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Recent investigations suggested pigeon associated Rotavirus Typ A genotype G18P[17] (RVA) as a causative agent of the classical 'young pigeon disease' (YPD). YPD was first described in the late 1980 s as an acute, mainly seasonally recurring disorder of mostly juvenile domestic pigeons (Columba livia) with clinical signs such as anorexia, dairrhea, vomiting, congested crops, weight loss and occasionally mortality. Various studies in the past indicated a multifactorial nature of YPD. Several pathogens, such as pigeon circovirus 1, avian adenoviruses and Escherichia coli were also suggested, but none of these could reproduce the disease experimentally. However, the impact of other pathogens on the clinical development of YPD cannot be excluded and requires further investigation. This present review summarizes available information on RVA-induced disease in pigeons, its association with YPD, the transmission, and diagnosis of the infection, and on prophylactic strategies to prevent RVA outbreaks.Jüngst veröffentlichte Studien belegen, dass Rotavirus A des Genotyps G18P[17] (RVA) Erkrankungen hervorrufen kann, die dem klinischen Bild der Jungtaubenkrankheit (JTK) entsprechen. Bei der Jungtaubenkrankheit handelt es sich um eine meist saisonal wiederkehrende Erkrankung junger Haustauben, die erstmals Ende der 1980er Jahre beschrieben wurde und durch Appetitlosigkeit, Durchfall, Erbrechen, mit Futter und Wasser angestaute Kröpfe sowie Gewichtsverlust und eine hohe Mortalität gekennzeichnet ist. In der Vergangenheit wurde spekuliert, dass es sich bei der JTK um ein multifaktorielles Krankheitsgeschehen handeln könnte. Tauben-Circovirus 1, Adenoviren und Escherichia coli wurden beispielsweise als potenziell beteiligte Erreger diskutiert. Ein epidemiologischer Zusammenhang oder eine Reproduktion des Krankheitsbildes konnte jedoch nicht belegt werden. Ein Einfluss anderer Erreger auf den Verlauf der YPD kann jedoch zum jetzigen Zeitpunkt nicht ausgeschlossen werden und bedarf weiterer Untersuchungen.Dieser Übersichtsartikel fasst die bisherigen Kenntnisse zur Geschichte der JTK, das durch Rotavirus Typ A induzierte Krankheitsbild, Übertragungsmöglichkeiten des RVA sowie Diagnose und Präventionsstrategien zur Vorbeugung von RVA-Ausbrüchen zusammen.

Studies indicate that the evaluation of animal welfare in birds may be carried out with the measurement of the stress-related hormone corticosterone in feathers. However a standardized procedure for corticosterone measurements in feathers is lacking, a validation needs to be carried out for each new species before implementation. The aim of the present study was to establish a valid method to measure corticosterone concentrations in feathers of laying hens in a precise and repeatable manner using an established and commercially available ELISA. Validation was performed with feather pools of tail and interscapular feathers of commercial Lohmann Brown laying hens. Assessment groups, consisting of 5 replicates, were created. All replicates of an assessment group were processed at the same time. Each replicate was run in 4 repetitions by ELISA. Intra-assay and interassay CV was 7.5 and 6.4%, respectively. The serial dilution showed linearity and parallelism. Examining the hormone extraction efficiency by using different methanol volumes resulted in no statistical differences (P > 0.05). Pulverized feathers showed higher corticosterone values than minced feathers (P > 0.05). Differences were shown between 2 feather types (tail vs. interscapular feathers; P 0.05). With the present study, a valid protocol, feasible for analyzing feather pools of laying hens, was developed. It may provide fundamentals for further investigations on corticosterone in feathers as a noninvasive indicator to evaluate aspects of animal welfare.

Infectious bursal disease virus (IBDV) induces one of the most important immunosuppressive diseases in chickens leading to high economic losses due to increased mortality and condemnation rates, secondary infections and the need for antibiotic treatment. Over 400 publications have been listed on PubMed.gov in the last 5 years pointing out the research interest in this disease and the development of improved preventive measures. While B cells are the main target cells of the virus, other immune and non-immune cell populations are also affected, leading to a multifaceted impact on the normally well-orchestrated immune system in IBDV-infected birds. Recent studies clearly revealed the contribution of innate immune cells as well as T cells to a cytokine storm and subsequent death of affected birds in the acute phase of the disease. Transcriptomics identified differential regulation of immune-related genes between different chicken genotypes as well as virus strains, which may be associated with a variable disease outcome. The recent availability of primary B cell culture systems allowed a closer look into virus-host interactions during IBDV infection. The new emerging field of research with transgenic chickens will also open up new opportunities to understand the impact of IBDV on the host under

Mycoplasma (M.) gallisepticum is the most pathogenic mycoplasma species in poultry. Infections cause mild to severe clinical symptoms associated with respiratory epithelial lesion development. Adherence, biofilm formation, and cell invasion of M. gallisepticum contribute to successful infection, immune evasion, and survival within the host. The important M. gallisepticum membrane-bound proteins, GapA and CrmA, are key factors for host cell interaction and the bacterial life-cycle, including its gliding motility, although their precise role in the individual infection step is not yet fully understood. In this study, we investigated the correlation between the host-pathogen interaction and the GapA/CrmA expression in an environment that represents the natural host's multicellular compartment. We used an in vitro tracheal organ culture (TOC) model, allowing the investigation of the M. gallisepticum variants, Rlow, RCL1, RCL2, and Rhigh, under standardised conditions. In this regard, we examined the bacterial adherence, motility and colonisation pattern, host lesion development and alterations of mucociliary clearance. Compared to low virulent RCL2 and Rhigh, the high virulent Rlow and RCL1 were more efficient in adhering to TOCs and epithelium colonisation, including faster movement from the cilia tips to the apical membrane and subsequent cell invasion. RCL2 and Rhigh showed a more localised invasion pattern, accompanied by significantly fewer lesions than Rlow and RCL1. Unrelated to virulence, comparable mucus production was observed in all M. gallisepticum infected TOCs. Overall, the present study demonstrates the role of GapA/CrmA in virulence factors from adherence to colonisation, as well as the onset and severity of lesion development in the tracheal epithelium.

The global turkey industry is confronted with emerging challenges regarding health and welfare. Performance and disease resilience are directly linked to gut health. A clear definition of a healthy gut is a prerequisite to developing new strategies for improved gut health and, thus, general health, welfare and productivity. To date, detailed knowledge about gut health characteristics, especially during the critical fattening period, is still lacking for turkeys. Therefore, the goal of this study was to describe the morphology, microbiota, and metabolome along the intestinal tract of clinically healthy Salmonella- and Campylobacter-free commercial turkey hens throughout the fattening period from 7 to 10 wk posthatch, and obtain information on the stability of the investigated values over time. Feed changes were avoided directly preceding and during the investigation period. Investigation methods included histomorphometric measurement of intestinal villi and crypts, Illumina-sequencing for microbiota analysis, and proton nuclear magnetic resonance spectroscopy for metabolite identification and quantification. Overall, the study demonstrated a high repeatability across all 3 experiments and gut section differences observed coincided with their functions. It was demonstrated that gut maturation, defined by gut microbiota stability, is reached earlier in the ceca than any other intestinal section where morphological changes are ongoing throughout the fattening period. Therefore, the present study provides valuable information necessary to advise future studies on the development and implementation of measures to support gut maturation and establish a protective microbiota in commercial turkeys.

