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Recent studies have suggested the potential of innovative serologic tests for accurate and rapid detection of bovine tuberculosis (bTB). Dual Path Platform (DPP) technology has been used to develop rapid animal-side antibody tests for Mycobacterium bovis infection in a range of livestock and wildlife host species. The present study evaluated diagnostic performance of DPP BovidTB IgM/IgG assay designed for differential detection of bovine IgM and IgG antibodies against two chimeric antigens, DID38 and TBf2, respectively, using 662 well-characterized serum samples from M. bovis-infected and bTB-free cattle collected in the United States, Great Britain, France, and South Africa. Test sensitivity and specificity ranged from 71% to 100% and from 95% to 100%, respectively, depending on the country, with overall accuracy of 83%. No significant risk of cross-reactivity with serum samples from cattle infected with most relevant species of mycobacteria other than M. bovis was found. The DPP BovidTB IgM/IgG assay may be suitable for use in multi-test algorithms to improve current strategies for bTB surveillance.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection are crucial for effective surveillance and management, especially in wildlife populations. There are, however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection, and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB-endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevalence of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB-endemic ecosystems and evaluate serological and other diagnostic tools in this species.

The present study highlights the transboundary nature of tuberculosis (TB) in alpacas and the failure of current antemortem testing protocols to identify TB-free alpaca herds and individuals for exportation. The tuberculin skin test (TST) failed to identify Mycobacterium bovis-infected animals prior to movement from the United Kingdom (UK) to Poland. This study describes the use of four serological assays [Enferplex Camelid TB, dual-path platform (DPP) VetTB and BovidTB assays, and multi-antigen print immunoassays (MAPIAs)] to detect TB in an alpaca herd with negative TST results. The breeding in Poland purchased alpacas for several years from the UK with the last group arriving in May 2018. In July 2018, two sick alpacas from the centre were hospitalized in a veterinary clinic and both died of TB a few weeks later. In November 2018, 20 alpacas remaining in this M. bovis-affected herd were euthanized and samples were collected. The study population included 20 M. bovis-infected and 20 uninfected alpacas, but only 15 infected animals were tested by all serology tests. The DPP VetTB and DPP BovidTB assays detected antibodies in 14 of the 20 infected alpacas, with results confirmed by MAPIA, and in none (MAPIA and DPP BovidTB) or one (DPP VetTB) of the 20 uninfected animals. None of the infected alpacas tested positive using the Enferplex assay. In addition, the group included three orphans and two cria-dam pairs, which provided an opportunity to analyse immune aspects of cria-mother relationships in this herd. The results suggest high susceptibility of this host species to M. bovis infection and rapid progression to disease. The serological tests used in this study offer useful tools for the detection of M. bovis infection in TST and Enferplex test non-reactive alpacas. These tests should be further evaluated for implementation into TB management and control strategies for camelid species.

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Bovine tuberculosis (bTB) control programs can be improved by implementation of advanced ante-mortem testing algorithms. Serodiagnostic methods using traditional blood or blood-derived specimens may benefit from the use of less invasive alternative biological fluids, provided those mirror systemic antibody responses. In the present study, we used Dual Path Platform (DPP) and Multiantigen Print Immunoassay (MAPIA) to compare antibody levels in ten sample types including whole blood (fresh and hemolyzed), plasma (fresh and leftover from Bovigam testing), serum, saliva, broncho-alveolar lavage, urine, diaphragm extract, and bile collected from cattle aerosol-infected with Mycobacterium bovis. High correlation (r = 0.97-0.99) in measurements of IgG antibodies to MPB70/MPB83 fusion antigen by DPP assay was found between all blood-derived specimens, supporting matrix equivalency. Broncho-alveolar lavage and diaphragm extract yielded positive results in all the infected animals tested, showing high correlation with matching serum data (r = 0.94 and r = 0.95, respectively) and suggesting their potential use in antibody assays. Characterized by MAPIA, the antigen reactivity patterns obtained with paired sera and alternative specimens were nearly identical, with slight differences in intensity. Antibodies were also found by DPP assay in saliva, urine, and bile from some of the infected animals, but the titers were relatively low, thus reducing the diagnostic value of such specimens. The proposed approach was evaluated in a pilot field study on warthogs diagnosed with M. bovis infection. Relative levels of antibody in tissue fluid obtained from lymph nodes or lungs were consistent with those detected in sera and detectable in all infected warthogs. The findings support the diagnostic utility of non-traditional biological fluids and tissue samples when used as alternative test specimens in serologic assays for bTB.

