Assessment of an oral Mycobacterium bovis BCG vaccine and an inactivated M. bovis preparation for wild boar in terms of adverse reactions, vaccine strain survival, and uptake by nontarget species

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.

Limitations of spoligotyping and variable-number tandem-repeat typing for molecular tracing of Mycobacterium bovis in a high-diversity setting

This study describes the attempt to trace the first Mycobacterium bovis outbreak in alpacas (Lama pacos) in Spain by spoligotyping and variable-number tandem-repeat (VNTR) analysis. Due to high genotype diversity, no matching source was identified, but local expansion of a clonal group was found and its significance for molecular tracing is discussed.

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.

Tuberculosis in alpacas (Lama pacos) caused by Mycobacterium bovis

We report three cases of tuberculosis in alpacas from Spain caused by Mycobacterium bovis. The animals revealed two different lesional patterns. Mycobacterial culture and PCR assay yielded positive results for M. bovis. Molecular typing of the isolates identified spoligotype SB0295 and identical variable-number tandem repeat (VNTR) allele sizes.

Single-nucleotide polymorphism in two representative multidrug-resistant Mycobacterium bovis isolates collected from patients in a Spanish hospital harboring a human infection outbreak

Mycobacterium bovis is the etiological agent of tuberculosis in domestic and wild animals. Its involvement as a human pathogen has been highlighted again with the recent descriptions of transmission through dairy products (18), reactivation or primary infection in human immunodeficiency virus-infected patients (5), and association with meat industry workers, animal keepers, or hunters (3). Strains resistant to antituberculous drugs (M. bovis is naturally resistant to pyrazinamide) pose an additional risk (2). Several studies have demonstrated that mutations in target genes are associated with resistance to antituberculous drugs (4, 7, 10, 11, 16). However, most of them have been developed in Mycobacterium tuberculosis strains and limited data are available regarding M. bovis isolates. The aim of this study was to characterize by sequencing the main genes involved in antibiotic resistance in two multidrug-resistant (MDR) M. bovis isolates in a human outbreak detected in a hospital in Madrid that subsequently spread to several countries (5, 6, 15). The isolates were resistant to 11 drugs, but only their rpoB and katG genes have been analyzed so far (1, 14). We studied the first (93/R1) and last (95/R4) M. bovis isolates of this nosocomial outbreak, characterized by spoligotyping as SB0426 (hexacode 63-5F-5E-7F-FF-60 in the database at www.mbovis.org) (1, 13). Several genes involved in resistance to isoniazid (katG, ahpC, inhA, and the oxyR-ahpC intergenic region), rifampin (rpoB), streptomycin (rrs, rpsL), ethambutol (embB), and quinolones (gyrA) were studied. These genes, or fragments of genes, were amplified and sequenced as previously described (12). The sequence analysis revealed polymorphisms in five (ahpC, rpoB, rpsL, embB, and gyrA) out of nine analyzed genes (Table 1). Nucleotide substitutions in four genes cause a change in the encoded amino acid. Two additional synonymous mutations in ahpC and rpsL differentiated the first and last isolates from the outbreak.

Protection against Tuberculosis in Eurasian Wild Boar Vaccinated with Heat-Inactivated Mycobacterium bovis

Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines

Infection of the central nervous system by Mycobacterium tuberculosis in patients infected with human immunodeficiency virus (the new neurotuberculosis)

Neurologic complications of HIV infection are numerous. This review focuses on the clinical presentation, diagnostic particularities and therapeutic issues of neurotuberculosis. The pertinent literature describing this important infection is succinctly summarized with a particular emphasis on the discussion of differences in clinical presentation between HIV-infected and -uninfected patients.

Mycobacterium Bovis em queijo coalho artesanal em Parnaíba, Piauí.

O objetivo desse estudo foi detectar Mycobacterium bovis em queijo de coalho artesanal comercializado em Parnaíba, Piauí, por meio de cultivo microbiológico e pela Reação em Cadeia de Polimerase em Tempo Real.

