Avian haemosporidian parasites can cause severe disease in their hosts due to excessive exo-erythrocytic merogony and anaemia caused by blood stages. Notably, the development of megalomeronts by species of Haemoproteus and Leucocytozoon has been associated with mortalities in birds. Diagnosis of lethal infections is currently accomplished by the detection of parasites’ tissue stages in histological sections combined with PCR and sequencing. However, sequences frequently are not reliably obtained and the generic discrimination of exo-erythrocytic tissue stages based on morphological characters is challenging. Therefore, the present study aimed at developing specific molecular probes for the identification of Haemoproteus spp. and Leucocytozoon spp. in histological sections using chromogenic in situ hybridization. Parasite subgenus-specific oligonucleotide probes were designed to target the 18S ribosomal RNA of Haemoproteus species (subgenus Parahaemoproteus) and Leucocytozoon spp. (subgenus Leucocytozoon) and were in situ hybridized to sections from formalin-fixed, paraffin-embedded tissue samples determined positive for these parasites by PCR and histopathology. To confirm the presence of parasites at sites of probe hybridization, consecutive sections were stained with haematoxylin–eosin and examined. Parahaemoproteus- and Leucocytozoon-specific probes labelled erythrocytic and exo-erythrocytic stages of Haemoproteus spp. and Leucocytozoon spp., respectively. Binding of probes to parasites was consistent with detection of the same exo-erythrocytic meronts in consecutive haematoxylin–eosin-stained sections. Cross-reactivity of the probes was ruled out by negative chromogenic in situ hybridization when applied to samples positive for a parasite of a genus different from the probes’ target. Chromogenic in situ hybridization using 18S ribosomal RNA-specific oligonucleotide probes reliably identifies and discriminates Haemoproteus and Leucocytozoon parasites in tissue sections and enables unequivocal diagnosis of haemosporidioses.

We present a high resolution polarization sensitive optical coherence tomography (PS-OCT) system for ocular imaging in rodents. The system operates at 840 nm and uses a broadband superluminescent diode providing an axial resolution of 5.1 µm in air. PS-OCT data was acquired at 83 kHz A-scan rate by two identical custom-made spectrometers for orthogonal polarization states. Pigmented (Brown Norway, Long Evans) and non-pigmented (Sprague Dawley) rats as well as pigmented mice (C57BL/6) were imaged. Melanin pigment related depolarization was analyzed in the retinal pigment epithelium (RPE) and choroid of these animals using the degree of polarization uniformity (DOPU). For all rat strains, significant differences between RPE and choroidal depolarization were observed. In contrast, DOPU characteristics of RPE and choroid were similar for C57BL/6 mice. Moreover, the depolarization within the same tissue type varied significantly between different rodent strains. Retinal nerve fiber layer thickness, phase retardation, and birefringence were mapped and quantitatively measured in Long Evans rats in vivo for the first time. In a circumpapillary annulus, retinal nerve fiber layer birefringence amounted to 0.16°/µm ± 0.02°/µm and 0.17°/µm ± 0.01°/µm for the left and right eyes, respectively.

We present a multi-functional optical coherence tomography (OCT) imaging approach to study retinal changes in the very-low-density-lipoprotein-receptor (VLDLR) knockout mouse model with a threefold contrast. In the retinas of VLDLR knockout mice spontaneous retinal-chorodoidal neovascularizations form, having an appearance similar to choroidal and retinal neovascularizations (CNV and RNV) in neovascular age-related macular degeneration (AMD) or retinal angiomatous proliferation (RAP). For this longitudinal study, the mice were imaged every 4 to 6 weeks starting with an age of 4 weeks and following up to the age of 11 months. Significant retinal changes were identified by the multi-functional imaging approach offering a threefold contrast: reflectivity, polarization sensitivity (PS) and motion contrast based OCT angiography (OCTA). By use of this intrinsic contrast, the long-term development of neovascularizations was studied and associated processes, such as the migration of melanin pigments or retinal-choroidal anastomosis, were assessed in vivo. Furthermore, the in vivo imaging results were validated with histological sections at the endpoint of the experiment. Multi-functional OCT proves as a powerful tool for longitudinal retinal studies in preclinical research of ophthalmic diseases. Intrinsic contrast offered by the functional extensions of OCT might help to describe regulative processes in genetic animal models and potentially deepen the understanding of the pathogenesis of retinal diseases such as wet AMD.

Plasmodium species feature only four to eight nuclear ribosomal units on different chromosomes, which are assumed to evolve independently according to a birth-and-death model, in which new variants originate by duplication and others are deleted throughout time. Moreover, distinct ribosomal units were shown to be expressed during different developmental stages in the vertebrate and mosquito hosts. Here, the 18S rDNA sequences of 32 species of avian haemosporidian parasites are reported and compared to those of simian and rodent Plasmodium species. Almost the entire 18S rDNAs of avian haemosporidians belonging to the genera Plasmodium (7), Haemoproteus (9), and Leucocytozoon (16) were obtained by PCR, molecular cloning, and sequencing ten clones each. Phylogenetic trees were calculated and sequence patterns were analysed and compared to those of simian and rodent malaria species. A section of the mitochondrial CytB was also sequenced. Sequence patterns in most avian Plasmodium species were similar to those in the mammalian parasites with most species featuring two distinct 18S rDNA sequence clusters. Distinct 18S variants were also found in Haemoproteus tartakovskyi and the three Leucocytozoon species, whereas the other species featured sets of similar haplotypes. The 18S rDNA GC-contents of the Leucocytozoon toddi complex and the subgenus Parahaemoproteus were extremely high with 49.3% and 44.9%, respectively. The 18S sequences of several species from all three genera showed chimeric features, thus indicating recombination. Gene duplication events leading to two diverged main sequence clusters happened independently in at least six out of seven avian Plasmodium species, thus supporting evolution according to a birth-and-death model like proposed for the ribosomal units of simian and rodent Plasmodium species. Patterns were similar in the 18S rDNAs of the Leucocytozoon toddi complex and Haemoproteus tartakovskyi. However, the 18S rDNAs of the other species seem to evolve in concerted fashion like in most eukaryotes, but the presence of chimeric variants indicates that the ribosomal units rather evolve in a semi-concerted manner. The new data may provide a basis for studies testing whether differential expression of distinct 18S rDNA also occurs in avian Plasmodium species and related haemosporidian parasites.

Background: Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. Methods: For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin–eosin-stained sections. Results: By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. Conclusion: This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.