Investigation of the spread bovine tuberculosis in Southern Brazil by Whole-genome sequencing.

Mycobacterium bovis is the causal agent of bovine tuberculosis, one of the most important diseases currently facing the cattle industry worldwide. Tracing the source of M. bovis infections that result from movement of livestock is an important tool to understand the epidemiology of bovine tuberculosis (bTB) and defining control/eradication strategies. Whole genome sequencing (WGS) provides a higher resolution than other established typing methods and greatly improves the definition of the regional localization of M. bovis types. Cultures of M. bovis were isolated from 58 bovine granulomatous tissue using conventional methods (Stonebrink medium) from eight dairy farms of the State of Rio Grande do Sul, Southern Brazil. The isolates were sequenced using both llumina technologies NextSeq 500 System and HiSeqX System

Tuberculose bovina em cervídeos.

A tuberculose bovina (bTB) é uma enfermidade infectocontagiosa, causada por Mycobacterium bovis, que acomete animais domésticos, silvestres e o homem. Em animais silvestres mantidos em cativeiro, a bTB representa risco aos tratadores de animais e aos visitantes de zoológicos, e há também a possibilidade de disseminação da infecção para animais domésticos e pela venda de animais silvestres infectados. Cervídeos das espécies sambar (Cervus unicolor), nobre (Cervus elaphus) e dama (Dama dama) de um parque safári, do Rio Grande do Sul, apresentaram quadro clínico de bTB (dispneia e perda de peso). Alguns animais foram a óbito, sendo detectadas lesões sugestivas de tuberculose (LST), confirmadas por histopatológica. Com o impedimento de comercialização de animais, realizou-se eutanásia de 281 cervídeos com autorização do IBAMA. Foram coletados linfonodos retrofaríngeos, submandibulares e vísceras de 21 animais, que foram cultivados em meio Stonebrink por até 90 dias. Após extração de DNA das colônias, realizou-se PCR para alvos flanqueando a região de diferenciação 4 (RD4). Das 21 amostras, 14 (61,9%) apresentaram LST, com aspecto granulomatoso, coloração esbranquiçada, e consistência caseosa ou calcificada e sete (38,1%), não apresentaram lesões. No cultivo das 14 amostras com LST, 13 (92,8%) apresentaram crescimento bacteriano compatível com M. bovis. No cultivo das sete amostras sem LST, seis (92,8%) apresentaram colônias compatíveis com M. bovis. A PCR convencional detectou como positivos 19 cultivos bacteriológicos sugestivos de M. bovis, confirmando o surto de bTB nos cervídeos. Lesões também foram detectadas em seis lhamas, um camelo, uma anta e um antílope, as quais foram confirmadas por histopatologia. Este fato sugere que houve transmissão entre espécies, muito embora não tenha sido possível realizar isolamento nestes casos. Estudos de genotipagem por sequenciamento genômico total estão sendo realizados com os isolados de M. bovis dos cervídeos, o que permitirá comparações filogenéticas com outros isolados já sequenciados no estado.bitstream/item/213635/1/Tuberculose-bovina-em-cervideos.pd

False-negative reactions to the comparative intradermal tuberculin test for bovine tuberculosis.

According to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary tests could help identify such animals and increase the odds of diagnostic success.Título em português: Reações falso-negativas ao teste cervical comparativo para tuberculose bovina

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDITOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future

ELISA using a recombinant chimera of ESAT-6/MPB70/MPB83 for Mycobacterium bovis diagnosis in naturally infected cattle.

Bovine tuberculosis (bTB) control programs generally rely on intradermal tuberculin tests for the antemortem diagnosis of Mycobacterium bovis infection in cattle, but these tests detect only a portion of the infected animals. The aim of the present study was to evaluate the diagnostic coverage of a combination of the bTB antemortem techniques known as the comparative intradermal tuberculin test (CITT) and an ELISA based on a recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen in cattle. The results were compared to postmortem findings based on M. bovis culturing and PCR. Paired comparisons of all data (n=92) demonstrated that ELISA and LST results compared to the culturing results did not present significant differences (P=0.27 on McNemar?s test and P=0.12 on Fisher?s exact test, respectively). Using culturing as the gold standard, the sensitivity and specificity of ELISA were 79.5% (95% CI: 64.5?89.2%) and 75.5% (95% CI: 62.4?85.1%), respectively, whereas LST demonstrated 100% sensitivity (95% CI: 91.03? 100%) and 92.5% specificity (95% CI: 82.1?97.0%). The ELISA results did not reveal significant differences in relation to the LST results (P>0.99 on Fisher?s exact test). Using the latter as the gold standard, the sensitivity and specificity of ELISA were 79.1% (95% CI: 64.8?88.6%) and 79.6% (95% CI: 66.4?88.5%), respectively. The use of ELISA with the recombinant chimera of ESAT-6/MPB70/ MPB83 as the antigen complements the diagnostic coverage provided by CITT and increases the removal of infected animals from herds

