More than 150 species of mycobacteria are validly described, and most of them are opportunistic pathogens. All mycobacteria cause risk for human and animal health. Human infections due to environmental mycobacteria are increasing in both industrial and developing countries. The most susceptible risk groups are children, seniors and people or animals with immunosuppressive conditions. Drug therapy of mycobacteriosis is difficult and not always successful. Infections caused by drug-resistant mycobacteria can be life-threatening also for healthy adults and thus they are a relevant risk for humans. In pigs, environmental mycobacterial infections are usually without clinical signs and the lesions are mainly detected at slaughter. Mycobacterium-infected pork can pass for human consumption due to the poor specificity of visual meat control at slaughterhouses and mycobacteria in pigs also cause economical losses due to condemnation of carcasses. The main challenge is the evaluation of the hygienic risk due to the use of mycobacteria contaminated pork.
The majority of environmental mycobacteria species have been isolated from various sources, such as water, swimming-pools, soil, plants and bedding material. In our study mycobacterial growth in piggeries were found in all bedding materials, sawdust, straw, peat and wood chips in most cases, water and food samples in many cases, and only occasionally in dust and on wall surfaces. We found mycobacteria maximum almost 1x10exp9 per gram of used bedding materials, which are close of the maximum bacterial concentration in any growth media. Mycobacteria can multiply in piggeries and contaminate feed and water. Isolation of mycobacteria from pig faeces should be considered as indication of an infection risk to humans.
Environmental mycobacteriosis in humans and pigs are mainly caused by M. avium subsp. hominissuis. There is little evidence of direct transmission from animals to humans, but certain strain types can be recovered from both humans and pigs. In our studies identical mycobacteria RFLP and MIRU-VNTR fingerprints of porcine and human origins were found. Interspecies clusters were more common than intraspecies clusters with both methods. Therefore, we concluded that pigs may be as a reservoir of virulent M. avium strains and the vector for transmission of infections towards humans and vice versa or humans and pigs may have an identical infection source.
Culturing of mycobacteria is the gold standard for diagnosis, but detection of environmental mycobacteria based on cultivation and biochemical methods may take several weeks. Culture-independent rapid and accurate techniques for the detection of mycobacteria in food and feed chains are urgently needed. In this work we developed a rapid and accurate real-time quantitative PCR for detection of environmental mycobacteria in bedding materials and pig organs.
Conclusion: Mycobacteria can multiply in bedding materials and the heavy load mycobacterial contamination caused simultaneous infections of pigs. Mycobacterial DNA was found in pig organ samples also without lesions and similar strains was found from humans and pig organ samples. It cannot be ruled out, that mycobacteria are transmitted between pigs and humans.
List of Original Publications
I. Pakarinen J, Nieminen T, Tirkkonen T, Tsitko I, Ali-Vehmas T, Neubauer P, Salkinoja-Salonen M, 2007: Proliferation of mycobacteria in a piggery environment
revealed by Mycobacterium-specific real-time quantitative PCR and 16S rRNA sandwich hybridization. Veterinary Microbiology 120, 105-112.
II. Tirkkonen T, Pakarinen J, Moisander A-M, Mäkinen J, Soini H, Ali-Vehmas T, 2007: High genetic relatedness among Mycobacterium avium strains isolated from pigs and humans revealed by comparative IS 1245 RFLP analysis. Veterinary Microbiology 125, 175-181.
III. Tirkkonen T, Pakarinen J, Rintala E, Ali-Vehmas T, Marttila H, Peltoniemi O, Mäkinen J, 2010: Comparison of Variable-Number Tandem-Repeat Markers typing and IS 1245 Restriction Fragment Length Polymorphism fingerprinting of Mycobacterium avium subsp. hominissuis from human and porcine origins. Acta Veterinaria Scandinavica 52:21.
IV. Tirkkonen T, Nieminen T, Ali-Vehmas T, Peltoniemi O, Wellenberg G, Pakarinen J, 2013. Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR. Acta Veterinaria Scandinavica 2013, 55:26.
The author’s contribution
Paper I: TT planned and performed the relevant sampling procedure and participated in the writing of the paper especially regarding veterinary medicine and zoonotic aspects.
