Serologic tests for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa)

Boadella et al.New tools to detect exposure of free-range Eurasian wild boar (Sus scrofa scrofa) to pathogenic mycobacteria would be valuable for improved disease surveillance and wildlife management. Two hundred sera from wild boar of known Mycobacterium bovis infection status were used to evaluate test suitability for the detection of antibodies against M. bovis and Mycobacterium avium subsp. paratuberculosis (or crossreacting members of the M. avium complex). Two traditional enzyme-linked immunosorbent assays were evaluated using M. bovis purified protein derivative (bPPD) and paratuberculosis protoplasmatic antigen 3 (PPA3) as antigens, respectively, and a new point-of-care test format for bovine tuberculosis (bTB) that uses the innovative dual-path platform (DPP TB) test. The effect of individual factors (sex, age, lesions) on the diagnostic performance of the serologic tests was also determined. Although the DPP had a sensitivity of 89.6% and a specificity of 90.4%, for bPPD, the sensitivity was 79.2% and the specificity 100%. Both tests had a kappa agreement of 0.80. Sixty-five of 68 (95.6%) wild boar sera with antibodies against the PPA3 antigen corresponded to known M. bovis-infected wild boar. Significant differences were not observed in the bPPD and DPP readings among lesion categories or between age classes. A slight sex-related difference in sensitivity toward males in the DPP was found, but it was not detected in the bPPD enzyme-linked immunosorbent assay. The results support the use of antibody-based diagnostic tests for both large-scale and individual bTB testing of Eurasian wild boar and suggest that wild boar cannot be used as sentinels for infections caused by M. avium complex members.The current study is a contribution to MCINN Plan Nacional I+D+i research grant AGL2008-03875 and FEDER and to FP7 EU grant TB-STEP (212414). Studies on diseases shared between domestic animals and wildlife are also supported by Grupo Santander Fundacion Marcelino Botin. Tania Carta acknowledges a grant from Regione Sardegna.Peer Reviewe

Development of a Multidimensional Proteomic Approach to Detect Circulating Immune Complexes in Cattle Experimentally Infected With Mycobacterium bovis

Objective: To evaluate a high-resolution method to identify pathogen-specific biomarkers in serum of calves infected with Mycobacterium bovis.Methods: Serum samples from four calves infected with M. bovis were collected before and after infection at weeks 9, 14, 15, 31, and 36. Immune-complex-associated mycobacterial antigens in the serum were enriched using an immunochromatography method termed, dual path platform (DPP). All regions of antigen capture zones, that consisted of monospecific rabbit polyclonal antibodies raised against M. tuberculosis lysates, on DPP strips were excised and analyzed by multidimensional proteomics. The resulting proteins were then passed through 4 rigorous peptide quality filters-false-hits, decoys, non-M. tuberculosis complex proteins were all removed followed by individual quality check of those remaining. Peptides were then checked on NCBI's BLASTp for M. tuberculosis complex specificity.Results: Proteins in 2 of the animals passed the multipronged-highly stringent peptide quality analysis. Animal#54 had 7 unique M. tuberculosis complex proteins at week 14 post-infection, while animal#56 had 4 at week 36 post-infection along with 1 immunoglobulin.Conclusion:M. tuberculosis complex -specific peptides identified in this study were identified in 2 animals and at 2 separate time points post infection. Further studies with better enrichment protocols and using larger sample sizes and replications are required to develop a TB-specific diagnostic tool for bovine tuberculosis

Testing Eurasian wild boar piglets for serum antibodies against Mycobacterium bovis

A. Che’ Amat et al.Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar (. Sus scrofa). Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2-6 month-old piglets. In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture (. n=. 53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19% to 38%, reaching sensitivities between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2-6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis-infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account.This is a contribution to Spanish Government MINECO Plan Nacional I+D+I grant AGL2014-56305 and FEDER, to a contract between CDTI and Glenton, and to the EU FP7 grant WildTBvac #613779. Azlan Che Amat has a PhD grant from the Malaysian Government, and José Angel Barasona and Iratxe Diéz-Delgado acknowledge PhD grants from the Spanish Government.Peer Reviewe

Experimental Infection of Captive Red Foxes (Vulpes vulpes) with Mycobacterium bovis

