Sabrina Rodríguez

High spoligotype diversity within a Mycobacterium bovis population: Clues to understanding the demography of the pathogen in Europe

Mycobacterium bovis is the main causative agent of bovine tuberculosis. This zoonotic disease produces important economic losses and must be considered a threat to endangered animal species and public health. This study was performed (1) to assess the degree of diversity of the Spanish M. bovis isolates and its effect on the epidemiology of the infection, and (2) to understand the connection of M. bovis populations within a European context. In this report we resume the DVR-spoligotyping results of 6215 M. bovis isolates collected between 1992 and 2007 from different hosts. The isolates clustered into 252 spoligotypes which varied largely in frequency, geographical distribution and appearance in different animal species. In general, the most frequent spoligotypes were found all over the country and in different animal species, though some were restricted to a geographical area. Among our most often isolated spoligotypes, SB0121 and SB0120 (BCG-like) are a common feature between mainland European countries, however, the spoligotypes differ with those found in the UK, the Republic of Ireland and abroad. A comparison of spoligotypes reported from other countries reveals hints for the M. bovis demography in Europe and suggests a common ancestor strain. This study gives insight into the usefulness of the standardized DVR-spoligotyping technique for epidemiological studies in a country with a high degree of strain diversity.

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Mycobacterium caprae Infection in Livestock and Wildlife, Spain

Mycobacterium caprae is a pathogen that can infect animals and humans. To better understand the epidemiology of M. caprae, we spoligotyped 791 animal isolates. Results suggest infection is widespread in Spain, affecting 6 domestic and wild animal species. The epidemiology is driven by infections in caprids, although the organism has emerged in cattle.

African 2, a Clonal Complex of Mycobacterium bovis Epidemiologically Important in East Africa

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

Humans as source of Mycobacterium tuberculosis infection in cattle, Spain.

To the Editor: Mycobacterium tuberculosis is the main causative agent of tuberculosis in humans. However, little attention has been paid to its transmission from humans to animals. We report M. tuberculosis infections in 3 cattle farms in Spain. The epidemiologic investigation traced humans as the source of infection, with 1 of the strains showing multidrug resistance. Recent studies have reported isolation of M. tuberculosis in cattle with prevalences of 4.7%–30.8% in African and Asian countries (1–3). In cattle, this infection occurs in countries with the highest incidence of human tuberculosis in the world. In Europe, only 14 cases of M. tuberculosis infection have been described in 3 eastern countries since implementation of eradication programs (4,5). The only reported cases of M. tuberculosis in cattle in western Europe were described in Great Britain and date back to the 1950s (6). During 2007–2009, three cases of tuberculosis caused by M. tuberculosis were detected in 3 unrelated cattle farms, 2 of them free of tuberculosis (farms 1 and 2). As part of the surveillance system of bovine tuberculosis, a pool of tissue samples from each cow (respiratory lymph nodes and lung) were homogenized with sterile distilled water, and culture was carried out by the BACTEC mycobacteria growth indicator tube 960 system (Beckton Dickinson, Madrid, Spain). Members of the M. tuberculosis complex were identified and genotyped by direct variable repeat spacer olignucleotide typing and mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) typing (7). The 3 M. tuberculosis–infected animals were <9 months of age (Table). As described (6), the possibility of infection in young animals could be more probable than infection in older cows. Table Relevant information about Mycobacterium tuberculosis infection in 3 cattle farms in Spain* M. tuberculosis–infected animals from farms 1 and 3 were detected by the intradermal tuberculin test (Table). The animal without immunologic response (farm 2) was detected because an M. bovis infection was confirmed in the herd, and all animals were slaughtered. Confirmation of infection by culture without immunologic response is rare, although the high sensitivity of the mycobacteria growth indicator tube system could detect a low bacterial load in the initial stages of infection. Recent implementation of liquid systems in animal health laboratories has enabled detection of M. tuberculosis when it is compared with results using only conventional methods. Moreover, no tuberculosis-compatible lesions were observed in the 3 animals, similar to previous studies (6). On the basis of these facts, M. tuberculosis transmission was not detected among cattle in the following intradermal tuberculin tests. Co-infection with other mycobacteria (M. avium subsp. hominissuis) was found in the same animal from farm 1 (Table). This co-infection suggested the immunocompromised status of the animal and hence a high susceptibility to M. tuberculosis infection. Moreover, M. bovis was isolated from 52% (16/31) of all animals from farm 2 that showed a positive reaction to the intradermal tuberculin skin test, making remarkable the absence of co-infection with M. bovis in the M. tuberculosis–infected animal. Therefore, the lack of M. tuberculosis transmission within this herd contrasts with the M. bovis dissemination. The veterinary services reported these findings to the National Public Health System, and an epidemiologic investigation was conducted on the cattle farms to determine the source of infection. In all cases, staff of the farms had active tuberculosis (Table). Three different strains were characterized: SIT2537 (octal code 777617777720771), 253533233433236252211423 (farm 1); SIT1564, 3′52334232455457251213423 (farm 2); and SIT58, 254343243232325262213423 (farm 3) (Table). The MIRU-VNTR pattern and spoligotype are shared by Spanish human and cattle isolates from farm 1; SIT2537 is an uncommon profile that has been detected in Brazil and Spain (according to the SITVIT2 database). The human strain showed multidrug resistance to isoniazid, rifampin, and ethionamide. In cattle and human isolates, genes associated with isoniazid and rifampin resistance were studied (8) and rpoB analysis confirmed rifampin resistance (Ser531Leu). In farm 2, the origin of the farm worker was eastern Europe and the cattle isolate showed an SIT1564 profile, which is found only in 6 human isolates in the SpolDB4 database, all from Poland, Bulgaria, and Russia. On farm 3, human and cattle isolates from Spain shared identical spoligotype and MIRU-VNTR patterns. The profile SIT58 is frequent in Spain (9) and other countries with historical links to Spain, mainly the south American countries (79/114 according to SpolDB4). A well-designed program for eradicating bovine tuberculosis helps to detect M. tuberculosis infection by immune response or by bacteriologic culture. The use of liquid systems and results of epidemiologic studies (Spanish Database of Animal Mycobacteriosis, mycoDB.es) (S. Rodriguez, unpub. data) are recommended for prompt confirmation of the M. tuberculosis complex infection and for enhancing the sensitivity of culture. In addition, the Spanish Ministry of Environment, Rural and Marine Affairs has reinforced the need to improve cooperation between human and animal health systems to minimize the risk for M. tuberculosis complex transmission from animals to humans or vice versa and to control infection in all susceptible animal species (10).