Paper II: TT performed strain selection for the porcine originating strains for the RFLP typing, interpreted the results and wrote the paper together with the co-authors.
Paper III: TT participated in the study design, sampling, analysis and interpretation of the results, TT wrote the paper and was the corresponding author.
Paper IV: TT participated in the study design, sampling, analysis and interpretation of the results, TT wrote the paper and was the corresponding author.
AIDS acquired immune deficiency syndrome
DI discriminatory index
DNA deoxyribonucleic acid
DSM Deutsche Sammlung von Mikroorganismen
ELISA-test Enzyme-Linked Immunosorbent Assay
IS1245 insertion sequence 1245
IS 1245 RFLP IS 1245 Restriction Fragment Length Polymorphism fingerprinting
MAA Mycobacterium avium subsp. avium
MAC Mycobacterium avium complex
MAH Mycobacterium avium subsp. hominissuis
MIRU genetic interspersed repetitive units of Mycobacteria
NTM nontuberculous mycobacteria
PCR polymerase chain reaction
qPCR quantitative polymerase chain reaction
RNA ribonucleic acid
rRNA ribosomal ribonucleic acid
spp. subspecies (plural)
VNTR typing Variable-Number Tandem-Repeat Marker typing
1. LITERATURE REVIEW
1.1.Description of the mycobacteria
The genus Mycobacterium consists of over 150 species. Mycobacteria are aerobic, non-spore-forming, non-motile, and rod shaped acid-fast bacilli. The mycobacteria include diverse species ranging from environmental saprophytes and opportunistic invaders to obligate pathogens. M. africanum, M. bovis, M. canettii, M. caprae, M. microti, M. pinnipedii, M. tuberculosis, M. leprae and M. lepraemurium are obligate pathogens to humans or animals (Portaels 1995, Vaerewijck et al 2005). Although some pathogenic mycobacteria exhibit a particular host preference, they can occasionally infect other species. Mycobacteria can multiply intracellularly and diseases in humans and animals are usually chronic granulomatous and progressive infections (Hibiya et al 2011, Koh et al 2002). Obligate pathogens, shed by infected animals, can also survive in the environment for extended periods (Portaels 1995, Vaerewijck et al 2005).
1.1.1. Environmental mycobacteria
Environmental mycobacteria are a heterogeneous group of slowly-growing species including saprophytes and opportunistic pathogens (El-Sayed et al 2013, Portaels 1995, Salem et al 2012, Vaerewijck et al 2005). Glycopeptidolipids in walls render mycobacteria hydrophobic and resistant to adverse environmental influences and disinfections such as chlorine. Some Mycobacterium spp. are resistant or even multiply at high temperatures (Vaerewijk et al 2005). Lipid-rich layer of the cell wall increases biofilm formation in some mycobacteria species (Freeman et al 2006, Johansen et al 2009, Recht et al 2000, Recht et al 2001,Yamazaki et al 2006). The ability to form biofilms is linked to virulence and resistance of bacteria (Carter et al 2003, Johansen et al 2009). Mycobacteria have been isolated from various environments such as soil, water, aerosols, protozoa, deep litter and fresh vegetation all over the world (Biet et al 2005, Eisenberg et al 2010). Opportunist environmental mycobacteria may cause tuberculous lesions and disseminated infections in humans and animals (Kunze et al 1992). Human infections due to environmental mycobacteria are increasing in both industrial and developing countries The most susceptible risk groups are children, seniors and people or animals with immunosuppressive conditions (Falkinham 1996, Hiller et al 2013, Vaerewijk et al 2005). Drug therapy of mycobacteriosis is difficult and not always successful. Infections caused by drug-resistant mycobacteria can be life-threatening also for healthy adults and thus they are a relevant risk for humans (Eriksson et al 2001, Hiller et al 2013, Nylen et al 2000). Generalized disease in birds and poultry (Pavlic et al 2000), pigs (Eisenberg et al 2012), cattle (Möbius et al 2006), cats and dogs (Thorel et al 2001), horses, foxes, cervids, game (Moser et al 2011) small rodenrs, insectivores (Fischer et al 2000) and other species caused by members of the M. avium complex have been reported (Pavlik et al 2005, Thorel 1997).
