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Background Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. Methods Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. Results For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. Conclusions We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

Herein, we developed a single and a duplex TaqMan quantitative qPCR for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wt-dhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

Oral infection with Toxoplasma gondii results in dysbiosis and enteritis, both of which revert to normal during chronic infection. However, whether infection leaves a lasting impact on mucosal responses remains uncertain. Here we examined the effect of the chemical irritant dextran sodium sulfate (DSS) on intestinal damage and wound healing in chronically infected mice. Our findings indicate that prior infection with T. gondii exacerbates damage to the colon caused by DSS and impairs wound healing by suppressing stem cell regeneration of the epithelium. Enhanced tissue damage was attributable to inflammatory monocytes that emerge preactivated from bone marrow, migrate to the intestine, and release inflammatory mediators, including nitric oxide. Tissue damage was reversed by neutralization of inflammatory monocytes or nitric oxide, revealing a causal mechanism for tissue damage. Our findings suggest that chronic infection with T. gondii enhances monocyte activation to increase inflammation associated with a secondary environmental insult.

Oral infection with Toxoplasma gondii results in dysbiosis and enteritis, both of which revert to normal during chronic infection. However, whether infection leaves a lasting impact on mucosal responses remains uncertain. Here we examined the effect of the chemical irritant dextran sodium sulfate (DSS) on intestinal damage and wound healing in chronically infected mice. Our findings indicate that prior infection with T. gondii exacerbates damage to the colon caused by DSS and impairs wound healing by suppressing stem cell regeneration of the epithelium. Enhanced tissue damage was attributable to inflammatory monocytes that emerge preactivated from bone marrow, migrate to the intestine, and release inflammatory mediators, including nitric oxide. Tissue damage was reversed by neutralization of inflammatory monocytes or nitric oxide, revealing a causal mechanism for tissue damage. Our findings suggest that chronic infection with T. gondii enhances monocyte activation to increase inflammation associated with a secondary environmental insult.

In contrast to the importance of type II interferon-γ (IFN-γ) in control of toxoplasmosis, the role of type I IFN is less clear. We demonstrate here that TgIST, a secreted effector previously implicated in blocking type II IFN-γ signaling, also blocked IFN-β responses by inhibiting STAT1/STAT2-mediated transcription in infected cells. Consistent with a role for type I IFN in cell intrinsic control, ∆ Tgis t mutants were more susceptible to growth inhibition by murine and human macrophages activated with IFN-β. Additionally, type I IFN was important for production of IFN-γ by natural killer (NK) cells and recruitment of inflammatory monocytes at the site of infection. Mice lacking type I IFN receptors (Ifnar1 −/− ) showed increased mortality following infection with wild-type parasites and decreased virulence of ∆Tgist parasites was restored in Ifnar1 −/− mice. The findings highlight the importance of type I IFN in control of toxoplasmosis and illuminate a parasite mechanism to counteract the effects of both type I and II IFN-mediated host defenses.

Background Osteomyelitis is relatively frequent in young pigs and a few bacterial species have been postulated to be potential causative agents. Although Actinobacillus (A.) pleuropneumoniae has been sporadically described to cause osteomyelitis, typically, actinobacillosis is characterized by respiratory symptoms. Nevertheless, subclinical infections are a challenging problem in pig herds. To the authors’ knowledge, this is the first case description that reports clinical, diagnostic imaging, pathological and histopathological findings of vertebral osteomyelitis in a pig and first describes A. pleuropneumoniae as the causative agent identified by advanced molecular methods. Case presentation An eight-week-old female weaner was presented with a non-ambulatory tetraparesis. The neurological signs were consistent with a lesion in the C6-T2 spinal cord segments. Imaging studies revealed a collapse of the seventh cervical vertebral body (C7) with a well demarcated extradural space-occupying mass ventrally within the vertebral canal severely compressing the spinal cord. Post-mortem examination identified an abscess and osteomyelitis of C7 and associated meningitis and neuritis with subsequent pathological fracture of C7 and compression of the spinal cord. In the microbiological analysis, A. pleuropneumoniae was identified using PCR and DNA sequence analysis. Conclusions A. pleuropneumoniae can be responsible for chronic vertebral abscess formation with subsequent pathological fracture and spinal cord compression in pigs.

A juvenile Little Owl (Athene noctua) was diagnosed with granulomatous encephalitis and muscular sarcocysts. Sarcocystis halieti was identified in the brain and muscle tissue by PCR and subsequent sequencing. This is the first report of S. halieti as a potential encephalitis-causing pathogen in birds.

