Fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in bovine sera

Veterinary Microbiology 120 (2007) 113–121 www.elsevier.com/locate/vetmic

Fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in bovine sera Michael E. Jolley a,*, Mohammad S. Nasir a, Om P. Surujballi b, Anna Romanowska b, Tomas B. Renteria c, Alfonso De la Mora c, Ailam Lim d, Steven R. Bolin d, Anita L. Michel e, Miladin Kostovic a, Edward C. Corrigan a a

b

Diachemix LLC, 683 E. Center Street Unit H, Grayslake, IL 60030, USA Animal Diseases Research Institute, Canadian Food Inspection Agency, 3851 Fallowfield Road, Nepean, Ontario, K2H 8P9 Canada c Instituto de Investigaciones en Ciencias Veterinarias, Universidad Autonoma de Baja California, Mexicali, Mexico d Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI 48910, USA e Department of Bacteriology, ARC-Onderstepoort Veterinary Institute, Private Bag x05, Onderstepoort 0110, South Africa Received 9 August 2006; received in revised form 16 October 2006; accepted 18 October 2006

Abstract The performance of a fluorescence polarization assay (FPA) that detects antibodies to Mycobacterium bovis in bovine sera is described. The FPA reported here is a direct binding primary screening assay using a small polypeptide derived from the M. bovis MPB70 protein. A secondary inhibition assay confirms suspect or presumed positive samples. Specificity studies involved five different veterinary laboratories testing 4461 presumed negative bovine samples. FPA specificity was 99.9%. The FPA was used to identify herd status as either M. bovis infected or non-infected. Herd surveillance studies (nine herds) were performed in Mexico and South Africa. The FPA had a specificity of 100% (two negative herds), and correctly identified six of seven infected herds. Finally, sera from 105 slaughter animals that had gross lesions in lymph nodes similar to those seen with bovine tuberculosis were tested by the FPA. Thin sections from the associated formalin-fixed paraffin-embedded samples of lymph nodes were stained using hematoxylin and eosin (H&E) for morphologic examination and using the Ziehl–Neelsen (ZN) method for detection of acid-fast bacilli. Of the 105 animals, 78 were classified as TB suspect based on lesion morphology, 21 were positive by ZN, 9 were positive by FPA and 13 were positive by PCR for the tuberculosis group of Mycobacterium. Among the 21 ZN positives, 11 (52.4%) were PCR positive. Among the 9 FPA positives, 8 (88.9%) were PCR positive. For the 13 PCR positives, 8 (61.5%) were FPA positive and 11 (84.6%) were ZN positives. These results show that use of the FPA for detection of M. bovis infection of cattle has value for bovine disease surveillance programs. # 2006 Elsevier B.V. All rights reserved. Keywords: Mycobacterium bovis; Tuberculosis; Fluorescence polarization; Immunoassay; FPA

* Corresponding author. Tel.: +1 847 548 2339; fax: +1 847 548 2984. E-mail address: [email protected] (M.E. Jolley). 0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.10.018

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1. Introduction M. bovis is the cause of bovine tuberculosis, an important disease that affects animal health and the economic value of cattle and food products derived from cattle. Furthermore, this disease is transmissible to other species of animals and humans. Bovine tuberculosis can progress insidiously through a herd of cattle without any obvious early stage clinical indications. Veterinarians rely on several diagnostic methods to detect M. bovis infection (De la Rua-Domenech et al., 2006). The M. bovis surveillance testing is performed on cattle at strategic control points such as before international transport, breeding services, sale or exchange, and carcass inspections at slaughterhouses. The various diagnostic methods used may be well suited for one stage of the disease progression but not necessarily others. Each diagnostic method or point of surveillance offers some advantage but has associated disadvantages. 1.1. Cell mediated immune system diagnostic methods The most frequently used diagnostic methods for bovine tuberculosis rely on the cell mediated immune (CMI) response to test for exposure to M. bovis. Diagnostic tests for CMI include the caudal fold test (CFT), the comparative cervical test (CCT), and the gamma interferon test (GIT). CMI tests offer the advantage of early detection of tuberculosis, as the CMI response against M. bovis is more readily detected in the initial stages of the disease than is the humoral immune response. A disadvantage of diagnostic tests that are based on the CMI response is specificity, as false positive tests occur in cattle exposed with organisms related to M. bovis (Hope et al., 2005). In addition to problems with specificity, the CMI diagnostic methods have a relatively high total cost per test. The CFT and CCT require at least two visits to the farm by an accredited veterinarian with a 3-day interval between visits. Further, cattle must be restrained for each visit; interpretation of the results is subjective; and tuberculin materials vary in formulation throughout the world, raising the issue of comparability or equivalency of testing results. For the GIT, there is a relatively high expense for the test kit,

