The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p < 0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.     

The use of MPB70-ELISA for the diagnosis of caprine tuberculosis in Brazil

After one clinical case that evidenced the outbreak, a complete screening by intradermal tuberculin test was performed in one goat herd in Brazil. The herd was composed by 500 animals and 83 of them (16.6%) showed to be reactive to the comparative double cervical intradermal test. Four months after the test, all the 83 reactive animals were slaughtered and blood samples were collected from 45 of them, for serological assays. From those 45, 32 were randomly chosen for necropsy and histopathological and bacteriological procedures were conducted. Histopathology evidenced at least one characteristic lesion of tuberculosis in each animal, with typical granulommas where acid-fast bacilli (AFB) could be observed. Bacteriology was positive for Mycobacterium bovis in 22 samples (68.7%), therefore confirming the etiology of the outbreak. Sera of 45 animals plus 20 other from a certified free tuberculosis farm were tested in an ELISA using the recombinant M.bovis protein MPB70 as capture antigens. From those, 43 were reactive to the test, with high ODs results, considering a cut-off point established by ROC curve analyzing results (cut-off = 0.8; mean = 0.55; range: 0.157–1.357). These results suggest that MPB70-ELISA can be considered as a reliable tool to diagnose tuberculosis in goat herds, since this assay was capable to correctly detect 95.6% of the animals here examined.     

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.     

Comparing recombinant MPB70/SahH and native 20-kDa protein for detecting bovine tuberculosis using ELISA

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. Test-and-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurization have been implemented to prevent the spread of tuberculosis from animals to humans worldwide. Despite the importance of precise and rapid diagnostic tests, conventional methods including intradermal skin tests and γ-interferon assays are limited by the high rate of false-negative results for cattle in the late infectious stage and due to laborious and time-consuming procedures. Therefore, antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) are urgently needed to supplement the established approaches and expand the diagnostic window. This study was conducted to develop a bTB ELISA by evaluating recombinant and native proteins and various assay parameters. We produced recombinant MPB70 and SahH (M70S) and a native 20-kDa protein (20K) and optimized the ELISA protocol. The 20K ELISA showed 94.4% sensitivity and 98.2% specificity with an optimal sample-to-positive ratio cut-off of 0.531. The sensitivity and specificity of M70S ELISA were 94.4% and 97.3%, respectively, with an optimal sample-to-negative ratio cut-off of 1.696. Both assays showed acceptable diagnostic efficiency and could be used for bTB diagnosis in combination with established methods for herd screening and to expand the diagnostic window.     

Evaluation of an enzyme-linked immunosorbent assay in the diagnosis of bovine tuberculosis

The behavior of the immunoenzymatic system was tested in the serologic diagnosis of bovine tuberculosis in different populations from areas free of the disease, and in other areas with different prevalence. The specificity registered in the 4009 samples showed 94.4%, while the range of sensitivity increased proportionally to the number and distribution of lesions found in the anatomopathological test. Overall the sensitivity of the ELISA was 47%. When animals from groups with different prevalence of the disease were studied, a significant correlation (78%) between the ELISA system and the Intradermal Tuberculin Test (IT) was observed in the groups with low prevalence of the disease, animals reactive to both tests were found in all the units. The results of the work and the inherent advantages of the ELISA technique allow recommending this ELISA in programs for control and/or eradication of bovine tuberculosis.     

Evaluation of MPB70, bovine PPD and lipoarabinomannan as antigens in ELISA for the serodiagnosis of bovine tuberculosis

The performance of the secretory protein MPB70 of Mycobacterium bovis, bovine PPD, and lipoarabinomannan (LAM) were evaluated as antigens in ELISA for detection of tuberculosis (TB) infected cattle. Sera were from 120 M. bovis infected cattle and 223 cattle from a TB free herd. ELISA results were analyzed using receiver operating characteristic (ROC) curves in relation to culture results. The areas under the three ROC curves were 71 ± 49% SE (MPB70), 71 ± 27% SE (bovine PPD), and 56 ± 4% SE (LAM).     

An abattoir study of tuberculosis in a herd of farmed elk.

The purpose of this study was to examine the prevalence and distribution of grossly visible lesions of tuberculosis in a herd of 344 North American elk (Cervus elaphus) depopulated during a three-month period in 1991. Abattoir inspection detected mycobacterial lesions in 134 (39.8%) of the 337 animals received for slaughter. The prevalence of lesions increased with the age of the animals. Lesions were predominantly suppurative rather than caseous, and mineralization was less evident than in tuberculous lesions in cattle. Lesions occurred predominantly in lymph nodes, and lungs were the only organs in which mycobacterial lesions were found. The distribution of lesions suggested that aerosol transmission was the most significant means of spread of the disease within the herd. Giant liver flukes (Fascioloides magna) were observed in approximately 80% of the adult elk.     

