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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 in vivo conditions, which will help to understand the complex virus-host interactions further.

Enterococcus cecorum (EC) is known to cause skeletal lesions in broiler chickens and also systemic infections in Pekin ducks. Despite the importance of the pathogen, there is still a lack of serological diagnostic tools for the detection of EC infections. Here we describe the development of an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the detection of EC-specific antibodies and its application by examination of 67 sera from experimentally infected Pekin ducks, 710 field samples from four Pekin duck breeder flocks previously vaccinated with inactivated vaccines, and 80 samples from commercial Pekin ducks coming from vaccinated parent flocks. All groups that had been experimentally inoculated via the air sac route were positive in the new ELISA, with significantly ( P  ≤ 0.05) increased mean sample/positive (S/P) ratios of 0.71-2.70 at days 7, 14 and 21 post-infection, while orally inoculated ducks and the EC-free control group remained negative with mean S/P ratios of 0.0-0.15. Antibodies were also detected in each of four vaccinated Pekin duck breeder flocks; 67.8% of the samples were antibody positive. The highest S/P ratios were found between 16 and 26 weeks (median S/P ratios from 0.15 to 1.03), but antibodies were still detected in some serum samples in weeks 61-67 post-hatch. No antibodies were detected in the commercial Pekin ducks. Antibody development in the ducks may be influenced by the composition of the inactivated vaccine. The new ELISA provides a useful tool for investigations of response to EC infections and vaccinations.

Bordetella avium (BA) is a respiratory pathogen of particular importance for turkeys. Specific adherence and damage to the respiratory epithelia are crucial steps of the pathogenesis, but knowledge about the mechanisms and the variety of virulence in field strains is limited. We analysed 17 BA field strains regarding their in vitro virulence-associated properties in tracheal organ cultures (TOC) of turkey embryos, and their genetic diversity. The TOC adherence assay indicated that BA field strains differ considerably in their ability to adhere to the tracheal mucosa, while the TOC ciliostasis assay illustrated a high degree of diversity in ciliostatic effects. These two virulence-associated properties were associated with each other in the investigated strains. Three of the investigated strains displayed significantly ( P  > 0.05) lower in vitro virulence in comparison to other strains. Genetic diversity of BA strains was analysed by core genome multilocus sequence typing (cgMLST). We applied a cgMLST scheme comprising 2667 targets of the reference genome (77.3% of complete genome, BA strain 197N). The results showed a broad genetic diversity in BA field strains but did not demonstrate a correlation between sequence type and virulence-associated properties. The cgMLST analysis revealed that strains with less marked virulence-associated properties had a variety of mutations in the putative filamentous haemagglutinin gene. Likewise, amino acid sequence alignment indicated variations in the protein. The results from our study showed that both adherence and ciliostasis assay can be used for virulence characterization of BA. Variations in the filamentous haemagglutinin protein may be responsible for reduced virulence of BA field strains.

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 5 , 10 7 or 10 9 colony forming units (CFU) of BA per bird or exposed to BA by aerosol with 10 5 -10 8  CFU/m 3 . The respiratory tract of all inoculated birds was BA-colonized, which was confirmed by choanal swabs and samples of trachea and lung, showing the highest prevalence in the aerosol-inoculated group. BA-specific humoral immune response was detected in the form of IgG in serum from five days post infection (dpi) and IgA in lacrimal fluid from seven dpi. In the second experiment, the model was tested in a vaccination trial. Twenty-one-day-old turkeys were vaccinated with a formalin-inactivated BA vaccine intramuscularly and challenged 21 days post vaccination with 10 7  CFU per bird oculonasally. BA-specific IgG antibodies were detected in serum and in lacrimal fluid 14 days post vaccination. As in the first experiment, secretory BA-specific antibodies of the IgA isotype were only detected in the inoculated groups from seven dpi. Despite the lack of clinical signs or pathological alterations in both experiments, vaccine efficacy was demonstrated by significant reduction in BA colonization of the trachea (P ≤ 0.05). In our study, a reliable model for BA infection has been established and has been demonstrated to be suitable for evaluation of vaccine efficacy.

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 < 0.05). Therefore, vHVT-IBD may be a suitable alternative to conventional IBD live vaccines, and may be applied even in the presence of maternally derived IBD antibodies without induction of detectable humoral immunosuppression.

