Letters in Applied Microbiology ISSN 0266-8254

Development of a simple and rapid method for the specific identification of organism causing anthrax by slide latex agglutination T.G. Sumithra1, V.K. Chaturvedi2, P.K. Gupta2, S.C. Sunita2, A.K. Rai2, M.V.H. Kutty2, U. Laxmi2 and M.S. Murugan2 1 College of Veterinary and Animal Sciences, Pookode, Kerala, India 2 Indian Veterinary Research Institute, Izatnagar, U.P., India

Significance and Impact of the Study: The article presents the first report of a latex agglutination test for the specific identification of the cultures of bacteria causing anthrax. As the test is targeting one of anthrax toxic protein (PA), this can also be used to determine virulence of suspected organisms. At the same time, the same LAT can be used directly on whole blood or sera samples under field conditions for the specific diagnosis of anthrax.

Keywords Bacillus anthracis, diagnosis, protective antigen, slide latex agglutination test. Correspondence Vinod Kumar Chaturvedi, Principal scientist & Head, Division of Biological Products, Indian Veterinary Research Institute, Izatnagar, U.P. 243122, India. E-mail: [email protected]

Abstract A specific latex agglutination test (LAT) based on anti-PA (protective antigen) antibodies having detection limit of 5 9 104 formalin treated Bacillus anthracis cells or 110 ng of PA was optimized in this study. The optimized LAT could detect anthrax toxin in whole blood as well as in serum from the animal models of anthrax infection. The protocol is a simple and promising method for the specific detection of bacteria causing anthrax under routine laboratory, as well as in field, conditions without any special equipments or expertise.

Present address V.K. Chaturvedi, Principal scientist & Head, Division of Biological Products, Indian Veterinary Research Institute, Izatnagar, U.P. 243122, India 2013/1647: received 13 August 2013, revised 25 November 2013 and accepted 26 November 2013 doi:10.1111/lam.12204

Introduction Anthrax, a fulminating disease that can strike almost all warm-blooded animals including humans, is usually caused by Bacillus anthracis, a spore-forming, Grampositive bacterium. The disease is still endemic in several countries of southern Africa, central Asia, small pockets of USA and certain regions of South America (Beyer and Turnbull 2009). Apart from the natural occurrence, B. anthracis remains as an appealing biological weapon. Therefore, development of a quick, less risky and reliable

detection method for the bacterium causing anthrax is a necessity in both medical and veterinary fields. With traditional methods, confirmation of B. anthracis cultures requires several days (Irenge and Gala 2012). The challenges posed by coincidental or noncoincidental presence of pathogenic micro-organisms together with closely related nonpathogenic micro-organisms of the same genus further complicate the specific identification method B. anthracis (Irenge and Gala 2012). As anthrax toxin is the essential prerequisite for the development of anthrax and, nonpathogenic isolates of B. anthracis lacking plas-

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PA-based LAT for anthrax

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mid encoding anthrax toxin have been reported, anthrax toxin is the most suitable target for the diagnosis of the disease (Turnbull et al. 1992; Beyer and Turnbull 2009). In addition, recently, strains of Bacillus cereus causing anthrax by the production of anthrax toxin were isolated (Hoffmaster et al. 2004), proving the necessity of anthrax toxin-based assays for the specific identification of organisms causing anthrax. Although several advanced techniques have been recently formulated for the same purpose, these tend to require expertise, expensive devices or complicated protocols (Liu et al. 2013), and thus are not suitable for routine laboratory conditions. Being simple, rapid, easy to perform and inexpensive, latex agglutination test (LAT) has been developed for the routine detection of many other infectious agents (Yap 1994). Keeping all these facts in view, an attempt was made to develop anthrax toxin-based LAT as a sensitive, rapid and easy-to-use tool technique for the specific identification of bacteria causing anthrax. Results and discussion Zoonotic potential, ability to be used as a weapon for bioterrorism, capacity to persist in environment for decades, quick onset and rapid lethal progression of the disease, coincidental or noncoincidental presence of pathogenic micro-organisms together with closely related nonpathogenic micro-organisms of the same genus etc. necessitate the development of rapid, reliable and specific detection methods for the organisms causing anthrax. Therefore, in the present study, an attempt was made to develop LAT as an inexpensive, simple and rapid test for the specific identification of the bacteria having potential to produce anthrax. Although methods targeting spores and vegetative cells are developed for the detection of B. anthracis, owing to their disadvantages in specificity and clinical practicality, these methods are far from satisfaction (Liu et al. 2013). Recently, AuCoin et al. (2009) developed LAT for detection of capsular antigen of B. anthracis in serum. However, toxin proteins will be a more suitable target for anthrax diagnosis as loss of pXO1 (encoding anthrax toxin) in environmental sample is rare and loss of pXO2 (encoding anthrax capsule) is common (Mock and Fouet 2001). Additionally, Sterne strain lacking pXO2 plasmid retains virulence for humans (Wang and Roehrl 2005) and certain species of animals (Cartwright et al. 1987). At the same time, B. anthracis lacking pXO1 plasmid is nonpathogenic (Turnbull et al. 1992). In addition, recently, strains of B. cereus causing anthrax by the production of anthrax toxin were isolated (Hoffmaster et al. 2004), proving the necessity of anthrax toxin-based assays. Accordingly, for detecting all the bacteria that can cause anthrax including Sterne strain of 402

