Diagnosis of Mycobacterium bovis infection in cattle by use of the gamma-interferon (Bovigam) assay

The strategic use of the gamma-interferon (IFN-gamma) assay (Bovigam) can provide a means for the early identification of Mycobacterium bovis infected cattle, thus ensuring their removal from an infected herd. When used in parallel with the tuberculin test, it is capable of identifying infected cattle, which might otherwise not be detected until later, if at all. The early detection and removal of these animals reduces the risk that they will become a source of infection for other cattle. When targeted in herds of high prevalence the benefits to the herd owner directly concerned can be considerable as the assay provides a means of shortening the period of restriction for such herds. This serves to generate confidence among herd owners and other stakeholders that effective schemes, based on sound scientific principles, can be developed to eradicate tuberculosis from infected cattle populations.     

The effect of repeated tuberculin skin testing of cattle on immune responses and disease following experimental infection with Mycobacterium bovis

The comparative intradermal skin test, in which a delayed type hypersensitivity (DTH) response to purified protein derivative of tuberculin (PPD) from Mycobacterium bovis and M. avium is assessed and compared, may be used repeatedly on non-infected animals on farms where bovine tuberculosis (TB) has occurred. A skin test is known to affect subsequent skin tests in infected animals. The reported study was to determine whether repeated skin testing prior to infection with M. bovis might affect the development of the comparative skin test and IFNgamma response subsequent to exposure to virulent M. bovis. The comparative intradermal skin test was applied to one group of six calves five times at 8-week intervals. These and six control calves were subsequently inoculated intratracheally with a dose of M. bovis that produced mild disease. The development of the DTH reaction, IFNgamma, IL-10 and proliferative responses were compared in the two groups of animals. No differences in IFNgamma, IL-10 and proliferative responses were seen between the two groups of calves prior to challenge. After infection with M. bovis no differences in the development of the DTH and IFNgamma responses to PPD were noted as a consequence of the repeated skin testing prior to challenge. No differences between the groups were evident when ESAT-6 was used as antigen and IFNgamma was assayed, although two animals that responded to PPD did not respond with ESAT-6. However, there did appear to be subtle effects of repeated skin testing on the immune response post-challenge that did not affect the diagnostic tests. After challenge control animals showed greater proliferative responses than animals given repeated skin tests prior to challenge, indicating that the procedure did have consequences for immune responses following infection. In both groups a marked reduction in the intensity of the skin test and in the number of animals that would be recognized as reactors was evident when animals were tested 15 weeks post-infection compared to their responses 8 weeks earlier that could have consequences for diagnosis of TB. An antibody response was not evident as a result of repeat skin testing prior to infection but was seen in both groups of calves following skin testing performed 7 weeks after infection.     

The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle: conditions affecting the production of gamma-interferon in whole blood culture

The recently developed gamma-interferon (IFN-gamma) assay system for the diagnosis of bovine tuberculosis in cattle has been accredited by the Standing Committee on Agriculture for use in Australia. In this test system, whole blood is incubated with tuberculin purified protein derivative (PPD) antigens for 16 to 24 h. The plasma is then collected and assayed for IFN-gamma production using an enzyme immunoassay (EIA). The assay system has proven to be a rapid, sensitive and inexpensive method for measuring antigen specific cell-mediated reactivity when compared with the more traditional lymphocyte proliferation assay. The IFN-gamma assay is the first in-vitro cellular assay to be used as a routine diagnostic test in veterinary medicine. While the IFN-gamma EIA has been optimised, several conditions affecting the production of IFN-gamma in whole blood culture needed investigation. We determined that optimal IFN-gamma production required the use of heparinised blood, cultured with 20 micrograms/ml of PPD within 8 h of collection. The use of blood collected post mortem resulted in reduced sensitivity for the assay. The kinetics of IFN-gamma release were established as were the effects of intradermal tuberculin testing on the IFN-gamma assay.     

Mycobacterium bovis infection and tuberculosis in cattle

This review considers the possible events that can occur when cattle are exposed to Mycobacterium bovis and, where appropriate, draws on principles accepted for tuberculosis infection in humans and laboratory animal models. Consideration is given to the many complex factors which influence the outcome of challenge with tubercle bacilli. These include features inherent to the mycobacterium, the host and the environment. It is apparent that clinical disease probably occurs only in a relatively small, but undetermined, proportion of cattle that are exposed to Al. bovis. The majority of animals may clear infection or control the bacilli, possibly in a condition of latency. It is concluded that a better understanding of the dynamics of the events following M. bovis exposure and subsequent infection in cattle would be of significant benefit in developing new tools appropriate for disease control and to designing optimal approaches for their application.     

