Exploiting multiple-locus variable number tandem repeat analysis to evaluate strain diversity of M. avium Complex in Argentina

GIOFFRÉ ; CORREA; PINEDO; MONTENEGRO; VACA; MORSELLA; SOLEDAD BARANDIARAN; FIORENTINO; MARCELA MARTINEZ VIVOT; MORCILLO; PAOLICHI; MARTIN JOSE ZUMARRAGA; ROMANO

Congreso; 11th International Colloquium on Paratuberculosis; 2012

EXPLOITING MULTIPLE-LOCUS VARIABLE NUMBER TANDEM REPEAT ANALYSIS TO EVALUATE STRAIN DIVERSITY OF M. AVIUM COMPLEX IN ARGENTINAM. AVIUM COMPLEX IN ARGENTINA Gioffré A1, Correa M2, Alvarado Pinedo MF3, Montenegro V1, Vaca J3, Morsella C4, Barandiaran S5, Imperiale B6, Fiorentino MA4, Martínez Vivot M5, Morcillo N6, Paolicchi F4, Zumárraga M1, Travería GE3, Romano MI11, Correa M2, Alvarado Pinedo MF3, Montenegro V1, Vaca J3, Morsella C4, Barandiaran S5, Imperiale B6, Fiorentino MA4, Martínez Vivot M5, Morcillo N6, Paolicchi F4, Zumárraga M1, Travería GE3, Romano MI16, Fiorentino MA4, Martínez Vivot M5, Morcillo N6, Paolicchi F4, Zumárraga M1, Travería GE3, Romano MI11 1 Instituto de Biotecnología CICVyA INTA-Castelar, Buenos Aires. Argentina. 2 Universidad Autónoma de Baja California, Mexicali, México. 3 CEDIVE y Cátedra de Zootecnia Especial II. Fac.de Cs. Veterinarias. Universidad Nacional de La Plata, Argentina. 4 Laboratorio de Bacteriología EEA-INTA Balcarce, Argentina. 5 Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Argentina. 6 Hospital Dr. A. Cetrángolo, Vicente López, Buenos Aires, Argentina. Introduction & Aim. The Mycobacterium avium complex (MAC) includes major human and animal pathogens. MAC related diseases have a significant impact on human health, mainly in immunocompromised patients. M. avium subsp. paratuberculosis (MAP) is the most economically important subspecies due to severe economic losses in infected herds, affecting the sustainability of agriculture even at familiar scale in developing countries. There is still lack of nationwide, reliable epidemiological data. Serological surveys in Buenos Aires province, one of the most important productive regions of the country, reported a prevalence between 0.8%- 19.6% in breeding herds and between 16.7%-39.3% in dairy herds of different locations (INTA-Balcarce). The impact of other subspecies such as M. avium subsp. silvaticum (MAS), M. avium subsp. hominissuis (MAS) and M. avium subsp. avium (MAA) in animal health is largely unknown, and frequently underestimated as a consequence of a poor diagnosis. Molecular typing has contributed with the correct identification and the intra-species differentiation of isolates, helping to understand the epidemiology of these bacteria. The PCR-based methods for molecular typing analysis represent an important technical advance and have simplified the molecular typing of microorganisms. The aim of this study was to determine the genetic diversity of Mycobacterium avium complex isolates through multiple-locus variable number tandem repeat analysis.The Mycobacterium avium complex (MAC) includes major human and animal pathogens. MAC related diseases have a significant impact on human health, mainly in immunocompromised patients. M. avium subsp. paratuberculosis (MAP) is the most economically important subspecies due to severe economic losses in infected herds, affecting the sustainability of agriculture even at familiar scale in developing countries. There is still lack of nationwide, reliable epidemiological data. Serological surveys in Buenos Aires province, one of the most important productive regions of the country, reported a prevalence between 0.8%- 19.6% in breeding herds and between 16.7%-39.3% in dairy herds of different locations (INTA-Balcarce). The impact of other subspecies such as M. avium subsp. silvaticum (MAS), M. avium subsp. hominissuis (MAS) and M. avium subsp. avium (MAA) in animal health is largely unknown, and frequently underestimated as a consequence of a poor diagnosis. Molecular typing has contributed with the correct identification and the intra-species differentiation of isolates, helping to understand the epidemiology of these bacteria. The PCR-based methods for molecular typing analysis represent an important technical advance and have simplified the molecular typing of microorganisms. The aim of this study was to determine the genetic diversity of Mycobacterium avium complex isolates through multiple-locus variable number tandem repeat analysis.M. avium subsp. paratuberculosis (MAP) is the most economically important subspecies due to severe economic losses in infected herds, affecting the sustainability of agriculture even at familiar scale in developing countries. There is still lack of nationwide, reliable epidemiological data. Serological surveys in Buenos Aires province, one of the most important productive regions of the country, reported a prevalence between 0.8%- 