PMID- 29608918 OWN - NLM STAT- MEDLINE DCOM- 20191014 LR - 20191014 IS - 1096-0805 (Electronic) IS - 0022-2011 (Linking) VI - 154 DP - 2018 May TI - A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus. PG - 37-41 LID - S0022-2011(18)30019-3 [pii] LID - 10.1016/j.jip.2018.03.015 [doi] AB - Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics. A battery of primers, designed to simultaneously amplify fragments of the RNA polymerase II large subunit (RPB1) of L. passim, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of C. mellificae and the DNA topoisomerase II (TOPII) of C. bombi, was tested for target specificity under single and mixed template conditions using DNA extracted from cell cultures (L. passim ATCC PRA403; C. mellificae ATCC 30254) and from a bumblebee specimen infected with C. bombi only (14_349). Once validated, the performance of the method was assessed using DNA extractions from seven Apis mellifera (Linnaeus, 1758) and five Bombus terrestris (Linnaeus, 1758) field samples infected with trypanosomatids whose identity had been previously determined by PCR-cloning and sequencing (P-C-S). The new method confirmed the results obtained by P-C-S: two of the honeybee samples were parasitized by L. passim, C. mellificae and C. bombi at the same time, whereas the other five were infected with L. passim only. The method confirmed the simultaneous presence of L. passim and C. mellificae in two B. terrestris, where these parasites had not previously been reported. CI - Copyright (c) 2018 Elsevier Inc. All rights reserved. FAU - Bartolome, Carolina AU - Bartolome C AD - Grupo de Medicina Xenomica, CIMUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain; Grupo de Xenomica Comparada de Parasitos Humanos, IDIS, Santiago de Compostela, Galicia, Spain. Electronic address: carolina.bartolome@usc.es. FAU - Buendia, Maria AU - Buendia M AD - Laboratorio de Patologia Apicola, Centro de Investigacion Apicola y Agroambiental, Instituto Regional de Investigacion y Desarrollo Agroalimentario y Forestal (CIAPA-IRIAF), JCCM, 19180 Marchamalo, Guadalajara, Spain. Electronic address: mbuendia92@gmail.com. FAU - Benito, Maria AU - Benito M AD - Laboratorio de Patologia Apicola, Centro de Investigacion Apicola y Agroambiental, Instituto Regional de Investigacion y Desarrollo Agroalimentario y Forestal (CIAPA-IRIAF), JCCM, 19180 Marchamalo, Guadalajara, Spain. Electronic address: marmes16@hotmail.com. FAU - De la Rua, Pilar AU - De la Rua P AD - Departamento de Zoologia y Antropologia Fisica, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia, Spain. Electronic address: pdelarua@um.es. FAU - Ornosa, Concepcion AU - Ornosa C AD - Departamento de Biodiversidad, Ecologia y Evolucion, Facultad de Ciencias Biologicas, Universidad Complutense, 28040 Madrid, Spain. Electronic address: paddy@bio.ucm.es. FAU - Martin-Hernandez, Raquel AU - Martin-Hernandez R AD - Laboratorio de Patologia Apicola, Centro de Investigacion Apicola y Agroambiental, Instituto Regional de Investigacion y Desarrollo Agroalimentario y Forestal (CIAPA-IRIAF), JCCM, 19180 Marchamalo, Guadalajara, Spain; Instituto de Recursos Humanos para la Ciencia y la Tecnologia, Fundacion Parque Cientifico Tecnologico de Albacete, Spain. Electronic address: rmhernandez@jccm.es. FAU - Higes, Mariano AU - Higes M AD - Laboratorio de Patologia Apicola, Centro de Investigacion Apicola y Agroambiental, Instituto Regional de Investigacion y Desarrollo Agroalimentario y Forestal (CIAPA-IRIAF), JCCM, 19180 Marchamalo, Guadalajara, Spain. Electronic address: mhiges@jccm.es. FAU - Maside, Xulio AU - Maside X AD - Grupo de Medicina Xenomica, CIMUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain; Grupo de Xenomica Comparada de Parasitos Humanos, IDIS, Santiago de Compostela, Galicia, Spain; Departamento de CC. Forenses, Anatomia Patoloxica, Xinecoloxia e Obstetricia, e Pediatria, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain. Electronic address: xulio.maside@usc.es. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20180330 PL - United States TA - J Invertebr Pathol JT - Journal of invertebrate pathology JID - 0014067 SB - IM MH - Animals MH - Bees/*parasitology MH - Euglenozoa Infections/diagnosis MH - Multiplex Polymerase Chain Reaction/*methods MH - Trypanosomatina/*genetics/isolation & purification OTO - NOTNLM OT - Apis mellifera OT - Bombus terrestris OT - Crithidia bombi OT - Crithidia mellificae OT - Lotmaria passim OT - Multiplex PCR EDAT- 2018/04/03 06:00 MHDA- 2019/10/15 06:00 CRDT- 2018/04/03 06:00 PHST- 2018/01/23 00:00 [received] PHST- 2018/03/23 00:00 [revised] PHST- 2018/03/29 00:00 [accepted] PHST- 2018/04/03 06:00 [pubmed] PHST- 2019/10/15 06:00 [medline] PHST- 2018/04/03 06:00 [entrez] AID - S0022-2011(18)30019-3 [pii] AID - 10.1016/j.jip.2018.03.015 [doi] PST - ppublish SO - J Invertebr Pathol. 2018 May;154:37-41. doi: 10.1016/j.jip.2018.03.015. Epub 2018 Mar 30.