Young pigeon disease syndrome (YPDS) is characterized as a seasonally occurring, acute and primarily enteric medical condition of mainly juvenile domestic pigeons (Columba livia) with highly variable mortality reaching more than 50%. Although the syndrome has been known in Europe for almost three decades, its aetiology remains largely obscure. Recently, a previously unknown pigeon-associated clade of Rotavirus A (RVA) genotype G18P[17] was detected in Europe and Australia in association with fatal diseases resembling YPDS. Here we show for the first time, that peroral inoculation of healthy juvenile homing pigeons with two genetically different cell culture isolates of RVA G18P[17] (106.3 foci-forming units per bird) induces an acute and self-limiting YPDS-like disease in all infected birds. Clinical signs included regurgitation, diarrhoea, congested crops, anorexia and weight loss, as described for naturally RVA-infected pigeons. In agreement with the original outbreaks, RVA isolate DR-7 induced more pronounced clinical signs as compared to isolate DR-5, indicating strain-dependent virulence factors to contribute to variable disease outcomes observed in the field. All inoculated birds developed rotavirus-reactive antibodies starting at seven days after inoculation. High levels of viral RNA and infectious virus were detectable in cloacal swabs and faecal samples already three days after inoculation. While shedding of infectious virus subsided within few days, moderate viral RNA levels were still detectable in cloacal swabs, faeces, and tissue samples at the end of the experiment three weeks after inoculation. Histopathological analysis at this time point revealed inflammatory lesions in spleens and livers of pigeons from both infected groups. In summary, we fulfilled Henle-Koch's postulates and confirmed RVA G18P[17] as a primary cause of YPDS-like diseases in domestic pigeons. By establishing an infection model, we provide a crucial tool for future research, such as identification of transmission routes and establishing vaccination regimes.

In recent years, pathogenic strains of Enterococcus cecorum (EC) have emerged as a causing agent of septicemia and skeletal infection in broiler chickens with a high economic impact worldwide. Although research has been conducted, many aspects of the pathogenesis of the EC-associated disease are still unknown. In the present study, an experimental infection model was established in broiler chickens. Two different EC strains (EC14 and EC15) were compared in two different concentrations of each strain (2 × 106 and 2 × 108 colony-forming units per milliliter (CFU/mL)) after oral infection of one-day-old chicks. Clinical signs and gross lesions of the EC-associated disease were monitored in the following seven weeks. Although both EC strains were originally isolated from clinical disease outbreaks and had a high embryonic lethality, only EC14 successfully induced the typical course of the EC-associated disease with characteristic clinical signs and gross lesions. In total, 23% of the birds in the two EC14-groups were EC-positive in extraintestinal organs on culture, and no differences were found between the two infectious doses. EC14 was frequently detected via real-time PCR in the free thoracic vertebra (FTV) and femoral heads without any detectable gross lesions. The number of EC positive spleens from infected broilers was comparable using bacterial isolation and a specific real-time PCR. Interestingly, EC15 was not detected in extraintestinal organs, although birds in the EC15 groups were colonized by EC in the ceca after experimental infection. The present study represents first proof that virulence differs among EC strains in experimentally infected chickens, and emphasizes the need to further characterize virulence factors and pathogenic mechanisms of EC. The strain EC14 at a dose of 106 CFU is suitable for reproduction of the EC-associated disease. The experimental infection model reported here provides the basis for further research on the EC pathogenesis and possible prevention and intervention strategies.

Aims Our aim was to analyse the survival of Enterococcus cecorum (EC) at various temperatures, relative air humidities and on different substrates commonly existing in broiler houses. Methods and results A pathogenic EC isolate (EC14) was used to inoculate sterile litter, polyvinyl chloride (PVC) and dust samples. Incubation at 37, 25 or 15°C with either 32% relative humidity (RH) or 78% RH followed. At defined time points (0-4272 h post-inoculation), samples were examined in triplicate for the total viable count. Selected combinations were repeated for a non-pathogenic and two additional pathogenic EC strains. For EC14, the measured survival time ranged from 48 to 4272 h (178 days) depending on the substrate-humidity-temperature combination. The longevity was the highest on litter, followed by dust and then PVC. Lower temperatures facilitated its survival, lower relative air humidity favoured the survival only in combination with 25 or 15°C. All three pathogenic strains showed longer survival times (up to 432 h, 18 days) compared to the non-pathogenic EC strain (168 h, 7 days) under the same conditions. Conclusions Enterococcus cecorum demonstrates a high persistence in the environment especially at 15°C and 32% RH. Significance and impact of the study Hygiene management plans should consider the durability of EC and the risk of a carry-over to control consecutive EC outbreaks.

The role of intestinal epithelial cells (IECs) in the physiology of the gastrointestinal tract (GIT) of chickens and pathogenesis of various diseases in chickens is still poorly understood. IECs line the GIT and represent the border between the unsterile environment and the sterile internal tissues. Bacterial, viral, fungal, or parasitic pathogens are able to invade or pass IECs under certain circumstances and cause various diseases. Pathogen-host interactions in the chicken gut are poorly understood because of the lack of suitable in vitro and ex vivo models. In this context, there is a need to optimize the cell isolation and culture conditions to be able to provide reproducible IEC cultures with defined epithelial characteristics. We compared different mechanical IEC isolation protocols and cell culture media and established a reproducible primary intestinal epithelial cell culture model from specific-pathogen-free layer-type chickens. By using isolated crypts from the duodenum of 5- to 12-wk-old birds to create the starting material, we were able to culture replicating cells between 7 and 10 days. Cells built an almost closed monolayer and showed epithelial-like characteristics, such as the expression of cytokeratin and epithelial cadherin. The primary IEC cultures described in this study represent a suitable model with which to investigate in vitro pathogen-host interactions relevant to the chicken gut.

Background Although Enterococcus cecorum (EC) infection is one of the most important bacterial diseases in modern broiler chickens today, many aspects of epidemiology and pathogenesis are still unknown. There is a need for better detection methods for EC than classical cultivation. In the present study, we describe the validation and application of a newly developed quantitative TaqMan real-time PCR (qPCR) assay based on the 16S–rRNA-gene for the detection of EC. Results Fifty EC strains isolated from 12 different animal species were detected with the assay, while none of the other 26 examined bacterial species were tested positive during validation procedure. The detection limit of the PCR was 6.25 CFU/ml PBS. The qPCR assay was also considerably more sensitive using intestine and organ samples than the classical cultivation method. Field application of the PCR setup was tested comparing two different broiler production cycles on one farm: in cycle I broilers showed signs of enterococcal spondylitis (ES) from day 24 post hatch onwards while broilers in cycle II developed no ES. Two totally different colonization patterns were found in the two cycles with the qPCR using cloacal swabs. Animals in cycle I showed significantly (P ≤ 0.05) higher detection rates of EC at the day of placement and throughout the cycle than broilers of cycle II. Additionally, significantly higher detection rates were found in the cecum compared to duodenum, jejunum and ileum within one cycle. Conclusions The new qPCR for EC is highly specific, more sensitive than classical cultivation and was able to show differences in colonization in a broiler cycle with later EC disease outbreak compared to a healthy cycle. These findings may be explained by infection with different strains, pathogenic EC isolates are probably more effective in colonization than commensal isolates. A high correlation was found between qPCR results from cecum and cloacal swabs in this study, indicating that cloacal swabs can be used to examine intestinal colonization of broilers with EC. The new qPCR significantly improves the diagnostic of EC infections and may help to answer open questions concerning epidemiology and pathogenesis.

Despite the application of live hemorrhagic enteritis virus (HEV) vaccines, HEV field outbreaks are suspected to still occur in turkey flocks in Germany. Increasing secondary bacterial infections in HEV-vaccinated flocks suggest that vaccines may be losing efficacy or, possibly, that vaccine strains are causing disease. Thus, the goal of the current study was to investigate the diversity of HEV isolates from fattening turkey flocks between 2008 and 2012 by characterizing the open reading frame (ORF)1 gene at its 5' and 3' ends. Analyses of ORF1 sequences of field isolates and comparison with sequences present in databases revealed that in many cases (13 out of 16 samples), vaccine (avirulent) strains were present. In addition, data indicated the circulation of suspected virulent field isolates and these isolates (3 out of 16) cluster with an early isolate from Germany in the 1980s, but show some mutations in the predicted amino acid (aa) sequences of ORF1 compared to the early isolate. These virulent isolates clearly differ from the spleen-derived avirulent Domermuth vaccine strain used in Germany. In this study, a unique isolate was identified and showed unusual nucleotide mutations that resulted in aa exchanges at the 5' end of ORF1 between aa positions 34 and 174. This genetic drift suggests evolution of HEV including virulent and vaccine-derived strains in the field. This may lead to evasion of vaccinal immunity by drifted viruses and/or an increase in the virulence of field strains.

