Background Tuberculosis (TB) in non-human primates (NHPs) is highly contagious, requiring efficient identification of animals infected with Mycobacterium tuberculosis. Tuberculin skin test is usually used but lacks desirable sensitivity/specificity and efficiency. Methods We aimed to develop an immunoassay for plasma antibodies against M. tuberculosis. A key challenge is that not all infected animals contain antibodies against the same M. tuberculosis antigen. Therefore, a multiplex panel of 28 antigens (Luminex

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (αIg) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The αIg control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly. Video LinkThe video component of this article can be found at https://www.jove.com/video/3715/ Protocol 1. Explanation of the MFIA Procedure (Figure 1) 1. The MFIA requires Charles River's antigen coated polystyrene microspheres (beads), test sera, labeled reagents (BAG, SPE) and buffers (Primary Diluent, Assay buffer). 2. The reagents are added stepwise to the wells of 96-well filter-bottom microtiter plates. 3. The MFIA is performed as a heterogeneous test meaning incubations are followed by filter-wash steps to remove unbound serum constituents or labeled reagents. Wash solution added to plate wells is removed by aspiration through well filter-bottoms, which retain the beads. 4. The MFIA assays are performed at room temperature (27°C+/-2°C). 5. Antigen-antibody complexes formed during the test serum incubation step are detected by incubations with biotinylated goat anti-species conjugates (BAG) followed by R-phycoerythrin-labeled streptavidin (SPE). 6. In the assay reader, the beads pass one at a time through a detector where they are exposed to two lasers. One laser excites the internal dyes that identify the bead's color set, which corresponds to an assay; the othe...

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KEYWORD herpesvirusesW e recently read a paper by Patel et al. (1) in which the authors present a novel murine herpesvirus that can induce severe thymic necrosis and T cell depletion in neonatal mice and has not been found before. The authors named this virus murine roseolovirus (MRV) on the basis of its homology to human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7.However, in the laboratory animal medicine field, another murine herpesvirus, a mouse thymic lymphotropic virus (MTLV; also known as MTV; International Committee on Taxonomy of Viruses [ICTV] designation, murid herpesvirus 3) affecting neonatal mouse T lymphocyte development and causing thymic necrosis, has been investigated and studied for decades (2). In our laboratory, which develops assays for murine pathogens, we have developed diagnostic tools that utilize the same virus isolate originating from the NIH described in the original paper (3, 4). As part of some recent efforts to develop recombinant antigens for MTLV, last fall, we completed a nearly complete sequence of the MTLV genome. At the time we sequenced it, there were no closely related viruses sequences deposited in GenBank. However, in a recent analysis, we found that the sequence we determined for MTLV is 100% identical to the newly reported MRV sequence, at its reverse complementary position of bp 7491 to 161219. Therefore, we conclude that MRV is the same virus that has been historically known as MTLV. In the abstract, there is reference to MRV having phenotypes similar to those of MTV, but it appears that the authors did not pursue a reference stock of MTLV to determine if the viruses were, in fact, the same.Having said this, we feel that it is important that the virology community is aware that MRV and MTLV are the same virus so that previous publications where MTLV has been investigated will also be associated with studies that investigate MRV. A task falling in the realm of the ICTV will be to attain a consensus on the nomenclature used for this virus. REFERENCES

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