and sample handling requirements are demanding. The harvested animal blood must be transported to a central laboratory within 30 h for further processing, and skilled technicians are needed to conduct the test. For all three of the diagnostic methods that rely on the CMI response there is the risk that as M. bovis disease advances, a severely infected animal may become anergic and fail to mount a detectable CMI response (Ritacco et al., 1991; Surujballi et al., 2002). Thus, conventional CMI methods may fail to detect infected animals with long standing M. bovis infection. 1.2. Histopathologic diagnostic methods Another important diagnostic method for detection of tuberculosis is histopathologic examination conducted post mortem. This requires considerable technical skill, an equipped histopathology laboratory, and substantial time to perform. This method is not well suited to large volume surveillance testing and is limited in that definitive identification of M. bovis is not possible. Consequently, animals and herds may be identified incorrectly as infected with M. bovis if other tests are not used for diagnostic confirmation. This false diagnostic result could lead to economic losses for producers. 1.3. ‘‘Gold standard’’ diagnostic methods: microbiological culture and polymerase chain reaction (PCR) The diagnostic ‘‘gold standard’’ methods for M. bovis detection are culture of the organism from affected tissues followed by confirmation of identity of the organism by PCR or, in some cases, PCR from fresh tissue. Culture offers isolation of the organism for further testing to achieve unambiguous identification of M. bovis, while PCR identifies the Mycobacterium tuberculosis complex that includes M. bovis. Both methods are more specific than CMI assays or histopathologic methods. However, a serious limitation of both culture and PCR is that they can only be performed reliably on samples collected post mortem. Culture of M. bovis requires stringent sample handling and advanced technical skill. Furthermore, culture methods can require 4 months for results. PCR requires sophisticated laboratory equipment, facilities,

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and advanced technical skills. Generally, a sample found to be positive by either culture or PCR is regarded as a true positive. However, a negative result by either method means that M. bovis was not detected in the sample of tissue that was tested. Other samples of tissue from the same animal could harbor M. bovis. Therefore, a negative test result from either culture or PCR does not provide conclusive proof that an animal is not infected with M. bovis, especially if the animal originated from an infected or suspect herd. While use of the ‘‘gold standard’’ methods is critical for confirmation of diagnosis, those methods are not well suited for large volume surveillance, where ease of use, assay speed, and large volume testing capabilities are important. 1.4. Serological diagnostic methods Serological assays provide an important and needed tool for large volume testing for exposure to M. bovis. They offer the important advantages of ease of use, assay speed and relatively low cost. A serological assay for M. bovis testing would complement the other established M. bovis diagnostic methods and facilitate diagnosis of the disease (Lin et al., 1996; De la Rua-Domenech et al., 2006). There have been several reports of the development of low cost serological tests that might provide a more definitive diagnosis of M. bovis infection (Wood et al., 1992; Wood and Rother, 1994; Lin et al., 1996; Surujballi et al., 2002). A new antibody-based test has recently been described which offers the potential for the early detection of M. bovis infection in cattle (Waters et al., 2006). Fluorescence polarization assay (FPA) detects the binding of a fluorescent low molecular weight moiety (tracer) to its high molecular weight binding partner by determining the tracer’s fluorescence polarization (FP). When bound (the binding partner is present), the tracer exhibits a high FP; when free (the binding partner is absent), the tracer has a low FP (Nasir and Jolley, 1999). FPA has been applied to serological immunodiagnostics for various veterinary diseases (Jolley and Nasir, 2003; Nasir and Jolley, 1999). FPA is well suited for animal disease general surveillance because of its rapidity, ease of use and high sensitivity and specificity (Jolley and Nasir, 2003; Gall and Nielsen, 2004). As a homogeneous assay, FPA is a

