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Abstract Background Campylobacter (C.) jejuni is one of the most important bacterial foodborne pathogens worldwide. Probiotics such as Lactobacillus or Bacillus species are considered one option for reducing the colonization rate and magnitude in poultry, the most frequent source of human infections. Due to the lack of suitable avian in vitro models such as chicken intestinal cell lines, especially those derived from the cecum, most in vitro studies on C. jejuni host interaction have been conducted with human intestinal cell lines. In this study, we compared C. jejuni-cell interactions between primary chicken cecal cells and the human intestinal cell line Caco-2, which is derived from colorectal adenocarcinoma, and investigated possible interfering effects of selected probiotic candidates. Results We detected differences in adhesion and invasion between the two tested gut cell types and between different C. jejuni strains. The probiotic inhibition of C. jejuni adhesion and invasion of human and avian gut cells was affected by host cell type, investigated C. jejuni strain and time points of probiotic treatment. Additionally, our results suggest a possible correlation between C. jejuni invasion and the detected increase in IL-6 mRNA expression. Conclusions Our results indicate distinct differences between avian and human gut cells in their interaction with C. jejuni. Therefore, data obtained in one host species on C. jejuni-host interaction may not easily be transferrable to another one. The factors influencing the variable efficacy of probiotic intervention in chicken and human derived cells should be investigated further.

While B cell development in the birds’ primary B cell organ, the bursa Fabricius, is relatively well understood, very little is known about post bursal B cell differentiation into plasma and memory cells though these cells are essential for a protecting antibody response and so far, no specific markers for these cells were available. Since immunoglobulin class switch is one part of the B cell differentiation process, our objective was to conduct a first detailed investigation of class-switched chicken B cells. As only very few IgY and IgA expressing cells were detected in lymphoid organs of young chickens, we used CD40L and IL-10 to establish a prolonged in vitro culture system, which induces B cell proliferation, class switch to IgY and IgA and enhanced antibody secretion. This enabled a phenotypic analysis of differentiating B cells. Importantly, these cells lost surface expression of the B cell markers chB6 and BAFF-R. B cell receptor surface expression remained unchanged, showing that while differentiating toward plasma cells, B cells can be addressed by L chain staining. Newly generated potential plasma cell markers CD138 and TACI showed only a transient expression on cultured cells and rather act as markers for B cell activation than plasma/memory cells in general. CD57 upregulation was connected to activation and blast formation but not to class switch. We also examined potential changes in class-switched cells in different age groups and post vaccination. Surprisingly, bursa involution, laying and age had no distinct effects on the presence of class-switched cells, but we detected significantly more class-switched B cells post vaccination. Hence, we are now able to generate class-switched plasmablasts in vitro for a more detailed characterization and can address them under different conditions in chickens for further analysis of their B cell response.

Abstract Enterococcus cecorum (EC) is one of the most relevant bacterial pathogens in modern broiler chicken production from an economic and animal welfare perspective. Although EC pathogenesis is generally well described, predisposing factors are still unknown. This study aimed to understand the effect of heat stress on the caecal microbiota, intestinal integrity, and EC pathogenesis. A total of 373 1-day-old commercial broiler chicks were randomly assigned to four groups: (1) noninoculated, thermoneutral conditions (TN); (2) noninoculated, heat stress conditions (HS); (3) EC-inoculated, thermoneutral conditions (TN + EC); and (4) EC-inoculated, heat stress conditions (HS + EC). Birds were monitored daily for clinical signs. Necropsy of 20 broilers per group was performed at 7, 14, 21, and 42 days post-hatch (dph). A trend towards enhanced and more pronounced clinical disease was observed in the EC-inoculated, heat-stressed group. EC detection rates in extraintestinal tissues via culture were higher in the HS + EC group (~19%) than in the TN + EC group (~11%). Significantly more birds were colonized by EC at 7 dph in the HS + EC group (100%) than in the TN + EC group (65%, p

Abstract 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.

Abstract Background Campylobacter (C.) species are the most common bacterial cause of foodborne diarrhea in humans. Despite colonization, most animals do not show clinical signs, making recognition of affected flocks and disruption of the infection chain before slaughter challenging. Turkeys are often cocolonized with C. jejuni and C. coli. To understand the pathogen-host-interaction in the context of two different Campylobacter species, we compared the colonization patterns and quantities in mono- and co-colonized female commercial turkeys. In three repeated experiments we investigated the impact on gut morphology, functional integrity, and microbiota composition as parameters of gut health at seven, 14, and 28 days post-inoculation. Results Despite successful Campylobacter colonization, clinical signs or pathological lesions were not observed. C. coli persistently colonized the distal intestinal tract and at a higher load compared to C. jejuni. Both strains were isolated from livers and spleens, occurring more frequently in C. jejuni- and co-inoculated turkeys. Especially in C. jejuni-positive animals, translocation was accompanied by local heterophil infiltration, villus blunting, and shallower crypts. Increased permeability and lower electrogenic ion transport of the cecal mucosa were also observed. A lower relative abundance of Clostridia UCG-014, Lachnospiraceae, and Lactobacillaceae was noted in all inoculated groups compared to controls. Conclusions In sum, C. jejuni affects gut health and may interfere with productivity in turkeys. Despite a higher cecal load, the impact of C. coli on investigated parameters was less pronounced. Interestingly, gut morphology and functional integrity were also less affected in co-inoculated animals while the C. jejuni load decreased over time, suggesting C. coli may outcompete C. jejuni. Since a microbiota shift was observed in all inoculated groups, future Campylobacter intervention strategies may involve stabilization of the gut microbiota, making it more resilient to Campylobacter colonization in the first place.

