Improved PCR-RFLP for the Detection of Diminazene Resistance in Trypanosoma congolense under Field Conditions Using Filter Papers for Sample Storage

Animal African trypanosomiasis (AAT) is caused by different species of the pro- tozoan parasite Trypanosoma and affects a wide range of domestic animals. Trypano- soma congolense is widespread in the whole of sub-Saharan Africa and is the species causing considerable losses in livestock pro- duction, often affecting the health status of humans through endangering the food supply of rural communities. It is estimated that 50 million cattle are at risk of the disease and that the direct and indirect annual losses related to AAT reach US$4.5 billion [1]

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

<div><p>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 <i>Amblyomma variegatum</i>, <i>Hyalomma truncatum</i>, <i>Hyalomma marginatum rufipes</i>, <i>Rhipicephalus (Boophilus) microplus</i>, <i>Rhipicephalus evertsi evertsi</i> and <i>Rhipicephalus sanguineus sl</i>. 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 <i>Rickettsia</i> spp., <i>Coxiella burnetii</i>, <i>Anaplasmataceae</i> and <i>Borrelia</i> 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.</p></div

Protocol design of ticks treatment for MALDI- TOF MS analyses.

<p>“De-alcoholization” (A), “dry” (B) and “direct” (C) protocols for sample preparation were illustrated.</p

The number of ticks used to perform the blind test and percentage of correct identification.

<p>The number of ticks used to perform the blind test and percentage of correct identification.</p

Tick species selected to create a MALDI-TOF MS reference database, identified by molecular biology.

<p>Tick species selected to create a MALDI-TOF MS reference database, identified by molecular biology.</p

List of the arthropod species present in the MALDI-TOF MS database 1.

<p>List of the arthropod species present in the MALDI-TOF MS database 1.</p

Primers and probes used for real-time quantitative and standard PCR in this study.

<p>Primers and probes used for real-time quantitative and standard PCR in this study.</p

Comparison of MALDI-TOF MS spectra from legs of same tick using “dry” and “direct” protocol.

<p>Representation of MS profiles by the superimposition of average MS profiles of “dry” and “direct” protocol (A), the gel view tool of “dry” and “direct” protocol (B) and comparison by Principal Component Analysis between “dry” and “direct” protocol (C). Assessment of spectra reproducibility for two protocols using composite correlation index (CCI) (D): The rainbow colours indicate the degree of similarity between pair mass spectra comparisons ranging from red (very similar) to blue (very dissimilar). The numbers 1 to 10 are tick numbers treated by “direct” protocol and 11 to 20 those treated by “dry” protocol.</p

Specific MALDI-TOF MS spectra of six species of ticks using for database creation.

<p>(A) Representation of leg MS spectra from <i>Rh</i>. <i>sanguineus sl</i> (1, 2), <i>Rh</i>. <i>(B) microplus</i> (3, 4) <i>Rh</i>. <i>e</i>. <i>evertsi</i> (5, 6), <i>Hy</i>. <i>truncatum</i> (7, 8), <i>Hy</i>. <i>m</i>. <i>rufipes</i> 9, 10), <i>Am</i>. <i>variegatum</i> (11, 12). (B) Dendrogram constructed using 2 to 5 representative MS spectra from 6 distinct tick species. (C) Principal Component Analysis performed with 20 specimens of six tick species; a.u., arbitrary units; m/z, mass-to-charge ratio.</p