Diarrhea in calves is one of the most important cattle diseases in Switzerland. The diagnosis and treatment of calf diarrhea represent a major challenge. Single-celled Cryptosporidium parasites are the most prevalent causative agents of calf diarrhea besides rotavirus in the first weeks of life, and are responsible for about 50% of diarrheal cases. Cryptosporidium parvum has been described as a cause of diarrhea in one to three weeks old calves since the 1970s. Oral ingestion of persistent environmental oocysts results in severe diarrhea lasting four to six days and shedding of large numbers of infectious oocysts. A tiny amount of 10 oocysts is already sufficient to cause disease. Detailed knowledge about the epidemiology and virulence of the different C. parvum strains is still lacking. In addition, current diagnostic tests cannot reliably distinguish between non-pathogenic (e.g. C. bovis) and pathogenic Cryptosporidium species. Until now, no effective therapeutic drug or vaccine against calf cryptosporidiosis has been found. Water-borne epidemics and the zoonotic potential of Cryptosporidium in immunodeficient patients are of great medical importance. The increasing number of cryptosporidiosis cases associated with high infant mortality in less industrialized and impoverished regions (including South-East Asia and sub-Saharan Africa) has intensified the research in recent years. The recent discoveries of new therapeutics against C. parvum may benefit calf medicine in the near future. This review article reports on these new developments, highlights calf cryptosporidiosis in Switzerland and draws attention to a new research project.Durchfall bei Kälbern ist eine der häufigsten Rinderkrankheiten in der Schweiz. Die ursächliche Diagnose und Behandlung des Kälberdurchfalls stellen eine grosse Herausforderung dar. Cryptosporidien sind in der Schweiz neben Rotaviren die am häufigsten vorkommenden Erreger des Kälberdurchfalls in den ersten Lebenswochen und für etwa 50% der Fälle verantwortlich. Der einzellige Parasit Cryptosporidium parvum wird seit den 1970er Jahren als Durchfallerreger bei ein bis drei Wochen alten Kälbern beschrieben. Nach oraler Aufnahme von in der Umwelt persistierenden Oocysten kommt es nach wenigen Tagen zu einem vier bis sechs Tage dauernden starken Durchfall mit massiver Ausscheidung von bereits infektiösen Oocysten. Bereits wenige in der Umwelt persistierende Oocysten können krankheitsauslösend sein. In der Epidemiologie herrschen noch grössere Erkenntnislücken zu der vermuteten unterschiedlichen Virulenz verschiedener C. parvum-Stämme. Zudem kommen auch als apathogen angesehene Spezies (unter anderem Cryptosporidium bovis) in Kälbern vor, die von den gängigen diagnostischen Tests nicht verlässlich unterschieden werden können. Bisher wurden kein therapeutisch wirksames Medikament und keine Vakzine gegen die Kälbercryptosporidiose gefunden. In der Human­medizin spielen wasservermittelte Epidemien und die zoonotische Bedeutung von Cryptosporidien bei im­mundefi­zienten Menschen eine grössere Rolle. Die hohe Krank­heitslast der Cryptosporidiose assoziiert mit einer hohen Kindersterblichkeit in wenig industrialisierten und verarmten Regionen (u.a. in Süd-Ostasien und in Afrika südlich der Sahara) hat in den letzten Jahren erneut die Forschung an dem Parasiten befeuert. Besonders die Entdeckung neuer Wirkstoffe gegen C. parvum dürfte in näherer Zukunft auch der Kälbermedizin zu Gute kommen. In diesem Übersichtsartikel wird über diese neuen Entwicklungen berichtet, vor allem aber die Kälbercryptosporidiose in der Schweiz beleuchtet und auf ein neues Forschungsprojekt aufmerksam gemacht.La diarrhée chez les veaux est l’une des maladies du bétail les plus courantes en Suisse. Le diagnostic de la cause et le traitement de la diarrhée des veaux représentent un défi majeur. En Suisse, les cryptosporidies sont, avec les rotavirus, l’agent causal le plus fréquent de diarrhée du veau dans les premières semaines et elles sont responsables d’environ 50% des cas. Le parasite unicellulaire Cryptosporidium parvum a été décrit depuis les années 1970 comme un agent de diarrhée chez les veaux d’une à trois semaines. Après ingestion orale d’oocystes persistants dans l’environnement, il se produit après quelques jours une diarrhée sévère de quatre à six jours avec excrétion massive d’oocystes déjà infectieux. Même quelques oocystes persistants dans l’environnement peuvent être pathogènes. Du point de vue épidémiologique, il existe encore de grandes lacunes dans la connaissance de la variabilité suspectée dans la virulence de diverses souches de C. parvum. En outre, des espèces non pathogènes (entre autres Cryptosporidium bovis) peuvent être présentes chez les veaux, qui ne se distinguent pas de C. parvum avec les tests diagnostiques actuels. Jusqu’à présent, aucun médicament efficace sur le plan thérapeutique et aucun vaccin contre la cryptosporidiose du veau n’ont été trouvés. En médecine humaine, les épidémies transmises par l’eau (en particulier aux États-Unis) et l’importance zoonotique des cryptosporidies comme pathogènes opportunistes chez les personnes immunodéficientes jouent un rôle de premier plan. La forte morbidité de la cryptosporidiose associée à une forte mortalité infantile dans les régions les moins industrialisées et les plus pauvres (entre autres en Asie du Sud-Est et en Afrique subsaharienne) ont relancé la recherche sur ces parasites au cours des dernières années. En particulier, la découverte de nouveaux médicaments contre C. parvum est susceptible de bénéficier à la médecine du veau dans un proche avenir. Cet article de synthèse fait le point sur ces nouveaux développements mais surtout sur la cryptosporidiose du veau en Suisse et attire l’attention sur un nouveau projet de recherche.La diarrea nei vitelli è una delle più frequenti malattie dei bovini in Svizzera. La diagnosi causale e il trattamento della diarrea dei vitelli sono una grande sfida. I criptosporidi con i rotavirus sono i più comuni agenti patogeni della diarrea dei vitelli nella prima settimana di vita e sono la causa del 50% dei casi. Il parassita unicellulare Cryptosporidium parvum viene descritto sin dagli anni 70 come l’agente patogeno della diarrea nei vitelli di età tra 1-3 settimane. Dopo alcuni giorni dall’ingestione orale di persistenti oocisti dell’ambiente circostante si verifica una forte diarrea della durata di quattro a sei giorni con una massiccia escrezione di oocisti già infetti. Solo poche oocisti persistenti nell’ambiente possono causare malattie. In epidemiologia ci sono ancora molte lacune nelle conoscenze della sospetta diversa virulenza dei diversi ceppi di C. parvum. Inoltre, nei vitelli si aggiungono delle specie non patogene (tra cui il Cryptosporidium bovis) che non si distinguono con i test diagnostici. Finora non si sono trovati né farmaci efficaci dal punto di vista terapeutico né vaccini contro la criptosporidiosi nel vitello. Nella medicina umana, le epidemie mediate dall’acqua (specialmente negli Stati Uniti) e l’importanza zoonotica dei criptosporidi come agenti patogeni opportunisti svolgono un ruolo importante per gli esseri umani immunodeficienti. L’alto onere della patologia della criptosporidiosi associato all’alto tasso di mortalità infantile nelle regioni meno industrializzate e povere (tra cui l’Asia sud-orientale e l’Africa sub-sahariana) ha stimolato negli ultimi anni la ricerca sul parassita. In particolare la scoperta di nuovi farmaci contro C. parvum dovrebbe portare in un futuro prossimo dei benefici anche nella medicina per i vitelli. In questa recensione si riporta di questi nuovi sviluppi e in particolare elucida sulla criptosporidiosi del vitello in Svizzera e richiama l’attenzione su un nuovo progetto di ricerca.

