- Email: [email protected]
The use of a Gamma-Interferon assay to confirm a diagnosis of bovine tuberculosis in Brazil
Acta Tropica 113 (2010) 199–201
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
The use of a Gamma-Interferon assay to confirm a diagnosis of bovine tuberculosis in Brazil Carla D. Marassi, Luciana Medeiros, Walter Lilenbaum ∗ Veterinary Bacteriology Laboratory, Universidade Federal Fluminense, R. Hernani Mello, 101, Niterói-RJ 24210-130, Brazil
a r t i c l e
i n f o
Article history: Received 20 July 2009 Received in revised form 16 September 2009 Accepted 3 October 2009 Available online 9 October 2009 Keywords: Diagnostic Tuberculosis IFN assay Cattle
a b s t r a c t Control of bovine tuberculosis (TB) is currently based on slaughter of cattle deemed positive on the basis of tuberculin testing; although the test has been broadly used for several years, there are some disadvantages such as the need for holding animals for 72 h. Besides it, test can lack both sensitivity and specificity depending on the interpretation and the site of the PPD inoculation. Therefore, the objective of the current study was to evaluate the use of the Gamma-Interferon (IFN) assay as a confirmatory test in Brazil. A Comparative Intradermal Tuberculin Test (CITT) was performed in 50 cows from a dairy herd known to be infected with TB. Blood samples for IFN testing were collected concurrent with the CITT, as well as seven and 21 days later. At 30 days after the CITT, all cattle deemed reactive to this test were slaughtered and samples were processed by both bacteriological culture and PCR. The sensitivity of IFN as a diagnostic tool was 91.4%, whereas specificity was 86.7%. When applied seven or 21 days after injection of PPD, sensitivity was 74.3 and 71.4%, respectively, whereas specificity was 86.7 and 80%. There was no significant difference between the reliability of the IFN assay and the other standards, on all three days of sampling. Therefore, we concluded that the IFN assay could be effectively used as a confirmatory test seven or 21 days after injection of PPD. © 2009 Elsevier B.V. All rights reserved.
Despite numerous control programs applied over many years, bovine tuberculosis (TB) remains a worldwide concern, affecting many countries, particularly those in the tropics, including Brazil (WHO, 2009). The most common diagnostic method, based on intradermal tests, has limitations regarding both sensitivity and specificity (Monaghan et al., 1994). Therefore, a reliable diagnostic method is required, not only for initial detection of infected animals, but also to confirm suspect cases. In some cases when intradermal tests are inconclusive, a definitive diagnosis is done by culture or PCR; however, those methods require tissue samples that could only be obtained post-mortem (Lilenbaum et al., 1999). Therefore, there is a clear need for noninvasive confirmatory tests. Immune responses against mycobacterial infections are predominantly cellular, at least initially (Wood and Jones, 2001). The Gamma-Interferon assay (IFN) measures the Gamma-IFN released in vitro in response to specific antigens in whole-blood culture (Wood et al., 1990). This assay has been evaluated as a primary diagnostic method in many countries (Wood and Jones, 2001), including Brazil (Lilenbaum et al., 1999), and has some important advantages. Since lymphocyte stimulation is done in vitro, it is not necessary to wait 60–90 days to repeat the test when the initial test is incon-
∗ Corresponding author at: Rua Hernani Mello, 101 sala 309, Niterói-RJ 24210130, Brazil. Tel.: +55 21 2629 2435; fax: +55 21 2629 2432. E-mail address: [email protected] (W. Lilenbaum). 0001-706X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2009.10.002
clusive (Lilenbaum et al., 1999). Unfortunately, this test is much more expensive than skin tests (Lilenbaum et al., 1999; Gormley et al., 2004). It has been recommended that cattle have not been given PPD for at least 60 days before IFN is conducted (Thom et al., 2004; Pollock et al., 2005), due to potential anamnestic effects. Nevertheless, Ryan et al. (2000) demonstrated that this assay could also be done with blood collected from tuberculin-sensitized cattle 8–28 days after the inoculation of PPD and that it could be used to complement the skin test. These apparent differences in results may be due to the variable conditions under which the studies were conducted (Palmer et al., 2006), including the nature and stage of infection, numbers of cattle used, skin protocols, and interpretation criteria (Gormley et al., 2004). Therefore, the purpose of the current study was to evaluate the use of the IFN assay, conducted on blood samples collected seven and 21 days after treatment with PPD, as a confirmatory test in a naturally infected dairy herd in Brazil. Study design: This study was conducted on a dairy herd comprised of 270 adult crossbred Holstein and Gir cows. Prior to the study, 21 adult cows had positive reactions to a Single Intradermal Tuberculin Test (SITT) and were kept in quarantine for 90 days, waiting for confirmatory tests to be conducted. After 90 days, a Comparative Intradermal Tuberculin Test (CITT) was performed in these 21 cows (Group A), plus 29 randomly selected cows that were negative to the first SITT test (Group B). Blood samples (for IFN testing) were collected from these 50 cows, at the time of the CITT, and seven and 21 days later. At 30 days after injection of PPD, all CITT-
