Genetic diversity of Mycobacterium avium complex strains isolated in Argentina by MIRU-VNTR.

Mycobacterium avium sp. avium (MAA), M. avium sp. hominissuis (MAH), and M. avium sp. paratuberculosis (MAP) are the main members of the M. avium complex (MAC) causing diseases in several hosts. The aim of this study was to describe the genetic diversity of MAC isolated from different hosts. Twenty-six MAH and 61 MAP isolates were recovered from humans and cattle, respectively. GenoType CM® and IS1311-PCR were used to identify Mycobacterium species. The IS901-PCR was used to differentiate between MAH and MAA, while IS900-PCR was used to identify MAP. Genotyping was performed using a mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) scheme (loci: 292, X3, 25, 47, 3, 7, 10, 32) and patterns (INMV) were assigned according to the MAC-INMV database (http://mac-inmv.tours.inra.fr/). Twenty-two (22/26, 84·6%) MAH isolates were genotyped and 16 were grouped into the following, INMV 92, INMV 121, INMV 97, INMV 103, INMV 50, and INMV 40. The loci X3 and 25 showed the largest diversity (D: 0·5844), and the global discriminatory index (Hunter and Gaston discriminatory index, HGDI) was 0·9300. MAP (100%) isolates were grouped into INMV 1, INMV 2, INMV 11, INMV 8, and INMV 5. The HGDI was 0·6984 and loci 292 and 7 had the largest D (0·6980 and 0·5050). MAH presented a higher D when compared with MAP. The MIRU-VNTR was a useful tool to describe the genetic diversity of both MAH and MAP as well as to identify six new MAH patterns that were conveniently reported to the MAC-INMV database. It was also demonstrated that, in the geographical region studied, human MAC cases were produced by MAH as there was no MAA found among the human clinical samples.

The genomics of mycobacteria.

The species Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis are the causal agents, respectively, of tuberculosis and paratuberculosis in animals. Both mycobacteria, especially M. bovis, are also important to public health because they can infect humans. In recent years, this and the impact of tuberculosis and paratuberculosis on animal production have led to significant advances in knowledge about both pathogens and their host interactions. This article describes the contribution of genomics and functional genomics to studies of the evolution, virulence, epidemiology and diagnosis of both these pathogenic mycobacteria.

Les mycobactéries Mycobacterium bovis et Mycobacterium avium subsp. paratuberculosis sont les agents étiologiques de la tuberculose et de la paratuberculose, respectivement. En outre, les deux mycobactéries (mais plus particulièrement M. bovis) peuvent infecter l'être humain et jouent donc un rôle en santé publique. En raison de cette importance et des effets de la tuberculose et de la paratuberculose sur la production animale, de grands efforts ont été déployés pour faire avancer nos connaissances sur ces deux agents pathogènes et sur leurs interactions avec leurs hôtes. Les auteurs décrivent la contribution de la génomique et de la génomique fonctionnelle dans les études sur l'évolution, la virulence, l'épidémiologie et le diagnostic de ces deux mycobactéries pathogènes.

Las especies Mycobacterium bovis y Mycobacterium avium subsp. paratuberculosis son los agentes causales de la tuberculosis y la paratuberculosis en animales, respectivamente. Además, ambas micobacterias, pero fundamentalmente M. bovis, son importantes para la salud pública, ya que pueden infectar a los humanos. Debido a esto último y al impacto de la tuberculosis y la paratuberculosis en la producción animal, en los últimos años se ha producido un avance significativo en los conocimientos de ambos agentes patógenos y de la interacción con sus hospedadores. En este artículo describiremos la contribución de la genómica y la genómica funcional a los estudios de evolución, virulencia, epidemiología y diagnóstico de ambas micobacterias patógenas.

Genomic comparison of two strains of Mycobacterium avium subsp. paratuberculosis with contrasting pathogenic phenotype.