19.6% in breeding herds and between 16.7%-39.3% in dairy herds of different locations (INTA-Balcarce). The impact of other subspecies such as M. avium subsp. silvaticum (MAS), M. avium subsp. hominissuis (MAS) and M. avium subsp. avium (MAA) in animal health is largely unknown, and frequently underestimated as a consequence of a poor diagnosis. Molecular typing has contributed with the correct identification and the intra-species differentiation of isolates, helping to understand the epidemiology of these bacteria. The PCR-based methods for molecular typing analysis represent an important technical advance and have simplified the molecular typing of microorganisms. The aim of this study was to determine the genetic diversity of Mycobacterium avium complex isolates through multiple-locus variable number tandem repeat analysis.M. avium subsp. silvaticum (MAS), M. avium subsp. hominissuis (MAS) and M. avium subsp. avium (MAA) in animal health is largely unknown, and frequently underestimated as a consequence of a poor diagnosis. Molecular typing has contributed with the correct identification and the intra-species differentiation of isolates, helping to understand the epidemiology of these bacteria. The PCR-based methods for molecular typing analysis represent an important technical advance and have simplified the molecular typing of microorganisms. The aim of this study was to determine the genetic diversity of Mycobacterium avium complex isolates through multiple-locus variable number tandem repeat analysis.subsp. avium (MAA) in animal health is largely unknown, and frequently underestimated as a consequence of a poor diagnosis. Molecular typing has contributed with the correct identification and the intra-species differentiation of isolates, helping to understand the epidemiology of these bacteria. The PCR-based methods for molecular typing analysis represent an important technical advance and have simplified the molecular typing of microorganisms. The aim of this study was to determine the genetic diversity of Mycobacterium avium complex isolates through multiple-locus variable number tandem repeat analysis.Mycobacterium avium complex isolates through multiple-locus variable number tandem repeat analysis. Methodology. We selected a scheme based on eight MIRU-VNTR loci according to Thibault et al.,(2007). We analyzed 95 isolates of MAP (bovine, n=76; caprine, n=16 and ovine, n=3) from farms of different regions of Buenos Aires province. The isolates were previously confirmed by IS900-PCR (Collins et al., 1993). Additionally, 25 isolates typed by IS1245-PCR as positive (Guerrero et al., 1995) and characterized by hsp65-PRA (Telenti et al., 1993) as M. avium type I were also analyzed: Six human samples from patients with non-tuberculous mycobacteriosis (HIV+, Cetrángolo Hospital, Buenos Aires), 2 isolates from cows, 15 isolates from pigs (sampled at the abattoir settled in the same province) and 2 from captive birds. All of the human isolates were identified as MAH since the IS901-PCR (Slana et al., 2010) was negative. All the bird, cattle and pig isolates were IS901 positive. This panel of 120 isolates was subjected to the scheme based on repetitive elements proposed by Thibault and coworkers to type. We selected a scheme based on eight MIRU-VNTR loci according to Thibault et al.,(2007). We analyzed 95 isolates of MAP (bovine, n=76; caprine, n=16 and ovine, n=3) from farms of different regions of Buenos Aires province. The isolates were previously confirmed by IS900-PCR (Collins et al., 1993). Additionally, 25 isolates typed by IS1245-PCR as positive (Guerrero et al., 1995) and characterized by hsp65-PRA (Telenti et al., 1993) as M. avium type I were also analyzed: Six human samples from patients with non-tuberculous mycobacteriosis (HIV+, Cetrángolo Hospital, Buenos Aires), 2 isolates from cows, 15 isolates from pigs (sampled at the abattoir settled in the same province) and 2 from captive birds. All of the human isolates were identified as MAH since the IS901-PCR (Slana et al., 2010) was negative. All the bird, cattle and pig isolates were IS901 positive. This panel of 120 isolates was subjected to the scheme based on repetitive elements proposed by Thibault and coworkers to type.,(2007). We analyzed 95 isolates of MAP (bovine, n=76; caprine, n=16 and ovine, n=3) from farms of different regions of Buenos Aires province. The isolates were previously confirmed by IS900-PCR (Collins et al., 1993). Additionally, 25 isolates typed by IS1245-PCR as positive (Guerrero et al., 1995) and characterized by hsp65-PRA (Telenti et al., 1993) as M. avium type I were also analyzed: Six human samples from patients with non-tuberculous mycobacteriosis (HIV+, Cetrángolo Hospital, Buenos Aires), 2 isolates from cows, 15 isolates from pigs (sampled at the abattoir settled