Development and Evaluation of a Test for Tuberculosis in Live European Badgers (<i>Meles meles</i>) Based on Measurement of Gamma Interferon mRNA by Real-Time PCR

Sawyer, Jason; Mealing, D.; Dalley, Deanna; Davé, Dipesh; Lesellier, Sandrine; Palmer, Si; Bowen-Davies, J.; Crawshaw, T. R.; Chambers, Mark A.

doi:10.1128/jcm.00292-07

Cited by 22 publications

(17 citation statements)

References 24 publications

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“…Further studies are necessary to assess positive test interpretation based upon increased IFN-␥ responses with M. bovis tuberculin relative to that observed with M. avium tuberculin analogous to commercial bovine TB ELISA assays. Actual application to any particular species requires confirmation by independent study as was recently performed for European badgers (27). Ultimately, the main usage of such tests may be to monitor the prevalence of disease in various wildlife populations, to screen animals prior to entry into disease-free herds, or to monitor the spread of infection in zoological parks.…”

Section: Discussionmentioning

confidence: 99%

“…Several genes, including IFN-␥ gene, have been cloned and sequenced for a variety of nontraditional domestic and wildlife species (29). The quantification of IFN-␥ mRNA levels therefore provides an alternative approach to IFN-␥ proteinbased assays for the detection of M. bovis-infected animals (27). Such an in vitro assay would be particularly useful for wildlife species, as it would require only a single handling event.…”

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Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

Harrington

Surujballi

Waters

et al. 2007

Clin Vaccine Immunol

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Tuberculosis of free-ranging and captive wildlife, including species implicated in the maintenance and transmission of Mycobacterium bovis, is a difficult disease to diagnose and control. Historically, diagnosis of tuberculosis has relied largely upon assays of cell-mediated immunity (CMI), such as tuberculin skin testing. This approach, however, is problematic or impractical for use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-␥)-based assays, are replacing or complementing skin testing of cattle and humans. Analogous assays are unavailable for most wildlife because of a lack of species-specific immunological reagents. This report describes the development and validation of a wholeblood assay to quantify antigen-specific IFN-␥ mRNA expression by quantitative real-time reverse transcription-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several susceptible species of interest with respect to tuberculosis infection. The assay was subsequently optimized to quantify the IFN-␥ mRNA expression in elk and red deer (Cervus elaphus) and was evaluated for its ability to detect mycobacterial antigen-specific responses of experimentally tuberculosis-infected animals. The assay was a simple, rapid, and sensitive measure of antigen-specific CMI. The IFN-␥ mRNA responses correlated well with IFN-␥ protein production and showed performance in determining an animal's infection status superior to that of either lymphocyte proliferation or IFN-␥ protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN-␥ expression by using consensus sequences of closely related species or of other species for which IFN-␥ sequence information is available.Mycobacterium bovis, the etiological agent of bovine tuberculosis (TB), has an extraordinarily broad mammalian host range that includes humans, domestic livestock, and wildlife. Globally, several wildlife species have been implicated in the maintenance and transmission of M. bovis: the badger (Meles meles) in Great Britain and Ireland (1), the brushtail possum (Trichosurus vulpecula) in New Zealand (2), white-tailed deer (Odocoileus virginianus) in Michigan (28), elk (Cervus elaphus) in Manitoba, Canada (17), and buffalo (Syncerus caffer) and kudu (Tragelaphus strepsiceros) in southern Africa (15). These wildlife hosts are difficult to control, and the transmission of M. bovis to domestic livestock may result in regulatory action, movement restrictions, or isolation of infected individuals and may become a significant barrier to the success of national eradication and control programs. Transmission among freeranging and captive wildlife also creates problems in the management of zoological collections; the risk of infection to other valuable animals is increased, directly affecting the value of the collection, and in some cases, the value of tourism or related economic benefits to a country (15).New control str...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

Harrington

Surujballi

Waters

et al. 2007

Clin Vaccine Immunol

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“…All sera were stored frozen at À20 8C until used for testing. Each badger from the RBCT was subjected to routine post-mortem examination and culture for the presence of M. bovis as described in Sawyer et al (2007). The infection status of Woodchester Park badgers was determined by bacterial culture of clinical specimens (feces, urine, sputum, purulent exudate from abscesses, and bite wound swabs) collected from anaesthetized animals (Clifton-Hadley et al, 1993;Delahay et al, 2000).…”

