Evidence of low prevalence of mycobacterial lymphadenitis in wild boars (Sus scrofa) in Poland
© The Author(s) 2017
Received: 9 June 2016
Accepted: 13 January 2017
Published: 25 January 2017
Mycobacterium spp. and Rhodococcus equi are generally regarded as the main causes of lymphadenitis in pigs and wild boars. In Poland, mycobacterial submandibular lymphadenitis was first diagnosed in a wild boar in 2012 but Mycobacterium spp. infections are also present in the Polish population of European bison (Bison bonasus). The prevalence of lymphadenitis in Polish wild boars has been found to 8.4% (95% CI 6.2–11.3%) and it has been proved that R. equi is not an important cause of purulent lesions in these animals. The current study was carried out to assess the prevalence of mycobacterial lymphadenitis in the Polish wild boar population. Submandibular lymph nodes with purulent lesions collected from 38 wild boars in 2010/2011 and negative for R. equi were included. Calculations based on the hypergeometric approximation were used to determine the probability that at least one positive individual would be detected if the infection had been present at a prevalence greater than or equal to the design prevalence. All 38 samples were negative for Mycobacterium spp. [0% (95% CI 0, 9.2%)]. Epidemiological analysis showed that the true prevalence was 95% likely to be lower than 10%. In conclusion, mycobacterial lymphadenitis seems to occur rarely in wild boars in Poland. Due to the presence of Mycobacterium spp. infections in other wildlife, the surveillance of mycobacterial infections in wild animals in Poland remains an important issue.
KeywordsMycobacterium spp. Abscess Wildlife
Wild animals play an important role in the epidemiology of infectious diseases as reservoirs of several zoonotic and non-zoonotic diseases. Tuberculosis (TB) is one of the most important diseases affecting wild and domestic animals and also humans . TB in wild boars and feral pigs is a growing problem in some European countries. These animals are much more sensitive TB-sentinels than other wildlife species and are considered to be not only a spill-over but also reservoir hosts or even super-shedders excreting significantly higher amounts of Mycobacterium spp. bacteria than standard shedders .
In Europe, the prevalence of TB in wild boars differs among countries and even within regions [3–8]. Tuberculosis in wild boars has been reported in several European countries such as Spain , Italy , Portugal , Great Britain , France  and recently in Poland .
Tubercular lesions in wild boars are typically caseocalcareus. They consists of tubercles with diameters up to 5 cm with a dry yellow content or greenish pus or as 1 mm sized miliary foci, located mostly in the lymph nodes of the head, usually the submandibular lymph nodes [3, 6].
Tuberculosis caused by Mycobacterium bovis or M. caprae in wild boars have been reported most frequently [2, 3, 5–8], while M. microti  and non-tuberculous, potentially pathogenic environmental mycobacteria, have been reported less often [9, 10].
The diagnosis of Mycobacterium spp. infection in free-ranging wildlife is relatively difficult and relies on post-mortem examination. Laboratory diagnosis is based mainly on microscopic examination of Ziehl-Neelsen stained slides and bacterial cultivation. Histopathology may be ambiguous as lesions caused by various mycobacterial species are difficult to distinguish. Different targeted polymerase chain reaction (PCR) assays as “IS6110” sequence are useful and reliable for the detection of mycobacteria in clinical specimens . However, their sensitivity varies and may be low . Therefore, culture is considered the gold standard [13, 14] due to the highest specificity of all available methods. It may however produce false-negative results and its sensitivity has been estimated at approximately 80% .
Tuberculous-like lesions in lymph nodes in livestock and wild animals can be caused not only by Mycobacterium spp. and R. equi but also by other aerobic and anaerobic bacteria including Staphylococcus spp., Streptococcus spp., Corynebacterium spp. or Trueperella pyogenes [9, 16–18]. In the last decade, R. equi has raised considerable interest because of its zoonotic potential and the similarity to tubercular lesions. In domestic pigs, R. equi has been recognized as the main cause of lymphadenitis  but it has also been isolated from lymphadenitis in wild boars in Brazil [9, 10] and from purulent lesions in American bison (Bison bison) co-infected with Mycobacterium spp. . On the other hand, R. equi did not prove an important cause of lymphadenitis in Polish wild boars  but was isolated from the lymph nodes of apparently healthy wild boars intended for human consumption . According to the data of the Polish Hunting Association in the 2010/2011 season, the population of adult wild boars in Poland was estimated at 226,936 heads while 211,331 animals (both young and adult) were hunted.