in the same province) and 2 from captive birds. All of the human isolates were identified as MAH since the IS901-PCR (Slana et al., 2010) was negative. All the bird, cattle and pig isolates were IS901 positive. This panel of 120 isolates was subjected to the scheme based on repetitive elements proposed by Thibault and coworkers to type900-PCR (Collins et al., 1993). Additionally, 25 isolates typed by IS1245-PCR as positive (Guerrero et al., 1995) and characterized by hsp65-PRA (Telenti et al., 1993) as M. avium type I were also analyzed: Six human samples from patients with non-tuberculous mycobacteriosis (HIV+, Cetrángolo Hospital, Buenos Aires), 2 isolates from cows, 15 isolates from pigs (sampled at the abattoir settled in the same province) and 2 from captive birds. All of the human isolates were identified as MAH since the IS901-PCR (Slana et al., 2010) was negative. All the bird, cattle and pig isolates were IS901 positive. This panel of 120 isolates was subjected to the scheme based on repetitive elements proposed by Thibault and coworkers to typeet al., 1993). Additionally, 25 isolates typed by IS1245-PCR as positive (Guerrero et al., 1995) and characterized by hsp65-PRA (Telenti et al., 1993) as M. avium type I were also analyzed: Six human samples from patients with non-tuberculous mycobacteriosis (HIV+, Cetrángolo Hospital, Buenos Aires), 2 isolates from cows, 15 isolates from pigs (sampled at the abattoir settled in the same province) and 2 from captive birds. All of the human isolates were identified as MAH since the IS901-PCR (Slana et al., 2010) was negative. All the bird, cattle and pig isolates were IS901 positive. This panel of 120 isolates was subjected to the scheme based on repetitive elements proposed by Thibault and coworkers to typehsp65-PRA (Telenti et al., 1993) as M. avium type I were also analyzed: Six human samples from patients with non-tuberculous mycobacteriosis (HIV+, Cetrángolo Hospital, Buenos Aires), 2 isolates from cows, 15 isolates from pigs (sampled at the abattoir settled in the same province) and 2 from captive birds. All of the human isolates were identified as MAH since the IS901-PCR (Slana et al., 2010) was negative. All the bird, cattle and pig isolates were IS901 positive. This panel of 120 isolates was subjected to the scheme based on repetitive elements proposed by Thibault and coworkers to type901-PCR (Slana et al., 2010) was negative. All the bird, cattle and pig isolates were IS901 positive. This panel of 120 isolates was subjected to the scheme based on repetitive elements proposed by Thibault and coworkers to type. All the bird, cattle and pig isolates were IS901 positive. This panel of 120 isolates was subjected to the scheme based on repetitive elements proposed by Thibault and coworkers to type M. avium subsp. paratuberculosis and further employed to type other subspecies (Radomski et al., 2010). PCR conditions were used as described previously with minor modifications. PCR products corresponding to different loci (292-X3-25-47-3-7-10-32) were resolved by 4% agarose gel electrophoresis and tandem repeats were determined using the BioNumerics software (Applied Maths, Belgium). The numerical profiles were classified according of INMV (INRA, Nouzilly, MIRU-VNTR) combinations suggested previously. (Thibault et al., 2007; Radomski et al., 2010).subsp. paratuberculosis and further employed to type other subspecies (Radomski et al., 2010). PCR conditions were used as described previously with minor modifications. PCR products corresponding to different loci (292-X3-25-47-3-7-10-32) were resolved by 4% agarose gel electrophoresis and tandem repeats were determined using the BioNumerics software (Applied Maths, Belgium). The numerical profiles were classified according of INMV (INRA, Nouzilly, MIRU-VNTR) combinations suggested previously. (Thibault et al., 2007; Radomski et al., 2010).loci (292-X3-25-47-3-7-10-32) were resolved by 4% agarose gel electrophoresis and tandem repeats were determined using the BioNumerics software (Applied Maths, Belgium). The numerical profiles were classified according of INMV (INRA, Nouzilly, MIRU-VNTR) combinations suggested previously. (Thibault et al., 2007; Radomski et al., 2010).et al., 2007; Radomski et al., 2010). Results Profiles & Frequencies. Eight different INMV MIRU-VNTR patterns were identified for the MAPisolates. The INMV1 pattern grouped 64.2% of the isolates and was the most frequent. The distribution of the other patterns was: INMV2, 10.52%; INMV16, 8.42%; INMV33, 7.3%; INMV13, 3.16%; and INMV11, 2.1%. One cattle isolate presented a pattern that, to our knowledge, has not been previously identified. On the other hand, in three isolates we could not obtain the PCR product for the locus 292 and 7. However the alleles in the rest of the loci indicated a