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Clinical and Vaccine Immunology, May 2009, p. 605-612, Vol. 16, No. 5
1071-412X/09/$08.00+0     doi:10.1128/CVI.00038-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Highly Accurate Antibody Assays for Early and Rapid Detection of Tuberculosis in African and Asian Elephants

Rena Greenwald,¹ Olena Lyashchenko,¹ Javan Esfandiari,¹ Michele Miller,² Susan Mikota,³ John H. Olsen,⁴ Ray Ball,⁴ Genevieve Dumonceaux,⁴ Dennis Schmitt,⁵ Torsten Moller,⁶ Janet B. Payeur,⁷ Beth Harris,⁷ Denise Sofranko,⁸ W. Ray Waters,⁹ and Konstantin P. Lyashchenko^1*

Chembio Diagnostic Systems, Inc., Medford, New York,¹ Disney's Animal Programs, Lake Buena Vista, Florida,² Elephant Care International, Hohenwald, Tennessee,³ Busch Gardens Tampa Bay, Tampa, Florida,⁴ Missouri State University, Springfield, Missouri,⁵ Kolmarden Zoo and Wildlife Park, Kolmarden, Sweden,⁶ National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, United States Department of Agriculture, Ames, Iowa,⁷ Animal Care, Animal and Plant Health Inspection Service, United States Department of Agriculture, Fort Collins, Colorado,⁸ National Animal Disease Center, United States Department of Agriculture, Ames, Iowa⁹

Received 27 January 2009/ Returned for modification 23 February 2009/ Accepted 26 February 2009

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Tuberculosis (TB) in elephants is a reemerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current methods for screening and diagnosis rely on trunk wash culture, which has serious limitations due to low test sensitivity, slow turnaround time, and variable sample quality. Innovative and more efficient diagnostic tools are urgently needed. We describe three novel serologic techniques, the ElephantTB Stat-Pak kit, multiantigen print immunoassay, and dual-path platform VetTB test, for rapid antibody detection in elephants. The study was performed with serum samples from 236 captive African and Asian elephants from 53 different locations in the United States and Europe. The elephants were divided into three groups based on disease status and history of exposure: (i) 26 animals with culture-confirmed TB due to M. tuberculosis or Mycobacterium bovis, (ii) 63 exposed elephants from known-infected herds that had never produced a culture-positive result from trunk wash samples, and (iii) 147 elephants without clinical symptoms suggestive of TB, with consistently negative trunk wash culture results, and with no history of potential exposure to TB in the past 5 years. Elephants with culture-confirmed TB and a proportion of exposed but trunk wash culture-negative elephants produced robust antibody responses to multiple antigens of M. tuberculosis, with seroconversions detectable years before TB-positive cultures were obtained from trunk wash specimens. ESAT-6 and CFP10 proteins were immunodominant antigens recognized by elephant antibodies during disease. The serologic assays demonstrated 100% sensitivity and 95 to 100% specificity. Rapid and accurate antibody tests to identify infected elephants will likely allow earlier and more efficient treatment, thus limiting transmission of infection to other susceptible animals and to humans.

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Tuberculosis (TB) in captive elephants has been recognized as a reemerging zoonotic disease since at least the 1960s (22, 24, 26). In the past decade, growing numbers of elephant TB cases due to Mycobacterium tuberculosis or Mycobacterium bovis have been reported, presumably as a result of increased surveillance (11, 16, 25, 28). North American populations of African (Loxodonta africana) and Asian (Elephas maximus) elephants are declining, while captive breeding is historically poor. Efforts to maintain a self-sustaining captive population are hindered by TB-related issues. Many infected elephants may never exhibit clinical signs, whereas others with progressive disease may do so only in the terminal stages, thus making it difficult to recognize TB early (22, 24). Shedding of organisms during the preclinical period results in environmental contamination and presents a high risk of transmission to humans, elephants, and other mammals (11, 20, 27, 31).

