Isospora and Lankesterella Parasites (Eimeriidae, Apicomplexa) of Passeriform Birds in Europe: Infection Rates, Phylogeny, and Pathogenicity.

Wild birds are common hosts to numerous intracellular parasites such as single-celled eukaryotes of the family Eimeriidae (order Eucoccidiorida, phylum Apicomplexa). We investigated the infection rates, phylogeny, and pathogenicity of Isospora and Lankesterella parasites in wild and captive passerine birds. Blood and tissue samples of 815 wild and 15 deceased captive birds from Europe were tested using polymerase chain reaction and partial sequencing of the mitochondrial cytochrome b and cytochrome c oxidase I and the nuclear 18S rRNA gene. The infection rate for Lankesterella in wild birds was 10.7% compared to 5.8% for Isospora. Chromogenic in situ hybridization with probes targeting the parasites' 18S rRNA was employed to identify the parasites' presence in multiple organs, and hematoxylin-eosin staining was performed to visualize the parasite stages and assess associated lesions. Isospora parasites were mainly identified in the intestine, spleen, and liver. Extraintestinal tissue stages of Isospora were accompanied by predominantly lymphohistiocytic inflammation of varying severity. Lankesterella was most frequently detected in the spleen, lung, and brain; however, infected birds presented only a low parasite burden without associated pathological changes. These findings contribute to our understanding of Isospora and Lankesterella parasites in wild birds.

RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

Novel phylogenetic clade of avian Haemoproteus parasites (Haemosporida, Haemoproteidae) from Accipitridae raptors, with description of a new Haemoproteus species.

Avian haemosporidian parasites (order Haemosporida, phylum Apicomplexa) are blood and tissue parasites transmitted by blood-sucking dipteran insects. Three genera (Plasmodium, Haemoproteus and Leucocytozoon) have been most often found in birds, with over 270 species described and named in avian hosts based mainly on the morphological characters of blood stages. A broad diversity of Haemoproteus parasites remains to be identified and characterized morphologically and molecularly, especially those infecting birds of prey, an underrepresented bird group in haemosporidian parasite studies. The aim of this study was to investigate and identify Haemoproteus parasites from a large sample comprising accipitriform raptors of 16 species combining morphological and new molecular protocols targeting the cytb genes of this parasite group. This study provides morphological descriptions and molecular characterizations of two Haemoproteus species, H. multivacuolatus n. sp. and H. nisi Peirce and Marquiss, 1983. Haemoproteus parasites of this group were so far found in accipitriform raptors only and might be classified into a separate subgenus or even genus. Cytb sequences of these parasites diverge by more than 15% from those of all others known avian haemosporidian genera and form a unique phylogenetic clade. This study underlines the importance of developing new diagnostic tools to detect molecularly highly divergent parasites that might be undetectable by commonly used conventional tools.

Title: Nouveau clade phylogénétique de parasites de rapaces Accipitridae du genre Haemoproteus (Haemosporida, Haemoproteidae), avec description d'une nouvelle espèce d'Haemoproteus. Abstract: Les parasites hémosporidies aviaires (ordre Haemosporida, phylum Apicomplexa) sont des parasites sanguins et tissulaires transmis par des insectes diptères hématophages. Trois genres (Plasmodium, Haemoproteus et Leucocytozoon) ont été le plus souvent trouvés chez les oiseaux, avec plus de 270 espèces décrites et nommées chez les hôtes aviaires en fonction principalement des caractères morphologiques des stades sanguins. Une grande diversité des Haemoproteus reste à identifier et à caractériser morphologiquement et génétiquement, en particulier ceux qui infectent les oiseaux de proie, un groupe d'oiseaux sous-représenté dans les études sur les hémosporidies. Le but de cette étude était d'étudier et d'identifier les Haemoproteus à partir d'un large échantillon comprenant des rapaces accipitriformes de 16 espèces, en combinant des protocoles morphologiques et de nouveaux protocoles moléculaires ciblant les gènes cytb de ce groupe de parasites. Cette étude fournit des descriptions morphologiques et des caractérisations moléculaires de deux espèces d'Haemoproteus, H. multivacuolatus n. sp. et H. nisi Peirce and Marquiss, 1983. Les Haemoproteus de ce groupe n'ont jusqu'à présent été trouvés que chez les rapaces accipitriformes et pourraient être classés dans un sous-genre ou même un genre distinct. Les séquences cytb de ces parasites divergent de plus de 15 % de celles de tous les autres genres d'hémosporidies aviaires connus et forment un clade phylogénétique unique. Cette étude souligne l'importance de développer de nouveaux outils de diagnostic pour détecter des parasites moléculairement très divergents qui pourraient être indétectables par les outils conventionnels couramment utilisés.