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quantitative method performed either in a single tube or in a microtiter plate format, with short incubation times and no washing or separation steps. FPA is being used for surveillance as part of Brucellosis Eradication and Control programs following recent United States Department of Agriculture (USDA) and World Organisation for Animal Health (OIE) approvals and European Union pending regulatory review (Nielsen et al., 1996, 1998, 2004; Nielsen and Gall, 2001; Gall and Nielsen, 2004). The MPB70 protein, secreted by M. bovis and other members of the M. tuberculosis complex, is a major immunodominant antigen (Wood et al., 1988; Lin et al., 1996). This protein is not present in Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis which are other members of the genus having epidemiological importance. A FPA for the detection of antibodies to M. bovis using the whole MPB70 protein has been described (Lin et al., 1996; Surujballi et al., 2002). Here we describe the use of a FPA employing a polypeptide-based tracer, derived from the whole MPB70 protein (Jolley and Nasir, 2003) as a diagnostic tool for M. bovis surveillance in bovine populations. The peptide sequence corresponds to certain amino acids of the MPB70 protein (amino acids 51–78), showing the most reactivity with control sera from positive cattle, with a C-terminal lysine added to improve solubility. The tracer (F-733) was peptide 733 N-terminally labeled with 6-carboxyfluorescein. The study was organized in three parts: (a) FPA general specificity studies carried out in the United States, Mexico, and South Africa; (b) FPA herd classification studies performed in Mexico and South Africa; (c) a Mexican slaughterhouse survey of carcasses that compared results of FPA on serum with results of testing lymph nodes with lesions, using acid-fast staining and PCR.

2. Materials and methods 2.1. Assay reagents and instrumentation All chemicals were obtained from Sigma–Aldrich (St. Louis, Missouri) unless stated otherwise. The PolarionTM (Tecan AG) microtiter plate reader,

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configured for fluorescence polarization (FP) was used for FP measurements. 2.2. Blood samples For the specificity studies, blood samples were collected at slaughterhouse facilities (United States), or by field veterinarians (Mexico and South Africa), and the sera shipped to participating laboratories. For the herd classification studies, blinded samples came from Mexican and South African defined status herds and also from farms with no historical evidence of tuberculosis. 2.3. Tissue samples Tissue samples were collected at the municipal slaughterhouse in Tijuana, Mexico, from cattle following carcass inspection for suspicious granulomatous lesions in lungs and lymph nodes from head and thorax for TB-like lesions. Tissue was formalinfixed, and transported to the School of Veterinary Medicine at the Universidad Autonoma de Baja California (UABC, Mexicali, Mexico) Pathology Laboratory for routine preparation for histopathologic examination by senior pathologists. Sections of tissue were stained with hematoxylin and eosin (H&E) for morphologic examination and the Ziehl–Neelson (ZN) method for acid-fast bacilli. 2.4. PCR assay for M. tuberculosis complex Formalin-fixed, paraffin-embedded samples were processed for extraction of DNA and PCR as described by Miller et al., 1997, with some modifications. Briefly, a section of tissue that was approximately 20 mm thick was placed in a sterile 2.0 ml screw cap tube containing several copper coated ball bearings (4.5 mm diameter), and approximately 200 ml of a 0.5% mixture of polyoxyethylene-sorbitan monolaurate (Tween 20) in DNase and RNase free molecular biology grade water was added. The contents of the tube were agitated in a Fast Prep FP 120 (Thermo Electron Corp., Waltham, MA) at a speed setting of 6.5 for 20 s. The tube was then sonicated for 5 min at a power setting of 60 (Model 2510 Ultrasonic Cleaner, Branson Ultrasonic Corp., Danbury, CT), incubated at 100 8C for 10 min, and snap-frozen by immersion in a

