Asta Krizanauskiene

Nested Cytochrome B Polymerase Chain Reaction Diagnostics Underestimate Mixed Infections of Avian Blood Haemosporidian Parasites: Microscopy is Still Essential

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.

Detecting shifts of transmission areas in avian blood parasites - a phylogenetic approach

We investigated the degree of geographical shifts of transmission areas of vector‐borne avian blood parasites (Plasmodium, Haemoproteus and Leucocytozoon) over ecological and evolutionary timescales. Of 259 different parasite lineages obtained from 5886 screened birds sampled in Europe and Africa, only two lineages were confirmed to have current transmission in resident bird species in both geographical areas. We used a phylogenetic approach to show that parasites belonging to the genera Haemoproteus and Leucocytozoon rarely change transmission area and that these parasites are restricted to one resident bird fauna over a long evolutionary time span and are not freely spread between the continents with the help of migratory birds. Lineages of the genus Plasmodium seem more freely spread between the continents. We suggest that such a reduced transmission barrier of Plasmodium parasites is caused by their higher tendency to infect migratory bird species, which might facilitate shifting of transmission area. Although vector‐borne parasites of these genera apparently can shift between a tropical and a temperate transmission area and these areas are linked with an immense amount of annual bird migration, our data suggest that novel introductions of these parasites into resident bird faunas are rather rare evolutionary events.

DIVERSITY AND PHYLOGENY OF MITOCHONDRIAL CYTOCHROME B LINEAGES FROM SIX MORPHOSPECIES OF AVIAN HAEMOPROTEUS (HAEMOSPORIDA: HAEMOPROTEIDAE)

Species of Haemoproteus (Haemosporida: Haemoproteidae), avian haemosporidians, have traditionally been described based on morphology of their gametocytes and on limited experimental information on their vertebrate host specificity. We investigated to what extent the morphological species are represented by monophyletic groups based on DNA sequence data using 2 different fragment lengths of the cytochrome b (cyt. b) gene. Phylogenetic reconstructions of obtained cyt. b lineages from 6 morphospecies of Haemoproteus showed that all lineages formed monophyletic clusters matching the morphospecies. Comparing our data with a recently published study showed that this is not always the case; the morphospecies H. belopolskyi consists of 2 distinct clusters of lineages that apparently have converged in morphology. However, the overall broad congruence between the molecular and morphological clustering of lineages will facilitate the integration of the knowledge obtained by traditional and molecular parasitology. Mean between morphospecies variation was 10-fold higher than the within species variation (5.5% vs. 0.54%), suggesting that Haemoproteus lineages with a genetic differentiation >5% are expected to be morphologically differentiated in most cases. When investigate the utility of 2 different fragment sizes of the cyt. b gene, the partial, 479-bp, cyt. b protocol picked up all mitochondrial (mt)DNA lineages that are found when using the full cyt. b gene, 1073 bp, suggesting that this protocol is sufficient for identification of most mtDNA lineages. All of the mtDNA lineages were associated with unique alleles when amplification was possible at a nuclear locus, strengthening the hypothesis that the designation of lineages based on mtDNA is largely genome-wide representative. We, therefore, propose the use of a cyt. b fragment of this length as a standard gene fragment for a DNA bar-coding system for avian Haemoproteus species.

Polymerase chain reaction-based identification of Plasmodium (Huffia) elongatum, with remarks on species identity of haemosporidian lineages deposited in GenBank

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only 11 morphospecies of Plasmodium have been linked to these lineages. Such linking is important because it provides opportunities to combine the existing knowledge of traditional parasitology with novel genetic information of these parasites obtained by molecular techniques. This study linked one mitochondrial cytochrome b (cyt b) gene lineage with morphospecies Plasmodium (Huffia) elongatum, a cosmopolitan avian malaria parasite which causes lethal disease in some birds. One species of Plasmodium (mitochondrial cyt b gene lineage P-GRW6) was isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to one naive juvenile individual of the same host species. Heavy parasitaemia developed in the subinoculated bird, which enabled identification of the morphospecies and deposition of its voucher specimens. The parasite of this lineage belongs to P. elongatum. Illustrations of blood stages of this parasite are given. Other lineages closely related to P. elongatum were identified. The validity of the subgenus Huffia is supported by phylogenetic analysis. Mitochondrial cyt b gene lineages, with GenBank accession nos. AF069611 and AY733088, belong to Plasmodium cathemerium and P. elongatum, respectively; these lineages have been formerly attributed to P. elongatum and P. relictum, respectively. Some other incorrect species identifications of avian haematozoa in GenBank have been identified. We propose a strategy to minimise the number of such mistakes in GenBank in the future.

