevalence of production-limiting diseases in wild pigs. Vet. Rec. 168, 564.
Dubosson, C.R., Conzelmann, C., Miserez, R., Boerlin, P., Frey, J., Zimmermann, W., Ha¨ni, H., Kuhnert, P., 2004. Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples. Vet. Microbiol. 102, 55–65. Kuhnert, P., Overesch, G., Belloy, L., 2011. Genotyping of Mycoplasma hyopneumoniae in wild boar lung samples. Vet. Microbiol. 152, 191–195. Le Carrou, J., Laurentie, M., Kobisch, M., Gautier-Bouchardon, A.V., 2006. Persistence of Mycoplasma hyopneumoniae in experimentally infected pigs after marbofloxacin treatment and detection of mutations in the parC gene. Antimicrob. Agents Chemother. 50, 1959–1966. Mayor, D., Zeeh, F., Frey, J., Kuhnert, P., 2007. Diversity of Mycoplasma hyopneumoniae in pig farms revealed by direct molecular typing of clinical material. Vet. Res. 38, 391–398. Mayor, D., Jores, J., Korczak, B.M., Kuhnert, P., 2008. Multilocus sequence typing (MLST) of Mycoplasma hyopneumoniae: a diverse pathogen with limited clonality. Vet. Microbiol. 127, 63–72. Otake, S., Dee, S., Corzo, C., Oliveira, S., Deen, J., 2010. Long-distance airborne transport of infectious PRRSV and Mycoplasma hyopneumoniae from a swine population infected with multiple viral variants. Vet. Microbiol.. Sibila, M., Pieters, M., Molitor, T., Maes, D., Haesebrouck, F., Segales, J., 2009. Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection. Vet. J. 181, 221–231. Sibila, M., Mentaberre, G., Boadella, M., Huerta, E., Casas-Diaz, E., Vicente, J., Gortazar, C., Marco, I., Lavin, S., Segales, J., 2010. Serological, pathological and polymerase chain reaction studies on Mycoplasma hyopneumoniae infection in the wild boar. Vet. Microbiol. 144, 214–218. Simionatto, S., Marchioro, S.B., Maes, D., Dellagostin, O.A., 2013. Mycoplasma hyopneumoniae: from disease to vaccine development. Vet. Microbiol. 165, 234–242. Stark, K.D.C., Nicolet, J., Frey, J., 1998. Detection of Mycoplasma hyopneumoniae by air sampling with a nested PCR assay. Appl. Environ. Microbiol. 64, 543–548. Stark, K.D., Miserez, R., Siegmann, S., Ochs, H., Infanger, P., Schmidt, J., 2007. A successful national control programme for enzootic respiratory diseases in pigs in Switzerland. Rev. Sci. Technol. 26, 595–606. Vengust, G., Valencak, Z., Bidovec, A., 2006. A serological survey of selected pathogens in wild boar in Slovenia. J. Vet. Med. B 53, 24–27.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Molecular epidemiology of Mycoplasma hyopneumoniae from outbreaks of enzootic pneumonia in domestic pig and the role of wild boar Peter Kuhnert *, Gudrun Overesch Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 May 2014 Received in revised form 15 August 2014 Accepted 22 August 2014
Mycoplasma hyopneumoniae is the major cause of enzootic pneumonia (EP) in domestic pigs, a disease with low mortality but high morbidity, having a great economic impact for producers. In Switzerland EP has been successfully eradicated, however, sporadic outbreaks are observed with no obvious source. Besides the possibility of recurrent outbreaks due to persisting M. hyopneumoniae strains within the pig population, there is suspicion that wild boars might introduce M. hyopneumoniae into swine herds. To elucidate possible links between domestic pig and wild boar, epidemiological investigations of recent EP outbreaks were initiated and lung samples of pig and wild boar were tested for the presence of specific genotypes by multilocus sequence typing (MLST). Despite generally different genotypes in wild boar, outbreak strains could be found in geographically linked wild boar lungs after, but so far not before the outbreak. Recurrent outbreaks in a farm were due to the same strain, indicating unsuccessful sanitation rather than reintroduction by wild boar. In another case outbreaks in six different farms were caused by the same strain never found in wild boar, confirming spread between farms due to hypothesized animal transport. Results indicate the presence of identical lineages of wild boar and domestic pig strains, and possible transmission of M. hyopneumoniae between wild boar and pig. However, the role of wild boar might be rather one as a recipient than a transmitter. More important than contact to wild boar for sporadic outbreaks in Switzerland is apparently persistence of M. hyopneumoniae within a farm as well as transmission between farms. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Swine Disease MLST Epidemiology Transmission Genotyping
