Experimental evidence of early-phase transmission of Yersinia pestis by Synopsyllus fonquerniei and Xenopsylla brasiliensis

BACKGROUND: Flea-borne transmission of the plague bacillus, Yersinia pestis, occurs through two regurgitation-based mechanisms. One, called early-phase transmission (EPT), takes place within 24-96 hours post-infection through a poorly understood mechanism. The second requires an intrinsic incubation period and relies on the formation of a biofilm obstructing the flea's proventriculus. Although these mechanisms have been described in different flea species, they have never been characterized in Xenopsylla brasiliensis and Synopsyllus fonquerniei, which are two flea vectors suspected and confirmed to contribute to Y. pestis transmission in Madagascar. Because EPT may facilitate rapid pathogen spread, this study aimed to experimentally assess the ability of both flea species to transmit Y. pestis via EPT. METHODOLOGY: Cohorts of starved X. brasiliensis and S. fonquerniei fleas were artificially infected with Y. pestis. From day 1 (D1) to day 4 (D4) post-infection (p.i.), pools of ten infected fleas were fed on sterile mouse blood. After feeding, bacterial loads in both the blood and the fleas were determined by Colony Forming Unit (CFU) counts on blood agar to assess bacterial transmission. Fleas were also observed under a binocular microscope to determine whether the foregut was blocked, as indicated by the presence of fresh blood in the foregut but not in the midgut. PRINCIPAL FINDINGS: The results showed that X. brasiliensis and S. fonquerniei were able to transmit Y. pestis as early as 24 hours p.i., although transmission events were infrequent. No correlation was found between flea bacterial load (ranging from 4.0 x 101 to 4.4 x 105 CFU) and transmission success. Blockage was observed in 1% of S. fonquerniei (D3 and 4 p.i) and 0.75% of X. brasiliensis (D4 p.i.), without detectable transmission by these blocked fleas during the experimental window. CONCLUSION: This study provides the first experimental evidence that X. brasiliensis and S. fonquerniei can support early-phase transmission of Y. pestis and are capable of developing proventricular blockage under laboratory conditions. These findings refine our understanding of the potential contribution of these flea species to plague transmission dynamics in Madagascar.