Molecular analysis
By PCR, 194 (63.4%) of 306 birds were positive for Plasmodium, Haemoproteus, Leucocytozoon or a combination of these genera (Table 2). Plasmodium showed the highest prevalence with 177 infected birds, Haemoproteus and Leucocytozoon were less prevalent with only 15 and 17 positive birds, respectively.
Of 194 positive birds, 40 (20.6%) showed mixed infections, including 35 double and 5 triple infections. The mixed infection rate was highest among Leucocytozoon-positive birds, with 15 (88.2%) of 17 showing a co-infection with either Plasmodium (9 cases), Haemoproteus (5 cases) or multiple Leucocytozoon lineages (1 case). Thirty-four (19.2%) of 177 Plasmodium-infected birds harboured multiple Plasmodium lineages (24 cases) or were co-infected with Leucocytozoon (9 cases) or Haemoproteus (1 case). Six (40.0%) of 15 Haemoproteus-infected birds showed mixed infections with Leucocytozoon (5 cases) or Plasmodium (1 case).
By sequencing, 26 unique cytb lineages (Plasmodium: 12, Leucocytozoon: 10, and Haemoproteus: 4) were identified, 13 of which were 100% identical to previously reported lineages in MalAvi or Genbank (Table 2).
Approximate position of Table 2.
The by far most prevalent lineages were LINN1, a lineage of P. matutinum, recorded in 99 (51.0%) of 194 infected birds, and SYAT05, a lineage of P. vaughani, detected in 89 birds (45.9%). Eight birds featured lineages showing 99% matches with SYAT05 (TUMER13, TUMER14, TUMER15, TUMER16) and LINN1 (TUPHI08, TUPHI09, TUPH10). Among Leucocytozoon, TUMER01 was most prevalent, detected in seven Eurasian blackbirds. From the genus Haemoproteus, the three lineages TUPHI01, TURDUS2 and TUCHR01, all linked to the morphospecies Haemoproteus minutus, were identified. One blackbird harboured a sequence (TUMER17) matching 99% with TURDUS2.
Chromogenic in situ hybridization
All 194 birds (179 Eurasian blackbirds and 15 song thrushes) positive for haemosporidian parasites by PCR and sequencing were analyzed by CISH, including 154 single and 40 mixed infections. CISH revealed parasites in tissue sections of 178 cases (91.8%) whereas 16 (8.2%) remained negative. The detected parasite stages were classified as blood or tissue stages based on the shape, size and location of signals. Blood stages typically showed oval to roundish signals not larger than blood cells and were located in capillaries and larger vessels. Signals exceeding the size of blood cells and showing variable shapes were considered exo-erythrocytic meronts (Additional file 1). In unclear cases, e.g., when the signals were all similar and in the size of blood cells, the corresponding HE-stained sections were checked for exo-erythrocytic meronts.
Detection of Plasmodium
CISH detected Plasmodium parasites in 165 (93.2%) of 177 PCR-positive birds, but not all birds exhibited exo-erythrocytic stages of the parasites in the tissues. 113 (68.5%) cases showed both erythrocytic and exo-erythrocytic stages of parasites, whereas 46 (27.9%) cases only showed exo-erythrocytic but no blood stages. Conversely, six cases (3.6%) presented blood stages but no exo-erythrocytic meronts.
Parasite load, in terms of exo-erythrocytic merogony, varied extensively among individuals, ranging from zero to high numbers of detectable meronts. Representative photographs of low-, moderate- and high-grade parasite loads are shown for the brain and the spleen (Additional file 2). Of 159 cases that presented exo-erythrocytic stages, 74 (46.6%) exhibited low numbers of meronts in at least one organ, 34 (21.4%) had moderate numbers of meronts in the tissues and 51 (32.1%) showed high loads of exo-erythrocytic meronts (Table 3). Comparing Eurasian blackbirds and song thrushes, the overall percentage of exo-erythrocytic-positive cases was similar with 89.8% (149/166) and 90.9% (10/11), respectively. However, in contrast to blackbirds, which showed various degrees of parasite merogony in their tissues, most song thrushes only exhibited low numbers of exo-erythrocytic meronts.
Approximate position of Table 3.
The distribution of exo-erythrocytic stages in different tissues ranged among individuals from single affected organs to a generalized occurrence. Regardless of the parasite lineage, certain organs exhibited exo-erythrocytic stages more frequently than others (Figure 1). The spleen was most commonly affected with a rate of 86.7% (144/166), followed by lung (65.0%, 93/143) and brain (61.0%, 100/164) (Figure 1). In about 50% of the cases, tissue meronts were detected in the heart (87/168), liver (80/152), gizzard (68/129) and bursa of Fabricius (13/25), whereas kidney (40.4%, 55/136), intestine (34.7%, 33/95) and skeletal muscle (18.2%, 2/11) were less commonly affected.