In a previous study, we evaluated the degree of virulence of Mycobacterium avium subsp. paratuberculosis (Map) strains isolated from cattle in Argentina in a murine model. This assay allowed us to differentiate between high-virulent MapARG1347 and low-virulent MapARG1543 strains. To corroborate whether the differences in virulence could be attributed to genetic differences between the strains, we performed Whole Genome Sequencing and compared the genomes and gene content between them and determined the differences related to the reference strain MapK10. We found 233 SNPs/INDELS in one or both strains relative to Map K10. The two strains share most of the variations, but we found 15 mutations present in only one of the strains. Considering NS-SNP/INDELS that produced a severe effect in the coding sequence, we focus the analysis on four predicted proteins, putatively related to virulence. Survival of MapARG1347 strain in bMDM was higher than MapARG1543 and was more resistant to acidic pH and H2O2 stresses than MapK10. The genomic differences between the two strains found in genes MAP1203 (a putative peptidoglycan hydrolase), MAP0403 (a putative serine protease) MAP1003c (a member of the PE-PPE family) and MAP4152 (a putative mycofactocin binding protein) could contribute to explain the contrasting phenotype previously observed in mice models.

New and rapid strategies for the diagnosis of bovine paratuberculosis "in situ" using latex particles.

The chemical coupling of a protoplasmatic antigen from Mycobacterium avium subsp. paratubeculosis onto core-shell carboxylated particles was investigated with the aim of producing latex-protein complexes to be used in immunoagglutination assays capable of detecting bovine paratuberculosis disease. For this purpose, sensitizations were carried out using both colored and not colored carboxylated latexes as well as the protoplasmatic antigen at pH close to its isoelectric point to favor the antigenic protein to approach the particle surface. In all cases, higher fractions of proteins were chemically-bound to carboxyl groups on the surface of the particles. The assessment of the performance of the visual immunoagglutination assays consisted of evaluating 111 sera from healthy and infected bovines with Mycobacterium avium subsp. paratuberculosis. Complexes obtained from the colored latex allowed an acceptable visual discrimination between the studied positive and negative sera. Most of the positive samples showed strong to very strong agglutination and only a few samples reacted weakly, i.e. a sensitivity of 70%. The specificity of the assay, on the other hand, was 86%. Therefore, this rapid detection technique allows an easy and inexpensive identification of animals possibly infected with paratuberculosis "in situ" in the herds.

Development and diagnostic validation of an ELISA based on an antigenic mixture for the detection of bovine tuberculosis.

Programs for the eradication of bovine tuberculosis (bTB) focus on the tuberculin skin test (TST) and slaughter of reactor cattle. However, the disease remains an animal health concern in several countries and improving the efficiency of the TST has become a critical issue. The detection of Mycobacterium bovis antibodies in serum, within weeks after the TST, may be a rapid and inexpensive way to improve bTB control. This study reports the validation of an enzyme-linked immunosorbent assay (ELISA) to detect bovine tuberculosis as an ancillary test to TST in dairy farms in Argentina. The estimated validation parameters were within the established requirements of the World Organization for Animal Health (OIE). The test demonstrated high repeatability, with coefficients of variation <25%. High test reproducibility through interlaboratory testing was also found, with an estimated Pearson coefficient of 0.9648 (95% confidence intervals 0.9315-0.9820). The ELISA detected tuberculous cattle unidentified by the TST. Of 43 animals sent to slaughterhouses that were ELISA positive 15-17 days after a negative TST, 36 were confirmed as infected with M. bovis by histopathology and IS6110 PCR. According to ROC curve analysis of results of 145 cattle from M. bovis-free herds and the 36 M. bovis-infected cattle, at a corrected optical density cut-off point of 0.3853, specificity was 95.95% and the positive predictive value at this cut-off was 83.72%. The ELISA detection test validated in this study could be readily applied in dairy farms, to complement a prior TST and improve livestock health.

Comparison between two in situ methods for Mycobacterium avium subsp. paratuberculosis detection in tissue samples from infected cattle.

