There is increasing evidence that avian haemosporidian parasites (genera of Plasmodium, Haemoproteus and Leucocytozoon) are not only benign commensals but are frequently responsible for lethal infections in birds. Pathogenicity is predominantly inferred by tissue stages of these parasites, which develop in different cell types and lead to blockage of blood vessels and tissue damage. Currently, there is only fragmented knowledge on long-term persistence of these infections and the existence of dormant parasite stages, on the cell types which are targets of replicative tissue stages, on the effect of mixed infections on tissue damage, and finally the question whether 18S ribosomal RNA is differentially expressed in certain developmental stages of the avian malaria parasites.

This study sets out to test the following hypotheses: (1) In analogy to primate malaria parasites, avian haemosporidia also exhibit genetically different 18S ribosomal RNA variants in hosts (birds) and vectors (mosquitoes or biting midges). (2) Relapses in avian malaria are caused by dormant tissue stages which can be visualized by sufficiently sensitive molecular detection methods. (3) Exo-erythrocytic merogony of avian haemosporidian parasites is restricted to few specialized host cell types in different tissues and is more destructive in co-infections.

First, differentially expressed 18 rRNA variants will be labelled by variant-specific probes in avian hosts and arthropod vectors. These results form the basis for the consecutive project parts. The search for dormant stages will be pursued by a highly sensitive in situ hybridization procedure in experimentally infected canaries. The host cell types supporting propagation of parasites in different tissues will be identified by double labelling approaches with antibodies for certain cell markers and molecular probes for haemosporidian meronts. In addition, the effect of mixed infections with two or more haemosporidian species will be tested by double staining procedures.

The majority of the proposed research questions have not been addressed previously (dormant stages and differential expression of rRNAs in avian malaria) or were not explored in a comparably convincing manner, like the identification of cell types allowing propagation of avian malaria parasites, or the effects of mixed infections on the severity of tissue damage.

Project staff:
Univ.-Prof. Dr. Herbert Weissenböck, Dipl.ECPHM, PI
Dr. Josef Harl, Postdoc
MMag. Tanja Himmel PhD, Postdoc

Cooperation partners:
Dr. Gediminas Valkiunas, Mikas Ilgunas, PhD, Nature Research Centre, Vilnius, Lithuania

Project period: 10/2020 - 09/2023

Funding:
FWF - Austrian Science Fund

Pneumocystis spp. are a group of highly diversified opportunistic fungi which are adapted to the lungs of a large variety of mammals. High numbers of Pneumocystis carriers have been described in rats and humans, but also in preliminary studies of pigs prevalences between 51% in pigs with pneumonia and 73% in pigs without respiratory disorders were observed. In humans, Pneumocystis colonization has several potentially important clinical effects as development of acute pneumonia, transmission to other hosts, or involvement in the progression of certain lung diseases as cofactor.

As different age classes of pigs may be kept together in a single facility and thus share the same microbiological environment, these animals are predisposed to polymicrobially caused diseases and in modern pig production systems respiratory disorders pose one of the main health problems. Preliminary data have shown that Pneumocystis can essentially contribute to polymicrobially caused respiratory diseases and in case of infections especially of young pigs the economic impact can be quite high, because developmental deficits early in life can hardly be compensated for.

Currently, the genomic landscape of Pneumocystis carinii f. sp. suis (referred as Pneumocystis suis in this proposal) is almost completely unknown. Whole genome sequencing of Pneumocystis suis will reveal its genome content and structure, and metabolic and other biological pathways, and provide insights into its unique characteristics compared to other Pneumocystis species. This work will contribute to a better understanding of the disease process and pathogenesis of Pneumocystis pneumonia in pigs, and potentially guide the development of new strategies for diagnosis, treatment and prevention.

Studies of Pneumocystis jirovecii in humans have shown that fungal loads depend on the respective co-infections and as a matter of fact differ significantly between Pneumocystis jirovecii genotypes. Besides high-throughput sequencing we have planned to genotype Pneumocystissuis with single nucleotid polymorphism and multilocus sequence typing analyses. The examination of samples from different farms will provide an excellent opportunity to examine if different Pneumocystis suis genotypes exist and if they are associated with Pneumocystis suis organism loads, pig’s ages, clinical signs and co-infections and to study the Pneumocystis suis epidemiology in Austrian pig herds.

Project staff:
Univ.-Prof. Dr. Herbert Weissenböck
Priv.-Doz. Dr. Christiane Weissenbacher-Lang

Cooperation partners:
Univ.-Prof. Dr. Wolfgang Sipos, University Clinic for Swine, University of Veterinary Medicine Vienna
Prof. Dr. Liang Ma, Critical Care Medicine Department, NIH Clinical Center, Bethesda, Maryland, USA
Dr. Ousmane Cissé, Critical Care Medicine Department, NIH Clinical Center, Bethesda, Maryland, USA

Project period: 09/2018 – 08/2021

Funding:
FWF - Austrian Science Fund

Avian haemosporidian parasites belonging to the three genera Plasmodium, Haemoproteus and Leucocytozoon, have a nearly worldwide distribution and affect many bird species. Our primary aim was to look at these parasites from the perspective of their ability of causing diseases and mortalities in wild birds. Disease in birds is predominantly induced by tissue stages of the parasites that cannot be distinguished by morphological methods. PCR assays and subsequent sequence analysis have lead to significant advances in molecular barcoding of the parasites and have generated more than 3500 individual lineages. However, this technique does not allow to reliably assess the identity of a specific parasite responsible for tissue lesions due to frequently occurring mixed infections. Thus a method allowing to specifically identifying tissue stages at genus or species level was highly desired.

