Tuberculosis in cattle is of major economic importance because of livestock trade barriers, resources expended on bovine tuberculosis surveillance, indemnity payments for the depopulation of tuberculosis-infected herds, and costs for other measures of tuberculosis eradication.1,2 The causative agent of tuberculosis in cattle, Mycobacterium bovis, is an important zoonotic pathogen and is one of the most important public health issues globally.1,3–6 Because of challenges to the global economy, there are concerns that budget cuts to bovine tuberculosis surveillance programs could hinder efforts to eradicate tuberculosis in cattle. Therefore, it has become important to develop bovine tuberculosis surveillance strategies that are inexpensive. A cost-saving approach would be to adapt bovine tuberculosis surveillance at POCs of cattle, such as slaughterhouses or livestock markets, with the goal of conducting regional surveillance at a single location instead of making visits to numerous farms. This approach would save considerable time and money.
Intradermal tuberculin testing and the IFN-γ assay are the most commonly used tuberculosis surveillance tests in cattle.7 Intradermal tuberculin testing requires that 2 contacts are made with each animal by a veterinarian before a presumptive diagnosis is made, which makes this a costly screening method2 and not suitable for POC screening. However, the IFN-γ assay requires only 1 contact with an animal, which makes it adaptable for POC screening.
The IFN-γ assay is an internationally accepted ante-mortem in vitro screening test for tuberculosis that measures IFN-γ released by lymphocytes in response to antigen stimulation.8 In addition to being adaptable to POC screening, the IFN-γ assay has some other advantages. Single-contact testing with the IFN-γ assay can reduce the cost of labor and other resources involved in tuberculosis screening,7 and several reports7,9–17 indicate that the IFN-γ assay is equivalent to or better than intradermal skin tests for detection of early-stage infection with M bovis in cattle.
A convenient place for POC screening for diseases in livestock is at slaughter. For example, in the United States, tuberculosis screening in cattle is conducted at slaughter by gross examination of carcasses and blood is collected during exsanguination for brucellosis screening.18 Use of the IFN-γ assay on blood collected during slaughter might facilitate screening for tuberculosis. It has been reported19 that the bovine IFN-γ response to stimulation with the tuberculosis antigen decreases by at least 50% when blood is collected from an exposed jugular vein ≥ 5 minutes after slaughter, compared with the response when blood is collected before slaughter. However, it is unclear from that study19 the specific time point after slaughter at which the sample with the decreased IFN-γ response was collected, whether the decrease affected the subsequent characterization of the animal as having positive or negative results for tuberculosis, and whether the slaughtering process was conducted in accordance with the standard operating protocol of slaughter facilities. Filling these knowledge gaps would aid understanding of any usefulness of the IFN-γ assay for POC screening of cattle for tuberculosis at slaughter.
We hypothesized that in cattle immunologically challenged with inactivated M bovis, the IFN-γ response after antigen stimulation would not differ significantly between blood samples collected at slaughter before stunning and at commencement of exsanguination. The objective of the study reported here was to determine whether the IFN-γ response to M bovis sufficient to categorize cattle as positive for tuberculosis can be detected in blood collected at commencement of exsanguination. The specific purposes were to determine whether there were categorical changes in interpretation of results for the IFN-γ assay between samples of blood collected before stunning or at commencement of exsanguination and to determine whether there were significant differences between the mean corrected OD450 values of the IFN-γ assay conducted on blood collected at these time points.
Materials and Methods
Animals—Fifteen healthy nonpregnant adult Holstein cows (3 to 5 years old) were used in the study. All study subjects had negative test results for the caudal fold tuberculin test for tuberculosis during the year preceding the study and negative results for the IFN-γ assay immediately before the start of the study. In addition, all cattle had negative test results for Mycobacterium avium subsp paratuberculosis as determined via serum ELISA and bacteriologic culture of feces. The cows were housed at the Michigan State University Beef Cattle Research and Teaching Center for the duration of the study. The study protocol was approved by the Michigan State University Institutional Animal Care and Use Committee.
