Abstract
BACKGROUND: Haemosporidian parasites are transmitted by dipteran blood-sucking insects but certain vectors remain unidentified for the great majority of described species. Sensitive PCR-based methods are often used for the detection of haemosporidian infection in wild-caught insects. However, this approach alone cannot distinguish between different sporogonic stages and thus is insufficient to demonstrate that the parasites produce the infective stage (sporozoite), which is essential for transmission. To prove that PCR-positive insects could act as vectors, the record of sporozoites is needed. We developed a methodology for the determination of natural vectors of avian Haemoproteus species and other haemosporidians. The essence of this approach is to apply PCR-based and microscopic diagnostic tools in parallel for sporozoite detection in insects. METHODS: Culicoides biting midges transmit avian Haemoproteus parasites, but certain insect species, which are involved in transmission, remain insufficiently investigated. Biting midges were collected in the wild and identified; parous females were dissected and preparations of thorax content containing salivary glands were prepared. Remnants of the dissected midges were screened using PCR-based methods. Only thorax preparations of PCR-positive biting midges were examined microscopically. RESULTS: In total, 460 parous females belonging to 15 species were collected and dissected. DNA of haemosporidians was detected in 32 (7%) of dissected insects belonging to 7 species. Of the thorax samples PCR-positive for Haemoproteus parasites, two preparations were microscopically positive for sporozoites. Both biting midges were Culicoides kibunensis. Haemoproteus pallidus (hPFC1) was identified, indicating that transmission of this infection occurs at the study site. It was proved that seven species of biting midges take bird blood meals naturally in the wild. CONCLUSIONS: Culicoides kibunensis is a new vector species of avian haemoproteids and is a natural vector of H. pallidus. Numerous studies have identified vectors of Haemoproteus parasites experimentally; however, this is the first direct identification of a natural vector of Haemoproteus infection in the Old World. We suggest using the described methodology for vector research of Haemoproteus and other haemosporidians in the wild.