Building on our experience with in situ hybridization and the successful design of an oligonucleotide probe for labelling parasites of the Plasmodium genus, we successfully generated and tested probes for identification of the genera Haemoproteus and Leucocytozoon. This was not a straightforward task, because the gene sequences of the ribosomal RNA of these parasites were unknown and had to be cloned and sequenced. Comparable to Plasmodium species causing human malaria several distinct variants of rRNA genes were identified in representatives of all three haemosporidian genera. However, based on these sequences robust and specific probes could be designed and were successfully used for identification of different types of blood and tissue stages. In addition, several species-specific probes were made. With the aid of these probes it was possible to find out that two common Plasmodium species in blackbirds, Plasmodium vaughani and P. matutinum were of different virulence for these birds. Infections with P. matutinum led to much more widespread and destructive multiplication of parasites in the birds´ tissues. While a large number of different Haemoproteus lineages seen in various bird species were mostly associated with low-grade tissue merogony, suggesting low pathogenic potential, several Leucocytozoon lineages, seen either in passerine birds as well as in owls or raptors were associated with megalomeront formation and significant tissue lesions. Thus it seems rewarding for future studies to place increasing emphasis on Leucocytozoon infections in wild birds in order to elucidate a larger number of cases of so far unknown causes of death.

The current project served as basis for a related citizen science project and a three-year follow-up project with the aims to investigate the previously unknown tissue localizations of the enigmatic dormant stages of avian hemosporidiosis, to test experimentally whether different variants of rRNA can be found in the vector and in the host, and to investigate with contemporary  methods whether traditional concepts for cellular localization of meronts prove to be correct.

Project staff:
Univ.-Prof. Dr. Herbert Weissenböck, Dipl.ECPHM, PI
Dr. Josef Harl, Postdoc
MMag. Tanja Himmel PhD

Cooperation partners:
Dr. Gediminas Valkiunas, Nature Research Centre, Vilnius, Lithuania
A.o. Prof. Dr. Alexandra Scope, Clinical Unit of Medicine Small Animals, Med.Uni.Vienna
Univ.-Prof. Dr. Leonida Fusani, KLIVV, Med.Uni.Vienna

Project period: 03/2017 - 09/2020 

Funding:
FWF - Austrian Science Fund
 

Publications:

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden.
Tanja Himmel, Josef Harl, Simone Pfanner, Nora Nedorost, Norbert Nowotny, Herbert Weissenböck
Malaria Journal 2020; 19:69
Link: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-020-3147-6

Molecular probes for the identification of avian Haemoproteus and Leucocytozoon parasites in tissue sections by chromogenic in situ hybridization
Tanja Himmel, Josef Harl, Anna Kübber‑Heiss, Cornelia Konicek, Nuhacet Fernández, Carles Juan‑Sallés,
Mikas Ilgūnas, Gediminas Valkiūnas, Herbert Weissenböck
Parasites & Vectors2019; 12:282
Link: https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-019-3536-2

The nuclear 18S ribosomal DNAs of avian haemosporidian parasites
Josef Harl, Tanja Himmel, Gediminas Valkiūnas, Herbert Weissenböck Malaria Journal 2019; 18:305
Link: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-019-2940-6


Geographic and host distribution of haemosporidian parasite lineages from birds of the family Turdidae.
Josef Harl, Tanja Himmel, Gediminas Valkiūnas, Mikas Ilgūnas, Támas Bakonyi, Herbert Weissenböck
Malaria Journal 2020; 19:335
Link: https://malariajournal.biomedcentral.com/track/pdf/10.1186/s12936-020-03408-0

With different molecular and histochemical techniques we perform cell cycle studies on nerve cells of brain tissue of cats. Investigations on nerve cell cultures and slice cultures of hippocampus and other brain regions should enable insights into the potential mitotic activity of adult neurons and yield broader approaches for the research of neuronal metabolism.

Involved scientists:
Dr. Angelika Url
Dr. Barbara Rebel-Bauder
Graduate and postgraduate students

Mycoplasma are often isolated from the respiratory tract of animals with respiratory diseases. Frequently their pathogenic role remains unclear.
With the aid of immunohistochemistry using antibodies against a number of different Mycoplasma species we intend to analyse whether these bacteria are concentrated in pneumonic lesions and thus likely to play a causative role. Currently, pneumonias of pigs, cats, dogs and different bird species are investigated.

Involved scientists:
Univ.-Prof. Dr. Herbert Weissenböck, Dipl.ECPHM
Dr. Barbara Richter, Dipl.ECVP
Ingrid Friedl
Graduate and postgraduate students

Cooperation partners:
Institute of Bacteriology und Hygiene, University of Veterinary

Funding:
Hochschuljubiläumsstiftung der Stadt Wien

 

By using "custom-made" oligonucleotide probes and the chromogenic in situ hybridization we develop novel diagnostic approaches for a number of protozoa and other single-celled eukaryotes. The probes are subjected to rigorous tests for specificity and represent novel tools in diagnostics and pathogenesis research.

Involved scientists:
Univ.-Prof. Dr. Herbert Weissenböck, Dipl.ECPHM
Dr. Barbara Richter, Dipl.ECVP
Mag. Nora Dinhopl
Karin Fragner

Funding:
FWF - Austrian Science Fund