Sample size—A sample size calculationa for paired categorical attributes (McNemar test) was conducted. On the basis of results for another study,19 the following conditions were set for the calculation: probability of type 1 error (ie, α) = 5%, power (ie, 1 − β) = 80%, expected proportion that would change from positive to negative results for IFN-γ = 60%, and expected proportion that would change from negative to positive results for IFN-γ = 1%. The study was focused on determining whether results of the IFN-γ assay would change from positive in blood samples collected before stunning to negative in blood samples collected at commencement of exsanguination; therefore, 12 cattle were required. Three additional cattle were included to increase the power of the study.
Study design—The 15 cattle were allocated into 3 groups (5 cows/group) via a stratified randomization method. The groups were stratified on the basis of age and placed in 3 adjacent pens. After a 1-week acclimation period, 4 cows from each group were selected via a simple randomization process and were immunologically challenged with inactivated M bovis. The remaining cow in each group served as a nonsensitized control animal. Each of the 12 sensitized cows was injected SC in the brisket with 0.1 mL of inactivated M bovisb in mineral oil (day of injection was designated as day 0). The 3 nonsensitized control cows were injected SC in the same region as the sensitized cows with 0.1 mL of mineral oil alone. On day 45, a blood sample was collected from a jugular vein of each of the 15 cows and assayed for an IFN-γ response. All sensitized cows had positive results for tuberculosis by use of the cutoff value established by the USDA for the IFN-γ assay; nonsensitized control cows had negative results for tuberculosis.
After testing on day 45, all cows were scheduled for slaughter at the Michigan State University Department of Animal Science Meats Laboratory. Blood samples for IFN-γ assay were collected from each cow immediately before stunning and at commencement of exsanguination. Prior to stunning, a blood sample (6 to 8 mL) was collected from the coccygeal vein or artery of each cow; each sample was collected with a 20-gauge needle into a 10-mL evacuated tube containing sodium heparin. Cows were then stunned via captive bolt and exsanguinated in accordance with the slaughter facility's standard operating protocols. A blood sample was collected from each cow immediately after the jugular and carotid arteries were severed and blood was flowing freely from the severed vessels. The interval from stunning to blood collection at commencement of exsanguination ranged from 60 to 90 seconds. At commencement of exsanguination, blood was collected into the wide end of a 10-mL syringe from which the plunger had been removed. After the syringe was filled, the syringe plunger was replaced and blood was immediately transferred via an 18-gauge needle into two 10-mL evacuated tubes containing sodium heparin. Blood samples were placed on ice in a cooler and delivered to the Michigan State University Diagnostic Center for Population and Animal Health within 1 hour after collection.
IFN-γ testing—The Diagnostic Center for Population and Animal Health has been approved by the USDA to conduct IFN-γ testing in support of eradication of tuberculosis in cattle. The IFN-γ assay was conducted by use of a commercially available antigen-capture ELISAc performed in accordance with the manufacturers' recommended protocol. In brief, aliquots (1.5 mL) of each blood sample were dispensed into each of 4 wells of a 24-well tissue culture plate and mixed with 100 μL of sterile PBS solution (no-antigen control stimulant), avian PPD (Mycobacterium avium), bovine PPD (M bovis), or pokeweed mitogen (nonspecific positive antigen control stimulant). The plates were incubated for 16 to 24 hours at 38°C in a humidified chamber. After incubation, plates were centrifuged at 1,730 × g for 10 minutes and the plasma harvested. Each plasma sample was then assayed in duplicate by use of the ELISA for detection of the IFN-γ content. The OD450 was used to detect IFN-γ content in each culture plate.
Results were expressed as the background-corrected OD450, with the response for the no-antigen control sample subtracted from the responses of both M bovis (bovine PPD) and M avium (avian PPD). Production of IFN-γ in response to pokeweed mitogen (nonspecific positive antigen control stimulant) was used to confirm lymphocyte viability. For an assay to be considered valid, the following condition needed to be met: (nonspecific positive antigen control response – no-antigen control response) ≥ 0.1. An animal was considered to have a positive result for tuberculosis when the difference between the bovine PPD and no-antigen control responses and the difference between the bovine PPD and avian PPD responses were both ≥ 0.1; when either of these differences was < 0.1, the animal was considered to have a negative result for tuberculosis.