Fluorescence polarization assay for the diagnosis of bovine brucellosis: adaptation to field use

A fluorescence polarization assay (FPA) was used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. abortus) with sera from animals with confirmed (bacteriologically) infection. A cut-off value between negative and positive values was initially established to be 87.2mP. This value was changed to 95mP to increase assay specificity without loss of sensitivity when testing blood samples from negative animals. The FPA technology was applied to whole blood samples in the field and to stored whole blood samples using two diluent buffers. Relative sensitivity and specificity values for the FPA performed in the field, based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95.3 and 97.3%, respectively. However, to obtain maximum sensitivity and specificity, a cut-off value of 105mP was determined for fresh whole blood samples. The relative sensitivity and specificity values of the FPA when testing stored whole blood samples were 100% each using a 95mP cut-off.The usefulness of the FPA for testing whole blood samples in the field was demonstrated.     

Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis

The sensitivity and specificity of an ELISA for the detection of bovine IgG anti-Mycobacterium bovis antibodies were 73.6% and 94.1%, respectively, as determined in 53 bacteriologically confirmed tuberculous cattle and 101 healthy cattle from a tuberculosis-free area. In addition, the results of ELISA and tuberculin tests in 149 cattle were compared with those of subsequent necropsy studies. Both tests failed to detect 2 animals with tuberculous lesions and positive culture; 3/12 cattle with M. bovis isolation and no lesions, and 2/7 with atypical mycobacterial infection reacted to tuberculin, but none had antibodies; in 128 cattle with neither lesions nor mycobacterial isolation, 6 were tuberculin reactors and 7 others had antibodies. Negative results were obtained by ELISA in 21/22 paratuberculous cattle. Antibodies were not detected in 88.9% to 96.4% of 697 cattle from two tuberculin negative herds of an endemic area. In a herd with proved M. bovis infection, distribution of seropositive animals in tuberculin and non-tuberculin reactors was similar. Antibody responses to cutaneous tuberculin stimuli were observed in 4 experimentally infected cattle, but only in 2/10 healthy controls after repeated PPD stimuli. Nine controls which had either received a single tuberculin dose or none showed no increase in antibody levels. The low sensitivity of this ELISA limits its usefulness as a diagnostic tool for bovine tuberculosis eradication campaigns. However, it could be helpful in epidemiological surveillance if its efficiency to identify infected herds is demonstrated.     

Mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis

Skin test negative cattle from a herd containing an unusually high proportion (194/382) of tuberculin skin test positive cattle were investigated for remaining Mycobacterium bovis infected animals. Blood samples from the skin test negative cattle, analysed by an antibody ELISA and an interferon-gamma assay, were mostly test negative for M. bovis. Radiometric culture of nasal mucus samples from 48 of the cattle yielded 22 culture positives with acid-fast bacilli and cording in 6 of these. Subculture on solid media was successful for 7, including 2 with cording of the 22 radiometric culture positives. Mycobacterium tuberculosis complex DNA probe testing using the Accuprobe (Gen-Probe, Inc.) and M. tuberculosis complex-specific PCR amplification, performed on the solid media subcultures, were negative. 16S rRNA PCR and sequence analysis were successful for 6 of the 7 solid media subcultures obtained and revealed the presence of Mycobacterium nonchromogenicum in all 6 subcultures. This is the first report of M. nonchromogenicum in nasal mucus of cattle. The observation highlights the importance of integrating definitive tests such as the PCR for diagnosis of bovine tuberculosis and indicates a possible zoonotic risk.     

Potential use of lymphocyte blastogenic responses in diagnosis of bovine tuberculosis

A comparison of in vitro lymphocyte responses and delayed type tuberculin skin test responses was made in an animal experimentally exposed to a Mycobacterium bovis-infected animal and in cattle naturally infected with M. bovis. Tuberculin skin tests did not suppress in vitro lymphocyte responses to M. bovis PPD and to M. avium PPD tuberculin. The whole blood test used in these studies provided for considerable savings in time as compared to use of purified lymphocytes for evaluating in vitro cellular responses. Variations in the responsiveness of lymphocytes to specific mycobacterial antigens was observed, therefore, it is recommended that profiles be established using three or more tests conducted at 14-day intervals.     