Background: Haemoproteus and Plasmodium species are widespread avian blood parasites. Several Plasmodium species are known for their high virulence and have caused significant declines in naïve bird populations. The impact of closely related Haemoproteus parasites is largely unknown. Recently we reported a lethal disease in two parrot aviaries caused by Haemoproteus parasites. Results: Here we show that the causative pathogen Haemoproteus minutus is responsible for further 17 lethal outbreaks in parrot aviaries in Denmark, Germany and Great Britain. All affected parrots are endemic to Australasia and South America. We sequenced the cytochrome b gene from megalomeront-infected muscle tissue of 21 parrots and identified the two lineages TUPHI01 and TURDUS2 as causative agents, commonly naturally infecting the common blackbird (Turdus merula) and the song thrush (Turdus philomelos), respectively, in the Palaearctic. No intraerythrocytic parasite stages were found in any of the parrots. We failed to detect H. minutus in invasive Indian ring-necked parakeets (Psittacula krameri) in Germany. Together this suggests that abortive infections with two virulent lineages of H. minutus are lethal for naïve parrot species from Australasia and South America. We asked whether we could detect H. minutus in New Zealand, where its Turdus hosts were introduced in the 1800s. We therefore tested invasive blackbirds and song thrushes, and the co-existing endemic red-fronted parakeet (Cyanoramphus novaezelandiae) population on three New Zealand islands. No Haemoproteus spp. DNA was detected in all blood samples, indicating absence of transmission. Conclusions: The results of this study show that captive parrots in Europe are threatened by two lineages of an otherwise benign parasite of Turdus spp. Aviary collections of parrots should be protected from Culicoides spp. vectors in Europe. Animal trade and climate changes extending the current vector and parasite distribution have to be considered as potential risk factors for the introduction of the disease in naïve parrot populations.

Bioluminescent reporter assays have been widely used to study the effect of Toxoplasma gondii on host gene expression. In the present study, we extend these studies by engineering novel reporter cell lines containing a gamma-activated sequence (GAS) element driving firefly luciferase (FLUC). In RAW264.7 macrophages, T. gondii type I strain (GT1) infection blocked interferon gamma (IFN-γ)-induced FLUC activity to a significantly greater extent than infection by type II (ME49) and type III (CTG) strains. Quantitative trait locus (QTL) analysis of progeny from a prior genetic cross identified a genomic region on chromosome XII that correlated with the observed strain-dependent phenotype. This QTL region contains two isoforms of the T. gondii enzyme nucleoside triphosphate hydrolase (NTPase) that were the prime candidates for mediating the observed strain-specific effect. Using reverse genetic analysis we show that deletion of NTPase I from a type I strain (RH) background restored the higher luciferase levels seen in the type II (ME49) strain. Rather than an effect on IFN-γ-dependent transcription, our data suggest that NTPase I was responsible for the strain-dependent difference in FLUC activity due to hydrolysis of ATP. We further show that NTPases I and II were not essential for tachyzoite growth in vitro or virulence in mice. Our study reveals that although T. gondii NTPases are not essential for immune evasion, they can affect ATP-dependent reporters. Importantly, this limitation was overcome using an ATP-independent Gaussia luciferase, which provides a more appropriate reporter for use with T. gondii infection studies.

Interferon gamma (IFN-γ) is an essential mediator of host defense against intracellular pathogens, including the protozoan parasite Toxoplasma gondii. However, prior T. gondii infection blocks IFN-γ-dependent gene transcription, despite the downstream transcriptional activator STAT1 being activated and bound to cognate nuclear promoters. We identify the parasite effector that blocks STAT1-dependent transcription and show it is associated with recruitment of the Mi-2 nucleosome remodeling and deacetylase (NuRD) complex, a chromatin-modifying repressor. This secreted effector, toxoplasma inhibitor of STAT1-dependent transcription (TgIST), translocates to the host cell nucleus, where it recruits Mi-2/NuRD to STAT1-dependent promoters, resulting in altered chromatin and blocked transcription. TgIST is conserved across strains, underlying their shared ability to block IFN-γ-dependent transcription. TgIST deletion results in increased parasite clearance in IFN-γ-activated cells and reduced mouse virulence, which is restored in IFN-γ-receptor-deficient mice. These findings demonstrate the importance of both IFN-γ responses and the ability of pathogens to counteract these defenses.