The effect of tuberculin testing on the development of cell-mediated immune responses during Mycobacterium bovis infection

Protection against tuberculosis (TB) is associated with Th1-type cell-mediated immunity (CMI). Whilst the intradermal injection of partially purified derivatives of tuberculin (PPD) represents the classic test assessing the delayed type hypersensitivity (DTH) response used in both humans and cattle for diagnosing TB, it has been suggested that the test may modulate host CMI responses. To investigate the kinetics of the development of the DTH response and its subsequent effect on CMI responses, groups of 6-month old calves were inoculated intranasally with 8 x 10(4) cfu of Mycobacterium bovis, subjected to the comparative intradermal tuberculin test (TT) using bovine and avian PPD (PPD-B, PPD-A) at various time intervals post-infection, and immune responses compared. These included DTH, lymphocyte proliferation, IgG production, and synthesis of the cytokines: IFNgamma, IL-10, IL-4, IL-6, and IL-13. All animals were subjected to post-mortem examination. The kinetics of the development of the DTH response assessed in the TT was such that infected cattle could be identified as early as 3 weeks post-infection, which correlated with the detection of an antigen-specific IFNgamma response. Transient increases in plasma-derived IFNgamma as a result of TT during an established TB infection were more pronounced when blood was stimulated with PPD-A compared with PPD-B stimulation. This has the potential to mask diagnosis of infection as a result of the stronger avian-bias if the IFNgamma test is used the week following TT. Disease pathology was not affected by TT. A transient failure to a second TT was observed in 1 of 30 animals and the time (post-infection) at which the TT is administered may be of significance. In serum, IgG responses to PPD-B, which were undetectable prior to TT, were elevated after TT and were most pronounced in cattle that were TT at 6 weeks post-infection. Other cytokines were also affected by the TT; IL-4 mRNA levels increased and IL-6 mRNA levels decreased, whilst PPD-B specific IL-10 protein synthesis was enhanced. These observations may offer the potential for further diagnostic assays that could complement the TT and IFNgamma test.     

The transmission of Mycobacterium bovis infection to cattle

The prevalence of Mycobacterium bovis infection in cattle is increasing rapidly in some countries, including the UK and Ireland. The organism infects a wide range of mammalian hosts, and eradication of the disease is difficult if there is an extensive reservoir in the wildlife population. Existing evidence suggests that wildlife vectors include the European badger in the UK and Ireland, the brush-tailed possum and ferret in New Zealand and ungulates in some other countries. Cattle grazing field boundaries or short swards are at particularly high risk, since the chance of contact with the intermediate host or their excreta is increased. There is evidence that the transmission of the disease between cattle following movement accounts for 10-15% of outbreaks in the British Isles and that transmission can occur across farm boundaries. The prevalence the prevalence of single reactors in herds suggested that within-herd transmission was not common. In herds with infected cattle, spreading slurry is a risk factor, which can be minimised by prolonged storage of the slurry, by spreading it on fields not used for grazing or by soil injection. M. bovis also survives in water and may enter the respiratory tract during drinking. It is concluded that M. bovis infection in cattle can be transmitted by a number of routes, some of which can be controlled by appropriate husbandry, but that circumstantial evidence suggests that the existence of a widespread intermediate host is the greatest contributor to infection in cattle.     

A fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in cattle sera

A fluorescence polarization assay (FPA) utilizing fluorescein-labelled MPB70 protein as the antigen was developed and evaluated for its ability to detect antibodies to Mycobacterium bovis in cattle sera. Three panels of sera were examined in this study. These included: (A) sera (n=28) obtained from cattle from which M. bovis was cultured; (B) sera (n=5666) from Canadian field cattle which were presumed to be free from M. bovis; (C) sera (n=10) from cattle infected with Mycobacterium paratuberculosis and known to contain antibodies to this organism. Receiver operating characteristic (ROC) curve analysis of the results of panels A and B yielded an area under the curve value of 0.975 (95% confidence interval=0.971-0.979), which indicated that this FPA is an accurate indicator of M. bovis infection. At the cut-off point recommended by the ROC curve analysis, the FPA sensitivity and specificity estimates were 92.9% (95% confidence interval=76.5-98.9%) and 98.3% (95% confidence interval=97.9-98.6%) respectively. The FPA results were compared to the results of the single intradermal (SID) test for the 28 infected cattle. Fifteen of these animals were scored positive with the SID test (sensitivity=53.6%). The FPA detected 15/15 (100%) of the SID test-positive animals and 11/13 (84.6%) of the SID test-negative animals. Two of the culture-positive cattle were not detected by either test. None of the sera that were obtained from the M. paratuberculosis-infected animals cross-reacted in this assay.     