T-cell immune responses were shown to play an important role in the regulation of infectious bursal disease virus (IBDV) replication and development of lesions in the bursa of Fabricius (BF) (bursal lesions) but also in the recovery from the infection. Studies suggested that the host-genotype influences T-cell responses during the acute phase of infection. Genotype-related differences in the recovery phase were not investigated so far. The present study used commercial broiler- (BT), layer- (LT), dual-purpose type (DT) chicken lines as well as a specific pathogen free (SPF) LT chicken as a reference for comparison of T-cell related differences in IBDV-immunopathogenesis not only in the early phase post inoculation (pi) but also in the recovery phase. The Deventer formula was used to determine the optimal time point of inoculation with an intermediate plus IBDV strain when maternally derived antibody (MDA) titers were below the calculated breakthrough level of the virus for all genotypes. Differences in the bursal lesion development, intrabursal CD4+ and CD8+ T-cell accumulation and numbers of IBDV-positive cells were determined. In addition, anti-IBDV antibody development and the relative amount of anti-inflammatory cytokine mRNA were recorded until 28 days post IBDV inoculation. Differences between the genotypes were observed in the duration and magnitude of bursal lesions, CD4+ and CD8+ T-cell infiltration as well as the presence of anti-inflammatory Interleukin (IL)-10 and Transforming growth factor (TGF) β4 cytokine mRNA (P < 0.05). While the investigated immune parameters were comparable between the genotypes at seven days pi, during 14, 21 and 28 days pi a delayed recovery process in LT and DT chickens compared to BT chickens was observed (P < 0.05). Furthermore, the age and residual MDA levels had a genotype-dependent influence on the onset of the anti-IBDV specific humoral and T-cell mediated immune responses. This study suggests, that the impact of T-cell immunity on the recovery process after IBDV infection may need to be considered further for the development of new breeding programs for disease resistant chicken lines.

The genotype of chickens is assumed to be associated with variable immune responses. In this study a modern, moderate performing dual-purpose chicken line (DT) was compared with a high-performing layer-type (LT) as well as a broiler-type (BT) chicken line. One group of each genotype was vaccinated in ovo with a recombinant herpesvirus of turkeys expressing the virus protein VP2 of the infectious bursal disease virus (HVT-IBD) while one group of each genotype was left HVT-IBD unvaccinated (control group). Genotype associated differences in innate and adapted immune responses between the groups were determined over five weeks post hatch. HVT-IBD vaccination significantly enhanced humoral immune responses against subsequently applied live vaccines compared to non-HVT-IBD vaccinated groups at some of the investigated time points (P < 0.05). In addition HVT-IBD vaccination had depending on the genotype a significant impact on splenic macrophage as well as bursal CD4+ T-cell numbers (P < 0.05). On the other hand, the detectable genotype influence on Interferon (IFN) γ and nitric oxide (NO) release of ex vivo stimulated spleen cells was independent of HVT-IBD vaccination. The results of our study suggest considering a genotype specific vaccination regime in the field.

More than 100 isolates of a Gram-stain-negative, curved rod-shaped, so far uncultured bacterium were isolated from chicken caecal content. The strains were obligate anaerobes which formed endospores. They were catalase-positive, cytochrome-oxidase-negative and did not hydrolyse aesculin. The detected predominant metabolic end product in reinforced clostridial bedium broth was acetate. The DNA G+C content was 62.2 mol%. The cell fatty acid composition was dominated by C15 : 0 iso, C15 : 0anteiso, C16 : 0, C13 : 0 iso, C14 : 0 iso and C16 : 0DMA. Analysis of the 16S rRNA gene sequences of the novel isolates showed 99 % similarity to each other. Phylogenetically they are placed in Clostridium cluster IV. They exhibited 99 % 16S rRNA gene sequence similarity to uncultured bacterial clone SJTU_C_03_83 (GenBank accession no. EF404038.1) and to three other so far uncultured strains. The closest related type strains, Hydrogenoanaerobacterium saccharovorans SW51T and Anaerotruncus colihominis WAL 14565T, exhibited a 16S rRNA gene sequence divergence of at least 8 % to the newly described isolates. In addition, morphological and biochemical characteristics allowed differentiation of the novel isolates from these and other related and validly published species, including Clostridium methylpentosum and Acetanaerobacterium elongatum. Therefore, a new genus, Harryflintia gen. nov., is proposed for the isolated hitherto unknown strains with V20-281aT (=DSM 100433T =CECT 8892T) as the type strain of its type species, Harryflintia acetispora gen. nov., sp. nov.

Campylobacter jejuni (C. jejuni)-host-interaction may be affected by the maturation stage of the chicken's immune system and the developing gut microbiota composition. We compared these parameters between birds C. jejuni-inoculated at day one, 10, 22 and 31 post hatch. The highest C. jejuni-colonization rate and numbers of colony forming units (CFU) were detected in caecal content of day-one-inoculated birds while the lowest was detected in 22-days-old birds. The low bacterial colonization of 22-days-old chickens correlated with the most prominent immune reactions in this age group in comparison to other age groups. Age and C. jejuni-inoculation had a significant effect on lymphocyte numbers and cytokine expression levels in caecum as well as on gut flora composition. Overall, the immune response to C. jejuni is significantly influenced by the age of the infected chickens leading to differences in C. jejuni-colonization pattern between age goups.

The blood cell counting methods used for diagnostic and research purposes in turkeys are, up to date, inferior to the techniques established for mammals and chickens. While microscopic counts are time consuming, previous flow cytometric approaches did not cover all blood cell types of interest due to the lack of turkey-specific markers for the different cell populations. Moreover, it is unknown to what extent the different leukocyte populations are affected by host and environmental factors including age, breed of the bird, and housing environment, respectively. In this study we established a whole blood based flow cytometric analysis method for turkeys. This method was used to determine baseline values depending on the age of the birds as well as under consideration of variations between trials and animal room effects. During three trials whole blood samples of B.U.T. 6 female turkeys were collected to analyze different leukocyte concentrations (cells/μl whole blood). In the first trial one group and in the second and third trial two groups with 22 birds/group were housed. Blood samples were collected at days one, 23, 43, 60, and 88 post hatch and concentrations of monocytes, MHC class II-positive, CD4+, and CD8+ lymphocytes, as well as thrombocytes and granulocytes were determined by flow cytometric analysis. Concentrations of all identified populations were not only influenced by the bird's age (p < 0.05), they varied also among trials (p < 0.05) and even for some of the populations between animal rooms within the same trial despite comparable housing and management conditions. Therefore, for the establishment of baseline values for leucocyte concentrations in whole blood effects of age and housing have to be considered. In addition, our data emphasize the importance of the establishment of baseline values for different age groups, as age had the strongest effect on the blood cell numbers in this study.