Paratuberculosis or Johne's disease is a chronic infectious disorder caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease produces diarrhea and weight loss in cattle and other animal species, and it is characterized by granulomatous enteritis and lymphadenitis. Histopathology and in situ techniques can be used as a diagnostic test, but the performance of these methods was not previously compared. The aim of this paper was to evaluate the ability of immunohistochemistry and in situ hybridization to detect Map in formalin-fixed tissue samples from infected cattle. Samples (ileum or ileocecal lymph node) from four animals that had positive Map culturing, lesions and detectable acid fast bacilli, as well as from two control animals, were tested by immunohistochemistry and in situ hybridization. Immunostaining and positive hybridization were observed in areas with lesions from infected animal samples, inside the cytoplasm of macrophages, epithelioid and giant cells. Immunostaining was intense in three samples and weak in one, while hybridization was weak in all cases. In situ hybridization was positive in negative areas of tissues analyzed by immunohistochemistry, which could be related to spheroplast detection as it was previously described for this method. Control samples resulted negative by these two methods. Both techniques were able to detect Map in formalin fixed and paraffin embedded tissues, however immunohistochemistry produced higher intensity staining and was easier to perform. Therefore, we believe that immunohistochemistry and in situ hybridization to be useful for the post-mortem diagnosis and research of Paratuberculosis.

[Identification of immunoreactive antigens of Leptospira interrogans]. / Identificación de antígenos inmunorreactivos de Leptospira interrogans.

A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutination (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.

Mutation in the P36 gene of Mycobacterium bovis provokes attenuation of the bacillus in a mouse model.

P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.

Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America.

All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.

Prolactin release, oestrogens and proliferation of prolactin-secreting cells in the anterior pituitary gland of adult male rats.

Relationships among the release of prolactin, the effect of oestrogens and the proliferation of prolactin-secreting cells were studied under several experimental conditions. Administration of sulpiride or oestradiol released prolactin and stimulated cell proliferation in the anterior pituitary gland of adult male rats. Clomiphene completely abolished the rise in cell proliferation, but did not interfere with the sulpiride-induced release of prolactin. Treatment with oestradiol plus sulpiride significantly increased serum prolactin concentrations and the mitotic index compared with the sum of the stimulation produced by both drugs separately. Bromocriptine abolished the stimulatory effect of oestradiol on the serum prolactin concentration and on cell proliferation. In oestradiol- and/or sulpiride-treated rats, 80% of the cells in mitoses were lactotrophs. The remaining 20% did not stain with antisera against any of the pituitary hormones. The number of prolactin-secreting cells in the anterior pituitary gland significantly increased after the administration of oestradiol or sulpiride. The results demonstrate that treatment with sulpiride and/or oestradiol increases the proliferation and the number of lactotrophs in the anterior pituitary gland of the rat.

Effect of ovariectomy and clomiphene on the in-vitro incorporation of [3H]thymidine into pituitary DNA and on prolactin synthesis and release in rats.

In the anterior pituitary gland changes in prolactin synthesis and in the incorporation of [3H]thymidine into DNA are coincident under several experimental conditions. We investigated whether these changes are obligatory, thus indicating a regulatory mechanism common to the synthesis of both macromolecules. Alternatively, the parallel changes may represent similar responses to various stimuli operating through different pathways. The administration of alpha-methyl-p-tyrosine (alpha MpT) to rats stimulated the incorporation of [3H]leucine into prolactin and [3H]thymidine into DNA. When the effectiveness of oestrogen was suppressed by ovariectomy or by blockage of oestrogen receptors by the antioestrogen clomiphene, alpha MpT stimulated the synthesis of prolactin but not the incorporation of [3H]thymidine into pituitary DNA. The results clearly indicate two independent mechanisms regulating the synthesis of prolactin and DNA in the anterior pituitary gland.

Correlation between LH secretion in castrated rats with cellular proliferation and synthesis of DNA in the anterior pituitary gland.

The relationship between the release of LH and the synthesis of DNA was studied in the anterior pituitary gland of castrated rats. Cell types were characterized immunocytochemically. Castration significantly (P less than 0.01) increased the concentration of LH in serum (1326%) and the incorporation of [3H]thymidine into pituitary DNA (72%). This was accompanied by an increment in the activity of the enzyme DNA polymerase-alpha (58%) and in the number of mitoses (from 2 +/- 0.1/mm2 in intact rats to 21 +/- 0.8/mm2 15 days after castration). Only 20% of the mitoses found in the pituitary gland of castrated rats were positively stained with the antiserum against the beta-subunit of LH. The other 80% did not stain either with LH antiserum or with antisera against the other pituitary hormones. There was a significant (P less than 0.01) increase in the number of LH cells in castrated rats (48%). All the changes produced in the anterior pituitary gland after castration were prevented by the administration of dihydrotestosterone. The results demonstrate that a stimulation of LH release is followed by an increase of DNA synthesis and cell proliferation of gonadotrophs in the anterior pituitary gland.