B. anthracis, protective antigen (PA), the most essential anthrax toxin component in pathogenesis is selected as target. As pathogenicity of all bacteria causing anthrax is mainly due to their toxin (Cella et al. 2010), detection methods targeting anthrax toxin have added advantage for determining the virulence of suspected organisms. Traditional approaches to prepare PA from the culture supernatant are tedious, risky and are not suitable for large-scale production. Therefore, we produced rPA using pQE Trisystem vector for the preparation of specific hyperimmune sera. As the 1683-bp-sized carboxyl-terminal portion of pag gene (encoding PA) is highly conserved and necessary for the formation of active anthrax toxin (Mourez et al. 2003), the particular region was targeted. For the cloning of pag into the expression vector, PCR amplification of the gene was performed as the first step with self-designed primers, which yielded the 1683-bpsized product (Fig. 1). The cloning and transformation into Escherichia coli M15 competent cells were followed, and the recombinant clones were finally confirmed by colony PCR, plasmid PCR and double restriction enzyme (RE) analysis (Fig. 2). The accuracy of construct was further verified by DNA sequencing. Homologies of nucleotide sequences in the construct with published pag sequences (Accession no. CP001216.1, CP001747 and AF306782.1) were 100%. The sequence obtained was deposited in NCBI GenBank database with the accession number JQ798178.1. Induction of recombinant clones

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Figure 1 PCR amplification of pag gene. Lane M: GeneRulerTM 1 kb DNA Ladder; Lane 1, 2: amplification of pag (1683 bp).

Letters in Applied Microbiology 58, 401--407 © 2013 The Society for Applied Microbiology

T.G. Sumithra et al.

PA-based LAT for anthrax

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subsequently verified by Western blotting (Fig. 3b). The rPA was found to have higher molecular weight compared with the original truncated PA, which was due to the expression of PA as 8 X-histidine-tagged (encoded by the vector) fusion protein. Concentration of purified rPA after dialysis was 047 mg ml 1. Hyperimmune sera production using rPA yielded sera with good antibody titre as evidenced by protein concentration of purified gamma globulin (40 mg ml 1) and ELISA P/N value (1362 at 1 : 800 dilution of serum) and used for sensitization of latex beads. As a large number of variables can affect the coating of polystyrene particles by ligands, it is important to determine the optimum conditions for the performance of test (Yap 1994). As commercial latex particles are usually supplied in medium containing various inhibitors for the attachment of ligands, cleaning of latex beads is necessary. Therefore, repeated washing (total 5 washings) in distilled water and dispersal by vigorous pipetting, and vortexing after centrifugation were carried out and found that this treatment was adequate for particle clean-up even though Seradyn (1988) reported that this method might not eliminate all removable surfactant group. This is in accordance with the interpretation of Yap (1994) that maximum adsorption which needs totally clean particles is not required in the slide agglutination test. A study of antibody concentration, incubation temperature, time and coating buffer type and pH revealed that optimum reaction could be achieved by coating 25 mg latex particles with 2 mg of purified gamma globulin in glycine-saline buffer of pH-96 at incubation temperature of 37°C for 6 h. In the proper LAT, anti-PA-sensitized latex reagent could give definite positive agglutination pattern (large sized bacterial clumps) with the formalin treated cultures