Statistical analysis—An independent t testd was used to analyze whether age and body weight differed significantly (P < 0.05) between the sensitized and nonsensitized cows. The Pearson correlatione, was used to analyze relationships between body weight and the IFN-γ response. Conditional probabilityf calculation that a sensitized animal would still have a positive result for tuberculosis in samples collected at commencement of exsanguination was conducted on all sensitized cattle. Paired t testd analysis was used to test for significant (P < 0.05) differences in the IFN-γ responses at the 2 measured time points. Changes in the IFN-γ responses were diagrammed in box-and-whiskers plots.g Data analysis was performed with statistical software.20
Results
Cows were 3 to 5 years old and weighed between 611 and 914 kg. Age and body weight did not differ significantly between the 12 sensitized and 3 nonsensitized control cows. There was not a significant (P = 0.127) correlation between body weight and IFN-γ response to bovine PPD (ie, bovine PPD response – no-antigen control response). All assayed blood samples met the condition for assay validity (ie, nonspecific positive antigen control response – no-antigen control response ≥ 0.1).
All 12 sensitized cows were categorized as positive for tuberculosis (ie, bovine PPD response – no-antigen control response ≥ 0.1 and bovine PPD response – avian PPD response ≥ 0.1) on the basis of the corrected OD450 values for IFN-γ responses for blood samples collected before stunning, and 9 still had positive results for tuberculosis on the basis of test results for blood samples obtained at commencement of exsanguination. The probability that cattle that had positive results for tuberculosis for blood samples collected before stunning would still have positive results for blood samples collected at commencement of exsanguination was 75% (95% confidence interval, 47% to 91%); all 3 nonsensitized control cows had negative results for tuberculosis on the basis of IFN-γ response for blood samples collected before stunning and at commencement of exsanguination (Table 1).
The IFN-γ response for blood samples collected at slaughter before stunning and at commencement of exsanguination after stunning from 12 cows sensitized by SC injection of Mycobacterium bovis and 3 nonsensitized control cows.
| Variable | At commencement of exsanguination | Total | |
|---|---|---|---|
| Tuberculosis positive | Tuberculosis negative | ||
| Before stunning | |||
| Tuberculosis positive | 9 | 3 | 12 |
| Tuberculosis negative | 0 | 3 | 3 |
| Total | 9 | 6 | 15 |
The IFN-γ responses were determined as OD450. An animal was considered to have a positive result for tuberculosis when the difference between the bovine PPD and no-antigen control responses and the difference between the bovine PPD and avian PPD responses were both ≥ 0.1; when either of these differences was < 0.1, the animal was considered to have a negative result for tuberculosis.
The corrected OD450 values for IFN-γ were higher for most cows in samples collected before stunning than in comparable samples of blood collected at commencement of exsanguination. Among the sensitized cattle, there was a significant decrease in mean corrected OD450 for the differences between the bovine PPD response and no-antigen control response, bovine PPD response and avian PPD response, avian PPD response and no-antigen control response, and nonspecific positive antigen control response and no-antigen control response (Figures 1 and 2; Table 2). No significant change in mean corrected OD450 was detected among the control cows (Table 3).
Box-and-whiskers plot illustrating the background-corrected IFN-γ response observed for blood samples collected from 12 cows sensitized by SC injection with inactivated Mycobacterium bovis and 3 nonsensitized control cows. Blood samples were collected at slaughter before stunning and at commencement of exsanguination after stunning. The background-corrected response was calculated as the response for blood samples stimulated with M bovis PPD minus the response for the no-antigen control stimulation; PBS solution was the no-antigen control stimulant. Each box represents the interquartile range (25th to 75th percentiles), the horizontal line in each box represents the median, and the vertical lines (whiskers) represent the range.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.847
Box-and-whiskers plot illustrating the IFN-γ response for blood samples collected from 12 sensitized cows and 3 nonsensitized control cows at slaughter before stunning and at commencement of exsanguination after stunning. Results represent the IFN-γ response for blood samples stimulated with M bovis PPD minus the IFN-γ response for blood samples stimulated with Mycobacterium avium PPD. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.847
The IFN-γ response for blood samples collected at slaughter before stunning and at commencement of exsanguination after stunning from 12 sensitized cows.