Unexpected absence of exo-erythrocytic merogony during high gametocytaemia in two species of Haemoproteus (Haemosporida: Haemoproteidae), including description of Haemoproteus angustus n. sp. (lineage hCWT7) and a report of previously unknown residual bodies during in vitro gametogenesis.

Neglected avian blood parasites of the genus Haemoproteus (Haemoproteidae) have recently attracted attention due to the application of molecular diagnostic tools, which unravelled remarkable diversity of their exo-erythrocytic (or tissue) stages both regarding morphology and organ tropism levels. The development of haemoproteids might result in pathologies of internal organs, however the exo-erythrocytic development (EED) of most Haemoproteus species remains unknown. Seven individual birds - Curruca communis (1) and Phylloscopus trochilus (6) - with high gametocytaemia (between 1% and 24%) of Haemoproteus angustus n. sp. (hCWT7) and Haemoproteus palloris (lineage hWW1) were sampled in Lithuania, and their internal organs were examined extensively by parallel application of histology and chromogenic in situ hybridization methods. Tissue stages were apparently absent, suggesting that the parasitaemia was not accompanied by detectable tissue merogony. Haemoproteus angustus n. sp. was described and characterized morphologically and molecularly. Sexual process and ookinete development of the new species readily occurred in vitro, and a unique character for Haemoproteus parasites was discovered - the obligatory development of several tiny residual bodies, which were associated with intracellular transformation of both macrogametocytes and microgametocytes before their escape from the host cells and formation of gametes. A DNA haplotype network was constructed with lineages that cluster in one clade with the lineage hCWT7. This clade consists of lineages mostly found in Curruca birds, indicating specificity for birds of this genus. The lineage hCWT7 is mainly a parasite of C. communis. Most reports of this lineage came from Turkey, with only a few records in Europe, mostly in birds wintering in Africa where transmission probably occurs. This study highlights unexpected difficulties in the research of EED even when using sensitive molecular diagnostic tools and extends information about transformation in early stages of gametogenesis in haemosporidian parasites.

Co-infecting Haemoproteus species (Haemosporida, Apicomplexa) show different host tissue tropism during exo-erythrocytic development in Fringilla coelebs (Fringillidae).

Avian haemosporidians of the genera Plasmodium, Haemoproteus, and Leucocytozoon are common blood parasites in wild birds all over the world. Despite their importance as pathogens potentially compromising host fitness and health, little is known about the exo-erythrocytic development of these parasites, particularly during co-infections which predominate in wildlife. This study aimed to address this issue using Haemoproteus parasites of Fringilla coelebs, a common bird species of the Western Palearctic and host to a variety of haemosporidian parasite lineages. Blood and tissue samples of 20 F. coelebs, positive for haemosporidians by blood film microscopy, were analysed by PCR and sequencing to determine cytochrome b lineages of the parasites. Tissue sections were examined for exo-erythrocytic stages by histology and in situ hybridization applying genus-, species-, and lineage-specific probes which target the 18S rRNA of the parasites. In addition, laser microdissection of tissue stages was performed to identify parasite lineages. Combined molecular results of PCR, laser microdissection, and in situ hybridization showed a high rate of co-infections, with Haemoproteus lineages dominating. Exo-erythrocytic meronts of five Haemoproteus spp. were described for the first known time, including Haemoproteus magnus hCCF6, Haemoproteus fringillae hCCF3, Haemoproteus majoris hCCF5, Haemoproteus sp. hROFI1, and Haemoproteus sp. hCCF2. Merogonic stages were observed in the vascular system, presenting a formerly unknown mode of exo-erythrocytic development in Haemoproteus parasites. Meronts and megalomeronts of these species were distinct regarding their morphology and organ distribution, indicating species-specific patterns of merogony and different host tissue tropism. New pathological aspects of haemoproteosis were reported. Furthermore, phylogenetic analysis of Haemoproteus spp. with regard to their exo-erythrocytic stages points towards separation of non-megalomeront-forming species from megalomeront-forming species, calling for further studies on exo-erythrocytic development of haemosporidian parasites to explore the phylogenetic character of this trait.