Approximate position of Figure 1.
Evaluation of histological lesions associated with the presence of meronts was difficult because tissues of the submitted dead birds showed varying degrees of autolysis and freezing artefacts. Histological alterations such as necrosis and inflammatory cell infiltration were most often observed in spleen, liver, lung, bursa of Fabricius and intestine and were in some cases associated with excessive exo-erythrocytic merogony of the parasites (Figure 2a). Apart from ‘regular’ tissue stages, some peculiar observations were made during histological examination of tissues. In a few P. matutinum and P. vaughani-infected birds, clusters of several exo-erythrocytic meronts were noticed in the brain. Some of the meronts had an untypical appearance as they seemed to divide into multiple smaller parts of similar size, which contained numerous merozoites (Figure 2b). Another frequent observation was the accumulation of erythrocytic parasite stages in the microvasculature of fat tissue and the serosal layers of visceral organs, most frequently in the heart and the gastrointestinal tract (Figure 2c and 2d).
Approximate position of Figure 2.
To evaluate whether infection intensity depended on parasite lineage, birds with single infections of the two most prevalent lineages P. matutinum (n = 65) and P. vaughani (n = 62) were compared. Because of the low number of song thrushes in the data set and to rule out host species as a confounding factor, only blackbirds were included in this analysis. The P. matutinum-infected group showed higher percentages of moderate and high-grade infections in all organs compared to the P. vaughani-infected group with a significant difference found for the heart, lung, kidney, brain and gizzard, but not the liver, spleen and intestine (Fisher’s exact, heart (n = 121): p = 0.016; lung (n = 102): p = 0.026; kidney (n = 97): p = 0.043; brain (n = 117): p < 0.001; gizzard (n = 92): p < 0.001; liver (n = 109): p = 0.896; spleen (n = 120): p = 0.30; intestine (n = 64): p = 0.066) (Figure 3). In P. vaughani-infected birds high-grade parasite load was noticed only in the heart, lung, liver and spleen, whereas in P. matutinum-infected birds high-grade parasite load was observed in almost all organs, with brain (25%, 16/64), lung (22%, 12/59) and heart (18%, 11/67) being most frequently affected.
Approximate position of Figure 3.
According to the PCR and sequencing results, nineteen birds (all Eurasian blackbirds) harboured co-infections with both P. matutinum LINN1 and P. vaughani SYAT05. Using the general Plasmodium probe, CISH detected exo-erythrocytic stages in 17 of these birds, showing moderate to high parasite loads in twelve cases and low parasite loads in five birds. Application of probes specific for either P. matutinum or P. vaughani to discern their relative roles in co-infections yielded ambiguous CISH-results. Meronts of P. matutinum were detected in all 17 cases, whereas CISH using the P. vaughani-specific probe resulted in only eight positive cases, seven of which exhibited tissue stages. In three of the seven cases that yielded positive signals with both probes, meronts of P. vaughani and P. matutinum were equally abundant with few differences regarding tissue distribution, whereas in four cases meronts of P. vaughani were more abundant in most organs. Interestingly, in almost half of the cases, the overall abundance of meronts detected by both species-specific probes was lower compared to the general Plasmodium probe, indicating lower sensitivity of the species-specific probes.
Detection of Haemoproteus
In nine of 15 Haemoproteus-infected birds, parasites were detected by CISH. In all cases, signals were few in number and restricted to larger vessels and capillaries of different organs, corresponding to blood stages of the parasites, indicating low parasitaemia. In none of the cases, exo-erythrocytic meronts were observed.
Detection of Leucocytozoon
CISH revealed parasite stages in all Leucocytozoon-infections with exception of birds infected with Leucocytozoon sp. ASOT06 (three birds). In most cases, however, only blood stages were detected. Two of seven blackbirds that were infected with Leucocytozoon sp. TUMER01 showed large tissue stages (megalomeronts) in various organs (Figure 4). In one bird, megalomeronts were detected in the heart, bursa of Fabricius and kidney. They appeared as densely packed homogenous masses of merozoites which were enclosed by thin capsules and surrounded by cells of unclear origin (Figure 4a). In the other bird, several megalomeronts were detected in the kidney, which appeared either intact or disintegrated. In the HE-stained sections, inflammatory reactions and necroses were associated with megalomeronts.
Approximate position of Figure 4.