different pattern to those mentioned above. ThisEight different INMV MIRU-VNTR patterns were identified for the MAPisolates. The INMV1 pattern grouped 64.2% of the isolates and was the most frequent. The distribution of the other patterns was: INMV2, 10.52%; INMV16, 8.42%; INMV33, 7.3%; INMV13, 3.16%; and INMV11, 2.1%. One cattle isolate presented a pattern that, to our knowledge, has not been previously identified. On the other hand, in three isolates we could not obtain the PCR product for the locus 292 and 7. However the alleles in the rest of the loci indicated a different pattern to those mentioned above. Thislocus 292 and 7. However the alleles in the rest of the loci indicated a different pattern to those mentioned above. Thisloci indicated a different pattern to those mentioned above. This Proceedings 11ICP Genotyping and Mycobacterium avium subsp. paratuberculosis diversityMycobacterium avium subsp. paratuberculosis diversity 265 uncomplete pattern represented 3.15% of the isolates. INMV1 and INMV2 patterns were the unique shared by bovine, caprine and ovine strains. Four out of six farms were co-infected with two or more genetically unrelated strains (2 to 6 different patterns). Co-infection at individual level was also demonstrated through the analysis of different isolates from the same animal (n=1). In spite of the low number of MAH isolates studied, we could observe that all the loci were polymorphic (5 and 3 lociloci were polymorphic (5 and 3 loci resulted polymorphic in the other subspecies). Five human isolates were grouped in 3 patterns not previously described and one human isolate was identified with a pattern already reported: INMV51. Three patterns were found in pig isolates and cattle isolates shared the same profile. These 4 patterns were not previously reported. In spite of the difficulties to determine the allele corresponding to locus 7 in bird isolates (a difference of approximately 6bp was observed), these isolates were classified as INMV100. The presence of polymorphisms in one or more repeats could generate PCR products of different sizes to those expected as previously reported for the same locus (Castellanos et al., 2010). We could not establish epidemiological link between different hosts, this was probably due to the reduced number of isolates studied here.locus 7 in bird isolates (a difference of approximately 6bp was observed), these isolates were classified as INMV100. The presence of polymorphisms in one or more repeats could generate PCR products of different sizes to those expected as previously reported for the same locus (Castellanos et al., 2010). We could not establish epidemiological link between different hosts, this was probably due to the reduced number of isolates studied here.locus (Castellanos et al., 2010). We could not establish epidemiological link between different hosts, this was probably due to the reduced number of isolates studied here. Allelic diversity and Discriminatory index. The allelic diversity and the discriminatory power were estimated as described previously by Castellanos et al. (2009). Epidemiologically unrelated strains were selected to this end. The locus 292 showed the maximum allelic diversity for MAP strains (0.53) whereas X3 (0.68) and 25 (0.6) were the markers most informative for MAA and MAH respectively. The results obtained for MAP and MAA were coincident with the previous obtained by Radomski et al. (2010) and others. We obtained a Hunter and Gaston discriminatory index (HGDI) of 0.86 for all subspecies. The HGDI for MAP was 0.68, lower than the discriminatory index obtained by Thibault et al., (0.75), but similar to that obtained by Stevenson et al.,( 0.64). A novel panel of MAA and MAH isolates would be suitable to obtain more accurate data in these subspecies.The allelic diversity and the discriminatory power were estimated as described previously by Castellanos et al. (2009). Epidemiologically unrelated strains were selected to this end. The locus 292 showed the maximum allelic diversity for MAP strains (0.53) whereas X3 (0.68) and 25 (0.6) were the markers most informative for MAA and MAH respectively. The results obtained for MAP and MAA were coincident with the previous obtained by Radomski et al. (2010) and others. We obtained a Hunter and Gaston discriminatory index (HGDI) of 0.86 for all subspecies. The HGDI for MAP was 0.68, lower than the discriminatory index obtained by Thibault et al., (0.75), but similar to that obtained by Stevenson et al.,( 0.64). A novel panel of MAA and MAH isolates would be suitable to obtain more accurate data in these subspecies.et al. (2009). Epidemiologically unrelated strains were selected to this end. The locus 292 showed the maximum allelic diversity for MAP strains (0.53) whereas X3 (0.68) and 25 (0.6) were the markers most