Wildlife tuberculosis is a major economic and conservation concern globally. Bovine tuberculosis (bTB), caused byMycobacterium bovis(M. bovis), is the most common form of wildlife tuberculosis. In South Africa, to date,M. bovisinfection has been detected in 24 mammalian wildlife species. The identification ofM. bovisinfection in wildlife species is essential to limit the spread and to control the disease in these populations, sympatric wildlife species and neighboring livestock. The detection ofM. bovis-infected individuals is challenging as only severely diseased animals show clinical disease manifestations and diagnostic tools to identify infection are limited. The emergence of novel reagents and technologies to identifyM. bovisinfection in wildlife species are instrumental in improving the diagnosis and control of bTB. This review provides an update on the diagnostic tools to detectM. bovisinfection in South African wildlife but may be a useful guide for other wildlife species.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection is crucial for effective surveillance and management, especially in wildlife populations. There are however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and to identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevelance of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation, and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB endemic ecosystems and evaluate serological and other diagnostic tools in this species.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, infects humans and animals causing lesions and disease like that of Mycobacterium bovis. The aim of this study was to evaluate antibody responses in European Bison (EB, Bison bonasus; a vulnerable species) naturally infected with M. caprae using dual path platform (DPP) BovidTB test and multi-antigen print immunoassay (MAPIA). Study cohorts consisted of naturally M. caprae-infected EB (n = 4), M. caprae-exposed but uninfected (n = 3), EB infected with non-tuberculous mycobacteria or other respiratory pathogens (n = 3), and negative controls (n = 19). M. caprae-infected EB were seropositive by both DPP and MAPIA; 3/4 were seropositive by DPP; and 4/4 were seropositive by MAPIA. One M. caprae-infected animal that developed generalized disease with most advanced gross lesions in the group produced the most robust antibody response. All 25 EB with no culture-confirmed M. caprae infection, including three animals exposed to M. caprae and three other animals infected with non-tuberculous pathogens, were seronegative on both tests. Antibody responses to M. caprae infection included IgM antibodies against MPB70/MPB83 and IgG antibodies to both MPB70/MPB83 and CFP10/ESAT-6. This study demonstrates the potential for use of serological assays in the ante-mortem diagnosis of M. caprae infection in EB.

Using multi-antigen print immunoassay and DPP VetTB Assay approved in the United States for testing captive cervids and elephants, we analyzed antibody recognition of MPB83 and CFP10/ESAT-6 antigens in Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis and in white-tailed deer (Odocoileus virginianus), fallow deer (Dama dama), elk (Cervus elaphus), and cattle (Bos taurus) infected with Mycobacterium bovis. Serum IgG reactivity to MPB83 was found in the vast majority of tuberculous cattle and cervid species among which white-tailed deer and elk also showed significant CFP10/ESAT-6 recognition rates with added serodiagnostic value. In contrast, the infected elephants developed antibody responses mainly to CFP10/ESAT-6 with MPB83 reactivity being relatively low. The findings demonstrate distinct patterns of predominant antigen recognition by different animal hosts in tuberculosis., This work was partially supported by the Small Business Innovation Research Program of the USDA National Institute of Food and Agriculture (Award No. 2016-33610-25688).

Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis-infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis-infected and 57 non-infected cattle showed the best accuracy (∼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis.

Tuberculosis (TB) is a pathogenic disease that affects a range of wildlife species, including African elephants (

