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 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.

Summary 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.

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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.

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.

BackgroundHaemoproteus 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.ResultsHere 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.ConclusionsThe 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.

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, ∆Tgist 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.

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