1. Introduction Enzootic pneumonia (EP) of swine, also called mycoplasmal pneumonia, is caused by Mycoplasma hyopneumoniae and is one of the most important causes of diseaseassociated losses in swine production (Sibila et al., 2009;
* Corresponding author at: Institute of Veterinary Bacteriology, University of Bern, Laenggassstr. 122, 3001 Bern, Switzerland. Tel.: +41 31 6312485; fax: +41 31 6312634. E-mail address: [email protected] (P. Kuhnert). http://dx.doi.org/10.1016/j.vetmic.2014.08.022 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
Simionatto et al., 2013). Infection with M. hyopneumoniae is characterized by a sporadic, dry and non-productive cough, retarded growth rate and inefficient utilization of feed. The disease has almost no mortality but a high morbidity. Infection occurs generally through direct contact with respiratory secretions from carrier animals. However, an airborne transmission over several kilometers is also reported (Stark et al., 1998; Otake et al., 2010). Despite the success in combating the disease in Switzerland, sporadic outbreaks are observed with no obvious source of infection (Stark et al., 2007). Recurrent outbreaks could be due to insufficient sanitation since
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M. hyopneumoniae can persist in the organs of pigs even after antibiotic treatment (Le Carrou et al., 2006). Besides, there is an increasing suspicion that wild boar transmit M. hyopneumoniae to the swine population, especially since outdoor pig production has become more popular, allowing contacts with wild animals. Nevertheless, the role of wild boar as a reservoir for M. hyopneumoniae has been poorly investigated so far and mainly focused on serological analyses (Vengust et al., 2006; Sibila et al., 2010; Baker et al., 2011). Thereby seroprevalences of 21% in Slovenia and Spain as well as 32% in parts of the US were reported. Genotyping as a molecular tool for epidemiological investigations has proved useful for EP outbreak investigations in pig and can be directly applied on clinical material (Mayor et al., 2007, 2008). For Switzerland a high variability of M. hyopneumoniae genotypes from various outbreaks was shown. However, a single strain was found responsible for an individual outbreak on a farm and farms in close geographical or organizational contacts had identical strains. The genotyping approach was also shown applicable to wild boar with some limitations (Kuhnert et al., 2011). Even though prevalence is rather high (>40%), wild boar harbor fewer amounts of M. hyopneumoniae than domestic pig, reducing the number of typeable wild boar samples. Moreover, the lungs show less macroscopic lesions and therefore deciding on the precise sampling spot is difficult. Finally, in contrast to pig more than one strain might be present in wild boar. In order to assess the potential role of wild boar as a reservoir and a transmitter of M. hyopneumoniae, genotypes derived from lung swab samples from EP outbreak cases and wild boar were investigated. Resulting genotypes were compared including pre- and post-outbreak samples from wild boar in the vicinity. Identical genotypes were found in pig and wild boar, indicating no difference in lineages and therefore possible transmission of M. hyopneumoniae between these two hosts. 2. Materials and methods 2.1. Samples and sample preparation Lungs from three pigs of a slaughter batch were received from the abattoirs for routine diagnosis in case of typical EP lesions. They resulted from 10 farm outbreaks during 2010–2013 (farms A–J). In two farms (farms A and C) recurrent outbreaks were observed after sanitation. Wild boar lungs were organized in coordination with the cantonal veterinarians and received from game wards as soon as possible after shooting of the animals. A few lungs were obtained in the framework of a preliminary study on the prevalence of M. hyopneumoniae in wild boar. A total of 47 individual wild boar lung samples were included in the genotyping study. All samples are indicated in Fig. 1 and their geographic distribution is shown in Fig. 2. Lysates were prepared from bronchial swabs from pig and wild boar lungs as previously described, tested by realtime PCR for presence of M. hyopneumoniae and positive samples were stored at 20 8C until further analysis by genotyping (Dubosson et al., 2004; Kuhnert et al., 2011).