200
C.D. Marassi et al. / Acta Tropica 113 (2010) 199–201
reactive cattle were slaughtered. From all 50 cattle, milk samples and nasal swabs were collected (on the day that PPD was injected) and subjected to bacteriological culture and PCR. Intradermal tests: Intradermal tests (both SITT and CITT) for tuberculosis diagnosis were performed on all 50 cows, in accordance with the regulations of the Brazilian Department of Agriculture. For SITT, 0.1 mL of bovine PPD (bovPPD–M. bovis strain AN5, 1 mg protein/mL; Instituto Biologico, SãoPaulo, SP, Brazil) was inoculated in the cervical area of each cow. After 72 h, the site of inoculation was measured with calipers, and the cow was considered reactive if a swelling >4.0 mm occurred at the injection site. The CITT consisted of the same procedure, plus an inoculation of 0.1 mL avium PPD (M. avium strain D4, 0.5 mg protein/mL; Instituto Biologico) in the cervical area, approximately 20 cm from the bovPPD inoculation. Cattle were considered reactive if the difference between the thicknesses of both sites of inoculation were >4.0 mm. Interferon-gamma assay (IFN): Heparinized blood samples from the 50 cows were collected for IFN testing just prior to injection of PPD, as well as seven and 21 days later. The assay was performed according to the manufacturer’s instructions (Bovigam, Prionics, Zurich, Switzerland), including the modifications recommended by Ryan et al. (2000) and with the same PPDs that had been used to perform the cattle tuberculinization. Evidence of M. bovis: Nasal swabs and milk samples were collected from all 50 cows on the day of the inoculation of PPD. All CITT-reactive cows were killed 30 days after injection of PPD, and a thorough necropsy was done. Mediastinal, scapular and retropharyngeal lymph nodes, as well as samples of lung, were collected. All samples were processed for both bacteriology and PCR. For bacteriologic culture to detect M. bovis, samples of tissue, milk and swabs were decontaminated according to standard methods and inoculated on two slopes of solid, egg-based media Lowenstein-Jensen with 0.5% pyruvate, and two slopes of Stonebrink media, which were incubated at 37 ◦ C and observed once weekly for 12 weeks. For evidence of M. bovis-specific DNA, a multiplex PCR targeting the RvD1Rv2031c genomic sequence was employed (Figueiredo et al., 2009). Statistical analysis: Sensitivity and specificity of IFN assay were calculated, considering culture isolation or PCR as standards. Alternatively, CITT results were also utilized in those cases where bacteriological culture was not conducted. Concordance between IFN and the standards was calculated by the kappa test. The total number of infected cows, according to the standards, was 35 (21/21 from Group A and 14/29 from Group B). Of these cattle, 27 were positive on the basis of bacterial culture or PCR (18 and nine from Groups A and B, respectively), whereas the remaining eight cows (three from Group A and five from Group B) were considered infected based exclusively on the CITT. At the moment of the first reading of the PPD inoculation (72 h post-inoculation), the CITT was positive for 21 and 11 cows in Groups A and B, respectively. For positive cows in these two groups, 20 of 21, and all 11, were IFN reactive. Additionally, IFN was positive on three cows from Group B which were CITT-negative; however, these three cows were positive on milk culture. The CITT-pos/IFNneg cow from Group A was culture positive and considered infected. Isolates of M. bovis were recovered from the milk of two CITTneg/IFN-neg cows from Group B. After seven days, 17 of 21 Group A cows were IFN reactive; the remaining four, including one that had been negative on first IFN testing, were all confirmed infected by culture, PCR, or both. Furthermore, 11 of 29 Group B cows were IFN positive; nine were considered infected, including five on the basis of culture, PCR, or both, and the other four based exclusively on CITT. Twenty-one days after injection of PPD, 15 of 21 Group A cows were IFN reactive. Of the remaining six, five were confirmed
