Evaluation of PCR, DNA hybridization and immunomagnetic separation – PCR for detection of Burkholderia mallei in artificially inoculated environmental samples.
Abstract: Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridization method was standardized for 823 bp specific dNA sequence of B. mallei. To enable the quicker and direct enrichment of B. mallei bacteria from environmental samples, an immunomagnetic separation (IMS) method was also standardized. Water, husk, grass and gram samples were artificially contaminated by B. mallei bacteria and after enrichment of B. mallei in BHIB-SS, detection was carried out by PCR and DNA hybridization. PCR was found to be a better method of the two with a detection limit of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices. Detection by PCR in the above samples without enrichment in BHIBSS was carried out following IMS where the detection limit was about 1-2 log higher than PCR following enrichment in BHIB-SS. We recommend PCR for 823 bp for detection of B. mallei from environmental samples either following enrichment in BHIB-SS or IMS. IMS-PCR method may be preferred in situations where numbers of B. mallei bacteria are expected to be high and results are required in short time.
Publication Date: 2010-03-05 PubMed ID: 23100824PubMed Central: PMC3450331DOI: 10.1007/s12088-010-0003-3Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research article outlines the development of a rapid detection strategy for Burkholderia mallei, a bacteria that causes glanders in equines and can be transmitted to humans, in environmental samples. The researchers found that polymerase chain reaction (PCR) is the most effective method, particularly when using a specific 823 bp DNA sequence of B. mallei.
Selection of Media for Bacterial Growth
- The researchers began by selecting a suitable medium to support the growth of the B. mallei bacteria. They evaluated several different bacteriological media.
- They found that the brain heart infusion broth medium with selective supplements (BHIB-SS) successfully supported the maximum growth of B. mallei. This was true even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus.
Standardization of PCR and DNA Hybridization Method
- The team then worked on developing a method to identify and isolate the bacteria. They standardized a Polymerase Chain Reaction (PCR) and a DNA hybridization method for a specific 823 base pair DNA sequence of B. mallei.
- The PCR method was deemed superior as it provided a lower detection limit, meaning the method could detect the presence of B. mallei even in lower concentrations.
Implementation of Immunomagnetic Separation Method
- To directly and quickly isolate B. mallei from environmental samples, an immunomagnetic separation (IMS) method was also standardized by the researchers.
- The rationale behind using IMS was to bring down the detection limit still further, allowing for quicker and more efficient detection of the bacteria.
Testing of Detection Methods on Artificially Contaminated Samples
- The researchers then contaminated water, husk, grass, and gram samples with B. mallei bacteria for testing.
- After letting the bacteria grow in BHIB-SS medium, they then observed and analyzed the samples using the PCR and DNA hybridization methods.
- The researchers found that PCR was superior to DNA hybridization, with an ability to detect concentrations of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices.
Conclusion and Recommendations
- The research concluded that PCR is the most effective method for detecting B. mallei in environmental samples, either following enrichment in BHIB-SS or after IMS.
- They recommended the IMS-PCR method in situations where high numbers of B. mallei bacteria are expected and results need to be obtained quickly.
Cite This Article
APA
Merwyn S, Kumar S, Agarwal GS, Rai GP.
(2010).
Evaluation of PCR, DNA hybridization and immunomagnetic separation – PCR for detection of Burkholderia mallei in artificially inoculated environmental samples.
Indian J Microbiol, 50(2), 172-178.
https://doi.org/10.1007/s12088-010-0003-3 Publication
Researcher Affiliations
- Division of High Containment Facility, Defence Research and Development Establishment, Gwalior, 474 002 India.
References
This article includes 25 references
- Fritz DL, Vogel P, Brown DR, Deshazer D, Wagg DM. Mouse model for sublethal and lethal intraperitoneal glanders (B. mallei). Vet Pathol 2000;37:626–636.
- Quinn P.J., Markey B.K., Carter M.E., Donnelly W.T.C., Leonard F.C., editors. Veterinary Microbiology and Microbial Disease. Oxford, UK: Blackwell Science Publication; 2002. pp. 237–251.
- Srinivasan A, Kraus CN, DeShazer D, Becker PM, Dick JD, Spacek L, Bartlett JG, Byrne WR, Thomas DL. Glanders in a military research microbiologist. N Engl J Med 2001;345:256–258.
- Zijderveld FG, Bongers J. Manual for Standards for Diagnostic Tests and Vaccines. 4th edition. Paris: Office International des Epizootics; 2000. Glanders; pp. 576–591.
- Arun S, Neubauer H, Gurel A, Ayyildiz G, Kuscu B, Yesildere T, Meyet H, Hermanns W. Equine glanders in Turkey. Vet Rec 1999;144:255–258.
- Krishna L, Gupta VK, Masand MR. Pathomorphological study of possible glanders in solipeds in Himachal Pradesh. Ind Vet J 1992;69:211–214.