Domestic pigs and wild suids are susceptible to Mycobacterium bovis (M. bovis) infection and may even serve as reservoir hosts in some situations. Therefore, detection of infected animals is important for understanding their role in the epidemiology of the disease as well as for management and control of bovine tuberculosis. Infected suids develop strong humoral responses, making serological screening a feasible approach to disease surveillance. However, to optimize sensitivity of the antibody assays, it is necessary to identify and incorporate immunodominant antigens recognized by the target species. The objective of this study was to characterize the antigen recognition by three suid species in a commercially available serological test, DPP VetTB Assay. Serum samples from naturally M. bovis-infected domestic pigs, wild boar and common warthogs were tested. MPB83 protein appeared to be the immunodominant antigen recognized by antibodies in all three species. Overall test sensitivity was increased in wild suids when seroreactivity to CFP10/ESAT-6 antigen was included. Infected animals with visible lesions showed more robust antibody responses than those without gross lesions. The high sensitivity and specificity of the DPP VetTB Assay demonstrated in the present study supports the utility of antibody tests employing these antigens in serological screening of the suid species for M. bovis infection., Financial support (MAM, EOR) was provided by the South African Medical Research Council, NRF South African Research Chair Initiative (SARChI, grant 86949) and the American Association of Zoological Medicine Wild Animal Health Fund (#7-2016).

We assessed the suitability of targeted removal as a means for tuberculosis (TB) control on an intensely managed Eurasian wild boar (Sus scrofa) hunting estate. The 60 km large study area included one capture (treatment) site, one control site, and one release site. Each site was fenced. In the summers of 2012, 2013 and 2014, 929 wild boar were live-captured on the treatment site. All wild boar were micro-chipped and tested using an animal side lateral flow test immediately after capture in order to detect antibodies to the Mycobacterium tuberculosis complex (MTC). The wild boar were released according to their TB status: Seropositive individuals onto the release site (hunted after summer), and seronegative individuals back onto the treatment site. The annual summer seroprevalence of antibodies to the MTC declined significantly in live-captured wild boar piglets from the treatment site, from 44% in 2012 to 27% in 2013 (a reduction of 39%). However, no significant further reduction was recorded in 2014, during the third capture season. Fall-winter MTC infection prevalence was calculated on the basis of the culture results obtained for hunter-harvested wild boar. No significant changes between hunting seasons were recorded on either the treatment site or the control site, and prevalence trends over time were similar on both sites. The fall-winter MTC infection prevalence on the release site increased significantly from 40% in 2011–2012 to 64% in 2012–2013 and 2013–2014 (60% increase). Recaptures indicated a persistently high infection pressure. This experiment, the first attempt to control TB in wild boar through targeted removal, failed to reduce TB prevalence when compared to the control site. However, it generated valuable knowledge on infection pressure and on the consequences of translocating TB-infected wild boar., This is a contribution to Spanish Government MINECO Plan Nacional grant AGL2014-56305 and FEDER, to CDTI, and to the EU FP7 WIldTBvac#613779 grant. Azlan Che’Amat has a PhD grant from the Malaysian Government.

The diagnosis of bovine tuberculosis (BTB) in living wildlife remains a complex problem, and one of particular importance in endangered species like European bison (Bison bonasus). To identify infection and avoid the unnecessary culling of such valuable individuals, current best practice requires the collection and culture of material from living animals, as mycobacteria isolation remains the gold standard in BTB diagnosis. However, such isolation is challenging due to the need for the immobilization and collection of appropriate clinical material, and because of the sporadic shedding of mycobacteria. In the present study, we evaluated the potential of sampling for the detection of BTB in a group of seven living European bison suspected of being infected with Mycobacterium caprae. The specimens were collected both as swabs from the nasal and pharyngeal cavities, tracheobronchial aspirates (TBA), ultrasound-guided biopsies from lateral retropharyngeal lymph nodes, and post mortem, from mandibular, retropharyngeal and mediastinal lymph nodes. Clinical samples were tested for mycobacterial species via mycobacteriological culture and PCR. M. caprae was isolated from collected material in two out of four living infected individuals (TBA, biopsy) and mycobacterial DNA was detected in three out of four (TBA, pharyngeal swab) bison. This is the first report of isolation of M. caprae in living European bison. Our findings demonstrate the value of diagnostic tests based on both molecular testing and culture in European bison and confirm the respiratory shedding of viable M. caprae in this host species.