1.1.2. Porcine related mycobacteria
Mycobacterium avium subsp. hominissuis belong to the Mycobacterium avium complex and is the most common environmental mycobacteria in infections of humans and pigs (Agdestein et al 2011, Agdestein et al 2012, Domingos et al 2009, Iwamoto et al 2012, Johansen et al 2007, Koh et al 2002, Mijs et al 2002, Pavlik et al 2003, Stepanova et al 2012). The condition of the host may differ between human and swine. Human hosts often have the infection in their lungs (Ashford et al 2001, Jarzembowski et al 2008, Johansen et al 2009) where as pigs are infected usually through oral ingestion. In pigs tubercles can be found usually in the retropharyngeal, sub maxillary and cervical lymph nodes ( Matlova et al 2005, Thorel et al 2001). Infections in aborted fetuses and in the genital organs of pigs and decrease in growth rate as well as increased mortality have been reported (Bille et al 1973, Eisenberg et al 2012, Wellenberg et al 2010). Hepatic lesions are observed in systemically infected porcine animals. However, its pathogenesis is not well understood (Hibiya et al 2008, Hibiya et al 2010). Johansen et al 2009 speculated that swine will get infected only when a large infective dose of the M. avium strains are in their living environment. Mycobacteria in pigs are reported to be multiple variants within the same pig, because mycobacteriosis is environmentally induced (Eisenberg et al 2012, Wellenberg et al 2010). In pigs M. avium infections can be persistent also without any clinical signs, but may anyway cause economic losses for the farmer, because meat from infected animals is considered unsuitable for human consumption and condemned (Pavlik et al 2003). In the last decade, the prevalence of M. avium subsp. hominissuis in slaughtered pigs has increased worldwide (El-Sayed et al 2013, Möbius et al 2006). Prevalence of mycobacterial like lesions in Finnish slaughterpigs during years1998-2012 is showing in fig 1.
Fig.1. Prevalence of mycobacterial like lesions in Finnish slaughter pigs during years1998-2012. Finnish Food Safety Authority, EVIRA, meat control results database 2013.
1.2. Disease risk of mycobacteria from an epidemiological viewpoint
The reservoirs of infective environmental mycobacteria are unclear but the majority of these species have been isolated from various aquatic and terrestrial environments (Arqueta et al 2000, Engel et al 1978, Falkinham III 1996, Falkinham III 2002, Matlova et al 2004, Matlova et al 2005, von, Reyn et al 1993, Yajko et al 1995, Vaerewijk et al 2005 ). Drinking water has been shown to be a possible source of environmental mycobacteria for both human and animals (Falkinham III et al 2001, Hilborn et al 2008, Johansen et al 2009, Nishiuchi et al 2007). Environmental mycobacteria survive in water distribution systems, because they are usually resistant to water treatment with ozone and chlorine especially when growing as multicellular aggregates (Hilborn et el al 2006, Johansen et al 2009, Steed et al 2006, Taylor et al 2000, Vaerewijck et al 2005). Environmental mycobacteria may also survive in water within amoebas (Hilborn et al 2006, Johansen et al 2009, Vaerewijck et al 2005).
It has been suggested that drinking water may be an important reservoir of infective mycobacteria especially for humans (Aronson et al 1999, Falkinham III 1996) whereas bedding materials has proved to be important sources of porcine infections (Alvarez et al 2011, Matlova et al 2004, Matlova et al 2005). The mycobacterial infections have a zoonotic character and similar types of mycobacteria strains have been found in pigs and humans (Komijn et al 1999, Möbius et al 2006). It cannot be ruled out that mycobacteria of humans may originate from piggeries (Komijn et al 1999, Komijn et al 2000, Möbius et al 2006). Contaminated fecal material in beddings and mycobacteria in raw pork meat is a potential food safety hazard (Alvarez et al 2011, Komijn et al 1999, Matlova et al 2005, Matlova et al 2004, Möbius et al 2006, Johansen et al 2014).
