A method for assessing the vectorial competence of field collectedCulicoidesspp. for bluetongue virus

C. Ninio, D. Augot, H. Ferté, E. Breard, T. Lilin, S. Zientara, P.S. Mellor, B. Dufour, J. Depaquit
2010 Parasite  
culicoides (Diptera: Ceratopogonidae) transmit Bluetongue virus (BtV). the identity of the competent species in the northern europe is still uncertain. to help resolve this issue, two sheep were experimentally infected with BtV8. Viremia was confirmed by quantitative rt-PCr: Ct = 32 on day 4 post infection (p.i.) and 26 on day 5 p.i. on day 4 p.i. the sheep were placed individually into 4 m³ netted cages under P3. 192 field collected females captured at two cattle holdings in northern France
more » ... e released into one cage, and 139 culicoides, that emerged in the laboratory from breeding site material, such as manure, mud around trough and pounds, collected from both holdings, into the second. After 24 hours surviving culicoides were collected and the blood-fed females were transferred into individual boxes. the blood engorged females were placed in an incubator (21 °C, 80 % humidity), supplied with a damp filter paper for egg laying and fed with sucrose solution. Four field collected c. obsoletus s. l., had taken a blood meal but none of the culicoides that emerged in the laboratory was blood-fed. A blood-fed female laid eggs six days after blood feeding and was assayed for virus eight days post feeding. the other blood-fed midges were killed before egg laying. All surviving culicoides were dissected for identification, thorax and abdomen being stored individually at -80 °C, then were crushed in 300 µl MeM. 100 µL was used for qrt-PCr, and 100 µL was inoculated intravenously into 10 days old embryonated chicken eggs. three c. sonorensis, a major vector of BtV in the uSA, were used as positive controls. they come from a colony kept in the institute of Animal Health in Pirbright (uK) and were intrathoracically infected with BtV8. the female which laid eggs was weakly positive for BtV antigen using the qrt-PCr assay (Ct = 36.9) but live virus was not isolated via the chicken egg inoculation assay. All other experimental culicoides were negative in both assays, but all * usc Vecpar-AFSSA, Je 2533,
doi:10.1051/parasite/2010172172 pmid:20597446 fatcat:6uj5aoebuje4joh2nyxptqjl34