Immunological memory is a central feature of adaptive immunity. Memory B cells are generated upon stimulation with antigen presented by follicular dendritic cells in the peripheral lymphoid tissues. This process typically involves class-switch recombination and somatic hypermutation and it can be dependent or independent on germinal centers or T cell help. The mature B cell memory pool is generally characterized by remarkable heterogeneity of functionally and phenotypically distinct sub-populations supporting multi-layer immune plasticity. Memory B cells found in human patients infected with Mycobacterium tuberculosis include IgD+ CD27+ and IgM+ CD27+ subsets. In addition, expansion of atypical memory B cells characterized by the lack of CD27 expression and by inability to respond to antigen-induced re-activation is documented in human tuberculosis. These functionally impaired memory B cells are believed to have adverse effects on host immunity. Human and animal studies demonstrate recruitment of antigen-activated B cells to the infection sites and their presence in lung granulomas where proliferating B cells are organized into discrete clusters resembling germinal centers of secondary lymphoid organs. Cattle studies show development of IgM+, IgG+, and IgA+ memory B cells in M. bovis infection with the ability to rapidly differentiate into antibody-producing plasma cells upon antigen re-exposure. This review discusses recent advances in research on generation, re-activation, heterogeneity, and immunobiological functions of memory B cells in tuberculosis. The role of memory B cells in post-skin test recall antibody responses in bovine tuberculosis and implications for development of improved immunodiagnostics are also reviewed.

Mycobacterium orygis, a newly identified member of the Mycobacterium tuberculosis complex, has been isolated predominantly from hoofstock in eastern Africa and the Arabian Peninsula, and sporadically in cattle (Bos taurus indicus), rhesus monkeys (Macaca mulatta), humans, and a greater one-horned rhinoceros (Rhinoceros unicornis) in South Asia. In rhinoceros, tuberculosis typically presents as a chronic progressive respiratory disease. The report describes the postmortem diagnosis of tuberculosis caused by Mycobacterium orygis in a greater one-horned rhinoceros with hind limb paresis due to neural granulomatosis. Serologic assays for detection of antibodies to M. tuberculosis complex proteins before culture results allowed for appropriate herd management protocols to be initiated. Mycobacterium genus-specific polymerase chain reaction assays with direct sequencing allowed timely confirmation of the serologic results. This is the first isolation of M. orygis in the western hemisphere, showing the need for mycobacterial testing of rhinoceros before international shipments and the urgency for validated antemortem M. tuberculosis complex screening assays in rhinoceros species.

Tuberculosis (TB) was diagnosed in four Asian elephants ( Elephas maximus) in a zoo in the United States. The first case was detected by isolation of Mycobacterium tuberculosis during routine trunk wash (TW) culture testing of a herd of eight elephants. Retrospective antibody analyses revealed seroconversion 1 yr before diagnosis. Serological testing of the whole elephant herd identified two additional suspect bulls with detectable antibody, but which remained culture-negative and had no clinical signs of disease. In the following months, M. tuberculosis, identical to the isolate from the index case, was isolated from TW samples of these two elephants. A fourth elephant seroconverted nearly 4 yr after the first TB case was detected, and M. tuberculosis was isolated from a TW sample collected 1 mo later. All four infected elephants received anti-TB therapy. Two treated elephants were eventually euthanized for reasons unrelated to M. tuberculosis and found to be culture-negative on necropsy, although one of them had PCR-positive lung lesions. One infected animal had to be euthanized due to development of a drug-resistant strain of M. tuberculosis; this animal did not undergo postmortem examination due to risk of staff exposure. The fourth animal is currently on treatment. Serial serological and culture results of the other four herd mates have remained negative.

Tuberculosis (TB) in humans is a global public health concern and the discovery of animal cases of Mycobacterium tuberculosis (Mtb) infection and disease, especially in multi-host settings, also has significant implications for public health, veterinary disease control, and conservation endeavors. This paper describes a fatal case of Mtb disease in a free-ranging African elephant (Loxodonta africana) in a high human TB burden region. Necropsy revealed extensive granulomatous pneumonia, from which Mtb was isolated and identified as a member of LAM3/F11 lineage; a common lineage found in humans in South Africa. These findings are contextualized within a framework of emerging Mtb disease in wildlife globally and highlights the importance of the One Health paradigm in addressing this anthroponotic threat to wildlife and the zoonotic implications.

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.

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.

