Abstract
Various microbial pathogens have been found in ticks such as Ixodes ricinus. However, most studies assessed tick microbiomes without prior decontamination of the tick surface, which may alter the results and mislead conclusions regarding the composition of the tick-borne microbiome. The aim of this study was to test four different decontamination methods, namely (i.) 70% ethanol, (ii.) DNA Away, (iii.) 5% sodium hypochlorite and (iv.) Reactive Skin Decontamination Lotion (RSDL), which have been previously reported for tick surface and animal or human skin decontamination. To test the efficiency of decontamination, we contaminated each tick with a defined mixture of Escherichia coli, Micrococcus luteus, Pseudomonas fluorescens, dog saliva and human sweat. No contamination was used as a negative control, and for a positive control, a no decontamination strategy was carried out. After nucleic acid extraction, the recovery rate of contaminants was determined for RNA and DNA samples by qPCR and tick-borne microbiome analyses by bacterial 16S rRNA and 16S rRNA gene amplicon sequencing. Ticks treated with 5% sodium hypochlorite revealed the lowest number of contaminants followed by DNA Away, RSDL and 70% ethanol. Moreover, tick microbiomes after 5% sodium hypochlorite decontamination clustered with negative controls. Therefore, the efficiency of decontamination was optimal with 5% sodium hypochlorite and is recommended for upcoming studies to address the unbiased detection of tick-borne pathogens.
Highlights
Ticks are widespread obligate bloodsucking ectoparasites with diverse wildlife, livestock, domestic animals and humans as hosts [1]
M. luteus was significantly less efficiently removed for cDNA samples compared to E. coli (p = 0.024), but not compared to P. fluorescens (p = 0M.9ic9ro9o)rg(aFniisgmus 2r0e201,B8,)9.8T7 he decontamination efficiency of P. fluorescens was similar (p = 06 .o0f5167) for cDNAwsaasmsipmlielsarco(pm=p0a.0r5e7d) tfoorDcNDNAAsasmamppleless, wcohmepreaaresdEt.ocoDliN(Ap =sa0m.0p3le1s),awnhdeMrea.sluEt.ecuosli((pp== 00..003111))adnidffered for boMth. lsuatmeups differed for both sample types
Our decontamination efficiency tests revealed that 5% sodium hypochlorite was the most efficient agent for tick surface decontamination followed by DNA Away, Reactive Skin Decontamination Lotion (RSDL), and by 70% ethanol (Figures 2 and 3)
Summary
Ticks are widespread obligate bloodsucking ectoparasites with diverse wildlife, livestock, domestic animals and humans as hosts [1]. Ixodes ricinus is the most common tick species in Germany and the vector for a vast range of TBP, including Borrelia, Rickettsia or Coxiella that can cause Lyme disease, rickettsial disease or Q fever [3,4,5]. The diversity and quantity of TBPs have been assessed by a variety of molecular methodologies, including next-generation sequencing (NGS) such as amplicon sequencing [1]. The 16S ribosomal RNA (rRNA) and its gene have been frequently used as a target for amplicon sequencing to identify bacterial taxa [1,6,7]. Nine hypervariable regions (V1–V9) in the rRNA gene have been assessed of which regions V1–V4 are most commonly used to explore the bacterial sequence diversity in ticks [8,9,10]
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