The diagnostic value of the only existing antemortem testing method (i.e., culture of trunk wash samples) officially recommended by the United States Department of Agriculture (USDA) is limited by poor accuracy, slow turnaround time for sample processing, variable specimen quality, and sample acquisition logistics (11, 16, 25). Antibody detection assays have shown promising potential for identification of elephants infected with M. tuberculosis or M. bovis (10, 16). The new version of the Guidelines for the Control of Tuberculosis in Elephants 2008 (4) was recently approved by the United States Animal Health Association TB Committee. The document, including certain serologic tests, along with trunk wash culture for routine surveillance, is currently under USDA review to be adopted. This study describes the use of three novel serologic techniques (the ElephantTB Stat-Pak kit, multiantigen print immunoassay [MAPIA], and the dual-path platform [DPP] test) designed for early and accurate detection of TB in elephants.

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Animals. The study was performed with samples from 236 captive African and Asian elephants 2 to 68 years of age from over 53 different locations in the United States and Europe. The elephants were divided into three groups based on disease status and history of exposure (Table 1). The TB-infected group included 26 animals from 17 herds with culture-confirmed TB due to M. tuberculosis (n = 25) or M. bovis (n = 1). Of the 26 elephants, 7 died and 11 were humanely euthanized. TB was not necessarily the cause of death or the reason for euthanasia. Disease was diagnosed antemortem by trunk wash culture (n = 15; 58%) or only postmortem by isolating M. tuberculosis or M. bovis from various tissues (n = 11; 42%). Ten elephants were treated with first-line anti-TB drugs as recommended by the Guidelines for the Control of Tuberculosis in Elephants (33). The TB-exposed group included 63 elephants originating from 14 infected herds. Although no positive culture was ever isolated from trunk washes, some of these elephants could have been infected, as all of them had been in direct or indirect contact with known TB cases. A proportion of this group may have received prophylactic treatment, but the exact number of treated animals remains unknown due to nondisclosure policies for treatment regimens. The noninfected group consisted of 147 elephants without clinical symptoms suggestive of TB, with consistently negative trunk wash culture results, and with no history of potential TB exposure in the past 5 years. This group included 134 healthy animals and 13 with alternative diagnoses, such as chronic wasting syndrome, osteomyelitis, glomerulonephritis, chronic arthritis, or mycobacteriosis other than TB (MOTT). The last subgroup of four elephants included lung infection with Mycobacterium intracellulare, lymph node infection with an unidentified atypical mycobacterium, and two cases of fatal mycobacteriosis caused by Mycobacterium szulgai (9). Eighteen elephants were confirmed TB negative postmortem, as they died or were euthanized for other reasons and no evidence of TB was found at necropsy. Banked sera and freshly collected blood specimens were used for both retrospective and prospective serologic studies. All serum samples were stored frozen at –20°C or –70°C until they were tested.

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TABLE 1. Study population

Culture. The procedure for collecting triple trunk wash samples for culture was performed as described previously (25). Feces, urine, vaginal discharge, and various other tissues obtained at necropsy were also submitted for culture testing. Isolation and identification of M. tuberculosis and other mycobacteria were performed at the National Veterinary Services Laboratories (Ames, IA) and other certified laboratories, in accordance with the Guidelines for the Control of Tuberculosis in Elephants (33). Briefly, Middlebrook 7H10 with glycerol, Middlebrook 7H11 with glycerol, Stonebrinks, and BBL Mycobactosel L-J media, as well as BACTEC 12B vials, were inoculated with 0.5 ml of sample supplemented with PANTA (Becton Dickinson) and erythromycin (32 µg/ml). Processed specimens were inoculated on media and incubated at 37°C and 10% CO₂ for up to 8 weeks. All isolates and Bactec bottles with a growth indicator value of >300 were confirmed with the AccuProbe Mycobacterium tuberculosis Complex Culture Identification Test (Gen-Probe, San Diego, CA). If the DNA probe was positive, the niacin-nitrate reduction test was performed to confirm M. tuberculosis. MOTT species were identified by Gen-Probe AccuProbe Culture Identification Tests, according to the manufacturer's recommended procedures.