| Variable | Before stunning | At commencement of exsanguination | P value* |
|---|---|---|---|
| No-antigen control response† | 0.037 ± 0.012 | 0.040 ± 0.035 | 0.831 |
| Difference between the bovine PPD response and the no-antigen control response† | 1.260 ± 0.822 | 0.566 ± 0.391 | 0.015 |
| Difference between the bovine PPD response and the avian PPD response | 0.801 ± 0.543 | 0.397 ± 0.317 | 0.022 |
| Difference between the avian PPD response and the no-antigen control response† | 0.459 ± 0.308 | 0.168 ± 0.101 | 0.009 |
| Difference between the nonspecific positive antigen control response‡ and the no-antigen control response† | 2.955 ± 0.914 | 2.253 ± 0.711 | 0.010 |
Values are reported as mean ± SD OD450.
Values represent results of a t test and are significant at P < 0.05.
Sterile PBS solution was used as the stimulant for the no-antigen control response.
Pokeweed mitogen was used as the stimulant for the nonspecific positive control response.
The IFN-γ response for blood samples collected at slaughter before stunning and at commencement of exsanguination after stunning from 3 nonsensitized control cows.
| Variable | Before stunning | At commencement of exsanguination | P value* |
|---|---|---|---|
| No-antigen control response† | 0.055 ± 0.033 | 0.031 ± 0.005 | 0.361 |
| Difference between the bovine PPD response and the no-antigen control response† | 0.011 ± 0.031 | 0.016 ± 0.025 | 0.734 |
| Difference between the bovine PPD response and the avian PPD response | −0.146 ± 0.184 | −0.043 ± 0.035 | 0.381 |
| Difference between the avian PPD response and the no-antigen control response† | 0.157 ± 0.212 | 0.059 ± 0.055 | 0.401 |
| Difference between the nonspecific positive antigen control response‡ and the no-antigen control response† | 2.850 ± 0.516 | 2.193 ± 0.914 | 0.232 |
See Table 2 for key.
The reduction in the qualitative IFN-γ response in samples collected at commencement of exsanguination decreased the sensitivity of the assay. For samples collected at commencement of exsanguination, the background-corrected OD450 (ie, bovine PPD response – no antigen control response) of all 12 sensitized cows was ≥ 0.1 (Figure 3). However, for 3 cows, the difference between the bovine PPD response and avian PPD response was < 0.1 (Figure 4). These 3 cows were classified as negative for tuberculosis at exsanguination because each failed to satisfy both conditions for classifying a cow as positive for tuberculosis. One of the 3 cows had an increased OD450 in the sample collected at exsanguination for the difference between the bovine PPD response and avian PPD response, compared with the OD450 in the sample collected before stunning, whereas the other 2 cows similarly had an increased OD450 for the difference between the bovine PPD response and no-antigen control response and between the bovine PPD response and avian PPD response.