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 < 0.05), providing circumstantial evidence that indeed turkeys may be more susceptible. The interferon-α response was down-regulated from 2 to 144 hours post infection in both species compared to virus-free controls (P < 0.05); this was more significant for CTOC than TTOC. Inducible nitric oxide synthase expression was significantly up-regulated in aMPV-A-infected TTOC and CTOC compared to virus-free controls (P < 0.05). However, the results suggest that NO may play a different role in aMPV pathogenesis between turkeys and chickens as indicated by differences in apoptosis rate and lesion development between species. Overall, our study reveals differences in innate immune response regulation and therefore may explain differences in aMPV - A replication rates between infected TTOC and CTOC, which subsequently lead to more severe clinical signs and a higher rate of secondary infections in turkeys.

Clinically healthy racing pigeons may harbour notifiable pathogens and serve as an unnoticed reservoir. Thus, 3480 healthy racing pigeons from 172 different lofts were monitored over a period of 2 years for the presence of avian influenza virus (AIV) and avian paramyxovirus-1 (APMV-1). Pharyngeal and cloacal swabs as well as blood samples were collected from juvenile and adult pigeons. Pools of five pharyngeal swabs per loft and age group were initially screened by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). Pharyngeal and cloacal samples from lofts that were positive or suspect in the AIV rRT-PCR or the APMV-1 rRT-PCR were inoculated into embryonated chicken eggs for virus isolation. In addition, sera were examined for antibodies against AIV by enzyme-linked immunosorbent assay. The antibody levels after vaccination against APMV-1 were determined by haemagglutination inhibition assay. Of the investigated lofts, 0.0 to 1.4% were positive by rRT-PCR for APMV-1 and 0.0 to 6.7% for AIV during this 2-year period with a total of four samplings. No sample yielded replicating virus in egg culture. No antibodies against AIV were detected. Haemagglutination inhibition test of vaccinated racing pigeons indicated age-dependent APMV-1 titres. The results suggest that the examined racing pigeons may have had contact with AIV, but virus replication may have been too low to induce detectable circulating antibody levels. Only a low percentage of samples were positive for APMV-1, but two outbreaks were observed in monitored flocks, indicating ongoing circulation of APMV-1 in the racing pigeon population. These observations highlight the relevance of APMV-1 vaccination and indicate the importance of flock immunity.

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.

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.

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 <0.001). No other influence of age or season was detected. For the first time, pigeon-derived C. jejuni isolates (n=15) were characterized for their ability to invade human enterocytes in vitro. All isolates were invasive with an invasion index between 0.4 and 34.1 (human reference strain: average 11.3). Of 50 C. jejuni isolates tested for antimicrobial susceptibility, 46.0% were resistant to ciprofloxacin. All isolates were sensitive to erythromycin and tetracycline. The analysis of risk factors in association with the infection status of lofts for C. jejuni and C. psittaci suggested that biosecurity measures reduce the risk of infection. This study indicated a zoonotic potential of pigeon-derived C. jejuni. However, clinically healthy homing pigeons pose only a low risk for transmission of the investigated pathogens to humans.

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.

Avian metapneumovirus (aMPV) is an important respiratory pathogen of turkeys with considerable economic impact on poultry production. Although vaccination is widely used for the control of the disease, questions regarding vaccine safety and efficacy remain to be elucidated. This report describes the problems associated with reproducibility of the aMPV-vaccine response, comparing T-lymphocyte-compromised and T-cell-intact turkeys. In three consecutive experiments, turkeys partially depleted of T-lymphocytes by treatment with cyclosporin A as well as untreated turkeys were vaccinated with a commercial live aMPV subtype A (aMPV-A) vaccine at 2 weeks of age. Two weeks later they were challenged with a virulent aMPV-A strain. Despite similar genetic background of the turkeys, comparable housing conditions under isolation and the application of the same aMPV-A vaccine, considerable variation was observed among the experiments regarding replication of the vaccine virus, vaccine-induced clinical signs and protection against challenge infection. The results indicate that differences in the outcome of aMPV-A vaccination may be associated with T-lymphocyte suppression and additionally with an interfering aMPV-B vaccine exposure at the hatchery in two of the experiments. Our study provides possible explanations for the variable protection provided by aMPV vaccines under field conditions.