Infectious bursal disease (IBD) is an economically important disease affecting poultry production worldwide. Previous experimental studies indicated that IBD live vaccination may induce transient immunosuppression, leading to suboptimal vaccine responses and therefore insufficient protection against other pathogens. Layer pullets are commonly not only vaccinated against IBD within their rearing period, but also against a variety of other pathogens. Therefore, it is of interest to investigate the effects of different IBD vaccination regimes on conventionally applied vaccines against other pathogens, and possible protection against widely spread very virulent IBD-virus (vvIBDV). A commercially available Herpesvirus of turkey vector vaccine (vHVT-IBD) expressing viral protein 2 of IBDV, and two IBD live vaccines were compared in commercial pullets for their effects on circulating B cell numbers, the ability of vaccinated birds to mount a humoral immune response against different antigens as well as their ability to induce protection against vvIBDV challenge. The results of this study demonstrate a clear immunosuppressive effect of the intermediate plus IBD live vaccine on the humoral branch of the immune system. On the other hand, no detectable effects of vHVT-IBD vaccination on these parameters were observed. All tested IBD vaccines protected against clinical IBD, although none induced sterile immunity in commercial layer pullets. vHVT-IBD-vaccinated birds showed significantly less lesions after vvIBDV challenge than IBD live-vaccinated or non-vaccinated birds (P

Respiratory infections are a common cause for increased mortality rates in poultry worldwide. To improve intervention strategies, circulating pathogens have to be identified and further characterized. Because of the lack of diagnostic tools, it was not known what pathogens contribute to the high mortality rates in association with respiratory disease in Algeria. Our objective was to determine if primary pathogens including Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), avian influenza virus (AIV), infectious bronchitis virus (IBV), and avian metapneumovirus (aMPV), known to be present in neighboring countries, can also be detected in Algerian chicken and turkey flocks. Results demonstrate the circulation of the investigated pathogens in Algerian poultry flocks as multi-infections. Phylogenetic characterization of the Algerian IBV strains confirmed the circulation of nephropathogenic viruses that are different from the strains isolated in neighboring countries. This could suggest the existence of a new IBV genotype in North Africa. Additionally, we detected for the first time an aMPV subtype B field strain and avian influenza virus. Interestingly, all viral pathogens were present in co-infections with MG, which could exacerbate clinical disease. Additional pathogens may be present and should be investigated in the future. Our results suggest that multiple respiratory infections may be responsible for high mortality in Algerian poultry flocks and very probably also in other regions of the world, which demonstrates the need for the establishment of more comprehensive control strategies.

Highlights • Infection of differentiated intestinal epithelial cells, influenza virus, receptors, sialic acid, cell surface. • PCIS can be used for virus cultivation. • PCIS can be used to analyze infection of target cells by intestinal viruses., Many viruses infect and replicate in their host via the intestinal tract, e.g. many picornaviruses, several coronaviruses and avian influenza viruses of waterfowl. To analyze infection of enterocytes is a challenging task as culture systems for differentiated intestinal epithelial cells are not readily available and often have a life span that is too short for infection studies. Precision-cut intestinal slices (PCIS) from chicken embryos were prepared and shown that the epithelial cells lining the lumen of the intestine are viable for up to 4 days. Using lectin staining, it was demonstrated that α2,3-linked sialic acids, the preferred receptor determinants of avian influenza viruses, are present on the apical side of the epithelial cells. Furthermore, the epithelial cells (at the tips) of the villi were shown to be susceptible to infection by an avian influenza virus of the H9N2 subtype. This culture system will be useful to analyze virus infection of intestinal epithelial cells and it should be applicable also to the intestine of other species.

Despite the importance of Bordetella avium (BA) as a respiratory pathogen of young turkeys, no infection model for the evaluation of BA-vaccine efficacy is available. The objective of this study was to evaluate the influence of route and dose of infection on the establishment of a BA-challenge model. In our first experiment, 28-day-old turkeys were either inoculated oculonasally with 10

Strains of a Gram-stain-negative, rod-shaped and immotile bacterium were isolated from broiler chicken caecal content. The isolates required strict anaerobic conditions for growth, formed spores, were catalase-positive and oxidase-negative. They produced butyrate as the major metabolic end product in reinforced clostridial medium broth. The genomic DNA G+C content of the isolated strains was 32.5–34.6 mol%. The major cellular fatty acids were C16 : 0 FAME, C14 : 0 FAME, C19 : 0CYC 9,10DMA and C16 : 0DMA. The fatty acid composition of the cell wall showed no similarity to any strain in the midi database. 16S rRNA gene sequence analysis showed that the nearest phylogenetic neighbours were Anaerostipes hadrus and Clostridium populeti (92 % sequence similarity) within Clostridium cluster XIVa of the phylum Firmicutes . Therefore, a novel genus is proposed, with the name Caecibacterium sporoformans gen. nov., sp. nov. The type strain of Caecibacterium sporoformans is LMG 27730T=DSM 26959T.

Campylobacter jejuni (C. jejuni) is a predominant cause of foodborne illness in humans, while its colonization in chickens is usually asymptomatic. Antibiotics are not routinely used to treat chickens against C. jejuni, but in the face of other bacterial diseases, C. jejuni may be exposed to antibiotics. In this study, chickens were treated with antibiotics (AT) to modify the gut microbiota composition and compared with untreated chickens (Conv) with respect to changes in C. jejuni-colonization and bacterial-intestine interaction. Groups of AT and Conv chickens were inoculated after an antibiotic-withdrawal time of eight days with one of three different C. jejuni isolates to identify possible strain variations. Significantly higher numbers of colony forming units of C. jejuni were detected in the cecal content of AT birds, with higher colonization rates in the spleen and liver compared to Conv birds independent of the inoculated strain (p < 0.05). Clinical signs and histopathological lesions were only observed in C. jejuni-inoculated AT birds. For the first time we demonstrated C. jejuni invasion of the cecal mucosa in AT chickens and its inter- and intracellular localization by using antigen-straining, and electronic microscopy. This study provides the first circumstantial evidence that antibiotic treatment with lasting modification of the microbiota may provide a suitable environment for C. jejuni invasion also in chickens which may subsequently increase the risk of C. jejuni-introduction into the food chain.

Enterococcus cecorum (EC) was thus far only known as a pathogen for broilers and broiler breeders. Recently there was evidence of EC field outbreaks in Pekin duck flocks in Germany. In this study we experimentally reproduced an EC infection in Pekin ducks. At 12 days post hatch, groups of Pekin ducks were infected orally, via the thoracic air sac or intravenously with 1.5 × 10(9) colony-forming units (CFU) of EC per bird or via the air sac with 8.5 × 10(5) or 8.5 × 10(7) CFU per bird. Ducks of the intravenously infected group showed 100% mortality after 2 days post infection. The air sac inoculated high-dose group exhibited a mortality rate of 67%. Birds that were infected with 8.5 × 10(5) and 8.5 × 10(7) CFU showed 6.7% mortality after 7 days post infection. Dead birds displayed pneumonia, airsacculitis, pericarditis and splenitis and EC was re-isolated from these organs. Surviving birds of all groups apart from the orally infected ducks demonstrated clinical signs such as huddling, reduced mobility and diarrhoea. Furthermore, they showed gross pathological lesions including airsacculitis and splenitis and lower bodyweights than the control group at necropsy on days 7, 14 and 21 post infection. The present study clearly confirms that EC is pathogenic for Pekin ducks after experimental infection via the intravenous route or the respiratory tract. EC therefore has to be considered as an emerging avian pathogen not only in broilers but also in Pekin ducks.

The young pigeon disease syndrome (YPDS) affects mainly young pigeons of less than one year of age and leads to crop stasis, vomitus, diarrhea, anorexia and occasionally death. This disease is internationally a major health problem because of its seasonal appearance during competitions such as homing pigeon races or exhibitions of ornamental birds. While the etiology of YPDS is still unclear, adenoviruses are frequently discussed as potential causative agents. Electron microscopy of feces from a YPDS outbreak revealed massive shedding of adenovirus-like particles. Whole genome sequencing of this sample identified a novel adenovirus tentatively named pigeon adenovirus 2 (PiAdV-2). Phylogenetic and comparative genome analysis suggest PiAdV-2 to belong to a new species within the genus Aviadenovirus, for which we propose the name Pigeon aviadenovirus B. The PiAdV-2 genome shares 54.9% nucleotide sequence identity with pigeon adenovirus 1 (PiAdV-1). In a screening of further YPDS-affected flocks two variants of PiAdV-2 (variant A and B) were detected which shared 97.6% nucleotide identity of partial polymerase sequences, but only 79.7% nucleotide identity of partial hexon sequences. The distribution of both PiAdV-2 variants was further investigated in fecal samples collected between 2008 and 2015 from healthy or YPDS-affected racing pigeons of different lofts. Independent of their health status, approximately 20% of young and 13% of adult pigeon flocks harbored PiAdV-2 variants. Birds were free of PiAdV-1 or other aviadenoviruses as determined by PCRs targeting the aviadenovirus polymerase or the PiAdV-1 fiber gene, respectively. In conclusion, there is no indication of a correlation between YPDS outbreaks and the presence of PiAdV-2 or other aviadenoviruses, arguing against an causative role in this disease complex.