infected by culture, PCR, or both. In Group B cows, IFN was positive in 13 of 29; ten were considered infected, six by culture, PCR, or both, and the remaining four by CITT only (Table 1). The sensitivity of IFN, when applied as a diagnostic tool, was 91.4%, whereas specificity was 86.7%. When this assay was applied seven or 21 days after the inoculation of the PPD, sensitivity was 74.3 and 71.4%, respectively, whereas specificity was 86.7 and 80%. Based on the Kappa test, there was no significant difference between the reliability of the IFN assay and the other standards, on all three days of sampling. Failure to detect all infected cattle is an important cause of persistence of TB in some herds (Gormley et al., 2004). When results of intradermal tests are inconclusive, it is necessary to wait at least 60 days before repeating the SITT or applying a CITT. This mandatory interval requires cattle to be kept in quarantine, and it increases the risk of spreading the disease to herdmates and potentially to humans. Even when comparative tests are employed as primary tests, false-positive results still occur (Monaghan et al., 1994). Due to federal laws requiring the slaughter of CITTreactive cattle, there is a need for a reliable confirmation test that could conducted soon after the first test. Ideally, the confirmatory test should be highly specific, thereby minimizing the unnecessary slaughtering of non-infected animals. Although tuberculin tests can lead to false-positive results, they are currently the official standard in Brazil and many other countries. Therefore, despite the possibility of including some non-infected animals in the study, it has been used in the present study as an alternative standard in order to reproduce the real conditions on filed trials. M. bovis was prevalent in the study herd, based on intradermal tests and IFN assay, and confirmed by culture and PCR. Nevertheless, although highly specific, neither bacterial culture, nor PCR from milk samples or nasal swabs, had acceptable sensitivities, and therefore cannot be recommended as confirmatory tests in live animals. Although bacteriology was positive on seven milk samples, bacteriological culture was negative for all nasal swabs of the 35 infected cows, whereas PCR was positive on only five and two, respectively. The latter results were not unexpected, since the sensitivity of PCR for detecting M. bovis infection in clinical specimens is limited, due to the requirement of samples with high bacillary concentrations (de la Rua Domenech et al., 2006). Unfortunately, milk and nasal swabs collected from live cattle tend to have low numbers of organisms, thereby limiting sensitivity (Mishra et al., 2005). When IFN was used as a confirmatory test in the present study, herd sensitivity was 74.3 and 71.4%, whereas specificity was 86.7 and 80% at seven and 21 days, respectively. Although the specificity was not high enough to be definitive, results were encouraging, especially if considered in context. From the 15 cows that were considered non-infected, following the standards (CITT or culture/PCR), two of them were reactive to the three IFN tests, on days zero, seven, and 21, although negative to standards. According to current knowledge regarding IFN, these cows may have been in the very early stages of the disease. In that regard, IFN detected infected cattle 90–150 days before they were SITT-reactive (Lilenbaum et al., 1999), and could detect M. bovis infection of cattle as soon as 14 days after inoculation (Buddle et al., 1995). Similarly, Scacchia et al. (2000) reported that 34% of IFN-positive, M. bovis culture positive cattle were skin test negative. Assuming that these cattle were recently infected, then specificity of IFN could be as high as 100% on day 7 and 92.3% on day 21. It is noteworthy that IFN was more sensitive and had a shorter interval from infection to testing positive than intradermal tests (Lilenbaum et al., 1999; Scacchia et al., 2000; Wood and Jones, 2001). However, due to its high cost, this assay has been recommended as an adjunct to the tuberculin test, particularly in herds
C.D. Marassi et al. / Acta Tropica 113 (2010) 199–201
201
Table 1 Results of SITT, CITT, ␥-IFN test (0, 7 and 21 days p.i.), bacteriological culture and PCR of 21 tuberculous cows from one herd in Rio de Janeiro, Brazil. Cow
SITT day (−) 90
CITT day zero
␥-IFN day zero
␥-IFN day 7 p.i.
␥-IFN day 21 p.i.
Culture
PCR
Conclusion
53 370 343 67 162 19 87 61 129 143 156 179 135 50 125 6 23 121 165 130 11
Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Reactive Negative Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Reactive Negative Negative Negative Negative Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Reactive Negative Negative Reactive Reactive Negative Reactive Reactive Reactive Negative Reactive Negative Reactive Negative Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Positive Positive Negative Positive Positive Negative Positive Positive Positive Positive Positive Negative Positive Negative Negative Negative Negative Negative Negative Negative Positive
Positive Negative Positive Positive Positive Positive Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive Negative Positive Positive Negative Positive
Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected
SITT: Single Intradermal Tuberculin Test; CITT: Comparative Intradermal Tuberculin Test; ␥-IFN: Gamma-Interferon assay; p.i.: post-inoculation.
