Abstract
The availability of next-generation sequencing techniques provides an unprecedented opportunity for the assignment of gene function. Streptococcus equi subspecies equi is the causative agent of strangles in horses, one of the most prevalent and important diseases of equids worldwide. However, the live attenuated vaccines that are utilized to control this disease cause adverse reactions in some animals. Here, we employ transposon-directed insertion-site sequencing (TraDIS) to identify genes that are required for the fitness of S. equi in whole equine blood or in the presence of H2O2 to model selective pressures exerted by the equine immune response during infection. We report the fitness values of 1503 and 1471 genes, representing 94.5 and 92.5 % of non-essential genes in S. equi , following incubation in whole blood and in the presence of H2O2, respectively. Of these genes, 36 and 15 were identified as being important to the fitness of S. equi in whole blood or H2O2, respectively, with 14 genes being important in both conditions. Allelic replacement mutants were generated to validate the fitness results. Our data identify genes that are important for S. equi to resist aspects of the immune response in vitro, which can be exploited for the development of safer live attenuated vaccines to prevent strangles.
Highlights
Strangles, caused by Streptococcus equi subspecies equi, is one of the most frequently diagnosed infectious diseases of equids worldwide, and is responsible for considerable economic and welfare cost to the horse industry [1]
The barcoded mutant libraries were each exposed to whole equine blood and the genes contributing to fitness in this environment were identified by transposon-d irected insertion-site sequencing (TraDIS)
We describe the genome-wide identification of genes required by S. equi subsp. equi for survival in the presence of whole equine blood and H2O2, conditions that mimic an interaction with the equine immune response
Summary
Strangles, caused by Streptococcus equi subspecies equi, is one of the most frequently diagnosed infectious diseases of equids worldwide, and is responsible for considerable economic and welfare cost to the horse industry [1]. Equi within lymph nodes induces infiltration of polymorphonuclear leukocytes, leading to swelling and abscessation, which may cause dysphagia, lending to this disease's common name of strangles [3]. The earliest vaccines against strangles used heat-killed bacteria, but whilst conferring significant levels of protection, these vaccines led to severe adverse reactions [4,5,6,7,8]. Cell-free- extract vaccines have been developed, but published data suggested that the protection conferred by such vaccines was short-lived at best and that adverse reactions at the injection site remained a problem [9]. Recombinant subunit vaccines benefit from enhanced safety, and a new multi-component vaccine, Strangvac 4, has been shown to confer significant levels of protection against S. equi subsp. Strangvac 4 is not yet available for use in horses
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