Patterns of Plasmodium homocircumflexum virulence in experimentally infected passerine birds.

BACKGROUND: Avian malaria parasites (genus Plasmodium) are cosmopolitan and some species cause severe pathologies or even mortality in birds, yet their virulence remains fragmentally investigated. Understanding mechanisms and patterns of virulence during avian Plasmodium infections is crucial as these pathogens can severely affect bird populations in the wild and cause mortality in captive individuals. The goal of this study was to investigate the pathologies caused by the recently discovered malaria parasite Plasmodium homocircumflexum (lineage pCOLL4) in four species of European passeriform birds. METHODS: One cryopreserved P. homocircumflexum strain was multiplied and used for experimental infections. House sparrows (Passer domesticus), common chaffinches (Fringilla coelebs), common crossbills (Loxia curvirostra) and common starlings (Sturnus vulgaris) were exposed by subinoculation of infected blood. Experimental and control groups (8 individuals in each) were observed for over 1 month. Parasitaemia, haematocrit value and body mass were monitored. At the end of the experiment, samples of internal organs were collected and examined using histological and chromogenic in situ hybridization methods. RESULTS: All exposed birds were susceptible, with similar average prepatent period and maximum parasitaemia, yet virulence was different in different bird species. Mortality due to malaria was reported in chaffinches, house sparrows and crossbills (7, 5 and 3 individuals died respectively), but not in starlings. Exoerythrocytic meronts (phanerozoites) were observed in the brain of all dead experimental birds. Blockage of blood vessels in the brain led to cerebral ischaemia, invariably causing brain damage, which is likely the main reason of mortality. Phanerozoites were observed in parenchymal organs, heart and muscles of all infected individuals, except starlings. CONCLUSION: This study shows that P. homocircumflexum is generalist and the same lineage caused similar parasitaemia-related pathologies in different host species. Additionally, the mode of exo-erythrocytic development is different in different birds, resulting in different mortality rates. This should be taken into consideration in studies addressing pathology during avian malaria infections.

Protozoan parasites in Culex pipiens mosquitoes in Vienna.

Avian malaria (Plasmodium spp.) and kinetoplastid (Trypanosoma spp.) parasites are common vector-borne pathogens in birds worldwide; however, knowledge about vector competence of different mosquito species is currently lacking. For a pilot project examining vector competence of mosquitoes of the Culex pipiens complex and Culex torrentium for protozoan parasites in the city of Vienna, 316 individual mosquitoes were sampled in the months June-August 2017 around the campus of the Veterinary University of Vienna. Since vector competence for avian Plasmodium can only be ascertained by finding infectious sporozoites in mosquito salivary glands, special emphasis was on examining these, or at least insect thoraxes, which contain the salivary glands. After species identification, the mosquitoes were processed in three different ways to determine the best method of visually detecting protozoan parasites in salivary glands: (1) microscopic examination of individual, fixed and Giemsa-stained salivary glands, (2) microscopic examination of stained sections of individually fixed and embedded mosquito thoraxes and (3) stained sections of individual whole insects. Material from all three groups was also subjected to PCR to detect avian haemosporidian and trypanosomatid parasite DNA. PCR was performed on all 316 collected mosquitoes, with 37 pools (n = 2-10) of 263 individuals and 53 single individuals in all together 90 PCR reactions. Avian Plasmodium was found in 18 (20%) and trypanosomatid parasites were found in 10 (11.1%) of the examined samples and pools yielded a higher proportion of positives than did individual samples. Six different species of protozoan parasites were identified, namely Plasmodium vaughani SYAT05 which was the most common, P. elongatum GRW6, P. relictum SGS1, Trypanosoma avium, T. culicavium and Crithidia dedva. Seventy-seven mosquito salivary glands were dissected and stained with Giemsa solution. Of these, one (1.3%) featured sporozoites and one (1.3%) trypanosomatid parasites. While the trypanosomes were identified as T. avium, the avian Plasmodium species were present in a mixed infection with P. vaughani SYAT05 as the dominant species. In conclusion, mosquitoes of the Culex pipiens complex are very likely vectors of different avian Plasmodium and Trypanosoma species and PCR was the most successful and reliable method for parasite detection in mosquito samples, delivering higher rates and more accurate results. The visual detection of parasite stages in the salivary glands was more difficult and only a few specimens were detected using Giemsa stain and chromogenic in situ hybridization. For further studies on vector competence of different protozoan parasites in mosquitoes, the use of PCR-based methods would be preferable.