Graphical abstract A: Fattening pigs in a pig barn with outdoor access. B: Echinococcus multilocularis (E.m.) liver lesion in a naturally infected pig. C: histology of a E.m. liver lesion in a naturally infected pig., Highlights • Minimal prevalence for Echinococcus multilocularis infection in Swiss domestic pigs was established. • Minimal prevalence was 0.008% for fattening pigs and 0.11% for breeding pigs, respectively. • All but one metacestode were degenerated; no protoscoleces were detected in histopathology. • Main risk factors were foxes or other animals in the barns, no hygiene barrier, outdoor feeding and grass feeding. • No geographical clusters of higher infection with E. multilocularis were evident., The canid tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE) in humans and other intermediate hosts. Depending on the permissiveness of the intermediate host, the larval form of E. multilocularis (metacestode) may be either fertile, e.g. in rodents, and thus supporting the life cycle of the parasite, or infertile, e.g. in pigs, and thus interrupting the life cycle. Pigs have been shown to act as aberrant hosts for the metacestode and consequently develop liver lesions but represent a dead-end for the parasite. Routine liver inspection at slaughter provided the basis for a large-scale surveillance study on E. multilocularis infection in pigs. The aim of this combined cross-sectional and case-control study was to estimate the minimal prevalence of E. multilocularis in pigs in Switzerland, to find factors associated with infection, and to assess potential regional clusters of infection. During the 12-month-study period, approximately 85% of all pigs slaughtered in Switzerland were assessed. In total, 450 pig livers with macroscopic lesions suggestive of E. multilocularis infection were analysed. Of those, 200 samples were positive by E. multilocularis-PCR. Thus, the overall minimal prevalence detected by molecular means was 0.009% in all slaughter pigs (200 of 2'143'996), 0.008% in finishing pigs (177 of 2'123'542), and 0.11% in breeding pigs (22 of 20'454). Histology revealed the unique presence of a laminated layer in 105 cases, and an additional germinal layer detected in a single case. Protoscoleces could not be observed in any of the lesions. Factors positively associated with infection were "foxes seen in the pig shed", "foxes on premises", "presence of other animals in the shed", "absence of a hygiene barrier", "outdoor feeding", "feeding grass", "lack of rodent control", "not having own dogs on the farm" and "infrequent deworming of sows". Infection was present in all regions sampled and was representative of the important pig rearing areas of Switzerland, without evidence of any obvious geographical cluster. Conclusively, our study provided further evidence of widespread environmental contamination with E. multilocularis eggs in Switzerland. Furthermore, the absence of protoscoleces in any of the lesions supported the concept that pigs act only as a dead-end host and thus do not contribute to the life cycle of the parasite. Factors associated with E. multilocularis infection were in-line with parasite biology, and many can be addressed by increasing hygiene and management standards.

Background Hepatozoon silvestris is an emerging apicomplexan parasite discovered in European wild cats from Bosnia and Herzegovina and blood samples of a domestic cat from Southern Italy in 2017. It has also been identified in Ixodes ricinus collected from a domestic cat in Wales, UK, in 2018. The clinical relevance, pathogenesis and epidemiology of this novel Hepatozoon species are not yet understood. Thus, the objective of this paper was to report and describe the first fatal case of an H. silvestris infection in a domestic cat. Results The cat, which originated from Switzerland, died shortly after presenting clinical signs of lethargy, weakness and anorexia. At necropsy, no specific lesions were observed. Histopathology of the heart revealed a severe lympho-plasmacytic and histiocytic myocarditis. Mature and developing protozoal meronts morphologically compatible with Hepatozoon species were observed associated with the myocardial inflammation. No other lesions were present in any other organ evaluated, and the cat tested negative for retroviral and other immunosuppressive infectious agents. Polymerase chain reaction from the myocardium resulted in a specific amplicon of the Hepatozoon 18S rRNA gene. Sequencing and BLAST analysis revealed 100% sequence identity with H. silvestris. Conclusions The severity of the infection with fatal outcome in an otherwise healthy animal suggests a high virulence of H. silvestris for domestic cats. The presence of this emerging parasite in a domestic cat in Switzerland with no travel history provides further evidence for a geographical distribution throughout Europe.

The protozoan parasite Sarcocystis calchasi causes a severe neurologic disease in domestic pigeons (Columba livia f. dom.) named pigeon protozoal encephalitis. Recently, the parasite has also been reported in psittacines causing a virtually identical disease with fatal outcome. So far, an etiological treatment of S. calchasi infections in pigeons or psittacines is unknown. The present study evaluates the effectiveness of the anticoccidian drug toltrazuril against S. calchasi and the influence of the timepoint of treatment. Therefore, nine domestic pigeons were inoculated with 400 S. calchasi sporocysts and treated with toltrazuril (25 mg/kg) in groups of three pigeons each at dpi 10/11 and dpi 40/41 and on two consecutive days at the onset of neurologic signs. After euthanasia at dpi 73, tissue samples including brain and skeletal muscles were examined by histology and S. calchasi-specific real-time PCR. All pigeons independent of the group developed neurologic signs from dpi 49 onwards. Histology identified sarcocysts in the skeletal muscles and a granulomatous encephalitis in the brains. The relative amount of S. calchasi DNA was on a comparable level in all pigeons. Consequently, toltrazuril was demonstrated to be not effective against S. calchasi with the applied treatment regime. Longer treatment periods or agents other the toltrazuril may be considered for further investigations. So far, preventive measures like roofing of aviaries for prevention of infection and regular disinfection remain the most important factor in the control of S. calchasi infections.