More knowledge about the routes of transmission in both animals and humans are required for the control of mycobacterioses (Agdestein et al 2012, Biet et al 2005, Thegerström et al 2005). Effective methods for identification, genetic profiling, and rapid real-time quantification of environmental mycobacteria are needed for the tracing of environmental reservoirs of human and animal mycobacteriosis and the risk assessment of these.
1.3. Detection and identification of mycobacteria
The acid-fast staining method is used to differentiate mycobacteria from other bacteria. In pigs, acid-fast bacilli can be found from caseous malformations in lymph nodes, kidneys, liver and spleen (Eisenberg et al 2012, Hiller et al 2013, Van Inger et al 2010, Offermann et al 1999), but also from Rhodococcus equi and other acid-fast bacilli infections (Dvorska et al 1999, Eisenberg et al 2012, Hiller et al 2013, Komijn et al 2007, Pate et al 2004). On the other hand, mycobacteria can be found in porcine lymph nodes without any visible lesions (Dvorska et al 1999, Hiller et al 2013). Differentiation of pathogenic mycobacteria relies on cultural characteristics, biochemical tests, animal inoculation, chromatographic analyses and molecular techniques. In addition, mycobacteria associated with opportunistic infections can be differentiated on the basis of pigment production, optimal incubation temperature and growth rate. Pathogenic mycobacteria grow slowly and colonies are not evident until cultures have been incubated for at least three weeks (Fig. 2).
KUVA 2 TULEE TÄHÄN VÄLIIN
Fig. 2. Mycobacteria avium growing three weeks on Löwenstein-Jensen agar.
In contrast, the colonies or growth in broth of rapidly growing saprophytes are visible within days (Fig. 3 A & B)
KUVA 3 A&B TULEE TÄHÄN VÄLIIN
Fig 3 (A&B). M. chelonae growth curve in Middledbrook broth (Fig 3A). The linear regression line is drawn by least-squares principle (including 95% confidence intervals) (Fig 3B), Ali-Vehmas 1998 (unpublished data).
However, identification of the members belonging to the M. avium complex by biochemical testing can take up to several weeks (Slana et al 2010, Springer et al 1996). Some mycobacterial isolates cannot be indentified using biochemical differentiation as their biochemical profiles are very difficult to interpret (Gunn-Moore et al 1996).
Culturing of mycobacteria is the golden standard of diagnosis, but molecular tests are also used (Agdestein et al 2011, Inderlied et al 1993, Thorel et al 2001, Turenne et al 2007). DNA probes, complementary to species-specific sequences of rRNA, are commercially available for some mycobacterium species. Nucleic acid amplification procedures, including the polymerase chain reaction, were and are being developed for the detection of mycobacteria in environmental and tissue samples (Khan et al 2 004, Kox et al 1995, Pakarinen 2008, Shrestha et al 2003, Talaat et al 1997, Telenti et al 1993) and DNA restriction endonuclease analyses (DNA fingerprinting), were used in epidemiological studies during last decades by several authors (Bauer et al 1999, Collins et al 1994, Johansen et al 2007). However, most of these methods are complex, laborous, expensive or not sensitive enough.
1.4.Current methods and recommendations in porcine meat control
European law (EU/854/2004) describes the procedure for meat inspection at slaughter, such as palpation and incision of the lymph nodes including the procedures of detection of porcine mycobacterial like infections (Fig. 4, Fig. 5).
KUVA 4 TULEE TÄHÄN
Fig. 4. Palpation of porcine livers at slaughterhouse meat control facility.
KUVA 5 TULEE TÄHÄN
Fig.5. A presumptive mycobacterial lesion in a slaughter pig liver.
The detection is not sensitive or accurate (Hiller et al 2013, Komijn et al 2007, Wisselink et al 2010). Similar lesions can be caused by other food safety hazardous microbes (Faldyna et al 2012, Hamilton et al 2002, Hiller et al 2013, Komijn et al 2007, Pavlik et al 2003) and mycobacteria can also be isolated from lymph nodes without any visible lesions (Offermann et al 1999, Wisselink et al 2006). The tuberculin test has been used to detect mycobacteria-positive pigs before slaughter, but this test is not sensitive (Faldyna et al 2012, Francis et al 1978). Therefore, more accurate screening methods are needed for the detection of mycobacteriosis in pigs (Faldyna et al 2012).