with a high probability of containing infected cattle (Gormley et al., 2004). The use of IFN on skin-reactive cattle was suggested by Ryan et al. (2000). In their study, IFN had a sensitivity of 85% and specificity of 93% when employed 8–28 days after skin testing. Following that study, IFN was approved in New Zealand for this purpose, seven to 30 days after caudal fold testing; this substantially reduces the interval of mandatory confinement, and thereby reduces the risk of spread (Wood and Jones, 2001). Nevertheless, few other countries have adopted this approach, and the use of IFN as a confirmatory test for bovine TB remains restricted. In conclusion, based on the outcome of the current study, the IFN assay could be used in Brazil, and potentially other tropical countries, as a confirmatory test on skin-tested animals seven or 21 days after PPD inoculation (with apparently slightly better efficiency for the earlier test date). Due the results achieved in our study, we suggest that this test could be applied in cows with inconclusive results to an intradermal test, or in any cases when a reliable confirmation of the infection is required, thereby reducing the 60-day quarantine, minimizing spread of the disease, and enhancing the efficacy of programs to control TB. Further investigations in a larger group of animals must be performed in order to clarify possible logistic problems, and evaluate the financial advantages or costs before introducing the test in a routine of diagnosis in countries where bovine tuberculosis is still an endemic infection. Acknowledgements The authors are thankful to Prof. W. Oelemann and R. Duarte (UFRJ, Brazil) for their help with laboratory analyses, to Dr. J. Schetinni for assistance with field tests, and to Prof. J. Kastelic (Canada) for critical review and correction of the manuscript. The Bovigam kit was kindly provided by Dr. P. Wood (Pfizer). This study was supported by CNPq and FAPERJ, Brazil. References Buddle, B.M., de Lisle, G.W., Pfeffer, A., Aldwell, F.E., 1995. Immunological responses and protection against Mycobacterium bovis in calves vaccinated with a low dose of BCG. Vaccine 13 (12), 1123–1130.
de la Rua Domenech, R., Goodchild, A.T., Vordemeier, H.M., Hewinson, R.G., Christiansen, K.H., Clifon-Hadley, R.S., 2006. Ante mortem diagnosis in cattle: a review of the tuberculin tests, Gamma-interferon assay and other ancillary diagnostic techniques. Research in Veterinary Science 81, 190–210. Figueiredo, E., Silvestre, F.G., Campos, W.N., Furlanetto, L., Medeiros, L., Lilenbaum, W., Fonseca, L.S., Silva, J.T., Paschoalin, V., 2009. Identification of Mycobacterium bovis isolates by a multiplex PCR. Brazilian Journal of Microbiology 40, 231–233. Gormley, E., Doyle, M.B., McGill, K., Costello, E., Good, M., Collins, J.D., 2004. The effect of the tuberculin test and the consequences of a delay in blood culture on the sensitivity of a gamma-interferon assay for the detection of Mycobacterium bovis infection in cattle. Veterinary Immunology and Immunopathology 102 (4), 413–420. Lilenbaum, W., Schettinni, J., Souza, G.N., Ribeiro, E.R., Moreira, E.C., 1999. Comparison between a Gamma-IFN assay kit and intradermal tuberculin test for the diagnosis of bovine tuberculosis on field trials in Brazil. Journal of Veterinary Medicine: Series B 46 (5), 353–358. Mishra, A., Singhal, A., Chauhan, D.S., Katoch, V.M., Srivastava, K., Thakral, S.S., Bharadwaj, S.S., Sreenivas, V., Prasad, H.K., 2005. Direct detection and identification of Mycobacterium tuberculosis and Mycobacterium bovis in bovine samples by a novel nested PCR assay: correlation with conventional techniques. Journal of Clinical Microbiology 43 (11), 5670–5678. Monaghan, M.L., Doherty, M.L., Collins, J.D., Kazda, J.F., Quinn, P.J., 1994. The tuberculin test. Veterinary Microbiology 40 (1–2), 111–124. Palmer, M.V., Waters, W.R., Thacker, T.C., Greenwald, R., Esfandiari, J., Lyashenko, K.P., 2006. Effects of different tuberculin skin-testing regiments on gamma interferon and antibody responses in cattle experimentally infected with Mycobacterium bovis. Clinical and Vaccine Immunology (March), 387– 394. Pollock, J.M., Welsh, M.D., McNair, J., 2005. Immune response in bovine tuberculosis: towards new strategies for the diagnosis and control of disease. Veterinary Immunology and Immunopathology 108, 37–43. Ryan, T.J., Buddle, B.M., De Lisle, G.W., 2000. An evaluation of the gamma interferon test for detecting bovine tuberculosis in cattle 8 to 28 days after tuberculin skin testing. Research in Veterinary Science 69, 57–61. Scacchia, M., Lelli, R., Petrini, A., Prencipe, V., Calistri, P., Giovannini, A., 2000. Use of innovative methods in the eradication of bovine tuberculosis. Journal of Veterinary Medicine: Series B 47, 321–327. Thom, M., Morgan, J.H., Hope, J.C., Villarreal-Ramos, B., Martin, M., Howard, C.J., 2004. The effect of repeated tuberculin skin testing of cattle on immune responses and disease following experimental infection with Mycobacterium bovis. Veterinary Immunology and Immunopathology 102 (4), 399–412. WHO-World Health Organization, http://www.who.int/zoonoses/neglected zoonotic diseases/en/ (accessed on June/02/2009). Wood, P.R., Jones, S.L., 2001. BOVIGAMTM : an in vitro cellular diagnostic test for bovine tuberculosis. Tuberculosis 81 (1–2), 147–155. Wood, P.R., Rothel, J.S., McWathers, P.G.D., Jones, S.L., 1990. Production and characterization of monoclonal antibodies specific for bovine gamma-interferon. Veterinary Immunology and Immunopathology 25, 37–46.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
