Mortality and pathology in birds due to Plasmodium (Giovannolaia) homocircumflexum infection, with emphasis on the exoerythrocytic development of avian malaria parasites

Birds were caught using mist nets and big funnel traps [17], and experiments were carried out at the Biological Station of the Zoological Institute of the Russian Academy of Sciences on the Curonian Spit in the Baltic Sea (55°05′ N, 20°44′ E) between 23 May and 16 July, 2014. The birds were exposed to experimental P. homocircumflexum (pCOLL4) infection and kept until they died. Blood samples and organs were collected and examined for blood stages and phanerozoites.

The Eurasian siskin and the common crossbill were kept indoors in a vector-free room. The common starling was kept outside in a cage covered with a cover made of fine-mesh bolting silk, which prevented penetration of blood-sucking insects in the cage. All birds were kept at a natural light–dark photoperiod. Before experiments, they were examined for possible presence of natural infections by microscopic examination of blood films and later by polymerase chain reaction (PCR)-based methods (see description below). All birds were non-infected with haemosporidian parasites before the experiment.

The isolate of P. homocircumflexum (lineage pCOLL4, GenBank accession no. KC884250), which was originally obtained from a naturally infected red-backed shrike was used. This isolate was cryopreserved and is available at the P. B. Šivickis Laboratory of Parasitology, Nature Research Centre, Vilnius, Lithuania [14]. One sample of this isolate (parasitaemia intensity of 4 %) was thawed and used to infect one Eurasian siskin, as described by Palinauskas et al. [18] with slight modifications. Briefly, the frozen tube containing infected blood was thawed and mixed with 12 % NaCl (one-third of the thawed sample amount). After equilibration for 5 min at room temperature, one volume of 1.6 % NaCl was added, followed by centrifugation at 200 g for 5 min. After centrifugation, the supernatant was removed and 1.6 % NaCl (one-third of the original sample) was added and centrifuged again. After removing the supernatant, the same procedure was repeated three times with 0.9 % NaCl solution. The final mixture was diluted with 0.9 % NaCl and sub-inoculated into one Eurasian siskin, as described by Palinauskas et al. [18].

One common crossbill and one common starling were infected using blood (parasitaemia intensity of 50 %) collected from the exposed Eurasian siskin. Briefly, the brachial vein of the donor Eurasian siskin was punctured using a needle. About 100 µl of blood was mixed with 25 µl sodium citrate and 125 µl 0.9 % saline solution. The mixture was sub-inoculated into the pectoral muscle of the experimental birds, as described by Palinauskas et al. [18]. Six wild caught non-infected Eurasian siskins were used as controls. The birds were maintained in the same room as the experimental birds.

All birds were observed until the experimental birds died [30–38 days post exposure (dpe)]. Blood was taken for microscopic examination and PCR-based testing during the course of the experiment (see Fig. 1). Approximately 50 µl of blood was collected in heparinized microcapillaries by puncturing the brachial vein. A drop of blood was used to make three blood films, which were air-dried, fixed with absolute methanol, stained with Giemsa and examined microscopically as described by Valkiūnas et al. [19]. Approximately 35 µl of the blood left in the capillary was fixed in non-lysis SET buffer (0.05 M Tris, 0.15 M NaCl, 0.5 M EDTA, pH 8.0) for molecular analysis; these samples were kept at room temperature in the field and at −20 °C in the laboratory.

Brain, heart, kidney, liver, lungs, spleen, and a piece of the pectoral muscle of the experimental birds were dissected after the birds’ death. The samples were fixed in 10 % neutral formalin and embedded in paraffin blocks. Histological sections of 4 µm were prepared, stained with haematoxylin-eosin (H&E) [4] and examined microscopically.