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Figure 2 Characterization of pag-pQE plasmid. Lane M: GeneRulerTM 1 kb DNA Ladder; Lane 1: RE digestion of pag-pQE plasmid with XhoI and BamHI showing the release of 1683-bp-sized insert; Lane 2: RE digestion of pag-pQE plasmid with XhoI showing the linearized plasmid having 7483 bp size (5800 bp vector + 1683 bp insert); Lane 3: undigested pag-pQE plasmid.

was followed with 1 mmol l 1 IPTG, which successfully expressed rPA as evidenced by SDS-PAGE analysis (Fig. 3a). Then, rPA was successfully purified from bacterial lysate using metal chelate affinity chromatography (Fig. 3a). The identity and immunogenicity of rPA were (a)

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50 40 Figure 3 SDS-PAGE and Western blot analysis of rPA. (a) SDS-PAGE analysis. Lane M: SpectraTM multicolour broad range protein ladder; Lane 1: uninduced colonies; Lane 2: induced colonies; Lane 3: purified rPA. (b) Western blotting. Lane M: SpectraTM multicolour broad range protein ladder; Lane 1–2: purified rPA.

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4·1 (b)

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Table 1 Different bacteria used in the study Bacteria

Source

Bacillus anthracis Sterne’s Strain

Biological Standardization (BS) Division, IVRI, Izatnagar BS Division, IVRI, Izatnagar MTCC, Chandigarh MTCC, Chandigarh MTCC, Chandigarh National Salmonella Centre, IVRI, Izatnagar Biological Products (BP) Division, IVRI, Izatnagar BP Division, IVRI, Izatnagar BP Division, IVRI, Izatnagar BP Division, IVRI, Izatnagar MTCC, Chandigarh MTCC, Chandigarh

B. anthracis IVRI Strain Bacillus cereus MTCC 7409 Bacillus subtilis MTCC 1133 Bacillus megaterium MTCC 1684 Salmonella Typhimurium E2393 Staphylococcus aureus Brucella abortus Strain 19 Pasteurella multocida Strain P52 Escherichia coli DH5a Shigella flexneri MTCC 1457 Vibrio cholerae MTCC 3904 (Classical O1) Yersinia enterocolitica ssp. enterocolitica MTCC 4858 Clostridium perfringens type D (IVRI strain)

MTCC, Chandigarh BP Division, IVRI, Izatnagar

of both strains of B. anthracis (field strain as well as Sterne strain) within 1–2 min (Fig. 4.1a, 4.2a) while agglutination reaction was negative (uniformly distributed blue coloured fluid) with B. cereus culture (Fig. 4.1b). The sterility checking of formalin treated cultures proved the ability of the followed protocol to inactivate both vegetative cells and spores of B. anthracis. As the optimized LAT can be performed in these inactivated solutions, there is no chance of exposure to the active culture other than during initial inoculation of suspected sample. Therefore, this method will contribute significantly in 404

Figure 4 Slide latex agglutination test. 4.1: slide LAT with inactivated bacterial cultures. 4.1: (a): positive agglutination with Bacillus anthracis culture; 4.1: (b): negative agglutination with Bacillus cereus culture. 4.2: sensitivity evaluation of slide LAT. 4.2: (a): LAT with 5 9 107 CFU of B. anthracis cells; 4.2: (b): LAT with 5 9 105 CFU of B. anthracis cells; 4.2: (c): LAT with 5 9 104 CFU of B. anthracis cells; 4.2: (d): LAT with 5 9 103 CFU of B. anthracis cells. 4.3: slide LAT using whole blood. 4.3: (a): positive agglutination with whole blood of anthrax-infected guinea pig; 4.3. (b): negative agglutination with whole blood of normal animal.

reducing the risk of laboratory-acquired anthrax infections during the identification of suspected cultures. The sensitivity testing (Fig. 4.2a-d) revealed that a minimum of 5 9 104 CFU of vegetative cells was sufficient for producing the positive agglutination. Therefore, the test was proved to be 10 times more sensitive than the detection limit of other immunological assays used by King et al. (2003) for B. anthracis. The test was confirmed to be very specific for anthrax toxin as there was no false-positive reaction with any other tested bacteria (Table 1) even at 1000 times higher concentrations than the detection limit of the test for B. anthracis cells. The sensitized latex beads were found to be stable for at least 4 months after storage at 4°C. In the evaluation of the repeatability of the optimized test, the specificity, sensitivity and stability of the LAT reagent tested on different occasions appeared identical. Additionally, the optimized LAT produced agglutination with expressed PA preparation (containing 047 mg ml 1 concentration of PA in PBS) although the time for the appearance of agglutination was slightly longer (2–5 min). During evaluation of sensitivity, the test was found to be sensitive up to 110 ng of expressed PA (10 ll of a preparation containing 11 lg ml 1 of PA concentration). It had been documented that concentration of PA in the serum of anthrax-infected animals at the time of death varied from 103 to 105 ng ml 1 (Kobiler et al. 2006), indicating the suitability for the direct application of the optimized LAT to specifically diagnose anthraxinfected cases. To assess this utility, an experimental anthrax infection was established in two guinea pigs. The whole blood and serum collected from these animals after death produced definite positive agglutination (Fig. 4.3a),