In this study, inflamed submandibular lymph nodes from 38 wild boars hunted in the 2010/2011 season (Fig. 1) were analyzed. These samples have previously been used in another study . All samples were negative for R. equi and most of the lesions were apparently indistinguishable from typical Mycobacterium-associated lesions. The samples were stored at -20 °C for approx. 24 months. A standard procedure according to the Manual of the World Organization for Animal Health (OIE) was used for the identification of Mycobacterium spp. Briefly, the thawed tissue samples were soaked and homogenized in 5% oxalic acid. The suspension was incubated at 37 °C for 10–15 min and centrifuged at 11 000×g for 10 min. The pellets were washed with 0.9% saline and inoculated onto Stonenbrink’s and Loewenstein–Jensen’s media supplemented with glycerin and pyruvate respectively (Oxoid, Postfach, Germany). The samples were incubated at 37 °C for 12 weeks with weekly readings. Media containing M. caprae and M. avium were used as positive controls. Mycobacteria were identified on the basis of colony growth and morphology according to . Additionally, the part of thawed tissue samples were cultured on Columbia Agar supplemented with 5% sheep blood (bioMerieux, Grenoble, France) and incubated at 37 °C in microaerophilic conditions.
Given that lymphadenitis, regardless of its cause, was found in 6–11% of Polish wild boars  and the general population of wild boars in Poland consists of approximately 200,000 adults, the population of lymphadenitis-affected wild boars was estimated at 20,000 animals. For the needs of epidemiological analysis, culture sensitivity and specificity were assumed to be 80% and 100%, respectively [7, 13]. Calculations based on the hypergeometric approximation were used to determine the probability (level of confidence of population freedom, LoC) that at least one positive individual would have been detected if the disease had been present at a prevalence greater or equal to the design prevalence. The following formula was used: LoC = 1 − (1 − TSe × n/N)DP where n denotes a sample size, N—population size, DP—design prevalence and TSe—test sensitivity of 80% .
The design prevalence included in the study ranged from 1 to 20%. The epidemiological analysis was performed in EpiTools . A 95% confidence interval (95% CI) for prevalence was calculated using Wilson score method .
Data regarding different pathogens isolation from wild boar lymph nodes with purulent lesions are scarce and limited to the two reports from Brazil [9, 10] in which Mycobacterium spp. were isolated in 8.4% of the cases, R. equi in 6.6%, T. pyogenes in 5.4%, and Staphylococcus sp., Streptococcus sp. and other bacteria in 2–3%. Interestingly, 18.6% of investigated samples were negative for any bacteria as were all samples investigated in this study. However, contrary to Europe, wild boars in Brazil are not wildlife, but exotic for the local fauna, kept on commercial farms in semi-extensive conditions and the results should be compared with caution.
This study has several limitations. Freezing of the tissues precluded histopathological examination and the long storage time at −20 °C could potentially have influenced the viability of the bacteria although it has been shown that the time of storage at −20 °C had no significant effect on the rate of M. tuberculosis recovery . In this study, samples of purulent lesions typical for Mycobacterium spp. infection were investigated and a high number of culture positive samples was expected. Influence of sample storage of other bacterial pathogens survival could not be excluded. PCR was not performed because the samples, which were left from a previous project, were intended for culture.
Differences in the prevalence of Mycobacterium spp. infection in various wild boar populations in other countries may have several explanations. One can be different prevalence of other infections positively linked to TB severity in wild boar  such as infections with porcine circovirus type 2, Aujeszky’s disease virus and Metastrongylus spp., which all are also present in wild boar population in Poland . The different prevalences can also be influenced by environmental factors such as high density of wildlife, contact with livestock or presence of the known TB-reservoir species . In Poland wild boar population is growing (from 120,000 in 1999/2000 to 285,000 in 2014/2015) and contact with livestock is possible. So far TB in wild animals in Poland has been restricted to the Bieszczady Region and other TB-reservoir species than the European bison population remain unknown [8, 15]. Poland is officially free of bovine TB since 2009 (Commission Decision 2009/342/EC).