Histological classification of canine and feline lymphoma using a modular approach based on deep learning and advanced image processing.

Histopathological examination of tissue samples is essential for identifying tumor malignancy and the diagnosis of different types of tumor. In the case of lymphoma classification, nuclear size of the neoplastic lymphocytes is one of the key features to differentiate the different subtypes. Based on the combination of artificial intelligence and advanced image processing, we provide a workflow for the classification of lymphoma with regards to their nuclear size (small, intermediate, and large). As the baseline for our workflow testing, we use a Unet++ model trained on histological images of canine lymphoma with individually labeled nuclei. As an alternative to the Unet++, we also used a publicly available pre-trained and unmodified instance segmentation model called Stardist to demonstrate that our modular classification workflow can be combined with different types of segmentation models if they can provide proper nuclei segmentation. Subsequent to nuclear segmentation, we optimize algorithmic parameters for accurate classification of nuclear size using a newly derived reference size and final image classification based on a pathologists-derived ground truth. Our image classification module achieves a classification accuracy of up to 92% on canine lymphoma data. Compared to the accuracy ranging from 66.67 to 84% achieved using measurements provided by three individual pathologists, our algorithm provides a higher accuracy level and reproducible results. Our workflow also demonstrates a high transferability to feline lymphoma, as shown by its accuracy of up to 84.21%, even though our workflow was not optimized for feline lymphoma images. By determining the nuclear size distribution in tumor areas, our workflow can assist pathologists in subtyping lymphoma based on the nuclei size and potentially improve reproducibility. Our proposed approach is modular and comprehensible, thus allowing adaptation for specific tasks and increasing the users' trust in computer-assisted image classification.

Exo-erythrocytic development of Leucocytozoon parasites (Haemosporida, Leucocytozoidae) in song thrushes Turdus philomelos.

Leucocytozoon parasites (Haemosporida, Leucocytozoidae) are haemosporidians whose diversity, exo-erythrocytic development and potential vectors are the least studied. The knowledge about their exo-erythrocytic development and pathogenicity is fragmentary, resulting in an incomplete comprehension of the impact of these parasites on avian hosts. For a long time, Leucocytozoon infections were considered benign to wild birds, even though they were virulent in poultry and responsible for some wild bird population declines. This study aimed to investigate the presence of Leucocytozoon species exo-erythrocytic stages in song thrushes Turdus philomelos using conventional histological techniques (sections stained by H&E) and chromogenic in situ hybridization (CISH). Tissues from ten birds (seven naturally infected and three opportunistic samplings) were examined using both methods. Parasite lineages were identified from blood samples using PCR-based techniques. Leucocytozoon species meronts were found in five individuals (in four birds using H&E staining protocol, and in three in CISH-treated histological sections). Meronts were found mainly in the kidneys, but some meronts were also present in the lungs. It was possible to observe different maturation stages of meronts in the same bird individual, indicating an asynchronous development. Cytomeres were readily visible in developing meronts. One megalomeront-like structure was present close to a blood vessel in the heart. It was covered with a prominent capsular-like wall. No inflammatory reaction or necrosis was seen in the tissues surrounding the meronts or the megalomeront-like structure. We could confirm the transmission of three Leucocytozoon lineages (lTUPHI14, lSTUR1 and lTUPHI13) in Europe, and add evidence of the transmission of two Plasmodium lineages, including Plasmodium circumflexum (pTURDUS1), and Haemoproteus asymmetricus (hTUPHI01). We call for further research to better understand Leucocytozoon parasite exo-erythrocytic development.

The 18S rRNA genes of Haemoproteus (Haemosporida, Apicomplexa) parasites from European songbirds with remarks on improved parasite diagnostics.

