Seroepidemiology of tuberculosis in sheep in southern Spain.

Tuberculosis (TB) is a multi-host infectious disease caused by members of the Mycobacterium tuberculosis complex (MTC). In Mediterranean ecosystems, where multiple animal hosts of TB are present, identifying the role of the different species involved in the epidemiology of TB is a key point to be able to implement proper control measures. Sheep are susceptible to MTC infection but have traditionally been considered a spillover host. However, the occurrence of outbreaks involving sheep in recent years evidences the need to better understand the role of this small ruminant species in the epidemiology of the disease. Here, we aimed to determine the seroprevalence and risk factors associated with MTC seropositivity in sheep in Andalusia (southern Spain), a region with one of the highest prevalence of MTC infection in both cattle and wild ungulates. A total of 2266 sheep from 83 flocks were tested for antibodies against MTC using an in-house indirect ELISA. Anti-MTC antibodies were detected in 16 (0.7%) of the 2266 sheep (adjusted true prevalence 0.29%, 95% posterior probability interval 0.01-1.05). Seropositivity was found in 14.5% (12/83; 95%CI: 6.9-22.0) of the sheep farms analyzed. A semi-extensive management system was identified as a risk factor associated with MTC seropositivity in sheep farms (OR = 3.7; p < 0.038; 95%CI: 1.1-12.4) in the study area. To the best of the authors' knowledge, this is the first active TB surveillance study carried out to assess MTC exposure in sheep. Our results indicate MTC circulation in sheep farms in southern Spain. However, the low individual seroprevalence obtained suggests that sheep may play a limited role in the epidemiology of TB in this region. Serosurveillance programs could be a valuable tool to detect MTC circulation in sheep in risk scenarios or target farms, in order to optimize control measures on TB animal in multi-host Mediterranean ecosystems.

P22 protein complex in the serodiagnosis of animal tuberculosis: Antigenic stability and cross-reactivity with Corynebacterium pseudotuberculosis infection.

The P22 ELISA was recently developed for the serodiagnosis of animal tuberculosis. Herein, the stability of the P22 antigen in different presentations and storage conditions, and the cross-reactivity with Corynebacterium pseudotuberculosis infection in small ruminants were evaluated. For the stability assay, serum samples from cows, sheep, goats, alpacas, badgers, and wild boar were used in the P22 ELISA. The cross-reactivity analysis used sera from sheep and goats with caseous lymphadenitis (CLA). Differences in the immune recognition of P22 were found when the antigen was stored at 40 °C, but without altering the negative or positive status of each sample. P22 ELISA presented 5.71 % cross-reactivity when CLA-positive sheep were evaluated, but no cross-reaction was observed among CLA-positive goat serum samples. This study showed that the P22 protein complex is stable under different formulations and temperatures, and that the assay presents a low cross-reactivity with CLA.

Effect of the Inoculation Site of Bovine and Avian Purified Protein Derivatives (PPDs) on the Performance of the Intradermal Tuberculin Test in Goats From Tuberculosis-Free and Infected Herds.

The single and comparative intradermal tuberculin (SIT and CIT) tests are used for the ante-mortem diagnosis of caprine tuberculosis (TB). The tuberculin injection site has been associated with a different performance of the test in cattle. In contrast to that required in cattle in Europe (cervical injection), it can be carried out in the scapular region in goats. Nevertheless, there are no previous data concerning the effect of the injection site on the performance of the test in goats. The aim of the present study was to evaluate the effect of two different inoculation sites (cervical and scapular) on the performance of the SIT/CIT tests. This was done by intradermally inoculating 309 goats from two infected herds and one TB-free herd with both avian and bovine PPDs in the mid-cervical and scapular regions. None of the animals from the TB-free herd had positive reactions, and the number of reactors was not significantly higher, regardless of the inoculation site, in the high and low prevalence herds. However, significantly higher increases in skin fold thickness were observed on the cervical site when compared to the scapular site after the avian and bovine PPD inoculations in the TB-free herd (p < 0.001) and after the bovine PPD injection in the high prevalence herd (p = 0.003). The presence of clinical signs was also more evident on the cervical site when using avian and bovine PPDs in the high prevalence herd (p < 0.01). In contrast, increases in higher skin fold thickness were observed on the scapular site when compared to the cervical site after the bovine and avian PPD inoculations were employed in the low prevalence herd (p < 0.01). These results suggest that the cervical injection of PPDs may improve the sensitivity of the intradermal tuberculin test in high TB prevalence caprine herds, mainly owing to the increased presence of local clinical signs and a better performance of the CIT test. Moreover, specificity was not affected when using standard interpretations, although further analyses in a great number of herds are required in order to confirm these findings.