Several serological tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when used after the injection of purified protein derivative (PPD) for skin testing, which significantly boosts M. bovis -specific antibody responses. The present findings demonstrate the onset and duration of boosted antibody responses after the injection of M. bovis PPD for the caudal fold test (CFT) and Mycobacterium avium and M. bovis PPDs for the comparative cervical test (CCT), administered in series in cattle experimentally infected with M. bovis . While skin tests boosted the responses to certain antigens (i.e., MPB83 and MPB70), they did not affect the responses to other antigens (e.g., ESAT-6, CFP10, MPB59, and MPB64). Administration of the CCT 105 days after the CFT resulted in an even greater secondary boost in antibody responses to MPB83 and MPB70 and to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis . Both IgM and IgG contributed to the initial boost in the MPB83/MPB70-specific antibody response after the CFT. The secondary boost after the CCT was primarily due to increased IgG levels. Also, the avidity of antibodies to MPB83 and MPB70 increased after the CCT in M. bovis -infected cattle. The avidity of antibodies to the WCS-PK antigens increased in the interval between the CFT and the CCT but did not increase further after the CCT. Together, these findings demonstrate that the administration of PPDs for skin tests results in additive enhancement (i.e., when the CFT and CCT are performed in series), both qualitative and quantitative, of MPB83/MPB70-specific antibody responses.

A comprehensive elephant tuberculosis (TB) survey using culture and four serological screening tests was conducted in Nepal in response to concern raised by wildlife officials that TB could threaten wild populations of elephants, rhinos, and other susceptible species. Captive elephants come into close contact with wild animals during conservation and tourism activities inside Nepal’s national parks. Private and government-owned male and female captive Asian elephants (Elephas maximus) were included in the study. The mean reported age was 38 years (range 5–60 years). A total of 289 samples from 120 elephants were collected for mycobacterial culture. Culture samples were processed at the National Tuberculosis Centre (NTC) in Nepal and the National Veterinary Services Laboratories (NVSL) in Ames, IA. Acid-fast organisms were observed in 11 and 21 samples processed at NTC and NVSL, respectively, and nontuberculous mycobacteria (NTMs) were isolated from six elephants. There were no isolations of Mycobacterium tuberculosis or Mycobacterium bovis. Blood samples were also collected from 115 of the elephants for serological testing using the Chembio ElephantTB STAT-PAK®, the Chembio MultiAntigen Print Immunoassay test, a multi-antigen ELISA, and an immunoblot assay. Culture and serological results were variable and required careful interpretation to develop criteria to assess TB risk. Elephants were assigned to one of four disease risk groups (high, moderate, low, and undetermined), and management recommendations for each group were made to government authorities. Serological results were prioritized in developing recommendations because of culture limitations and inconclusive culture results. This strategy was based on evidence for the early predictive value of serological tests and the urgent need expressed by wildlife authorities in Nepal to protect their captive elephants, mitigate TB at the captive-wild interface, and safeguard tourism.

Bovine tuberculosis (TB), caused by Mycobacterium bovis , remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (∼95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.

The presence of circulating antigen in cattle experimentally infected with Mycobacterium bovis was demonstrated using dual-path platform (DPP) technology. The antigen capture immunoassays employed rabbit polyclonal antibody recognizing predominantly M. tuberculosis complex-specific epitopes and were able to detect soluble substances and whole cells of mycobacteria. The antigen found in serum appeared to be mostly bound to IgM, but not to IgG, within the immune complexes formed at early stages of M. bovis infection. The antigen was also detected in bile and urine, indicating possible clearance pathways. The data correlation analyses supported the idea of the role of IgM responses in antigen persistence during M. bovis infection. The antigen was detectable in serum months prior to detectable antibody seroconversion. This proof-of-concept study suggested the potential for improved immunodiagnostics for bovine tuberculosis.

Over the past two decades, highly virulent strains of Mycobacterium tuberculosis have emerged and spread rapidly in man, suggesting a selective advantage based on virulence. A similar scenario has not been described for Mycobacterium bovis infection in cattle (i.e. bovine tuberculosis). An epidemiological investigation of a recent outbreak of bovine tuberculosis in a USA dairy indicated that the causative strain of M. bovis (strain 10-7428) was particularly virulent, with rapid spread within the herd. In the present study, the virulence of this strain (10-7428) was directly compared in the target host with a well-characterized strain (95-1315) of relevance to the USA bovine tuberculosis eradication programme. Aerosol inoculation of 10(4) colony forming units of M. bovis 95-1315 (n = 8) or 10-7428 (n = 8) resulted in a similar distribution and severity of gross and microscopical lesions of tuberculosis as well as mycobacterial colonization, primarily affecting the lungs and lung-associated lymph nodes. Specific cell-mediated and antibody responses, including kinetics of the response, as well as antigen recognition profiles, were also comparable between the two treatment groups. Present findings demonstrate that M. bovis strains 95-1315 and 10-7428 have similar virulence when administered to cattle via aerosol inoculation. Other factors such as livestock management practices likely affected the severity of the outbreak in the dairy.