BACKGROUND: The nuclear ribosomal RNA genes of Plasmodium parasites are assumed to evolve according to a birth-and-death model with new variants originating by duplication and others becoming deleted. For some Plasmodium species, it has been shown that distinct variants of the 18S rRNA genes are expressed differentially in vertebrate hosts and mosquito vectors. The central aim was to evaluate whether avian haemosporidian parasites of the genus Haemoproteus also have substantially distinct 18S variants, focusing on lineages belonging to the Haemoproteus majoris and Haemoproteus belopolskyi species groups. METHODS: The almost complete 18S rRNA genes of 19 Haemoproteus lineages of the subgenus Parahaemoproteus, which are common in passeriform birds from the Palaearctic, were sequenced. The PCR products of 20 blood and tissue samples containing 19 parasite lineages were subjected to molecular cloning, and ten clones in mean were sequenced each. The sequence features were analysed and phylogenetic trees were calculated, including sequence data published previously from eight additional Parahaemoproteus lineages. The geographic and host distribution of all 27 lineages was visualised as CytB haplotype networks and pie charts. Based on the 18S sequence data, species-specific oligonucleotide probes were designed to target the parasites in host tissue by in situ hybridization assays. RESULTS: Most Haemoproteus lineages had two or more variants of the 18S gene like many Plasmodium species, but the maximum distances between variants were generally lower. Moreover, unlike in most mammalian and avian Plasmodium species, the 18S sequences of all but one parasite lineage clustered into reciprocally monophyletic clades. Considerably distinct 18S clusters were only found in Haemoproteus tartakovskyi hSISKIN1 and Haemoproteus sp. hROFI1. The presence of chimeric 18S variants in some Haemoproteus lineages indicates that their ribosomal units rather evolve in a semi-concerted fashion than according to a strict model of birth-and-death evolution. CONCLUSIONS: Parasites of the subgenus Parahaemoproteus contain distinct 18S variants, but the intraspecific variability is lower than in most mammalian and avian Plasmodium species. The new 18S data provides a basis for more thorough investigations on the development of Haemoproteus parasites in host tissue using in situ hybridization techniques targeting specific parasite lineages.

Rustrela Virus-Associated Encephalomyelitis ('Staggering Disease') in Cats from Eastern Austria, 1994-2016.

Clinical cases of 'staggering disease', a nonsuppurative encephalomyelitis associated with gait abnormalities in cats, have been documented for decades in Sweden. In Austria, an increased incidence was observed in the 1990s. Only recently, rustrela virus (RusV) was identified as the causative agent of this clinicopathologic disease entity. In this retrospective study, we analyzed a total of 23 brain and spinal cord samples from Austrian cats with the pathohistological diagnosis of nonsuppurative encephalomyelitis and clinical signs consistent with staggering disease from 1994 to 2016 using reverse transcription real-time polymerase chain reaction (RT-qPCR) and in situ hybridization. We were able to detect RusV nucleic acids in seven of the examined samples. Borna disease virus 1 (BoDV-1) could be excluded in all cases via immunohistochemistry and RT-qPCR. This study confirms that RusV has been a relevant etiological agent of nonsuppurative encephalomyelitis of cats in a geographically and temporally limited disease cluster in Austria, mainly in the 1990s. The geographic distribution of the positive samples in this study is consistent with earlier reports on 'staggering disease' in Austria. Further studies are necessary to confirm the reservoir host of 'staggering disease' in Austria, as well as investigations on the disappearance of this disease and its possible zoonotic potential.

Comparative Analysis of the Exo-Erythrocytic Development of Five Lineages of Haemoproteus majoris, a Common Haemosporidian Parasite of European Passeriform Birds.

Haemoproteus parasites (Apicomplexa, Haemosporida) are widespread pathogens of birds, with a rich genetic (about 1900 lineages) and morphospecies (178 species) diversity. Nonetheless, their life cycles are poorly understood. The exo-erythrocytic stages of three Haemoproteus majoris (widespread generalist parasite) lineages have been previously reported, each in a different bird species. We aimed to further study and compare the development of five H. majoris lineages-hCCF5, hCWT4, hPARUS1, hPHSIB1, and hWW2-in a wider selection of natural avian hosts. A total of 42 individuals belonging to 14 bird species were sampled. Morphospecies and parasitemia were determined by microscopy of blood films, lineages by DNA-barcoding a 478 bp section of the cytochrome b gene, and exo-erythrocytic stages by histology and chromogenic in situ hybridization. The lineage hCWT4 was morphologically characterized as H. majoris for the first time. All lineage infections exclusively featured megalomeronts. The exo-erythrocytic stages found in all examined bird species were similar, particularly for the lineages hCCF5, hPARUS1, and hPHSIB1. Megalomeronts of the lineages hWW2 and hCWT4 were more similar to each other than to the former three lineages. The kidneys and gizzard were most often affected, followed by lungs and intestines; the site of development showed variation depending on the lineage.

A single, episodic event of unilateral/bilateral scrotal swelling in a group of adult boars at an Austrian boar stud.