Electrophoresis and immunoblot assay. The antibody responses of the elephants were evaluated over time by immunoblot analysis using procedures described previously (6). Briefly, 115 µg of a whole-cell sonicate (WCS) of M. bovis strain 95-1315 was electrophoresed through preparative 12% (wt/vol) polyacrylamide gels. Electrophoretic transfer of proteins onto pure nitrocellulose was accomplished with the Bio-Rad Trans Blot Cell (Bio-Rad Laboratories, Mississauga, Ontario, Canada) using sodium phosphate buffer (25 mM; pH 7.8) at 0.8 Å for 90 min. After transfer, the filters were blocked with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (Sigma) and 2% (wt/vol) bovine serum albumin (PBST-BSA). After being blocked, the filters were placed into a 20-slot miniprotean II multiscreen device (Bio-Rad), and individual sera (diluted 1:200 in PBST-BSA) were added to independent slots. After 2 h of incubation at 24°C with gentle rocking, the blots were washed three times with PBST and incubated with peroxidase-conjugated protein L (Sigma) diluted 1:2,500 in PBST-BSA for 1.5 h. The blots were again washed three times with PBST and developed for chemiluminescence in SuperSignal detection reagent (Pierce Chemical Co.).

MAPIA. The MAPIA test was performed as previously described (15) using a panel of 12 proteins of M. tuberculosis and peroxidase-conjugated protein G (Sigma), along with 3,3',5,5'-tetramethyl benzidine (Kirkegaard & Perry Laboratories). The following recombinant antigens were immobilized on nitrocellulose membranes: ESAT-6 and CFP10 proteins, as well as the hybrids CFP10/ESAT-6 and Acr1/MPB83 produced at the Statens Serum Institut (Copenhagen, Denmark); MPB59, MPB64, MPB70, and MPB83 produced at the Veterinary Sciences Division (Stormont, United Kingdom); alpha-crystallin (Acr1) and the 38-kDa protein purchased from Standard Diagnostics (Seoul, South Korea); native MPB83 protein supplied by the Veterinary Laboratories Agency (Weybridge, United Kingdom); and Mtb8 and Mtb48 proteins and polyepitope fusion TBF10 developed by Corixa Corp. (Seattle, WA), as described previously (8). MAPIA results were scored by two independent operators who did not know the true infection status of the elephants, with a band of any intensity being read as a positive reaction.

ElephantTB Stat-Pak assay. The one-step lateral-flow test ElephantTB Stat-Pak (Chembio Diagnostic Systems, Inc., Medford, NY) employed selected M. tuberculosis antigens and a blue latex signal detection system for rapid detection of antibodies, as previously described (16). The test required 30 µl of elephant serum or plasma and 3 drops of sample buffer (included in the kit), which were added to the device sequentially. The results were read visually 20 min later by two independent operators who did not know the true infection status of the elephants. Any visible band in the test area, in addition to the control line, was considered an antibody-positive result, whereas no test band was considered a negative result.

DPP VetTB assay. A new-generation point-of-care test for TB in elephants was developed using Chembio innovative DPP technology. Unlike the ElephantTB Stat-Pak kit, which employed a conventional lateral-flow method, the DPP VetTB assay has two nitrocellulose strips that are connected in a "T" shape inside the device to allow independent delivery of test sample and antibody-detecting reagent. The first strip receives a serum sample and buffer solution via the sample well. The diluted sample migrates toward the second strip, containing two test lines (MPB83 and CFP10/ESAT-6 printed as separate bands) and one control line. Adding buffer to the conjugate well releases dried colloidal gold particles coupled with protein A/G and facilitates its migration along the second strip to the test area. If antibody is present in the sample, it binds to the immobilized test antigen, and the gold particles then react with this immune complex, thus making the test band visible. In the absence of detectable antibody, no specific immune complex would be formed on the test line, and therefore, no visible band would appear in the test area. The control band would develop, as the gold particles continue migrating along the second strip irrespective of the presence of antibody, ensuring correct performance of the test. The DPP VetTB assay was performed using 5 µl of elephant serum, 2 drops of buffer in the sample well, and 4 drops of buffer in the conjugate well. The results were read at 15 min visually by two independent operators who did not know the true infection status of the elephants or by using a DPP optical-reader device measuring reflectance in relative light units (RLU). Reactivity of CFP10/ESAT-6 and/or MPB83 above the cutoff value of 3.0 RLU was considered a positive result for the presence of antibody. No measurable reactivity with either of the two test antigens was taken as an antibody-negative result.

