Philipp Olias

Avian Malaria Deaths in Parrots, Europe

To the Editor: Avian malaria is an insect-borne disease induced by a so far unknown number of protozoan blood parasites of the genera Plasmodium and Haemoproteus (hematozoa) (1,2). The unintentional introduction of P. relictum to the Hawaiian Islands, USA, has had fatal effects for the native bird fauna (3). In Europe, asymptomatic blood infections by hematozoa have been regularly observed, with an especially high prevalence in songbirds (4). However, numerous outbreaks of fatal protozoan infections have been reported over the past 40 years, mainly among psittacines of Australia that have been kept in aviaries (5,6). Diagnosis in all these cases was based on histopathologic detection of protozoan cyst-like structures of unexplained origin in the heart and skeletal muscles and, to a lesser extent, in other organs. In most cases, the protozoans were identified as members of the genus Leucocytozoon because of their morphologic features. Recent studies suggest that these cases may, in fact, have been infections of Besnoitia spp. (Sarcocystidae) or other unknown hematozoa (5); however, genetic evidence is lacking. In August 2010, sudden deaths of parrots were noticed in 2 separate aviaries in northern Germany and Switzerland (Technical AppendixTable). Nine yellow-crowned parakeets (Cyanoramphus auriceps), 3 barred parakeets (Bolborhynchus lineola), and 2 budgerigars (Melopsittacus undulatus) died within 2–5 days after a history of reduced general condition and reduced activity and food intake before death. In addition, 2 budgerigars and 1 barred parakeet in the aviary in Germany showed lethargy and reduced food intake for 2 weeks but fully recovered. About half of the birds were juvenile. No new birds had been introduced into the aviaries during the previous 24 months. Necropsy and histologic examination of 7 animals with fatal disease showed numerous large cyst-like protozoan structures (size up to 800 µm in diameter; Technical Appendix Figure) in myocardial and skeletal muscles and, to a lesser extent, in the lung and the smooth muscles of the intestinal tract without obvious signs of inflammation. The cyst-like structures had a thick eosinophilic outer wall, were partly compartmented by internal septae, and were filled with many merozoites. Surrounding muscle fibers were degenerated or necrotic and, in some cases, associated with hemorrhage. Blood smears of clinically affected animals screened for ≈5 × 105 cells each did not show parasites. To further characterize the parasites, we carried out a nested PCR and subsequent DNA sequencing as described (7). Notably, phylogenetic comparison of 479 bp of the mitochondrial cytochrome b gene derived from protozoan cyst-like structures with known sequences of avian hematozoa found 99%–100% homology of parasites from both outbreaks with the avian malaria parasites (Haemoproteus spp.) of European songbirds (Figure). Identical cytochrome b sequences were detected in a yellow-crowned parakeet from Switzerland (CYAUR1), a budgerigar from Germany (MEUND1), and a Haemoproteus sp. (TUPHI1) previously found in the blood of a song thrush (Turdus philomelos) in Bulgaria. The sequence derived from the barred parakeet (BOLIN1) of the German outbreak was identical with H. minutus of the common blackbird (T. merula). In fact, different psittacine species of the German outbreak were infected with different Haemoproteus spp. Because all affected parrots had been bred in Europe and had no contact to imported birds, these results suggest that infection was the result of previously unknown cross-species transmission of Haemoproteus spp. between birds of only distantly related orders (8,9). Figure Phylogenetic relationships based on alignment of 479 bp of the cytochrome b gene of Haemoproteus spp. isolated from megalomeronts (m) of infected muscles and blood (b) of parrots with related hematozoan parasites in GenBank and the database MalAvi (http://mbio-serv4.mbioekol.lu.se/avianmalaria ... Blood samples from surviving, asymptomatic animals from the German outbreak were tested cytologically and by nested PCR for the presence of Haemoproteus spp. PCR identified Haemoproteus sequences in the blood of 3 of 26 psittacines, although parasitic structures were not identifiable in blood smears. Retrieved sequences were identical with that of MEUND1, except for a single-nucleotide polymorphism in 1 sequence (MEUND3; Figure). A latent infection of these animals therefore seems possible and may constitute a potential risk for further horizontal transmission in aviaries by blood-sucking insects such as biting midges (Culicoides), the vectors for Haemoproteus spp. of passerine birds in Europe (2). In conclusion, we identified the cause of a previously unexplained lethal disease of captive parrots in Europe, induced by numerous large cyst-like megalomeronts in several organs, including the heart. Morphologically, the parasitic structures were strikingly similar to yet undetermined parasites of numerous previous outbreaks (5,6). Genetically, the parasites had 99%–100% homology to known Haemoproteus spp. from wild European songbirds. The avian malaria parasites identified are highly prevalent in the native songbird population but generally do not cause overt disease or death in their natural hosts. In contrast, the cases reported here suggest that these parasites that have adapted to European songbirds may cause fatal outbreaks in native psittacines of Australia, New Zealand, and South America that are raised in captivity. These findings also show that preexisting pathogens may be a potential hazard for invading species. Avian malaria should therefore be considered a threat for exotic parrots in Europe until results of further epidemiologic and experimental studies are available. Because many European bird species have been introduced to the native range of the psittacines studied here, a concern has been expressed that these parasites already have become established in these areas and are affecting the natural populations.

