The Many Hosts of Mycobacteria 9 (MHM9): A conference report.

The Many Hosts of Mycobacteria (MHM) meeting series brings together basic scientists, clinicians and veterinarians to promote robust discussion and dissemination of recent advances in our knowledge of numerous mycobacterial diseases, including human and bovine tuberculosis (TB), nontuberculous mycobacteria (NTM) infection, Hansen's disease (leprosy), Buruli ulcer and Johne's disease. The 9th MHM conference (MHM9) was held in July 2022 at The Ohio State University (OSU) and centered around the theme of "Confounders of Mycobacterial Disease." Confounders can and often do drive the transmission of mycobacterial diseases, as well as impact surveillance and treatment outcomes. Various confounders were presented and discussed at MHM9 including those that originate from the host (comorbidities and coinfections) as well as those arising from the environment (e.g., zoonotic exposures), economic inequality (e.g. healthcare disparities), stigma (a confounder of leprosy and TB for millennia), and historical neglect (a confounder in Native American Nations). This conference report summarizes select talks given at MHM9 highlighting recent research advances, as well as talks regarding the historic and ongoing impact of TB and other infectious diseases on Native American Nations, including those in Southwestern Alaska where the regional TB incidence rate is among the highest in the Western hemisphere.

Cephem-Pyrazinoic Acid Conjugates: Circumventing Resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading source of infectious disease mortality globally. Antibiotic-resistant strains comprise an estimated 10 % of new TB cases and present an urgent need for novel therapeutics. ß-lactam antibiotics have traditionally been ineffective against M. tuberculosis (Mtb), the causative agent of TB, due to the organism's inherent expression of ß-lactamases that destroy the electrophilic ß-lactam warhead. We have developed novel ß-lactam conjugates, which exploit this inherent ß-lactamase activity to achieve selective release of pyrazinoic acid (POA), the active form of a first-line TB drug. These conjugates are selectively active against M. tuberculosis and related mycobacteria, and activity is retained or even potentiated in multiple resistant strains and models. Preliminary mechanistic investigations suggest that both the POA "warhead" as well as the ß-lactam "promoiety" contribute to the observed activity, demonstrating a codrug strategy with important implications for future TB therapy.

The Bewildering Antitubercular Action of Pyrazinamide.

Pyrazinamide (PZA) is a cornerstone antimicrobial drug used exclusively for the treatment of tuberculosis (TB). Due to its ability to shorten drug therapy by 3 months and reduce disease relapse rates, PZA is considered an irreplaceable component of standard first-line short-course therapy for drug-susceptible TB and second-line treatment regimens for multidrug-resistant TB. Despite over 60 years of research on PZA and its crucial role in current and future TB treatment regimens, the mode of action of this unique drug remains unclear. Defining the mode of action for PZA will open new avenues for rational design of novel therapeutic approaches for the treatment of TB. In this review, we discuss the four prevailing models for PZA action, recent developments in modulation of PZA susceptibility and resistance, and outlooks for future research and drug development.

Impact of the host environment on the antitubercular action of pyrazinamide.

Pyrazinamide remains the only drug in the tuberculosis pharmacopeia to drastically shorten first-line therapy from nine to six months. Due to its unparalleled ability to sterilize non-replicating bacilli and reduce relapse rates, PZA is expected to be irreplaceable in future therapies against tuberculosis. While the molecular target of PZA is unclear, recent pharmacokinetic studies using small animal models and patient samples have highlighted the importance of host metabolism and immune responses in PZA efficacy. Delineating which host factors are important for PZA action will be integral to the design of next-generation therapies to shorten current TB drug regimens as well as to overcome treatment limitations in some patients. In this review, we discuss evidence for influence of the host environment on PZA activity, targets for PZA mechanism of action, recent studies in PZA pharmacokinetics, PZA antagonism and synergy with other first-line anti-TB drugs, and implications for future research.

Major histocompatibility complex I of swine respiratory cells presents conserved regions of influenza proteins.

Influenza A virus in swine (IAV-S) is a prevalent respiratory pathogen in pigs that has deleterious consequences to animal and human health. Pigs represent an important reservoir for influenza and potential mixing vessel for novel gene reassortments. Despite the central role of pigs in recent influenza outbreaks, much remains unknown about the impact of swine immunity on IAV-S transmission, pathogenesis, and evolution. An incomplete understanding of interactions between the porcine immune system and IAV-S has hindered development of new diagnostic tools and vaccines. In order to address this gap in knowledge, we identified swine leukocyte antigen (SLA) restricted IAV-S peptides presented by porcine airway epithelial cells using an immunoproteomics approach. The majority of MHC-associated peptides belonged to matrix 1, nucleoprotein and nonstructural 1 proteins. Future investigation of the potential cross-reactive nature of these peptides is needed to confirm antigen recognition by cytotoxic T lymphocytes and their utility as vaccine candidates.