Evaluation of P22 ELISA for the Detection of Mycobacterium bovis-Specific Antibody in the Oral Fluid of Goats.

The ante-mortem diagnosis of tuberculosis (TB) in ruminants is based mainly on the intradermal tuberculin test and the IFN-γ assay. Antibody (Ab)-based tests have emerged as potential tools for the detection of TB infected animals using serum, plasma, or even milk samples. Oral fluids have also been evaluated as alternative samples with which to detect specific Abs against Mycobacterium bovis in pigs or wild boars, but not in ruminants. The objective of this study was, therefore, to evaluate the performance of an in house-ELISA for TB diagnosis (P22 ELISA) in goats as an experimental model for the diagnosis of TB using oral fluid samples. Oral fluid samples from 64 goats from a TB-infected herd (n = 197) and all the animals from a TB-free herd (n = 113) were analyzed using the P22 ELISA. The estimated sensitivity (Se) and specificity (Sp) were 34.4% (95% CI: 22.4-45.6) and 100% (95% CI: 97.4-100), respectively. The optimal cut-off point was set at 100% according to the ROC analysis. Those animals with a higher level of Abs in their oral fluid attained a higher lesion score (p = 0.018). In fact, when taking into account only the setting of the animals with severe lesions (n = 16), the ELISA showed a Se of 75% (95% CI: 53.7-96.2). Results of the present study suggest that the P22 ELISA is highly specific but has a limited value detecting infected animals in oral fluid samples. Nevertheless, its performance is significantly higher in the presence of severe lesions.

Evaluation of the immunogenicity and efficacy of BCG and MTBVAC vaccines using a natural transmission model of tuberculosis.

Effective vaccines against tuberculosis (TB) are needed in order to prevent TB transmission in human and animal populations. Evaluation of TB vaccines may be facilitated by using reliable animal models that mimic host pathophysiology and natural transmission of the disease as closely as possible. In this study, we evaluated the immunogenicity and efficacy of two attenuated vaccines, BCG and MTBVAC, after each was given to 17 goats (2 months old) and then exposed for 9 months to goats infected with M. caprae. In general, MTBVAC-vaccinated goats showed higher interferon-gamma release than BCG vaccinated goats in response to bovine protein purified derivative and ESAT-6/CFP-10 antigens and the response was significantly higher than that observed in the control group until challenge. All animals showed lesions consistent with TB at the end of the study. Goats that received either vaccine showed significantly lower scores for pulmonary lymph nodes and total lesions than unvaccinated controls. Both MTBVAC and BCG vaccines proved to be immunogenic and effective in reducing severity of TB pathology caused by M. caprae. Our model system of natural TB transmission may be useful for evaluating and optimizing vaccines.

Development and characterization of mouse monoclonal antibodies to eight human complement components: Analysis of reactivity with orthologs of nine mammalian genera.

To study complement function in mammalian leishmanioses, we developed mouse monoclonal antibodies to the human complement system components C1q, C4, factor D, factor H, factor B, properdin, C5 and C9. Antibody specificity was determined by indirect and capture ELISA and by Western blot. In flow cytometry analysis, seven antibodies recognized the cognate component on human serum-opsonized Leishmania promastigotes. Antibody reactivity was screened against promastigotes opsonized with sera of nine mammalian genera: pig, guinea pig, goat, rabbit, cat, dog, hamster, jird and rat. No antibody recognized jird epitopes on promastigotes. Anti-C4, -properdin, and -C5b reacted with the orthologous protein of all other mammals tested except cat (anti-properdin) and hamster (anti-C5b); anti-C9 only recognized the rabbit ortholog, and anti-C1q, -factor B and -factor H did not react with any of the nine orthologs. Such interspecies crossreactive antibodies can be valuable tools for analysis of mammalian complement function in infectious diseases.

New serological platform for detecting antibodies against Mycobacterium tuberculosis complex in European badgers.