SUMMARY Early electron microscopy studies revealed the elaborate cellular features that define the unique adaptations of apicomplexan parasites. Among these were bulbous rhoptry (ROP) organelles and small, dense granules (GRAs), both of which are secreted during invasion of host cells. These early morphological studies were followed by the exploration of the cellular contents of these secretory organelles, revealing them to be comprised of highly divergent protein families with few conserved domains or predicted functions. In parallel, studies on host-pathogen interactions identified many host signaling pathways that were mysteriously altered by infection. It was only with the advent of forward and reverse genetic strategies that the connections between individual parasite effectors and the specific host pathways that they targeted finally became clear. The current repertoire of parasite effectors includes ROP kinases and pseudokinases that are secreted during invasion and that block host immune pathways. Similarly, many secretory GRA proteins alter host gene expression by activating host transcription factors, through modification of chromatin, or by inducing small noncoding RNAs. These effectors highlight novel mechanisms by which T. gondii has learned to harness host signaling to favor intracellular survival and will guide future studies designed to uncover the additional complexity of this intricate host-pathogen interaction.

Pigeon protozoal encephalitis is a biphasic, neurologic disease of domestic pigeons (Columba livia f. domestica) caused by the apicomplexan parasite Sarcocystis calchasi. Despite severe inflammatory lesions of the brain, associated parasitic stages have only rarely been identified and the cause of the lesions is still unclear. The aim of this study was therefore to characterize the tissue distribution of S. calchasi within pigeons between the two clinical phases and during the occurrence of neurological signs. For this purpose, a semi-quantitative real-time polymerase chain reaction (PCR) was developed. Forty-five domestic pigeons were infected orally (via a cannula into the crop) with 200 S. calchasi sporocysts and euthanized in groups of three pigeons at intervals of 2 to 10 days over a period of 61 days. Tissue samples including brain and skeletal muscle were examined by histology, immunohistochemistry, and PCR. Schizonts were detected in the liver of one pigeon at day 10 post infection. A mild encephalitis was detected at day 20 post infection, around 4 weeks before the onset of neurological signs. At the same time, immature sarcocysts were present in the skeletal muscle. In seven pigeons a few sarcocysts were identified in the brain, but not associated with any lesion. These results suggest that the encephalitis is induced at a very early stage of the S. calchasi lifecycle rather than in the chronic phase of pigeon protozoal encephalitis. Despite the increasing severity of lesions in the central nervous system, the amount of sarcocysts did not increase. This supports the hypothesis of a delayed-type hypersensitivity response as the cause of the encephalitis. The study also demonstrated that S. calchasi DNA is detectable in tissues negative by histological methods, indicating a higher sensitivity of the real-time PCR.

Several studies have shown differences in the course of histomonosis, the infection with the trichomonad parasite Histomonas meleagridis, in different chicken breeds. In the present study, 10 specific-pathogen-free (SPF) layer-type (LT) chickens and twelve SPF meat-type (MT) chickens were infected intracloacally with 200,000 H. meleagridis trophozoites. One and two weeks postinfection (p.i.), three birds of each group were euthanatized. The remaining birds were euthanatized 3 wk p.i. Infected birds showed severe gross lesions typical for histomonosis in ceca at the first and second week p.i., while livers showed necrotic foci at 2 and 3 wk p.i., but only very rarely at 1 wk p.i. Differences between groups in the severity of lesions were statistically insignificant. In histopathology, LT chickens showed a significantly more-severe necrosis and ablation of the cecal epithelium 1 wk p.i. Parasites without inflammation were also found in most investigated spleens and lungs but only in a few kidneys. Investigation of these organs for histomonal DNA by real-time PCR confirmed these results. In addition, the humoral immune response against histomonal actinin 1 and 3 was measured by an ELISA. The humoral immune response against actinin 1 started sooner and was significantly higher in LT chickens than in MT chickens. In conclusion, the results of the present study suggest that the genetic background of the birds influences the reaction to infection with H. meleagridis.

Feline lung tumours are currently subclassified according to the criteria of the World Health Organisation, but this scheme contains overlap in the tumour phenotype. The aims of the present study were to re-evaluate the histological features of feline lung tumours and to correlate these with expression of the markers thyroid transcription factor (TTF)-1 and Ki67. TTF-1 was found to be a highly specific marker for neoplastic and non-neoplastic lung tissue and thyroid tissue, but was expressed only weakly in invasive lung tumours. A combined semiquantitative score for Ki67 and TTF-1 expression correlated well with differentiation and invasive behaviour of the tumours and may thus be of potential value for evaluation of feline lung tumours.

“ 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.

Aspergillus terreus is emerging as a causative agent of life-threatening invasive aspergillosis. Prognosis for affected patients is often worse than for A. fumigatus infections. To study A. terreus–mediated disease, we developed 3 infection models. In embryonated hen’s eggs and leucopenic mice, the outcome of invasive aspergillosis was similar to that described for A. fumigatus. However ,1 0 2 - and 10 3 -fold higher conidia concentrations were required for 100% lethality. In corticosteroid-treated mice, only 50% mortality was observed, although bioluminescence imaging revealed transient disease in all infected animals. In surviving animals, we observed persistence of ungerminated but viable conidia. Cytokine levels in these mice were comparable to uninfected controls. In contrast to A. fumigatus infections, all mice infected with A. terreus developed fatty liver degeneration, suggesting the production of toxic secondary metabolites. Thus, at least in mice, persistence and subclinical liver damage are unique features of A. terreus infections.

