W. Ray Waters

Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment.

ABSTRACT Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with M. tuberculosis and one was in an African elephant with Mycobacterium bovis. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of M. tuberculosis from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively.

Bovine tuberculosis vaccine research: Historical perspectives and recent advances

The emergence of wildlife reservoirs of Mycobacterium bovis infection in cattle as well as increased inter-regional trade with associated spread of M. bovis has led to renewed interest in the use of vaccines for the control of bovine tuberculosis (TB). Field efficacy trials performed in the early 20th century demonstrated the partial effectiveness of bacilli Calmette-Guerin (BCG) for the control of bovine TB. Recent experimental trials with cattle have demonstrated that: (1) subunit vaccines may boost immunity elicited by BCG in cattle, (2) T cell central memory immune responses evoked by protective vaccines correlate with protection upon subsequent M. bovis challenge, (3) BCG is particularly protective when administered to neonates, and (4) differentiation of infected from vaccinated animals (DIVA) is feasible in cattle using in vitro or in vivo methods. In regards to wildlife reservoirs, the efficacy of BCG delivered orally has been demonstrated for brushtail possums (in field trials) as well as Eurasian badgers, wild boar, and white-tailed deer (each in experimental challenge studies). Vaccine delivery to wildlife reservoirs will primarily be oral, although a parenteral route is being deployed for badgers in England. Vaccine efficacy trials, both experimental challenge and field studies, with cattle and their wildlife reservoirs represent a primary example of the one health approach, with outcomes relevant for both veterinary and medical applications.

Characterization of Bovine Homologues of Granulysin and NK-lysin

Granulysin and NK-lysin are antimicrobial proteins found in the granules of human and swine cytotoxic lymphocytes. A murine counterpart to granulysin has not been identified to date, indicating the importance of additional models to fully characterize the role of granulysin-like molecules in the immune response to infectious disease. Two partial nucleotide sequences corresponding to the complete functional domain of granulysin and NK-lysin were amplified from bovine PBMC mRNA. Following stimulation with phorbol ester and calcium ionophore, expression of the bovine gene was detected in CD3+ T cells, CD4+ T cells, CD8+ T cells, WC1+ γδ T cells, and PBMC depleted of CD3+ T cells, but was absent in CD21+ cells and CD14+ cells. Intracellular flow cytometry and immunoblotting confirmed the presence of protein corresponding to the bovine granulysin homologue in activated T lymphocytes and PBMC. Synthetic human, bovine, and swine peptides corresponding to the C terminus of helix 2 through helix 3 region of granulysin displayed potent antimicrobial activity against Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Mycobacterium bovis bacillus Calmette-Guérin. Human and bovine peptides corresponding to helix 2 displayed antimycobacterial activity against M. bovis bacillus Calmette-Guérin. Expression of the bovine gene was detected in laser microscopy-dissected lymph node lesions from an M. bovis-infected animal. The identification of a biologically active bovine homologue to granulysin demonstrates the potential of the bovine model in characterizing the role of granulysin in the immune response to a variety of infectious agents.

Tuberculosis immunity: Opportunities from studies with cattle

Mycobacterium tuberculosis and M. bovis share >99% genetic identity and induce similar host responses and disease profiles upon infection. There is a rich history of codiscovery in the development of control measures applicable to both human and bovine tuberculosis (TB) including skin-testing procedures, M. bovis BCG vaccination, and interferon-γ release assays. The calf TB infection model offers several opportunities to further our understanding of TB immunopathogenesis. Recent observations include correlation of central memory immune responses with TB vaccine efficacy, association of SIRPα + cells in ESAT-6:CFP10-elicited multinucleate giant cell formation, early γδ T cell responses to TB, antimycobacterial activity of memory CD4+ T cells via granulysin production, association of specific antibody with antigen burden, and suppression of innate immune gene expression in infected animals. Partnerships teaming researchers with veterinary and medical perspectives will continue to provide mutual benefit to TB research in man and animals.

Analysis of the Immune Response to Mycobacterium avium subsp. paratuberculosis in Experimentally Infected Calves

ABSTRACT Johnes disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4+ T cells with a memory phenotype (CD45R0+) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8+ T cells proliferated in response to antigens until 18 months p.i. γδ T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1+ CD2− and a few WC1− CD2+ γδ T cells expressed CD25 at time zero. By 18 months, however, subsets of γδ T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3− non-T non-B null cells, CD2+ and CD2−, proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.

