Aims of the study
1) Examination and quantification of environmental samples from piggeries for the presence of hmycobacteria. To develop and apply DNA and rRNA-based methods for the detection of potentially hazardous mycobacteria in the piggery environment.
2) Sequencing (16S rDNA) and typing (RFLP, MIRU-VNTR) of the mycobacterial isolates from porcine origin and compared them to isolates of humans for examination the similarity of the M. avium strains originating from slaughter pigs and human cases in regards of public health aspects in Finland.
3) To investigate the possibilities of parallel application of Restricted Fragment Length Polymorphism (RFLP) patterns and Variable-Number Tandem Repeat (VNTR) typing of genetic interspersed repetitive units of Mycobacteria (MIRUs).
4) Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR.
3.Materials and methods
3.1. Samples and experimental design
3.1.1.Piggery environmental samples and experimental design
Piggery environmental samples were collected from birth to slaughter farms with high condemnation rates for environmental mycobacteria . The total viable mycobacteria contents were analyzed from environmental samples taken from five piggeries, about 15-20 samples per piggery, total 94 samples, 2 parallels. The prevalence of tuberculous lesions in meat control, selected 5 piggeries was more than 4% during years 2002 to 2004
(Table XX). TABLE XX,PAKARINEN 2007, PAPER I OLI TÄSSÄ VÄLISSÄ, JÄTETÄÄNKÖ POIS ?
Experimental design for piggery environmental samples is showing in Fig.6. The results were confirmed by 16S rRNA sandwich hybridization.
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Fig 6. Experimental design for piggery environmental samples.
3.1.2.Pig organs and humans samples
Pig organ samples were collected from the slaughter-line after meat inspection in Finland, 16 M. avium positive pig organs (paper 2 and 3). 7 pig organs samples from Netherlands was kindly provided by Gerard Wellenberg (paper 4).
Thirteen clinical M. avium isolates collected from Finnish human patients in 2001-2004 were randomly selected. The origin of the isolates was sputum, bronchial washings and lung biopsies (papers 2 and 3)
M. avium strains from pigs and humans as well as reference strains were stored at the culture collection of Mycobacterial Reference Laboratory, National Public Health Institute in Turku, Finland.
3.2.Typing of mycobacteria
Mycobacteria strains (16 pigs and 13 humans) were identified by direct sequence determination of 16S rRNA gene fragments and typed both by IS1245 restriction fragment length polymorphism (RFLP) and Variable-Number Tandem Repeat (VNTR) of genetic interspersed repetitive units of mycobacteria (MIRUs). Identification and RFLP and MIRU-VNTR typing were carried out in Mycobacterial reference laboratory, National Public Health institute in Turku, Finland. The discriminatory index (DI) of RFLP and MIRUs were calculated.
3.3. Mycobacteria specific real-time qPCR
Description of the samples in real-time qPCR is shown in table 1. Samples kindly provided by G.Wellenberg.
TABLE 1 TULEE TÄHÄN VÄLIIN
Table 1. Samples for mycobacterial quantification by qPCR. (Tirkkonen 2013, paper IV)
First we tried to isolate the Mycobacterial DNA from tissue specimens according to standard protocol. With this method the amount of isolated mycobacterial DNA was poor. Therefore, we modified the protocol to increase the mycobacterial cell wall lysis by digesting the tissue at 65°C with Proteinase K under agitation at 160 rpm for 16 h. This novel modification improved significantly the amount of isolated mycobacterial DNA. The detailed protocol is described in paper IV.
4.Results and discussion
4.1.Viable mycobacteria in piggeries
Mycobacterial growth was found in all bedding materials: sawdust, straw, peat and wood chips in most cases, water and food samples in many cases, and rare in dust and spider webs. (Fig. 7 A&B, 8 A&B, 9A&B, 10 A&B, 11 A,B&C).
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Fig.7 (A&B). Mycobacterial growth was found in over 60% in used bedding material samples inside the piggeries.
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Fig. 8. (A&B) Mycobacterial growth was found around 35% in unused bedding material samples outside the piggeries.
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Fig 9. (A&B) Mycobacteria grew around 25% of feed samples inside the piggeries.
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Fig 10. (A&B) Mycobacteria grew in approximately 20% of water samples.
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Fig 11. (A,B&C) No mycobacterial growth was found within 8 weeks of cultivation in ventilation dust, colostrum or spider webs, but was some growth found in spider webs and ventilation dust after 3 months of cultivation.
