Dirofilariasis in stray dogs in southern Russia: improvement of laboratory studies

Methods: Using the software of the Bruker Daltonics Germany mass spectrometer, we analyzed the protein spectra of dirofilaria (58 specimens of D. repens and of 118 D. immitis).Specimen of D. repens were surgically removed from 52 patients and six dogs.D. immitis were removed at autopsy from the hearts and major pulmonary vessels of 7 dogs, 2 wolves, 1 fox and 1 jackal.Sample preparation was carried out in accordance with the original method.According to Bruker standards, to obtain reliable identification results, material from each sample was applied to 6 target wells of MALDI-TOF mass spectrometer.To obtain a single mass spectrum, 40 laser pulses (frequency 60 Hz) were used; the analyzed mass/charge range was from 20 0 0 to 20,0 0 0 kDa.From 1 sample, 1,440 spectra were collected, from 176 specimens-253,440 spectra (from 118 D. immitis -169,920, from 58 D. repens -83,520).Statistical processing of the results was carried out using Statistica Microsoft Excel.Results: To create a database, reliably repeating peaks of digital mass/charge values with repetition frequency 50 1% were selected.A comparative analysis of digital values in groups of D. repens and D. immitis specimens revealed identical mass/charge indicators were constant.They characterized the entire genus Dirofilaria with mass/charge values of 2016.5 kDa; 2084.6 kDa; 2313.0 kDa with a slight difference in the number of replicates.Based on the registration of reliably repeating peaks in mass/charge values, variable proteins were identified depending on the species of dirofilaria.The species peaks of mass/charge values for D. immitis were: 2002.9kDa (repeat frequency 57%); 2029.4 kDa (66%); 2056.4 kDa (51%); 2099.3 kDa (51%); 2106.7 kDa (49%); 2707.2 kDa (58%).The species peaks (variable proteins) of mass/charge values for D. repens were: 20 6 6.3 kDa (repeat frequency 64%); 4 817.7 kDa (4 9%).Discussion: The obtained digital values were used as reference (superspectra) for the identification of dirofilaria.Based on the presented digital values, a database was formed.Database management system type and version: Bruker Daltonics Biotyper.Database size 562 kB (0.562 MB): D. repens