informative for MAA and MAH respectively. The results obtained for MAP and MAA were coincident with the previous obtained by Radomski et al. (2010) and others. We obtained a Hunter and Gaston discriminatory index (HGDI) of 0.86 for all subspecies. The HGDI for MAP was 0.68, lower than the discriminatory index obtained by Thibault et al., (0.75), but similar to that obtained by Stevenson et al.,( 0.64). A novel panel of MAA and MAH isolates would be suitable to obtain more accurate data in these subspecies.locus 292 showed the maximum allelic diversity for MAP strains (0.53) whereas X3 (0.68) and 25 (0.6) were the markers most informative for MAA and MAH respectively. The results obtained for MAP and MAA were coincident with the previous obtained by Radomski et al. (2010) and others. We obtained a Hunter and Gaston discriminatory index (HGDI) of 0.86 for all subspecies. The HGDI for MAP was 0.68, lower than the discriminatory index obtained by Thibault et al., (0.75), but similar to that obtained by Stevenson et al.,( 0.64). A novel panel of MAA and MAH isolates would be suitable to obtain more accurate data in these subspecies.et al. (2010) and others. We obtained a Hunter and Gaston discriminatory index (HGDI) of 0.86 for all subspecies. The HGDI for MAP was 0.68, lower than the discriminatory index obtained by Thibault et al., (0.75), but similar to that obtained by Stevenson et al.,( 0.64). A novel panel of MAA and MAH isolates would be suitable to obtain more accurate data in these subspecies.et al., (0.75), but similar to that obtained by Stevenson et al.,( 0.64). A novel panel of MAA and MAH isolates would be suitable to obtain more accurate data in these subspecies.et al.,( 0.64). A novel panel of MAA and MAH isolates would be suitable to obtain more accurate data in these subspecies. Conclusions and Perspectives The most predominant patterns in M. avium subsp. paratuberculosis strains (INMV1 and INMV2) were also described as the most frequent in other countries from Europe (Thibault et al., 2007, Stevenson et al., 2009). This result could be an indicative of higher transmission rate or infectivity of these clones. This study suggests that co-infection with different strains could be a common feature of paratuberculosis infection in Argentina and may point out trading of animals between herds and highlights the lack of monitoring programs in our country. Despite the low number of isolates processed in the rest of the members of the MAC, we obtained 9 patterns, most of them not described previously. This highlights the high degree of MIRU-VNTR diversity of M. avium subsp. avium and hominissuis, being congruent with previous studies. Several authors have used the MIRU-VNTR typing scheme proposed by Thibault and coworkers to type MAC isolates from different countries of the world: France, Germany, Czech Republic, Finland, Greece, The Netherlands, Norway, Scotland, Spain, Japan and more recently Colombia and Argentina among others (Möbius et al., 2008; Stevenson et al., 2009; Inagaki et al., 2009; Castellanos et al., 2010; Radomski et al., 2010; Fernández-Silva et al., 2011, this work). Several studies have included new markers and/or omitted others or have used a different pattern nomenclature. In this way, the results from different studies are very difficult to compare. Hence, the standardization of protocols must be encouraged. Finally, a MAC database could be a suitable tool for understanding global epidemiology of these bacteria. Collaborative work should focus to these endsM. avium subsp. paratuberculosis strains (INMV1 and INMV2) were also described as the most frequent in other countries from Europe (Thibault et al., 2007, Stevenson et al., 2009). This result could be an indicative of higher transmission rate or infectivity of these clones. This study suggests that co-infection with different strains could be a common feature of paratuberculosis infection in Argentina and may point out trading of animals between herds and highlights the lack of monitoring programs in our country. Despite the low number of isolates processed in the rest of the members of the MAC, we obtained 9 patterns, most of them not described previously. This highlights the high degree of MIRU-VNTR diversity of M. avium subsp. avium and hominissuis, being congruent with previous studies. Several authors have used the MIRU-VNTR typing scheme proposed by Thibault and coworkers to type MAC isolates from different countries of the world: France, Germany, Czech Republic, Finland, Greece, The Netherlands, Norway, Scotland, Spain, Japan and more recently Colombia and Argentina among others (Möbius et al., 2008; Stevenson et al., 2009; Inagaki et al., 2009; Castellanos et al., 2010; Radomski et al., 2010; Fernández-Silva et al., 2011, this work). Several studies have included new mar