Javier Bezos

Bovine Tuberculosis (Mycobacterium bovis) in Wildlife in Spain

ABSTRACT Mycobacterium bovis infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovis through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (Cervus elaphus), fallow deer (Dama dama), wild boar (Sus scrofa), Iberian lynx (Lynx pardina), hare (Lepus europaeus), and cattle (Bos taurus). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovis strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures.

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.

Comparison of Four Different Culture Media for Isolation and Growth of Type II and Type I/III Mycobacterium avium subsp. paratuberculosis Strains Isolated from Cattle and Goats

ABSTRACT Culture is considered the definitive technique for Johnes disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrolds egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and Löwenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.

Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation

The role of wildlife in tuberculosis epidemiology is being widely studied since it can affect the effectiveness of eradication campaigns in cattle. The health problem is enhanced when it concerns also wildlife welfare and biodiversity conservation. This study was performed to understand the epidemiology of Mycobacterium bovis population affecting livestock and wild animals in the Doñana National Park using bacteriology and molecular characterisation techniques. Tuberculosis research was performed on 1209 cattle and wild animals (artiodactyla and carnivore) collected over 6 years in the Park. One hundred and sixty-three animals were found to be infected with M. bovis, comprising 7.96% of the cattle and 20.53% of the wild animals tested. Spoligotyping revealed nine patterns, being SB1232 and SB1230 the most prevalent (77.30% and 15.34% of infected animals, respectively). MIRU-VNTR analysis of a selected panel of 92 isolates showed eight different profiles, including several spoligotypes within the same MIRU-VNTR profile. The discriminatory capacity of both techniques in this panel was similar. The results obtained by combination of both techniques corroborate that wildlife species are infected with the M. bovis strains which are more prevalent in cattle and reveal their persistence. Genotype variation between isolates strongly suggests micro-evolutionary events in the M. bovis population in the same area. This study in the Doñana National Park exposes the risk of introduction of domestic animals into wildlife areas when there is not a warranty of disease freedom, appropriate diagnostic techniques and control measures.

Evaluation of the sensitivity and specificity of bovine tuberculosis diagnostic tests in naturally infected cattle herds using a Bayesian approach.

Test-and-slaughter strategies have been the basis of bovine tuberculosis (BT) eradication programs worldwide; however, eradication efforts have not succeeded in certain regions, and imperfect sensitivity and specificity of applied diagnostic techniques have been deemed as one of the possible causes for such failure. Evaluation of tuberculosis diagnostic tools has been impaired by the lack of an adequate gold standard to define positive and negative individuals. Here, a Bayesian approach was formulated to estimate for the first time sensitivity (Se) and specificity (Sp) of the tests [single intradermal tuberculin (SIT) test, and interferon-gamma (IFN-γ) assay] currently used in Spain. Field data from the first implementation of IFN-γ assay (used in parallel with SIT test 2-6months after a first disclosure SIT test) in infected beef, dairy and bullfighting cattle herds from the region of Castilla and Leon were used for the analysis. Model results suggested that in the described situation: (i) Se of SIT test was highly variable (40.1-92.2% for severe interpretation, median=66-69%), and its Sp was high (>99%) regardless interpretation criteria; (ii) IFN-γ assay showed a high Se (median=89-90% and 83.5% for 0.05 and 0.1 cut-off points respectively) and an acceptable Sp (85.7% and 90.3% for 0.05 and 0.1 thresholds) and (iii) parallel application of both tests maximized the combined Se (95.6% using severe SIT and 0.05 cut-off point in the IFN-γ assay). These results support the potential use of the IFN-γ assay as an ancillary technique for routine BT diagnosis.

Effect of paratuberculosis on the diagnosis of bovine tuberculosis in a cattle herd with a mixed infection using interferon-gamma detection assay

Interferon-gamma (IFN-gamma) detection assay is being applied as an ancillary test to tuberculin tests in the diagnosis of bovine tuberculosis to detect the maximum number of infected animals. Among possible factors influencing the performance of tuberculosis-diagnostic tests, paratuberculosis, a widespread disease in Spain and other European countries, has been pointed out as a cause of false positive reactions. Still, its effect on the sensitivity of these tests in cattle has yet to be fully characterized. The impact of paratuberculosis in the apparent sensitivity of IFN-gamma assay was studied in a bullfighting cattle herd with a mixed tuberculosis-paratuberculosis infection, using culture of Mycobacterium bovis and Mycobacterium avium paratuberculosis as the gold standard to determine the infection status of every animal. A total of 218 animals were slaughtered and sampled for bacteriology after blood sampling. IFN-gamma assay showed a lower apparent sensitivity in animals with a mixed infection (50%) compared to all animals suffering tuberculosis (78.3%). This finding indicates that the presence of paratuberculosis in tuberculosis-infected herds could imply a serious impairment in the sensitivity of IFN-gamma detection test.

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.

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.