Section: Animals and Samplesmentioning

confidence: 99%

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

Lyashchenko¹,

Greenwald²,

Esfandiari³

et al. 2008

Veterinary Microbiology

116

108

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Lyashchenko, K.P.; Greenwald, R.; Esfandiari, J.; Chambers, M.A.; Vicente, J.; Gortazar, C.; Santos, N.; Correia-Neves, M.; Buddle, B.M.; Jackson, R.; O'Brien, D.J.; Schmitt, S.; Palmer, M. V.; Delahay, R.J.; and Waters, W.R., "Animal-side serologic assay for rapid detection of Mycobacterium Bovis infection in multiple species of free-ranging wildlife" (2008 AbstractNumerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n = 1532), white-tailed deer (n = 463), brushtail possums (n = 129), and wild boar (n = 177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT This article is a U.S. government work, and is not subject to copyright in the United States.may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock. #

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“…The difference in sensitivity was again highly significant (P Ͻ 0.0001, Fisher exact test). We have recently demonstrated improved detection rates of TB in the badger using quantitative real-time PCR (16) and an enzyme immunoassay (5) to detect the antigen-specific production of gamma interferon (IFN-␥). For both the IFN-␥ enzyme immunoassay and the Brock Test, the sensitivity was also significantly higher in badgers with VL than in those with NVL (5).…”

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Validation of the BrockTB Stat-Pak Assay for Detection of Tuberculosis in Eurasian Badgers ( Meles meles ) and Influence of Disease Severity on Diagnostic Accuracy

Chambers¹,

Crawshaw²,

Waterhouse³

et al. 2008

J Clin Microbiol

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A lateral-flow immunoassay (BrockTB Stat-Pak) for detecting tuberculosis in Eurasian badgers was 49% sensitive and 93% specific against culture for M. bovis (n ‫؍‬ 1,464) at necropsy. However, the sensitivity was significantly higher (66 to 78%) in animals with more severe tuberculosis, indicating that the BrockTB Stat-Pak may be useful for the detection of badgers with the greatest risk of transmitting disease.Despite attempts to control bovine tuberculosis (TB), the average incidence of disease in cattle in Great Britain increased an estimated 225% between the years 1996 and 2006 (7), causing considerable economic loss to the government and the farming community. In parts of Great Britain and Ireland, Eurasian badger (Meles meles) populations constitute a reservoir of infection with Mycobacterium bovis and a potential source of infection to cattle (8,14,15). In an effort to simplify the serodetection of badger TB, we developed a lateral-flow immunoassay (13) that is now manufactured as the BrockTB Stat-Pak assay (Chembio Diagnostic Systems, Inc., Medford, NY). We report here the results of ongoing evaluation of the assay, in particular the influence of disease severity on diagnostic accuracy.Badger sera were obtained in Great Britain from two sources and stored frozen at Ϫ20°C until testing (i) 1,464 animals killed as part of the defra/Independent Scientific Group Randomized Badger Culling Trial (RBCT) (8) and (ii) 68 animals captured, sampled, and then released as part of an ongoing ecological and epidemiological study in Woodchester Park (WP), southwest England (6). Each badger from the RBCT was subjected to routine necropsy examination and culture for the presence of M. bovis. Carcasses were stored at 4°C and examined by a standardized necropsy protocol within 3 days of submission. A range of tissues and lymph nodes were examined for evidence of TB. In badgers with no lesions the bronchial, mediastinal, and retropharyngeal lymph nodes were placed in 1% cetyl-pyridinium chloride prior to bacterial culture. In badgers with lesions the lesion tissue was always submitted for culture. Bite wounds were sampled separately. Tissues were processed within 72 h of sampling. The cetyl-pyridinium chloride was discarded, and the tissues were rinsed in saline and then emulsified before inoculation onto six slopes of modified Middlebrook 7H11 agar. The slopes were incubated at 37 Ϯ 2°C. Any growth of organisms characteristic of mycobacteria was identified by spoligotyping (9). The infection status of WP badgers was determined by bacterial culture of clinical samples of feces, urine, sputum, pus from abscesses, and bite wound swabs as described previously (3).Compared to the culture results from tissues taken at necropsy (n ϭ 1,464), the findings were as follows: BrockTB StatPak positive, culture negative, n ϭ 74; BrockTB Stat-Pak negative, culture negative, n ϭ 1,004; BrockTB Stat-Pak positive, culture positive, n ϭ 190; and BrockTB Stat-Pak negative, culture positive, n ϭ 196. Thus, the BrockTB Stat-Pak had a sensitivity ...

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Development and Evaluation of a Test for Tuberculosis in Live European Badgers (Meles meles) Based on Measurement of Gamma Interferon mRNA by Real-Time PCR

Cited by 22 publications

References 24 publications

Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

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

Validation of the BrockTB Stat-Pak Assay for Detection of Tuberculosis in Eurasian Badgers ( Meles meles ) and Influence of Disease Severity on Diagnostic Accuracy

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