DBS. BloodStain cards (Whatman, Inc., Florham Park, NJ) were used according to the manufacturer's protocol to make dried blood spots (DBS) with elephant whole blood, serum, and plasma. Two antibody-positive and two negative control samples of each type from the same group of elephants were applied in duplicate to BloodStain cards (100 µl within each circle) and dried in the open air for 2 h at room temperature, and then the cards were sealed in plastic bags with desiccant and stored at +4°C. One and 5 weeks later, samples were extracted from the DBS by incubating each circle, cut into several pieces, in 200 µl of PBS overnight at +4°C. The eluted specimens were tested by serologic assays. For reference samples, aliquots of the positive and negative sera used for making DBS were kept frozen at –20°C until they were tested.

Data analysis. The TB-infected and noninfected groups were used for evaluation of the diagnostic sensitivity and specificity of the serologic assays. The TB-exposed group was considered only for seroprevalence analysis. The diagnostic performance of the serologic assays was evaluated against the gold standard of M. tuberculosis or M. bovis culture using available software (http://faculty.vassar.edu/lowry/VassarStats.html). The test sensitivity and specificity, as well as disease prevalence, were calculated for each assay and are presented with the 95% confidence interval (CI).

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Antibody responses in elephant TB. An initial indication that elephants can produce robust antibody responses to infection with M. tuberculosis was obtained by immunoblotting of sera collected over the course of >9 years from an elephant diagnosed with TB (Fig. 1). Antibody responses to a crude mycobacterial antigen preparation (i.e., M. bovis WCS) were detected 4 years prior to the isolation of M. tuberculosis from trunk wash samples. Notable in these responses were (i) a complex and progressive pattern of reactivity, (ii) consistency in responses to antigens of a distinct mass (e.g., 24 kDa, 32kDa, and 52 kDA), and (iii) a rapid decrease in reactivity to multiple antigens after initiation of antimycobacterial chemotherapy. To further analyze these findings, patterns of reactivity to a panel of recombinant proteins were evaluated by MAPIA (Table 2 and Fig. 2) using samples from the TB-infected and noninfected groups. All 26 of the infected elephants produced detectable serum immunoglobulin G (IgG) against one or more M. tuberculosis proteins, displaying variable profiles of antibody reactivity (Fig. 2). Among single proteins, ESAT-6 and CFP10 were the most frequently recognized molecules (92% and 81%, respectively), followed by MPB83 (58%) and others (4 to 19%) (Table 2). CFP10/ESAT-6 fusion protein was reactive with sera from all 26 infected elephants. Samples from all 147 noninfected elephants did not react with ESAT-6 or CFP10 alone or with the fusion protein. Sera from three of the four elephants with MOTT reacted with MPB83 (i.e., 2% of the noninfected group), but these sera did not react with ESAT-6 or CFP10 antigens. Thus, ESAT-6 and CFP10 proteins appeared to be predominant and specific serological targets; use of a cocktail or fusion of the two proteins may provide an accurate antibody test for elephant TB.

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FIG. 1. Immunoblot to M. bovis WCS. Archived serum samples from an elephant infected with M. tuberculosis were evaluated for reactivity to M. bovis WCS by standard immunoblotting techniques. The samples are arranged sequentially, with the serum collection date (year) indicated at the top and molecular mass (Kilodaltons) on the left. The arrow indicates the time when the culture of M. tuberculosis was isolated from trunk wash samples and treatment was initiated.