A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival.

The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc(2) 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

A combined enrichment and aptamer pulldown assay for Francisella tularensis detection in food and environmental matrices.

Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1-106 CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology.

Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Mannosylated lipoarabinomannan in serum as a biomarker candidate for subclinical bovine tuberculosis.

BACKGROUND: Early and unambiguous detection of bovine tuberculosis (bTB), a significant disease of cattle worldwide, is necessary to control the spread of infection to other animals and humans. Current testing strategies are laborious, time consuming and heavily reliant on host responses that do not distinguish bTB from other mycobacteria. We report the presence of a pathogen signature, liparabinomannan (LAM), as a potential biomarker for bTB infection. FINDINGS: Fifty-five animals (uninfected [n = 33], bTb [n = 10] and exposed cases [n = 12]) from a well characterized bovine serum repository were screened for the presence of LAM using a commercially available ELISA. Analysis showed that LAM had a sensitivity of 100% and a specificity of 91.7% for bTB detection (bTB positive versus bTB exposed animals). CONCLUSION: LAM detection easily separated bTB infected animals from bTB exposed and negative controls. We propose that pathogen related markers, such as LAM, should be included with current testing strategies as a battery diagnostic for bTB.

Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model.

Vaccination remains a major tool for prevention and progression of Johne's disease, a chronic enteritis of ruminants worldwide. Currently there is only one licensed vaccine within the United States and two vaccines licensed internationally against Johne's disease. All licensed vaccines reduce fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP) and delay disease progression. However, there are no available vaccines that prevent disease onset. A joint effort by the Johne's Disease Integrated Program (JDIP), a USDA-funded consortium, and USDA-APHIS/VS sought to identify transposon insertion mutant strains as vaccine candidates in part of a three phase study. The focus of the Phase I study was to evaluate MAP mutant attenuation in a well-defined in vitro bovine monocyte-derived macrophage (MDM) model. Attenuation was determined by colony forming unit (CFUs) counts and slope estimates. Based on CFU counts alone, the MDM model did not identify any mutant that significantly differed from the wild-type control, MAP K-10. Slope estimates using mixed models approach identified six mutants as being attenuated. These were enrolled in protection studies involving murine and baby goat vaccination-challenge models. MDM based approach identified trends in attenuation but this did not correlate with protection in a natural host model. These results suggest the need for alternative strategies for Johne's disease vaccine candidate screening and evaluation.

Circulating Mycobacterium bovis peptides and host response proteins as biomarkers for unambiguous detection of subclinical infection.

Bovine tuberculosis remains one of the most damaging diseases to agriculture, and there is also a concern for human spillover. A critical need exists for rapid, thorough, and inexpensive diagnostic methods capable of detecting and differentiating Mycobacterium bovis infection from other pathogenic and environmental mycobacteria at multiple surveillance levels. In a previous study, Seth et al. (PLoS One 4:e5478, 2009, doi:10.1371/journal.pone.0005478) identified 32 host peptides that specifically increased in the blood serum of M. bovis-infected animals). In the current study, 16 M. bovis proteins were discovered in the blood serum proteomics data sets. A large-scale validation analysis was undertaken for selected host and M. bovis proteins using a cattle serum repository containing M. bovis (n = 128), Mycobacterium kansasii (n = 10), and Mycobacterium avium subsp. paratuberculosis (n = 10), cases exposed to M. bovis (n = 424), and negative controls (n = 38). Of the host biomarkers, vitamin D binding protein (VDBP) showed the greatest sensitivity and specificity for M. bovis detection. Circulating M. bovis proteins, specifically polyketide synthetase 5, detected M. bovis-infected cattle with little to no seroreactivity against M. kansasii- and M. avium subsp. paratuberculosis-infected animals. These data indicate that host and pathogen serum proteins can serve as reliable biomarkers for tracking M. bovis infection in animal populations.

Host-Mycobacterium avium subsp. paratuberculosis interactome reveals a novel iron assimilation mechanism linked to nitric oxide stress during early infection.

