Development of quantitative Real-Time Polymerase Chain Reaction coupled with High-Resolution Melting (HRM- qPCR) analysis for the Diagnosis of Trypanosoma evansi in Canis lupus familiaris

LUCERO, RAÚL HORACIO; SCHIJMAN, ALEJANDRO G.; MUÑOZ CALDERON ARTURO; FORMICHELLI, LAURA; BETTINA, BRUSES

Mar del Plata

Congreso; Sociedad Argentina de Parasitologia; 2019

Sociedad Argentina de Parasitologia

The Trypanosomiasiscaused by Trypanosoma evansi affectsa wide diversity of mammals being zoonotic potential in man, with a casereported in 2005 in India. This haemoflagellate protozoa can parasitize mostdomestic mammals, being horses, dogs and cattle the most affected species. Diagnostictools for this parasitic infection are scarce, even though this trypanosomiasiscan be very lethal if the animals are not treated. This work reports the developmentof a multi-diagnosis assay based on qPCR coupled to HRM that differentiatesinfections with diverse species of trypanosomatids and Leishmanias withzoonotic potential in peripheral blood samples from canines. The molecularmarker selected was the Internal Transcribed Spacer (ITS1) present in the ribosomalRNA locus. This marker is highly conserved and present size variability among trypanosomesspecies. The results using as a template gDNA of different trypanosomatidspecies, showed specific amplification with distinctive patterns in Melting Curvesfor T. evansi, T. cruzi, T. brucei, T. rangeli and different species ofLeishmanias. This was confirmed in agarose gels, resulting in single ormultiple bands with a size range from 250 to 480 bp. Its clinical validationwas carried out on 14 peripheral blood samples from domestic canines from northeasternArgentina. The results showed positivity for infection with T. evansi in 36% of the samples. Additionally, through this standardizedtechnique, in one sample it could be detected infection with Leishmania infantum with lowparasitaemia, confirmed by sequencing and subsequent alignment of the ITS1region with reference sequences. Therefore, molecular diagnosis of animaltrypanosomiasis by HRM-qPCR represents a viable tool for wide-scale epidemiologicalstudies, which may be used to report the true prevalence of the infection andallow implementation strategies to control these zoonotic diseases in Argentina,as well as the rest of South America, Africa and Asia.<!-- /* Font Definitions */ @font-face{font-family:"MS Mincho";panose-1:2 2 6 9 4 2 5 8 3 4;mso-font-alt:"ＭＳ 明朝";mso-font-charset:128;mso-generic-font-family:modern;mso-font-format:other;mso-font-pitch:fixed;mso-font-signature:-536870145 1791491579 134217746 0 131231 0;}@font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:3 0 0 0 1 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:swiss;mso-font-pitch:variable;mso-font-signature:-1610611985 1073750139 0 0 159 0;}@font-face{font-family:Cambria;panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:-1610611985 1073741899 0 0 159 0;}@font-face{font-family:"\@MS Mincho";panose-1:2 2 6 9 4 2 5 8 3 4;mso-font-charset:128;mso-generic-font-family:modern;mso-font-format:other;mso-font-pitch:fixed;mso-font-signature:-536870145 1791491579 134217746 0 131231 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin:0cm;margin-bottom:.0001pt;mso-pagination:widow-orphan;font-size:12.0pt;font-family:"Cambria",serif;mso-fareast-font-family:"MS Mincho";mso-bidi-font-family:"Times New Roman";mso-ansi-language:ES-TRAD;mso-fareast-language:ES;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:10.0pt;mso-ansi-font-size:10.0pt;mso-bidi-font-size:10.0pt;font-family:"Cambria",serif;mso-ascii-font-family:Cambria;mso-fareast-font-family:"MS Mincho";mso-hansi-font-family:Cambria;}size:612.0pt 792.0pt;margin:70.85pt 3.0cm 70.85pt 3.0cm;mso-header-margin:36.0pt;mso-footer-margin:36.0pt;mso-paper-source:0;}div.WordSection1{page:WordSection1;}