The use of a Gamma-Interferon assay to confirm a diagnosis of bovine tuberculosis in Brazil Carla D. Marassi, Luciana Medeiros, Walter Lilenbaum ∗ Veterinary Bacteriology Laboratory, Universidade Federal Fluminense, R. Hernani Mello, 101, Niterói-RJ 24210-130, Brazil
a r t i c l e
i n f o
Article history: Received 20 July 2009 Received in revised form 16 September 2009 Accepted 3 October 2009 Available online 9 October 2009 Keywords: Diagnostic Tuberculosis IFN assay Cattle
a b s t r a c t Control of bovine tuberculosis (TB) is currently based on slaughter of cattle deemed positive on the basis of tuberculin testing; although the test has been broadly used for several years, there are some disadvantages such as the need for holding animals for 72 h. Besides it, test can lack both sensitivity and specificity depending on the interpretation and the site of the PPD inoculation. Therefore, the objective of the current study was to evaluate the use of the Gamma-Interferon (IFN) assay as a confirmatory test in Brazil. A Comparative Intradermal Tuberculin Test (CITT) was performed in 50 cows from a dairy herd known to be infected with TB. Blood samples for IFN testing were collected concurrent with the CITT, as well as seven and 21 days later. At 30 days after the CITT, all cattle deemed reactive to this test were slaughtered and samples were processed by both bacteriological culture and PCR. The sensitivity of IFN as a diagnostic tool was 91.4%, whereas specificity was 86.7%. When applied seven or 21 days after injection of PPD, sensitivity was 74.3 and 71.4%, respectively, whereas specificity was 86.7 and 80%. There was no significant difference between the reliability of the IFN assay and the other standards, on all three days of sampling. Therefore, we concluded that the IFN assay could be effectively used as a confirmatory test seven or 21 days after injection of PPD. © 2009 Elsevier B.V. All rights reserved.
Despite numerous control programs applied over many years, bovine tuberculosis (TB) remains a worldwide concern, affecting many countries, particularly those in the tropics, including Brazil (WHO, 2009). The most common diagnostic method, based on intradermal tests, has limitations regarding both sensitivity and specificity (Monaghan et al., 1994). Therefore, a reliable diagnostic method is required, not only for initial detection of infected animals, but also to confirm suspect cases. In some cases when intradermal tests are inconclusive, a definitive diagnosis is done by culture or PCR; however, those methods require tissue samples that could only be obtained post-mortem (Lilenbaum et al., 1999). Therefore, there is a clear need for noninvasive confirmatory tests. Immune responses against mycobacterial infections are predominantly cellular, at least initially (Wood and Jones, 2001). The Gamma-Interferon assay (IFN) measures the Gamma-IFN released in vitro in response to specific antigens in whole-blood culture (Wood et al., 1990). This assay has been evaluated as a primary diagnostic method in many countries (Wood and Jones, 2001), including Brazil (Lilenbaum et al., 1999), and has some important advantages. Since lymphocyte stimulation is done in vitro, it is not necessary to wait 60–90 days to repeat the test when the initial test is incon-
∗ Corresponding author at: Rua Hernani Mello, 101 sala 309, Niterói-RJ 24210130, Brazil. Tel.: +55 21 2629 2435; fax: +55 21 2629 2432. E-mail address: [email protected] (W. Lilenbaum). 0001-706X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2009.10.002
clusive (Lilenbaum et al., 1999). Unfortunately, this test is much more expensive than skin tests (Lilenbaum et al., 1999; Gormley et al., 2004). It has been recommended that cattle have not been given PPD for at least 60 days before IFN is conducted (Thom et al., 2004; Pollock et al., 2005), due to potential anamnestic effects. Nevertheless, Ryan et al. (2000) demonstrated that this assay could also be done with blood collected from tuberculin-sensitized cattle 8–28 days after the inoculation of PPD and that it could be used to complement the skin test. These apparent differences in results may be due to the variable conditions under which the studies were conducted (Palmer et al., 2006), including the nature and stage of infection, numbers of cattle used, skin protocols, and interpretation criteria (Gormley et al., 2004). Therefore, the purpose of the current study was to evaluate the use of the IFN assay, conducted on blood samples collected seven and 21 days after treatment with PPD, as a confirmatory test in a naturally infected dairy herd in Brazil. Study design: This study was conducted on a dairy herd comprised of 270 adult crossbred Holstein and Gir cows. Prior to the study, 21 adult cows had positive reactions to a Single Intradermal Tuberculin Test (SITT) and were kept in quarantine for 90 days, waiting for confirmatory tests to be conducted. After 90 days, a Comparative Intradermal Tuberculin Test (CITT) was performed in these 21 cows (Group A), plus 29 randomly selected cows that were negative to the first SITT test (Group B). Blood samples (for IFN testing) were collected from these 50 cows, at the time of the CITT, and seven and 21 days later. At 30 days after injection of PPD, all CITT-
200
C.D. Marassi et al. / Acta Tropica 113 (2010) 199–201