Riemerella anatipestifer (RA) is the causative agent of septicaemic and exudative diseases in a variety of bird species. Despite numerous outbreaks, little is known about the pathogenicity of RA for turkeys. We investigated the development of RA-induced disease in commercial turkey poults following RA inoculation via different respiratory routes. Inoculation by aerosol or injection into the abdominal air sac led to systemic infection and mild gross lesions, including pericarditis, epicarditis and airsacculitis, which were less pronounced compared with field outbreaks. It was speculated, that viral pathogens, such as the avian metapneumovirus (aMPV), may exacerbate RA pathogenesis under field conditions. We inoculated turkey poults with virulent aMPV. Subsequently, aMPV-infected and virus-free birds were exposed 3 to 5 days later to a high dose of RA by aerosol (>10(10) colony-forming units/ml in 8 ml aerosol per 11 or 12 birds) or were inoculated 4 days later with a low RA dose (10(4.9) colony-forming units per bird) via the intranasal route. Intranasal RA inoculation with the low bacterial dose led to a respiratory and systemic RA infection in aMPV-infected birds, while virus-free birds remained RA-negative. Following exposure to a high RA dose by aerosol, aMPV-infected groups showed slightly enhanced incidences of gross lesions and RA re-isolation. The present study clearly confirms that RA is pathogenic for turkeys after experimental inoculation via respiratory routes, which are speculated to be the natural route of infection. However, experimental models in this study did not reproduce the severity of RA-related disease as observed under field conditions, which emphasizes the importance of other contributing factors. aMPV-induced respiratory lesions may serve as a predisposing factor for the establishment of RA infection, since they favour colonization of the bacterium.

The avian metapneumovirus (aMPV) is the causative agent of an acute respiratory disease in turkeys, which causes considerable economic losses to the poultry industry. Currently attenuated live and inactivated vaccines are widely used to control the disease, but vaccine breaks are frequently observed. For improvement of current vaccination strategies it is necessary to gain enhanced knowledge of the immune mechanisms against aMPV infection. Field observations suggest that vaccine-induced aMPV-specific antibodies are not indicative for protection. In the present study we investigated the role of antibodies in protection of turkeys against aMPV. In two experiments, commercial turkey poults received aMPV-specific antibodies by intravenous injection. The antibody transfer resulted in increased antibody levels in the sera. Virus-specific antibodies were also detected on mucosal surfaces such as the trachea, conjunctivae and gall bladder. Turkeys were subsequently challenged with a virulent aMPV subtype A strain. Development of clinical signs, virus detection by polymerase chain reaction and histopathological changes of tracheal mucosa in challenged turkeys with and without passively transferred antibodies were comparable with each other. Our results suggest that humoral immunity does not provide sufficient protection against aMPV infection. Thus, the measurement of vaccine-induced aMPV antibody response may not be considered as an adequate indicator of vaccine efficacy. Further research on the protective role of cell-mediated immune mechanisms is necessary to improve current vaccine strategies.

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.

The right strategy for infectious bursal disease (IBD) control and its success rate under field conditions depends on hygiene management, IBD field pressure, level and variation in maternally derived IBD antibodies, and the IBD vaccine strains to be used. Usually, standard vaccination programmes are used, which are not always adapted to the specific conditions on the farm and to the immune status of chickens. Employing the "Deventer formula" may help to estimate the optimal time for vaccination for a specific flock based on the maternally derived antibody level, its variation, the genetic background of the chicken, and the IBD vaccine strain. Two field studies with 16 or 20 commercial broiler flocks were conducted, applying an intermediate IBD vaccine before, at the best, and after the estimated optimal vaccination time estimated by the "Deventer formula". These studies showed that flocks IBD-vaccinated between 1 day before, at, or up to 3 days after the estimated optimal time point developed detectable humoral immunity up to 14 days post vaccination. If birds had been vaccinated more than 1 day before the calculated optimal vaccination date, the humoral immune response was delayed or non-detectable until slaughter. The induction of humoral immunity correlated with the incidence of bursa lesions and IBDV detection by reverse transcriptase-polymerase chain reaction. As indicated in this study, under field conditions bursa lesions may develop later than predicted based on experimental experiences. The late incidence of bursa lesions after vaccination may be confused with field virus-induced lesions, in which case sequencing may offer a valuable tool for differentiation.