Adaptation of IFN-gamma ELISA and ELISPOT tests for feline tuberculosis.

There are currently no reliable immunodiagnostic tests for feline tuberculosis. Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for Mycobacterium bovis, and rodents for M. microti. In the African National Parks, where M. bovis infection of Bovidae is an increasing problem, transmission to big cats is occurring via their ingestion of infected carcasses. We have adapted feline ELISA and ELISPOT assays to potentially provide the first cell-based diagnostic test for the detection of tuberculosis in cats. We tested peripheral blood mononuclear cell antigen-specific IFN-gamma responses of 18 cats suspected of mycobacterial infection for which biopsy material was co-submitted to the Veterinary Laboratories Agency for mycobacterial culture and identification. Seventeen cats were tested by ELISA while seven cats were tested by ELISPOT (six cats were tested by both ELISA and ELISPOT). Six healthy control cats provided baseline data for these tests. Responses to bovine and avian tuberculins (PPDB and PPDA) and a protein cocktail of ESAT6 and CFP10 were measured, together with positive mitogen (PMA and calcium ionophore) and negative (medium) controls. Overall, both ELISPOT and ELISA tests were found to be suitable for generating rapid results (2 and 4 days, respectively), which provided good predictive information for M. bovis and M. microti infections, but were unable to reliably discern M. avium infection.

Assessment of cross-reactivity between Mycobacterium bovis and M. kansasii ESAT-6 and CFP-10 at the T-cell epitope level.

Cross-reactivity between Mycobacterium kansasii ESAT-6 and CFP-10 homologues and their M. bovis counterparts can confound the interpretation of immunodiagnostic tests for tuberculosis. M. kansasii is a nontuberculous mycobacterial species cultured from skin test-positive cattle in Great Britain. Using peptides derived from M. bovis and M. kansasii ESAT-6 and CFP-10 regions that differ between these species, we investigated the species specificity and cross-reactivity at the level of individual bovine T-cell epitopes. Our results demonstrated that all peptides tested are fully cross-reactive, with the exception of one ESAT-6-derived peptide that harbored an M. bovis-specific epitope(s) when it was recognized in the context of bovine leukocyte antigen (BoLA)-DQ but that was cross-reactive with its M. kansasii homologues when it was restricted by BoLA-DR. This observation further highlights that prediction of species specificity by comparing sequence identity/homology alone is not sufficient and that individuals with diverse major histocompatibility complex constellations need to be tested to characterize the cross-reactivity or species specificity of peptide-based reagents.

Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR.

BACKGROUND: We have evaluated a sensitive screening assay for Mycobacterium tuberculosis (MTB) complex organisms and a specific assay for detecting Mycobacterium bovis DNA in lymph nodes taken from cattle with evidence of bovine tuberculosis. Underlying these series of experiments was the need for a versatile DNA extraction protocol which could handle tissue samples and with the potential for automation.The target for the screening assay was the multi-copy insertion element IS1081, present in 6 copies in the MTB complex. For confirmation of M. bovis we used primers flanking a specific deletion in the genome of M. bovis known as region of difference 4 (RD4). The sensitivity and specificity of these PCRs has been tested on genomic DNA from MTB complex reference strains, mycobacteria other than tuberculosis (MOTT), spiked samples and on clinical material. RESULTS: The minimum detection limits of the IS1081 method was < I genome copy and for the RD4 PCR was 5 genome copies. Both methods can be readily adapted for quantitative PCR with the use of SYBR Green intercalating dye on the RotorGene 3000 platform (Corbett Research). Initial testing of field samples of bovine lymph nodes with visible lesions (VL, n = 109) highlighted two shortfalls of the molecular approach. Firstly, comparison of IS1081 PCR with the "gold standard" of culture showed a sensitivity of approximately 70%. The sensitivity of the RD4 PCR method was 50%. Secondly, the success rate of spoligotyping applied directly to clinical material was 51% compared with cultures. A series of further experiments indicated that the discrepancy between sensitivity of detection found with purified mycobacterial DNA and direct testing of field samples was due to limited mycobacterial DNA recovery from tissue homogenates rather than PCR inhibition. The resilient mycobacterial cell wall, the presence of tissue debris and the paucibacillary nature of some cattle VL tissue may all contribute to this observation. Any of these factors may restrict application of other more discriminant typing methods.A simple means of increasing the efficiency of mycobacterial DNA recovery was assessed using a further pool of 95 cattle VL. Following modification of the extraction protocol, detection rate with the IS1081 and RD4 methods increased to 91% and 59% respectively. CONCLUSION: The IS1081 PCR is a realistic screening method for rapid identification of positive cases but the sensitivity of single copy methods, like RD4 and also of spoligotyping will need to be improved to make these applicable for direct testing of tissue extracts.

