A Case Report of Avian Malaria (Plasmodium spp.) in Pen-Reared Pigeons (Columba livia).

One dead 6-wk-old male racing pigeon (Columba livia) was submitted for postmortem evaluation after presenting with weight loss, anorexia, dry shanks, dehydration, and lethargy. The bird belonged to a confined flock with 12 other pigeons raised by a hobbyist. Two pigeons in the flock reportedly had died with a history of similar clinical signs. On gross examination, the liver and the spleen were diffusely dark brown to black. Histopathology revealed moderate to large amounts of anisotropic, intracytoplasmic black pigment, compatible with hemozoin, in the spleen, liver, lung, and kidneys, with small amounts in the heart and meninges of the brain. Marked plasmacytic infiltrates were observed in liver, lungs, heart, and kidneys. Blood smears from a clinically affected concomitant pigeon from the flock revealed numerous light-blue, round to oval, intraerythrocytic trophozoites and meronts suggestive of Plasmodium spp. PCR and sequencing tests were performed from spleen and ceca with fragments of the 18S ribosomal RNA and the mitochondrial cytochrome b (cytB) genes. Sequencing results confirmed the presence of Plasmodium in the affected pigeon. Although an exact genetic match could not be determined, the most similar species to the isolate from this study are Plasmodium relictum, Plasmodium matutinum, Plasmodium lutzi, and Plasmodium homocircumflexum.

Reporte de caso­Reporte de un caso de malaria aviar (Plasmodium spp.) en palomas criadas en corrales (Columba livia) Una paloma mensajera macho de 6 semanas muerta (Columba livia) fue remitido a evaluación post mortem después de presentar pérdida de peso, anorexia, patas secas, deshidrataciœn y letargo. El pájaro pertenecía a una parvada confinada con otras 12 palomas criadas por un criador aficionado. Dos palomas de la parvada habían muerto con antecedentes de signos clínicos similares. En el examen macroscópico, el hígado y el bazo se observaron de color marrón oscuro a negro. La histopatología reveló cantidades moderadas a abundantes de pigmento negro intracitoplasmático y anisotrópico, compatible con hemozoína, en el bazo, hígado, pulmón y riñones, con pequeñas cantidades en el corazón y en las meninges del cerebro. Se observaron marcados infiltrados plasmocíticos en hígado, pulmones, corazón y riñones. Los frotis de sangre de otra paloma clínicamente afectada de la parvada revelaron numerosos trofozoítos intraeritrocíticos y esquizontes de color azul claro, redondos a ovalados, que sugerían Plasmodium spp. Se realizaron pruebas de PCR y secuenciación a partir del bazo y el ciego con fragmentos de los genes de ARN ribosómico 18S y del citocromo b mitocondrial (cytB). Los resultados de la secuenciación confirmaron la presencia de Plasmodium en la paloma afectada. Aunque no se pudo determinar una identidad genética exacta, las especies más similares al aislado de este estudio son Plasmodium relictum, Plasmodium matutinum, Plasmodium lutzi y Plasmodium homocircumflexum.

Physiological and morphological correlates of blood parasite infection in urban and non-urban house sparrow populations.

In the last decade, house sparrow populations have shown a general decline, especially in cities. Avian malaria has been recently suggested as one of the potential causes of this decline, and its detrimental effects could be exacerbated in urban habitats. It was initially thought that avian malaria parasites would not have large negative effects on wild birds because of their long co-evolution with their hosts. However, it is now well-documented that they can have detrimental effects at both the primo- and chronical infection stages. In this study, we examined avian malaria infection and its physiological and morphological consequences in four populations of wild house sparrows (2 urban and 2 rural). We did not find any relationship between the proportions of infected individuals and the urbanisation score calculated for our populations. However, we observed that the proportion of infected individuals increased during the course of the season, and that juveniles were less infected than adults. We did not detect a strong effect of malaria infection on physiological, morphological and condition indexes. Complex parasite dynamics and the presence of confounding factors could have masked the potential effects of infection. Thus, longitudinal and experimental studies are needed to understand the evolutionary ecology of this very common, but still poorly understood, wild bird parasite.

First report of avian malaria in a Manx shearwater (Puffinus puffinus).