BACKGROUND: Scrotal swelling is a clinical situation which can be caused by different aetiologies. In this case report, we describe a multi-week episode of unilateral and bilateral scrotal swelling in boars at an Austrian boar stud and its diagnostic work-up. CASE PRESENTATION: In the summer of 2020, the herd veterinarian of an Austrian boar stud reported that over a period of six weeks, five out of 70 boars presented with unilateral severe swelling of the left scrotum and three out of 70 boars with bilateral severe swelling of the left and moderate swelling of the right scrotum, respectively. A complete history was obtained and an on-site evaluation of the facility was done. Five boars were necropsied, and a variety of samples harvested for further diagnostic investigations. Infectious differential diagnoses associated with unilateral swelling of the scrotum or the testis were excluded through serological and tissue testing. In three of the five boars, histopathology revealed complete acute haemorrhagic necrosis of the left testis concurrent with strongly congested blood vessels. Review of the collected information with a group of experts in the field of boar stud management resulted with consensus that, most likely, trauma was the etiologic event causing the clinical signs and pathology. Coincident with discussion of implementing video recording cameras in the boar housing area, no further clinical cases followed. As this case occurred during the first lockdown of the COVID-19 pandemic, we propose that the distress and travelling restrictions may have contributed to frustration among boar stud workers, which was consequently expressed as misbehaviour against boars. CONCLUSIONS: Once all known infectious causes of unilateral swelling of the scrotum were excluded, a critical diagnostic work-up focused on non-infectious causes. Non-infectious causes, such as trauma, need to be carefully evaluated, as it may also include human misbehaviour against boars. Summarizing all findings of this case report, the authors hypothesize that a blunt trauma was the reason for the series of mainly unilateral swelling of the scrota of boars.

Exo-erythrocytic development of two Haemoproteus species (Haemosporida, Haemoproteidae), with description of Haemoproteus dumbbellus, a new blood parasite of bunting birds (Emberizidae).

Avian haemosporidians are widespread parasites categorized into four families of the order Haemosporida (Apicomplexa). Species of the subgenus Parahaemoproteus (genus Haemoproteus) belong to the Haemoproteidae and are transmitted by Culicoides biting midges. Reports of death due to tissue damage during haemoproteosis in non-adapted birds have raised concerns about these pathogens, especially as their exo-erythrocytic development is known for only a few Haemoproteus spp. More research is needed to better understand the patterns of the parasites' development in tissues and their impact on avian hosts. Yellowhammers Emberiza citrinella (Emberizidae) and common house martins Delichon urbicum (Hirundinidae) were screened for Haemoproteus parasites by microscopic examination of blood films and PCR-based testing. Individuals with single infection were selected for histological investigations. H & E-stained sections were screened for detection and characterization of the exo-erythrocytic stages, while chromogenic in situ hybridization (CISH) and phylogenetic analysis were performed to confirm the Haemoproteus origin and their phylogenetic relationships. Haemoproteus dumbbellus n. sp. was discovered in Emberiza citrinella single-infected with the lineage hEMCIR01. Meronts of H. dumbbellus n. sp. developed in various organs of five of six tested individuals, a pattern which was reported in other Haemoproteus species clustering in the same clade, suggesting this could be a phylogenetic trait. By contrast, in Delichon urbicum infected with the Haemoproteus lineage hDELURB2, which was linked to the more distantly related parasite Haemoproteus hirundinis, only megalomeronts were found in the pectoral muscles of two of six infected individuals. All exo-erythrocytic stages were confirmed to be Haemoproteus parasites by CISH using a Haemoproteus genus-specific probe. While the development of meronts seems to be typical for species of the clade containing H. dumbbellus, further investigations and data from more species are needed to explore whether a phylogenetic pattern occurs in meront or megalomeront formation.

Mystery of fatal 'staggering disease' unravelled: novel rustrela virus causes severe meningoencephalomyelitis in domestic cats.

'Staggering disease' is a neurological disease entity considered a threat to European domestic cats (Felis catus) for almost five decades. However, its aetiology has remained obscure. Rustrela virus (RusV), a relative of rubella virus, has recently been shown to be associated with encephalitis in a broad range of mammalian hosts. Here, we report the detection of RusV RNA and antigen by metagenomic sequencing, RT-qPCR, in-situ hybridization and immunohistochemistry in brain tissues of 27 out of 29 cats with non-suppurative meningoencephalomyelitis and clinical signs compatible with'staggering disease' from Sweden, Austria, and Germany, but not in non-affected control cats. Screening of possible reservoir hosts in Sweden revealed RusV infection in wood mice (Apodemus sylvaticus). Our work indicates that RusV is the long-sought cause of feline 'staggering disease'. Given its reported broad host spectrum and considerable geographic range, RusV may be the aetiological agent of neuropathologies in further mammals, possibly even including humans.