reactive cattle were slaughtered. From all 50 cattle, milk samples and nasal swabs were collected (on the day that PPD was injected) and subjected to bacteriological culture and PCR. Intradermal tests: Intradermal tests (both SITT and CITT) for tuberculosis diagnosis were performed on all 50 cows, in accordance with the regulations of the Brazilian Department of Agriculture. For SITT, 0.1 mL of bovine PPD (bovPPD–M. bovis strain AN5, 1 mg protein/mL; Instituto Biologico, SãoPaulo, SP, Brazil) was inoculated in the cervical area of each cow. After 72 h, the site of inoculation was measured with calipers, and the cow was considered reactive if a swelling >4.0 mm occurred at the injection site. The CITT consisted of the same procedure, plus an inoculation of 0.1 mL avium PPD (M. avium strain D4, 0.5 mg protein/mL; Instituto Biologico) in the cervical area, approximately 20 cm from the bovPPD inoculation. Cattle were considered reactive if the difference between the thicknesses of both sites of inoculation were >4.0 mm. Interferon-gamma assay (IFN): Heparinized blood samples from the 50 cows were collected for IFN testing just prior to injection of PPD, as well as seven and 21 days later. The assay was performed according to the manufacturer’s instructions (Bovigam, Prionics, Zurich, Switzerland), including the modifications recommended by Ryan et al. (2000) and with the same PPDs that had been used to perform the cattle tuberculinization. Evidence of M. bovis: Nasal swabs and milk samples were collected from all 50 cows on the day of the inoculation of PPD. All CITT-reactive cows were killed 30 days after injection of PPD, and a thorough necropsy was done. Mediastinal, scapular and retropharyngeal lymph nodes, as well as samples of lung, were collected. All samples were processed for both bacteriology and PCR. For bacteriologic culture to detect M. bovis, samples of tissue, milk and swabs were decontaminated according to standard methods and inoculated on two slopes of solid, egg-based media Lowenstein-Jensen with 0.5% pyruvate, and two slopes of Stonebrink media, which were incubated at 37 ◦ C and observed once weekly for 12 weeks. For evidence of M. bovis-specific DNA, a multiplex PCR targeting the RvD1Rv2031c genomic sequence was employed (Figueiredo et al., 2009). Statistical analysis: Sensitivity and specificity of IFN assay were calculated, considering culture isolation or PCR as standards. Alternatively, CITT results were also utilized in those cases where bacteriological culture was not conducted. Concordance between IFN and the standards was calculated by the kappa test. The total number of infected cows, according to the standards, was 35 (21/21 from Group A and 14/29 from Group B). Of these cattle, 27 were positive on the basis of bacterial culture or PCR (18 and nine from Groups A and B, respectively), whereas the remaining eight cows (three from Group A and five from Group B) were considered infected based exclusively on the CITT. At the moment of the first reading of the PPD inoculation (72 h post-inoculation), the CITT was positive for 21 and 11 cows in Groups A and B, respectively. For positive cows in these two groups, 20 of 21, and all 11, were IFN reactive. Additionally, IFN was positive on three cows from Group B which were CITT-negative; however, these three cows were positive on milk culture. The CITT-pos/IFNneg cow from Group A was culture positive and considered infected. Isolates of M. bovis were recovered from the milk of two CITTneg/IFN-neg cows from Group B. After seven days, 17 of 21 Group A cows were IFN reactive; the remaining four, including one that had been negative on first IFN testing, were all confirmed infected by culture, PCR, or both. Furthermore, 11 of 29 Group B cows were IFN positive; nine were considered infected, including five on the basis of culture, PCR, or both, and the other four based exclusively on CITT. Twenty-one days after injection of PPD, 15 of 21 Group A cows were IFN reactive. Of the remaining six, five were confirmed
infected by culture, PCR, or both. In Group B cows, IFN was positive in 13 of 29; ten were considered infected, six by culture, PCR, or both, and the remaining four by CITT only (Table 1). The sensitivity of IFN, when applied as a diagnostic tool, was 91.4%, whereas specificity was 86.7%. When this assay was applied seven or 21 days after the inoculation of the PPD, sensitivity was 74.3 and 71.4%, respectively, whereas specificity was 86.7 and 80%. Based on the Kappa test, there was no significant difference between the reliability of the IFN assay and the other standards, on all three days of sampling. Failure to detect all infected cattle is an important cause of persistence of TB in some herds (Gormley et al., 2004). When results of intradermal tests are inconclusive, it is necessary to wait at least 60 days before repeating the SITT or applying a CITT. This mandatory interval requires cattle to be kept in quarantine, and it increases the risk of spreading the disease to herdmates and potentially to humans. Even when comparative tests are employed as primary tests, false-positive results still occur (Monaghan et al., 1994). Due to federal laws requiring the slaughter of CITTreactive cattle, there is a need for a reliable confirmation test that could conducted soon after the first test. Ideally, the confirmatory test should be highly specific, thereby minimizing the unnecessary slaughtering of non-infected animals. Although tuberculin tests can lead to false-positive results, they are currently the official standard in Brazil and many other countries. Therefore, despite the possibility of including some non-infected animals in the study, it has been used in the present study as an alternative standard in order to reproduce the real conditions on filed trials. M. bovis was prevalent in the study herd, based on intradermal tests and IFN assay, and confirmed by culture and PCR. Nevertheless, although highly specific, neither bacterial culture, nor PCR from milk samples or