Instituto de Biotecnología, CICV/INTA, Buenos Aires, Argentina1
Departamento de Micobacterias, DILAB SENASA, C. C. 77 Morón (1708), Buenos Aires, Argentina2
Fundación Mundo Marino, San Clemente del Tuyú, Argentina3
Laboratorio de Tuberculosis de la Dirección de Laboratorios Veterinarios (DILAVE) Miguel C. Rubino, Montevideo, Uruguay4
Departamento de Microbiología, Salud Pública y Medicina Preventiva, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, Spain5
Author for correspondence: M. I. Romano. Tel: +54 1 621 1447. Fax: +54 1 481 2975. e-mail: mromano{at}cicv.inta.gov.ar
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ABSTRACT |
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Keywords: tuberculosis, Mycobacterium tuberculosis complex, spoligotyping, RFLP
Abbreviations: DR, direct repeat; PZA, pyrazinamide; Pzase, pyrazinamidase; TB, tuberculosis
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INTRODUCTION |
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A new technique called spoligotyping was designed as a tool for molecular epidemiology of the M. tuberculosis complex (Kamerbeek et al., 1997 ). The method is based on the in vitro amplification of the DNA sequence of the highly polymorphic direct repeat (DR) locus in the chromosome of members of the M. tuberculosis complex. This region flanks an IS6110 copy, and has a characteristic organization, with conserved 36 bp DR sequences interspersed with variable spacers (Hermans et al., 1991
). The polymorphism is due to these spacers, which are variable in length, sequence and number. The spoligotyping technique detects the spacers present in the strains, and allows each to be characterized by its spacer content (Kamerbeek et al., 1997
). Spoligotyping has been used to type isolates of M. tuberculosis (Goguet de la Salmonière et al., 1997
) and M. bovis (Aranaz et al., 1996
; Blázquez et al., 1997
; Cousins et al., 1998
; Zumárraga et al., 1999
).
Currently, strains of M. bovis and M. tuberculosis are distinguished by several biochemical parameters, including niacin accumulation, pyrazinamide (PZA) susceptibility, pyrazinamidase (Pzase) activity, nitrate reduction and thiophenecarboxylic acid hydrazide susceptibility (Collins et al., 1985 ). An immunological method for differentiating between M. bovis and M. tuberculosis based on detection of the protein antigen MPB70 in M. bovis has been reported (Harboe & Nagai, 1984
), but its use and reliability are limited because this protein is also present in M. tuberculosis. This protein is highly expressed in M. bovis and minimally expressed in M. tuberculosis (Harboe & Nagai, 1984
). The mtp40 gene, considered to be present in M. tuberculosis but absent in M. bovis, was used to differentiate M. tuberculosis from M. bovis (Del Portillo et al., 1991
). However, this method has recently been invalidated, as the gene has been shown not to be present in all M. tuberculosis strains, and not absent in all M. bovis strains (Weil et al., 1996
). In many mycobacteria, 16S rRNA and the heat-shock protein gene (hsp65) contain variable sequences that allow mycobacterial differentiation at the species level. However, within the M. tuberculosis complex, these genes are not variable. (Kirschner et al., 1993
; Telenti et al., 1993
). M. tuberculosis complex organisms have multiple mutations in the oxyR gene, a homologue of the extensively studied central regulator of peroxide stress response in enteric bacteria (Deretic et al., 1997
). Because oxyR in M. tuberculosis complex isolates probably does not encode a functional protein, it is referred to as a pseudogene. In M. bovis the oxyR pseudogene has an adenine residue at nucleotide 285, while the remaining M. tuberculosis complex strains have a guanine residue (Sreevatsan et al., 1996
). In addition, the Pzase gene (pncA) has a guanine instead of a cytosine residue at nucleotide position 169 in M. bovis. This results in production of an inactive Pzase, thus conferring resistance to PZA in these strains (Scorpio & Zhang, 1996
). Therefore, polymorphisms in the oxyR and pncA genes allow differentiation of M. bovis from the other mycobacteria of the complex.