Letters in Applied Microbiology 58, 401--407 © 2013 The Society for Applied Microbiology

T.G. Sumithra et al.

PA-based LAT for anthrax

while blood and serum collected from the control animals showed negative reaction (Fig. 4.3b), illustrating the potential of the optimized LAT for the specific diagnosis of anthrax-infected cases under field conditions. Although various advanced approaches have been proposed for the specific identification of bacteria that can cause anthrax with much more sensitivity, all of these require expensive devices or complicated protocols and thus are not suitable under routine laboratory conditions (Liu et al. 2013). Although ELISA for detection of anthrax toxin can be more sensitive, the test is tedious and requires expertise with careful quality control of reagents. At the same time being an inexpensive, simple, rapid and easy-to-use test, the optimized LAT can be used for the specific diagnosis of anthrax under field conditions. In addition, the test can be used for the specific identification and virulence determination of suspected cultures under routine laboratory conditions.

of Platinum Pfx polymerase. Subsequently, pag was cloned in frame into pQE Trisystem expression vector, which was then transformed into E. coli M15 competent cells. Positive clones were confirmed by PCR, double RE digestion and sequence analysis. The expression of histidine-tagged rPA was subsequently induced from the positive clones by 1 mmol l 1 IPTG (Sambrook and Russell 2003). Afterwards, histidine-tagged rPA was purified from induced culture under denaturing conditions by nickel-chelating affinity chromatography. Purity, identity and immunogenicity of the eluted rPA were confirmed by SDS-PAGE and Western blotting using hyperimmune sera raised against sonicated culture of B. anthracis. Urea from purified PA was subsequently removed by stepwise dialysis. Concentration of purified rPA after dialysis was estimated spectrophotometrically (Nanodropâ; Thermo Scientific, Wilmington, DE) at 280 nm.

Materials and methods

Preparation of hyperimmune sera for coating into latex beads

Bacterial strains A virulent field strain (initially obtained from Bacteriological Laboratory, Mukteshwar in 1975) and uncapsulated 34 F2 (Sterne) strain of B. anthracis were obtained from Biological Standardization Division, Indian Veterinary Research Institute (IVRI), Izatnagar. Animals Two healthy rabbits and guinea pigs were procured from Lab Animal Resource section, IVRI and maintained under standard conditions of nutrition and management. The animal ethics committee of IVRI, Deemed University, approved this study. Preparation of recombinant protective antigen (rPA) Based on the information from GenBank and Blast database of NCBI specific primers (RE sites were underlined), PA-F: 5′-aaaggatccggttccagaccgtgacaatga-3′; were PA-R: 5′-ggggctcgagtcctatctcatagccttttttagaa-3′ designed for the amplification of pag and got synthesized from Integrated DNA Technologies, Coralville, Iowa. PCR amplification of pag was performed with the following parameters: predenaturation for 5 min at 94°C; 30 cycles of 94°C for 1 min, 55°C for 1 min and 68°C for 2 min; postextension for 10 min at 68°C; and cooling to 4°C. Reaction mixture (25 ll) consisted of 200 lmol l 1 of each dNTPs, 375 ll of 109 Pfx reaction buffer, 20 mmol l 1 MgSO4, 50 pmol of each primers, 1 ll of pX-O1 plasmid (isolated from Sterne strain) and 10 U