nasal swabs, had acceptable sensitivities, and therefore cannot be recommended as confirmatory tests in live animals. Although bacteriology was positive on seven milk samples, bacteriological culture was negative for all nasal swabs of the 35 infected cows, whereas PCR was positive on only five and two, respectively. The latter results were not unexpected, since the sensitivity of PCR for detecting M. bovis infection in clinical specimens is limited, due to the requirement of samples with high bacillary concentrations (de la Rua Domenech et al., 2006). Unfortunately, milk and nasal swabs collected from live cattle tend to have low numbers of organisms, thereby limiting sensitivity (Mishra et al., 2005). When IFN was used as a confirmatory test in the present study, herd sensitivity was 74.3 and 71.4%, whereas specificity was 86.7 and 80% at seven and 21 days, respectively. Although the specificity was not high enough to be definitive, results were encouraging, especially if considered in context. From the 15 cows that were considered non-infected, following the standards (CITT or culture/PCR), two of them were reactive to the three IFN tests, on days zero, seven, and 21, although negative to standards. According to current knowledge regarding IFN, these cows may have been in the very early stages of the disease. In that regard, IFN detected infected cattle 90–150 days before they were SITT-reactive (Lilenbaum et al., 1999), and could detect M. bovis infection of cattle as soon as 14 days after inoculation (Buddle et al., 1995). Similarly, Scacchia et al. (2000) reported that 34% of IFN-positive, M. bovis culture positive cattle were skin test negative. Assuming that these cattle were recently infected, then specificity of IFN could be as high as 100% on day 7 and 92.3% on day 21. It is noteworthy that IFN was more sensitive and had a shorter interval from infection to testing positive than intradermal tests (Lilenbaum et al., 1999; Scacchia et al., 2000; Wood and Jones, 2001). However, due to its high cost, this assay has been recommended as an adjunct to the tuberculin test, particularly in herds
C.D. Marassi et al. / Acta Tropica 113 (2010) 199–201
201
Table 1 Results of SITT, CITT, ␥-IFN test (0, 7 and 21 days p.i.), bacteriological culture and PCR of 21 tuberculous cows from one herd in Rio de Janeiro, Brazil. Cow
SITT day (−) 90
CITT day zero
␥-IFN day zero
␥-IFN day 7 p.i.
␥-IFN day 21 p.i.
Culture
PCR
Conclusion
53 370 343 67 162 19 87 61 129 143 156 179 135 50 125 6 23 121 165 130 11
Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Reactive Negative Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Reactive Negative Negative Negative Negative Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Reactive Negative Negative Reactive Reactive Negative Reactive Reactive Reactive Negative Reactive Negative Reactive Negative Reactive Reactive Reactive Reactive Reactive Reactive Reactive
Positive Positive Negative Positive Positive Negative Positive Positive Positive Positive Positive Negative Positive Negative Negative Negative Negative Negative Negative Negative Positive
Positive Negative Positive Positive Positive Positive Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive Negative Positive Positive Negative Positive
Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected Infected
SITT: Single Intradermal Tuberculin Test; CITT: Comparative Intradermal Tuberculin Test; ␥-IFN: Gamma-Interferon assay; p.i.: post-inoculation.
with a high probability of containing infected cattle (Gormley et al., 2004). The use of IFN on skin-reactive cattle was suggested by Ryan et al. (2000). In their study, IFN had a sensitivity of 85% and specificity of 93% when employed 8–28 days after skin testing. Following that study, IFN was approved in New Zealand for this purpose, seven to 30 days after caudal fold testing; this substantially reduces the interval of mandatory confinement, and thereby reduces the risk of spread (Wood and Jones, 2001). Nevertheless, few other countries have adopted this approach, and the use of IFN as a confirmatory test for bovine TB remains restricted. In conclusion, based on the outcome of the current study, the IFN assay could be used in Brazil, and potentially other tropical countries, as a confirmatory test on skin-tested animals seven or 21 days after PPD inoculation (with apparently slightly better efficiency for the earlier test date). Due the results achieved in our study, we suggest that this test could be applied in cows with inconclusive results to an intradermal test, or in any cases when a reliable confirmation of the infection is required, thereby reducing the 60-day quarantine, minimizing spread of the disease, and enhancing the efficacy of programs to control TB. Further investigations in a larger group of animals must be performed in order to clarify possible logistic problems, and evaluate the financial advantages or costs before introducing the test in a routine of diagnosis in countries where bovine tuberculosis is still an endemic infection. Acknowledgements The authors are thankful to Prof. W. Oelemann and R. Duarte (UFRJ, Brazil) for their help with laboratory analyses, to Dr. J. Schetinni for assistance with field tests, and to Prof. J. Kastelic (Canada) for critical review and correction of the manuscript. The Bovigam kit was kindly provided by Dr. P. Wood (Pfizer). This study was supported by CNPq and FAPERJ, Brazil. References Buddle, B.M., de Lisle, G.W., Pfeffer, A., Aldwell, F.E., 1995. Immunological responses and protection against Mycobacterium bovis in calves vaccinated with a low dose of BCG. Vaccine 13 (12), 1123–1130.