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TABLE 2. Seroreactivity rates of M. tuberculosis proteins in MAPIA with serum samples from culture-confirmed and control elephants

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FIG. 2. Differential antigen reactivity by serum IgG obtained from M. tuberculosis-positive elephants. The images represent MAPIA strips developed with sera from a noninfected elephant (sample 1), an elephant infected with M. szulgai (sample 2), and four elephants infected with M. tuberculosis (samples 3 to 6). The printed antigens are listed on the right.

Diagnostic performance of serologic assays. The diagnostic potentials of the ElephantTB Stat-Pak and MAPIA were initially demonstrated in a proof-of-concept study with a small number of samples (16). Recently, we have developed a novel point-of-care test based on the innovative DPP technology (Fig. 3). Reader device-generated data demonstrated clear-cut discrimination between the TB-infected and noninfected elephants using the DPP VetTB assay (Fig. 4). Good agreement was observed between test results obtained with the ElephantTB Stat-Pak, MAPIA, and DPP VetTB test. All 26 elephants in the TB group were CFP10/ESAT-6 antibody positive, thus yielding a sensitivity of 100% (95% CI, 84.0 to 100%) for each serologic test. None of the sera from noninfected elephants (n = 147) reacted with CFP10/ESAT-6 in MAPIA or the DPP VetTB assay, demonstrating a specificity of 100% (95% CI, 96.8 to 100%) for each test. As demonstrated with MAPIA, sera from three elephants with MOTT were reactive only with MPB83 antigen in the DPP VetTB test; however, this antibody reactivity was clearly distinguishable from the response to CFP10/ESAT-6 using the two-line format of the DPP VetTB (Fig. 3). The ElephantTB Stat-Pak kit showed seven false-positive results in the control group, resulting in a specificity of 95.2% (95% CI, 90.1 to 97.9%). Three of the seven reactors in the ElephantTB Stat-Pak assay were MOTT cases (presumably due to cross-reactivity with MPB83, as demonstrated with MAPIA and DPP VetTB), whereas three of the remaining four false-positive results were obtained with sera from elephants with arthritis. The diagnostic significance of the latter observation remains to be confirmed in future studies with larger sample numbers.

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FIG. 3. Antibody detection by DPP VetTB assay. The images represent examples of typical results obtained for elephant TB (A and B), MOTT due to M. szulgai (C), or noninfected control (D). Reflectance values in RLU generated by a DPP reader device for the MPB83 test band (gray bars) and the CFP10/ESAT-6 test band (black bars) are shown for each result. The proposed DPP VetTB test interpretation algorithm is shown on the right.

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FIG. 4. Quantitative detection of serum IgG antibodies by DPP VetTB in culture-confirmed elephants. Reflectance values in RLU generated by a DPP reader with a CFP10/ESAT-6 test band are shown for sera from the 26 elephants with TB (solid circles) and for 100 randomly selected noninfected controls (open circles). A cutoff value of 3.0 RLU (dotted line) was established with the control sera as the mean plus 5 standard deviations.

The specificities of the assays were additionally assessed with sera from four clinically healthy Asian elephants from which various species of atypical mycobacteria had been isolated during routine trunk wash culture testing. Sampling was performed every 3 to 6 months for each elephant over a period of 3 years. Trunk wash and blood specimens were obtained during the same week. Overall, 19 trunk wash samples collected from the four animals on different occasions were positive for mycobacteria. Two to seven different mycobacterial species were collected from each elephant, including Mycobacterium avium complex (n = 5), M. intracellulare (n = 2), Mycobacterium gordonae (n = 2), Mycobacterium fortuitum (n = 2), Mycobacterium abscessus, Mycobacterium asiaticum, Mycobacterium chelonae, Mycobacterium flavescens, Mycobacterium mucogenicum, Mycobacterium nonchromogenicum, Mycobacterium simiae, and Mycobacterium terrae. The matching 19 serum samples collected from the four elephants were tested by ElephantTB Stat-Pak, DPP VetTB, and MAPIA. No positive result was obtained with any of the sera, demonstrating that the presence of atypical mycobacteria in trunk wash specimens does not interfere with interpretation of the serologic assays for TB.

