Field comparison of the interferon-gamma assay and the intradermal tuberculin test for the diagnosis of bovine tuberculosis

An extensive field comparison of the gamma interferon (IFN-gamma) assay and the single intradermal tuberculin test for the diagnosis of bovine tuberculosis was conducted in Australia. The specificity of the IFN-gamma assay was determined by testing more than 6000 cattle from tuberculosis-free herds and varied from 96.2% to 98.1%, depending on the cut-off point chosen to define a positive reactor. For the sensitivity trial, cattle from herds being de-populated because of bovine tuberculosis were examined with both assays. The sensitivity of the IFN-gamma assay was shown to be significantly higher than the single intradermal tuberculin test and varied from 76.8% to 93.6% depending on the method of interpretation. A maximum overall sensitivity of 95.2% was obtained by testing with the IFN-gamma and the tuberculin test in parallel. The superior sensitivity of the IFN-gamma assay and the ability to adjust the sensitivity of the system depending on the task involved, will provide the Australian Tuberculosis Eradication Campaign with a valuable additional test to enable it to accomplish its goals.     

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.     

Evaluation of single and comparative intradermal tuberculin tests for tuberculosis eradication in caprine flocks in Castilla y León (Spain)

Goats can act as reservoirs for tuberculosis (TB) infection. The main etiological agents of TB in goats are Mycobacterium caprae and Mycobacterium bovis and they infect also a wide range of domestic and wild animals and humans. Control programmes based mainly on the application of single and comparative intradermal tuberculin (SIT and SCIT respectively) tests are being implemented in certain regions of Spain with a high density of caprine flocks as Castilla y León, including goats with epidemiological relationship with cattle. The aim of this study was to evaluate the performance of the intradermal tests in naturally TB-infected caprine flocks from this region. The study was performed using data from 17,450 goats in 54 different flocks that were classified as TB-infected in the control programmes executed in 2010 and 2011. Data from 1237 goats from 7 dairy flocks depopulated after the first intradermal testing were used to estimate the sensitivity (Se) using bacteriology as the gold-standard. Overall Se of the SIT test using the severe interpretation was 43.9% (CI 95%, 40.4–47.4) and decreased to 38.8% (CI 95%, 35.5–42.3) using the standard interpretation. Overall Se of the SCIT test ranged between 21.3% (CI 95%, 17.6–25.4) and 7% (CI 95%, 4.9–9.8) depending of the interpretation criteria. A significant weak positive correlation was found between age and skin fold thickness (Spearman’s test p < 0.05). Results from this study yielded, in general, low Se values probably due the systematic detection and slaughter of reactors as a consequence of the eradication programme in previous years or the presence of factors that may interfere in the diagnosis. Therefore, these results suggest the necessity of including ancillary diagnostic tools and/or strict interpretation criteria to maximize detection of positive animals in infected settings.     

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.     

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.     

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.     

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.     

Appraisal of interpretation criteria for the single intra-dermal comparative cervical tuberculin test for the diagnosis of tuberculosis in dromedary camels in Ethiopia

Tropical Animal Health and Production - Dromedary camels are the main sources of milk, meat and income for the Ethiopian pastoralists as they withstand the harsh environments of the regions of the...     

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.     

Performance of a whole blood interferon-gamma assay for detection and eradication of caseous lymphadenitis in sheep

Caseous lymphadenitis (CLA), a chronic bacterial disease of sheep and goats caused by Corynebacterium pseudotuberculosis, could be controlled by eradication of infected carriers. This study aimed at validation of a whole blood interferon-gamma (IFN-gamma) enzyme immunoassay (EIA) (Bovigam, Pfizer) in naturally infected sheep for use in eradication of infection from a flock. This assay used formalin-inactivated whole bacterial cells as antigen. The sensitivity of the whole cell assay was improved by increasing both the volume of blood and the number of bacterial cells. The assay was validated in experimentally infected sheep and in a flock of known-negative sheep, as well as in a naturally infected flock, a proportion of which was vaccinated with a commercial CLA vaccine. An optical density (540nm) (OD) cut-off of 0.09 was effective in classifying animals as test positive or negative in the naturally infected flock, although there was variation in OD between visits, notably with weakly reacting animals. The test had a sensitivity of 91% and a specificity of 98%. Postmortem data supported the results in test-negative animals. Visit-to-visit variation in IFN-gamma EIA OD in the naturally infected flock as well as CLA disease status was used to develop an algorithm for the eradication of CLA from a known infected flock. The whole blood IFN-gamma assay shows promise for eradication of caseous lymphadenitis from sheep flocks.     