2.2. Genotyping Multilocus sequence typing (MLST) was done according to Mayor et al. (2008). For this purpose the three housekeeping genes adk, rpoB and tpiA were amplified by PCR and subsequently sequenced. Sequences were edited using Sequencher (GeneCodes, Ann Arbor, MI, USA) and proofread sequences were entered into Bionumerics v7.1 (Applied Maths, Sint-Martens-Latem, Belgium). Cluster analyses were performed in Bionumerics by UPGMA using individual similarity matrices from the three genes. 3. Results 3.1. Genotypes Successful genotyping was generally achieved with all three individual animal lung samples from real-time PCR positive pig farms. Genotypes from all three animals from a single farm were always identical. With wild boar samples the rate of successful genotyping was much lower and the Ct-values from the real-time PCR were much higher than with positive pig samples (data not shown). About half of the PCR positive wild boar samples could be effectively genotyped. Given an observed prevalence in our sample set of about 50% in wild boar based on testing bronchial swabs, roughly one-quarter of all investigated wild boar lungs resulted in a genotype. A cluster analysis of EP outbreak genotypes and all observed wild boar genotypes generated in the study is given in Fig. 1. In total 18 clusters were observed, five of them containing pig and 16 containing wild boar samples. Two clusters were only formed by pig genotypes whereas 13 only by wild boar genotypes. The wild boar clusters formed by multiple entries were generally formed by samples that were also geographically clustered (Fig. 2). 3.2. Outbreak analysis The geographic location of outbreak and wild boar samples analyzed are shown in Fig. 2. From all outbreak scenarios wild boar samples were available. In the Geneva region (GE) no outbreak occurred during the analyzed time period but wild boar samples were available from this part. Looking in more detail at the EP outbreaks there was one segregated case (farm B). This was a sentinel farm in the Jura right at the border to France and the outbreak did not show any connection with other cases nor with any wild boar samples. Furthermore, the genotype retrieved in farm B was not previously observed in Switzerland (Mayor et al., 2008). It therefore remained a completely independent outbreak in contrast to the others. In another outbreak scenario in the Eastern part the identical genotype was found in six different farms (farms D–I). Three of these farms were in close proximity (D, F, and G), two of them actually neighbors. The others were in some distance southwards from these, with farm E 20 km and farms H and I more than 40 km away. A similar but still divergent genotype to this cluster (no. 48) was found in a wild boar from the region of the probable start of the outbreak. Another farm 40 km northwards (farm J) showed a clearly different genotype not related to the one found in the previous six farms. This outbreak continued for at least
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Fig. 1. Samples analyzed and genotypes determined in the study. The sample number, date of sampling, origin, ZIP code and the state are indicated. Samples from pig are indicated by farm the ones from wild boar by WB. Cluster analysis was done in Bionumerics v7.1 based on partial sequences of adk, rpoB and tpiA using UPGMA.
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Fig. 2. Geographic map of outbreak farms and wild boar samples analyzed in the study. Numbered white circles represent wild boar samples, black dots indicate domestic pig outbreak farms. Panel A: overview of localization of samples including the most Western Geneva region (GE). Panel B: samples from the Bern (BE) and Jura (JU/BL) region. Panel C: samples from Eastern Switzerland (SG/TG/SH).
P. Kuhnert, G. Overesch / Veterinary Microbiology 174 (2014) 261–266
two months and the same genotype was later found in the wild boar population in the same region (SH), including all seven wild boar samples that were typed from this region (nos. 51–57). Finally, in two of the outbreaks recurrent infection was observed (farms A and C). In the case of farm A (BE), three wild boar samples taken after the first outbreak showed the identical genotype as the outbreak strain (nos. 2, 5 and 6). The most abundant dataset was obtained in the context of the outbreak in farm C (JU). In this scenario wild boar samples from the region were available before and after the outbreak. Whereas the outbreak genotype was not observed in any wild boar sample before, it was frequently present in the wild boar population in this region after the outbreak (nos. 36–39). 4. Discussion This is the first epidemiological study comparing M. hyopneumoniae genotypes from EP outbreaks with genotypes found in temporally and geographically related wild boar samples. About half of the investigated wild boar lungs were positive in the M. hyopneumoniae specific realtime PCR, indicating a relatively high prevalence around 50% in the investigated set of wild boar samples. In some cases not only indirect detection, but also actually isolation of M. hyopneumoniae from wild boar lungs was possible (e.g. no. 14), providing final proof that this organism is in fact present and can colonize wild animals. Moreover, the genotype of the isolate corresponded to the one obtained by direct analysis of clinical material, what confirms the validity of the direct genotyping approach also for wild boar lung samples. These isolates available will allow for more detailed analyses including whole genome sequencing and comparative genomics with pig strains. Genotyping directly from wild boar lung swabs was less successful than from pig, as has been previously observed (Kuhnert et al., 2011). The relative amount of M. hyopneumoniae DNA in wild boar was lower than in affected pig and Ct-values below 30 in the REP assays were necessary to provide enough genetic material for successful genotyping. Sibila et al. (2010) showed, that