Molecular analysis of human and bovine tubercle bacilli from a local setting in Nigeria.

To establish a molecular epidemiological baseline for the strains causing tuberculosis in Nigeria, a survey of isolates from humans and cattle was carried out. Spoligotyping and variable-number tandem-repeat analysis revealed that the majority of tuberculosis disease in humans in Ibadan, southwestern Nigeria, is caused by a single, closely related group of Mycobacterium tuberculosis strains. Using deletion typing, we show that approximately 13% of the disease in humans in this sample was caused by strains of Mycobacterium africanum and Mycobacterium bovis rather than M. tuberculosis. Molecular analysis of strains of M. bovis recovered from Nigerian cattle show that they form a group of closely related strains that show similarity to strains from neighboring Cameroon. Surprisingly, the strains of M. bovis recovered from humans do not match the molecular type of the cattle strains, and possible reasons for this are discussed. This is the first molecular analysis of M. tuberculosis complex strains circulating among humans and cattle in Nigeria, the results of which have significant implications for disease control.

Inactivation of the Mycobacterium bovis homologue of the polymorphic RD1 gene Rv3879c (Mb3909c) does not affect virulence.

The RD1 locus is deleted from all strains of Mycobacterium bovis BCG but present in virulent isolates of M. bovis and Mycobacterium tuberculosis. The RD1 gene Rv3879c encodes a proline- and alanine-rich protein that shows sequence polymorphism across members of the M. tuberculosis complex. The role of this protein in virulence was investigated by deleting the Rv3879c homologue from M. bovis (Mb3909c) and testing the virulence of the mutant in the guinea pig model. The M. bovis Delta Mb3909c mutant was not attenuated in the guinea pig model, showing that this gene does not encode a virulence factor and plays no role in the attenuation caused by loss of RD1.

Evaluation of adjuvants for protein vaccines against tuberculosis in guinea pigs.

Subunit vaccines against tuberculosis show promise but require administration with adjuvants to stimulate relevant immune responses for protection. Guinea pigs are the model of choice for evaluating protective immunity to aerogenic challenge with virulent mycobacteria, but few studies have been undertaken to identify suitable adjuvants for vaccine screening in this species. Here, we compare the efficacy of several adjuvants to induce T cell responses to culture filtrate protein in guinea pigs. We report that of several adjuvants tested, the most promising was CpG ODN formulated in an aqueous emulsion. This adjuvant induced type 1 T cell responses equivalent to that of FIA, as measured by delayed-type hypersensitivity reactions (DTH), antigen-specific T cell proliferation and antigen-specific IgG1 and IgG2 responses. These data demonstrate the potential for CpG motif based adjuvants for use in TB vaccine screening in guinea pigs, and other diseases where a type 1 T cell response is required.

Simple objective measurement of the cutaneous delayed-type hypersensitivity reaction to tuberculin using spectrophotometry.

BACKGROUND/AIMS: A number of subjective methods have been used to quantify the extent of the cutaneous delayed-type hypersensitivity (DTH) reaction. However, because of their subjective nature, significant differences in measurements may be seen between individual observers or laboratories unless thorough training is given to each observer. METHODS: Objective measurement of the DTH reaction using a hand-held spectrophotometer is described. Guinea pigs were primed using inoculation with Mycobacterium bovis Bacille Calmette-Guerin and challenged five weeks later in the shaved flank with three doses of bovine purified protein derivative. The extent of the ensuing DTH reaction was measured 24 and 48 h later. Spectrophotometric measurement of the reaction site was compared with a control region of skin on each animal and expressed as the change within a standard colour space. Data obtained with the spectrophotometer was compared with the subjective measurement of the area of the DTH reaction by an experienced operator. RESULTS: The measurements obtained with the spectrophotometer correlated very closely with conventional measurement of the reaction area by a trained operator. The reaction size in square mm and changes along the red/green colour axis was correlated most strongly. CONCLUSION: Spectrophotometric measurement of the DTH reaction had advantages over conventional measuring techniques in terms of speed, reproducibility and reduced operator to operator variation. We conclude that the cutaneous DTH reaction may be simply and objectively quantified with the use of a hand-held spectrophotometer.