1 infected with Mycobacterium bovis 2 3 Konstantin P. Lyashchenko,* Rena Greenwald, Javan Esfandiari, Daniel J. O’Brien, 4 Stephen M. Schmitt, Mitchell V. Palmer, and W. Ray Waters c 5 6 Chembio Diagnostic Systems, Inc., Medford, NY, USA; Wildlife Disease Laboratory, 7 Michigan Department of Natural Resources, Lansing, MI, USA; National Animal 8 Disease Center, Agricultural Research Service, United States Department of Agriculture, 9 Ames, IA, USA 10 11 12 13 *Corresponding author: Chembio Diagnostic Systems, Inc., 3661 Horseblock Road, 14 Medford, NY 11763, USA. Phone: 631-924-1135. Fax: 631-924-6033. E-mail: 15 klyashchenko@chembio.com. 16 17 18 19 Running title: DPP VetTB antibody assay for deer 20 21 22 Copyright © 2013, American Society for Microbiology. All Rights Reserved. Clin. Vaccine Immunol. doi:10.1128/CVI.00120-13 CVI Accepts, published online ahead of print on 17 April 2013

Ten South American sea lions (Otaria flavescens) were presented for clinical evaluation and diagnosis of tuberculosis following known exposure to Mycobacterium pinnipedii. CT was used to determine whether foci of calcification in mediastinal lymph nodes, typically associated with pinniped tuberculosis, could be detected and whether CT was a useful diagnostic modality, in conjunction with other tests, for the diagnosis of tuberculosis in this species. Blood was collected from the caudal gluteal vein of each animal for serological testing using commercially available serological tests (ElephantTB STAT-PAK and DPP Vet; Chembio Diagnostic Systems) and a multiantigen print immunoassay (MAPIA), carried out at Chembio to verify the in-house kits. In four of nine animals that underwent CT scanning, lesions consistent with pinniped tuberculosis were apparent and these were confirmed at subsequent postmortem examination. The five remaining animals did not show any abnormalities on CT, with three being negative on serological tests, which were considered to be normal and potentially used as reference images for healthy sea lions. One animal could not be CT scanned due to its large size and weight (510 kg).

A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis is the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n = 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n = 5). The results from this study demonstrated a rapid initiation of M. avium subsp. paratuberculosis-specific gamma interferon (IFN-γ) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovis purified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovis recombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. avium subsp. paratuberculosis antibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovis antigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis.

Published Ahead of Print 19 October 2011. 10.1128/CVI.05386-11. 2011, 18(12):2143. DOI: Clin. Vaccine Immunol. Patrik Zanolari Rua-Domenech, Mireille Meylan, HMartin Vordermeier and Esfandiari, Shelley Rhodes, Gillian Dean, Ricardo de la Konstantin P. Lyashchenko, Rena Greenwald, Javan Camelids microti Infection in South American Mycobacterium bovis or Mycobacterium Assays for Rapid Detection of Diagnostic Value of Animal-Side Antibody

Johne’s disease (JD) is caused by infection of mostly ruminants (including dairy cattle) with Mycobacterium avium ssp. paratuberculosis and is responsible for a significant economic loss to the US dairy industry. Diagnosis of JD is currently conducted in diagnostic laboratories, creating dairy farmers costly expenses for veterinary service, sample handling, and shipping. An automated on-site diagnostic device for JD would reduce the diagnosis-related costs. Lab-on-a-chip (LOC) technology has been used in various analytical processes and is offering opportunities for the development of on-site diagnostic devices. In this report, we developed and tested a LOC immunoassay system based on AC electrothermal (ACET) effect for detection of JD-specific antibodies in bovine serum samples. The LOC used in this study was composed of poly-dimethylsiloxane microchannels sealed over an ACET electrode chip. The surface of the ACET electrode chip was coated with M. paratuberculosis antigen and, after blocking uncoated surface, reacted sequentially with bovine serum sample and fluorescently labeled secondary antibody. Liquid flow was electrically controlled by ACET micropumping effect. The level of antibody binding was then measured by using a LED-induced fluorescence with a low cost mini-spectrometer. JD-positive and JD-negative serum samples were tested with this LOC immunoassay system. Antibody binding in JD-positive serum was detected by this ACET-based system after loading the serum and secondary antibody for 3 min each. Without the ACET effect, the antibody binding was not detectable after the 3-min reactions. The level of antibody binding in the JD-positive serum was greater than that of JD-negative serum. Higher antibody binding was observed at the electrode edges and we assumed that ACET caused a swirl of liquid around the electrode and thereby accelerated the antibody-antigen interaction. This assumption was supported by our theoretical prediction and numerical simulation. This ACET-based LOC immunoassay may form a basis for the development of an on-site JD diagnostic method.

