Blechomonas campbelli from Ctenocephalides felis: in vitro development, thermotolerance, and phylogenetic insights.

BACKGROUND: Blechomonas spp. are flea-specific monoxenous trypanosomatids, whose biology, host range, and geographic distribution remain poorly understood, even for species inhabiting ubiquitous cat and dog fleas. OBJECTIVES: To isolate and characterise blechomonads from fleas of domestic dogs in Brazil, focusing on culture growth, morphology, and phylogenetic relationships. METHODS: Fleas collected from dogs in Sabará, Brazil, were morphologically identified and screened by cultivation. The isolate FL-1 was analysed for temperature-dependent growth, morphology, and phylogeny using 18S rRNA and gGAPDH sequences, including those mined from public metagenomic datasets. FINDINGS: Of 340 fleas (four per pool), only one culture tested positive. Growth was optimal at 25ºC, reduced at 30ºC, and absent at 35ºC. Four cell types were revealed, including a predominant "uromonad" stage specialised for attachment and aggregation. Phylogenetic analyses placed the isolate within Blechomonas campbelli and indicated that this species and Blechomonas lauriereadi occur in Ctenocephalides spp. from multiple continents. MAIN CONCLUSIONS: Blechomonas campbelli exhibits developmental adaptations to the flea gut and limited tolerance to elevated temperatures, constraining its potential to persist in warm-blooded hosts. These findings highlight the value of combining culture-based and in silico approaches to investigate flea-associated trypanosomatids.