The IFN-γ response for blood samples collected at slaughter before stunning (black diamonds) and at commencement of exsanguination after stunning (white squares) from 12 sensitized cows (A through L). Results represent the response for blood samples stimulated with M bovis PPD minus the response for the no-antigen control stimulation. The horizontal dashed line represents the cutoff value for the IFN-γ assay; cattle with values below the cutoff are classified as negative for tuberculosis.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.847
The IFN-γ response for blood samples collected at slaughter before stunning (black diamonds) and at commencement of exsanguination after stunning (white squares) from 12 sensitized cows. Results represent the IFN-γ response for blood samples stimulated with M bovis PPD minus the IFN-γ response for samples stimulated with M avium PPD. See Figure 3 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.847
Discussion
To our knowledge, the study reported here was the first in which investigators determined the possibility of obtaining valid results for an IFN-γ assay by use of blood samples collected at commencement of exsanguination at slaughter. Nine of 12 sensitized cattle had positive results for blood samples collected during exsanguination. This finding supports the need to further develop the IFN-γ assay for POC screening of cattle at slaughter as part of a regional or national program to eradicate tuberculosis in cattle. A potential application of this assay in a POC screening strategy would be to enhance existing visual slaughter surveillance as a tool to identify suspected tuberculosis-infected herds for subsequent targeted whole-herd testing. This strategy would be similar to that currently used for slaughter surveillance for brucellosis in the United States. Collection of blood samples during exsanguination for screening with the IFN-γ assay would be unlikely to introduce a delay in the slaughter process, given that blood samples are already collected during exsanguination for brucellosis surveillance. If used in parallel with routine visual slaughter surveillance, the use of the IFN-γ assay in samples obtained at slaughter could enhance detection of tuberculosis-infected cattle herds because the assay is capable of detecting M bovis infection before the onset of gross lesions.7,9–17 This strategic application of the IFN-γ assay for bovine tuberculosis surveillance would be economical because it could be used to reduce the amount of whole-herd testing performed via individual-animal screening tests (eg, caudal fold tuberculin tests), which would potentially reduce surveillance costs and likely facilitate tuberculosis control and eradication efforts.
Although blood samples collected during exsanguination were found capable of yielding IFN-γ in response to antigen stimulation, there was a significant decrease in the OD450 for each stimulant (avian PPD, bovine PPD, and pokeweed mitogen) in samples collected before versus after stunning. Test interpretation for cattle that had a comparatively higher OD450 before slaughter was not affected by the decrease in OD450 detected in samples collected at commencement of exsanguination. In cattle that had borderline positive results before slaughter, decrease in the OD450 after stunning resulted in a change to a negative tuberculosis classification. For those cattle, the difference between the bovine PPD response and avian PPD response for the IFN-γ assay was < 0.1 (Figure 4). The background-corrected OD450 (ie, bovine PPD response – no-antigen control response) also decreased but remained > 0.1 (Figure 3). Given the decrease in the IFN-γ response for blood samples collected at commencement of exsanguination, 1 approach to correct this problem and increase the sensitivity of the IFN-γ assay would be to lower the cutoff value to a number that does not compromise the specificity of the assay. For example, in the present study, decreasing the cutoff value for the difference between the bovine PPD response and the no-antigen control response and between the bovine PPD response and avian PPD response to 0.04 would have resulted in the correct diagnostic classification of 11 of the 12 sensitized cows without any false-positive classification of the 3 nonsensitized control cows. Prospectively, such reduction in the cutoff value could decrease the specificity of the assay and would need to be evaluated in future studies.
The decrease in the IFN-γ response in blood samples collected at commencement of exsanguination in the present study is in agreement with results of another study19 in which blood samples collected from experimentally infected cattle 5 minutes after slaughter had a decreased response for IFN-γ. Although it was unclear from that study19 whether the decrease in the IFN-γ response affected the diagnostic classification of the cattle, the authors considered the decrease important and expressed concern that blood samples collected after death would not be useful for conducting the IFN-γ assay. However, blood samples in that study19 were collected ≥ 5 minutes after the cattle were slaughtered, compared with 1 to 2 minutes after stunning in the present study. Although the difference in time of blood collection after stunning between the studies was small, a few minutes may be critical for samples used in the assay. Therefore, it can be inferred that the IFN-γ response decreases after stunning and that time lapse between stunning and blood collection is a critical factor for use of the IFN-γ assay.
