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Bartonella bacilliformis  is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrión’s disease. After constructing a low-passage GFP-labeled strain of  B .  bacilliformis , we artificially infected  Lutzomyia verrucarum  and  L .  longipalpis  populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48h,  B .  bacilliformis  transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72h when  B .  bacilliformis  was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in  L .  verrucarum , but colonization of  L .  longipalpis  was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of  B .  bacilliformis  in  L .  longipalpis  was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that  L .  longipalpis  could potentially serve as a vector. The capacity of  L .  longipalpis  to transmit viable  B .  bacilliformis  from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by  B .  bacilliformis  and also demonstrates the utility of  L .  longipalpis  as a user-friendly, live-vector model system for studying this severely neglected tropical disease.

plos neglected tropical diseases

Colonization of Lutzomyia verrucarum and Lutzomyia longipalpis Sand Flies (Diptera: Psychodidae) by Bartonella bacilliformis, the Etiologic Agent of Carrión’s Disease