1.5.Serological response and monitoring of porcine mycobacterial infections
Several authors have suggested that Mycobacterium avium can cause porcine infections and may be a potential food safety hazard for humans. Diagnosis of the mycobacterial infections of pigs is usually done only after slaughtering by palpation and visual observation of the lesions in the lymph nodes and livers. Caseous lesions can also be found in porcine kidneys and spleen (Hiller et al 2013, Van Ingen et al 2010, Offermann et al 1999, Thoen et al 2006) but the formation of the lesions caused by M.avium infections may take several months in pigs (Wisselink et al 2006). Ulceration or necrosis of the skin have been also observed in M. avium infections (Agdestein et al 2012). The post mortem visually inspections of the lymph nodes and livers give a high number of both false positive and negative results being a non-sensitive and non-specific test (Eisenberg et al 2012, Faldyna et al 2012, Hiller et al 2013, Komijn et al 2007, Wisselink et al 2006, Wisselink et al 2010). The sensitivity of visual meat inspection has been found to be highest in pigs infected by M. avium at an age between 2.5 and 4.5 weeks but low in pigs infected at the age of 18 weeks (Wisselink et al 2006). Mycobacterial infections without any visible lesions have also been found (Brown et al 1979, Dvorska et al 1999, Hiller et al 2013, Offermann et al 1999, Wisselink et al 2006). Repeated infections by M. avium may cause an altered immune response and inhibit the formation of lesions in pigs. It has been reported that pigs infected three times had a low number of lesions in their lymph nodes (Wisselink et al 2006). In that case M. avium infected pigs can pass the post mortem visual inspection (Wisselink et al 2006). Also other bacteria such as Rhodococcus have been isolated from pathological, mycobcteria like lesions in pigs (Dvorska et al 1999, Faldyna et al 2012, Hiller et al 2013, Komijn et al 2007, Pavlik et al 2003). Cross-contamination with other pathogens, for example salmonella (Hamilton et al 2002 has also been reported). Even two third of the found granulomatous malformations in porcine lymph nodes are caused by other non mycobacterial reasons (Hiller et al 2013).
Tuberculin skin test is the standard method when mycobacterial infections are diagnosed in living animals, but the sensitivity of the test is low (Monagham et al 1994, Stepanova et al 2011). Immunological responses detected by tuberculin skin test can be applied on an herd level only and they are quite inaccurate also in that case (Eisenberg et al 2012). Rather poor correlations have been reported between the ELISA-test (Eisenberg et al 2012) as well as gamma interferon release assay (Faldyna et al 2012) and the tuberculin skin test results (Eisenberg et al 2012, Faldyna et al 2012). Immunological host responses detected by the tuberculin skin test and ELISA correlated positively at herd level only (Eisenbe Stepanova et al 2011rg et al 2012 Komijn et al 2007).
Several methods based on serological response of mycobacterial infections have been published ( Boadella et al 2011, Faldyna et al 2012). Immunological response such as interferon release assay may have a higher sensitivity than the tuberculin test and the interferon assay can be used for diagnosis of M. avium infections in live and naturally infected pigs (Faldyna et al 2012). M. avium induced central memory cells in porcine infections but at least a six months period was needed to detect cell-mediated immunity to M. avium in pigs (Stepanova et al 2011). This is problematic because most finisher pigs are being slaughtered around the age of six months. Moreover the in vitro re-stimulation interferon gamma production was decreased (Stepanova et al 2011). Some lymphocyte release may cause long-term immunity in M. avium infected pigs (Stepanova et al 2011). Recently, sever mycobacteria specific tests have been applied to describe the correlation of abortions, re-breedings or stillbirths and mycobacterial infections (Eisenberg et al 2012.). Stepanova et al (2011) concluded that interferon gamma and lymphocyte transformation may represent a specific method for the identification of individual M. avium infections in pigs, but more detailed studies are needed (Stepanova et al 2011). The results of Stepanova et al (2011) indicate that interferon gamma release assay and lymphocyte transformation test can be used for the identification of M. avium infected pigs (Stepanova et al 2011).