Characterization of Plasmodium relictum, a cosmopolitan agent of avian malaria.

BACKGROUND: Microscopic research has shown that Plasmodium relictum is the most common agent of avian malaria. Recent molecular studies confirmed this conclusion and identified several mtDNA lineages, suggesting the existence of significant intra-species genetic variation or cryptic speciation. Most identified lineages have a broad range of hosts and geographical distribution. Here, a rare new lineage of P. relictum was reported and information about biological characters of different lineages of this pathogen was reviewed, suggesting issues for future research. METHODS: The new lineage pPHCOL01 was detected in Common chiffchaff Phylloscopus collybita, and the parasite was passaged in domestic canaries Serinus canaria. Organs of infected birds were examined using histology and chromogenic in situ hybridization methods. Culex quinquefasciatus mosquitoes, Zebra finch Taeniopygia guttata, Budgerigar Melopsittacus undulatus and European goldfinch Carduelis carduelis were exposed experimentally. Both Bayesian and Maximum Likelihood analyses identified the same phylogenetic relationships among different, closely-related lineages pSGS1, pGRW4, pGRW11, pLZFUS01, pPHCOL01 of P. relictum. Morphology of their blood stages was compared using fixed and stained blood smears, and biological properties of these parasites were reviewed. RESULTS: Common canary and European goldfinch were susceptible to the parasite pPHCOL01, and had markedly variable individual prepatent periods and light transient parasitaemia. Exo-erythrocytic and sporogonic stages were not seen. The Zebra finch and Budgerigar were resistant. Neither blood stages nor vector stages of all examined P. relictum lineages can be distinguished morphologically. CONCLUSION: Within the huge spectrum of vertebrate hosts, mosquito vectors, and ecological conditions, different lineages of P. relictum exhibit indistinguishable, markedly variable morphological forms. Parasites of same lineages often develop differently in different bird species. Even more, the variation of biological properties (parasitaemia dynamics, blood pathology, prepatent period) in different isolates of the same lineage might be greater than the variation in different lineages during development in the same species of birds, indicating negligible taxonomic value of such features. Available lineage information is excellent for parasite diagnostics, but is limited in predictions about relationships in certain host-parasite associations. A combination of experiments, field observations, microscopic and molecular diagnostics is essential for understanding the role of different P. relictum lineages in bird health.

Plasmodium delichoni n. sp.: description, molecular characterisation and remarks on the exoerythrocytic merogony, persistence, vectors and transmission.

Malaria parasite Plasmodium (Novyella) delichoni n. sp. (Haemosporida, Plasmodiidae) was found in a widespread Eurasian songbird, the common house martin Delichon urbicum (Hirundinidae). It is described based on the morphology of its blood stages and segments of the mitochondrial cytochrome b and apicoplast genes, which can be used for molecular identification of this species. Erythrocytic meronts and gametocytes are strictly nucleophilic, and mature gametocytes possess pigment granules of markedly variable size, including large ones (1 µm in length). Due to these features, P. delichoni can be readily distinguished from all described species of avian malaria parasites belonging to subgenus Novyella. Additionally, mature erythrocytic merozoites contain a dense clump of chromatin, a rare character in avian malaria parasites. Erythrocytic merogony is asynchronous. Illustrations of blood stages of the new species are given, and phylogenetic analysis identifies DNA lineages closely related to this parasite. Domestic canary Serinus canaria and Eurasian siskin Carduelis spinus were infected after subinoculation of infected blood obtained from the house martin. Parasitemia was long lasting in both these hosts, but it was high (up to 70 %) in Eurasian siskins and low (up to 1 %) in canaries. Mortality was not observed, and histological examination and chromogenic in situ hybridisation did not reveal secondary exoerythrocytic meronts (phanerozoites) in the exposed birds. It is likely that persistence of this infection occurs due to long-lasting parasitemia in avian hosts. Sporogony was abortive in mosquitoes Culex pipiens pipiens form molestus, Culex quinquefasciatus and Aedes aegypti at gametogenesis or ookinete stages. The new species is absent from juvenile birds at breeding sites in Europe, indicating that transmission occurs at African wintering grounds.