Riemerella anatipestifer (RA) is an important avian pathogen with considerable impact on poultry production worldwide. However, the diagnosis of RA infections may be difficult, mainly due to problems with unequivocal differentiation of RA from other Flavobacteriaceae and a lack of standardized methods and reagents. The aim of the present study was therefore to complement the routine diagnostic strategies for RA by design and evaluation of alternative diagnostic tools. We designed and validated a new RA-specific polymerase chain reaction assay, which proved to be a valuable tool for the identification of RA isolates as well as for rapid and sensitive RA detection directly from diagnostic samples. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry fingerprinting of whole bacterial cells was also demonstrated to identify RA isolates efficiently. Furthermore, this method may also provide opportunities for RA subtyping. In our study, a stable subcluster was formed by the mass spectroscopy profiles of a group of RA isolates originating from turkey flocks in northern Germany, suggesting an epidemiological relationship of these isolates. Serotyping is a further important measure to characterize RA isolates. We tested a set of commercially available anti-RA sera with RA serotype reference strains and field isolates to allow comparison between these sera and reference sera. In summary, this report contributes to the improvement of present microbiological and molecular strategies for the diagnosis of RA infections by providing new tools as well as enhanced knowledge on existing methods.

Infectious bursal disease virus (IBDV) is an important immunosuppressive pathogen of chickens worldwide. The introduction and evolution of IBDV in most African countries, especially in Ethiopia, remains unclear. We have investigated IBDV isolates obtained from commercial broilers, indigenous chickens, and pullets. The hypervariable region of the virus protein (VP) 2 and the 5' two-thirds of VP1 of 11 IBDV isolates were characterized by RT-PCR and further sequencing. All isolates were identified as very virulent (vv) IBDV based on the predicted amino acid (aa) sequences of the VP2 protein. Interestingly, the sequence analysis of the 5' two-thirds of VP1 indicated that the Ethiopian IBDV strains have aa residues typical for vvIBDV and for attenuated IBDV strains. Among all IBDV strains included in this study for phylogenetic comparison of VP2 nucleotide sequences, Ethiopian strains form a cluster within the vvIBDV lineage. We have also shown that Ethiopian IBDV strains have mutations in the VP1 region. Their roles in IBDV virulence may require further in vivo studies. As depicted in this study, the nucleotide and aa sequence analysis of VP1 in addition to VP2 is necessary to obtain a clear picture of the molecular evolution of IBDV.

Little is known about the induction and modulation of gut-associated immune reactions after nematode infection in the chicken. The objective of the present study was to compare the pathogenesis, induction of immune reactions and electrophysiological changes of the gut after mono-infection with Heterakis gallinarum and after dual infection with H. gallinarum and Histomonas meleagridis in layer chickens. In two experiments 3-week-old chickens were inoculated with embryonated H. gallinarum eggs, which were positive for H. meleagridis. While birds of the first experiment were left untreated, those of the second experiment were treated with dimetridazol to prevent H. meleagridis co-infection. Mild to moderate histological lesions and local immune reactions with a significant increase in CD4(+), CD8α(+), TCRαβ(+) and TCRδγ(+) cells in the lamina propria and induction of the T-helper type 2 (Th2) cytokine interleukin-13 dominated the H. gallinarum immune response at 2 weeks post infection. Co-infection with H. gallinarum and H. meleagridis induced an increase in mRNA expression of the T-helper type 1 (Th1) cytokine interferon-γ, a decrease in splenic CD4(+) cells and severe destruction of the caecal mucosa in association with strong T-cell infiltration in the caecal lamina propria. There was no obvious effect on the chloride secretion of the caecal epithelium, which was investigated once the mucosa had almost recovered from the infection, in either experiment. These results suggest that the local T-cell reactions to nematode infections in chickens may be comparable with mammals and may be shifted from a Th2-dominated to a Th1-dominated response when accompanied by a protozoan infection.

Infections with avian Metapneumovirus (aMPV) are often associated with swollen head syndrome in meat type chickens. Previous studies in turkeys have demonstrated that local humoral and cell-mediated immunity plays a role in aMPV-infection. Previous experimental and field observations indicated that the susceptibility of broilers and their immune reactions to aMPV may differ from turkeys. In the presented study local and systemic immune reactions of broilers were investigated after experimental infections with subtypes A and B aMPV of turkey origin. Both virus subtypes induced a mild respiratory disease. The recovery from respiratory signs correlated with the induction of local and systemic aMPV virus-neutralizing antibodies, which began to rise at 6 days post infection (dpi), when the peak of clinical signs was observed. In a different manner to the virus neutralizing (VN) and IgG-ELISA serum antibody titres, which showed high levels until the end of the experiments between 24 and 28 dpi, the specific IgA-ELISA and VN-antibody levels in tracheal washes decreased by 10 and 14 dpi, respectively, which may explain the recurring aMPV-infections in the field. Ex vivo cultured spleen cells from aMPV-infected broilers released at 3 and 6 dpi higher levels of IFN-γ after stimulation with Concanavalin A as compared to virus-free birds. In agreement with studies in turkeys, aMPV-infected broilers showed a clear CD4+ T cell accumulation in the Harderian gland (HG) at 6 dpi (P

Nutrition of the host animal may not only influence interactions between the host and its parasites, but also relations between different parasites species residing on the same host. We investigated effects of insoluble and soluble non-starch polysaccharides (NSP) on establishment and development of Heterakis gallinarum in chicken being treated or left untreated against Histomonas meleagridis. Six groups of one-day-old birds were allocated to three diets, two on each diet. The birds were fed ad libitum either a basal diet (CON), or CON+insoluble NSP (I-NSP) or CON+soluble NSP (S-NSP) until an age of 11wk. At an age of 19d, one of each diet groups was prophylactically treated for 9d with dimetridazole (0.05%, w/v) via drinking water against histomonas. The remaining three groups were left un-treated. Two days after starting dimetridazole treatment (at 3wk), each of the 6 groups was divided into two sub-groups. One dimetridazole treated and one dimetridazole un-treated groups of birds on each diet (6 groups) were infected with 200 embryonated eggs of H. gallinarum that were previously harvested from histomonas-carrying H. gallinarum infected chickens. The remaining 6 groups of uninfected birds, either treated or left un-treated against H. meleagridis, served as controls. Worm burdens of infected birds were determined 8wk p.i. Treatment against H. meleagridis significantly increased incidence of H. gallinarum infection and average worm length in all infected groups independent of the diet consumed (p0.001). An interaction between effects of diet and dimetridazole treatment on worm burden (p0.001) indicated that the S-NSP diet resulted in lowest worm burden in dimetridazole un-treated birds, whereas it caused the highest worm burden in the treated birds (p0.05). Furthermore, the treatment resulted in higher worm burdens when compared to un-treated birds on the corresponding diets (p0.05). Infection with H. gallinarum impaired body weight (BW) of the chicks (p0.05) and H. meleagridis aggravated this effect (p0.05). Dimetridazole treated and un-treated uninfected birds developed similar BW (p0.05). Both NSP supplemented diets resulted in lower (p0.05) BW when compared with the CON diet, S-NSP being inferior to I-NSP (p0.05). It is concluded that H. meleagridis harms the definitive host as well as H. gallinarum. Both insoluble and soluble NSP supplemented diets favor H. gallinarum infection while S-NSP additionally intensifies histomonas infection, which then impairs establishment and development of H. gallinarum.