European badgers (Meles meles) have been identified as wildlife reservoirs for Mycobacterium bovis in the UK and Ireland, and may also have a role in the epidemiology of animal tuberculosis in other European regions. Thus, detection of M. bovis-infected badgers may be required for the purposes of surveillance and monitoring of disease levels in infected populations. Current serological assays to detect M. bovis infection in live badgers, while rapid and inexpensive, show limited diagnostic sensitivity. Here we describe and evaluate new ELISA platforms for the recognition of the P22 multiprotein complex derived from the purified protein derivative (PPD) of M. bovis. The recognition of IgG against P22 multiprotein complex derived from PPD-B was tested by ELISA in the serum of badgers from the UK, Ireland and Spain. TB infection in the badgers was indicated by the presence of M. bovis in tissues by culture and histology at post-mortem examination and TB-free status was established by repeated negativity in the interferon γ release assay (IGRA). In experimentally infected badgers, humoral antibody responses against P22 developed within 45 days post-infection. The ELISA tests showed estimated sensitivity levels of 74-82% in experimentally and naturally infected badgers with specificities ranging from 75% to 100% depending on the badger population tested. The P22 multi-antigen based ELISAs provide a sensitive and specific test platform for improved tuberculosis surveillance in badgers.

Validation of a new serological assay for the identification of Mycobacterium tuberculosis complex-specific antibodies in pigs and wild boar.

BACKGROUND AND OBJECTIVES: Animal tuberculosis (TB) is a multi-host disease involving a wide variety of domestic and wild mammals and causing a significant economic burden and sanitary problems. Wild boar and domestic pigs (Sus scrofa) are indicators of the circulation of the Mycobacterium tuberculosis complex (MTC) and can play a role in its maintenance. The proper diagnosis of MTC contact in these species is, therefore, a key factor as regards controlling TB. The objective of the current study is to evaluate the diagnostic performance of the protein complex P22 as a candidate for use in an in-house ELISA to identify M. tuberculosis complex-specific antibodies for the diagnosis of TB in comparison to the commonly used bPPD-based ELISA (bPPD ELISA) in suids. METHODS: We conducted a retrospective study. Sera were collected from wild boar during hunting season and from domestic pigs during routine handling, and all the animals underwent reference standard tests (detailed necropsy followed by bacteriological culture and isolation). Animal TB was confirmed to be positive in 277 animals and negative in 366 animals based on both reference standard tests. Sera from those animals were tested by P22 ELISA as well as bPPD ELISA. RESULTS: Both ELISAs yielded a good diagnostic value, however, a higher sensitivity (Se) and specificity (Sp) was achieved with the P22 ELISA (Se: 84.1%; CI95%: 79.3-88.2% / Sp: 98.4%; CI95%:96.5-99.4%) when compared to the bPPD ELISA (Se: 77.3%; CI95%: 71.9-82.2% / Sp: 97.3%; CI95%: 95-98.3%). An optimum Sp of 100% (CI95%: 98.54-100%) was attained with white pigs for both the bPPD and the P22 ELISA. DISCUSSION: The results suggest that serological tests for MTC-antibody detection, and particularly the P22 ELISA, are valuable tools in the diagnosis of TB in wild boar and domestic pigs when attempting to detect contact with MTC and thereby facilitate TB control and management.

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

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.

Evaluation of five serologic assays for bovine tuberculosis surveillance in domestic free-range pigs from southern Spain.

In countries where bovine tuberculosis (bTB) is still prevalent the contact among different animal species in extensive systems contributes to the circulation of Mycobacterium bovis (M. bovis) and other members of the Mycobacterium tuberculosis complex (MTC). Thus, free-range pigs can develop subclinical infections and may contribute to disease spread to bovine and wildlife. Serodiagnosis has been proposed as a screening tool for detecting infected pig herds; however, the value of this method to obtain an accurate diagnosis in this species is still not clear. In this study, sensitivity (Se) and specificity (Sp) estimates of four ELISAs and a lateral flow immunochromatographic antibody assay based on different M. bovis antigens, including MPB70 and MPB83 proteins, were evaluated in naturally infected domestic free-range pigs. For this purpose, submandibular lymph nodes and blood samples from 217 pigs from both TB-infected and historically negative farms were sampled at slaughterhouse and analysed by gross examination, histopathology, bacteriological culture and qPCR. Se and Sp estimates of the 5 evaluated assays ranged from 66.1% to 78% (CI95 from 52.6 to 87.7%) and from 98.9% to 100% (CI95 from 93.8 to 100%), respectively. Results of our study suggest that all the evaluated assays could be used as a first screening tool to conduct bTB surveillance in domestic pigs at population level; however, animals from seropositive herds should later be surveyed by other methods in order to reduce false negative results.