M. tuberculosis can be assigned to one of three distinct genotypic groups, based on the combinations of polymorphisms at the genes encoding catalase-peroxidase (katG) and the A subunit of gyrase (gyrA). M. tuberculosis group 1 has the allele combination katG codon 463 CTG (Leu) and gyrA codon 95 ACC (Thr); group 2 has katG codon 463 CGG (Arg) and gyrA codon 95 ACC (Thr); group 3 has katG codon 463 CGG (Arg) and gyrA codon 95 AGC (Ser). All isolates of M. bovis, M. microti and M. africanum studied have the combination of polymorphisms of M. tuberculosis group 1. The isolates of M. tuberculosis grouped in clusters were mainly of genotypic groups 1 and 2 (Sreevatsan et al., 1997 ).
The aim of this study was to evaluate the epidemiological relationship and genetic characteristics of the mycobacteria that cause TB in seals in South America.
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METHODS |
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The mycobacteria isolated from seals were susceptible to PZA, isoniazid, streptomycin, rifampicin, ethambutol and thiophene-2-carboxylic acid hydrazide, and relatively resistant to p-aminosalicylic acid. A few strains were positive for niacin production. Biochemical, drug-susceptibility and biological tests were performed by standard procedures (Wayne & Kubica, 1986 ).
RFLP and spoligotyping techniques.
The RFLP technique using IS6110 and mtp40 as genetic markers was described previously (Romano et al., 1995 ). Spoligotyping was performed according to Kamerbeek et al. (1997
). Clustering analysis using UPGMA and the Dice coefficient of spoligotypes was performed with the aid of the computer program GelCompar, version 2.1 (Applied Maths, Kortrijk, Belgium). The spoligotypes found in the mycobacteria isolated from seals were compared with the spoligotypes found in over 609 M. bovis isolates that originated from different sources (human, cattle, cat, goat, llama, buffalo and deer) and from different countries in Latin America and Europe.
Production of MPB70 antigen.
To prepare mycobacterial culture supernatant and sonic extracts, cultures were centrifuged for 30 min at 10000 g. For cell extracts, bacteria were resuspended in distilled water and sonicated for 10 cycles of 30 s followed by a 30 s interval. The culture supernatant was precipitated with trichloroacetic acid (10%) and resuspended in loading buffer (2% SDS, 0·125 M Tris/HCl pH 6·8, 1% 2-mercaptoethanol, 0·02% bromophenol blue, 10% glycerol).
Proteins (50 µg) were analysed by electrophoresis in 12% polyacrylamide-SDS gels and electrotransferred onto a nitrocellulose sheet by the semidry method. Transfer yield was visualized by transient staining with Ponceau Rouge. The membranes were incubated with monoclonal antibody 4C3/17 against MPB70 (CSL Laboratories, Victoria, Australia) overnight at 4 °C and with an alkaline-phosphatase-conjugated goat-antimouse antiserum (Sigma) for 2 h at 37 °C.
PCR and DNA sequencing
pncA.
The pncA gene was amplified by PCR, using primers PNCA-1 and PNCA-2. To determine the sequence of pncA (GenBank accession no. U59967) from various strains of mycobacteria isolated from seals, primer PNCA-1 (5'-ATGCGGGCGTTGATCATCGTC-3'), corresponding to bp 121 of the M. tuberculosis pncA gene, and primer PNCA-2 (5'-TCAGGAGCTGCAAACCAA-3'), corresponding to bp 561544 of the same gene (Scorpio & Zhang, 1996 ) were used. PCR was performed using PCR buffer composed of 10 mM Tris/HCl, pH 8·8; 50 mM KCl; 1·5 mM MgCl2; 0·125 µM of the above-mentioned primers; 0·125 mM dNTPs; 1·25 units Taq DNA polymerase (Advanced Biotechnologies); and 10100 ng DNA. PCR amplification was performed in a Perkin-Elmer Cetus DNA thermal cycler, set for 3 min at 94 °C, followed by 40 cycles at 94 °C for 1 min, 62 °C for 1 min, and 72 °C for 1 min. The pncA sequences from different mycobacterial strains were determined by PCR direct sequencing, using a Thermo Sequenase labelled primer cycle sequencing kit (Amersham) with fluorescent 5' Cy 5-labelled PNCA-1 and PNCA-2 primers, in an automatic DNA sequencer (ALFexpress, Pharmacia).
katG.