A western blot characterization of Mycobacterium bovis antigens recognized by cattle sera.

The immune response to Mycobacterium bovis in cattle was assessed by Western blot. The antibody recognition pattern to M. bovis whole cell extracts and culture supernatant antigens was studied by using sera from M. bovis-infected (n = 62) and healthy (n = 38) cattle. Although the recognition patterns were highly variable, some proteins were regularly detected, mainly those with molecular masses of 17, 23, 28, 42, 66, 71 and 80 kDa in cellular extracts, and with molecular masses of 23 and 33 kDa in supernatants. Whole cell extract antigens were more frequently recognized than culture supernatant antigens. Healthy controls produced only a weak antibody response. The antibody response was variable, depending on tuberculosis stage. In early stages very few antibodies were detected. A response against the 66-kDa stress protein was mounted in intermediate tuberculosis and remained stable in more advanced disease. In late diseases, the preferentially recognized antigens were a 28-kDa cellular protein and supernatant antigens. The 28-kDa protein was studied in some detail. As determined by using monoclonal antibodies, the 28-kDa protein is different from superoxide dismutase. This protein aggregated in stored cell extracts and was not totally transferred to nitrocellulose. The principal conclusions of this work are: (i) whole cell extract proteins are more frequently recognized than the secreted proteins and (ii) a 28-kDa protein is a major antigen in late disease.

Cloning of a novel polymorphic GC-rich repetitive DNA from Mycobacterium bovis.

A repetitive DNA from a wild-type Mycobacterium bovis isolate was cloned and characterized. The repeated segment was present in M. bovis and M. tuberculosis but was absent from the six other mycobacteria tested. Sequence analysis demonstrated that this repetitive element belonged to the polymorphic GC-rich repeat sequence type, a family of interspersed repeated DNA. This fragment, when used as a probe in restriction fragment length polymorphism analyses, was able to detect polymorphism in M. bovis genotypes that went undetected when the established IS6110 was used as a probe. This repetitive element should be useful in epidemiological studies of bovine tuberculosis.

An mpb-64 flanking sequence specific for Mycobacterium bovis.

A clone carrying a plasmid with the mpb-64 gene and 3' flanking sequences (plasmid pMBA122) was detected during the screening of a Mycobacterium bovis genomic library with sera from infected cattle. When the pMBA122 insert was used as a probe in Southern blots against PvuII-digested mycobacterial DNA, it distinguished the different M. tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis, a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains from wild seals. Smal genomic digestions enabled us to locate this polymorphic region downstream of the mpb-64 gene. In order to clone this particular region, we designed a pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; no amplification was seen in M. tuberculosis DNA. When the annealing temperature was lowered from 70 to 55 degrees C, an amplification product of the same size was obtained with M. tuberculosis. This product was cloned and sequenced, and showed partial homology to the M. bovis amplified fragment. Therefore, this region comprises M. bovis sequences with a lower homology with M. tuberculosis than other compared sequences. This suggests that a more precise differentiation method at the species level for the M. tuberculosis complex could be achieved using PCR directed to this region.

Evaluation of the polymerase chain reaction for the detection of Mycobacterium tuberculosis.

The development of nucleic acid-based technologies has improved the sensitivity, specificity and speed of detection of Mycobacterium tuberculosis in clinical samples. Both commercially available and 'in-house' polymerase chain reaction (PCR) systems are in use, and a significant number of reports compare such systems with more traditional diagnostic tools for tuberculosis. Few studies, however, have focused on the reproducibility of the results when submitting a sample batch to PCR in different laboratories, especially in developing countries. Consequently, PCR results obtained from six laboratories in six different Latin American countries for samples reconstituted with defined amounts of M. tuberculosis cells were evaluated. Each laboratory used specific conditions of sample processing, nucleic acid amplification and amplicon detection. Analysis of results allowed large differences in sensitivity and specificity to be observed. We conclude that in its present setting, in-house PCR cannot be used as a single diagnostic tool for tuberculosis, and that special care needs to be taken upon interpretation of results by inclusion of a proper number of positive and negative controls.