Two healthy New Zealand White rabbits were immunized intramuscularly each with 100 lg of rPA emulsified in Freund’s complete adjuvant. Animals were boosted with 100 lg of antigen at days 14, 21 and 42 with Freund’s incomplete adjuvant. After 5 days of the last injection, sera antibody titre was confirmed by dot ELISA. Animals were finally bled at 7 days of the last injection; serum was separated and stored at 20°C. The gamma globulin fractions of hyperimmune sera were isolated by 40% ammonium sulfate fractionation, and the precipitate was resuspended in phosphate-buffered saline (PBS), pH- 74. The solution was subsequently dialysed against PBS until all ammonium sulfate had been removed. To determine the strength of hyperimmune sera, protein concentration in purified gamma globulin was estimated spectrophotometrically at 280 nm and by plate ELISA of the serum using 1 : 800 dilution of rPA antigen and 1 : 5000 dilution of goat anti-rabbitHRP conjugate. ELISA result was expressed as positive/ negative (P/N) ratio (Briggs et al. 1986). Preparation of latex particles Deep blue-dyed latex bead particles (080 lm diameter, Sigma, USA) consisting of 25% solids in aqueous suspension were washed five times in distilled water. Each washing step was performed by vigorous mixing of latex particles in distilled water by vortexing and pipetting followed by centrifugation, decanting and resuspending in distilled water. Finally, the washed particles were resuspended in glycine-saline buffer (002 mol l 1 glycine,

Letters in Applied Microbiology 58, 401--407 © 2013 The Society for Applied Microbiology

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003 mol l 1 NaCl, pH 96) to the original volume and stored at 4°C. Sensitization of latex particles with anti-PA hyperimmune serum A 100 ll volume (25 mg solids) of washed latex beads in buffer was added to 100 ll of purified gamma globulins (20 mg ml 1). The mixture was vortexed for 1 min and then allowed to incubate with gentle stirring at 37°C for 6 h. Excess protein was removed by washing twice with 500 ll of glycine-saline buffer, and the coated beads were resuspended in 01 ml of the same buffer. Sodium azide at a final concentration of 005% was added, and the reagent was stored at 4°C. Modifications to this procedure such as change in antibody concentration, incubation temperature and coating buffer pH were carried out, and finally, the above conditions were found to be optimum. Slide LAT Both strains of B. anthracis were grown in 5 ml nutrient broth and incubated at 37°C for 24 h as stationary cultures. The cultures were then inactivated by 5% formalin treated followed by incubation at 37°C for 4 h. To check sterility of the obtained solutions, these were subcultured into 5 ml nutrient broth and incubated at 37°C for 24 h. For LAT, 10 ll of the inactivated culture was mixed with equal volume of sensitized latex beads on a glass slide and mixed thoroughly and observed for 1–2 min. A negative control reaction was simultaneously carried out by mixing the inactivated culture of B. cereus with equal volume of sensitized latex beads. Similarly, 10 ll of the latex beads was tested for agglutination with 10 ll of rPA (test) as well as 10 ll of PBS (negative control). Quality evaluation To verify the specificity, the optimized LAT was performed by employing 10 ll of the formalin treated overnight culture having 109 CFU ml 1 of various bacteria of same and different genera (Table 1). Briefly, each bacterium was grown in 5 ml nutrient broth and incubated at 37°C for 24 h. The amount of bacteria in the overnight culture was then determined in terms of CFU ml 1 by spread plate method (Heritage et al. 1996). The cultures were then inactivated by formalin treated, and each culture was diluted in PBS (pH-74) as 109 CFU ml 1. For LAT, 10 ll of each diluted culture was mixed with equal volume of sensitized latex beads on a glass slide and observed for 1–2 min. To measure assay sensitivity, serial tenfold dilutions of inactivated cultures from B. anthracis were prepared in 406

PBS and 10 ll from each dilution was used for the agglutination. The highest dilution that can produce distinct agglutination was found out. The amount of bacteria corresponding to this dilution was then determined in terms of CFU ml 1 by spread plate method (Heritage et al. 1996). Similarly, serial tenfold dilutions of purified rPA were prepared in PBS, and the highest dilution that can produce distinct agglutination within 5 min was found out. Protein concentration of this dilution was then found out using Nanodropâ (Thermo Scientific). For testing stability, sensitized latex beads stored at 4°C for 1–4 months were subjected to reactions with inactivated B. anthracis and other cultures at the end of each month. The repeatability of LAT was tested with 1-month interval for 1–4 months by the same lot of sensitized latex antigen. The specificity and sensitivity of LAT were also assayed with the latex reagents sensitized after storage of the purified gamma globulins at 80°C for 4 months. The specificity, sensitivity and stability evaluation of the test was also carried out using hyperimmune sera raised in another rabbit in the similar conditions. Experimental infection study Two guinea pigs were injected intraperitoneally with 05 ml of B. anthracis, field strain culture (108 CFU ml 1). The blood was collected from heart of both guinea pigs after death (within 24 h after the injection) in heparinized vial (for whole blood) and in nonheparinized vial (for sera). The resulted whole blood and serum were used for agglutination. Whole blood and serum collected in similar manner from normal guinea pigs were used as negative control. Acknowledgements The authors are grateful to the Director and Joint Director (Academic and Research) of the IVRI for providing the necessary facilities to carry out the present investigation. TGS thanks Indian Council of Medical Research for providing financial support in the form of Senior Research Fellowship. Conflict of interest No conflict of interest declared. References AuCoin, D.P., Sutherland, M.D., Percival, A.L., Lyons, C.R., Lovchik, J.A. and Kozel, T.R. (2009) Rapid detection of polyc-D-glutamic acid capsular antigen of Bacillus anthracis by latex agglutination. Diagn Microbiol Infect Dis 64, 229–232.

