A method to preserve low parasitaemia Plasmodium-infected avian blood for host and vector infectivity assays

Abstract

Background: Avian malaria vector competence studies are needed to understand more succinctly complex avian parasite-vector-relations. The lack of vector competence trials may be attributed to the difficulty of obtaining gametocytes for the majority of Plasmodium species and lineages. To conduct avian malaria infectivity assays for those Plasmodium spp. and lineages that are refractory to in vitro cultivation, it is necessary to obtain and preserve for short periods sufficient viable merozoites to infect naïve donor birds to be used as gametocyte donors to infect mosquitoes. Currently, there is only one described method for long-term storage of Plasmodium spp.-infected wild avian blood and it is reliable at a parasitaemia of at least 1%. However, most naturally infected wild-caught birds have a parasitaemia of much less that 1%. To address this problem, a method for short-term storage of infected wild avian blood with low parasitaemia (even ≤ 0.0005%) has been explored and validated.

Methods: To obtain viable infective merozoites, blood was collected from wild birds using a syringe containing the anticoagulant and the red blood cell preservative citrate phosphate dextrose adenine solution (CPDA). Each blood sample was stored at 4 °C for up to 48 h providing sufficient time to determine the species and parasitaemia of Plasmodium spp. in the blood by morphological examination before injecting into donor canaries. Plasmodium spp.--infected blood was inoculated intravenously into canaries and once infection was established, Culex stigmatosoma, Cx. pipiens and Cx. quinquefasciatus mosquitoes were then allowed to feed on the infected canaries to validate the efficacy of this method for mosquito vector competence assays.

Results: Storage of Plasmodium spp.--infected donor blood at 4 °C yielded viable parasites for 48 h. All five experimentally-infected canaries developed clinical signs and were infectious. Pathologic examination of three canaries that later died revealed splenic lesions typical of avian malaria infection. Mosquito infectivity assays demonstrated that Cx. stigmatosoma and Cx. pipiens were competent vectors for Plasmodium cathemerium.

Conclusions: A simple method of collecting and preserving avian whole blood with malaria parasites of low parasitaemia (≤ 0.0005%) was developed that remained viable for further experimental bird and mosquito infectivity assays. This method allows researchers interested in conducting infectivity assays on target Plasmodium spp. to collect these parasites directly from nature with minimal impact on wild birds.

Keywords: Avian malaria; Bird inoculation; Blood preservation; Culex spp. vectors; Experimental infection; Pathology; Plasmodium cathemerium.

Fig. 1

Parasite identification and bird blood inoculation scheme. The pictures are of parasites found in Giemsa-stained blood smears. Parasite pictures labelled A represent the donor blood, where SL stands for Stone Lakes (the location where the bird was collected) and the ID of the bird, BHGR stands for Black-headed Grosbeak (Pheucticus melanocephalus) and HOFI stands for House Finch (Haemorhous mexicanus). Pictures labelled B represent the blood taken six days post infection (dpi) from the experimental canaries. It is important to note that sample SL7 (BHGR) was originally infected with Haemoproteus spp., but P. cathemerium was not visualized during microscopic screening. Canaries 0502 and 0509 did not become infected with Haemoproteus as expected, but P. cathemerium successfully established an infection

Fig. 2

Bayesian phylogeny of 21 mitochondrial cytochrome b Plasmodium spp./lineages and two Haemoproteus spp. The Leucocytozoon sp. lineage was used as the out-group. Numbers at each node represent the Bayesian posterior probabilities. Lineage names used in experiments are given in bold; they were obtained from birds collected at the Stone Lakes National Wildlife Refuge. All Plasmodium species/lineages used for this analysis are delineated by the parasite name with the subgenus in parenthesis and followed by the Genbank accession number

Fig. 3

Flow chart showing the process for obtaining and preservation of Plasmodium spp. from wild birds. CPDA citrate phosphate dextrose adenine solution

Fig. 4

Infection status of mosquito thoraxes for (a) Culex pipiens (N = 11) and (b) Culex stigmatosoma (N = 14) that fed on canary 0506 infected with Plasmodium cathemerium-like lineage HOFI_CA_JSC_1P. Mosquitoes were dissected and processed at 10, 16, 20, and 25 dpi

Fig. 5

Ookinetes (A, B), oocysts (C, D), and sporozoites (E, F) of Plasmodium cathemerium-like (lineage HOFI_CA_JSC_1P) in Culex pipiens (A, C, and E) and Culex stigmatosoma (B, D, and F). 1000× oil immersion of Giemsa stain (A, C) and 400× of mercurochrome midgut stain (B)

Fig. 6

Micrographs of spleen (A), lung (B), and brain (C) from canary 0505 infected with Plasmodium cathemerium (lineage SPTO_CA_ELW_6P). Exoerythrocytic meronts (arrows) in histiocytes and endothelial cells (arrows). Haematoxylin and eosin stain, original magnification 1000× (oil). Bars = 10 µm