Detection of point mutations in the catalase-peroxidase gene (katG) (GenBank accession no. X68081) was performed as described by Uhl et al. (1996 ).
gyrA.
To determine the mutations of the quinolone-resistance-determining region of the gene gyrA (GenBank accession no. L27512) from M. tuberculosis strains, a region of 320 bp was amplified and sequenced. The forward primer GyrA1 (5'-CAGCTACATCGACTATGCGA-3'), corresponding to bp 23832402 of gyrA, and the reverse primer GyrA2 (5'-GGGCTTCGGTGTACCTCAT-3'), corresponding to bp 26842702 of this gene, were used. PCR and sequencing was performed as described above for pncA except that an annealing at 60 °C for 1 min was used in the PCR, and GyrA1 and GyrA2 were used as sequencing primers.
16S rRNA.
A 1030 bp fragment of 16S rRNA from M. tuberculosis (GenBank accession no. X52917) was amplified using 50 pmol each of primers 285 (5'-GAGAGTTTGATCCTGGCTCAG-3'), corresponding to bp 930 of the E. coli 16S rRNA, and 264 (5'-TGCACACAGGCCACAAGGGA-3'), corresponding to bp 10271046 of the E. coli 16S rRNA (GenBank accession no. U59967) per 50 µl of reaction, as described by Kirschner et al.. (1993 ). The cycling parametres were 95 °C for 5 min, followed by 40 cycles of 95 °C for 1 min, 68 °C for 1 min and 72 °C for 1 min. The reaction was performed in a Perkin-Elmer Cetus DNA thermal cycler.
The 16S rRNA sequence was determined by PCR direct sequencing using a Thermo Sequenase labelled primer cycle sequencing kit (Amersham), with the fluorescent 5' Cy 5-labelled 244 (5'-CCCACTGCTGCCTCCCGTAG-3') primer corresponding to bp 341361 of the E. coli 16S rRNA, in an automatic DNA sequencer (ALFexpress, Pharmacia).
PCR-restriction analysis
hsp65.
Specific amplification of a fragment of 439 bp of hsp65 (GenBank accession no. M15467) from each strain, and digestion by BstEII and HaeIII of the amplified fragments, were performed according to the procedure described by Telenti et al. (1993 ). Following digestion, 10 µl of the mixture was loaded onto a 4% agarose gel. The 10 bp DNA ladder (Gibco-BRL) was used as the molecular marker.
oxyR.
A 548 bp segment of the oxyR pseudogene of the M. tuberculosis complex (GenBank accession no. U16243) was amplified with the forward primer 5'-GGTGATATATCACACCATA-3' and the reverse primer 5'-CTATGCGATCAGGCGTACTTG-3', and restriction fragment length polymorphism of the amplified product with restriction endonuclease AluI was analysed as described by Sreevatsan et al. (1996 ), to differentiate M. bovis from other complex members. PCR was performed as described above for pncA except that the cycling conditions used were 30 cycles at 96 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min.
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RESULTS |
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Experimental inoculation of guinea pigs with the mycobacteria isolated from seals produced significant and generalized lesions, and the intradermal tuberculin test with M. bovis purified protein derivative (PPD: 50 IU), of the experimentally inoculated guinea pigs gave a strong positive reaction. There was an increase of 6 mm or more in skinfold thickness, and these reactions were 616 times greater than with M. avium PPD.