The reduction in the IFN-γ response in blood samples collected during exsanguination may have been attributable to other reasons, including a reduction in the number of lymphocytes in the blood or a loss of lymphocyte function. In other studies,21–23 investigators have determined that acute stress results in a significant decrease in the absolute number of T cells, B cells, and other cells in the blood. This decrease in blood leukocyte numbers represents a redistribution of leukocytes from the blood to organs such as the bone marrow and skin.24,25 The act of stunning, which renders an animal unconscious, could result in a physiologic state that mimics acute stress. Because IFN-γ is released by lymphocytes, any decrease in the absolute number of lymphocytes would also decrease the IFN-γ response. It also is possible that stunning could have affected lymphocyte function. Any degree of stress may inhibit T-cell activation and antigen presentation and suppress effector cell function.23
Another possible factor in the decrease in the IFN-γ response could have been the difference in methods of blood collection. Blood samples obtained before stunning were collected from the coccygeal vein or artery via an evacuated tube, whereas blood samples at exsanguination were collected from free-flowing jugular veins and carotid arteries into a syringe. In another study,26 significant differences in WBC counts were not found when cattle blood was collected directly into blood tubes from the coccygeal or jugular veins.26 However, we are not aware of any reports that would indicate free-flowing blood would affect cell numbers in blood samples. Determining the reasons for the decreased IFN-γ response in blood samples collected at commencement of exsanguination was outside the scope of the present study but would be useful in the development of strategies to improve accuracy for use in slaughtered animals.
Two concerns were identified for the future application of the IFN-γ assay. First, the decrease in the IFN-γ response in samples collected during exsanguination may lead to false-negative results in a few animals. This limitation could be addressed by reducing the cutoff value for test results of blood collected during exsanguination or by complementary visual slaughter surveillance, which is expected to be used in parallel with test results. Second, the implementation of at-slaughter surveillance by use of the IFN-γ assay could increase the burden on diagnostic laboratories and personnel at slaughter facilities. The added burden would need to be offset by decreased costs in other areas of bovine tuberculosis control programs, even as the accuracy of the surveillance program is maintained or even improved.
The use of sensitized cattle, as opposed to naturally infected cattle, was a limitation of the present study. The extent to which the results obtained in the present study apply to naturally infected cattle remains to be verified. Cattle inoculated with inactivated M bovis in mineral oil and cattle naturally infected with M bovis have similar immune responses, which has been exploited in many studies8,11,13,14,19,27–29 in which a tuberculosis immune response was desired.
Future studies should evaluate factors responsible for the reduction in the IFN-γ response in blood samples collected during exsanguination, impact of the duration between stunning and exsanguination on the IFN-γ response, and the effect of time of blood collection following slaughter on the IFN-γ response. In addition, it would be useful to understand how the ability of the assay to detect disease would differ with samples of blood obtained from cattle naturally infected with tuberculosis and at various stages of the disease progression. Furthermore, it is necessary to conduct a cost analysis of the proposed strategy or alternative strategies to determine whether there are economic advantages and disadvantages. Filling these knowledge gaps would aid in determining usefulness of the IFN-γ assay for blood samples obtained at slaughter.
The IFN-γ response to M bovis before slaughter of cattle can be detected in samples collected during exsanguination when blood samples are collected within 90 seconds after stunning. The present study supports further development of the IFN-γ assay for use in POC screening of cattle at slaughter as part of regional or national tuberculosis eradication efforts. Application of the IFN-γ assay for bovine tuberculosis surveillance of samples obtained at slaughter could be used to improve current bovine tuberculosis surveillance strategies and help support continued eradication efforts in the United States.
ABBREVIATIONS
| IFN | Interferon |
| OD450 | Optical density measured at 450 nm |
| POC | Point of concentration |
| PPD | Purified protein derivative |
StatTools. Programs for sample size estimation: McNemar test. Available at: www.stattools.net/SSizMcNemar_Pgm.php. Accessed May 15, 2010.
Sensitinogen, USDA APHIS National Veterinary Services Laboratories, Ames, Iowa.
Bovigam, Prionics AG, Zurich, Switzerland.
PROC TTEST, SAS, version 9.2, SAS Institute Inc, Cary, NC.
PROC PCORR, SAS, version 9.2, SAS Institute Inc, Cary, NC.
PROC FREQ AGREE, SAS, version 9.2, SAS Institute Inc, Cary, NC.
PROC BOXPLOT, SAS, version 9.2, SAS Institute Inc, Cary, NC.
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