Two experiments were carried out to examine the effects of feeding an uncontaminated control diet (CON) or a Fusarium toxin-contaminated diet (FUS; 10.7 mg deoxynivalenol [DON]/kg diet) to growing broilers, which were either uninfected or infected with infectious bursal disease virus (IBDV) beginning at 1 day post hatch. Broilers had been infected at three weeks post hatch with either a classical virulent infectious bursal disease virus (IBDV-IM, Exp. 1) or a very virulent IBDV (vvIBDV, Exp. 2) strain. The effects of the DON-contaminated diet in combination with the virus-infection on the bursa of Fabricius and spleen were determined at 3 and 6-7 days post infection. The transient development of the bursa oedema and the bursa atrophy was not significantly affected by the diet after infection with the different IBDV-strains. The histopathological lesions were more severe in IBDV-IM-infected birds at 6 days post infection when additionally exposed to the FUS diet as compared to the FUS-free feed. Most parameters of the bursa of Fabricius and spleen protein turnover (e.g. fractional protein synthesis rate, protein, DNA and RNA content and derived indices) were significantly and interactively influenced by infection and stage of infection. The vvIBDV-infected birds responded with a more pronounced depressing effect on the fractional protein synthesis rate after feeding the DON-containing FUS diet when compared to their IBDV-IM-infected counterparts, where the opposite effect was observed. It can be concluded that feeding a FUS diet to IBDV-infected broilers might modulate the virulence-dependent pathogenesis of an IBDV infection.

Previous studies and field observations have suggested that genetic background influences infectious bursal disease virus (IBDV) pathogenesis. However, the influence of the virulence of the infecting IBDV strain and the mechanisms underlying the differences in susceptibility are not known. In the present study IBDV pathogenesis was compared between specific-pathogen-free layer-type (LT) chickens, which are the most susceptible chicken for IBDV and have been used as the model for pathogenesis studies, and broiler-type (BT) chickens, which are known to be less susceptible to clinical infectious bursal disease (IBD). The innate and acquired immune responses were investigated after inoculation of an intermediate (i), virulent (v) or very virulent (vv) strain of IBDV. IBDV pathogenesis was comparable among genetic backgrounds after infection with iIBDV. After infection with vIBDV and vvIBDV, LT birds showed severe clinical disease and mortality, higher bursal lesion scores and IBDV-antigen load relative to BT birds. Circulating cytokine induction varied significantly in both timing and quantity between LT and BT birds and among virus strains (P

Highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 cause fatal disease in poultry (fowl plague) but also have zoonotic potential. Currently commercially available vaccines often do not provide sufficient protection and do not allow easy discrimination between vaccinated and infected birds. Therefore, vaccination of domestic poultry against H5 and H7 HPAIV is not allowed in many countries, or is only possible after special permission has been provided. We generated a recombinant marker vaccine based on non-transmissible vesicular stomatitis virus (VSV) expressing the HA antigen of HPAIV A/FPV/Rostock/34 (H7N1) in place of the VSV G gene. This virus, VSV*DeltaG(HA), was propagated on a helper cell line providing VSV G in trans. Since no progeny virus was produced after infection of non-complementing cells, the vector was classified as biosafety level 1 organism ("safe"). Chickens were immunized via the intramuscular route. Following booster vaccination with the same replicons high titers of serum antibodies were induced, which neutralized avian influenza viruses of subtypes H7N1 and H7N7 but not H5N2. Vaccinated chickens were protected against a lethal dose of heterologous HPAIV A/chicken/Italy/445/99 (H7N1). Secretion of challenge virus was short-term and significantly reduced. Finally, it was possible to discriminate vaccinated chickens from infected ones by a simple ELISA assay. We propose that VSV replicons have the potential to be developed to high-quality vaccines for protection of poultry against different subtypes of avian influenza viruses.

Swollen head syndrome (SHS) associated with avian metapneumovirus (aMPV) subtype A or subtype B in broilers and broiler breeders has been reported worldwide. Data about pathogenesis of aMPV subtypes A and B in broilers are scarce. It has been difficult to reproduce swollen sinuses in chickens with aMPV under experimental conditions. In the field, SHS in broilers is suspected to be induced by combined infections with different respiratory pathogens. The objectives of the present study were to compare the pathogenesis of subtypes A and B aMPV in commercial broilers and to investigate the reproducibility of clinical disease. In two repeat experiments, commercial broilers free of aMPV maternal antibodies were inoculated with aMPV subtypes A and B of turkey origin. The clinical signs such as depression, coughing, nasal exudates, and frothy eyes appeared at 4 days post inoculation, followed by swelling of periorbital sinuses at 5 days post inoculation. Higher numbers of broilers showed clinical signs in subtype-B-inoculated compared with subtype-A-inoculated groups. Seroconversion to aMPV was detectable from 10 to 11 days post inoculation. The appearance of serum aMPV enzyme-linked immunosorbent assay antibodies and the clearance of the aMPV genome coincided. Subtype B aMPV showed a broader tissue distribution and longer persistence than subtype A. Histopathological changes were observed in the respiratory tract tissues of aMPV-inoculated broilers, and also in paraocular glands, such as the Harderian and lachrymal glands. Overall, our study shows that representative strains of both aMPV turkey isolates induced lesions in the respiratory tract, accompanied by swelling of infraorbital sinuses, indicating the role of aMPV as a primary pathogen for broilers.

Multiple respiratory infections have a significant impact on health and economy. Pathogenesis of co-infecting viruses and bacteria and their interaction with mucosal surfaces are poorly characterized. In this study we established a co-infection model based on pre-incubation of tracheal organ cultures (TOC) with Mycoplasma (M.) gallisepticum and a subsequent infection with avian influenza virus (AIV). Mycoplasma gallisepticum modified the pathogenesis of AIV as demonstrated in TOC of two different avian species (chickens and turkeys). Co-infection promoted bacterial growth in tracheal epithelium. Depending on the interaction time of M. gallisepticum with the host cells, AIV replication was either promoted or suppressed. M. gallisepticum inhibited the antiviral gene expression and affected AIV attachment to the host cell by desialylation of α-2,3 linked sialic acids. Ultrastructural analysis of co-infected TOC suggests that both pathogens may attach to and possibly infect the same epithelial cell. The obtained results contribute to better understanding of the interaction dynamics between M. gallisepticum and AIV. They highlight the importance of the time interval between infections as well as the biological properties of the involved pathogens as influencing factors in the outcome of respiratory infections.

Avian metapneumovirus (aMPV) is a pathogen with worldwide distribution, which can cause high economic losses in infected poultry. aMPV mainly causes infection of the upper respiratory tract in both chickens and turkeys, although turkeys seem to be more susceptible. Little is known about virus-host interactions at epithelial surfaces after aMPV infection. Tracheal organ cultures (TOC) are a suitable model to investigate virus-host interaction in the respiratory epithelium. Therefore, we investigated virus replication rates and lesion development in chicken and turkey TOC after infection with a virulent aMPV subtype A strain. Aspects of the innate immune response, such as interferon-α and inducible nitric oxide synthase mRNA expression, as well as virus-induced apoptosis were determined. The aMPV-replication rate was higher in turkey (TTOC) compared to chicken TOC (CTOC) (P

Campylobacter jejuni (C. jejuni) is the most frequently reported bacterial food-borne pathogen. Poultry is regarded to be the main reservoir for human campylobacteriosis. By some authors C. jejuni is considered as a commensal of the chicken gut, but clinical signs may be observed indicating host-pathogen interaction. Little is known about C. jejuni strain dependent differences in stimulation of the immune response in chicken. Therefore we investigated the colonization pattern as well as humoral and cell-mediated immune parameters in three-week old specific pathogen-free (SPF) layer-type (LT) chicken after inoculation with different human and avian derived C. jejuni isolates between three and 21dpi. In a subsequent experiment we investigated earlier time points and additionally compared commercial broilers with SPF-LT chicken to identify possible differences after inoculation of selected C. jejuni strains from prior experiments. No clinical symptoms were observed during the experiments. The colonization pattern did not correlate with the strain origin or magnitude of circulating C. jejuni specific antibodies. Only minor changes were observed in caecal T cell populations after C. jejuni inoculation compared to non-inoculated controls. Interestingly the mRNA expression of IL-6 and IFN-gamma was down regulated at some time points after inoculation suggesting a possible immunomodulatory effect of some C. jejuni strains in the gut. Furthermore, broilers were colonized to a higher extend and the local immune cell response was different compared to the SPF -LT birds indicating an influence of genotype on C. jejuni colonization pattern. Overall, our study demonstrates that the outcome of C. jejuni infection in chicken is influenced by genotype and partially by the C. jejuni isolate, leading to differences in the early immune response and thereafter the control of colonization and infection.

