Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain

A field comparison of the interferon-gamma (IFN-gamma) assay and the single intradermal cervical tuberculin (SICT) test for the diagnosis of bovine tuberculosis was conducted. A total of 1136 cattle belonging to 85 herds placed in 'Castilla y León' (northwestern Spain) were chosen, and 21 of these herds were subjected to the diagnostic assays two or three times at intervals of at least 4 months. All the animals positive to any of the tests were slaughtered and tuberculosis was confirmed by culture isolation method (CIM) and further identification by means of PCR. Only 10.6% of cattle reacted with the bovine PPD in the SICT test, a percentage that increased to 12.8% in the IFN-gamma assay. The sensitivity of the IFN-gamma assay compared to CIM was shown to be higher (84.9%) than that of the SICT test (80.2%), but the combination of both tests offered the highest sensitivity (92.9%). The number of false positive reactors (those animals in which CIM was negative) was considerably higher for the IFN-gamma assay than for the SICT test and, conversely, the number of false negative animals (M. bovis isolation but negative immunological result) was higher for the skin test than for the interferon assay. In the herds tested twice, tuberculosis was eradicated after the second cycle of testing in 50%, and in 75% after the third cycle in herds tested three times. The combination of these two techniques instead of separately seems, therefore, to be useful in eradication programmes against bovine tuberculosis.     

Comparison between a gamma-IFN assay and intradermal tuberculin test for the diagnosis of bovine tuberculosis in field trials in Brazil.

Bovine tuberculosis is a major problem in Brazil. The intradermal tuberculin test is the standard test for detection of bovine tuberculosis in Brazil but can lack both sensitivity and specificity. The purpose of this study was to compare a bovine gamma-IFN assay with the tuberculin test under field conditions in Brazil. A total of 1632 animals from 13 dairy farms were tested using the single cervical tuberculin test (SCTT). Among those animals, about 15% of each herd, 220 in total, represented a high-risk group and were selected to be tested using the gamma-IFN test. Of the 1632 animals tested, 207 presented significant reactions representing 12.7% of the cattle studied. In the selected group the number of animals positive by the gamma-IFN assay was 126/220 (57.3%) and the total number of reactive cows on SCTT was 106/220 (48.2%). The real number of infected cattle, following standards, was 120/220 (54.5%). From these results the relative sensitivity rate of gamma-IFN test was 100% including the false-positive results and 88.3% for the SCTT--a significant (P < 0.01) difference in favour of the gamma-IFN test of 11.7%. The gamma-IFN assay also identified some positive animals 60-120 days earlier than the SCTT. In conclusion, we believe that the gamma-IFN assay can be used alone or in combination with the SCTT, as a valuable tool for the control of bovine tuberculosis in the Brazilian national herd.     

Validity of intradermal tuberculin testing for the screening of bovine tuberculosis in Madagascar.

A sample survey with the objective of determining the prevalence of bovine tuberculosis by means of an intradermal tuberculin test was conducted in Madagascar and it was found that the prevalence rate varied from 0-30% by veterinary district. In order to estimate the true prevalence, the validity of the test was investigated by assessing its sensitivity and specificity in two groups of animals from two different regions, which were destined for slaughter. In the first group where the probability of non-infected animals should have been the highest, sensitivity was estimated at 0.52 (n = 21) and specificity at 0.99 (n = 79). In the second group selected on the basis of apparent ill health of the animals in a high-prevalence bovine tuberculosis area, sensitivity was estimated at 0.8 (n = 10) and specificity at 1 (n = 12). The results obtained from both groups of cattle were not combined for statistical purposes because the sensitivity of the skin test seemed to fluctuate in relation to the chronicity of the disease. These fluctuations are discussed. However, since the first group of zebu cattle was more representative of the cattle population across the country as a whole, its results were retained as operational parameters for further screening.     

