Development and Evaluation of a Serological Assay for the Diagnosis of Tuberculosis in Alpacas and Llamas

Abstract

South American camelids are susceptible to tuberculosis, caused mainly by Mycobacterium bovis and M. microti. Despite the tuberculin skin test being the official test for tuberculosis, it has a very low sensitivity in these species (14–20%). Serological tests present the advantages of being rapid, easy to perform and facilitate analysis of large numbers of samples in a short period of time. Novel antigen discovery and evaluation would provide enhanced detection of specific antibodies against members of M. tuberculosis complex. Here, we describe the development and evaluation of an ELISA-type immunoassays to use in the diagnosis of tuberculosis in llamas and alpacas based on P22, a multiprotein complex obtained by affinity chromatography from bovine Purified Protein Derivative (bPPD), that showed high sensitivity and specificity in mice, cattle and goats. This work was performed in two stages. First, a preliminary panel of samples collected from tuberculosis-free (n = 396) and M. bovis-infected herds (n = 56) was assayed, obtaining high specificity (100%) and sensitivity ranging from 63 to 96%. Subsequently, the use of the serological assay was tested using samples from two herds suffering from clinical M. bovis (n = 88) and M. microti (n = 25) infection to evaluate the ability of the ELISA to detect infected animals. 11 out of 88 alpacas were positive to the ELISA in a M. bovis outbreak and 7 out of 25 in a M. microti outbreak. The P22 ELISA potentially provides a sensitive and specific platform for improved tuberculosis surveillance in camelids.

Protective Effect of Oral BCG and Inactivated Mycobacterium bovis Vaccines in European Badgers (Meles meles) Experimentally Infected With M. bovis

“…We therefore might conclude that sero-reactivity in those animals was unspecific due to cross-reaction with other environmental antigens. However, another reasonable possibility for these seroreactivities to P22 could be that these animals had a previous contact or were infected with M. microti, since infection with this species is also detectable using P22 ELISA (37). Mycobacterium microti isolation by culture, proven to be suitable for other species within the complex, can be quite difficult (37).…”

“…However, another reasonable possibility for these seroreactivities to P22 could be that these animals had a previous contact or were infected with M. microti, since infection with this species is also detectable using P22 ELISA (37). Mycobacterium microti isolation by culture, proven to be suitable for other species within the complex, can be quite difficult (37). In addition, if a mixed culture containing both M. microti and the challenge M. bovis or BCG strains is spoligotyped, the presence of M. microti would go unnoticed because the few spacers this species can harbor (if any) are also present in M. bovis and BCG strains (38).…”

Evaluation of P22 Antigenic Complex for the Immuno-Diagnosis of Tuberculosis in BCG Vaccinated and Unvaccinated Goats

“…Recently, the P22 antigenic complex has been evaluated for the detection of IgG in ELISA tests in different species: cattle goat, sheep, pigs, and wild boar ( 24 – 27 ), red deer ( 28 ), badgers ( 29 ), and alpacas and llamas ( 30 ). In the present study, the performance of the P22 antigenic complex for diagnostic tests based on CMI, namely, STs and IGRA, was evaluated for the first time in goats.…”

“…Recently, a new multiprotein complex called P22, obtained from purified protein derivative of M. bovis (PPD-B) by affinity chromatography, has been developed (23), yielding high Se and variable Sp, depending on the animal species and epidemiological contexts (24). To date, this antigen has been tested to detect humoral response against MTBC in different species (25)(26)(27)(28)(29)(30); however, there is a lack of information regarding its performance for cell-mediated immunity (CMI)-based diagnostics.…”

Study on a supplemental test to improve the detection of bovine tuberculosis in individual animals and herds

“…Over the last few years, the potential for use of an antibody assays to detect M. bovis infection in cattle is being consolidated (16,17,18,19,20,21,22,23,24,25). The ELISA have proven to be useful as ancillary serial (to enhance Sp) and parallel (to enhance Se) tests in several species (26,27,28,29,30). Moreover, a booster effect on the antibody response caused after injection of tuberculin has been reported and recommended as a strategic option to increase the sensitivity of serological assays (26,27,28).…”