Assessment of MALDI-TOF MS biotyping for Borrelia burgdorferi sl detection in Ixodes ricinus

Abstract

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been demonstrated to be useful for tick identification at the species level. More recently, this tool has been successfully applied for the detection of bacterial pathogens directly in tick vectors. The present work has assessed the detection of Borrelia burgdorferi sensu lato in Ixodes ricinus tick vector by MALDI-TOF MS. To this aim, experimental infection model of I. ricinus ticks by B. afzelii was carried out and specimens collected in the field were also included in the study. Borrelia infectious status of I. ricinus ticks was molecularly controlled using half-idiosome to classify specimens. Among the 39 ticks engorged on infected mice, 14 were confirmed to be infected by B. afzelii. For field collection, 14.8% (n = 12/81) I. ricinus ticks were validated molecularly as infected by B. burgdorferi sl. To determine the body part allowing the detection of MS protein profile changes between non-infected and B. afzelii infected specimens, ticks were dissected in three compartments (i.e. 4 legs, capitulum and half-idiosome) prior to MS analysis. Highly reproducible MS spectra were obtained for I. ricinus ticks according to the compartment tested and their infectious status. However, no MS profile change was found when paired body part comparison between non-infected and B. afzelii infected specimens was made. Statistical analyses did not succeed to discover, per body part, specific MS peaks distinguishing Borrelia-infected from non-infected ticks whatever their origins, laboratory reared or field collected. Despite the unsuccessful of MALDI-TOF MS to classify tick specimens according to their B. afzelii infectious status, this proteomic tool remains a promising method for rapid, economic and accurate identification of tick species. Moreover, the singularity of MS spectra between legs and half-idiosome of I. ricinus could be used to reinforce this proteomic identification by submission of both these compartments to MS.

Fig 1. Comparison of MALDI-TOF MS spectra from legs, capitula and half-idiosomes of adult I. ricinus pathogen-free (i) or infected by Borrelia afzelii (ii).

Representative MS spectra of legs (A, B), capitula (C, D) and half-idiosomes (E, F) from laboratory reared I. ricinus homogenized automatically using FastPrep-24 device with glass powder. a.u., arbitrary units; m/z, mass-to-charge ratio.

Fig 2. MSP dendrogram of MALDI-TOF MS spectra from legs, capitula and half-idiosomes of adult I. ricinus pathogen-free or infected by Borrelia afzelii.

Five specimens per body part and B. afzelii infectious status were used to construct the dendrogram. The dendrogram was created using Biotyper v3.0 software and distance units correspond to the relative similarity of MS spectra. The specimens infected by B. afzelii were indicated by asterisks (*).

Fig 3. Assessment of I. ricinus MS spectra reproducibility according to tick body parts and Borrelia infectious status using composite correlation index (CCI).

MS spectra from five specimens per body part and B. afzelii infectious status were analysed using the CCI tool. Body part and infectious status are indicated on the left side of the heat map. Levels of MS spectra reproducibility are indicated in red and blue revealing relatedness and incongruence between spectra, respectively. CCI matrix was calculated using MALDI-Biotyper v3.0. software with default settings (mass range 3.0–12.0 kDa; resolution 4; 8 intervals; auto-correction off). The values correspond to the mean coefficient correlation and respective standard deviations obtained for paired condition comparisons. CCI were expressed as mean ± standard deviation. BI, Borrelia-infected; PF, pathogen-free.

Fig 4. Principal component analysis (PCA) from MS spectra of idiosomes and legs of I. ricinus infected or not by Borrelia sp.

PCA dimensional image from MS spectra of I. ricinus idiosomes (A) and legs (B) Borrelia-free (red dots, n = 10), infected by B. afzelii (green dots, n = 6), B. burgdorferi (blue dots, n = 3), B. garinii (yellow dots, n = 2), co-infected by B. garinii and B. burgdorferi (purple dots, n = 1). (C) PCA dimensional image from the same MS spectra of I. ricinus idiosomes (red dots, n = 22) and legs (green dots, n = 22). The contributions of PC1, PC2 and PC3 were 38.4%, 15.5% and 7.2%, respectively. Among the Borrelia-free specimens, five were laboratory reared and the other five came from field collection.

Fig 5. Sensitivity of MALDI-TOF MS for Borrelia detection in mix protein extract.

Representative MS spectra from half-‡idiosome I. ricinus protein extract, without (A) or with the addition of 10⁴ (B), 10⁵ (C) or 10⁶ (D) Borrelia afzelii bacteria. MS profiles from 10⁶ Borrelia afzelii alone (E) and half-idiosome protein extract from I. ricinus infected by Borrelia afzelii (F) were shown. MS peaks commonly found between B. afzelii and half-idiosome I. ricinus protein extract with the addition of 10⁶ were indicated by dashed lines.

Fig 6. Comparison of LSVs from MS spectra of I. ricinus ticks according to body part, origin and Borrelia-infectious status.

Dashed line represent the threshold value for relevant identification (LSVs>1.8). LSV, log score value; NE, non-exposed; BF; Borrelia-free; BI; Borrelia-infeted.