Molecular characterization of Mycobacterium avium subspecies paratuberculosis Types II and III isolates by a combination of MIRU-VNTR loci

Mycobacterial interspersed repetitive units and variable number tandem repeats typing (MIRU-VNTR) is a useful technique that has been recently applied to characterize members of the Mycobacterium avium complex (MAC). The aim of this study was to examine the genetic variability among a collection of Spanish M. avium subspecies paratuberculosis (M. a. paratuberculosis) isolates with a combination of MIRU-VNTR loci. For this purpose we tested six MIRU-VNTR loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32, VNTR-292 and VNTR-259) in 70 M. a. paratuberculosis isolates of Types II and III that were recovered from 22 Spanish localities during a nine-year period (1998-2007). The combination of five loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32 and VNTR-259) enabled the differentiation of 12 allelic profiles, with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.84. Moreover, we obtained MIRU-VNTR patterns that were unique for each of the M. a. paratuberculosis types analyzed (II and III); other patterns were host-related or restricted to geographic areas. Therefore, this MIRU-VNTR approach could be a useful sub-typing molecular tool in order to get a better sense of the epidemiology of Johnes disease.

Polymorphisms in gyrA and gyrB genes among Mycobacterium avium subsp. paratuberculosis type I, II, and III isolates.

ABSTRACT The analysis of the gyrA and gyrB genes of a panel of Mycobacterium avium subsp. paratuberculosis isolates from types I, II, and III detected type-specific single nucleotide polymorphisms. Based on these results, we developed a PCR and restriction enzyme analysis to discriminate type I and III isolates. The application of this technique would be the unique strategy to characterize these strains when there is not enough bacterial growth to perform pulsed-field gel electrophoresis and IS900 restriction fragment length polymorphism.

Epidemiological investigation of a Mycobacterium avium subsp. hominissuis outbreak in swine.

Mycobacterium avium subsp. hominissuis (MAH) infection in swine may cause granulomatous lesions in lymph nodes that must undergo differential diagnosis with those caused by M. tuberculosis complex members. Moreover, MAH outbreaks can lead to severe economic losses due to condemnation of carcasses. A number of potential sources of infection for animals can usually be identified in contaminated farms. This report describes the application of several molecular characterization techniques in order to identify the possible environmental sources of MAH infection in an outbreak involving four breeding farms and six fattening farms. Molecular profiles obtained from MAH strains suggested a likely epidemiological link between clinical and environmental isolates cultured from sawdust and cooling systems from one breeding farm. These results highlight the potential risk posed by these environmental elements in the spread of infection and the need for implementation of adequate management practices in order to minimize this risk.

Single Nucleotide Polymorphisms in the IS900 Sequence of Mycobacterium avium subsp. paratuberculosis Are Strain Type Specific

ABSTRACT Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

Evaluation of specificity of tuberculosis diagnostic assays in caprine flocks under different epidemiological situations.

The aim of this study was to evaluate the specificity of the most widely used tuberculosis (TB) diagnostic tests, single intradermal tuberculin (SIT) and single comparative intradermal tuberculin (SCIT) tests and interferon-gamma (IFN-γ) assay in 937 animals from eight TB-free caprine flocks under different epidemiological situations. Maximum specificity was found using SCIT test (99.4-100% depending on the interpretation criteria) while SIT test and IFN-γ assay showed a slightly lower overall specificity (97.6-99.2% and 96.4-98.4% respectively). Specificity of the SIT test in a Corynebacterium pseudotuberculosis infected flock was significantly (P<0.05) lower if a severe interpretation criterion was applied. Similarly, specificity values of SIT test and particularly IFN-γ assay in a paratuberculosis (PTB)-vaccinated flock were lower than those observed in non-vaccinated flocks. Higher proportion of false positive reactors to TB tests (SIT and IFN-γ assay) were observed among animals positive in the PTB-ELISA in PTB vaccinated flock. These results demonstrate that TB diagnostic tests show an adequate specificity when performed in goats from TB-free flocks in most situations. However, certain factors such as C. pseudotuberculosis infection and paratuberculosis vaccination can have a negative impact in the most sensitive tests.