Plasmodium relictum (lineage P-SGS1): Further observation of effects on experimentally infected passeriform birds, with remarks on treatment with Malarone.

Plasmodium relictum (lineage P-SGS1) is a widespread malaria parasite that causes disease of different severity in different species of birds. However, experimental studies on the effects of this parasite on avian hosts are uncommon. We investigated development of this lineage in experimentally infected greenfinches Carduelis chloris and compared the obtained data with the literature information about the virulence of the same parasite lineage for phylogenetically closely related bird species. We also used an opportunity to test the efficacy of the antimalarial drug Malarone in treatment of the experimental infection. The cryopreserved strain of the lineage P-SGS1 was multiplied in 4 experimentally infected chaffinches. Light parasitemia developed in these birds; the parasites were then inoculated to 6 uninfected recipient greenfinches. Six uninfected greenfinches were used as negative controls. Light parasitemia developed in all experimental greenfinches. There were no significant effects of malaria on the body mass of greenfinches, but haematocrit value was slightly lower in experimental birds than in control ones; the infection did not cause mortality or morbidity in these birds. According to available data, all investigated fringillid birds are susceptible to P. relictum (P-SGS1), but the same malaria parasite develops markedly differently in different bird species, even closely related hosts. Thus, the observed effects of the same malaria lineage on one species of bird cannot be generalized to others, even closely related ones. The cure with Malarone was highly efficient for blood stages of P. relictum, but exoerythrocytic stages were unaffected.

A new method for isolation of purified genomic DNA from haemosporidian parasites inhabiting nucleated red blood cells.

During the last 10 years, whole genomes have been sequenced from an increasing number of organisms. However, there is still no data on complete genomes of avian and lizard Plasmodium spp. or other haemosporidian parasites. In contrast to mammals, bird and reptile red blood cells have nuclei and thus blood of these vertebrates contains high amount of host DNA; that complicates preparation of purified template DNA from haemosporidian parasites, which has been the main obstacle for genomic studies of these parasites. In the present study we describe a method that generates large amount of purified avian haemosporidian DNA. The method is based on a unique biological feature of haemosporidian parasites, namely that mature gametocytes in blood can be induced to exflagellate in vitro. This results in the development of numerous microgametes, which can be separated from host blood cells by simple centrifugation. Our results reveal that this straight forward method provides opportunities to collect pure parasite DNA material, which can be used as a template for various genetic analyses including whole genome sequencing of haemosporidians infecting birds and lizards.

Molecular characterization of Haemoproteus sacharovi (Haemosporida, Haemoproteidae), a common parasite of columbiform birds, with remarks on classification of haemoproteids of doves and pigeons

Haemoproteus (Haemosporida, Haemoproteidae) is the largest genus of avian haemosporidian parasites, some species of which cause lethal diseases in birds. Subgenera Parahaemoproteus and Haemoproteus are usually accepted in this genus; these parasites are transmitted by biting midges (Ceratopogonidae) and hippoboscid flies (Hippoboscidae), respectively. As of yet, species of Parahaemoproteus have not been reported to infect doves and pigeons (Columbiformes), parasites of these birds have not been reported to be transmitted by biting midges (Ceratopogonidae). Applying microscopy and PCR based methods, we identified mitochondrial cytochrome b (cyt b) sequences of Haemoproteus sacharovi, a wide-spread parasite of doves and pigeons. Phylogenetic relationships of dove haemoproteids, which traditionally have been classified in the subgenus Haemoproteus, showed that H. sacharovi and H. turtur, common parasites of doves, branch in the clade with Parahaemoproteus species, indicating that these haemoproteids may belong to this subgenus and are likely transmitted by biting midges. This study provides barcodes for H. sacharovi, clarifies the taxonomic positions of H. sacharovi and H. turtur, and indicates directions for development of classification of avian haemoproteid species. Our analysis shows that the current subgeneric classification of avian haemoproteids is generally effective, but the position of some species may need to be revised.