dry ice-ethanol bath for 3 min. Finally, the tube was incubated for an additional 5 min at 100 8C and centrifuged at 3000  g at 4 8C to pellet tissue debris and float the melted paraffin to the surface. The paraffin layer was removed with a sterile toothpick, and 2.5 ml of the liquid phase was used for PCR reaction. A real-time PCR assay was used to detect DNA from the M. tuberculosis complex of bacteria, using a primer set specific to the insertion sequence 6110. The primer set used was 50 -CTCGTCCAGCGCCGCTTCGG-30 and 50 -CCTGCGAGCGTAGGCGTCGG-30 . The reaction mixture was made from the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and contained 200 nM of forward primer, 100 nM of reverse primer, 2.5 ml DNA, and molecular biology grade water to a final volume of 25 ml. The PCR was done in an ABI 700 Sequence Detection System (Applied Biosystems, Foster City, CA) with reaction conditions of 95 8C for 10 min, 40 cycles of 95 8C for 15 s and 70 8C for 1 min, followed by standard post amplification melt curve analysis. The amplification product was 123 bp and the peak melting temperature was 86.0 8C. Using serial dilution of stock cultures of M. bovis as a measure, the sensitivity of the PCR assay is 1 colony forming unit of bacteria. 2.5. FPA serum testing Two FPAs were used in this study: (a) a direct binding primary screening assay using F-733 alone; (b) a confirmatory inhibition assay using F-733 in combination with a large excess of unlabeled 733 peptide. 2.5.1. Primary screening assay Assay buffer (100 ml; 0.01 M sodium phosphate, pH 7.5, containing 9 g/l sodium chloride, 1 g/l sodium azide and 4 g/l lithium dodecyl sufate; PBSA-LDS) was added to the wells of a 96-well flat bottomed black microtiter plate (Greiner Catalogue #655209 or equivalent). Serum sample (100 ml) was added and mixed well, avoiding air bubbles. Each plate contained four negative and two positive serum controls. After incubation at room temperature for 30 min a background fluorescence reading was made. An aliquot (10 ml) of a solution of F-733 (125 nM) in 0.01% sodium phosphate buffer, pH 7.5, containing 9 g/l

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Mexican and South African herd classification surveillance testing; (c) Mexican slaughterhouse surveillance, lesion inspection, and confirmation by ‘‘gold standard’’ methods.

sodium chloride, 1 g/l sodium azide, and 100 mg/l bovine gamma globulin (PBSA-BGG) was added and mixed well, avoiding bubbles. After 10 min the blanksubtracted FP of the tracer in each well was determined. A sample was considered ‘‘Presumed Positive’’ when the FP value of the sample was at least 40 mP (millipolarization units) higher than the mean of the negative controls (delta mP). Samples less than 20 delta mP were considered ‘‘Negative’’. Samples between 20 and 40 delta mP were considered ‘‘Suspect’’, and were re-tested in duplicate.

3.1. FPA specificity study All testing was performed on a blind sample basis under the supervision of the participating laboratories. For the United States samples, the source was presumed negative bovine sera from normal slaughterhouse surveillance testing programs. The South African and Mexican studies were performed on bovine sera from herds with no historical evidence of tuberculosis. Table 1 shows that the specificity with individual panels ranged from 99.5 to 100%. From the total 4461 presumed negative samples, the FPA determined 4456 to be negative, giving an overall specificity of 99.9%.