Genetic characterization
Mycobacterium species can be differentiated by amplification of a fragment of the hsp65 gene followed by restriction enzyme analysis (Telenti et al., 1993 ), and by sequencing of a region of the 16S rRNA (Kirschner et al., 1993
), because these genes exhibit some internal polymorphism within these regions. Mycobacteria from different seal isolates had DNA fragment sizes generated after amplification of hsp65 followed by digestion with BstEII and HaeIII identical to the organisms belonging to the M. tuberculosis complex (data not shown). The sequence of the amplified 16S rRNA gene of the different mycobacteria isolated from seals was identical to the sequence of the members of the M. tuberculosis complex (data not shown). According to their 16S rRNA gene sequence and the PCR-restriction characteristics of the hsp65 gene, the seal isolates were identified as belonging to the M. tuberculosis complex (Table 2
). The IS6110 element and DR region, both sequences characteristic of the M. tuberculosis complex, were found in the DNA of the mycobacteria isolated from seals (Fig. 1
and 2
, Tables 1
and 2
). These mycobacteria had spoligotypes different from those in M. tuberculosis, M. africanum, M. microti and M. bovis AN5 (Fig. 2
). All the seal isolates lacked the spacers 3943. The absence of these spacers is characteristic of M. bovis isolates. Clustering analysis using UPGMA and the Dice coefficient of spoligotypes was performed to compare the spoligotypes found in the mycobacteria isolated from seals with the spoligotypes of over 609 M. bovis isolates from different countries in Latin America and Europe, and with the spoligotypes of BCG and M. bovis AN5. The analysis of these spoligotypes showed that the spoligotypes of the mycobacteria isolated from seals were exclusive to these isolates (Fig. 3
).
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The MPB70 antigen, which is always detected in M. bovis, was not detected in the mycobacteria from seals (Table 2).
Epidemiological implications
Spoligotyping and IS6110-RFLP techniques were used to study the epidemiology of the TB in seals. Six IS6110-RFLP types were found among the 10 seal isolates (Table 1). These isolates contained three to seven copies of IS6110 (Fig. 1
). The IS6110-RFLP types were arbitrarily designated as AF. IS6110-RFLP type A contained three copies of this element (Fig. 1
), and was found in mycobacteria isolated from two seals (Table 1
). One of the animals belonged to the species Arctocephalus australis and was found stranded on the Argentine coast in 1992 (Table 1
, case 4); the second mycobacterial isolate was obtained in 1996 from an Arctocephalus tropicalis individual (Table 1
, case 8). IS6110-RFLP type B contained five copies of IS6110 (Fig. 1
), and was found in mycobacteria isolated from four seals (Table 1
): one Otaria flavescens individual, involved in an outbreak of TB in 1987 at the Montevideo Zoo, Uruguay (Table 1
, case 1), and three wild seals of the species A. australis found on the Argentine coast, in 1991, 1992 and 1996 (Table 1
, cases 2, 3 and 9, respectively). The remaining four RFLP types (C, D, E and F) were found in mycobacteria isolated from four different animals (Table 1
, cases 5, 6, 7 and 10, respectively).
Although there were six different IS6110 fingerprint patterns, they shared many of their IS6110-containing restriction fragments (Fig. 1). Mycobacteria isolated from nine animals had an identical spoligotype. Only one isolate showed a minor difference: it had two missing spacers in its spoligotype (Fig. 2
; Table 1
, case 10). This individual corresponded to the last isolate (September, 1996), which also showed a unique IS6110 RFLP pattern (F) (Fig. 1
; Table 1
, case 10).