Tuberculosis (TB) is a pathogenic disease that affects a range of wildlife species, including African elephants (Loxodonta africana). The recent discovery of fatal disease caused by infection with Mycobacterium tuberculosis in a bull elephant in the Kruger National Park (KNP), which is a bovine TB endemic area, emphasizes the importance this disease could have on both wild and captive elephant populations globally. Elephants with culture-confirmed TB have previously been shown to produce strong antibody-responses before the mycobacteria can be isolated. Therefore, we used two serologic assays that detect TB antibodies to retrospectively screen a cohort of 222 free-ranging African elephants sampled between 2004 and 2018 in KNP. The estimated TB seroprevalence for this free-roaming elephant population was between 6% (95% confidence interval [CI], 2-12%) and 9% (95% CI, 6-15%) based on the two tests. Overall, males had a higher TB seroprevalence than females, and adults (≥25 yr) had a higher TB seroprevalence than younger elephants (≤24 yr) on both rapid tests. The relatively high TB seroprevalence that we found highlighted the value of conducting retrospective studies in free-ranging wildlife populations in order to better understand the potential risk of disease.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis infection, causes morbidity and mortality in free-ranging lions in bTB-endemic areas of South Africa. However, the only currently used diagnostic test is the tuberculin skin test (TST). This test is logistically challenging to perform because it requires immobilization of lions twice in a 72-hr period. Blood-based diagnostic tests, such as serological assays, have been previously reported for M. bovis detection in lion populations, and have the advantage of only requiring a single immobilization. In addition, serological assays can be used for retrospective testing. Therefore, the aim of this study was to test free-ranging lions with the STAT-PAKt (Chembio Diagnostics Systems, Medford, NY 11763, USA) and DPPt VetTB (Chembio Diagnostics Systems) serological assays and compare those results with the tuberculin skin test. The serological assays were also used to determine prevalence in bTB-endemic and uninfected lion populations. The results showed that the serological assays could distinguish between M. bovis culture-positive and -negative lions. In addition, antigen-specific humoral responses were present in lions that had clinical signs of bTB disease or were shedding M. bovis antemortem. Although the seroprevalence of M. bovis infection in Kruger National Park lions was similar to that obtained from antemortem mycobacterial culture (4.8 and 3.3%, respectively), it was less than that estimated by the TST (72%). These findings support the hypothesis that assays based on cell-mediated immune responses are more sensitive than serology is in detecting M. bovis infection in lions. However, serological assays can have a role in bTB disease detection in lions and are especially useful for retrospective studies.

In 1997 a 26-yr-old gemsbok (Oryx gazelle gazelle) died of bovine tuberculosis in a zoo. Three remaining gemsbok were administered the comparative tuberculin skin test repeatedly over a period of 5 mo. Two animals showed inconclusive results on the second test. All three gemsbok were euthanatized. Mycobacterium bovis was isolated from one of those with an inconclusive skin test result, whereas Mycobacterium fortuitum was detected in the other gemsbok. Eight years later, an onager (Equus hemionus onager) died of bovine tuberculosis. This animal had been kept in the same building as the gemsbok. Three herd mates were culled after administering the comparative tuberculin skin test. They were all nonreactors and produced no evidence of tuberculosis at postmortem examination. Retrospectively, using plasma samples collected from the gemsbok and onagers, three antibody tests, Elephant TB STAT-PAK, multiantigen print immunoassay (MAPIA), and dual-path platform (DPP) VetTB (Chembio Diagnostic Systems Inc., Medford, New York, 11763, USA), were used to assess their diagnostic value for these species. The M. bovis-infected gemsbok tested strongly positive by Elephant TB STAT-PAK at the time of euthanasia and 5 mo earlier when the skin test was negative. This animal was not antibody reactive in MAPIA and DPP VetTB. No M. bovis-specific antibody was detected in the other two gemsboks by any of the immunoassays. Among the onagers, Elephant TB STAT-PAK, MAPIA, and DPP VetTB revealed gradually increasing antibody response in the animal that died of bovine tuberculosis, but not in the three disease-free herd mates euthanatized. Seroconversion in the M. bovis-infected onager was first noticed 5 yr before death when the tuberculin skin test was negative.