Attività enzimatica ed inibizione della tirosin-fosfatasi A (MptpA) di Mycobacterium tuberculosis in funzione del pH

La tubercolosi è una malattia infettiva causata dal batterio patogeno Mycobacterium tuberculosis. Tale micobatterio è in grado di secernere due importanti tirosin-fosfatasi, MptpA e MptpB, che esplicano un importante ruolo come fattori di virulenza permettendo la sopravvivenza del batterio all’interno dell’ospite mediante evasione della risposta immunitaria. In particolare, MptpA è un membro della famiglia delle tirosin-fosfatasi a basso peso molecolare ed è composta da 163 amminoacidi (17,9 kDa). L’enzima impedisce la normale maturazione del fagosoma e la fusione fagosoma-lisosoma, attraverso la defosforilazione della proteina VPS33B, inibendo così la risposta cellulare all’infezione. Obiettivo del seguente lavoro di tesi è stato analizzare il comportamento catalitico di MptpAW152F, variante caratterizzata dalla presenza di un solo triptofano, in funzione del pH. È stata inoltre valutata l’eventuale inibizione dell’attività enzimatica ad opera di alcuni composti naturali, i glucosinolati e di un inibitore di sintesi (L335-M34). Lo studio cinetico effettuato in presenza del substrato fosfotirosina ha evidenziato la presenza di un secondo sito allosterico, adiacente al sito attivo dell’enzima, che influenza l’attività di MptpAW152F. Questo risulta essere un promettente bersaglio per la progettazione di nuovi farmaci ed inibitori selettivi. La valutazione di alcuni tra questi, come l’inibitore sintetico L335-M34 ha riportato risultati promettenti. I saggi di attività in pre-steady-state in funzione del pH e le analisi SPR hanno permesso di indagare ulteriormente l’affinità di legame tra MptpAW152F e la fosfotirosina e di evidenziare il riarrangiamento strutturale dell’enzima da una forma aperta ad una forma chiusa, cataliticamente attiva.

Investigating the causes of late fruit drop in ‘Regina’ sweet cherry (Prunus avium)

The presented study aimed to correctly describe the (late) fruit drop pattern of sweet cherry cv. Regina grafted on ‘Gisela 5’ and investigate its internal causes. In the first season, a method to describe the fruit drop pattern was defined and validated. The second season was devoted to a province-based screening of the phenomenon to identify potential influences of environmental, physiological and management factors. The multisite trial involved 6 commercial orchards located at different elevations, from 225 up to 1175m a.s.l. The third season was dedicated to find confirmation of the hypothesis formulated during the previous year. The multisite comparison was maintained but reduced to only two orchards to allow more frequent samplings. It emerged that late fruit drop is a complex phenomenon showing variable intensity: the percentage of late fruit drop ranged from 7 to 76% of the fruitlets set, depending on the orchard and on the year considered. Two main waves of fruitlets drop have been observed: the first one was composed by unfertilized parthenocarpic fruitlets, probably caused by late or missing fertilization, that immediately after bloom already showed smaller diameters and symptoms of senescence; the second one (the focus of this study) was composed by fully developed fruits that at a certain point decreased their growth rate and got senescent. All the late dropped cherries showed an aborted embryo. This sudden change has been observed to be concomitant both with prolonged periods of low temperatures (or sudden severe decreases in the daily Growing Degree Hours accumulation) and with extraordinary high temperatures close to or above 30°C. Other factors, such as the position of the limb within the canopy, its orientation (sunny vs. shady side) or nutrition played only a marginal role. Excessive vigor can increase late fruit drop intensity but is not its main cause.

Sinergia entre o vírus HIV e o Mycobacterium tuberculosis

Unidade 2, atividade 1 do curso Manejo da coinfecção TB-HIV: videoaula em que a médica infectologista Denise Arakaki-Sanchez explica a associação sinérgica entre o Mycobacterium e o vírus da Imunodeficiência, e como juntos eles podem acarretar tantos distúrbios no corpo humano.

Sinergia entre o HIV e o Mycobacterium tuberculosis [transcrição da videoaula]

Unidade 2, atividade 1 do curso Manejo da coinfecção TB-HIV: transcrição da videoaula em que a médica infectologista Denise Arakaki-Sanchez explica a associação sinérgica entre o Mycobacterium e o vírus da Imunodeficiência, e como juntos eles podem acarretar tantos distúrbios no corpo humano.

Sinergia entre o vírus HIV e o Mycobacterium tuberculosis [versão para impressão]

Unidade 2, atividade 1 do curso Manejo da coinfecção TB-HIV: slides da aula em que a médica infectologista Denise Arakaki-Sanchez explica a associação sinérgica entre o Mycobacterium e o vírus da Imunodeficiência, e como juntos eles podem acarretar tantos distúrbios no corpo humano.