An Olympus BX51 light microscope equipped with Olympus DP12 digital camera and imaging software Olympus DP-SOFT was used to examine slides and prepare illustrations. To trace the parasitaemia, each blood slide was examined for 15–20 min at medium magnification (×400), and then at least 100 fields were studied at high magnification (×1000). Intensity of parasitaemia was estimated as a percentage by actual counting of the number of parasites per 1000 erythrocytes or per 10,000 erythrocytes if infections were light [20]. Microscopic examination was also used to determine possible presence of co-infections with other haemosporidian parasites. Histological preparations were examined using similar protocol, but they were also examined at low magnification (×200) for 10–15 min, followed by examination of the preparations for 10–15 min at medium magnification (×400) and then for another 20–30 min at high magnification (×1000). Statistical analyses were carried out using the ‘Statistica 7’ package.

Total DNA was extracted from blood samples using the standard ammonium-acetate protocol [21] with a minor modification: instead of 250 µl of fixed blood, 125 µl was used. A nested-PCR protocol [22] was applied for the molecular analysis. For the first PCR, the primer pair HaemFNI [5′-CATATATTAAGAGAAITATGGAG-3′] and HaemNR3 [5′-ATAGAAAGATAAGAAATACCATTC-3′] was used. This is a general primer pair that amplifies the mitochondrial cytochrome b (cyt b) gene of Plasmodium, Haemoproteus and Leucocytozoon species. The reaction mix for the first PCR consisted of 12.5 µl of Dreamtaq Master Mix (Fermentas, Lithuania), 8.5 µl of nuclease-free water, 1 µl of each primer and 2 µl of template DNA. The thermal conditions for the first PCR were according to Helgren et al. [22]. For the second PCR, the primer pair HAEMF [5′-ATGGTGCTTTCGATATATGCATG-3′] and HAEMR2 [5′-GCATTATCTGGATGTGATAATGGT-3′] was used. This primer pair amplifies a 479 bp fragment of cyt b gene [23]. The reaction mix for the second PCR was identical to the mix for the first PCR, only instead of extracted DNA, 2 µl of the first PCR products were used as templates. The conditions for the second PCR were the same as in the first PCR, but 35 cycles instead of 20 were run. Success of the amplification was evaluated by running 2 µl of the second PCR product on 2 % agarose gel. One negative control (nuclease-free water) and one positive control (a Plasmodium sample, which was positive by microscopic examination of blood films) were used to determine possible false amplifications. No case of false amplification was found.

The cyt b gene fragments were sequenced from the 5′ end using the HAEMF primer [23]. Dye terminator cycle sequencing (Big Dye) was used. Samples were loaded onto an ABI PRISM TM 3100 capillary sequencing robot (Applied Biosystems, USA). Sequences of parasites were edited and examined using the BioEdit program [24]. The ‘Basic Local Alignment Search Tool’ and the megablast algorithm were used to identify the cyt b lineages of detected DNA sequences.

Chromogenic in situ hybridization ISH was carried out according to Dinhopl et al. [8]. In brief, 3 μm paraffin wax-embedded tissue sections were subjected to proteolytic treatment with proteinase K (Roche, Basel, Switzerland) 6 μg/ml in Tris-buffered saline at 37 °C for 50 min. For hybridization, the slides were incubated overnight at 40 °C with hybridization mixture and a final probe concentration of 100 ng/ml. The used oligonucleotide probe (sequence: 5′-TTTAATAACTCGTTATATATATCAGTGTAGCAC-3′) was labelled with digoxigenin at the 3′ end (Eurofins MWG Operon, Ebersberg, Germany). The probe is aimed at 18S rRNA strand and is specific to detect avian Plasmodium spp. [9]. The digoxigenin-labelled hybrids were detected by incubating the slides with antidigoxigenin-AP Fab fragments (Roche) (1:200) for 1 h at RT. Visualization of the reaction was carried out using the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT) (Roche). Probe specificity has been extensively tested previously [8]. Tissues from a deceased wild Blackbird Turdus merula free of avian malaria parasites as well as application of an irrelevant oligonucleotide probe (designed for Leishmania spp.) on the experimental samples [see 8] were used as controls in this study.

Experimental procedures of this study were approved by the International Research Co-operation Agreement between the Biological Station Rybachy of the Zoological Institute of the Russian Academy of Sciences and Institute of Ecology of Nature Research Centre (25-05-2010). All efforts were made to minimize handling time and potential suffering of animals. None of the experimental birds suffered apparent injury during experiments.