2.5.2. Confirmatory assay The confirmatory assay was conducted similarly to the primary screen assay except that unlabeled peptide 733 (10 ml; 1 mg/l in PBSA-BGG) plus PBSA-LDS (90 ml) was used instead of 100 ml of PBSA-LDS in the inhibitor wells. Two wells with inhibitor and two without inhibitor were used for each sample. A sample was considered ‘‘Confirmed Positive’’ when the mean delta mP of the sample with inhibitor was less than 50% of the mean delta mP of the sample without inhibitor. A sample was considered ‘‘Negative’’ if the mean delta mP of the sample with inhibitor was greater than 75% of the mean of sample without inhibitor. A sample remained ‘‘Presumed Positive’’ when the delta mP of the mean with inhibitor was 25– 49% of the mean of sample without inhibitor.

3.2. Mexico and South Africa: FPA herd classification Table 2 reports the results for these two herd surveillance classification studies. FPA correctly classified eight out of nine herds. Both non-infected farms had no serological evidence of disease presence based on the 130 sera tested. Among the officially designated infected farms, the FPA found evidence of the disease in six of the seven farms based on 1965 sera tested.

3. Results 3.2.1. Mexican samples This study involved 447 bovine sera from four farms located in the Mexicali and Tijuana regions of the Baja California State of Mexico. Bovine sera

Results are discussed in three sections: (a) specificity studies using Mexican, South African and United States bovine surveillance testing; (b) Table 1 The specificity of FPA for the detection of antibodies to M. bovis Study panel name

Size

FPA negative

FPA specificity (%)

Missouri 2003 #1 (Missouri Diagnostic Lab) Missouri 2003 #2 (Missouri Diagnostic Lab) Texas 2003 (Texas Animal Health Commission) South Africa ARC Field Study 2004 (ARC Onderstepoort Veterinary Institute) Baja California Vet Control Farm 2005 (Universidad Autonoma de Baja California, Mexicali)

200 2061 2070 56

199 2059 2068 56

99.5 99.9 99.9 100.0

74

74

100.0

Total

4461

4456

99.9

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Table 2 FPA agreement with farm herd classification official status Positive farms

Negative farms

Farms total

Mexico

South Africa

Mexico

South Africa

FPA positive FPA negative

2 1

4 0

0 1

0 1

6 3

Total (no. of samples)

3 (373)

4 (1592)

1 (74)

1 (56)

9 (2095)

(n = 373) were tested from three farms identified as having M. bovis infection. A fourth farm was identified as the UABC Veterinary School ‘‘negative control’’ farm with 74 samples from non-infected cattle. For the Mexican herds, FPA correctly identified the non-infected farm. One infected farm had one FPA positive sample out of a total of 71 and another had two FPA positive samples out of a total of 150. The third infected farm had no FPA positive samples out of a total of 152. 3.2.2. South African samples This study used 1648 bovine sera from five farms located in the country’s Eastern Cape Province. Serum samples were grouped by herd with a masked farm identification code. Four farms were officially designated as being infected with M. bovis, and one farm was regularly tested without any history of infection. FPA did not provide any indication for the presence of infection in the non-infected farm (56 sera), and correctly identified the four infected farms (1592 sera). Among the infected farms there were 66 FPA positive cattle, of which 31 had tuberculous lesions upon inspection at slaughter. In addition, 12 FPA positive animals had tested positive on the CCT.

Table 3 Summary of test results on lesion positive animals Total carcass inspections Granulomatous lesions Tuberculosis suspecta ZN positive FPA positive PCR positive a

350 105 78 21 b 9b 13 c

After histopathologic examination and HE staining. Out of 105 lesion positive samples. c Out of 81 Tuberculosis suspect, FPA positive and ZN positive samples. b