Use of ESAT-6 in the interferon-gamma test for diagnosis of bovine tuberculosis following skin testing

The whole blood interferon-gamma (IFN-gamma) test has proven to be a practical ancillary test for re-testing cattle for bovine tuberculosis 8-28 days following tuberculin skin testing. An improvement in the specificity of the IFN-gamma test could further reduce culling of false positive animals. The primary aim of this study was to evaluate a single mycobacterial antigen, ESAT-6 in the IFN-gamma test for use in skin test-positive cattle. These skin test-positive cattle comprised 51 Mycobacterium bovis-infected animals from tuberculosis-infected herds and 85 non-infected animals from tuberculosis-free herds. The test based on ESAT-6 had a higher specificity than the test based on purified protein derivative (PPD) tuberculin, but this was offset by a small decrease in sensitivity. Use of a lower cut-off in the ESAT-6-based test improved the sensitivity, while still maintaining a very high specificity. A secondary aim in the study was to assess the ESAT-6 and PPD-based tests for detecting bovine tuberculosis in skin test-negative animals from a persistently infected herd. The PPD-based test detected the majority of the lesioned or M. bovis-culture positive animals, while the ESAT-6-based test detected a smaller proportion. The false negatives in the IFN-gamma test from both the skin test-negative and positive groups were predominantly M. bovis-culture positive animals with no visible lesions. The current study has shown that a defined specific antigen such as ESAT-6 can markedly improve the specificity of the IFN-gamma test for re-testing skin test-positive animals. An ESAT-6-based IFN-gamma test could be particularly useful to reduce the false positive rate, yet still maintain an acceptable level of sensitivity.     

Investigation of the possible role of the tuberculin intradermal test in the spread of enzootic bovine leukosis

The single intradermal comparative test was used with both avian and bovine tuberculin. Three cattle infected with bovine leukosis virus (BLV) were used as a source of infection. BLV-positive and susceptible animals were tuberculin tested alternately. Fifteen susceptible calves and 15 susceptible sheep were tested. A further three calves and three sheep were used as controls; the needles of the tuberculin syringes were deliberately contaminated with blood from the BLV-infected cattle, before being used in the test. Whereas all three calves and the three sheep inoculated intradermally with contaminated needles developed BLV infections, all of the other 30 animals have remained serologically negative to BLV for 10 months. Transmission of BLV with needles contaminated with BLV-infected blood was prevented by wiping the needles with absorbent cotton wool.     

The comparative performance of the single intradermal test and the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations of differing potencies

In national bovine tuberculosis (BTB) control programmes, testing is generally conducted using a single source of bovine purified protein derivative (PPD) tuberculin. Alternative tuberculin sources should be identified as part of a broad risk management strategy as problems of supply or quality cannot be discounted. This study was conducted to compare the impact of different potencies of a single bovine PPD tuberculin on the field performance of the single intradermal comparative tuberculin test (SICTT) and single intradermal test (SIT). Three trial potencies of bovine PPD tuberculin, as assayed in naturally infected bovines, namely, low (1192IU/dose), normal (6184IU/dose) and high (12,554IU/dose) were used. Three SICTTs (using) were conducted on 2102 animals. Test results were compared based on reactor-status and changes in skin-thickness at the bovine tuberculin injection site. There was a significant difference in the number of reactors detected using the high and low potency tuberculins. In the SICTT, high and low potency tuberculin detected 40% more and 50% fewer reactors, respectively, than normal potency tuberculin. Furthermore, use of the low potency tuberculin in the SICTT failed to detect 20% of 35 animals with visible lesions, and in the SIT 11% of the visible lesion animals would have been classified as negative. Tuberculin potency is critical to the performance of both the SICTT and SIT. Tuberculin of different potencies will affect reactor disclosure rates, confounding between-year or between-country comparisons. Independent checks of tuberculin potency are an important aspect of quality control in national BTB control programmes.     

