Molecular and MALDI-TOF identification of ticks and tick-associated bacteria in Mali

Abstract

Ticks are considered the second vector of human and animal diseases after mosquitoes. Therefore, identification of ticks and associated pathogens is an important step in the management of these vectors. In recent years, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of arthropods including ticks. The objective of this study was to improve the conditions for the preparation of tick samples for their identification by MALDI-TOF MS from field-collected ethanol-stored Malian samples and to evaluate the capacity of this technology to distinguish infected and uninfected ticks. A total of 1,333 ticks were collected from mammals in three distinct sites from Mali. Morphological identification allowed classification of ticks into 6 species including Amblyomma variegatum, Hyalomma truncatum, Hyalomma marginatum rufipes, Rhipicephalus (Boophilus) microplus, Rhipicephalus evertsi evertsi and Rhipicephalus sanguineus sl. Among those, 471 ticks were randomly selected for molecular and proteomic analyses. Tick legs submitted to MALDI-TOF MS revealed a concordant morpho/molecular identification of 99.6%. The inclusion in our MALDI-TOF MS arthropod database of MS reference spectra from ethanol-preserved tick leg specimens was required to obtain reliable identification. When tested by molecular tools, 76.6%, 37.6%, 20.8% and 1.1% of the specimens tested were positive for Rickettsia spp., Coxiella burnetii, Anaplasmataceae and Borrelia spp., respectively. These results support the fact that MALDI-TOF is a reliable tool for the identification of ticks conserved in alcohol and enhances knowledge about the diversity of tick species and pathogens transmitted by ticks circulating in Mali.

Author summary

Ticks are among the most important vectors and reservoirs of several animal and human pathogens such as viruses, bacteria and protozoa. However, very few studies have been done on ticks in Mali. At present, little information is available about tick species infesting livestock or human tick-borne diseases transmitted in Mali. The identification of tick species and the determination of pathogens associated are essential to evaluate epidemiology and risks of human and animal diseases: the One Health approach. Current identification methods are time consuming, expensive and laborious. Previous studies have shown that MALDI-TOF mass spectrometry analyses may allow accurate tick species identification. A recent study suggested that it was possible to identify ticks preserved in alcohol by MALDI-TOF MS. The aim of the present study was to improve tick leg sample preparation conditions for their identification by MALDI-TOF MS from Malian ethanol-preserved specimens collected in the field. This study provided 99.4% concordance between morphological and MALDI-TOF identification. The detection of microorganisms was also performed by molecular biology revealing the presence of the presence of Rickettsia spp., Coxiella burnetii, Borrelia spp. and Anaplasmataceae. These results support the use of MALDI-TOF MS in entomology, tick diseases epidemiology and improve the knowledge of tick species-diversity and tick-borne pathogens circulating in Mali.