Detection of Pneumocystis and Morphological Description of Fungal Distribution and Severity of Infection in Thirty-Six Mammal Species.

Pneumocystis spp. are thought to adapt to the lungs of potentially all mammals. However, the full host range, fungal burden and severity of infection are unknown for many species. In this study, lung tissue samples originating from 845 animals of 31 different families of eight mammal orders were screened by in situ hybridization (ISH) using a universal 18S rRNA probe for Pneumocystis, followed by hematoxylin and eosin (H&E) staining for determining histopathological lesions. A total of 216 (26%) samples were positive for Pneumocystis spp., encompassing 36 of 98 investigated mammal species, with 17 of them being described for the first time for the presence of Pneumocystis spp. The prevalence of Pneumocystis spp. as assessed by ISH varied greatly among different mammal species while the organism load was overall low, suggesting a status of colonization or subclinical infection. Severe Pneumocystis pneumonia seemed to be very rare. For most of the Pneumocystis-positive samples, comparative microscopic examination of H&E- and ISH-stained serial sections revealed an association of the fungus with minor lesions, consistent with an interstitial pneumonia. Colonization or subclinical infection of Pneumocystis in the lung might be important in many mammal species because the animals may serve as a reservoir.

Exo-Erythrocytic Development of Avian Haemosporidian Parasites in European Owls.

Avian haemosporidian parasites (Haemosporida, Apicomplexa) are globally distributed and infect birds of many orders. These pathogens have been much investigated in domestic and wild passeriform birds, in which they are relatively easy to access. In birds belonging to other orders, including owls (order Strigiformes), these parasites have been studied fragmentarily. Particularly little is known about the exo-erythrocytic development of avian haemosporidians. The goal of this study was to gain new knowledge about the parasites infecting owls in Europe and investigate their exo-erythrocytic stages. Tissue samples of 121 deceased owls were collected in Austria and Lithuania, and examined using polymerase chain reactions (PCR), histology, and chromogenic in situ hybridization (CISH). PCR-based diagnostics showed a total prevalence of 73.6%, revealing two previously unreported Haemoproteus and five novel Leucocytozoon lineages. By CISH and histology, meronts of several Leucocytozoon lineages (lASOT06, lSTAL5, lSTAL7) were discovered in the brains, heart muscles, and kidneys of infected birds. Further, megalomeronts of Haemoproteus syrnii (lineage hSTAL2) were discovered. This study contributes new knowledge to a better understanding of the biodiversity of avian haemosporidian parasites infecting owls in Europe, provides information on tissue stages of the parasites, and calls for further research of these under-investigated pathogens relevant to bird health.

Trichomonosis in Austrian Songbirds-Geographic Distribution, Pathological Lesions and Genetic Characterization over Nine Years.

In the early summer of 2012, sudden mass mortality among songbirds, particularly in greenfinches (Chloris chloris, syn: Carduelis chloris) was observed in Austria, which was caused by the protozoan parasite Trichomonas gallinae. This pathogen induced fibrinonecrotic ingluvitis and/or esophagitis, leading to impairment of food intake and ultimately death due to starvation. The pathogen was successfully detected within the lesions by polymerase chain reaction (PCR) and chromogenic in situ hybridization. The epizootic resulted in a significant decline in the Austrian greenfinch population. Continuing passive surveillance in the subsequent years (2013-2020) revealed that the condition occurred each year and was present in the entire country. Genetic characterization of the pathogen showed the presence of an identical strain irrespective of geographical location, bird species, and year.

Avian haemosporidian parasites of accipitriform raptors.

BACKGROUND: The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. METHODS: Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites' 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. RESULTS: Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. CONCLUSION: The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.

A novel enterovirus in lambs with poliomyelitis and brain stem encephalitis.

An Austrian organic dairy sheep farm experienced cases of recumbency and sudden deaths in 3- to 4-week-old lambs. Two animals were subjected to thorough clinical and pathological investigations. Pathohistological analysis identified severe nonsuppurative myelitis and mild nonsuppurative encephalitis. A reverse-transcription quantitative PCR (RT-qPCR) assay for the recently discovered ovine picornavirus causing comparable lesions scored negative. By next-generation sequencing-based metagenomics, a nearly complete genome of a novel enterovirus could be detected and assembled. In situ hybridization using a specifically designed probe revealed robust signals in affected motoneurons of the spinal cord suggesting a causative role of the novel virus.