Serology versus culture. Banked pre-TB diagnosis sera were available for 16 infected elephants, which had been tested at least annually by trunk wash culture, providing the opportunity to retrospectively determine seroconversion times relative to antemortem culture results. Nine of these elephants were first diagnosed with TB by positive trunk wash culture. The other seven infected elephants were always trunk wash culture negative yet were confirmed TB infected by culture at a postmortem examination. In all 16 elephants, antibody responses were detected by the three serologic assays prior to culture-based diagnoses. Figure 5 demonstrates the times (range, 0.75 to 10 years; median, 3 years) established between individual seroconversions detected by MAPIA and isolations of M. tuberculosis or M. bovis either from trunk wash samples or from tissue specimens at necropsy. The remaining 10 elephants in the TB-infected group, for which no prediagnosis sera were available for retrospective analysis, tested strongly seropositive at the time the first positive culture was obtained.

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FIG. 5. Seroconversion time to first positive culture. Individual times are shown in years between initial MAPIA-positive samples collected from each elephant with TB and the first positive cultures of M. tuberculosis or M. bovis isolated from trunk wash samples (gray bars) or at postmortem examination (black bars).

Specific antibody responses in exposed, potentially infected, but trunk wash culture-negative elephants. Of the 63 elephants in the TB-exposed group, sera from 22 animals were reactive by the ElephantTB Stat-Pak kit (35%), 10 of which were also positive for CFP10/ESAT-6 antibody by MAPIA or the DPP VetTB test (16%). The discrepancies between the assays might be partially due to ElephantTB Stat-Pak false-positive reactions, to the ability of the technique to detect antibodies of all immunoglobulin classes (thus proving potentially more sensitive), or to the fact that some of the exposed elephants had been subjected to antimycobacterial therapy that may have prevented shedding and reduced the antibody responses to select antigens. Anti-TB treatment is known to affect the performance of the serologic assays over time differently (16), so that a treated elephant could remain reactive by ElephantTB Stat-Pak but eventually become nonreactive by MAPIA or the DPP VetTB test.

Seroprevalence of TB in elephants. With the close correlation found between the presence of circulating CFP10/ESAT-6-reactive antibody and TB in elephants, we estimated the prevalence of disease in the study population by using either culture or serology data (Table 3). While the overall estimates were 11.0% and 15.3% by culture or antibody detection, respectively, a fivefold-higher rate of seropositivity (21.9%, a statistically significant increase) was found among Asian elephants than among African elephants. Unlike the Asian elephant population, African elephants had no difference between TB prevalence estimates based on culture and serology (Table 3). Surprisingly, none of the four African elephants diagnosed with TB had ever produced a positive culture from multiple trunk wash specimens, whereas 15/22 (68%) infected Asian elephants were trunk wash culture positive. Therefore, if only antemortem culture testing was taken into consideration, the prevalence of disease would be greatly underestimated, being only 6.4% in total (0% in African elephants and 10.3% in Asian elephants).

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TABLE 3. Estimates of TB prevalence in captive elephant populations based on culture and serology data

Use of DBS specimens. The use of DBS specimens may enable collection of samples in remote locales, thereby facilitating broader surveillance, including in low-resource settings. We performed a DBS feasibility study with serum, plasma, and fresh whole blood collected from two M. tuberculosis-positive and two noninfected control elephants. Cellulose-based filter kits, BloodStain cards (Whatman, Inc.), originally designed for human diagnostic applications (7), were used in this pilot experiment. By comparing the DBS-eluted samples to the standard negative and positive control sera, it was determined that elephant TB-specific antibodies remained stable in DBS for at least 5 weeks (Fig. 6). This was demonstrated by ElephantTB Stat-Pak, DPP VetTB, and MAPIA. Procedures to produce DBS did not cause nonspecific reactions that could potentially result in false-positive results. There was no difference between serum, plasma, and whole blood with respect to both quantitative and qualitative characteristics of antibody detection (Fig. 6).

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FIG. 6. MAPIA testing of DBS samples. The images represent MAPIA strips developed with serum (A), plasma (B), or whole-blood (C) samples eluted from DBS after 1 week (1) or 5 weeks (2) of storage, in comparison with the standard positive and negative sera used for making DBS (0). The printed antigens are listed on the right.