The comparative performance of the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations from different manufacturers

Ireland currently obtains its avian and bovine tuberculin purified protein derivatives (PPDs) from a single source. Because problems of supply or quality cannot be discounted, it is prudent that Ireland identify alternative supplier(s) as part of a broad risk management strategy. Therefore, the aim of this study was to compare the performance of a number of different tuberculin combinations (that is, pairings of bovine and avian PPD; with different manufacturers) in the single intradermal comparative tuberculin test (SICTT), as currently performed in Ireland. The study was randomised, controlled and double-blinded. A total of 2172 cattle were used in the study. Each animal was tested using two SICTTs, the first based on the tuberculin combination in current use, and the second using one of six trial tuberculin combinations. Analyses were conducted to compare both reactor-status and skin increase. For each control/trial tuberculin combination, there was good agreement between the control and trial reactor-status. Differences in skin increases were mainly confined to animals categorised as either negative or severe inconclusive. However, the measured differences were minor, and unlikely to have a significant impact on the actual test outcome, either for individual animals or for herds. In conclusion, while further studies determining sensitivity and specificity in Ireland would have to be done in the event of a change in tuberculin PPD there should be minimal disruption of the national programme if alternative tuberculin PPDs meeting WHO, OIE and EU regulations were used. In this study, the precision of the guinea pig bio-assay to assess tuberculin potency was low and therefore Ireland should maintain its practice of periodically assessing potency in naturally infected cattle, even though this is not currently required under WHO, OIE or EU Regulations.     

An evaluation of the comparative tuberculin skin test for detecting tuberculosis in farmed deer

A comparative cervical skin test using 1.0 mg/ml bovine purified protein derivative and 0.5 mg/ml avian purified protein derivative was evaluated as a method for detecting tuberculosis in farmed deer. A positive comparative cervical skin test reaction was defined as a bovine response with a 2 mm or greater increase in skin thickness which was greater or equal to the avian response. Estimates of the sensitivity of the comparative cervical skin test were obtained from a series of experiments conducted on 60 deer intratracheally inoculated with Mycobacterium bovis. Prior tuberculin skin testing was found to suppress the skin reactivity to a subsequent comparative cervical skin test. This effect was most pronounced at short intervals of 3-7 days, but could still be measured 60 days after the previous test. When the test interval was greater than 60 days, the sensitivity of the comparative cervical skin test was 91.4%. The specificity of the comparative cervical skin test was 98.7% when 1157 deer from 17 uninfected herds with a history of nonspecific skin test reactions were examined. There was no statistical difference in the mean skin thickness increases of three groups of infected animals tested with 2 mg/ml, 0.2 mg/ml and 0.02 mg/ml of bovine purified protein derivative respectively.     

An evaluation of the gamma interferon test for detecting bovine tuberculosis in cattle 8 to 28 days after tuberculin skin testing

The gamma interferon (IFN -gamma) test was evaluated for its ability to diagnose bovine tuberculosis in cattle that 8 to 28 days previously had a positive caudal fold skin test. The sensitivity of the test was determined in a group of 163 Mycobacterium bovis -infected cattle from 21 herds. The specificity was estimated in a group of 213 cattle which had reacted to a caudal fold test, but were from 82 herds that had no evidence of infection with M bovis. The sensitivity and specificity of the IFN -gamma test was 85 and 93 per cent respectively. No significant differences in the sensitivity and specificity of the test were observed between blood samples that were cultured on the day of collection and those cultured the day after collection. These findings support the use of the IFN -gamma test as a practical serial test that can be used to complement the caudal fold skin test.     

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.     

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.     

An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds

Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.