TB diagnosis in non-human primates: comparison of two interferon-gamma assays and the skin test for identification of Mycobacterium tuberculosis infection

To examine the effect of recombinant bovine interferon-gamma (rbIFN-gamma) on cattle persistently infected with bovine leukemia virus (BLV), BLV-infected cattle were inoculated intraperitoneally with IFN-gamma. All cattle were febrile after inoculation with IFN-gamma and then recovered within 48 h. Flow cytometric analysis showed that the numbers of CD4+ and CD8+ T cells were decreased for 2-3 days and then their numbers were recovered. The number of gammadelta T cells increased after the fever. In contrast, the number of IgM+ lymphocytes remained low for about 1 week. Moreover, the numbers of syncytia produced by peripheral blood lymphocytes decreased and remained low compared to that before IFN-gamma administration. These results suggest that IFN-gamma induces the up-regulation of gammadelta T cells, decreases the number of IgM+ lymphocytes and suppresses the growth of BLV in BLV-infected cattle in vivo.     

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.     

Assessment of an intradermal test for the detection of bovine brucellosis

Forty-eight cattle were sensitised toBrucella antigens either by vaccination withBrucella abortus strain 19 (S19) orB. abortus 45/20 (S45/20) and 24 of these and 12 unvaccinated cattle were subsequently challenged with virulentB. abortus strain 544 (S544). All these cattle (n=60), together with 12 control cattle which were neither vaccinated nor challenged, were subsequently subjected to an intradermal test using a S45/20 protein antigen. Reactions were interpreted subjectively by observation and palpation and were measured to the nearest mm with calipers at 48 and 72 hours after injection of protein antigen. Ten weeks later the cattle were slaughtered and tissues cultured for the presence ofB. abortus. Two of the 48 vaccinated cattle died, 40 of the remaining 46 gave a positive response to the intradermal test at 48 hours and 36 were positive at 72 hours. In the controls any increase in the skin thickness had disappeared by 72 hours. An increase in skin thickness was still present at 72 hours in all other cattle except those vaccinated with S19 only. The intradermal test was found to be sensitive but not specific in detecting infected cattle and both sensitive and highly specific if used (with the exception of S19) to detect exposure toBrucella antigen.     

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.     

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.     

Longer-term risk of Mycobacterium bovis in Irish cattle following an inconclusive diagnosis to the single intradermal comparative tuberculin test

In Ireland, new bovine tuberculosis (bTB) cases are detected using both field and abattoir surveillance. During field surveillance, an animal may be deemed a 'standard inconclusive reactor' (SIR) to the single intradermal comparative tuberculin test (SICTT) if the bovine response is >2mm, and from 1 to 4mm greater than the avian response. Little is known about the future infection risk posed by SIR animals that pass a subsequent retest, so-called 'transient SIR' (TIR) animals. The objective of this study was to critically evaluate the future bTB status of TIR animals, by examining the future risk of bTB diagnosis over the 4 years following initial SIR diagnosis and clearance at the subsequent retest. The study included all TIRs that were identified as SIRs in 2005 in otherwise free herds at tests with no other reactors at that test and that were clear at the subsequent retest. The analysis was restricted to cows that were neither sold, other than direct to slaughter, nor exported from the herd during the follow up period (to the end of 2009). Five control cows were randomly selected from each study herd. A parametric survival model with shared frailties, to account for clustering within herds, was developed to model time from passing a retest to future bTB diagnosis. The final parametric survival model contained the variables: TIR status in 2005, inconclusive status during the follow-up period, location, herd restricted during the study, time since last restriction within the herd and age. The time ratio for the TIR status variable was significant (p<0.001) indicating that on average the time to diagnosis with bTB for TIRs was 78% shorter compared to the non-TIRs. The frailty term was significant (p<0.001) indicating that animals within some herds were more likely to become reactors compared to other herds. These results have important implications for national policy and future management of TIR animals. Further, private veterinary practitioners and their clients should be aware of the increased risk associated with TIRs.     