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Beyer, W. and Turnbull, P.C. (2009) Anthrax in animals. Mol Aspects Med 30, 481–489. Briggs, D.J., Whitfill, C.E., Skeeles, J.K., Story, J.D. and Reed, K.D. (1986) Application of the positive/negative ratio method of analysis to quantitate antibody responses to infectious bursal disease virus using commercially available ELISA. Avian Dis 30, 216–218. Cartwright, M.E., McChesney, A.E. and Jones, R.L. (1987) Vaccination related anthrax in three llamas. J Am Vet Med Assoc 19, 715–716. Cella, L.N., Sanchez, P., Zhong, W., Myung, N.V., Chen, W. and Mulchandani, A. (2010) Nano aptasensor for protective antigen toxin of anthrax. Anal Chem 82, 2042–2047. Heritage, J., Evans, E.G.E. and Killington, R.A. (1996) Introductory Microbiology. Cambridge: Cambridge University Press. Hoffmaster, A.R., Ravel, J., Rasko, D.A., Chapman, G.D., Chute, M.D., Marston, C.K., De, B.K., Sacchi, C.T. et al. (2004) Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proc Natl Acad Sci 101, 8449–8454. Irenge, L.M. and Gala, J.L. (2012) Rapid detection methods for Bacillus anthracis in environmental samples: a review. Appl Microbiol Biotechnol 93, 1411–1422. King, D., Luna, V., Cannons, A., Cattani, J. and Amuso, P. (2003) Performance assessment of three commercial assays for direct detection of Bacillus anthracis spores. J Clin Microbiol 41, 3454–3455. Kobiler, D., Weiss, S., Levy, H., Fisher, M., Mechaly, A., Pass, A. and Altboum, Z. (2006) Protective antigen as a

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correlative marker for anthrax in animal models. Infect Immun 74, 5871–5876. Liu, X., Wang, D., Ren, J., Tong, C., Feng, E., Wang, X., Zhu, L. and Wang, H. (2013) Identification of the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. PLoS ONE 8, e57959. Mock, M. and Fouet, A. (2001) Anthrax. Annu Rev Microbiol 55, 647–671. Mourez, M., Yan, M., Lacy, D.B., Dillon, L., Bentsen, L., Marpoe, A., Maurin, C., Hotze, E. et al. (2003) Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis. Proc Natl Acad Sci 100, 13803–13808. Sambrook, J. and Russell, D.W. (2003) Molecular cloning-A laboratory manual, 3rd edn. New York: Cold spring harbor laboratory press. Seradyn. (1988) Particle Technology Division-Micro particle immunoassay techniques, 2nd edn. Indianapolis: Seradyn Inc. Turnbull, P.C.B., Hutson, R., Ward, M.J., Jones, M.N., Quinn, C.P., Finnie, N.J., Duggleby, C.J., Kramer, J.M. et al. (1992) Bacillus anthracis but not always anthrax. J Appl Bacteriol 72, 21–28. Wang, J.Y. and Roehrl, M.H. (2005) Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin. Med Immunol 4, 1–8. Yap, K.L. (1994) Development of a slide latex agglutination test for rotavirus antigen detection. Malaysian J Pathol 16, 49–56.

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Development of a simple and rapid method for the specific identification of organism causing anthrax by slide latex agglutination.

A specific latex agglutination test (LAT) based on anti-PA (protective antigen) antibodies having detection limit of 5 × 10(4) formalin treated Bacill...
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