We conclude that in the 2010/2011 hunting season, mycobacterial lymphadenitis in wild boars was less prevalent in Poland than in other European countries. Due to the presence of Mycobacterium spp. infection in the European Bison population in the Bieszczady Region, a growing wild boar population, and the presence of pathogens predisposing wild boars to TB, the surveillance of mycobacterial infections in this species is necessary.
- 95% CI:
95% confidence interval
polymerase chain reaction
participated in acquisition of funding, conceiving, designing and coordination of the study, general supervision of the research group, participation in material and data collection, participation in laboratory analysis, and drafting of the manuscript. BO assisted in conceiving and designing the study, participated in laboratory analysis, and drafting of the manuscript. MR assisted in conceiving and designing the study, participated in laboratory analysis, and drafting of the manuscript. MC participated in conceiving and designing the study, statistical analysis, and assisted in drafting the manuscript. MW assisted in conceiving and designing the study and participated in material and data collection. KA assisted in conceiving and designing the study, and assisted in drafting the manuscript. JK assisted in conceiving and designing the study and assisted in drafting the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and material
All data and materials are available in Laboratory of Veterinary Epidemiology and Economics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland.
Ethics approval and consent to participate
The study was approved by the 3rd Local Commission for Ethics in Animal Experiments (Decision No. 44/2009), Warsaw University of Life Sciences—SGGW, Ciszewskiego 8, 02-786 Warsaw, Poland.
The work was partially supported by the grant from the National Science Centre in 2010–2013 as a research project No. N N308 131638. Publication was funded by KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal—Safe Food”, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015.
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- Tompkins DM, Carver S, Jones ME, Krkosek M, Skerratt LF. Emerging infectious diseases of wildlife: a critical perspective. Trends Parasitol. 2015;31:149–59.View ArticlePubMedGoogle Scholar
- Nugent G, Gortazar C, Knowles G. The epidemiology of Mycobacterium bovis in wild deer and feral pigs and their roles in the establishment and spread of bovine tuberculosis in New Zealand wildlife. N Z Vet J. 2015;63:54–67.View ArticlePubMedPubMed CentralGoogle Scholar
- Risco D, Serrano E, Fernandez-Llario P, Cuesta JM, Goncalves P, Garcia-Jimenez WL, et al. Severity of bovine tuberculosis is associated with co-infection with common pathogens in wild boar. PLoS ONE. 2014. doi:10.1371/journal.pone.0110123.PubMedPubMed CentralGoogle Scholar
- Chiari M, Ferrari N, Giardiello D, Avisani D, Pacciarini ML, Alborali L, et al. Spatiotemporal and ecological patterns of Mycobacterium microti infection in wild boar (Sus scrofa). Transbound Emerg Dis. 2015. doi:10.1111/tbed.1231.Google Scholar
- Foyle KL, Delahay RJ, Massei G. Isolation of Mycobacterium bovis from a feral wild boar (Sus scrofa) in the UK. Vet Rec. 2010;166:663–4.View ArticlePubMedGoogle Scholar
- Zanella G, Duvauchelle A, Hars J, Moutou F, Boschiroli ML, Durand B. Patterns of lesions of bovine tuberculosis in wild red deer and wild boar. Vet Rec. 2008;163:43–7.View ArticlePubMedGoogle Scholar
- Santos N, Geraldes M, Afonso A, Almeida V, Correia-Neves M. Diagnosis of tuberculosis in the wild boar (Sus scrofa): a comparison of methods applicable to hunter-harvested animals. PLoS ONE. 2010. doi:10.1371/journal.pone.0012663.Google Scholar
- Krajewska M, Lipiec M, Zabost A, Augustynowicz-Kopec E, Szulowski K. Bovine tuberculosis in a wild boar (Sus scrofa) in Poland. J Wildl Dis. 2014;50:1001–2.View ArticlePubMedGoogle Scholar