Evaluation of the gamma-interferon assay for eradication of tuberculosis in a goat herd

Objective To evaluate the usefulness of the gamma-interferon assay in the diagnosis of caprine tuberculosis in comparison with a single intradermal tuberculin test, and to obtain a group of animals free from this infection in a herd with a high prevalence.
Design An immunological study involving four serial comparative gamma-interferon and single intradermal tuberculin tests.
Animals A herd of 87 goats of Guadarrama breed.
Procedure Serial testing and segregation of animals.
Results We found that the number of infections detected by the gamma-interferon test was considerably greater than the number detected by the single intradermal tuberculin test. A group of 10 animals was negative to both tests in two consecutive rounds and three kids were negative in the last round of testing.
Conclusions Gamma-interferon assay is appropriate for diagnosis and eradication of tuberculosis in goats. This test is able to detect early Mycobacterium bovis infection. Avian reactors with simultaneous increased reaction to bovine PPD in the gamma-interferon assay (designated as avian^B reactors) should be considered test positive for M bovis . By serial testing with the gamma-interferon and the single intradermal tuberculin tests, and a policy of segregation of kids at birth, it is possible to achieve a group of animals test negative for tuberculosis from a herd of goats with high immunoreactivity to this infection.     

Tuberculosis in domesticated red deer: Comparison of purified protein derivative and the specific protein MPB70 for in vitro diagnosis

The use of a Mycobacterium bovis-specific protein, mycobacterial protein bovis 70 (MPB70), was compared with complex, M bovis-derived purified protein derivative (bovine PPD), for its ability to improve the diagnostic precision of in vitro assays for tuberculosis in farmed deer. A combination of lymphocyte transformation and enzyme-linked immunosorbent assay (ELISA) was used to differentiate between specific M bovis reactivity and crossreactivity due to sensitisation with saprophytic mycobacteria such as Mycobacterium avium. In the lymphocyte transformation assay the response of mononuclear cells, from red deer, to MPB70 was found to be more specific, but less sensitive, as an indicator of infection by M bovis when compared with the complex antigen bovine PPD. When used in conjunction with bovine PPD alone, MPB70 was found to increase the specificity of the ELISA in diagnosing animals with disease.     

Comparison of fluorescence polarization assay with card and complement fixation tests for the diagnosis of goat brucellosis in a high-prevalence area

An evaluation of fluorescence polarization assay (FPA) to detect antibodies against Brucella melitensis according to the Mexican Official Norm (NOM) was performed. In this study, a total of 2582 goat serum samples from a high-prevalence area in northeast Mexico where vaccination is applied, were used. Of these, 1094 were classified as NOM negatives (card test (CT) negatives or CT positives/complement fixation test (CFT) negatives) and 1488 as NOM positives (CT and CFT positives). The receiver operator characteristics (ROC) curve analysis was used to obtain the FPA sensitivity (83.5%), specificity (82.2%) and accuracy (88.2%) compared with NOM criteria, using a cut-off value of 89mP for positive samples. In addition, FPA produced 84.1% of negative results versus 65.7% of CT using 1094 CFT negative samples, which indicated that FPA performance was better than CT to detect negative samples or differentiate samples from vaccinated animals. Finally, FPA showed 95.8% sensitivity when using 702 negative non-vaccinated samples. Taken together, these results suggested that FPA might replace CT as a screening test for its better performance compared with CFT, its adjustable cut-off useful in different epidemiological situations, and for its reliability, ease of performance, comparable cost with CT regimen, and potential application in field and high-throughput laboratories. The use of FPA as screening test will help to reduce the percentage of goats wrongly slaughtered because of brucellosis misdiagnosis. More studies on FPA are required for its approval as diagnostic tool for goat brucellosis.     

牛结核分枝杆菌MPB70基因的克隆及其在大肠杆菌中的表达

以牛分枝杆菌DNA为模板,克隆了牛分枝杆菌MPB70基因,构建了克隆载体pGEM-MPB70和表达载体pET30a-MPB70,经IPTG诱导在大肠杆菌BL-21中表达,用SDS-PAGE和免疫印迹分析表达产物并进行蛋白纯化。试验结果表明,牛分枝杆菌MPB70基因体外扩增产物与预期值相符,约582 bp;所构建表达质粒pET30a-MPB70经测序,结果与预期一致;SDS-PAGE分析表明,该融合蛋白以包涵体的形式表达,其分子质量约为29 kD,蛋白表达量约占菌体总蛋白的20%;该蛋白经电洗脱纯化后,纯度达85%以上;免疫印迹分析表明,原核表达的融合蛋白可与兔抗牛分枝杆菌多克隆抗体结合,并且具特异的免疫反应性。     

Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine brucellosis.