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.

Background Cervical Cancer is the second most common cancer among women. Nevertheless, similar tumours have only been rarely described in Great Apes. This report characterizes the pathological and molecular features of a metastatic endocervical adenocarcinoma in a Western lowland gorilla (Gorilla g. gorilla). Methods Necropsy and histopathology was performed to identify the cause of the disease in an cachectic 50-year-old western lowland gorilla. Immunohistochemistry for Ki67, oestrogen receptor alpha and ERBB2 was performed to characterize the tumor. In addition, Pan-herpesvirus and Pan-papillomavirus PCR were used to identify a possible viral cause. Results The endoccervical carcinoma showed a severe metastatic spread to the lung, brain and bone and was herpesvirus and papillomavirus-negative. Most tumor cells were ERBB2-positive, 15% of tumor cells were Ki67-positive and only few tumor cells had oestrogen receptor alpha expression. Conclusions Histopathologically and immunohistochemically, the tumour had striking similarities to human endocervicial adenocarcinomas of the common type. However, PCR analysis failed to identify herpes- or papillomaviral DNA in the tumor at the time of necropsy, thus leaving the question for cause of the disease open.

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 parasite’s 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.

Tissue cysts of a protozoan parasite were present in the skeletal muscle of a domestic pigeon (Columba livia f. domestica) with neurologic disease in Minnesota, USA. The animal had a severe granulomatous meningoencephalitis. The cysts were slender, up to 1 mm long and up to 0.03 mm in diameter. The cysts had a smooth wall without projections. Size and wall morphology were compatible with Sarcocystis calchasi. Polymerase chain reaction using S. calchasi-specific primers resulted in a specific amplicon from the skeletal muscle but not from the brain. Sequencing of the highly variable genomic regions ITS1 and D2 revealed 100% nucleic acid identity with the German strain of S. calchasi. Sarcocystis calchasi is the cause of an emerging lethal disease in pigeons in Germany. This is the first description of the parasite outside of Germany.

Iron storage diseases are rare conditions of dysregulated iron metabolism in man and animals. A genetic basis has been confirmed only for human haemochromatosis. Iron storage disease was diagnosed in six related, 2-year-old male red deer of the same herd. These animals presented with weight loss and rough hair coats. Haematological examination was unremarkable. At necropsy examination, gross lesions were restricted to cachexia. Microscopical examination revealed severe, diffuse hepatocellular necrosis and iron accumulation in hepatocytes, Kupffer cells, cardiac myocytes and renal tubular cells in all affected animals. Four animals also had moderate bridging fibrosis in the liver. Hepatic iron concentrations were increased (1108-2275 mg/kg wet weight; reference range 100-200 mg/kg). Drinking water in rusty iron tubs in the deer park contained eight times more iron than the accepted level for human drinking water. To test for a possible genetic basis of increased iron uptake and storage in red deer, the cervid haemochromatosis gene (HFE) was identified. Sequence comparisons between the six diseased animals and three healthy free-ranging unrelated animals failed to identify differences in the HFE sequences. Furthermore, the disease was not associated with common amino acid substitutions reported in human patients with haemochromatosis, including C282Y and H63D. Polymorphisms in other non-HFE genes involved in iron metabolism may have led to a higher sensitivity to iron and this, together with the high iron content of the drinking water, may have been the cause of the observed iron storage in these red deer.

Accurate species diagnosis in cases of fungal pneumonia may be hampered by environmental contamination and colonization resulting in false-positive results. Our novel approach for fungal species diagnostics combines fluorescent staining of mounted cryosections with the optical brightener Blankophor, laser capture microdissection and PCR amplification with subsequent sequencing of the first internal transcribed spacer region (ITS-1). Using clinical specimens from infected birds, we show that the procedure is suitable for species identification from single hyphae of intralesional filamentous fungi. Our data also suggest that multiple Aspergillus fumigatus strain infections may occur frequently in pulmonary aspergillosis of birds.

A novel Sarcocystis species has recently been reported in the domestic pigeon (Columba livia f. domestica) as intermediate host, causing severe central nervous signs similar to Paramyxovirus-1 or Salmonella Typhimurium var. cop. infection. Transmission of the parasite via the northern goshawk (Accipiter gentilis) as definitive host has been established. Experimental infection of domestic pigeons with sporocysts excreted by experimentally infected northern goshawks reproduced the natural infection in the pigeon, proving the causative role of the parasite in the disease. Here, we describe in greater detail the course of the fulminant biphasic disease depending on the infectious dose. Pigeons infected with 10(3) or 10(4) sporocysts showed clinical signs of polyuria and apathy around 10-11 days postinfection (dpi) and sudden neurological signs 51-57 dpi as a second phase of disease. Pigeons infected with higher doses died within 7-12 dpi, also showing polyuria and apathy but without nervous signs. At necropsy, livers and spleens had multifocal necroses and infestations with parasitic stages, namely, schizonts. Moreover, lesions and schizonts were also found in the lung, bone marrow, and next to blood vessels in the connective tissue of various organs. Pigeons infected with 102 sporocysts remained symptomless until 58-65 dpi, when sudden central nervous signs occurred. Major histopathologic findings of pigeons with neurological signs were encephalitis and myositis of virtually every skeletal muscle with high infestations of sarcocysts. Only mild myocarditis and very few cysts were found in the heart muscles. Importantly, a sentinel pigeon developed identical lesions when compared to those of low-dose infected pigeons, suggesting a risk of mechanical transmission of sporocysts from freshly infected to uninfected pigeons in a flock. By contrast, chickens failed to develop any clinical signs or pathologic lesions in the same experiment. The findings further characterize the new highly pathogenic disease in domestic pigeons, which clinically mimics paramyxovirosis and salmonellosis in both phases of the disease and exclude chickens as further intermediate host species.