This study reports the case of a Manx shearwater (Puffinus puffinus) that died from avian malaria while under care at a rehabilitation center in Espírito Santo, Brazil. The bird was rescued on October 2018, and remained under care until it died suddenly on January 2019. A blood smear produced 8 days before death was negative for parasites, whereas a blood smear produced post-mortem revealed a high parasitemia by a parasite resembling Plasmodium cathemerium. The sequence of a 412 bp segment of the cyt-b gene was identical to that of lineage PADOM09, and phylogenetic analysis corroborated that this parasite was closely-related to known lineages of P. cathemerium. The acuteness and severity of the infection documented in this case suggest that seabirds of the order Procellariiformes might be highly susceptible to Plasmodium infections, raising the concern that avian malaria may present a significant threat to their conservation.

Detection of avian malaria in wild birds at Trisik Beach of Yogyakarta, Java (Indonesia)

Avian haemosporidian (Plasmodium, Haemoproteus and Leucocytozoon) are abundant and widespread vector-borne parasites in birds. However, our knowledge is very limited. This study used a nested-PCR to detect the prevalence level of haematozoan parasites in wild bird at coastal area at Tisik Beach of Yogyakarta, Java Island, Indonesia. In total, 112 DNA samples of 22 species were used. Amplification of cyt-b mtDNA of birds at Trisik beach detected 11 out of 112 samples (9.8 %) of all the blood parasites. Only 5 species out of 22 wild bird species were infected by the avian malaria parasites. Meanwhile, only one out of 20 samples of domestic birds was infected. All positive samples sequenced consistently generated around 450 base pair nucleotides. Alignments of 12 sequences have revealed six parasite lineages in the wild bird at Trisik Beach of Yogyakarta, consist of five lineages for Plasmodium sp. and the rest respectively one lineages for Haemoproteus sp. and Leucocytozoon sp. The results of this study provide additional evidences for Plasmodium lineages that uniquely were only infected the Pintail Snipe, Javan Munia, and domestic duck, respectively one lineage, and two lineages in the Yellow Bittern. Meanwhile, Haemoproteus and Leucocytozoon were not uniquely infecting specific host.

Pathological and molecular characterization of avian malaria in captive Magellanic penguins (Spheniscus magellanicus) in South America.

Avian malaria is a mosquito-borne disease that affects multiple avian species and is caused by protozoans of the genus Plasmodium. An avian malaria infection caused by Plasmodium sp. in Magellanic penguins (Spheniscus magellanicus) with high mortality is described in a zoo in Southern Brazil. Clinically, three birds presented signs of inappetence, anorexia, pale mucosa, dyspnea, and opisthotonus, with death in a clinical course of 5-8 h. At the necropsy, all birds exhibited pale mucosa, marked splenomegaly and hepatomegaly, in addition to moderate leptomeningeal blood vessels ingurgitation in the brain. Microscopically, multiple exoerythrocytic meronts were observed in the cytoplasm of endothelial cells in the spleen, liver, heart, lungs, brain, kidneys, and pancreas. The spleen had a multifocal perivascular inflammatory infiltrate of lymphocytes, plasma cells, and macrophages, which also exhibited hemosiderosis and erythrophagocytosis. The liver had a multifocal periportal inflammatory infiltrate of lymphocytes, macrophages, and plasma cells, in addition to marked hemosiderosis in the hepatic sinusoids. Fragments of spleen, liver, brain, skeletal muscle, and lung were tested by the polymerase chain reaction technique for the detection of a fragment of the cytochrome B gene from haemosporidians, which resulted positive for Plasmodium spp. After sequencing, the samples were phylogenetically associated to Plasmodium sp. detected in Turdus albicollis (KU562808) in Brazil and matched to the lineage TURALB01 previously detected in T. albicollis. Avian malaria infections caused by Plasmodium sp. of lineage TURALB01 may occur in S. magellanicus with high mortality, and, thus, it is essential to detect and characterize the agent involved to obtain the differential diagnosis of the condition.

Cases of mortality in little penguins (Eudyptula minor) in New Zealand associated with avian malaria.