Hiller et al 2013 applied Mycobacterial specific enzyme-linked immunosorbent assay ( ELISA) to test the screening of the presence of M. avium antibodies in blood samples of slaughtered pigs in the Netherlands and Germany. The presence of M. avium antibodies was detected to estimate the prevalence of mycobacterial infections on herd level. M. avium ELISA test was validated to identify M. avium positive farms. The validation results showed in this research, that the sensitivity of an individual test was low and only 20% of the bacteriologically positive herds could be identified when 36 blood samples were tested. The low sensitivity on herd level were supposed to be due to the presence of infections with other M. avium serotypes that have lower immunity towards the antigens tested. Closely related M. avium subsp. avium and subsp. hominissuis showed different capacities to stimulate the porcine immune system. Mycobacterium avium subsp. hominissuis showed low cell-mediated immunity with high individual variability (Dvorska et al 2004, Hiller et al 2013, Stepanova et al 2012). However, Hiller et al (2013) concluded that serological screening by M. avium specific ELISA-test could be capable of identifying bacteriological M. avium positive herds and pig populations that are at risk for M. avium infections. The discrimination power between infected and non-infected farms using the ELISA test may be improved with additional mycobacterial antigens (Hiller et al 2013).
M.avium subsp. hominissuis is a weaker pathogen compared to M. avium subsp. avium (Faldyna et al 2012). As far as the author knows, are the developed serological tests not suitable for M. avium subsp. hominissuis (Domingos et al 2009, Faldyna et al 2012). Immunological parameters of M. avium subsp. avium and M. avium subsp. hominissuis have been compared and systemic immune response during experimental mycobacteriosis of pig has been measured. Releasing of Interferon gamma can be found after 5 weeks in M. avium subsp. avium and subsp. hominissuis infected pigs but not in every individual (Agdestein et al 2012). The cell-mediated Immunological response of M. avium subsp. hominissuis is significantly weaker when compared to M. avium subsp. avium. The release of mycobacterial specific antibodies or gamma interferon was low and variable in M. avium subsp. hominissuis infections compared to M. avium. subsp. avium infections (Stepanova et al 2012). The release of pro-inflammatory cytokines from macrophages was notably higher in vitro experiments when inductor was M. avium subsp. avium in comparison of M.avium subsp. hominissuis. Also so the amount of M. avium subsp. hominissuis was at least 1000 fold lower, than M. avium subsp. avium in infected gastro-intestinal tissues of pigs (Slana et al 2010, Stepanova et al 2012). The macrophage response to M. avium subsp. hominissuis infections have been reported to be significantly weaker than the response to M. avium subsp. avium infections in vitro. M. avium subsp. hominissuis infected macrophages showed also weaker induction of pro-inflammatory cytokines and chemokines (Stepanova et al 2012). The immunological response is also different between different M. avium subsp. hominissuis genotype (El-Sayed et al 2013, Stepanova et al 2012, Thegerström et al 2012). It can be concluded that M.avium subsp. hominissuis can induce only a weak cell-mediated immunity, however in some case positive results were obtained in the Interferon gamma release assay of M. avium subsp. hominissuis infections of pigs without specific antibodies detection (Stepanova et al 2012). Interferon gamma release assay may be an effective tool for discrimination of M. avium subsp. avium infected pigs, but too inaccurate for detection of M. avium subsp. hominissuis infections of pigs (Stepanova et al 2012).
Moreover most of the immunological tests for mycobacterial infections in pigs have been applied to Mycobacterium avium subsp.avium, but the majority of M avium strains in mycobacteriosis of pigs are M. avium subsp. hominissuis ( Domingos et al 2009, Garrido et al 2010, Shitaye et al 2006, Stepanova et al 2011, Stepanova et al 2012). In the porcine mycobateriosis caused by M. avium subsp. hominissuis the infected pigs produced significantly lower levels of mycobacterial specific antibodies and interferon gamma as compared to M. avium. subsp. avium infected pigs (Stepanova et al 2012). The immunodominant antigens in different mycobacterial species may not be cross-reactive (Faldyna et al 2012). Therefore, more reliable detection methods are required for the identification of mycobacterial infections in routine diagnosis of pigs (Faldyna et al 2012, Wisselink et al 2006).
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