Concentration of the mycobacteria in pig environmental samples was 10exp5 to 10exp7 in unused and 10exp8 cells at highest in used bedding materials per gram of dry weigh when Mycobacterium-specific hybridization probes were used for detection. Mycobacteria strains contain usually 1 or 2 16S rRNA gene copies per cell (Klappenbach et al 2001, Pakarinen 2008). Since rRNA is found mainly in living cells the results confirm that mycobacteria are viable and proliferate in bedding materials during use.
The routes of infective environmental mycobacteria are unclear, but several previously publishers reported earlier, that bedding materials may be the source of infections in pig (Matlova et al 2003, Matlova et al 2004, Matlova et al 2005, Windsor et al 1984, Pakarinen 2008) and mycobacteria from fecal materials of farms may found in drinking water (Bland et al 2005, Pakarinen 2008). Quantitative methods are required for detection of the high mycobacterial concentrations in environment which may be infective (Nichols et al 2004, Pakarinen 2008). In this work we quantified mycobacterial concentration from bedding materials of piggeries, which has not been done earlier.
4.2. Typing of mycobacteria by IS1234 RFLP and MIRU-VNTR methods
M. avium isolates from pig livers and clinical human samples were compared by IS 1245 RFLP analysis to evaluate the similarity of the strains from human and swine with regard to public health aspects in Finland. Almost similar IS1245 RFLP typing patterns were found from pig and human origins. The similarity was confirmed by MIRU-VNTR typing (Fig 12)
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Fig. 12. M. avium isolates MIRU-VNTR types and their diversity in IS 1245 RFLP analysis. Similar porcine and human strains are marked red.
One human and one porcine strain clustered identically together in both typing methods. It can be concluded that the strains are clonal. The same M. avium strains may infect both humans and pigs. In agreement with our results the other studies confirmed the high similarity in M. avium subsp. hominissuis of human and porcine origins (Komijn et al 1999, Möbius et al 2006, Thorel et al 2001). Similar RFLP-profiles of M. avium have also been found in peat and human and peat and swine (Agdestein et al 2011, Bauer et al 1999, Johansen et al 2009, Matlova et al 2005), strengthening the hypothesis that humans and pigs have the same environmental infection source. The results are in accordance with other studies (Johansen et al 2007, Johansen et al 2009, Möbius et al 2006, O`Grady et al 2000).
Four different mycobacteria strains were found in one pig which can be result of a heavy load of M. avium in the piggeries or high susceptibility of some pigs to M. avium infections. Different M. avium strains in one pig have also been reported by Eisenberg et al (2010). Our earlier study showed that piggery environments can be important mycobacteria infection sources for pigs and humans (Pakarinen et al 2007). This is confirmed by several authors (Agdestein et al 2011, Agdestein et al 2014, Arbeit et al 1993, Oliveira et al 2000, Pate et al 2008).
16S rRNA sequences are similar in M. avium strains of humans and pigs. They grouped together in different typing methods and are classified as Mycobacterium avium subsp. hominissuis ( Mijs et al 2002). The IS1245 insertion sequence is specific to M. avium subsp. hominissuis and strains have a high degree of IS1245-based polymorphism which can be used to detect the genetic diversity of the M. avium strains (Eisenberg et al 2012, El-Sayed et al 2013, Guerrero et al 1995, Johansen et al 2007, de Sequeira et al 2005).
Genotyping of M. avium isolates have been performed by many authors previously using RFLP (El-Sayed et al 2013, Moravkova et al 2008) and MIRU-VNTR typing (Despierres et al 2012, El-Sayed et al 2013, Pate et al 2011, Romano et al 2005). In our studies the major RFLP clusters grouped in to comparable MIRU-VNTR clusters. IS1245 RFLP patterns and MIRU-VNTR resulted in a majority of interspecies rather than intraspecies clusters. The discriminatory index for IS1245 RFLP was 0.98 and 0.92 for MIRU-VNTR typing. Our results from Finnish isolates are parallel with in other publications (Eisenberg et al 2012, Inagaki et al 2009, Pate et al 2011). RFLP and MIRU-VNTR typing provided high level of both, reproducibility and genetic diversity. RFLP and MIRU-VNTR typing methods show great potential for epidemiological mapping and transmission pathways of M. avium subspecies (Eisenberg et al 2012, El-Sayed et al 2013)