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DISCUSSION |
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In the present study, the spoligotypes of the mycobacteria isolated from seals were identical; only the most recent isolate showed a minor difference in its spoligotype. However, the isolates with the same spoligotype could be differentiated by RFLP analysis with the IS6110 probe. Thus the IS6110 element was more useful than spoligotyping to assess the epidemiological relationship between these mycobacteria. In a previous study, mycobacteria isolated from seals were also typed by RFLP analysis with DR and polymorphic GC-rich repetitive sequence (PGRS) probes. All strains showed the same DR and PGRS patterns, except for one strain which showed a minor difference with the DR probe (Romano et al., 1995 ), suggesting an epidemiological relationship between these isolates.
The similarities in IS6110-associated RFLPs among mycobacteria isolated from seals suggest that they may have recently diverged from a common ancestor. It is not possible to calculate exactly the time elapsed since the divergence from this putative common ancestor. Another group of strains, which share the majority of their IS6110-containing restriction fragments, are those M. tuberculosis strains which have been circulating in the Republic of China (Beijing family); in this case the authors proposed that they have evolved from recent clonal expansion that began less than a century ago (van Soolingen et al., 1995 ).
The cases of TB reported in this study were in animals that came from the rookeries situated on the Argentine and Uruguayan coasts. Most of the individuals found stranded on the Argentine coast were physiologically depressed, with a higher percentage of diverse illnesses than is generally seen in animals from natural colonies. This situation could contribute to the active transmission of the TB infection and the illness may be endemic among these animals. The results of van Soolingen et al. (1991 ) indicated that IS6110 patterns from M. tuberculosis strains from regions where TB is endemic are more related to each other than isolates from countries where the transmission rate is slow. Thus, similar IS6110 types between mycobacterial isolates from seals are a sign of an active transmission of TB between these animals.
IS6110-RFLP type B was found in a mycobacterial isolate, which caused tuberculosis in a colony of captive seals of the species O. flavescens, at Montevideo Zoo (Uruguay) (Castro Ramos et al., 1998 ). These captive seals were collected from wild colonies in Uruguay and at least one of them may have been ill at the time of the capture. This same RFLP type was found in mycobacterial isolates from TB cases in wild seals of the species A. australis, found stranded on the Argentine coast. This is probably due to the fact that seals of these two species inhabit the same islands in Uruguay. IS6110-RFLP type A was found in wild seals of the species A. australis and A. tropicalis, stranded on the Argentine coast. The presence of the latter species is incidental on this coast, because it comes from subantarctic islands near South Africa. These wandering specimens are swept north by the Benguela current, reach the equatorial current in the direction of South America, and then arrive at the Argentine coast pushed by the Brazil current (Rodríguez et al., 1995
).
Cases of TB in other species of marine mammals have also been described in Australia and New Zealand (Neophoca cinerea, Arctocephalus forsteri and A. pusillus doriferus) (Cousins et al., 1990 , 1993
; Forshaw & Phelps, 1991
; Thompson et al., 1993
), where these mycobacteria infected a seal trainer (Cousins et al., 1993
; Thompson et al., 1993
). The restriction endonuclease analysis and RFLP patterns of these seal isolates were different from those of other members of the M. tuberculosis complex, and the protein MPB70, present in M. bovis, was not detected in these seal isolates (Cousins et al., 1993
). Comparisons between Australian and Argentinian isolates would be necessary to determine whether there is a single or multiple origin of this animal disease.
The importance of the results presented here is that they indicate that there are new mycobacteria in the M. tuberculosis complex, which cause TB in marine mammals and appear to be specific to these animals, and which are actively transmitted within the animal colonies. It will be necessary to study the prevalence of this disease in the seal colonies of Uruguay and Argentina, in addition to controlling the human population that has contact with these animals.
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ACKNOWLEDGEMENTS |
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We are grateful to Haydee Gil for her excellent technical assistance, and to Mariana del Vas and Laura Boschiroli for their critical observations.
C.M., A.C. and M.I.R. are fellows of the RELACTB (Red Latinoamericana del Caribe de Tuberculosis). A.C. and M.I.R. are fellows of the National Research Council of Argentina (CONICET).
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Received 13 January 1999;
revised 10 May 1999;
accepted 12 May 1999.