- Muhammad G, Khan MZ, Athar M. Clinicomicrobiological and therapeutic aspects of glanders in equines. J Equine Sci 1998;9:93–96.
- Sanford JP. Pseudomonas species (including malioidosis and glanders). In: Mandell GL, Bennett JE, Dolin R, editors. Principles and Practice of Infectious Diseases. 4th edition. New York: Churchill Livingstone; 1995. pp. 2003–2009.
- Heine HS, England MJ, Wagg DM, Byrne WR. In vitro antibiotic susceptibilities of Burkholderia mallei (causative agent of glanders) determined by broth microdilution and E-test. Antimicrob Agents Chemother 2001;45:2119–2121.
- Wheelis M. First shots fired in biological warfare. Nature 1998;395:213.
- Verma RD and Mishra VC (1989) Research on epizootic diagnosis and control of glanders with a view to eradicate the disease from India. Project closure reported submitted ICAR, Ministry of Agriculture, Govt. of India, New Delhi, pp 1–54
- Hutyra F, Marek J, Manninger R. Special pathology and therapeutics of the diseases of domestic animals. 5th Eng Edn. London: Balliere Tindall and Cox; 1949. pp. 722–773.
- Boyden SV. Absorption by erythrocytes of antigens of Pf. mallei and Pf. whitmori. Proc Sac Exp Biol Med 1950;73:289–291.
- Sen GP, Singh G, Joshi TP. Comparative efficacy of serological tests in the diagnosis of glanders. Ind Vet J 1968;45:286–292.
- Jana AM, Gupta AK, Pandya G, Verma RD, Rao KM. Rapid diagnosis of glanders in equines by counterimmuno-electrophoresis. Ind Vet J 1982;59:5–9.
- Howe C, Miller WR. Human glanders: report of six cases. Ann Intern Med 1947;26:93–115.
- Palermo D, Puccini V, Sobrero L. Experimental studies of glanderd diagnosis. I. Identification of Actinobacillus mallei by immunofluoresence. Acta Med Vet Napoli 1974;20:177–181.
- Verma RD, Sharma JK, Venkateswaran KS, Batra HV. Development of an avidin-biotin dot enzymelinked immunosorbent assay and its comparision with other serological tests for diagnosis of glanders in equines. Vet Microbial 1990;25:77–85.
- Tomaso H, Scholz HC, Dahouk S, Pitt TL, Treu TM, Neubauer H. Development of 5′ nuclease real-time PCR assays for the rapid identification of the Burkholderia mallei/Burkholderia pseudomallei complex. Diagn Mol Pathol 2004;13:247–253.
- Scholz HC, Joseph M, Tomaso H, Dahouk S, Witte A, Kinne J, Hagen RM, Wernery U, Neubauer H. Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay. Diag Microbiol Infect Dis 2006;54:241–247.
- Kumar S, Balakrishna K, Singh GP, Batra HV. Rapid detection of Salmonella typhi in foods by combination of immunomagnetic separation and Polymerase chain reaction. World J Microbiol Biotechnol 2005;21:625–628.
- Bergeron MG, Boissinot M, Huletsky A, Menard C, Ovellette M, Picard FJ and Roy PH (2002) Highly conserved genes and their use to generate probes and primers for detection of microorganisms. Patent no:WO/2001/023604
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–275.
- Gupta S.K. Antisera — techniques of immunization, use of adjuvants. In: Talwar G.P., editor. A Hand Book of Practical Immunology. New Delhi: Vikas Publishing House Pvt. Ltd.; 1993. pp. 32–48.
- Batra HV, Chand P, Ganju L, Mukherjee R, Sadana JR. Dot-ELISA for the detection of antibodies in bovine brucellosis. Res Vet Sci 1989;46:143–146.
Citations
This article has been cited 3 times.- Gaspar EB, Santos LRD, Egito AAD, Santos MGD, Mantovani C, Rieger JDSG, Abrantes GAS, Suniga PAP, Favacho JM, Pinto IB, Nassar AFC, Santos FLD, Araújo FR. Assessment of the Virulence of the Burkholderia mallei Strain BAC 86/19 in BALB/c Mice. Microorganisms 2023 Oct 20;11(10).
- Knox A, Zerna G, Beddoe T. Current and Future Advances in the Detection and Surveillance of Biosecurity-Relevant Equine Bacterial Diseases Using Loop-Mediated Isothermal Amplification (LAMP). Animals (Basel) 2023 Aug 18;13(16).
- Abreu DC, Gomes AS, Tessler DK, Chiebao DP, Fava CD, Romaldini AHCN, Araujo MC, Pompei J, Marques GF, Harakava R, Pituco EM, Nassar AFC. Systematic monitoring of glanders-infected horses by complement fixation test, bacterial isolation, and PCR. Vet Anim Sci 2020 Dec;10:100147.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