Aspergillosis is an important cause of morbidity and mortality in birds. Turkey poults are known to be particularly susceptible to fungal infection. Although the respiratory tract is the most commonly affected, dissemination can occur into virtually any organ. Here, we report an unusual outbreak of articular aspergillosis in a flock of meat turkeys with clinical signs of lameness. Between 7 and 11 weeks of age, turkeys had severe granulomatous osteoarthritis of the hip joints with necrosis of the femur head. Fungal morphology and PCR amplification and sequencing of the first ITS1-5.8S-ITS2 rDNA region identified Aspergillus fumigatus as the infectious agent. Concurrently, Staphylococcus spp. was isolated from the hip joints, which may have promoted the tropism of the fungus. Mild respiratory tract aspergillosis was observed in only one case. The findings suggest that fungal arthritis may present a specific disease entity in turkeys and should be considered as further cause of lameness in turkeys.

A large number of Sarcocystis spp. infect birds as intermediate hosts, but pigeons are rarely affected. We identifi ed 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.

Pigeon protozoal encephalitis (PPE) is an emerging central nervous system disease of pigeons (Columba livia f. domestica) caused by the apicomplexan parasite Sarcocystis calchasi. The intermediate host specificity of S. calchasi had been considered high, as domestic chickens were resistant to experimental infection. Here, we have re-evaluated this concept and expanded the known host range of S. calchasi by experimental infection of cockatiels (Nymphicus hollandicus), a species distantly related to pigeons. In this work, a group of eight cockatiels were experimentally infected with S. calchasi, which resulted in a biphasic central nervous system disease that paralleled PPE in many aspects, albeit with a more diverse pathology. All cockatiels became lethargic and polyuric between days 7 and 13 pi and during that time schizonts of S. calchasi were found primarily in the liver and spleen accompanied by necrosis and inflammation. As with pigeons, neurological signs occurred during a chronic phase of the disease in three cockatiels between 57 and 63 dpi. However, all five cockatiels necropsied in that period, or at the end of the trial at 76 dpi, had a severe lymphohistiocytic and necrotizing encephalitis. No tissue cysts were found in the heart, and cockatiels infected with 10(5) sporocysts only had a negligible parasite load in skeletal muscles despite the presence of severe central nervous system lesions. Notably, intralesional schizonts were identified in the brain of one cockatiel. In contrast to previous results, intralesional schizonts were also identified in the brains of three of six naturally infected pigeons from Minnesota and Missouri examined as part of an epidemiological investigation. In both the cockatiel and the pigeons, tissue cysts were found concurrently with schizonts suggesting an uncommon phenomenon in the Sarcocystis life cycle. Based on the results of this study, transmission of S. calchasi to avian species other than the domestic pigeon is possible. These findings suggest a, so far, unmonitored prevalence of S. calchasi in avian populations and highlight a possible ongoing dissemination of this parasite in the Northern Hemisphere.

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease.

Haemochromatosis is one of the most common human hereditary diseases. It is defined as a pathological condition with normal iron-driven erythropoiesis, but toxic accumulation of iron in vital organs, which is caused by mutations in any gene that encodes a protein involved in limiting the entry of iron into the blood. Iron storage diseases have also been described in several mammalian and avian species and these have been proposed as comparative animal models for human haemochromatosis. Genetically engineered mouse strains with mutations in iron metabolism genes model several aspects of human haemochromatosis and study of these animals has facilitated understanding of the disease. Spontaneously arising iron storage diseases in non-murine species also overlap in some clinicopathological aspects with human haemochromatosis. However, the lack of conclusive information on the molecular biology of theses species-specific diseases and the common impact of dietary iron concentration on disease progression in most species limit their usefulness as comparative models.

The African black rhinoceros (Diceros bicornis) has adapted to a low iron diet during evolution and is thus prone to iron overload in captivity, which is associated with a number of serious disorders. A S88T polymorphism in the HFE gene has been suggested as a potential genetic basis of increased iron uptake in the black rhinoceros, while the Indian rhinoceros is thought to be unaffected by iron overload in captivity. In the present study, the histopathology and distribution of iron accumulations in five black rhinoceroses with iron overload syndrome were characterized and compared with three Indian rhinoceroses (Rhinoceros unicornis) and one African white rhinoceros (Ceratotherium simum). At necropsy examination, iron storage in black rhinoceroses was not associated with gross lesions. Microscopically, the most consistent and highest degree of iron load was found in the spleen, liver, small intestine and lung. There was minimal fibrosis and single cell necrosis in the liver. Endocrine organs, lymph nodes, heart and kidney were less often and less markedly affected. Unexpectedly, Indian rhinoceroses also showed iron load in the spleen and smaller amounts in organs similar to the black rhinoceros except for in the heart, while the white rhinoceros had only minor detectable iron storage in intestine, liver and lung. Sequence analysis confirmed the HFE S88T polymorphism in black but not in Indian rhinoceroses. The results indicate that Indian rhinoceroses may also be affected by iron storage in captivity, although in a milder form than the black rhinoceros, and therefore challenge the relevance of the S88T polymorphism in the HFE gene of black rhinoceroses as the underlying cause for iron overload.