A citizen science-based survey of avian mortality focusing on haemosporidian infections in wild passerine birds.

BACKGROUND: Haemosporidioses are common in birds and their manifestations range from subclinical infections to severe disease, depending on the involved parasite and bird species. Clinical haemosporidioses are often observed in non-adapted zoo or aviary birds, whereas in wild birds, particularly passerines, haemosporidian infections frequently seem to be asymptomatic. However, a recent study from Austria showed pathogenic haemosporidian infections in common blackbirds due to high parasite burdens of Plasmodium matutinum LINN1, a common parasite in this bird species, suggesting that virulent infections also occur in natural hosts. Based on these findings, the present study aimed to explore whether and to what extent other native bird species are possibly affected by pathogenic haemosporidian lineages, contributing to avian morbidity. METHODS: Carcasses of passerine birds and woodpeckers were collected during a citizen science-based survey for avian mortality in Austria, from June to October 2020. Tissue samples were taken and examined for haemosporidian parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon by nested PCR and sequencing the mitochondrial cytb barcode region, histology, and chromogenic in situ hybridization applying genus-specific probes. RESULTS: From over 160 dead bird reportings, 83 carcasses of 25 avian species were submitted for investigation. Overall haemosporidian infection rate was 31%, with finches and tits prevailing species counts and infections. Sequence analyses revealed 17 different haplotypes (4 Plasmodium, 4 Haemoproteus, 9 Leucocytozoon), including 4 novel Leucocytozoon lineages. Most infected birds presented low parasite burdens in the peripheral blood and tissues, ruling out a significant contribution of haemosporidian infections to morbidity or death of the examined birds. However, two great tits showed signs of avian malaria, suggesting pathogenic effects of the detected species Plasmodium relictum SGS1 and Plasmodium elongatum GRW06. Further, exo-erythrocytic tissue stages of several haemosporidian lineages are reported. CONCLUSIONS: While suggesting generally little contribution of haemosporidian infections to mortality of the investigated bird species, the findings indicate a possible role of certain haemosporidian lineages in overall clinical manifestation, either as main causes or as concurrent disease agents. Further, the study presents new data on exo-erythrocytic stages of previously reported lineages and shows how citizen science can be used in the field of haemosporidian research.

Investigations on the occurrence of a muscular disorder in Austrian slaughter pigs.

BACKGROUND: In slaughterhouse, veterinarians responsible for meat inspection are often confronted with abnormalities in carcasses, not only in pigs but in all livestock species. In 2017, a veterinarian responsible for meat inspection in a slaughterhouse in Styria, Austria, observed gluteal muscles infiltrated by fat and muscle tissue obviously being replaced by fat in two different slaughter pigs. These muscles were sent for further diagnostics to the University Clinic for Swine. RESULTS: The two muscle samples were investigated histopathologically and diagnosed with fatty muscular dystrophy. The results of routine histopathology were confirmed by dystrophin-specific immunohistochemistry. Sex of the two affected animals was determined retrospectively using a PCR-based protocol and resulted in one male and one female pig. A survey to determine the prevalence of fatty muscular disorders of pork revealed that this phenomenon gets frequently observed in Styria, but also occurs in Upper Austria and Lower Austria. Mostly gluteal and lumbal muscles were affected and approximately 20-40% of the affected muscles were replaced by fat. CONCLUSIONS: Fatty muscular dystrophy or muscular steatosis, as it was sometimes called in early literature, seems not to be an uncommon and rare event and is known to have several different causes. As it was detected in both sexes, our observations are different to the described case in Japan, where only one male individual was affected. To avoid further increase of such cases (fatty muscular dystrophy), it would be useful to clarify the cause. First, whether the cause is environmental or genetic, and in case it is genetic it would be key to disentangle the underlying genomic architecture. Having causal variants described-one could think about integrating this information (depending on the mode of inheritance and the number of loci involved) in the breeding program of pigs. Furthermore, the proportion of non-Austrian pig genetics used for commercial pig production in Austria should be reviewed in order to be able to make reliable statements about the spread of the disease not only in Austrian pig breeds, but also in pig breeds worldwide.