- Lara GH, Ribeiro MG, Leite CQ, Paes AC, Guazzelli A, da Silva AV, et al. Occurrence of Mycobacterium spp. and other pathogens in lymph nodes of slaughtered swine and wild boars (Sus scrofa). Res Vet Sci. 2011;90:185–8.View ArticlePubMedGoogle Scholar
- Ribeiro MG, Takai S, Guazzelli A, Lara GH, da Silva AV, Fernandes MC, et al. Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil. Res Vet Sci. 2011;91:478–81.View ArticlePubMedGoogle Scholar
- Sinha P, Gupta A, Prakash P, Anupurba S, Tripathi R, Srivastava GN. Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments. BMC Infect Dis. 2016. doi:10.1186/s12879-016-1450-1.Google Scholar
- Huyen MN, Tiemersma EW, Kremer K, de Haas P, Lan NT, Buu TN, et al. Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam. Int J Tuberc Lung Dis. 2013;17:1479–85.View ArticlePubMedGoogle Scholar
- de Lisle GW, Bengis RG, Schmitt SM, O’Brien DJ. Tuberculosis in free-ranging wildlife: detection, diagnosis and management. Rev Sci Tech. 2002;21:317–34.PubMedGoogle Scholar
- Gavier-Widen D, Cooke MM, Gallagher J, Chambers MA, Gortazar C. A review of infection of wildlife hosts with Mycobacterium bovis and the diagnostic difficulties of the ‘no visible lesion’ presentation. N Z Vet J. 2009;57:122–31.View ArticlePubMedGoogle Scholar
- Krajewska M, Zabost A, Welz M, Lipiec M, Orłowska B, Anusz K, et al. Transmission of Mycobacterium caprae in a herd of European bison in the Bieszczady Mountains, Southern Poland. Eur J Wildl Res. 2015;61:429–33.View ArticleGoogle Scholar
- Cardoso-Toset F, Gomez-Laguna J, Amarilla SP, Vela AI, Carrasco L, Fernandez-Garayzabal JF, et al. Multi-etiological nature of tuberculosis-like lesions in condemned pigs at the slaughterhouse. PLoS ONE. 2015. doi:10.1371/journal.pone.0139130.PubMedPubMed CentralGoogle Scholar
- Komijn RE, Wisselink HJ, Rijsman VM, Stockhofe-Zurwieden N, Bakker D, van Zijderveld FG, et al. Granulomatous lesions in lymph nodes of slaughter pigs bacteriologically negative for Mycobacterium avium subsp. avium and positive for Rhodococcus equi. Vet Microbiol. 2007;120:352–7.View ArticlePubMedGoogle Scholar
- Witkowski L, Rzewuska M, Takai S, Kizerwetter-Swida M, Kita J. Molecular epidemiology of Rhodococcus equi in slaughtered swine, cattle and horses in Poland. BMC Microbiol. 2016. doi:10.1186/s12866-016-0712-9.PubMedPubMed CentralGoogle Scholar
- Buergelt CD, Layton AW, Ginn PE, Taylor M, King JM, Habecker PL, et al. The pathology of spontaneous paratuberculosis in the North American bison (Bison bison). Vet Pathol. 2000;37:428–38.View ArticlePubMedGoogle Scholar
- Witkowski L, Rzewuska M, Cisek AA, Chrobak-Chmiel D, Kizerwetter-Swida M, Czopowicz M, et al. Prevalence and genetic diversity of Rhodococcus equi in wild boars (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) in Poland. BMC Microbiol. 2015. doi:10.1186/s12866-015-0445-1.PubMedPubMed CentralGoogle Scholar
- Sergeant ESG. Epitools epidemiological calculators. Ausvet Pty Ltd. 2017. http://epitools.ausvet.com.au.
- Altman D, Machin D, Bryant T, Gardner M. Statistics with confidence: confidence intervals and statistical guidelines. 2nd ed. London: BMJ Books; 2000.Google Scholar
- Tessema B, Beer J, Emmrich F, Sack U, Rodloff AC. Rate of recovery of Mycobacterium tuberculosis from frozen acid-fast-bacillus smear-positive sputum samples subjected to long-term storage in Northwest Ethiopia. J Clin Microbiol. 2011;49:2557–61.View ArticlePubMedPubMed CentralGoogle Scholar
- Fabisiak M, Podgorska K, Skrzypiec E, Szczotka A, Stadejek T. Detection of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in meat juice samples from Polish wild boar (Sus scrofa L.). Acta Vet Hung. 2013;61:529–36.View ArticlePubMedGoogle Scholar
- Vicente J, Barasona JA, Acevedo P, Ruiz-Fons JF, Boadella M, Diez-Delgado I, et al. Temporal trend of tuberculosis in wild ungulates from Mediterranean Spain. Transbound Emerg Dis. 2013;60(Suppl 1):92–103.View ArticlePubMedGoogle Scholar