The distribution and prevalence of infections with species of Sarcocystis in domestic fowl in Asia are poorly known. Here, ducks, pigeons, and chickens from Yunnan Province, China were examined for evidence of parasitic infection with Sarcocystis spp. One hundred and ninety one chickens, 514 ducks, and nine pigeons were investigated. Whereas the ducks and pigeons lacked tissue cysts in their muscle, brain or peripheral nervous system, cysts of Sarcocystis wenzeli were identified in 17 of 191 chickens (8.9%). Morphologically, the cysts were thread-like, ranging in size from 334-3169 × 41-117 μm (mean 1093 × 65 μm). Cysts were septate with dense, short finger-like protrusions which appeared radially striated. The cyst wall was 1.4-3.5 μm (mean 2.4 μm) thick. The bradyzoites were lancet shaped and measured 12.2-17.7 × 1.8-2.9 μm (mean 14.6 × 2.5 μm). Ultrastucturally, the primary sarcocyst wall had stubby villar protrusions, corresponding to the 'type 9' class previously designated. The protrusions measured 0.87-1.89 × 0.47-0.91 μm (mean 1.27 × 0.59 μm; n = 57). These findings confirm previous work from the vicinity of Kunming concerning the occurrence of S. wenzeli in chickens, and its use of both cats and dogs as definitive hosts, but indicate that corresponding infections may not occur in the regional domestic flocks of other types of fowl.

SummaryThis report describes a case of intraosseous lipoma in a two-year-old Leonberger. The dog was presented with a history of ten month lameness in the right forelimb. A massive swelling from the elbow to the carpus of the right forelimb was visible. Treatment with anti-inflammatory medications by the local veterinarian for ten months was unsuccessful and the dog was presented at the university clinic. Radiographic images showed that the diaphyseal part of the ulna was affected by extensive cyst-like osteolysis. Furthermore, the distal metaphysis of the radius showed cyst-like osteolytic changes. The soft-tissue mass and parts of the ulna periosteum were surgically resected. Histopathological analysis of the mass in combination with clinical, surgical and radiographic findings was diagnostic for an intraosseous lipoma. The dog had a good long-term outcome as it was free of any signs of recurrence at the follow-up examinations performed after 18 months and after five years. To the authors' knowledge, this is the first description of intraosseous lipoma in a dog.

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.

Aspergillus fumigatus is a common pathogen in poultry and captive wild birds and an emerging opportunistic fungal pathogen in immunocompromised humans. Although invasive aspergillosis is frequently reported in free-ranging wild birds, the incidence and epidemiology of the disease in a natural setting is unknown. We recently reported endemic outbreaks of invasive aspergillosis at white stork nesting sites close to human habitation in Germany with significant subsequent breeding losses. Therefore, we hypothesized that A. fumigatus strains with higher virulence in birds may have evolved in this environment and performed the first epidemiological analysis of invasive aspergillosis in free-ranging wild birds. Sixty-one clinical and environmental A. fumigatus isolates from six affected nesting sites were genotyped by microsatellite analysis using the STRAf-assay. The isolates showed a remarkable high genomic diversity and, contrary to the initial hypothesis, clinical and environmental isolates did not cluster significantly. Interestingly, storks were infected with two to four different genotypes and in most cases both mating types MAT-1.1 and MAT-1.2 were present within the same specimen. The majority of selected clinical and environmental strains exhibited similar virulence in an in vivo infection model using embryonated chicken eggs. Noteworthy, virulence was not associated with one distinct fungal mating type. These results further support the assumption that the majority of A. fumigatus strains have the potential to cause disease in susceptible hosts. In white storks, immaturity of the immune system during the first three weeks of age may enhance susceptibility to invasive aspergillosis.

White stork (Ciconia ciconia) chicks have previously been suggested to be particularly susceptible to environmental conditions such as climatic changes during their first 3 wk of life. However, limited data are available on causes of mortality in free-ranging birds prior to fledging in general. One hundred and one white stork chicks found dead in 2007 and 2008 were examined and the causes of death identified. Of these, 44.6% had fungal granulomatous pneumonia resulting in obstruction and compression of airways. Of note, 94.1% of pulmonary infections occurred in white stork chicks below 23 days of age. PCR amplification and sequencing of the fungal internal transcribed spacer region 1 identified Aspergillus fumigatus and various zygomycetes as primary causative agents. Thermomyces lanuginosus, previously unknown to cause pulmonary infections, was identified in one chick. The findings suggest that fungal pneumonia plays a major role in the loss of white stork chicks of up to 3 wk of age and represents a major threat, similar to the threat posed to young poultry.

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 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.

International audience; 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 Borna virus (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. Viremia was observed in a PDD affected bird whereas a restriction of ABV to nerve tissue was found in the not PDD affected bird. Healthy birds from the same aviary as the affected birds were also found to harbour the virus; 19/59 (32.2%) 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.

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 microm in width. These were subdivided into small chambers by fine septae and filled with lancet-shaped cystozoites (7.5 x 1.5 microm) 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.

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.