Sera from Canadian pigs (brucellosis free, n = 14037) and sera from pigs infected with Brucella suis (n = 401) were tested by the buffered antigen plate agglutination test, the complement fixation test, an indirect and a competitive enzyme immunoassay and a fluorescence polarization assay. The results were analysed and assay sensitivity and specificity estimates were calculated. The sensitivity and specificity of the tests were as follows: the buffered antigen plate agglutination test, 77.1 and 96.9%; the complement fixation test (considering anticomplementary sera as negative), 93.3 and 95.5%; the complement fixation test (considering anticomplementary sera as positive), 58.1 and 99.9%; the indirect enzyme immunoassay, 94.0 and 97.9%; the competitive enzyme immunoassay, 90.8 and 96.6%; and the fluorescence polarization assay, 93.5 and 97.2%; respectively. It was concluded that the fluorescence polarization assay was a valuable asset to the diagnosis of porcine brucellosis because of its accuracy, ease of performance and relative cost.     

A complementary diagnosis of naturally occurring tuberculosis in water buffaloes (Bubalus bubalis) in Rio de Janeiro using a MPB70-ELISA, Brazil

As tuberculosis is still a worldwide infection and buffalo breeding represents an important economic activity in various countries, the purpose of this study was to employ an enzyme-linked immunosorbent assay (ELISA) using MPB70 as a capture antigen for the diagnosis of naturally occurring tuberculosis in water buffaloes in Brazil. After the introduction of newly acquired cattle onto a tuberculosis (TB) free farm, an outbreak of TB was recorded in a mixed herd comprising water buffaloes (21) and cattle (46). The entire herd was tested by intradermal tuberculin injection (ITT) and positive animals were slaughtered and tested by culture, polymerase chain reaction (PCR), and ELISA. From the 21 buffaloes sampled, three were reactive by ITT. All the three had positive culture and ELISA, while PCR was positive in two of them. Besides that, one ITT-negative buffalo was slaughtered and presented positive results by both culture and ELISA, and was considered as anergic. Although there were only few animals, those findings demonstrate the diagnostic usefulness of an MPB70-ELISA to correctly detect Mycobacterium bovis tuberculosis in water buffaloes.     

Breakdown severity during a bovine tuberculosis episode as a predictor of future herd breakdowns in Ireland

A retrospective cohort study of Irish cattle herds investigated whether the severity of a herd’s bovine tuberculosis (BTB) breakdown was a predictor of the hazard of a future BTB breakdown in that herd. Data on 10,926 herds not having had BTB in 1995 (the “non-exposed” group) were obtained using a 10% random sample from all herds without BTB in 1995. Data on 6757 herds that had a new BTB breakdown in 1995 (the “exposed” group) were obtained and categorized into five increasing exposure-severity classes based on the total number of standard reactors (to the single intra-dermal comparative cervical tuberculin test) detected during the breakdown. Exposed herds were deemed to be free of BTB after they passed a 6-month check test; non-exposed herds were deemed free as of the date of the first negative herd-test in 1995.

In the 5 years after 1995, 18% of the non-exposed herds had a BTB breakdown, whereas 31% of the exposed herds had a subsequent breakdown. Relative to the hazard for non-exposed herds, the hazard for the first future singleton standard reactor breakdown, was 1.6-times higher for exposed herds with only 1 standard reactor in 1995, and 1.8-times higher for those exposed herds with 4–8 standard reactors during the 1995 episode. When the outcome for future breakdowns was 2 or more standard reactors, the hazard ratios ranged from 1.6 for exposed herds with only 1 standard reactor in 1995 up to 2.9 in exposed herds with 8 or more standard reactors during the 1995 episode. The latter hazard ratio varied over time, decreasing to 1.7 after 3 years of risk. The hazard of a future BTB breakdown increased directly with number of cattle in the herd, a positive history of previous BTB in the herd, and the local herd prevalence of BTB. The presence of confirmed BTB lesions in reactor cattle was not predictive of the future breakdown hazard when the effects of other factors were controlled.     

牛分枝杆菌MPB70基因的克隆及其在大肠杆菌中的表达

本研究以牛分枝杆菌Vallee111染色体DNA为模板,以MPB70成熟蛋白基因特异性引物进行PCR扩增,获得约500bp的DNA片段.通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,成功地构建出克隆载体pGEM-T-70.以BamH Ⅰ和EcoR Ⅰ双酶切pGEM-T-70和pET28a( ),并将纯化的MPB70基因亚克隆至pET28a( )中,构建出原核表达载体pET28a-70.将pET28a-70转化至感受态E.coli BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约25Ku外源蛋白带.Western blot分析发现,该蛋白具有牛分枝杆菌抗原性,从而为进一步研究MPB70的亚单位疫苗及DNA疫苗奠定基础.