Nature Research Centre, Akademijos 2, 08412, Vilnius, Lithuania.
Parasit Vectors. 2019 Jun 18;12(1):307. doi: 10.1186/s13071-019-3559-8.
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.
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.
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.
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.
血孢子虫寄生虫通过双翅目吸血昆虫传播，但对于绝大多数已描述的物种，仍有某些传播媒介尚未确定。基于 PCR 的敏感方法通常用于检测野外捕获的昆虫中的血孢子虫感染。然而，这种方法本身并不能区分不同的孢子发生阶段，因此不足以证明寄生虫产生感染阶段（孢子），而这对于传播是必不可少的。为了证明 PCR 阳性昆虫可以作为媒介，需要记录孢子。我们开发了一种用于确定禽血孢子虫物种和其他血孢子虫的自然媒介的方法。该方法的本质是并行应用基于 PCR 的和显微镜诊断工具来检测昆虫中的孢子。
致倦库蚊传播禽血孢子虫寄生虫，但某些参与传播的昆虫物种的研究仍不够充分。在野外采集致倦库蚊并进行鉴定；解剖产卵雌蚊，并制备含有唾液腺的胸内容物的切片。使用基于 PCR 的方法筛选解剖的致倦库蚊的残留物。仅对 PCR 阳性致倦库蚊的胸部分片进行显微镜检查。
共收集和解剖了 460 只属于 15 个种的产卵雌蚊。在属于 7 个种的 32 只（7%）解剖昆虫中检测到血孢子虫的 DNA。在 PCR 阳性的 Haemoproteus 寄生虫的胸部分片中，有两个准备好的标本在显微镜下为孢子阳性。这两种致倦库蚊均为库蠓。鉴定出的 Haemoproteus pallidus（hPFC1）表明，在研究地点发生了这种感染的传播。证明了七种致倦库蚊在野外自然吸食鸟类血液。
库蠓是禽血孢子虫的新媒介种，是 H. pallidus 的自然媒介。许多研究已经通过实验确定了 Haemoproteus 寄生虫的媒介；然而，这是首次在旧世界直接鉴定出 Haemoproteus 感染的自然媒介。我们建议在野外使用描述的方法研究 Haemoproteus 和其他血孢子虫的媒介。