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Elephant TB is a reemerging zoonotic disease caused primarily by M. tuberculosis (11, 22, 26). The Guidelines for the Control of Tuberculosis in Elephants define general principles and protocols for testing and treatment (33). Current diagnosis relies predominantly on trunk wash cultures, which is increasingly recognized to have serious limitations (11, 16, 25). This method, requiring three trunk wash samples collected on different days within 1 week, is labor-intensive, time-consuming, and expensive. Both the elephant and the caretaker or veterinarian must be well trained to obtain a trunk wash sample of acceptable quality. After sample collection, mycobacterial isolation and identification take 8 to 12 weeks, delaying notification of results. Increased numbers of trunk wash specimens for culture testing are required following a positive serologic assay for TB, as now recommended by the new Guidelines for the Control of Tuberculosis in Elephants 2008 (4). In the present study, only 58% of the elephants with necropsy-confirmed TB had positive antemortem cultures from trunk wash specimens. This finding does not imply that the remaining 42% of the elephants never shed M. tuberculosis, as excreting of the organism by infected animals is inherently intermittent (3, 25). Also, human studies have demonstrated that patients with sputum smear-negative pulmonary TB, commonly considered low-risk sources of infection, are capable of transmitting the disease (1, 32).

Most elephants with active TB display no clinical signs, making it even more difficult to suspect disease (22, 25). In fact, only 15% of the culture-positive cases reported in this study had TB-suggestive symptoms prior to the time of diagnosis. Therefore, rapid and more efficient detection of infected animals is crucial to improve control of TB in elephants and other zoo species, as early diagnosis allows timely initiation of chemotherapy and quarantine to prevent transmission. Using sera serially collected over multiple years from 236 captive elephants, we evaluated the diagnostic potentials of three serologic techniques designed for early and accurate identification of elephants infected with M. tuberculosis or M. bovis. The ElephantTB Stat-Pak, MAPIA, and the DPP VetTB test correctly identified all infected animals and produced no false-negative reactions, thus demonstrating a perfect negative predictive value and 100% sensitivity. Importantly, the new serologic assays appeared to provide antemortem testing tools superior to the existing methods. Many infected elephants showed specific seroconversion years before shedding was detectable by culture of trunk wash samples. Moreover, the serologic assays identified a number of elephants with TB (confirmed postmortem) that had never been trunk wash culture positive and had no clinical signs of disease. The diagnostic test specificities were 100% for MAPIA and the DPP VetTB test and 95% for the ElephantTB Stat-Pak assay.

Importantly, the results obtained with samples collected from culture-negative elephants that had been in contact with known TB cases indicate that serology may be a useful approach for more efficient surveillance of animals at risk for developing disease. Over the course of the present study, three TB-exposed elephants in different locations tested positive by ElephantTB Stat-Pak and MAPIA (with ESAT-6 antigen), although their trunk wash specimens were repeatedly culture negative. After one of these elephants died and other two were euthanized as strong suspects, M. tuberculosis strains (identical to those obtained from the source of infection in each case) were isolated from lung lesions collected at necropsy. These observations demonstrate the predictive value of highly sensitive antemortem tests for early diagnosis in "at-risk" groups. Thus, serology may be used to facilitate change in management practices in order to minimize infection risks to other animals, exhibition personnel, and the public (11, 27, 31).

