Fast and Simple Detection of Yersinia pestis Applicable to Field Investigation of Plague Foci

Stéphanie Simon,Elisabeth Carniel,Christian Demeure,Marc Plaisance,Patricia Lamourette,Hervé Volland,Christophe Créminon,Sofia Filali

doi:10.1371/journal.pone.0054947

Abstract

Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37°C. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20°C and 37°C, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples.

Highlights

Despite public health measures implemented to eradicate plague, the disease persists in several countries, and is even reemerging [1]
Purification of a plasminogen activator (PLA) Protein Produced by Recombinant Escherichia coli To produce the large amounts of purified PLA protein necessary to immunize mice, the pla gene of Y. pestis CO92 was cloned into the IPTG inducible pET22b plasmid, generating a pla gene carrying a poly-histidine tag sequence at its 39 end
Our results indicated that the sandwich PLA-ELISA using the Pla45/Pla35* monoclonal antibodies (mAbs) pair allows a specific and reliable recognition of Y. pestis strains grown at various temperatures, and

Summary

Introduction

Despite public health measures implemented to eradicate plague, the disease persists in several countries, and is even reemerging [1]. Y. pestis is commonly believed to be unable to survive outside a flea or a mammalian host [3], different works documented the concept of burrowing plague by showing that Y. pestis could survive for several years in the burrows of dead rodents [4,5]. This ‘burrow-rodent-burrow’ cycle may maintain the plague bacillus in some endemic foci. The plague bacillus can persist in environmental samples even outside animal bodies. There is the threat of a bioterrorist act, leading to the deliberate spread of Y. pestis in the environment [10]

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Results

Conclusion