Comparative evaluation of specific methods for labeling of Encephalitozoon cuniculi in paraffin wax-embedded tissue samples.

Detection of the microsporidian Encephalitozoon cuniculi in tissue samples is considered difficult. The aim of the current study was to determine whether immunohistochemistry (IHC) and in situ hybridization (ISH) represent reliable methods for the detection of E. cuniculi in postmortem tissue samples of rabbits. Paraffin-embedded tissue sections of brain and kidneys of 48 naturally infected pet rabbits, 10 negative controls, and the eyes of 3 further rabbits were used for all investigations. By IHC in 19 animals (37.3%), spores could be clearly detected and were all equally stained. By ISH using a digoxigenin-labeled oligonucleotide probe, only 6 animals (11.8%) proved undoubtedly positive. In these cases, many parasite-like objects revealed strong typical purple-black positive signals. However, several of the examined samples showed only partial staining of the pathogen or unclear results. Thus, in order to find an explanation for these inconsistent ISH results and to take a more detailed look at the different developmental stages of the organism, electron microscopy was applied. Empty spores, which had already discharged their polar filaments, prevailed in total number. Taken together, both techniques are rather insensitive, but under the condition that sufficient numbers of microsporidia are present, IHC can be recommended for specific identification of E. cuniculi in tissue samples. In contrast, ISH failed to detect some developmental stages of the organism, and, as such, ISH is therefore considered an inappropriate diagnostic method.

Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins.

In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.

Simultaneous detection of protozoa in the tissues of snakes by double in situ hybridization.

Different methods have been established for the simultaneous detection of different pathogens in tissue samples, each with certain advantages and disadvantages. Chromogenic in situ hybridization combines specific molecular pathogen detection with microscopic evaluation of pathogen quantity, morphology and distribution, as well as associated tissue damage. Furthermore, only a minimum of usually costly technical equipment is needed. The aim of our study was to detect two different protozoa simultaneously in tissue samples using exclusively digoxigenin (DIG)-labeled probes and alkaline phosphatase-coupled anti-DIG-antibodies and the chromogens Vector Red and NBT/BCIP with standard protocols. Gastrointestinal tissue samples from 15 snakes infected with either one or two protozoan species were investigated. All expected protozoa stained clearly dark purple or bright red, respectively, depending on the chromogen used. This technique can be used in pathogenicity studies of various pathogens in any kind of tissue.

In situ hybridization for Lawsonia intracellularis--specific 16s rRNA sequence in paraffin-embedded tissue using a digoxigenin-labeled oligonucleotide probe.

An in situ hybridization (ISH) procedure with a digoxigenin-labeled oligonucleotide probe for detection of Lawsonia intracellularis in paraffin-embedded tissue is described. This technique recognized 71% of PCR-positive cases and was thus superior to Warthin-Starry silver stain, which only detected 41%. The presented ISH is of comparable sensitivity to previously published immunohistochemical assays and is recommended for laboratories wishing to diagnose L. intracellularis infections in tissue sections but without access to antibodies.

Usutu virus activity in Austria, 2001-2002.

Usutu virus (USUV), a member of the mosquito-borne clade within the Flaviviridae family, was responsible for avian mortality in Austria in 2001. In 2002, the virus continued to kill birds, predominantly blackbirds. High numbers of avian deaths were recorded within the city of Vienna and in surrounding districts of the federal state of Lower Austria, while single die-offs were noticed in the federal states of Styria and Burgenland. A total of 72 birds were submitted for laboratory examination, 30 of which tested positive for USUV by immunohistochemistry and/or polymerase chain reaction. Laboratory-confirmed cases of USUV infection originated from the federal states of Vienna and Lower Austria only. The data show that (i) USUV has managed to overwinter and has been able to establish a transmission cycle in Austria, (ii) the virus seems to have become a resident pathogen of Austria with a tendency to spread to other geographic areas, and (iii) the surveillance of dead blackbirds is a useful sentinel system for monitoring USUV activity.