Overall, the diagnostic performance of the DPP VetTB assay was equal to that of MAPIA and superior (in specificity) to the ElephantTB Stat-Pak kit. With the antigens studied, specific elephant TB serodiagnosis was closely associated with the presence of antibodies to the ESAT-6 and CFP10 proteins of M. tuberculosis. The predominant serologic recognition of ESAT-6 has been reported for nonhuman primates infected with M. tuberculosis or M. bovis (2, 18), but not for other host species (5, 6, 12, 13, 14, 17, 19, 34-36). In contrast, several ElephantTB Stat-Pak false-positive results found in MOTT cases were due to the cross-reactive antibody responses against MPB83 protein, but not ESAT-6 or CFP10. Previous studies also demonstrated MPB83 seroreactivity in elephants infected with M. szulgai (9) or in cattle experimentally inoculated with Mycobacterium kansasii (37). Having this protein as a separate band in MAPIA or the DPP VetTB test appears to allow serological differentiation between elephant TB (antibodies against ESAT-6 and/or CFP10, alone or among others) and MOTT (anti-MPB83 antibody only) infections. Therefore, similar to MAPIA (16), the DPP VetTB assay can also be used under field conditions, if needed, as a faster and more convenient animal-side confirmatory tool for elephant TB.

The serologic assays performed well with blood specimens recovered from DBS, suggesting a useful sampling alternative for peripheral areas (e.g., field applications in Africa, Southeast Asia, etc.), where short-term storage or transportation of blood samples to a remote testing laboratory may be needed. This approach has been successfully utilized for serological surveillance of human viral infections in resource-limited countries (7, 29). For elephant-testing applications, a more extensive DBS validation with greater numbers of well-characterized samples will be required.

The high accuracy of elephant TB serodiagnosis was rather unexpected. Using similar immunoassays, we and others have reported much lower rates of TB detection in other species (5, 12, 17, 18, 34-36). The antibody test sensitivities ranged from 45% in brushtail possums, 49 to 51% in Eurasian badgers, or 73 to 75% in cervids to 77% in wild boar (3, 19), but they were never as high as the 100% found for the 26 infected elephants in the present study. This striking feature may stem from the complex biology of host-pathogen interactions, with variability in the immune responses between species. While the idea is only speculative, elephants normally have levels of peripheral blood monocytes significantly higher than those of ungulates, often in the range of 25 to 42% of circulating leukocytes (21, 23, 30), which may impact their immunity. The specific mechanisms for unusually robust antibody responses to TB in elephants remain unclear.

Despite their higher diagnostic potential, antibody assays are unlikely to replace culture methods in elephant testing. Isolation of mycobacteria from infected animals will always be useful to confirm the diagnosis, identify the strain (especially useful for molecular epidemiology studies), and generate drug susceptibility data (22). However, the management and control of TB in captive elephants and other nondomestic species will greatly benefit from early and rapid serodiagnosis. The cost of delayed diagnosis may be extremely high (16). Furthermore, undetected elephant TB may pose a serious zoonotic threat, with infection spillover from captive animals to free-ranging wildlife. This possibility is supported by findings of identical M. tuberculosis strains isolated from an infected elephant and an Addra gazelle housed in one facility (our unpublished observations) or from a group of elephants with TB and other species in the same zoo, including gibbon, tapir, and giraffe (11). Thus, timely recognition of disease followed by immediate and adequate interventions will likely prevent the spread of infection.

In conclusion, many African and Asian elephants with culture-confirmed TB produce robust antibody responses years before M. tuberculosis or M. bovis can be isolated from trunk wash samples. The serologic assays described in the present study have high diagnostic value for earlier detection of disease. The rapid and accurate identification of infected elephants will likely improve zoo TB control programs and allow more efficient treatment, thus limiting the transmission of infection to other susceptible animals and to humans.

 
We are grateful to Peter Andersen, Mark Chambers, Raymond Houghton, Jim McNair, and John Pollock for providing purified antigens, as well as to the numerous zoo veterinarians and elephant caretakers for supplying serum samples.

This work was supported by Busch Gardens Tampa Bay and Chembio Diagnostic Systems, Inc.

 
* Corresponding author. Mailing address: Chembio Diagnostic Systems, Inc., 3661 Horseblock Road, Medford, NY 11763. Phone: (631) 924-1135. Fax: (631) 924-6033. E-mail: klyashchenko{at}chembio.com

Published ahead of print on 4 March 2009.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Clinical and Vaccine Immunology, May 2009, p. 605-612, Vol. 16, No. 5
1071-412X/09/$08.00+0     doi:10.1128/CVI.00038-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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