[EN] In Europe, animal tuberculosis (TB) due to Mycobacterium bovis involves multi-host communities that include cattle and wildlife species, such as wild boar (Sus scrofa), badgers (Meles meles) and red deer (Cervus elaphus). Red fox (Vulpes vulpes) infections have also been recently reported in some TB endemic regions in the Iberian Peninsula and France, with some of the infected animals shedding M. bovis in urine and feces. In order to understand the pathogenesis of M. bovis infection in foxes and the associated risk of transmission, 12 captive foxes (6 females and 6 males) were inoc-ulated orally with 2 × 107 colony-forming units of a French field isolate of M. bovis. Clinical samples (urine, feces and oropharyngeal swabs) were collected every four weeks and tested for molecular diagnosis and bacteriology. Serological responses were measured by IDEXX M. bovis Ab Test and Multi Antigen Print Immunoassay (MAPIA). At a post-mortem examination performed 12 weeks post infection (wpi), tissues were tested for the presence of M. bovis and associated gross and microscopic TB-like lesions. M. bovis was detected by PCR in bladder swabs of 3 animals at 12 wpi. It was also detected pre-mortem at different time points of the experiment in the oropharyngeal mu-cus of three individuals and in the feces of nine foxes, with two of them confirmed by bacteriology. All 12 foxes had at least 4 PCR positive samples (out of the 23 tested), and all but 1 fox had at least 1 culture positive sample. The culture negative fox was PCR positive in both retropharyngeal and mesenteric lymph nodes, in line with the results of the other animals. Seroconversion was observed in all foxes except one during the experiment, and in nine at the final time point. No gross visible lesions were found in any animal at the post-mortem examination. The histology showed small granulomas within the lymph nodes, tonsils, liver and lungs from eight animals, with the presence of few acid-fast bacilli. These results confirmed that all orally-infected foxes developed mild TB lesions but they were able to shed mycobacteria in about 75% of cases, 1 month post-infection (9 out 12 foxes). These results show that it is possible to induce typical TB infection experimentally in captive foxes, with measurable M. bovis excretion; such an experimental system could be useful for future evaluations of diagnostics and vaccines in this speciesSIThe French Ministry of Agriculture mainly financed the sampling and the analyses in the framework of the RFSA call on TB projects (Anses-DGAl credit agreement RFSA 2017-326). The animals and the running cost of the BSL3 facilities and technical resources were financed by the European Commission in the context of Horizon 2020?Vetbionet Transnational Access Activities (TNA) call. This work is also partially the result of the I+D+i research project RTI2018-096010-B-C21, funded by the Spanish MCIN/AEI/10.13039/501100011033/ Ministry of Science, Innovation and the European Regional Development Funds (FEDER Una manera de hacer Europa), and of PCTI 2021? 2023 (GRUPIN: IDI2021-000102) funded by Principado de Asturias and FEDE

Tonsils of the soft palate do not mediate the response of pigs to oral vaccination with heat-inactivated Mycobacterium bovis

Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research.This study is a contribution to EU FP7 grant 613779 WildTBVac and to MINECO Plan Nacional grant AGL2011-30041 and FEDER.Peer Reviewe

Bovine Tuberculosis in a Nebraska Herd of Farmed Elk and Fallow Deer: A Failure of the Tuberculin Skin Test and Opportunities for Serodiagnosis

In 2009, Mycobacterium bovis infection was detected in a herd of 60 elk (Cervus elaphus) and 50 fallow deer (Dama dama) in Nebraska, USA. Upon depopulation of the herd, the prevalence of bovine tuberculosis (TB) was estimated at ∼71–75%, based upon histopathology and culture results. Particularly with elk, gross lesions were often severe and extensive. One year ago, the majority of the elk had been tested for TB by single cervical test (SCT), and all were negative. After initial detection of a tuberculous elk in this herd, 42 of the 59 elk were tested by SCT. Of the 42 SCT-tested elk, 28 were TB-infected with only 3/28 reacting upon SCT. After SCT, serum samples were collected from the infected elk and fallow deer from this herd at necropsy and tested by three antibody detection methods including multiantigen print immunoassay, cervidTB STAT-PAK, and dual path platform VetTB (DPP). Serologic test sensitivity ranged from 79 to 97% depending on the test format and host species. Together, these findings demonstrate the opportunities for use of serodiagnosis in the rapid detection of TB in elk and fallow deer

Oral re-vaccination of Eurasian wild boar with Mycobacterium bovis BCG yields a strong protective response against challenge with a field strain

Background: Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain.Results: BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge.Conclusions: Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions.Peer reviewedVeterinary Pathobiolog

Oral vaccination with heat inactivated Mycobacterium bovis activates the complement system to protect against tuberculosis

Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar.This research was supported by Plan Nacional I+D+I AGL2011-30041 from Ministerio de Economía y Competitividad (MINECO), Spain and FEDER. This is also a contribution to EU FP7 grant WildTBvac and the EU FP7 ANTIGONE project number 278976. R.C. Galindo was funded by MEC, Spain. B. Beltrán-Beck was supported by MINECO grant BES-2009-017401.Peer Reviewe

Potential for rapid antibody detection to identify tuberculous cattle with non-reactive tuberculin skin test results

Abstract Background Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. Results Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP®) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. Conclusions Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds

Potential for improved detection of bovine tuberculosis by targeting combined blood biomarkers in multi-test algorithms

Bovine tuberculosis (bTB) control programs can be improved by combined use of tests for humoral and cell-mediated immune responses targeting multiple biomarkers of Mycobacterium bovis. To further the diagnostic benefits of this approach, we used Dual Path Platform (DPP) technology to test sera from cattle with naturally acquired bTB in the United States (US) and Spain for the presence of M. bovis antigen, IgM and/or IgG antibodies to MPB70/MPB83 fusion antigen in conjunction with tuberculin skin tests (TST) or interferon-gamma release assays (IGRA). When TST was complemented with detection of IgM and IgG antibodies, the diagnostic sensitivity increased from 85.4% to 95.1% in the US and from 64.2% to 81.5% in Spain. Likewise, adding the DPP assays enhanced IGRA diagnostic sensitivity from 82.7% to 93.8% in Spain. Detection of circulating M. bovis antigen showed added value when used in combination with the DPP antibody assays but it was limited when analyzed in the context of TST or IGRA results. Present findings support the benefits of a multi-test approach for the ante-mortem diagnosis of bTB in cattle.This work was supported by the Small Business Innovation Research Program of the USDA National Institute of Food and Agriculture (Award No. 2016-33610-25688).Peer reviewe