wild boar harboring M. hyopneumoniae did not show macroscopic lesions and supposed, that wild boars are only subclinically infected by this swine pathogen. This is in agreement with our observation that wild boars are affected by fewer amounts of M. hyopneumoniae than diseased pig and could therefore be just carriers of the organism. The fact that less mycoplasmas are detected in wild boar than in diseased swine also means that there is less discharge of organisms given, compared to affected pigs. The environmental contamination by M. hyopneumoniae is therefore much higher in the surrounding of an outbreak farm than it is around an infected individual wild animal or even a positive wild boar horde. Whereas similar genotypes as in pigs were observed previously in wild boar (Kuhnert et al., 2011), this is the first time that identical genotypes in pig and wild boar could be repeatedly identified. This allowed for more detailed outbreak investigations. A total of 10 EP outbreaks in individual farms were investigated, with two farms having recurrent outbreaks after 8 (farm A) and 14 months (farm C). In both these cases
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as well as in the outbreak in farm J, identical pig and wild boar genotypes were found as shown by cluster analysis (Fig. 1). Whereas for farms A and J no wild boar samples were available from the region before the outbreak, several such samples were obtained from the same region (perimeter of <20 km) before the outbreak in farm C. In this case, all wild boar samples taken before showed a clearly different genotype to the outbreak strain of farm C. The observations in these three farms indicate that identical genotypes of M. hyopneumoniae can be found in pig and wild boar and transmission between the two certainly occurs. Data for farm C, showing that the outbreak genotype is only observed in wild boar after the outbreak but never before is an indication that M. hyopneumoniae is transmitted from an affected pig farm to the environment. It remains an open question if a transmission from wild boar to pig is equally possible. Free ranging or at least outdoor access in pig production became very popular if not legally demanded. Under these circumstances, interactions of pigs with wild animals and especially boar are observed and a transmission of the pathogen cannot be excluded. However, in our experience the mycoplasmal load in wild boar is much less than the one in affected pigs. Moreover, an affected and coughing pig herd sheds much more of the pathogen than an individual wild boar in the environment. Therefore, while for a transmission from wild boar to pig a direct contact might be necessary, the pathogen can easily be transmitted at high loads from an affected pig farm to the environment even in a closed building by mechanical ventilation. There was a clear geographic clustering of wild boar genotypes identical to outbreak genotypes around the corresponding pig farms. Moreover, the outbreak genotype was frequently found in wild boar and in the case of farm C never before in the numerous samples available from the region. This is another hint, that M. hyopneumoniae is probably released in high amounts from affected pig farms and transmitted to several wild boar animals in the vicinity. Farm B served as a sentinel farm located at the very border to France only 20 km East to farm C. However, the genotype responsible for this outbreak was clearly different from the one in farm C as well as from any wild boar genotype and this outbreak remained a single observation. Unfortunately, no wild boar or any pig samples from the French part of the Jura were available to check for this specific genotype. There were six farms from two different cantons affected by EP in the Eastern part of Switzerland. Genotyping revealed a single type being responsible for all six farm outbreaks. There was a geographic clustering of these farms, however, with 50 km between the most distant farms. Inquiries at the Cantonal Veterinary Service revealed that these outbreaks might have been related to animal transport what was supported by genotyping results and all six farms must be regarded as a single outbreak. This case illustrates the potential of genotyping to elucidate such epidemiological connections. Finally, the study confirmed previous findings, with geographic clustering of genotypes from wild boar indicating that a certain genotype circulates within a wild boar horde (Kuhnert et al., 2011).
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In conclusion the study showed that identical genotypes of M. hyopneumoniae can be found in wild boar and pig, indicating that there are no different lineages between the two. Furthermore, it shows that transmission from one to the other is possible with transmission from affected pig farms to wild boar being more probable than the other way. Transmission from farm to farm by (illegal) animal transport as well as incomplete sanitation has still to be considered as the major source and main reason for EP outbreaks observed in Switzerland. Genotyping allows tracing of such inter-farm outbreaks. In addition it would be the appropriate tool to more precisely elucidate the role of wild boar in sporadic cases by consequent monitoring of M. hyopneumoniae genotypes in the wild boar population before EP outbreaks occur. Acknowledgements We are grateful for the technical help of Amandine Ruffieux and Romie Jonas. We thank the cantonal veterinarians Dr. Urs-Peter Brunner and Peter Uehlinger (Canton of Schaffhausen), Dr. Anne Ceppi (Canton of Jura) and Dr. Paul Witzig (Canton of Thurgau) for organizing wild boar samples and/or providing information on EP outbreaks. This work was funded by the Federal Veterinary Office (grant 1.11.16). References Baker, S.R., O’Neil, K.M., Gramer, M.R., Dee, S.A., 2011. Estimates of the seroprevalence of production-limiting diseases in wild pigs. Vet. Rec. 168, 564.
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