Until now, Enterococcus cecorum (EC) has been known as a pathogen for broilers, broiler breeders, and Pekin ducks. In the present report, we describe a fatal systemic EC infection in a young racing pigeon (Columba livia forma domestica). EC was isolated from the heart, liver, spleen, and intestine of the bird in pure culture. In the pathologic examination, the pigeon showed enteritis and an ulcerative gastritis, which may have been predisposing factors for the development of the generalized EC infection. An accumulation of gram-positive cocci in spleen tissue was found in the histopathologic examination and confirms the presence of a systemic EC infection in the pigeon. Additionally, EC was isolated from cloacal swabs of other pigeons in the same loft, but no additional pigeons were submitted for necropsy. All EC isolates tested were negative by PCR for the enterococcal virulence factors cytolysin, enterococcal surface protein, aggregation substance, hyaluronidase, and gelatinase. Therefore, the reason for the enhanced virulence of the EC isolate remains unknown. Our report confirms EC as a disease-causing agent in pigeons and presents the first data concerning the analysis of EC for virulence factors.

Influenza A viruses (IAVs) are the most relevant and continual source of severe infectious respiratory complications in humans and different animal species, especially poultry. Therefore, an efficient vaccination that elicits protective and neutralizing antibodies against the viral hemagglutinin (HA) and neuraminidase (NA) is an important strategy to counter annual epidemics or occasional pandemics. With the help of plasmid-based reverse genetics technology, it is possible that IAV vaccine strains (IVVS) are rapidly generated. However, the genetic instability of some cloned HA-cDNAs after transformation into competent bacteria represents a major obstacle. Herein, we report efficient cloning strategies of different genetically volatile HA segments (H5- and H9-subtypes) employing either a newly constructed vector for reverse genetics (pMKPccdB) or by the use of the Escherichia coli strain HB101. Both approaches represent improved and generalizable strategies to establish functional reverse genetics systems preventing genetic changes to the cloned (HA) segments of IAV facilitating more efficient rescue of recombinant IAV for basic research and vaccine development.

The role of cell-mediated immunity (CMI) in pathogenesis of infectious bursal disease virus (IBDV) was investigated. One-day-old specific pathogen-free chickens were treated with 3 mg of cyclophosphamide (Cy) per chicken for 4 consecutive days and, 3 weeks later, infected with the IBDV-IM strain. Chickens were examined for: (a) mitogenic response of splenocytes to ConA, as an indicator of T-cell functions in vitro, (b) antibody against IBDV by ELISA, (c) IBDV genome in various tissues by RT-PCR and (d) immunological memory. At the time of IBDV infection, Cy-treated chickens had depleted bursal tissue (an avian primary B-cell lymphoid organ), severely compromised antibody-producing ability, but normal T-cell response to ConA. In primary infection, no detectable antibody against IBDV antigen in Cy-treated, IBDV-infected chickens was observed up to 28 days post-infection (PI), while IBDV genome was detected by RT-PCR in spleen, thymus, liver and blood until 10 days PI. Like intact control chickens infected with IBDV, Cy-treated, IBDV-infected chickens suppressed splenocytes responses to ConA from 5 to 10 days PI, suggesting that intact control as well as Cy-treated chickens responded similarly to IBDV infection in the early phase. Following re-infection with IBDV, no detectable secondary antibody response to IBDV as well as IBDV genome in tissues were observed in Cy-treated chickens, while intact control chickens developed vigorous secondary antibody response. Similar to intact control chickens infected with IBDV, Cy-treated chickens after second infection with IBDV did not suppress splenocyte response to ConA. These results suggested that in the absence of detectable anti-IBDV antibodies, protection of Cy-treated chickens from IBDV infection may occur via immunological memory mediated by CMI. We concluded that under normal conditions, IBDV induces a protective antibody response, however, in the absence of antibody, CMI alone is adequate in protecting birds against virulent IBDV.

A multivalent in ovo vaccine (MIV) was tested for safety and efficacy in a commercial broiler complex. The MIV comprised five replicating live viruses including serotypes 1, 2, and 3 of Marek's disease virus (MDV), an intermediate infectious bursal disease virus (IBDV) and a recombinant fowl poxvirus (FPV) vector vaccine containing HN and F genes of Newcastle disease virus (NDV). The performance of MIV-vaccinated broilers was compared with that of hatchmates that received turkey herpesvirus (HVT) alone (routinely used in ovo vaccine in the broiler complex). The chickens that hatched from the MIV-injected and HVT-injected eggs were raised under commercial conditions in six barns. Barn 1 housed 17,853 MIV-vaccinated chickens and each of the barns 2-6 housed 18,472-22,798 HVT-vaccinated chickens. The HVT-vaccinated chickens were given infectious bronchitis virus (IBV) and NDV vaccines at hatch and at 2 wk of age. The MIV-vaccinated chickens received IBV vaccine at hatch and IBV + NDV at 2 wk of age. The relative values of hatchability of eggs, livability and weight gain of chickens, and condemnation rates at processing were comparable between the MIV and the HVT groups (P > 0.05). Chickens from the MIV- and the HVT-vaccinated groups were challenged with virulent viruses under laboratory conditions. The resistance of vaccinated chickens against Marek's disease could not be assessed because of high natural resistance of unvaccinated commercial broilers to virulent MDV. The relative resistances of the MIV- and the HVT-vaccinated groups, respectively, against other virulent viruses were as follows: IBDV, 100% for both groups; NDV, 81% vs. 19%; FPV, 86% vs. 0%. The successful use of MIV under field conditions expands the usefulness of the in ovo technology for poultry.

SUMMARY. Avian pneumovirus (APV) is an immunosuppressive respiratory pathogen of turkeys. We examined the effect of APV infection on the vaccine efficacy of hemorrhagic enteritis virus (HEV) vaccines. APV was inoculated in 2-wk-old turkeys. Two or four days later, an attenuated HEV vaccine (HEVp30) or marble spleen disease virus (MSDV) vaccine were administered. Virulent HEV challenge was given 19 days after HEV vaccination. APV exposure compromised the ability of HEVp30 and MSDV to protect turkeys against virulent HEV. The protective index values were as follows: MSDV (100%) versus APV + MSDV (0%) (P < 0.05); HEVp30 (60%) versus APV + HEVp30 (30%) (P < 0.05) (Experiment I) and HEVp30 (56%) versus APV + HEVp30 (20%) (P < 0.05) (Experiment II). These data indicated that APV reduced the efficacy of HEV vaccines in turkeys.