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.

Veterinary RecordVolume 165, Issue 11 p. 323-324 Short Communication Use of serological techniques for diagnosis of Mycobacterium bovis infection in a llama herd G. S. Dean PhD, Corresponding Author G. S. Dean PhD [email protected] [email protected]Search for more papers by this authorH. M. Vordermeier PhD, H. M. Vordermeier PhD Veterinary Laboratories Agency (VLA) — Weybridge, New Haw, Addlestone, Surrey, KT15 3NBSearch for more papers by this authorT. R. Crawshaw BVetMed, MSc, MRCVS, T. R. Crawshaw BVetMed, MSc, MRCVSSearch for more papers by this authorD. F. Twomey MVB, MRCVS, D. F. Twomey MVB, MRCVS VLA — Starcross, Staplake Mount, Starcross, Exeter, EX6 8PESearch for more papers by this authorR. de la Rua-Domenech DVM, PhD, DipECVPH, MRCVS, R. de la Rua-Domenech DVM, PhD, DipECVPH, MRCVS TB Programme, Defra, Nobel House, 17 Smith Square, London, SW1P 3JRSearch for more papers by this authorL. Farrant BVetMed, MRCVS, L. Farrant BVetMed, MRCVS Animal Health, Clyst House, Winslade Park, Clyst St Mary, Devon, EX5 1DYSearch for more papers by this authorR. Greenwald MSc, R. Greenwald MScSearch for more papers by this authorK. Lyashchenko PhD, K. Lyashchenko PhD Chembio Diagnostic Systems, 3661 Horseblock Road, Medford, NY, 11763 USASearch for more papers by this authorR. J. Higgins BVM&S, MSc, MRCVS, R. J. Higgins BVM&S, MSc, MRCVS VLA — Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, EH26 0PZSearch for more papers by this author G. S. Dean PhD, Corresponding Author G. S. Dean PhD [email protected] [email protected]Search for more papers by this authorH. M. Vordermeier PhD, H. M. Vordermeier PhD Veterinary Laboratories Agency (VLA) — Weybridge, New Haw, Addlestone, Surrey, KT15 3NBSearch for more papers by this authorT. R. Crawshaw BVetMed, MSc, MRCVS, T. R. Crawshaw BVetMed, MSc, MRCVSSearch for more papers by this authorD. F. Twomey MVB, MRCVS, D. F. Twomey MVB, MRCVS VLA — Starcross, Staplake Mount, Starcross, Exeter, EX6 8PESearch for more papers by this authorR. de la Rua-Domenech DVM, PhD, DipECVPH, MRCVS, R. de la Rua-Domenech DVM, PhD, DipECVPH, MRCVS TB Programme, Defra, Nobel House, 17 Smith Square, London, SW1P 3JRSearch for more papers by this authorL. Farrant BVetMed, MRCVS, L. Farrant BVetMed, MRCVS Animal Health, Clyst House, Winslade Park, Clyst St Mary, Devon, EX5 1DYSearch for more papers by this authorR. Greenwald MSc, R. Greenwald MScSearch for more papers by this authorK. Lyashchenko PhD, K. Lyashchenko PhD Chembio Diagnostic Systems, 3661 Horseblock Road, Medford, NY, 11763 USASearch for more papers by this authorR. J. Higgins BVM&S, MSc, MRCVS, R. J. Higgins BVM&S, MSc, MRCVS VLA — Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, EH26 0PZSearch for more papers by this author First published: 12 September 2009 https://doi.org/10.1136/vr.165.11.323Citations: 5Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume165, Issue11September 2009Pages 323-324 RelatedInformation

ABSTRACT Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP against Mycobacterium tuberculosis to identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated with M. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP.