Shorter-term risk of Mycobacterium bovis in Irish cattle following an inconclusive diagnosis to the single intradermal comparative tuberculin test

In Ireland, new cases of bovine tuberculosis (bTB) are detected using both field and abattoir surveillance (More and Good, 2006). Field surveillance is conducted through annual testing of all cattle using the single intradermal comparative tuberculin test (SICTT). An animal may be deemed a 'standard inconclusive reactor' (SIR) to the SICTT if the bovine response is >2mm and between 1 and 4mm>the avian response. The herdowner then has three choices for the management of the SIR: option 1 is to have the animal retested after a minimum period of 42 days (an inconclusive reactor retest, IRR), option 2 is to slaughter the SIR and, provided the animal has no visible lesions, have a full herd test 42 days after the SIR leaves the herd, option 3 is to slaughter the SIR and have the lymph nodes examined using histology and/or culture for bTB. In the current study, we examine the bTB risk for SIRs both at slaughter prior to the IRR and at the IRR, and the future bTB risk of TIR animals (so-called 'transient SIRs'; SIR animals with a negative SICTT result at the subsequent IRR) that moved from the herd of disclosure within 6 months of the IRR. We also investigate factors associated with the future bTB status of SIRs at slaughter prior to the IRR and at the IRR. The study population included all SIRs identified in Ireland between 2005 and 2009 inclusive in a herd otherwise Officially TB free (OTF). Between 11.8% and 21.4% of SIRs slaughtered prior to the IRR were confirmed bTB positive at post mortem (using histology or culture if histology was not definitive), compared to 0.13-0.22% of SICTT -ve cohort animals. The post mortem bTB lesion rate of SIRs is lower than the lesion rate reported for reactor animals between 2005 and 2009 of between 34% and 39%, reflecting the doubtful infection status of these animals. Between 20.3% and 27.9% of herds were restricted at the IRR. The herd restriction rate amongst the national herd between 2005 and 2009 varied from 5.09% to 6.02%. TIRs that moved out of the disclosing herd within 6 months of the IRR were 12 times more likely to be bTB positive at the next test/slaughter compared to all animals in the national herd. The same increased risk did not apply to the SICTT -ve cohort animals that moved out of the same herds at the same time. Based on a range of measures, SIRs and TIRs are each at increased bTB risk into the future. Consequently, differential treatment of TIR animals would be justified.     

The specificity of the single cervical intradermal tuberculosis test in a population of Tasmanian fallow deer putatively free of bovine tuberculosis

The single cervical intradermal tuberculin test was used on 34 farmed fallow deer in Tasmania, to determine the specificity of this test when increases in skin thickness of 1 and 2 mm were used as arbitrary significant responses. This was to assess the response that could be expected if this test was used in monitoring or export testing programs of deer in Tasmania. Tasmania is free from bovine tuberculosis, thus providing a unique opportunity to undertake such a study. All deer were slaughtered following testing, and thoroughly inspected post-mortem. Deer that reacted to the skin test had lymph nodes cultured for the presence of Mycobacteria sp. With the reactor response set at 1 mm or more, 25 out of 34 deer tested did not react to the skin test, giving a specificity of 73.5% (95% CI 57.5–89.5%). With the reactor response set at 2 mm or greater, the specificity of the test was 100%.

One lymph node each from two reactors contained mycobacteria identified as Mycoplasma avium and Mycoplasma scrofulaceum. This trial indicates that despite being free of bovine tuberculosis for 20 years, Tasmania also experiences the same difficulties as other countries in the use of the single cervical skin test for certifying deer free from bovine tuberculosis.     

Effects of positive results for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA on results of caudal fold tuberculin test and interferon-gamma assay for tuberculosis in cattle

OBJECTIVE: To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis. ANIMALS: 1043 cattle from 10 herds in Michigan. PROCEDURE: Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium. RESULTS: Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.