Direct Analysis and Identification of Pathogenic Lichtheimia Species by Matrix-Assisted Laser Desorption Ionization–Time of Flight Analyzer-Mediated Mass Spectrometry

ABSTRACT Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor, Rhizopus, and Lichtheimia. Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)–time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant (Lichtheimia corymbifera, L. ramosa, and L. ornata) and irrelevant (L. hyalospora and L. sphaerocystis) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time.

Anatomical distribution of avian bornavirus in parrots, its occurrence in clinically healthy birds and ABV-antibody detection

Proventricular dilatation disease (PDD) is a fatal infectious disease of birds that primarily affects psittacine birds. Although a causative agent has not been formally demonstrated, the leading candidate is a novel avian bornavirus (ABV) detected in post-mortem tissue samples of psittacids with PDD from the USA, Israel and, recently, Germany. Here we describe the presence of ABV in a parrot with PDD as well as in clinically normal birds exposed to birds with PDD. In two ABV-positive post-mortem cases, the tissue distribution of ABV was investigated by quantitative real-time reverse transcription-polymerase chain reaction. Viraemia was observed in a PDD-affected bird whereas a restriction of ABV to nerve tissue was found in the non-PDD-affected bird. Healthy birds from the same aviary as the affected birds were also found to harbour the virus; 19/59 (32.2%) birds tested positive for ABV RNA in cloacal swabs, providing the first evidence of ABV in clinically healthy birds. In contrast, 39 birds from the same geographic area, but from two different aviaries without PDD cases in recent years, had negative cloacal swabs. ABV RNA-positive, clinically healthy birds demonstrated the same serological response as the animal with confirmed PDD. These results indicate that ABV infection may occur without clinical evidence of PDD and suggest that cloacal swabs can enable the non-invasive detection of ABV infection.

Molecular pathology, taxonomy and epidemiology of Besnoitia species (Protozoa: Sarcocystidae).

Until recently, besnoitiosis has been a neglected disease of domestic animals. Now, a geographic expansion of the causing protozoan parasite Besnoitia besnoiti in livestock has been recognized and the disease in cattle is considered emerging in Europe. Bovine besnoitiosis leads to significant economic losses by a decline in milk production, sterility, transient or permanent infertility of bulls, skin lesions and increase of mortality in affected cattle population. Phylogenetically, the Besnoitia genus is closest related to the well studied and medically important protozoans, Toxoplasma gondii and Neospora caninum. In contrast, discriminative molecular markers to type and subtype large mammalian Besnoitia species (B. besnoiti, B. caprae, B. tarandi, B. bennetti) on a relevant level of species and strains are lacking. Similarly, these cyst-forming parasites may use two hosts to fulfill their life cycle, but this has not been proven for all large mammalian Besnoitia species yet. Most important though, the final hosts and transmission routes of these Besnoitia species remain mysterious. Here, we review aspects of parasites pathology, speciation, phylogeny, epidemiology and transmission with a special focus on recent molecular studies of all to date known Besnoitia species. Using an integrated approach, we have tried to highlight some promising directions for future research.

Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species

Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

High prevalence of Sarcocystis calchasi sporocysts in European Accipiter hawks.

The emerging Sarcocystis calchasi induces a severe and lethal central nervous disease in its intermediate host, the domestic pigeon (Columba livia f. domestica). Experimental studies have identified the Northern goshawk (Accipiter g. gentilis) as final host. Phylogenetically closely related European sparrowhawks (Accipiter n. nisus) and wood pigeons (Columba palumbus) have been found to harbor genetically closely related Sarcocystis spp. However, data on the prevalence and potential interspecies occurrence of these parasites are lacking. Here, we report that European Accipiter hawks (Accipitrinae) are highly infected with S. calchasi, S. columbae and Sarcocystis sp. ex A. nisus in their small intestine. Thirty-one of 50 (62%) Northern goshawks necropsied during 1997-2008 were positive for S. calchasi in a newly established species-specific semi-nested PCR assay based on the first internal transcribed spacer region. Unexpectedly, 14 of 20 (71.4%) European sparrowhawks tested also positive. In addition, birds of both species were found to be infested with S. columbae and an, as yet, unnamed Sarcocystis sp. recently isolated from European sparrowhawks. These findings raise new questions about the host specificity of S. calchasi and its high virulence in domestic pigeons, since sparrowhawks only rarely prey on pigeons. Notably, isolated sporocysts from both infected Accipiter spp. measured 8 μm × 11.9 μm, precluding a preliminary identification of S. calchasi in feces of Accipiter hawks based on morphology alone. Importantly, three of four Northern goshawks used in falconry tested positive for S. calchasi. In conclusion, the results indicate that both European Accipter spp. in Germany serve as natural final hosts of S. calchasi and suggest that falconry and pigeon sport may serve as risk factors for the spread of this pathogen in domestic pigeons.