4.3. Quantification of Mycobacterium subspecies in pig tissues by real-time quantitative PCR.
M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing. So far, there have been very few reports on the identification of M. avium strains directly from infected tissue without previous culturing (Agdestein et al 2011, Slana et al 2010, Tell et al 2003). In this work we developed and applied culture-independent real-time quantitative PCR assays for the detection of M. avium strains in pig tissues. Concentration from about 10exp5 mycobacteria cells per gram of organs were found in tissue samples with or without lesions. Similar results have been reported earlier (Agdestein et al 2011). The response of the qPCR assay to the logarithmic amount of M. avium added to pig liver was linear approximately in the range of 10exp5 to 10exp 7 bacteria per gram (Fig 13.).The qPCR method was confirmed by microscopy calculation. Recently several qPCR methods have been developed for the detection of mycobacteria strains from humans, animals and environmental samples. However, less laborious and complex methods were needed (Kriz el al 2014). In this work we developed the accurate qPCR method for the identification of mycobacterial infections in pigs. Our protocol provides a novel efficient and simple protocol to the detection of total mycobacteria cells also in samples without visible lesions.
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Fig 13. Response of the Mycobacterium-specific qPCR assay to M. avium subspecies avium ATCC 25291 cells in pig liver. Mycobacterial DNA was quantified in five parallel extracts of pig liver spiked with M. avium subspecies avium (1 × 104 to 1 × 107 bacteria per gram). Open symbols denote qPCR results below the detection limit (4 × 104 mycobacteria per gram). The curve can be interpreted that the dispersion is less when the amount of colony forming units (CFU) increases
Environmental mycobacteria are regarded as a potential zoonotic risk and cause economical losses worldwide. This thesis shows that:
1) viable mycobacteria occur generally in piggeries and can multiply in bedding materials to concentrations that may cause a potential infection risk to humans and animals
2) Identical M. avium subsp. hominissuis fingerprints were obtained from human and porcine isolates suggesting that M. a. hominissuis is transmitted between pigs and humans, or that pigs and humans share common environmental sources of infection.
3) Several mycobacteria strains found in one pig can be the result of a heavy load of mycobacteria in the piggery environment, or susceptibility of some pigs to mycobacteria infections.
4) The developed real-time qPCR method was suitable for the identification of tuberculous infections of pigs with and also without visible lesions.
Future research of mycobacterial infections and epidemiology are needed to estimate of common sources and reservoirs of mycobacteria. However the epidemiology of mycobacteria is very complex and challenging.
This work was carried out in the Faculty of Veterinary Medicine of the University of Helsinki in collaboration with following laboratories: the Faculty of Agriculture and Forestry, Department of Applied Chemistry and Microbiology, University of Helsinki, Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering and Biocenter, University of Oulu, Mycobacterial Reference Laboratory, National Public Health Institute, Turku, Animal Health Service (GD-Deventer) Deventer, The Netherlands.
I wish to express my gratitude to my supervisors: Olli Peltoniemi, Hannu Saloniemi, Timo Soveri, Faik Atroshi, Anna-Maija Virtala, Hanna Soini and Terhi Ali-Vehmas.
I want to thank all my co-authors: Mirja Salkinoja-Salonen, Jaakko Pakarinen, Timo Nieminen, Hanna Soini, Johanna Mäkinen, Olli Peltoniemi, Anna-Maria Moisander, Elina Rintala, Irina Tsitko, Terhi Ali-Vehmas, Harri Marttila, Outi Hälli, Peter Neubauer and Gerard Wellenberg.
I thank Irmeli Sippola for the tissue samples of pigs and prof. Olav Tirkkonen for checking my discriminatory power calculations in IS1245 RFLP and MIRU-VNTR typing methods.
My warmest thanks also to my supervisors and colleagues in the Finnish meat industry: Veli-Matti Jäppilä, Stefan Saaristo, Veikko Tuovinen, Matti Perälä, Sari Eskelä, Elias Jukola and Seija Pihlajaviita.
Finally and most, I would like to thank my family for their love, tolerance and support during this work. Without you this work would never have been done.
This work was financed by the by the Academy of Finland (53305,119769), Mercedes Zachariassen`s foundation, Finnish veterinary foundation, TEKES (the Finnish Funding Agency for Innovation) grant no. 2265/31/03, the Finnish Graduate School of Applied Bioscience and Finnish Meat Industry Association.
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