Bovine tuberculosis (TB) in cervids remains a significant problem affecting farmed herds and wild populations. Traditional skin testing has serious limitations in certain species, whereas emerging serological assays showed promising diagnostic performance. The recently developed immunochromatographic dual-path platform (DPP) VetTB assay has two antigen bands, T1 (MPB83 protein) and T2 (CFP10/ESAT-6 fusion protein), for antibody detection. We evaluated the diagnostic accuracy of this test by using serum samples collected from groups of white-tailed deer experimentally inoculated with Mycobacterium bovis , M. avium subsp. paratuberculosis , or M. bovis BCG Pasteur. In addition, we used serum samples from farmed white-tailed deer in herds with no history of TB, as well as from free-ranging white-tailed deer culled during field surveillance studies performed in Michigan known to have bovine TB in the wild deer population. The DPP VetTB assay detected antibody responses in 58.1% of experimentally infected animals within 8 to 16 weeks postinoculation and in 71.9% of naturally infected deer, resulting in an estimated test sensitivity of 65.1% and a specificity of 97.8%. The higher seroreactivity found in deer with naturally acquired M. bovis infection was associated with an increased frequency of antibody responses to the ESAT-6 and CFP10 proteins, resulting in a greater contribution of these antigens, in addition to MPB83, to the detection of seropositive animals, compared with experimental M. bovis infection. Deer experimentally inoculated with either M. avium subsp. paratuberculosis or M. bovis BCG Pasteur did not produce cross-reactive antibodies that could be detected by the DPP VetTB assay. The present findings demonstrate the relatively high diagnostic accuracy of the DPP VetTB test for white-tailed deer, especially in the detection of naturally infected animals.

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.

We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-γ) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas fromMycobacterium bovisculture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities of the IFN-γ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN-γ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN-γ test was at least in part due to undisclosedMycobacterium microtiinfection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-γ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-γ test with two antibody tests in a “test package,” although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations.

Three serologic methods for antibody detection in elephant tuberculosis (TB), the multiantigen print immunoassay (MAPIA), ElephantTB STAT-PAK kit, and DPP VetTB test, were evaluated using serial serum samples from 14 captive elephants infected withMycobacterium tuberculosisin 5 countries. In all cases, serological testing was performed prior to the diagnosis of TB by mycobacterial culture of trunk wash or tissue samples collected at necropsy. All elephants produced antibody responses toM. tuberculosisantigens, with 13/14 recognizing ESAT-6 and/or CFP10 proteins. The findings supported the high serodiagnostic test accuracy in detecting infections months to years beforeM. tuberculosiscould be isolated from elephants. The MAPIA and/or DPP VetTB assay demonstrated the potential for monitoring antimycobacterial therapy and predicting TB relapse in treated elephants when continuously used in the posttreatment period. History of exposure to TB and past treatment information should be taken into consideration for proper interpretation of the antibody test results. Data suggest that the more frequent trunk wash culture testing of seropositive elephants may enhance the efficiency of the TB diagnostic algorithm, leading to earlier treatment with improved outcomes.