3.3. Mexican slaughterhouse granulomatous lesion testing A total of 350 cattle were inspected during slaughter for presence of suspicious granulomatous lesions in lymph nodes at the municipal slaughterhouse in Tijuana, Mexico (Table 3). Out of the 350 inspected carcasses, 105 (30%) had visible granulomatous-like lesions in retropharyngeal lymph nodes similar to gross lesions associated with TB. Of those 105 animals, 78 (74%) were classified as TB suspect following histopathologic examination of H&E stained tissue sections. Tissues from those 78 animals were further examined for presence of acid-fast organisms using ZN staining. On initial examination, 18 animals (17%) had acid-fast organisms within granulomatous lesions. The FPA was performed on all 105 samples and 9 (8.6%) were positive. Repeat FPA testing gave the same results. Tissues corresponding to 9 FPA positive samples were then retested by ZN and acid-fast organisms were found in lesions in lymph nodes from three additional cattle, bringing the total to 21 (20%) of the cattle that had lesions and acid-fast organisms within lesions. PCR was first performed on the 23 samples of formalin fixed, paraffin embedded tissue that were either positive by the ZN method and/or by FPA. Of those 23 samples of tissue, 12 were positive for bacterial DNA (Table 4). Subsequently, PCR was performed on the remaining 58 samples that were suspect after histopathologic examination of H&E stained tissue sections, but were negative for acid-fast organisms after examination of ZN stained sections of tissue. All were negative by PCR. In addition, samples (#51 and #200) that were FPA positive and PCR negative on initial testing, were tested again by PCR, using an additional section of tissue for extraction of DNA. Sample 51 was positive and sample 200 remained negative. Sample 116 was also retested as

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Table 4 Comparison of PCR results for the 23 samples positive by either FPA or ZN staining Sample no.

Lesion status

ZN first run

1 5 25 26 28 39 41 51 69 72 95 112 114 115 116 123 126 142 146 148 178 187 200

TBa EGb TB TB TB TB TB TB TB TB TB TB TB TB TB TB TB PGc TB TB TB TB EG

POS NEG POS POS POS POS POS NEG POS POS POS POS POS POS POS POS POS NEG POS POS POS NEG NEG

a b c

ZN repeat POS

POS

POS

POS POS NEG

NEG POS

FPA first run

FPA repeat

PCR first run

NEG POS NEG NEG POS NEG NEG POS NEG NEG NEG NEG NEG NEG POS POS POS POS NEG NEG NEG POS POS

NEG POS NEG NEG POS NEG NEG POS NEG NEG NEG NEG NEG NEG POS POS POS POS NEG NEG NEG POS POS

NEG POS NEG NEG POS POS POS NEG NEG NEG NEG POS POS POS POS POS POS POS NEG NEG NEG POS NEG

PCR repeat

POS

POS

NEG

Tuberculosis suspect. Eosinophilic granuloma. Parasitic granuloma.

a positive control and again was positive. Thus there were a total of 13 PCR positive tissues (Tables 3 and 4). Table 5 shows the FPA agreement with PCR. Among the nine FPA positives, eight (88.9%) were subsequently confirmed as positive by PCR. In addition, the sensitivity and specificity of FPA compared to PCR were 61.5 and 98.5%, respectively. Table 6 (first run) and Table 7 (first and second runs) show the ZN agreement with PCR. Among the 18 first run ZN positives, 9 (50%) were PCR positive Table 5 FPA comparison to PCR PCR+

Table 6 First run ZN comparison to PCR PCR

Total

ZN+ ZN

PCR+ 9 4

9 59

18 63

Total

13

68

81

ZN agreement: 83.4% (68/81); ZN sensitivity: 69.2% (9/13 PCR positives); ZN specificity: 86.8% (59/68 PCR negatives).

Table 7 Total ZN comparison to PCR PCR

Total

PCR+

PCR

Total

FPA+ FPA

8 5

1 67

9 72

ZN+ ZN

11 2

10 58

21 60

Total

13

68

81

Total

13

68

81

FPA agreement: 92.6% (75/81); FPA sensitivity: 61.5% (8/13 PCR positives); FPA specificity: 98.5% (67/68 PCR negatives).

ZN agreement: 85.2% (69/81); ZN sensitivity: 84.6% (11/13 PCR positives); ZN specificity: 85.3% (58/68 PCR negatives).