Abstract Background Enterococcus cecorum (EC) is one of the main reasons for skeletal disease in meat type chickens. Intervention strategies are still rare and focus mainly on early antibiotic treatment of the disease, although there are no data available concerning the effectivity of this procedure. The present study aimed to investigate the effectivity of early lincomycin-spectinomycin treatment during the first week of life after EC-infection. Furthermore, the impact of lincomycin-spectinomycin treatment and EC infection on the development of cecal microbiota was investigated. Methods A total of 383 day-old broiler chicks were randomly assigned to four groups (non-infected and non-treated, non-infected and treated, EC-infected and non-treated, and EC-infected and treated). The EC-infected groups were inoculated orally with an EC suspension at the day of arrival and at study day 3. The treatment groups were treated with lincomycin-spectinomycin via the drinking water for six consecutive days, starting two hours after the first inoculation. Necropsy of 20 chickens per group was performed at study days 7, 14, 21, and 42. Bacteriological examination via culture and real-time PCR was performed to detect EC in different extraintestinal organs. Cecal samples of nine chickens per group and necropsy day were analyzed to characterize the composition of the cecal microbiota. Results No clinical signs or pathologic lesions were found at necropsy, and EC was not detected in extraintestinal organs of the EC-infected and treated birds. Lincomycin-spectinomycin promoted the growth of the bacterial genus Escherichia/Shigella and reduced the amount of potentially beneficial Lactobacillus spp. in the ceca regardless of EC-infection. Unexpectedly, the highest abundances of the genus Enterococcus were found directly after ending antibiotic treatment in both treatment groups, suggesting the growth of resistant enterococcal species. EC was not detected among the most abundant members of the genus Enterococcus. Oral EC-infection at the first day of life did not influence the development of cecal microbiota in the present study. Conclusions Lincomycin-spectinomycin treatment during the first week of life can prevent the EC-associated disease in broiler type chickens and has a direct impact on the development of the cecal microbiota. The low abundance of EC in the ceca of infected chickens underlines the pathogenic nature of the disease-causing EC strains. Further research on alternative prevention and intervention strategies is needed with regard to current efforts on reducing the use of antibiotics in livestock animals.

ABSTRACT: 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.

Since the ban of effective feed additives and therapeutics, histomonosis has become an important disease and, subsequently, a welfare issue for turkey production. We conducted an interview-based case series study to generate hypotheses about possible disease-favouring conditions in 31 H. meleagridis-infected flocks. The determined parameters were related to the general farm (flock management, biosecurity measures, etc.) as well as the histomonosis-specific disease management. Some inadequate biosecurity measures were observed. An inappropriate usage of the hygiene lock and cleaning as well as the disinfection frequency of equipment, clothes, and the hygiene lock could possibly be histomonosis-favouring conditions. These factors could increase the risk for the introduction of H. meleagridis and the risk of a pathogen spread on an affected farm. Insects, wild birds, litter materials, and contaminated dung could be potential vectors of H. meleagridis. Predisposing gastrointestinal diseases were observed in 71% of the affected flocks. Additionally, stress events related to higher temperature, movement of birds, and vaccination were documented in association with clinical histomonosis. The results emphasise the need for both good disease control and health management to ensure sustainable animal health and welfare.

In poultry, several respiratory viral infections lead to a drop in egg production associated with high economic losses. While the virus–host interactions at the respiratory epithelium are well studied, less is known about these interactions in the oviduct. To investigate possible differences between virus infections at these epithelial structures, we compared the interactions of two important poultry viruses on turkey organ cultures. Two members of the order Mononegavirales, the Avian Metapneumovirus (AMPV) and the Newcastle disease virus (NDV), were selected to conduct the in vitro experiments since these viruses can infect both the trachea and oviduct. In addition, we used different strains of these viruses, a subtype A and a subtype B strain for AMPV and the NDV strains Komarow and Herts’33, to detect possible differences not only between the tissues but also between different viral strains. Turkey tracheal and oviduct organ cultures (TOC and OOC) were prepared to investigate viral replication, antigen localisation, lesion development, and the expression pattern of interferon-λ and importin-α isoforms. All viruses replicated more efficiently in the oviduct than in the tracheal epithelium (p < 0.05). In addition, we observed higher expression levels of both, IFN-λ and importin-α in OOCs compared to TOCs. Our results indicated strain-dependent differences, with the AMPV-B- and Herts’33 strains being more virulent in organ cultures than the AMPV-A- and Komarow strains, based on the higher viral genome loads, more severe histological lesions, and higher upregulation of IFN-λ. Overall, our findings reveal tissue- and virus strain-dependent differences, which may have consequences for disease development in the host tissue and, subsequently, possible treatment strategies.