CASE HISTORY A little penguin (Eudyptula minor) of wild origin, in captivity at Wellington Zoo, became inappetent and lethargic in March 2013. Despite supportive care in the zoo's wildlife hospital, the bird died within 24 hours. CLINICAL FINDINGS Weight loss, dehydration, pale mucous membranes, weakness, increased respiratory effort and biliverdinuria were apparent on physical examination. Microscopic evaluation of blood smears revealed intra-erythrocytic stages of Plasmodium spp. and a regenerative reticulocytosis in the absence of anaemia. PATHOLOGICAL FINDINGS Post-mortem findings included reduced body condition, dehydration, pulmonary congestion and oedema, hepatomegaly, splenomegaly, hydropericardium and subcutaneous oedema. Histopathological findings included protozoal organisms in sections of lung, liver and spleen. A marked, diffuse, sub-acute interstitial histiocytic pneumonia was present. Accumulation of haemosiderin was noted in the Kupffer cells of the liver and in histiocytic-type cells in the spleen. MOLECULAR TESTING DNA was extracted from frozen portions of the liver. Nested PCR results and DNA sequencing confirmed infection of the deceased little penguin with Plasmodium (Huffia) elongatum lineage GRW06. DIAGNOSIS Avian malaria due to Plasmodium (Huffia) elongatum GRW06 RETROSPECTIVE INVESTIGATION A retrospective analysis of 294 little penguin cases in the Massey University post-mortem database revealed three other potential avian malaria cases. Analysis of archived tissues using a nested PCR for Plasmodium spp. followed by DNA sequencing revealed that a little penguin which died at Auckland Zoo was infected with P. elongatum GRW06 and two wild little penguins found dead on New Zealand beaches were infected with P. relictum SGS1 and Plasmodium. sp. lineage LINN1. Therefore, the overall frequency of deaths in little penguins associated with avian malaria was 4/295 (1.36%). CLINICAL RELEVANCE Our results suggest that avian malaria is associated with sporadic mortality in New Zealand's little penguins both in the wild and in captivity, but there is no evidence of mass mortality events due to Plasmodium spp. infection.

Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand.

Although wildlife rehabilitation and translocations are important tools in wildlife conservation in New Zealand, disease screening of birds has not been standardized. Additionally, the results of the screening programmes are often difficult to interpret due to missing disease data in resident or translocating avian populations. Molecular methods have become the most widespread method for diagnosing avian malaria (Plasmodium spp.) infections. However, these methods can be time-consuming, expensive and are less specific in diagnosing mixed infections. Thus, this study developed a new real-time PCR (qPCR) method that was able to detect and specifically identify infections of the three most common lineages of avian malaria in New Zealand (Plasmodium (Novyella) sp. SYAT05, Plasmodium elongatum GRW6 and Plasmodium spp. LINN1) as well as a less common, pathogenic Plasmodium relictum GRW4 lineage. The assay was also able to discern combinations of these parasites in the same sample and had a detection limit of five parasites per microlitre. Due to concerns relating to the presence of the potentially highly pathogenic P. relictum GRW4 lineage in avian populations, an additional confirmatory high resolution (HRM) qPCR was developed to distinguish between commonly identified P. elongatum GRW6 from P. relictum GRW4. The new qPCR assays were tested using tissue samples containing Plasmodium schizonts from three naturally infected dead birds resulting in the identified infection of P. elongatum GRW6. Thus, these rapid qPCR assays have shown to be cost-effective and rapid screening tools for the detection of Plasmodium infection in New Zealand native birds.

Diversity and distribution of avian malaria and related haemosporidian parasites in captive birds from a Brazilian megalopolis

The role of zoos in conservation programmes has increased significantly in last decades, and the health of captive animals is essential to guarantee success of such programmes. However, zoo birds suffer from parasitic infections, which often are caused by malaria parasites and related haemosporidians. Studies determining the occurrence and diversity of these parasites, aiming better understanding infection influence on fitness of captive birds, are limited. Methods: In 2011–2015, the prevalence and diversity of Plasmodium spp. and Haemoproteus spp. was examined in blood samples of 677 captive birds from the São Paulo Zoo, the largest zoo in Latin America. Molecular and microscopic diagnostic methods were used in parallel to detect and identify these infections. Results: The overall prevalence of haemosporidians was 12.6%. Parasites were mostly detected by the molecular diagnosis, indicating that many birds harbour subclinical or abortive infections. In this project, birds of 17 orders (almost half of all the orders currently accepted in taxonomy of birds), 29 families, and 122 species, were tested, detecting positive individuals in 27% of bird species. Birds from the Anatidae were the most prevalently infected (64.7% of all infected animals)...

Improving detection of avian malaria from host blood: a step towards a standardised protocol for diagnostics.