Efficacy and immunogenicity of Mycobacterium bovis ΔRD1 against aerosol M. bovis infection in neonatal calves

An attenuated Mycobacterium bovisRD1 deletion (DeltaRD1) mutant of the Ravenel strain was constructed, characterized, and sequenced. This M. bovis DeltaRD1 vaccine strain administered to calves at 2 weeks of age provided similar efficacy as M. bovis bacillus Calmette Guerin (BCG) against low dose, aerosol challenge with virulent M. bovis at 3.5 months of age. Approximately 4.5 months after challenge, both DeltaRD1- and BCG-vaccinates had reduced tuberculosis (TB)-associated pathology in lungs and lung-associated lymph nodes and M. bovis colonization of tracheobronchial lymph nodes as compared to non-vaccinates. Mean central memory responses elicited by either DeltaRD1 or BCG prior to challenge correlated with reduced pathology and bacterial colonization. Neither DeltaRD1 or BCG elicited IFN-gamma responses to rESAT-6:CFP-10 prior to challenge, an emerging tool for modern TB surveillance programs. The DeltaRD1 strain may prove useful for bovine TB vaccine programs, particularly if additional mutations are included to improve safety and immunogenicity.

Biomarker discovery in subclinical mycobacterial infections of cattle

Background Bovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle. Methodology/Principal Findings We hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P≤0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals. Conclusions/Significance The discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions.

Bovine tuberculosis: Effect of the tuberculin skin test on in vitro interferon gamma responses

Bovine tuberculosis (bTB) is a disease of zoonotic and economic importance. In many countries, control is based on test and slaughter policies and/or abattoir surveillance. For testing, cell mediated immune- (CMI-) based assays (i.e., tuberculin skin test (TST) supplemented by the interferon gamma (IFN-gamma) assay) are the primary surveillance and disease control tests for bTB. The combined use of the in vivo and in vitro CMI assays to increase overall sensitivity has raised the question of whether the IFN-gamma response is influenced by injection of purified protein derivatives (PPDs) for TST. Published data on the influence of the TST, applied as the caudal fold test (CFT) or the comparative cervical test (CCT), on the IFN-gamma assay are contradictory. Reviewing published data and including additional data, the following conclusions can be drawn: (1) in naturally infected cattle, PPD administration for the single or repeated short-interval CCT neither boosts nor depresses PPD-specific IFN-gamma production. Disparate results have been concluded from some studies using experimental infections, emphasizing the importance of confirming initial experimental-based findings with studies using cattle naturally infected with Mycobacterium bovis. (2) In cattle experimentally infected with M. bovis, PPD administration for CFT boosts PPD-specific IFN-gamma production for up to 7 days without any effect on test interpretation. Importantly, in naturally infected cattle, CFT-related boosting selectively increases the in vitroM. bovis PPD (PPD-B) response 3 days after CFT, resulting in an increased PPD-B response relative to the response to Mycobacterium avium PPD (PPD-A). In non-infected cattle, it cannot be excluded that the CFT induces a mild boost of the PPD-specific response, particularly in animals sensitized to environmental, non-tuberculous mycobacteria, thus decreasing the specificity of the IFN-gamma assay. (3) In general, there is a lack of data clearly characterizing the effect of TSTs on the IFN-gamma assay. Further studies are required to clearly describe the effects of both CFT and CCT in non-infected animals and in naturally infected cattle, especially in low reacting infected cattle.

Mycobacterium bovis: A Model Pathogen at the Interface of Livestock, Wildlife, and Humans

Complex and dynamic interactions involving domestic animals, wildlife, and humans create environments favorable to the emergence of new diseases, or reemergence of diseases in new host species. Today, reservoirs of Mycobacterium bovis, the causative agent of tuberculosis in animals, and sometimes humans, exist in a range of countries and wild animal populations. Free-ranging populations of white-tailed deer in the US, brushtail possum in New Zealand, badger in the Republic of Ireland and the United Kingdom, and wild boar in Spain exemplify established reservoirs of M. bovis. Establishment of these reservoirs is the result of factors such as spillover from livestock, translocation of wildlife, supplemental feeding of wildlife, and wildlife population densities beyond normal habitat carrying capacities. As many countries attempt to eradicate M. bovis from livestock, efforts are impeded by spillback from wildlife reservoirs. It will not be possible to eradicate this important zoonosis from livestock unless transmission between wildlife and domestic animals is halted. Such an endeavor will require a collaborative effort between agricultural, wildlife, environmental, and political interests.

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.