ABSTRACT Vaccine development and our understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by definition of the immunological correlates of protection and/or pathology. In this study we analyzed humoral immune responses in Mycobacterium bovis BCG-vaccinated and control cattle (in particular, the relationship between the intradermal comparative tuberculin skin test and serum immunoglobulin G [IgG] responses) against a range of mycobacterial antigens (MPB59, MPB64, MPB70, MPB83, ESAT-6, CFP-10, Acr1, and PstS-1) by multiantigen print immunoassay and conventional enzyme-linked immunosorbent assay. Following M. bovis infection, the comparative tuberculin skin test strongly boosted IgG, IgG1, and IgG2 antibody responses, particularly against MPB83 and MPB70, in unvaccinated cattle but failed to boost these responses, or did so only weakly, in BCG-vaccinated calves. In addition, the skin test-induced increases in MPB83-specific IgG responses correlated positively with bacterial loads and ESAT-6-induced in vitro gamma interferon responses. In conclusion, both the negative correlation of skin test-enhanced MPB83-specific antibody responses with BCG-induced protection and their positive correlation with bacterial loads can serve as useful markers for vaccine efficacy after challenge.

Despite attempts to control bovine tuberculosis, the incidence of disease in Great Britain continues to rise. In GB, the European badger (Meles meles) is a reservoir of infection with Mycobacterium bovis. In an effort to improve the serodetection of badger tuberculosis, we examined sera from M. bovis culture-positive and culture-negative badgers for their ability to recognize M. bovis antigens, using a multi-antigen print immunoassay (MAPIA). Depending on the antigens used in the MAPIA, the assay had a sensitivity of 49–59% and a specificity of 84-88% Results from the MAPIA were used to select antigens for the development of a lateral-flow immunoassay. This so-called ‘Rapid Test’ used 5μl of serum and gave unambiguous results within 10 min. When applied to 178 badger sera, the Rapid Test had a sensitivity of 53% and a specificity of 95%. This represented an improvement over the performance of the existing ELISA Test, which had a sensitivity of 47% and a specificity of 89% on the same sera. This is the first report of a diagnostic test for badger tuberculosis that can be performed alongside the captive animal.

Antibodies against Mycobacterium tuberculosis antigens were detected by enzyme-linked immunosorbent assay in cerebrospinal fluid (CSF) samples obtained from 442 patients with tuberculous meningitis (TBM) and 102 control patients. Antibodies were found in the CSF of 87% of patients with clinical (culture-negative) TBM, 72% of patients with culture-positive TBM, and 65% of patients with autopsy-proven TBM. That anti-M. tuberculosis antibodies were detected in the CSF of patients with clinically diagnosed cases more frequently than in patients with culture-positive cases suggests that the detection of antibodies in CSF tends to decrease as bacillary load increases. Of the patients with clinical TBM who were coinfected with human immunodeficiency virus (HIV), 70% exhibited anti-M. tuberculosis antibody in CSF, which suggests that antibody responses in this group were substantially weaker than those in HIV-negative patients with clinical TBM. Some groups showed a stronger response to certain antigens, which suggests that antigen recognition patterns may be specific for the stage of disease.

MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.

ABSTRACT In a search for new skin test reagents specific for tuberculosis, we found that the antigen encoded by gene Rv3874 of Mycobacterium tuberculosis elicited delayed-type hypersensitivity in M. tuberculosis-infected guinea pigs but not in control animals immunized with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium avium. The antigen, which was named MTSA-10 (for M. tuberculosis-specific antigen 10), is a prime candidate for a component of a new tuberculin that will allow discrimination by a skin test of latent M. tuberculosis infection from vaccination with BCG or from sensitization with environmental, nontuberculous mycobacteria.