Avian malaria, caused by Plasmodium spp., has been linked to the mortality and population-level declines in native birds in some regions. While molecular diagnostic methods have greatly improved our ability to detect infections of both human and bird malaria, failing to identify false negatives remains an important handicap, particularly for avian malaria due to host DNA presence in the bird blood cells. In an attempt to improve the accuracy of diagnostics by PCR, we evaluated the performance of a commercial silica-membrane-based DNA extraction kit by modifying the protocol with four unpooled elution volume alternatives. Our results suggest that the best template is the DNA extract obtained from the second eluate of a first 50 µL elution step. In one case, the only band visible was from this second eluate and, thus, may not have been identified as positive for Plasmodium spp. if a different elution protocol had been followed. Our results are likely explained by the concept of size exclusion chromatography by which particles of different sizes will elute at different rates. Overall, first elution templates may consist of a lower ratio of parasite to host DNA, while second eluates may contain a higher parasite to host DNA ratio. A low ratio of parasite to host DNA is a concern in detecting chronic infections, in which birds typically carry low levels of parasitemia, making accurate diagnostics imperative when identifying reservoirs of disease that could lead to spillback events.

Malaria in penguins - current perceptions.

Avian malaria is a mosquito-borne disease caused by protozoans of the genus Plasmodium, and it is considered one of the most important causes of morbidity and mortality in captive penguins, both in zoological gardens and rehabilitation centres. Penguins are known to be highly susceptible to this disease, and outbreaks have been associated with mortality as high as 50-80% of affected captive populations within a few weeks. The disease has also been reported in wild penguin populations, however, its impacts on the health and fitness of penguins in the wild is not clear. This review provides an overview of the aetiology, life cycle and epidemiology of avian malaria, and provides details on the strategies that can be employed for the diagnostic, treatment and prevention of this disease in captive penguins, discussing possible directions for future research.

Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections.

Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.

Molecular detection of the avian malaria parasite Plasmodium gallinaceum in Thailand.

Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol-chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol-chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds.

Molecular detection of Plasmodium in free-ranging birds and captive flamingos (Phoenicopterus chilensis) in Chicago.

Frozen blood samples from 13 species of free-ranging birds (n = 65) and captive Chilean flamingos (Phoenicopterus chilensis) (n = 46) housed outdoors in the Chicago area were screened for Plasmodium. With the use of a modified polymerase chain reaction, 20/65 (30.8%) of free-ranging birds and 26/46 (56.5%) of flamingos were classified as positive for this parasite genus. DNA sequencing of the parasite cytochrome b gene in positive samples demonstrated that eight species of free-ranging birds were infected with five different Plasmodium spp. cytochrome b lineages, and all positive Chilean flamingos were infected with Plasmodium spp. cytochrome b lineages most closely related to organisms in the Novyella subgenus. These results show that Chilean flamingos may harbor subclinical malaria infections more frequently than previously estimated, and that they may have increased susceptibility to some Plasmodium species.

Malaria infections reinforce competitive asymmetry between two Ficedula flycatchers in a recent contact zone.

Parasites may influence the outcome of interspecific competition between closely related host species through lower parasite virulence in the host with which they share the longer evolutionary history. We tested this idea by comparing the prevalence of avian malaria (Haemosporidia) lineages and their association with survival in pied and collared flycatchers (Ficedula hypoleuca and F. albicollis) breeding in a recent contact zone on the Swedish island of Öland. A nested PCR protocol amplifying haemosporidian fragments of mtDNA was used to screen the presence of malaria lineages in 1048 blood samples collected during 6 years. Competitively inferior pied flycatchers had a higher prevalence of blood parasites, including the lineages that were shared between the two flycatcher species. Multistate mark-recapture models revealed a lower survival of infected versus uninfected female pied flycatchers, while no such effects were detected in male pied flycatchers or in collared flycatchers of either sex. Our results show that a comparatively new host, the collared flycatcher, appears to be less susceptible to a local northern European malarial lineage where the collared flycatchers have recently expanded their distribution. Pied flycatchers experience strong reproductive interference from collared flycatchers, and the additional impact of species-specific blood parasite effects adds to this competitive exclusion. These results support the idea that parasites can strongly influence the outcome of interspecific competition between closely related host species, but that the invading species need not necessarily be more susceptible to local parasites.

Evaluation of a restriction fragment length enzyme assay for differentiation of Haemoproteus and Plasmodium across a standard region of the mitochondrial genome.