Antigenic characterization of mycobacteria from South American wild seals.

Tuberculosis-producing mycobacteria have been previously described in marine mammals (Cousins et al., 1990, 1993; Romano et al., 1995; Bernardelli et al., 1996). The strains belonged to the M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti and M. africanum), but showed genetic and biochemical differences. The antigenic composition of mycobacteria isolated from wild seals was analyzed by Western blots, using antibodies against some selected antigens. The antigenic content was compared with that of M. bovis, M. tuberculosis and M. microti isolates. The lack of Hsp65 protein in supernatants suggested a low degree of cell lysis in the three-week cultures used. SOD, P27 lipoprotein, MPB64 and antigen 85 were observed in all the strains studied. The wild seal strains, as well as M. tuberculosis, did not produce MPB70 and MPB83. Only very weak bands of P36 antigen were observed in culture supernatants from wild seal mycobacteria. Summarizing, the antigenic composition of mycobacterial strains from wild seals is different from M. bovis strains.

Genetic characterization of mycobacteria from South American wild seals.

Tuberculosis has been recently diagnosed in four wild seals found stranded in the Atlantic coast of Argentina. By bacteriological studies and IS6110 hybridization, these isolates were characterized as belonging to the Mycobacterium tuberculosis complex. A genetic characterization using RFLP (Restriction fragment length polymorphism) and a species-specific probe of M. tuberculosis, called mtp40, showed hybridization with this probe on a single band. A similar band was also found in M. tuberculosis H37Rv. This showed a relationship between M. tuberculosis and the wild seal isolates. However these would also seem to belong to a different genetic group in the M. tuberculosis complex, since they do not grow on glycerol-egg containing medium (Lowenstein-Jensen) as typical M. tuberculosis strains usually do. Repeated sequences pMBA2, pTNB12, DR and IS6110 were used as probes to evaluate the epidemiological relationships between the 4 cases of tuberculosis. A low degree of polymorphism was observed, that suggested that these isolates were epidemiologically related.

Comparison of different genetic markers for molecular epidemiology of bovine tuberculosis.

In the present study three different genetic markers were used in an RFLP study to differentiate Mycobacterium bovis (M. bovis) isolated in Argentina. The markers were: the insertion sequence IS6110, the direct repeat (DR) sequence flanking IS6110, and a polymorphic GC-rich repetitive sequence (PGRS), called pMBA2. Two restriction enzymes were used, PvuII for IS6110 and DR (DR/PvuII) and AluI for DR (DR/AluI) and pMBA2. DNA from 85 of M. bovis isolates was digested with PvuII and hybridized with IS6110 and Dr. IS6110 was not useful to differentiate M. bovis because most of the isolates contain a single monomorphic copy. The use of DR allowed a limited degree of differentiation. DNA from 44 of these isolates was also digested with AluI and hybridized with DR and pMBA2. In this condition these probes differentiated the isolates in many different RFLP types. By combining the patterns generated with DR/AluI and pMBA2 it was possible to increase the differentiation up to obtain 30 different RFLP types and 54% of the isolates were differentiated because they showed a unique pattern. Six isolates of M. bovis involved in two different outbreaks of bovine tuberculosis were correctly identified. Thus, DR and pMBA2 could be, at the moment, the probes of choice for comparisons of M. bovis isolates in different regions and for epidemiological surveillance of bovine tuberculosis.

Molecular epidemiology of Mycobacterium bovis isolates from South America.

One hundred seventy-eight isolates of Mycobacterium bovis were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the direct repeat element (DR) and the polymorphic GC-rich repeat sequence (PGRS) as probes. By combining the patterns generated by the two repeat DNA elements, 93 different patterns were observed. One hundred-one isolates were grouped in clusters, which include 25 different clusters. One pattern was the most frequently observed, clustering 18.5% of isolates. It was only found in the Center and northeast regions of Argentina and in one isolate from Paraguay. The isolates from Brazil analyzed here presented exclusive patterns (only found in a particular region). The number of exclusive patterns was high in all argentine regions: northeast 78%, center 81%, and Buenos Aires 81%.