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and of the 21 first and second run ZN positives, 11 (52.4%) were subsequently confirmed as positive by PCR. The sensitivity and specificity of the first run ZN compared to PCR were 69.2 and 86.8%, respectively. The sensitivity and specificity of the first and second ZN runs combined were 84.6 and 85.3%, respectively. Two samples (#142 and #187) that were PCR positive, FPA positive, and ZN negative, were tested for a third time by the ZN method, and again, negative results were obtained.

4. Discussion The purpose of these studies was to evaluate the usefulness of the M. bovis antibody FPA for the rapid detection of exposure to M. bovis in cattle under conditions where skin testing or obtaining fresh tissues for culture are not feasible, such as herd surveillance, in sale barns and the slaughterhouse environment. A positive result by FPA should be a reliable indicator of the population’s exposure to M. bovis and would warrant further investigation. The FPA showed an excellent overall specificity of 99.9% with 4461 presumed negative bovine sera. This is critical for surveillance testing because of the waste of time, effort and expense incurred during the followup of false positive results. The herd surveillance study performed with 9 bovine herds (both infected and non-infected) demonstrates that FPA is a useful tool for herd classification by correctly identifying eight herds. The herd which FPA falsely identified as negative was from a recently infected farm with a presumably low prevalence. Since not every bovine was tested, it is possible that misidentification was due to no infected bovine sera being included in the samples tested, or infected animals having been already culled. Some cattle may have been infected but were at an early stage, with a humoral response insufficient for serological detection. Importantly, no herds were identified falsely as infected. The Mexican slaughterhouse study involving 105 suspect animals, following 350 carcass inspections, showed important results. Fresh tissue was not available for culture and so the sensitivity of the FPA was compared to the PCR of the formalin-fixed tissues. Although being able to detect one colony forming bacterium, the sensitivity of the PCR,

compared to culture, in tissues is not known. Of these 105, 23 cattle had lesions that contained acidfast organisms similar in appearance to Mycobacteria and/or contained DNA from the tuberculosis group of Mycobacteria. Of the 13 PCR positive animals, 11 were histopathologic TB suspects (84.6% sensitivity). Only one sample (#200) was positive by FPA and repeatedly negative by PCR. Interestingly, this sample was initially negative by ZN and only weakly positive on retesting. It is therefore very possible that sample #200 was a true positive and was negative by PCR due to a sampling insufficiency. Indeed, sample #51, a FPA positive, was initially negative by both ZN and PCR but was positive by both upon retesting. The sensitivity of FPA (61.5%) was comparable to the first ZN staining (69.2%) but the specificity was much better (98.5% versus 86.8%). The agreement of FPA with PCR (92.6%) was better than the ZN method, even after repeat testing (85.2%). The data reported here indicates that the FPA for the detection of bovine antibody to M. bovis, using the polypeptide tracer F-733 could play an important role as part of a disease surveillance program for the eradication and control of bovine M. bovis disease. In addition, being homogenous, and requiring no washing or separation steps, the FPA is rapid. The single tracer reagent is stable and there are no enzymes or substrates involved. There are also automation opportunities for high volume processing laboratories. FPA has these distinct performance advantages compared with other diagnostic methods for bovine M. bovis detection. Furthermore, FPA is specific for the Mycobacterium tuberculosis complex, with no known cross-reaction with the more frequent M. avium subsp. avium or M. avium subsp. paratuberculosis.

Acknowledgements The authors acknowledge the following laboratories and their personnel for blood collection and testing work in support of this research:  Dr. Mo Salman and Joan Triantis, Animal Health Population Diagnostic Laboratory at Colorado State University (Fort Collins, Colorado).  Dr. Chuck Massengill and Quintin Muenks, Animal Diagnostic Laboratory of the State of Missouri

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Department of Agriculture (Jefferson City, Missouri).  Dr. Max Coats and Rick Nabors, Animal Diagnostic Laboratory of the Texas Animal Health Commission (Austin, Texas).  Dr. Rosa Maria Bermudez, Animal Laboratory of Tuberculosis and Brucellosis of the Universidad Autonoma de Baja California (Mexicali, Mexico).

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