Avian hemosporidian parasites are a genetically diverse group of parasites with a near cosmopolitan distribution. Over the past 2 decades, several PCR protocols have been designed to detect these parasites. The majority of these protocols amplify part of or the entire mitochondrial cytochrome b gene. However, many of these protocols co-amplify 2 genera (Haemoproteus and Plasmodium), making it impossible to determine which genus is amplified without post-PCR analysis. A uniform database (MalAvi), containing sequences amplified with the primers HAEMF and HAEMR2, has been developed to increase comparability across studies. We analyzed sequences from the MalAvi database and new sequences and found that digestion with EcoRV could be used to distinguish Haemoproteus from the majority of Plasmodium sequences. In addition, we tested 220 wild birds from Costa Rica and the United States for avian hemosporidians and assessed the ability of EcoRV to distinguish these 2 genera. Thirty-six positive samples were sequenced to confirm the restriction profiles, and we also analyzed 63 new hemosporidian sequences from ongoing studies in the United States for the restriction site. Among these new samples, all of the 85 Haemoproteus (subgenus Parahaemoproteus) and 14 Plasmodium were distinguishable. Overall, 887 of 898 (98.8%) sequences from our studies and the MalAvi database were assigned to the correct genus. Of these samples, all Haemoproteus samples were correctly identified and all but 11 Plasmodium samples were correctly identified by the EcoRV assay. Overall, this restriction enzyme protocol is able to quickly and efficiently classify these 2 genera of avian malarial parasites and would be useful for researchers interested in identifying parasites to genus-level, studies focused on sequence analysis of only a single genus, or for detecting co-infections that would need cloning prior to sequence analysis.

A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene.

Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia . This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium ( Huffia ) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of <0.01%) in the same sample with P. polymorphum ; the latter parasite clearly predominated (3.5% parasitemia). However, experienced researchers were unable to detect sequences of mitochondrial cytochrome b gene (cyt b ) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)-based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co-infections, general PCR protocols tend to favor the amplification of the parasite with the higher parasitemia or the amplification with the best matching sequence to the primers. Because the parasitemia of P. polymorphum was >50-fold higher than that of P. relictum and several different primers were tested, we suggest that the failure to amplify P. polymorphum is a more complex problem than why co-infections are commonly overlooked in PCR-based studies. We suggest possible explanations of these results and call for additional research on evolution of mitochondrial genome of hemosporidian parasites.

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.

Fitness effects of endemic malaria infections in a wild bird population: the importance of ecological structure.

1. Parasites can have important effects on host populations influencing either fecundity or mortality, but understanding the magnitude of these effects in endemic host-parasite systems is challenging and requires an understanding of ecological processes affecting both host and parasite. 2. Avian blood parasites (Haemoproteus and Plasmodium) have been much studied, but the effects of these parasites on hosts in areas where they are endemic remains poorly known. 3. We used a multistate modelling framework to explore the effects of chronic infection with Plasmodium on survival and recapture probability in a large data set of breeding blue tits, involving 3424 individuals and 3118 infection diagnoses over nine years. 4. We reveal strong associations between chronic malaria infection and both recapture and survival, effects that are dependent on the clade of parasite, on host traits and on the local risk of infection. 5. Infection with Plasmodium relictum was associated with reduced recapture probability and increased survival, compared to P. circumflexum, suggesting that these parasites have differing virulence and cause different types of selection on this host. 6. Our results suggest a large potential survival cost of acute infections revealed by modelling host survival as a function of the local risk of infection. 7. Our analyses suggest not only that endemic avian malaria may have multiple fitness effects on their hosts and that these effects are species dependent, but also that adding ecological structure (in this case parasite species and spatial variation in disease occurrence) to analyses of host-parasite interactions is an important step in understanding the ecology and evolution of these systems.

The sensitivity of microscopy and PCR-based detection methods affecting estimates of prevalence of blood parasites in birds.

Inferences about the evolution of host-parasitic relationships are often made based on the prevalence of avian malaria, which is usually estimated in a large sample of birds using either microscopic or molecular screening of blood samples. However, different techniques often have variable accuracy; thus, screening methodology can raise issues about statistical bias if method sensitivity varies systematically across parasites or hosts. To examine this possibility, published information was collected on the prevalence of species in 4 genera of avian blood parasites ( Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma) from various sources that used different tools. The data were tested to determine if the application of different methods provided different estimates for the same hosts. In these comparisons between the main methodologies, the PCR-based molecular methods were generally found to provide higher estimates for Plasmodium spp. prevalence than microscopic tools, while there was no significant tendency for such a trend in species of Haemoproteus and Leucocytozoon. When analyzing intraspecific variance of prevalence within molecular studies, some studies provided consistently higher estimates for Haemoproteus spp. prevalence than others, indicating that differences between studies can affect detected estimates. Within microscopic studies, surveys that examined more microscopic fields were more likely to report higher prevalence for Plasmodium spp. than those relying on fewer microscopic fields. Consequently, studies making comparisons across parasite genera and/or host species from different sources need to consider several types of bias originating from variation in method sensitivity.