Infectious bursal disease virus (IBDV) induces an acute, highly contagious immunosuppressive disease in young chickens. We examined the role of T cells in IBDV-induced immunopathogenesis and tissue recovery. T cell-intact chickens and birds compromised in their T cell function by a combination of surgical thymectomy and Cyclosporin A treatment (Tx-CsA) were infected with an intermediate vaccine strain of IBDV (Bursine 2, Fort Dodge). Our data revealed that functional T cells were needed to control the IBDV-antigen load in the acute phase of infection at 5 days post infection. The target organ of IBDV, the bursa of Fabricius, of Tx-CsA-birds had a significantly higher antigen load than the one of T cell-intact birds (P0.05). Tx-CsA-treatment abrogated the IBDV-induced inflammatory response and significantly (P0.05) reduced the incidence of apoptotic bursa cells and the expression of cytokines such as interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) in comparison to T cell-intact birds. T cell-released IL-2 and IFN-gamma may have mediated the induction of inflammation and cell death in T cell-intact birds. The IBDV-induced upregulation of tumor necrosis like-factor (TNF) expression was comparable between T cell-intact and Tx-CsA-birds. Tx-CsA-birds showed a significantly faster resolution of IBDV-induced bursa lesions than T cell-intact birds (P0.05). This study suggests that T cells modulate IBDV pathogenesis in two ways: a) they limit viral replication in the bursa in the early phase of the disease at 5 days post infection, and b) intrabursal T cells promote bursal tissue damage and delay tissue recovery possibly through the release of cytokines and cytotoxic effects.

International audience; The avian coronavirus (AvCoV) infectious bronchitis virus (IBV) is a major poultry pathogen. A characteristic feature of IBV is the occurrence of many different strains belonging to different serotypes, which makes a complete control of the disease by vaccinations a challenging task. Reasons for differences in the tissue tropism and pathogenicity between IBV strains, e.g. a predilection for the kidneys or the oviduct are still an open question. Strains of the QX genotype have been major pathogens in poultry flocks in Asia, Europe and other parts of the world. They are the cause of severe problems with kidney disease and reproductive tract disorders. We analysed infectivity and binding properties of the QX strain and compared them with those of the nephropathogenic strain B1648. As most IBV strains do not infect permanent cell lines and show infection only in primary chicken cells of the target organs, we developed a culture system for chicken oviduct explants. The epithelial cells of the oviduct showed a high susceptibility to infection by the QX strain and were almost resistant to infection by the nephropathogenic B1648 strain. Binding tests with isolated primary oviduct epithelial cells and soluble S1 proteins revealed that S1 proteins of two IBV strains bound with the same efficiency to oviduct epithelial cells. This attachment was sialic acid dependent, indicating that the sugar binding property of IBV spike proteins is not the limiting factor for differences in infection efficiency for the oviduct of the corresponding viruses.

Congo African Grey Parrots (GP; Psittacus erithacus erithacus) from four different avicultures, presented in the Clinic for Exotic Pets, Reptiles and Birds, University of Veterinary Medicine Hannover, Foundation, showed choanal papillomas or hyperemia of the cloacal mucosa. Histologically, the mucosal choanal proliferations were diagnosed as exophytic papillomas and a mild hyperplasia of the cloacal mucosa with lympho-histiocytic inflammation with no visible inclusion bodies was found. Herpesvirus genome was detected by nested PCR in pooled choanal and cloacal swabs from clinically diseased parrots and healthy contact animals. Sequencing of parts of the herpesvirus DNA-polymerase gene indicated 98-100% homology of the detected herpesviruses with the Psittacid Herpesvirus 2 (PsHV-2). In one aviculture with cloacal inflammation papillomavirus-DNA was concurrently found to a PsHV-2 infection. In addition to the four avicultures with clinical symptoms 25 more flocks of grey parrots, in total 57 Congo-GP and 13 Timneh-GP, were examined for a herpesvirus infection. A total of six out of 29 studied parrot avicultures were tested positive for PsHV-2. The detection of this virus also in flocks of GP, which were bred in Europe, shows the establishment of this infection in the GP population in captivity. As indicated in the literature as well as in our study PsHV-2 could be only detected in Congo-GP, independently if they were kept either alone or in mixed avicultures with amazon and macaw species. These findings suggest that PsHV-2 is adapted to this Psittacus species.

Wild-type mammalian adenoviruses are known to inhibit programmed cells death in infected cells. This study demonstrated for the first time that an avian type II adenovirus, the hemorrhagic enteritis virus (HEV) of turkeys, induced apoptosis in turkey spleen cells at 3 and 4 days post infection. The increased apoptosis rate in spleens of HEV-infected turkeys was associated with increased virus replication. Increased apoptosis preceded extensive virus-induced cellular necrosis. At 3 days post infection, spleen cells from HEV-infected turkeys released tumor necrosis like factor and nitric oxide inducing factors after ex vivo stimulation with concanavalin A. Spleen cells from HEV-exposed turkeys also secreted an interleukin 6-like factor when cultured in vitro. These cytokines may have contributed to HEV-pathogenesis and HEV-induced apoptosis and necrosis in the spleen. Induction of apoptosis by an avian adenovirus but not by wild-type mammalian adenoviruses indicates that evolutionarily distant adenoviruses may have different pathogenic mechanisms.

In this study we show that a type I-IFN inducing compound, S-28828, modulated the pathogenesis of an avian type II adenovirus in turkeys. By itself, S-28828 induced a strong reaction in the spleen characterized by hyperplasia of the red and white pulps as well as an increase in lymphoid cell aggregations. Oral administration of S-28828 before the time of virus inoculation suppressed significantly (P

A synthetic imidazoquinolinamine, S-28828, has been shown to be an effective antiviral and antitumor agent in mammals. This immune modifier induces a number of cytokines such as interferons, tumor necrosis factor-alpha, interleukins and granulocyte-macrophage colony-stimulating factors in mammals. We showed that when turkeys were given S-28828 orally, high serum titers of IFN were induced in a dose-dependent manner. Turkeys, once stimulated by S-28828, became refractory to IFN production by repeated stimulation. S-28828 induced spleen, bone marrow and peripheral leukocytes to produce IFN in vitro. Splenic adherent cells were the main producers of IFN after in vitro stimulation. S-28828-induced IFN was identified as type I IFN that was pH-resistant but heat-labile. We examined the effect of a high dose (100 mg kg(-1) body weight) of S-28828 on the pathogenesis of E. coli in turkeys. Treatment with S-28828 increased mortality in infected birds and impaired E. coli clearance from the liver. The enhancement of the pathogenicity of E. coli by S-28828 may have been due to the massive release of cytokines inducing a shock-like syndrome in infected turkeys.

Clinically healthy homing pigeons may serve as an unnoticed reservoir for zoonotic bacteria. Hence, healthy pigeons from 172 different racing pigeon lofts were examined for Salmonella serovars, Campylobacter spp. and Chlamydophila (Chlamydia) psittaci. Two samplings were performed during the racing season in summer (1242 adult and 1164 juvenile pigeons) and two during winter (1074 adult pigeons). Each sampling was accompanied by a questionnaire to identify risk factors for positive lofts. Between 0.9 and 3.7%, 13.1 and 23.7%, and 12.8 and 42.6% of lofts were tested positive by cultural methods or polymerase chain reaction for Salmonella Typhimurium var. Copenhagen, Campylobacter jejuni and C. psittaci, respectively. The detection rate of C. psittaci was twice as high in samples from juvenile pigeons (29.1%) compared with samples from adult pigeons (15.0%, P

Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens. The virus protein (VP) 2 induces neutralizing antibodies, which protect chickens against the disease. The aim of this study was to develop a cationic poly(d,l-lactide-co-glycolide) (PLGA) microparticle (MP) based IBDV-VP2 DNA vaccine (MP-IBDV-DNA) for chickens to be delivered orally and by eye drop route. The tested IBDV-VP2 DNA vaccines were immunogenic for specific-pathogen-free chickens and induced an antibody response after intramuscular application. Co-inoculation with a plasmid encoding chicken IL-2 (chIL-2) or CpG-ODN did not significantly improve protection against IBDV challenge. However, the application of a MP-IBDV-DNA vaccine alone or in combination with a delayed oral and eye drop application of cationic MP loaded with CpG-ODN or chIL-2 improved protection against challenge. The MP-IBDV-DNA-vaccinated chickens showed less pathological and histopathological bursal lesions, a reduced IBDV antigen load as well as T-cell influx into the bursa of Fabricius (BF) compared to the other groups (p