BACKGROUND: The initial interaction between host cell and pathogen sets the stage for the ensuing infection and ultimately determine the course of disease. However, there is limited knowledge of the transcripts utilized by host and pathogen and how they may impact one another during this critical step. The purpose of this study was to create a host-Mycobacterium avium subsp. paratuberculosis (MAP) interactome for early infection in an epithelium-macrophage co-culture system using RNA-seq. RESULTS: Establishment of the host-MAP interactome revealed a novel iron assimilation system for carboxymycobactin. Iron assimilation is linked to nitric oxide synthase-2 production by the host and subsequent nitric oxide buildup. Iron limitation as well as nitric oxide is a prompt for MAP to enter into an iron sequestration program. This new iron sequestration program provides an explanation for mycobactin independence in some MAP strains grown in vitro as well as during infection within the host cell. Utilization of such a pathway is likely to aid MAP establishment and long-term survival within the host. CONCLUSIONS: The host-MAP interactome identified a number of metabolic, DNA repair and virulence genes worthy for consideration as novel drug targets as well as future pathogenesis studies. Reported interactome data may also be utilized to conduct focused, hypothesis-driven research. Co-culture of uninfected bovine epithelial cells (MAC-T) and primary bovine macrophages creates a tolerant genotype as demonstrated by downregulation of inflammatory pathways. This co-culture system may serve as a model to investigate other bovine enteric pathogens.

Infection with Mycobacterium avium subsp. paratuberculosis results in rapid interleukin-1ß release and macrophage transepithelial migration.

Pathogen processing by the intestinal epithelium involves a dynamic innate immune response initiated by pathogen-epithelial cell cross talk. Interactions between epithelium and Mycobacterium avium subsp. paratuberculosis have not been intensively studied, and it is currently unknown how the bacterium-epithelial cell cross talk contributes to the course of infection. We hypothesized that M. avium subsp. paratuberculosis harnesses host responses to recruit macrophages to the site of infection to ensure its survival and dissemination. We investigated macrophage recruitment in response to M. avium subsp. paratuberculosis using a MAC-T bovine macrophage coculture system. We show that M. avium subsp. paratuberculosis infection led to phagosome acidification within bovine epithelial (MAC-T) cells as early as 10 min, which resulted in upregulation of interleukin-1ß (IL-1ß) at transcript and protein levels. Within 10 min of infection, macrophages were recruited to the apical side of MAC-T cells. Inhibition of phagosome acidification or IL-1ß abrogated this response, while MCP-1/CCL-2 blocking had no effect. IL-1ß processing was dependent upon Ca(2+) uptake from the extracellular medium and intracellular Ca(2+) oscillations, as determined by EGTA and BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)] treatments. Thus, M. avium subsp. paratuberculosis is an opportunist that takes advantage of extracellular Ca(2+)-dependent phagosome acidification and IL-1ß processing in order to efficiently transverse the epithelium and enter its niche--the macrophage.

Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures.

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.

Monoclonal Antibodies Bind A SNP-Sensitive Epitope that is Present Uniquely in Mycobacterium avium Subspecies Paratuberculosis.

Due to a close genetic relatedness, there is no known antibody that detects Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne's disease in cattle and sheep, and does not cross-react with other M. avium subspecies. In the present study, a monoclonal antibody (MAb; 17A12) was identified from mice immunized with a cell membrane fraction of MAP strain K-10. This antibody is 100% specific as it detected a 25-kDa protein in all 29 MAP whole cell lysates, but did not bind to any of the 29 non-paratuberculosis strains tested in immunoblot assays. However, the antibody revealed variable reactivity levels in MAP strains as it detected higher levels in bovine isolates but comparably lower levels in ovine isolates of MAP. In order to identify the target binding protein for 17A12, a lambda phage expression library of MAP genomic fragments was screened with the MAb. Four reactive clones were identified, sequenced and all shown to be overlapping. Further analysis revealed all four clones expressed an unknown protein encoded by a sequence that is not annotated in the K-10 genome and overlapped with MAP3422c on the opposing DNA strand. The epitope of 17A12 was precisely defined to seven amino acids and was used to query the K-10 genome. Similarity searches revealed another protein, encoded by MAP1025, possessed a similar epitope (one-amino acid mismatch) that also reacted strongly to the antibody. A single nucleotide polymorphism (SNP) in MAP1025 was then identified by comparative sequence analysis, which results in a Pro28His change at residue 28, the first amino acid within the 17A12 epitope. This SNP is present in all MAP strains but absent in all non-MAP strains and accounts for the specificity of the 17A12 antibody. This new antibody is the first ever isolated that binds only to the paratuberculosis subspecies of M. avium and opens new possibilities for the specific detection of this significant ruminant pathogen.

A single DNA aptamer functions as a biosensor for ricin.