This study describes the comparison of the cell-based interferon-gamma (IFNγ) test with serological rapid antibody tests (STAT-PAK and DPP VetTB) for the ante mortem testing of tuberculosis in domestic cats. The antibody specificities of rapid antibody test-positive cats were further discerned using multi-antigen print immunoassay. A total of 62 cats with culture-confirmed Mycobacterium bovis, Mycobacterium microti, Mycobacterium avium and Mycobacterium malmoense, as well as negative controls and dangerous-contact cats were tested. Tests were also applied longitudinally to one further cat undergoing TB chemotherapy for suspected M. bovis infection. Our data from this small study show excellent test specificity (100% for all tests) and encouraging levels of test sensitivity for M. bovis and TB Complex infections (IFNγ 70–100% depending upon test interpretation criteria; rapid tests both 90% for M. bovis infection and up to 46.2% for M. microti infection). The differential diagnosis of very pathogenic TB Complex (M. bovis, Mycobacterium tuberculosis), as opposed to less-pathogenic TB Complex (M. microti) was possible where positive responses to the protein cocktail ESAT6/CFP10 were observed (80% of M. bovis-infected cats in this study showed positive IFNγ responses to ESAT6/CFP10, while 20% had antibody responses to ESAT6/CFP10 using MAPIA). Finally, preliminary data from a longitudinal study of one M. bovis-exposed cat with a positive IFNγ test pre-treatment suggest that a decrease in bacterial burden may be reflected in the IFNγ response, and thus the IFNγ test may provide a monitor for TB chemotherapy.

In 2009,Mycobacterium bovisinfection 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.

Tuberculosis (TB) is an emerging disease in elephants primarily caused by Mycobacterium tuberculosis (M. tb) and in some occassion by M. bovis. We performed culture and three serological teststhe Elephant TB STAT-PAK,(R) DPP VetTB (R) Assay, and MAPIA (multi-antigen print immunoassay)prospectively on samples from eight elephants in Nepal that died of suspected or confirmed tuberculosis (TB) between 2007 and 2013. Among them, all elephants were reactive to DPP VetTB (R) Assay, five to Elephant TB STAT-PAK,(R) and two were reactive to MAPIA. Similarly, six elephants were positive on culture on samples collected antemortem or postmortem. We observed antibody responses months to years before culture confirmation of TB which shows that serological tests can be highly useful for the early diagnosis of TB in elephants. Validated point-of-care serological tests are easily performed in the field and hold promise for improved TB surveillance in other non-domestic species.

This study aimed to maximize the sensitivity of bovine tuberculosis detection in living wild fallow deer (Dama dama) under field conditions. We evaluated the rapid test (RT; CervidTB STAT-PAK Assay, Chembio Diagnostic Systems, Inc., USA) in comparison with the comparative cervical skin test (CCT). A total of 134 fallow deer were captured between January and March 2008. At time 0, 0.1 ml of avian purified protein derivative (avian PPD; Cooper-Zeltia, Spain), 0.1 ml bovine PPD (Cooper-Zeltia, Spain), 0.1 ml negative control PBS and 0.1 ml of a positive control (the mitogen phytohaemagglutinin, PHA; containing 250 mg PHA, diluted in PBS) were injected intradermally at four shaved sites in the neck. The skin fold thickness at each injection site was measured at time 0 and 72 h (3 repeats each time). Animals with a skin test response of 2 mm or more at the bovine PPD injection site and animals with any visible reactivity in the RT were necropsied and tissues submitted for culture and for histopathology. A total of 36 fallow deer were considered reactors to bovine PPD or to the RT (apparent prevalence 27%). Regarding both bovine PPD reactivity and the skin fold increase at the PHA injection site, we found significant effects of age and sex by age interaction. Adult males had the largest responses. Mycobacterium bovis was isolated from lymphoid tissues of 21 fallow deer. Skin test sensitivity, as compared to M. bovis culture confirmed deer, was 80.1% (17/21). But, the CCT alone would have missed 4 of 21 culture confirmed animals. RT sensitivity, based on culture confirmed deer, was also 80.1% (17/21). Similarly, the RT alone would have missed another 4 of 21 culture confirmed deer. However, combining the CCT and the RT allowed for detecting all 21 culture positive fallow deer. We conclude that the combined application of the RT and the skin testing can maximize the sensitivity of bTB detection in living fallow deer, thus facilitating control programs for wildlife disease surveillance., This study was funded by Patrimonio Nacional (special thanks to M. Moreno and A. Jacoste) through contracts to IREC and TRAGSEGA (A. Rosón). This is a contribution to MCINN Plan Nacional Research grant AGL2008-03875 and FEDER, and to EU grant TB-STEP 212414. Studies on diseases shared between domestic animals and wildlife are also supported by grants and contracts from INIA and Ministerio de Medio Ambiente y Medio Rural y Marino (OAPN and SDGSPP), and Grupo Santander – Fundacion Marcelino Botin. MPM-H acknowledges a post-doctoral contract from Instituto de Salud Carlos III.