Assessment of an enzyme linked immunosorbent assay for the detection of cattle infected with Mycobacterium bovis

An indirect enzyme linked immunosorbent assay (ELISA) using an unpurified antigen was assessed for its accuracy in detecting Mycobacterium bovis infected cattle. The ELISA test recorded sensitivities of 88.7% and 63%, respectively, for infected cattle tuberculin tested positive and for infected cattle never tuberculin tested. Specificity was determined at 52.6% for cattle from confirmed free herds which had never been tuberculin tested. Significant differences in the mean ELISA values were recorded between the 3 groups. No evidence was found for long term effects of tuberculin testing upon the titre of antibodies detected by the ELISA in unaffected cattle. The indirect ELISA using the unpurified antigen of this assay was considered to be unsuitable as an alternative to tuberculin testing for the detection of M. bovis infected animals.     

Pulmonary lesions and Mycobacterium bovis excretion from the respiratory tract of tuberculin reacting cattle

Although it is generally recognised that tuberculous lesions are present in lymph nodes associated with the respiratory tract in approximately 90 per cent of reactors with confirmed infection, lung lesions are found in only 1 to 2 per cent of such cases during abattoir examination. When lung lesions are not detected, it has been claimed that such cattle are non-excretors and thus unimportant in the epidemiology of the disease. In this study the lungs of 55 reactor cattle were sliced into sections approximately 0.5 cm thick. Tuberculous lesions were evident in over 70 per cent of lungs from reactors with concurrent lesions in lymph nodes of the respiratory system. Further, M bovis was isolated from single samples of nasal and, or, tracheal mucus taken at slaughter in 19 per cent of confirmed cases. Several of these reactors had a clear tuberculin test less than six months previously indicating recent infection. This study confirms the continued importance of the infected bovine in the epidemiology and current eradication of bovine tuberculosis. It is suggested that all tuberculous cattle with lesions in respiratory lymph nodes, rather than being regarded as non-excretors, should be considered as possible excretors and thus important sources of infection for other cattle both within and between herds.     

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.     

Method for evaluation of Mycobacterium bovis purified protein derivative tuberculin in experimentally infected cattle.

A method for the evaluation of Mycobacterium bovis purified protein derivative (PPD) tuberculin in experimentally infected cattle is presented. The development of skin test responses in M bovis-infected cattle was determined for International Standard PPD-S, M bovis PPD-2, and M bovis PPD-5 at 24, 48, and 72 hours. Significantly larger reactions (dermal thickness) were observed at 48 and 72 hours than at 24 hours (P = 0.001). Statistically significant differences were not detected in the responses obtained with M bovis PPD-2, M bovis PPD-5, and International Standard PPD-S if comparisons were made at approximately the same concentrations in M bovis-infected cattle (P greater than 0.25). In Mycobacterium avium-infected cattle, M bovis PPD-2 produced skin test responses that were significantly smaller than responses obtained using M avium PPD-2 (P = 0.001). Significant variation was not observed in the PPD-S responses in 2 groups of M bovis-infected cattle (P greater than 0.1).     

Development of a loop-mediated isothermal amplification assay for the detection of Mycobacterium bovis

Bovine tuberculosis caused by Mycobacterium bovis is an important zoonosis. In this study, a simple, rapid method for detecting this organism was developed based on loop-mediated isothermal amplification of the mpt83 gene. The technique will be of value in the clinical and field-based diagnosis of M. bovis and can differentiate it from other bacteria such as Corynebacterium diphtheriae, Streptococcus pneumoniae, β-haemolytic streptococci, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Staphylococcus aureus, and Klebsiella pneumoniae. The limit of detection was 10 copies/μL and the results were corroborated by PCR. The method was highly specific and more sensitive than PCR.     

Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n=1532), white-tailed deer (n=463), brushtail possums (n=129), and wild boar (n=177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.     