The use of microorganisms or toxins as weapons of death and fear is not a novel concept; however, the modes by which these agents of bioterrorism are deployed are increasingly clever and insidious. One mechanism by which biothreats are readily disseminated is through a nation's food supply. Ricin, a toxin derived from the castor bean plant, displays a strong thermostability and remains active at acidic and alkaline pHs. Therefore, the CDC has assigned ricin as a category B reagent since it may be easily amendable as a deliberate food biocontaminate. Current tools for ricin detection utilize enzymatic activity, immunointeractions and presence of castor bean DNA. Many of these tools are confounded by complex food matrices, display a limited dynamic range of detection and/or lack specificity. Aptamers, short RNA and single stranded DNA sequences, have increased affinity to their selected receptors, experience little cross-reactivity to other homologous compounds and are currently being sought after as biosensors for bacterial contaminants in food. This paper describes the selection and characterization of a single, dominant aptamer, designated as SSRA1, against the B-chain of ricin. SSRA1 displays one folding conformation that is stable across 4-63 °C (ΔG = -5.05). SSRA1 is able to concentrate at least 30 ng mL(-1) of ricin B chain from several liquid food matrices and outcompetes a currently available ELISA kit and ricin aptamer. Furthermore, we show detection of 25 ng mL(-1) of intact ricin A-B complex using SSRA1 combined with surface enhanced Raman scattering technique. Thus, SSRA1 would serve well as pre-analytical tool for processing of ricin from liquid foods to aid current diagnostics as well as a sensor for direct ricin detection.

Primary transcriptomes of Mycobacterium avium subsp. paratuberculosis reveal proprietary pathways in tissue and macrophages.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) persistently infects intestines and mesenteric lymph nodes leading to a prolonged subclinical disease. The MAP genome sequence was published in 2005, yet its transcriptional organization in natural infection is unknown. While prior research analyzed regulated gene sets utilizing defined, in vitro stress related or advanced surgical methods with various animal species, we investigated the intracellular lifestyle of MAP in the intestines and lymph nodes to understand the MAP pathways that function to govern this persistence. RESULTS: Our transcriptional analysis shows that 21%, 8% and 3% of the entire MAP genome was represented either inside tissues, macrophages or both, respectively. Transcripts belonging to latency and cell envelope biogenesis were upregulated in the intestinal tissues whereas those belonging to intracellular trafficking and secretion were upregulated inside the macrophages. Transcriptomes of natural infection and in vitro macrophage infection shared genes involved in transcription and inorganic ion transport and metabolism. MAP specific genes within large sequence polymorphisms of ancestral M. avium complex were downregulated exclusively in natural infection. CONCLUSIONS: We have unveiled common and unique MAP pathways associated with persistence, cell wall biogenesis and virulence in naturally infected cow intestines, lymph nodes and in vitro infected macrophages. This dichotomy also suggests that in vitro macrophage models may be insufficient in providing accurate information on the events that transpire during natural infection. This is the first report to examine the primary transcriptome of MAP at the local infection site (i.e. intestinal tissue). Regulatory pathways that govern the lifecycle of MAP appear to be specified by tissue and cell type. While tissues show a "shut-down" of major MAP metabolic genes, infected macrophages upregulate several MAP specific genes along with a putative pathogenicity island responsible for iron acquisition. Many of these regulatory pathways rely on the advanced interplay of host and pathogen and in order to decipher their message, an interactome must be established using a systems biology approach. Identified MAP pathways place current research into direct alignment in meeting the future challenge of creating a MAP-host interactome.

Paradigm redux--Mycobacterium avium subspecies paratuberculosis-macrophage interactions show clear variations between bovine and human physiological body temperatures.

The physiological conditions encountered by pathogenic mycobacteria inside their hosts significantly influence their adaptation, virulence, and gene expression. Current in vitro models investigating host-pathogen interactions of Mycobacterium avium subsp. paratuberculosis use 37 degrees C, the normal body temperatures of mice and humans. However since the physiological temperature of MAP's natural host is 39 degrees C, we hypothesized that host and pathogen behavior to vary considerably in comparison to 37 degrees C. Our MAP-macrophage interaction studies show striking differences in regards to velocity of cell invasion of MAP as well as bacterial and host gene regulation at 39 degrees C compared with 37 degrees C. Upregulation of host genes (nod2, tlr2, mapkp38 and il-10) follow a similar trend at 37 degrees C and 39 degrees C; however, there is over a five-fold increase as early as 0.5 and 2 h in 39 degrees C treatments. While host signaling is completed by 48 h p.i. at 39 degrees C in MDMs cultures due to early cell death, signaling and infection is sustained at 37 degrees C. Surprisingly, transcription of MAP genes did not show a set pattern and were upregulated at different time points for both temperatures. Interestingly, MAP genes encoding a lipase (lipN) and an oxidoreductase (MAP3464) are staggered at 39 degrees C, while they increase steadily at 37 degrees C. In conclusion, infection and culture at a physiologically relevant temperature influences host-pathogen interaction, which may have far reaching ramifications including for currently used animal models, in vitro culture methods, bacterial pathogenesis and host responses, and vaccine candidate design and screening.

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 < or = 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.