Assessment of a Protein Cocktail-Based Skin Test for Bovine Tuberculosis in a Double-Blind Field Test in Cattle

Abstract

Bovine tuberculosis (bTB) is a worldwide zoonosis caused mainly by Mycobacterium bovis. The traditional diagnostic method used often is the tuberculin skin test, which uses bovine purified protein derivatives (PPD-B). However, it is difficult to maintain uniformity of PPD-B from batch to batch, and it shares common antigens with nonpathogenic environmental mycobacteria. To overcome these problems, M. bovis-specific antigens that showed good T cell stimulation, such as CFP-10, ESAT-6, Rv3615c, etc., have been used in the skin test, but there have been no large-scale clinical studies on these antigens. In this study, two combinations (CFP-10/ESAT-6/TB10.4 protein cocktail and CFP-10/ESAT-6/Rv3872/MPT63 protein cocktail) were developed and used as stimuli in the skin test. Cattle were double-blind tested to assess the efficiency of the protein cocktail-based skin tests. The results showed that the CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test can differentiate TB-infected cattle from Mycobacterium avium-infected ones and that it shows a high degree of agreement with the traditional tuberculin skin test ( ‫؍‬ 0.8536) and gamma interferon (IFN-␥) release assay ( ‫؍‬ 0.8154). Compared to the tuberculin skin test, the relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 87% and 97%, respectively., The relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 93% and 92%, respectively, on comparison with the IFN-␥ release assay. The correlation between the increases in skin thickness observed after the inoculation of stimuli was high ( (1, 2). M. bovis mainly infects cattle, causing bovine tuberculosis (bTB), and occasionally affects other species of mammals (3). M. bovis can also infect humans through the inhalation of aerosols or the ingestion of unpasteurized dairy products (4, 5). The eradication of bovine tuberculosis is based on reliable diagnostic methods.The traditional diagnostic method of identifying bTB is the tuberculin skin test, which is based on the detection of delayedtype hypersensitivity (DTH) induced by bovine purified protein derivative (PPD-B). The tuberculin skin test has been used worldwide and is economical and easily applied. However, PPD-B is a poorly defined mixture of proteins, lipids, and carbohydrates obtained from a virulent M. bovis culture, the uniformity of which is difficult to maintain from batch to batch (6). Moreover, the use of virulent M. bovis during the production of PPD-B may potentially harm workers. Importantly, shared antigenic components in PPD-B and nonpathogenic environmental mycobacteria can reduce the specificity of the tuberculin skin test (7,8). Although the gamma interferon (IFN-␥) release assay (IGRA) showed higher specificity than the single intradermal skin test, the high price of this kit limits its use in developing countries such as China (9, 10). To overcome the drawbacks of the traditional diagnostic methods, scientists have focused on screening new M. bovis-specific antige...

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

“…In vivo immunization experiments revealed that among five high-affinity peptides, two (MPT63 [18][19][20][21][22][23][24][25][26] and MPT6 ) triggered cytotoxic T-cell response whereas the remaining three high-affinity peptides failed to induce IFN-g-secreting T-cell responses. Conversely, three low-affinity peptides (MPT63 [10][11][12][13][14][15][16][17][18][19] , MPT63 [20][21][22][23][24][25][26][27][28] and MPT63 [29][30][31][32][33][34][35][36][37] ) all exhibited immunogenicity in HLA-A Ã 0201 transgenic mice. The present study has further confirmed that the binding affinity of peptides for HLA molecules is not the only factor determining their immunogenicity.…”

“…As shown in Figure 4 and Table 2, all patients responded to one or more immunogenic peptides, the percentage of responding patients varying between 45% and 73%. The percentages of TB patients responding to MPT63 [5][6][7][8][9][10][11][12][13][14] , MPT63 [10][11][12][13][14][15][16][17][18][19] , MPT63 [18][19][20][21][22][23][24][25][26] , MPT63 [20][21][22][23][24][25][26][27][28] and MPT63 [29][30][31][32][33][34][35][36][37] were 63%, 73%, 45%, 60% and 68%, respectively. The combined diagnostic sensitivity of these five immunogenic peptides was 93%.…”

Teste intradérmico com proteínas recombinantes de Mycobacterium bovis como antígenos em Cavia porcellus

“…Estudos prévios têm demonstrado que a utilização de antí-genos específicos do complexo Mycobacterium tuberculosis (CMT) em teste intradérmico pode proporcionar um diagnóstico mais específico e sensível , Xin et al 2013.…”

“…A proteína ESAT-6 confirmou seu potencial para o diagnóstico da tuberculose bovina, devido à capacidade demonstrada nesse estudo, quando purificada em condição nativa, em diferenciar os animais sensibilizados experimentalmente daqueles não sensibilizados, mesmo quando utilizada a menor concentração de 6 mg. Além disso, os resultados demonstraram não haver diferença significativa na magnitude das reações estimuladas por ESAT-6 nas dosagens de 6, 12, 24 e 48mg, indicando que não foi possível observar uma relação de dose dependência, assim como observado por Xin et al (2013), ao testar as concentrações de 20, 50 e 70mg de ESAT-6 juntamente com CFP10 e TB10.4 em teste intradérmico em bovinos. Por outro lado, Aggerbeck & Madsen (2006) ao avaliarem ESAT-6 recombinante em teste cutâneo em animais, observaram uma reação proporcional ao aumento da concentração das proteínas de 0,01, 0,1 e 1mg, mas que só foi verificado após sucessivas aplicações de seis doses da proteína no mesmo animal, o que estimulou a potencialização da resposta de hipersensibilidade.…”

“…Não existe uma vacina eficaz para a tuberculose bovina (OIE 2013) e os programas de controle e erradicação da enfermidade realizados na maioria dos países (Álvarez et al 2012) inclusive no Brasil (Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal-PNCEBT), são baseados no método tradicional de diagnóstico, o teste intradérmico, que visa a detecção de uma reação de hipersensibilidade tardia (DTH) induzida após a inoculação de derivado proteico purificado (PPD), que gera uma reação inflamatória local nos animais com infecção prévia pelo bacilo (Xin et al 2013).…”