In order to identify cytokines that may be useful as candidates for inclusion in diagnostic tests for Mycobacterium bovis infection in cattle, we compared the levels of gamma interferon (IFN-␥), interleukin 1␤ (IL-1␤), IL-4, IL-10, IL-12, macrophage inflammatory protein 1␤ (MIP-1␤), and tumor necrosis factor alpha (TNF-␣) in whole-blood cultures from tuberculosis (TB) reactor animals or TB-free controls following stimulation with M. bovis-specific antigens (purified protein derivative from M. bovis [PPD-B] or ESAT-6/CFP-10). In addition to IFN-␥ responses, the production of IL-1␤ and TNF-␣ was also statistically significantly elevated in TB reactor cattle over that in uninfected controls following stimulation with PPD-B or ESAT-6/CFP-10 peptides. Thus, we evaluated whether the use of these two additional readouts could disclose further animals not detected by measuring IFN-␥ alone. To this end, receiver operating characteristic (ROC) analyses were performed to define diagnostic cutoffs for positivity for TNF-␣ and IL-1␤. These results revealed that for ESAT-6/CFP-10-induced responses, the use of all three readouts (IFN-␥, TNF-␣, and IL-1␤) in parallel increased the sensitivity of detection of M. bovis-infected animals by 11% but also resulted in a specificity decrease of 14%. However, applying only IFN-␥ and IL-1␤ in parallel resulted in a 5% increase in sensitivity without the corresponding loss of specificity. The results for PPD-B-induced responses were similar, although the loss of specificity was more pronounced, even when only IFN-␥ and IL-1␤ were used as readout systems.In conclusion, we have demonstrated that the use of an additional readout system, such as IL-1␤, can potentially complement IFN-␥ by increasing overall test sensitivity for the detection of M. bovis infection in cattle.

M embers of the mycobacterial genus are renowned for their waxy, lipid-rich outer envelope. Under physiological conditions, this outer layer is likely to be the first point of contact between the bacterial cell and the host's immune system, and the outcome of this interaction is pivotal in the establishment of infection. One of the most important groups of membrane bound lipids consists of the phosphatidylinositol mannosides (PIMs). Interest in PIMs was stimulated since it was shown that phosphatidylinositol dimannoside (PIM 2 ) forms the phosphoglycolipid anchor which tethers a large array of glycolipids and lipoglycans, including lipomannan (LM) and lipoarabinomannan (LAM), to the cellular membrane (1). PIMs have been shown to interact with a variety of immune components and mediate significant effects on the host. Ever since the realization that NKT cells could respond to lipid antigens (2-4) and the subsequent discovery of the CD1d-restricted lipid antigen ␣-galactosylceramide (␣-GalCer) (5, 6), much research effort has been concentrated on understanding lipid antigens. Although work was initially focused on invariant NKT cells, it has since been shown that great diversity exists in the lipid-responsive T cell receptor (TCR) repertoire (7-9) and that these diverse NKT cells contribute to the Th1/Th2 balance (7, 10). It has been shown that CD1d-restricted NKT cells are capable of recognizing a variety of lipid antigens, including phospholipids (11). More recently, a CD1b-restricted subset of T cells has been found (12). Similarly to CD1d-restricted invariant NKT (iNKT) cells, the CD1b-restricted variant cells require CD1B for their development and produce proinflammatory cytokines in response to CD1b-expressing dendritic cells (DCs) (12). It is clear that NKT-like cells have a significant role to play in lipid-mediated responses, and the hunt for their antigens has continued (13-15).Given the ability of lipid molecules to generate responses in peripheral blood mononuclear cells (PBMC), we decided to assess the ability of individual, highly purified natural PIMs to activate bovine lymphocytes. In this study, we developed a novel extraction method which allowed us to extract and highly purify a variety of PIM molecules from virulent Mycobacterium tuberculosis H37Rv. The ability of these molecules to induce lymphocyte responses in Mycobacterium bovis-infected cattle was investigated by measuring lymphocyte proliferation and gamma interferon (IFN-␥) production. Furthermore, flow cytometry techniques were utilized to characterize responding cell populations. MATERIALS AND METHODS Extraction of PIMs.Using the novel methodology outlined below, highly pure phosphatidylinositol dimannoside (PIM 2 ), acylphosphatidylinositol dimannoside (AcPIM 2 ), diacyl-phosphatidylinositol dimannoside (Ac 2 PIM 2 ), acylphosphatidylinositol hexamannoside (AcPIM 6 ), and diacylphosphatidylinositol hexamannoside (Ac 2 PIM 6 ) were successfully isolated. Individual PIM molecules were analyzed by electrospray ionization mass spectrometr...

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