Sarcocystis calchasi is distinct to Sarcocystis columbae sp. nov. from the wood pigeon (Columba palumbus) and Sarcocystis sp. from the sparrowhawk (Accipiter nisus).

Sarcocystis calchasi has been identified as causative agent of a newly discovered central nervous disease in domestic pigeons (Columba livia f. domestica) observed for the first time in Germany in 2006. Initial studies have indicated that this parasite is highly pathogenic for domestic pigeons after ingestion of low doses of sporocysts shed by the Northern goshawk (Accipiter gentilis). Here we tested whether phylogenetically related birds might regularly harbor Sarcocystis species. Five wood pigeons (Columba palumbus) and five sparrowhawks (Accipiter nisus) from Northern Germany were examined. All birds were PCR negative for S. calchasi by universal primers. Instead, both avian species harbored two as yet undescribed Sarcocystis species. Light and transmission electron microscopy identified cysts in the skeletal muscle of wood pigeons of 56-156 microm in width. The cysts had a smooth surface without protrusions. Sporocysts derived from the small intestine of the sparrowhawks measured 11.88 micromx8.34 microm. Polymerase chain reaction amplification and sequencing of the first internal transcribed spacer region (ITS-1), the 18S rRNA and the 28S rRNA gene comprising the variable D2 and D3 domains further characterized them as two novel Sarcocystis species. S. calchasi displays a pairwise distance value of the ITS-1 region ranging between 0.165 and 0.195 with the Sarcocystis spp. from the wood pigeon and the sparrowhawk, respectively. A phylogenetic analysis further supported the existence of two new species.

A novel Sarcocystis-associated encephalitis and myositis in racing pigeons

Sarcosporidian cysts in the skeletal muscle of domestic pigeons (Columba livia f. domestica) have previously been attributed to infection with Sarcocystis falcatula, which is shed in the faeces of the opossum (Didelphis virginiana). Here, we describe fatal spontaneous encephalitis and myositis associated with Sarcocystis infections in three flocks of racing pigeons with 47 of 244 animals affected. The clinical course was characterized by depression, mild diarrhoea, torticollis, opisthotonus, paralysis and trembling. Histopathological examination of 13 pigeons revealed generalized severe granulomatous and necrotizing meningoencephalitis and myositis with sarcosporidian cysts. Light and transmission electron microscopy identified cysts in heart and skeletal muscle of 1 to 2 mm in length and 20 to 50 µm in width. These were subdivided into small chambers by fine septae and filled with lancet-shaped cystozoites (7.5×1.5 µm) and dividing metrocytes, which is characteristic for Sarcocystis. The cysts had smooth walls and were devoid of protrusions typical of S. falcatula. Polymerase chain reaction amplification and sequencing of the internal transcribed spacer region (ITS-1) and the complete 28S rRNA identified a novel Sarcocystis species with only 51% ITS-1 nucleotide sequence similarity with S. falcatula. A phylogenetic comparison of the 28S rRNA revealed close sequence homologies with Frenkelia microti, Frenkelia glareoli and Sarcocystis neurona. The clinical, histopathological, electron microscopic and genetic data are unlike any previously described protozoan infections in pigeons, suggesting a novel, severe disease due to an as yet undescribed Sarcocystis species.

A Novel High-Resolution Melt PCR Assay Discriminates Anaplasma phagocytophilum and “Candidatus Neoehrlichia mikurensis”

ABSTRACT “Candidatus Neoehrlichia mikurensis” (Anaplasmataceae) is an emerging pathogen transmitted by Ixodes ticks. Conventional PCR and the newly developed high-resolution melt PCR were used to detect and discriminate “Candidatus Neoehrlichia mikurensis” and Anaplasma phagocytophilum. Both bacterial species were frequently found in Ixodes ricinus and Ixodes hexagonus but virtually absent from Dermacentor reticulatus. In rodents, “Candidatus N. mikurensis” was significantly more prevalent than A. phagocytophilum, whereas in cats, only A. phagocytophilum was found.

Sarcocystis Species Lethal for Domestic Pigeons

A large number of Sarcocystis spp. infect birds as intermediate hosts, but pigeons are rarely affected. We identified a novel Sarcocystis sp. that causes lethal neurologic disease in domestic pigeons in Germany. Experimental infections indicated transmission by northern goshawks, and sequence analyses indicated transnational distribution. Worldwide spread is possible.