A panel of ten protein antigens of Mycobacterium tuberculosis was used to evaluate serum antibody responses to tuberculosis in patients co-infected with the human immunodeficiency virus (HIV) and in HIV-infected control individuals without tuberculosis. Most (70%) of the tuberculosis patients had serum reactivity to at least one antigen and maintained the diverse antibody repertoire previously observed in HIV-negative tuberculosis patients.

ABSTRACT Antibody responses during tuberculosis were analyzed by an enzyme-linked immunosorbent assay with a panel of 10 protein antigens of Mycobacterium tuberculosis . It was shown that serum immunoglobulin G antibodies were produced against a variety of M. tuberculosis antigens and that the vast majority of sera from tuberculosis patients contained antibodies against one or more M. tuberculosis antigens. The number and the species of serologically reactive antigens varied greatly from individual to individual. In a given serum, the level of specific antibodies also varied with the antigen irrespective of the total number of antigens recognized by that particular serum. These findings indicate that person-to-person heterogeneity of antigen recognition, rather than recognition of particular antigens, is a key attribute of the antibody response in tuberculosis.

ABSTRACT The tuberculin skin test currently used to diagnose infection withMycobacterium tuberculosis has poor diagnostic value, especially in geographic areas where the prevalence of tuberculosis is low or where the environmental burden of saprophytic, nontuberculous mycobacteria is high. Inaccuracy of the tuberculin skin test often reflects a low diagnostic specificity due to the presence in tuberculin of antigens shared by many mycobacterial species. Thus, a skin test specific for tuberculosis requires the development of new tuberculins consisting of antigens specific to M. tuberculosis. We have formulated cocktails of two to eight antigens of M. tuberculosis purified from recombinant Escherichia coli. Multiantigen cocktails were evaluated by skin testing guinea pigs sensitized with M. bovis BCG. Reactivity of multiantigen cocktails was greater than that of any single antigen. Cocktail activity increased with the number of antigens in the cocktail even when the same amount of total protein was used for cocktails and for each single antigen. A cocktail of four purified antigens specific for the M. tuberculosis complex elicited skin test responses only in BCG-immunized guinea pigs, not in control animals immunized with M. avium. These findings open the way to designing a multiantigen formulation for a skin test specific for tuberculosis.

Proteins that are actively secreted by Mycobacterium tuberculosis serve as major targets of immune responses in the infected host. To identify and purify novel proteins in the filtrates of M. tuberculosis cultures, a bacteriophage lambda library of M. tuberculosis H37Rv DNA was immunoscreened by using an anti-culture filtrate rabbit antiserum. Of 20 positive clones isolated, 6 were analyzed and found to express the genes for two known components of the early culture filtrate, the secreted 45/47-kDa antigen complex and the KatG protein, and two novel genes. Here we report the molecular cloning and nucleotide sequence of one of the new genes encoding a culture filtrate protein of 310 amino acid (aa) residues. We called this gene mtc28. The deduced polypeptide sequence contained an NH2-terminal, highly hydrophobic 32-aa region having properties of a secretion signal peptide. The putative 278-aa mature MTC28 protein was characterized at its NH2 and COOH termini by a high content of proline and alanine residues organized in an (AP)n motif. Thus, MTC28 is a new member of a group of proline-rich antigens found in M. tuberculosis and Mycobacterium leprae. As shown by DNA hybridization experiments, the mtc28 gene was present only in species of the M. tuberculosis complex. Purified recombinant MTC28 antigen evoked strong delayed-type hypersensitivity and antibody responses in guinea pigs immunized with Mycobacterium bovis BCG, but not in guinea pigs immunized with Mycobacterium avium. The strong immunological activity of MTC28 and the absence of B- and T-cell epitopes cross-reactive with a common environmental mycobacterial species, such as M. avium, make this novel antigen an attractive reagent for immunodiagnosis of tuberculosis.