Diagnóstico de tuberculose bovina: avanços na identificação de Mycobacterium bovis por espectrometria de massas MALDI-TOF.

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Variations on a theme: diversification of cuticular hydrocarbons in a clade of cactophilic Drosophila

<p>Abstract</p> <p>Background</p> <p>We characterized variation and chemical composition of epicuticular hydrocarbons (CHCs) in the seven species of the <it>Drosophila buzzatii </it>cluster with gas chromatography/mass spectrometry. Despite the critical role of CHCs in providing resistance to desiccation and involvement in communication, such as courtship behavior, mating, and aggregation, few studies have investigated how CHC profiles evolve within and between species in a phylogenetic context. We analyzed quantitative differences in CHC profiles in populations of the <it>D. buzzatii </it>species cluster in order to assess the concordance of CHC differentiation with species divergence.</p> <p>Results</p> <p>Thirty-six CHC components were scored in single fly extracts with carbon chain lengths ranging from C₂₉to C₃₉, including methyl-branched alkanes, <it>n</it>-alkenes, and alkadienes. Multivariate analysis of variance revealed that CHC amounts were significantly different among all species and canonical discriminant function (CDF) analysis resolved all species into distinct, non-overlapping groups. Significant intraspecific variation was found in different populations of <it>D. serido </it>suggesting that this taxon is comprised of at least two species. We summarized CHC variation using CDF analysis and mapped the first five CHC canonical variates (CVs) onto an independently derived <it>period </it>(<it>per</it>) gene + chromosome inversion + mtDNA COI gene for each sex. We found that the COI sequences were not phylogenetically informative due to introgression between some species, so only <it>per </it>+ inversion data were used. Positive phylogenetic signal was observed mainly for CV1 when parsimony methods and the test for serial independence (TFSI) were used. These results changed when no outgroup species were included in the analysis and phylogenetic signal was then observed for female CV3 and/or CV4 and male CV4 and CV5. Finally, removal of divergent populations of <it>D. serido </it>significantly increased the amount of phylogenetic signal as up to four out of five CVs then displayed positive phylogenetic signal.</p> <p>Conclusions</p> <p>CHCs were conserved among species while quantitative differences in CHC profiles between populations and species were statistically significant. Most CHCs were species-, population-, and sex-specific. Mapping CHCs onto an independently derived phylogeny revealed that a significant portion of CHC variation was explained by species' systematic affinities indicating phylogenetic conservatism in the evolution of these hydrocarbon arrays, presumptive waterproofing compounds and courtship signals as in many other drosophilid species.</p

Characterising the phenotypic diversity of Papilio dardanus wing patterns using an extensive museum collection

The history of 20th Century evolutionary biology can be followed through the study of mimetic butterflies. From the initial findings of discontinuous polymorphism through the debates regarding the evolution of mimicry and the step-size of evolutionary change, to the studies on supergene evolution and molecular characterisation of butterfly genomes, mimetic butterflies have been at the heart of evolutionary thought for over 100 years. During this time, few species have received as much attention and in-depth study as Papilio dardanus. To assist all aspects of mimicry research, we present a complete data-derived overview of the extent of polymorphism within this species. Using historical samples permanently held by the NHM London, we document the extent of phenotypic variation and characterise the diversity present in each of the subspecies and how it varies across Africa. We also demonstrate an association between “imperfect” mimetic forms and the transitional race formed in the area where Eastern and Western African populations meet around Lake Victoria. We present a novel portal for access to this collection, www.mimeticbutterflies.org, allowing remote access to this unique repository. It is hoped that this online resource can act as a nucleus for the sharing and dissemination of other collections databases and imagery connected with mimetic butterflies