de la Rua Domenech, R., Goodchild, A.T., Vordemeier, H.M., Hewinson, R.G., Christiansen, K.H., Clifon-Hadley, R.S., 2006. Ante mortem diagnosis in cattle: a review of the tuberculin tests, Gamma-interferon assay and other ancillary diagnostic techniques. Research in Veterinary Science 81, 190–210. Figueiredo, E., Silvestre, F.G., Campos, W.N., Furlanetto, L., Medeiros, L., Lilenbaum, W., Fonseca, L.S., Silva, J.T., Paschoalin, V., 2009. Identification of Mycobacterium bovis isolates by a multiplex PCR. Brazilian Journal of Microbiology 40, 231–233. Gormley, E., Doyle, M.B., McGill, K., Costello, E., Good, M., Collins, J.D., 2004. The effect of the tuberculin test and the consequences of a delay in blood culture on the sensitivity of a gamma-interferon assay for the detection of Mycobacterium bovis infection in cattle. Veterinary Immunology and Immunopathology 102 (4), 413–420. Lilenbaum, W., Schettinni, J., Souza, G.N., Ribeiro, E.R., Moreira, E.C., 1999. Comparison between a Gamma-IFN assay kit and intradermal tuberculin test for the diagnosis of bovine tuberculosis on field trials in Brazil. Journal of Veterinary Medicine: Series B 46 (5), 353–358. Mishra, A., Singhal, A., Chauhan, D.S., Katoch, V.M., Srivastava, K., Thakral, S.S., Bharadwaj, S.S., Sreenivas, V., Prasad, H.K., 2005. Direct detection and identification of Mycobacterium tuberculosis and Mycobacterium bovis in bovine samples by a novel nested PCR assay: correlation with conventional techniques. Journal of Clinical Microbiology 43 (11), 5670–5678. Monaghan, M.L., Doherty, M.L., Collins, J.D., Kazda, J.F., Quinn, P.J., 1994. The tuberculin test. Veterinary Microbiology 40 (1–2), 111–124. Palmer, M.V., Waters, W.R., Thacker, T.C., Greenwald, R., Esfandiari, J., Lyashenko, K.P., 2006. Effects of different tuberculin skin-testing regiments on gamma interferon and antibody responses in cattle experimentally infected with Mycobacterium bovis. Clinical and Vaccine Immunology (March), 387– 394. Pollock, J.M., Welsh, M.D., McNair, J., 2005. Immune response in bovine tuberculosis: towards new strategies for the diagnosis and control of disease. Veterinary Immunology and Immunopathology 108, 37–43. Ryan, T.J., Buddle, B.M., De Lisle, G.W., 2000. An evaluation of the gamma interferon test for detecting bovine tuberculosis in cattle 8 to 28 days after tuberculin skin testing. Research in Veterinary Science 69, 57–61. Scacchia, M., Lelli, R., Petrini, A., Prencipe, V., Calistri, P., Giovannini, A., 2000. Use of innovative methods in the eradication of bovine tuberculosis. Journal of Veterinary Medicine: Series B 47, 321–327. Thom, M., Morgan, J.H., Hope, J.C., Villarreal-Ramos, B., Martin, M., Howard, C.J., 2004. The effect of repeated tuberculin skin testing of cattle on immune responses and disease following experimental infection with Mycobacterium bovis. Veterinary Immunology and Immunopathology 102 (4), 399–412. WHO-World Health Organization, http://www.who.int/zoonoses/neglected zoonotic diseases/en/ (accessed on June/02/2009). Wood, P.R., Jones, S.L., 2001. BOVIGAMTM : an in vitro cellular diagnostic test for bovine tuberculosis. Tuberculosis 81 (1–2), 147–155. Wood, P.R., Rothel, J.S., McWathers, P.G.D., Jones, S.L., 1990. Production and characterization of monoclonal antibodies specific for bovine gamma-interferon. Veterinary Immunology and Immunopathology 25, 37–46.