Agreement between the caudal fold test and serological tests for the detection of Mycobacterium bovis infection in bison

The objective of this study was to estimate agreement between the caudal fold test (CFT) and different serological tests for the detection of Mycobacterium bovis infection in bison by using prevalence-adjusted bias-adjusted kappa (PABAK). A total of 212 of wild wood bison from Wood Buffalo National Park were tested with the CFT as well as several serological tests: fluorescent polarization assay (FPA), multiantigen print immunoassay (MAPIA), rapid lateral-flow test (RT) and dual path platform test (DPP). For RT, 3 variations were conducted using 30μl of serum (RT 30), 20μl of serum (RT 20) and 20μl of serum considering only a strong reaction as positive (RT 20 ST). The McNemar's χ(2) test was conducted to assess whether the proportion of positive test results to 2 different tests differed. Two measures of agreement between pair of tests were estimated: the Cohen's kappa statistic and PABAK. The apparent prevalence of tuberculosis in the sampled animals varied depending on the diagnostic test from 6.1% (FPA and DPP) to 47.2% (CFT). The prevalence estimated by CFT differed from the prevalence estimated by the other tests, whereas the prevalence estimated by FPA, MAPIA, RT 20 ST and DPP were not significantly different. The kappa and PABAK estimates calculated between CFT and the rest of the tests suggested poor to slight agreement between tests (k and PABAK<0.25 in all cases). The PABAK estimates for the pairwise combinations among serological tests were numerically greater than the kappa estimates (and significantly greater when FPA was compared to the rest of serological tests), and suggested substantial to almost perfect agreement (PABAK>0.75 in all cases). The disagreement between the skin and serological tests for the detection of M. bovis infection could be partly because the tests measure different immunological responses (cell-mediated vs. humoral) that are predominant at different stages of the infection, and partly due to inaccuracy of the tests. Further research is needed to evaluate the accuracy of diagnostic tests in order to establish a reliable case definition, combining different tests, to be used in the surveillance and control of tuberculosis in free-ranging bison populations.     

Development of a whole blood lymphocyte stimulation assay for detecting hypersensitivity to PPD in cattle experimentally infected with Mycobacterium bovis

A whole blood lymphocyte stimulation assay utilizing the uptake of tritiated thymidine was developed for the detection of Mycobacterium bovis sensitivity in cattle. Results on eight M. bovis infected animals (six to ten weeks after infection) and eight control animals show that satisfactory lymphocyte stimulation can be obtained using heparinized whole blood diluted 10-fold in tissue culture medium and cultured with purified protein derivative (PPD) for three to seven days. Infected animals exhibited significantly greater stimulation when cultured with PPD than did control animals.     

The development and evaluation of an enzyme-linked immunosorbent assay for the detection of Mycobacterium bovis

A double antibody sandwich layer enzyme-linked immunosorbent assay (ELISA) was used to detect Mycobacterium bovis. The ELISA detected M. bovis is pure culture at concentrations of 1 x 10(5) colony-forming units (CFU) ml-1 and greater, compared to a minimum detection level of 1 x 10(6) CFU ml-1 for isolation techniques. Neither technique detected M. bovis at 1 x 10(4) CFU ml-1. The ELISA did not cross-react with common mycobacterial contaminants such as Mycobacterium avium intracellulare-scrofulaceum complex serotypes 18 and 42, M. terrae, M. fortuitum, M. flavescens and with Escherichia coli or Rhodococcus equi. Further work is needed to evaluate this assay in detecting M. bovis in tissues and the environment.     

Enzyme-linked immunosorbent assay for the detection of antibodies to Hypoderma bovis in cattle

An adaptation of the enzyme-linked immunosorbent assay has been shown to detect antibodies in the circulation of cattle infected with Hypoderma bovis. Strong positive reactions were obtained from all infected cattle, even those which harboured only one larva. A strong cross reaction was observed in sera taken from cattle infected with H lineatum, but not in cattle infected with Fasciola hepatica or Ostertagia ostertagi.     

Intravenous johnin and tuberculin tests in cattle vaccinated with Mycobacterium paratuberculosis cells and subsequently inoculated with Mycobacterium bovis.

Calves at 30 days of age were vaccinated with a killed whole-cell Mycobacterium paratuberculosis vaccine. Four months later, these calves were inoculated with Mycobacterium bovis. The intravenous tuberculin and johnin tests were applied both before and after inoculation. The results of the hematologic investigation had extremes at both high and low values and were too unsuitable for statistical analysis. The intravenous tuberculin test is considered unsuitable for diagnosis of bovine tuberculosis in cattle vaccinated against paratuberculosis.