Nematode infections may induce immune-modulatory effects and influence host-immune responses to other pathogens. The aim of the study was to investigate whether a mixed nematode-infection influences non-specific and vaccine-induced humoral immunity against Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), and Avian Metapneumovirus (AMPV) in already vaccinated hens of a dual-purpose (Lohmann Dual, LD) or a layer genotype (Lohmann Brown Plus; LB). Until 17 weeks-of-age, LD (n = 70) and LB (n = 109) hens were vaccinated against major bacterial and viral diseases and coccidiosis. At 24 weeks-of-age, the hens received either a placebo or an oral inoculation of 1,000 infectious eggs of A. galli and H. gallinarum. Plasma total immunoglobulin (Ig) isotypes (IgY, IgM, IgA) levels and vaccine-induced antibody titers against NDV, IBV, and AMPV were determined from 2 to 18 weeks post-infection (wpi). Infections had no suppressing effect on total Ig isotypes IgY, IgM, and IgA as well as on vaccine-induced antibody titers against NDV, IBV, and AMPV (P > 0.05). Overall, LB hens had higher levels of IgY, IgM, and IgA than those of LD hens (P < 0.05). There were no differences between IBV titers of the two genotypes (P > 0.05). Independent of infection status of the hens, NDV titers were higher in LB hens than in LD hens at wpi 2 (P < 0.05), but not in following weeks (P > 0.05). Uninfected LD hens had lower AMPV titers than their infected counterparts at 6 and 14 wpi (P < 0.05). Regardless of nematode infection, LD hens revealed a higher risk of responding weak to vaccination against NDV (odds ratio = 5.45; P = 0.021) and AMPV (odds ratio = 13.99, P < 0.001) than did LB hens (P > 0.05). We conclude that nematode infections have no adverse effects on non-specific and vaccine-induced humoral immunity in either genotype. LB hens have higher levels of total immunoglobulin isotypes than LD hens. Except for IBV, vaccine-induced humoral immune responses show a dependency on genotype. Dual-purpose hens show lower responsiveness to vaccinations against NDV and AMPV, possibly due to factors associated with increased body fat reserves in this genotype.

ABSTRACT: Measuring corticosterone concentrations in feathers of poultry may be suitable to determine birds' exposure to stress. It is thinkable, that in laying hens such information could be helpful as an animal welfare indicator to evaluate adverse husbandry conditions and to predict the risk of developing behavioral disorders, such as feather pecking and cannibalism. Yet, there are some fundamental issues which remain unclear. Therefore, the objective of the current pilot study was to examine the inter- and intraindividual variation of pullets at the end of the rearing period, when most of the feathers are fully grown and animals are reaching sexual maturity. Flight feathers from both wings (n = 4), the tail (n = 2 – 3), and body feathers (n = 1 pool of 3 – 5 feathers) were taken from pullets (n = 10), genetics Lohmann Brown, at an age of 19 weeks who were reared in the same flock (N = 728). Corticosterone analysis was performed applying a validated protocol for laying hens. Results indicate not only high intraindividual, but also high interindividual variation. Mean over all samples was 75.2 pg/mg (± 38.58 pg/mg, n = 76), showing higher intraindividual variation (between feather types; SD: 23.75 pg/mg – 49.38 pg/mg; n = 10 pullets) than interindividual variation (within feather types; SD: 11.91 pg/mg – 49.55 pg/mg; n = 6 feather types). The variation between different feather types within one bird was higher than the variation within one feather type between different birds, indicating that birds a) may respond differently when exposed to stressors and b) corticosterone measurements should be done with the same feather type.

The Common Eider (Somateria mollissima) inhabits the entire northern hemisphere. In northern Europe, the flyway population reaches from the southern Wadden Sea to the northern Baltic coast. The European population is classified as endangered due to declines in Common Eider numbers across Europe since 1990. In this study, we assessed 121 carcasses of Common Eiders, captured incidentally in gillnets in the Western Baltic between 2017 and 2019. The most common findings were parasitic infections of the intestine by acanthocephalans in 95 animals, which correlated with enteritis in 50% of the cases. Parasites were identified as Profilicollis botulus in 25 selected animals. Additionally, oesophageal pustules, erosions, and ulcerations, presumably of traumatic origin, were frequently observed. Nephritis and hepatitis were frequent, but could not be attributed to specific causes. Lung oedema, fractures and subcutaneous haemorrhages likely resulted from entangling and drowning. Two Common Eiders had mycobacterial infections and in one of these, Mycobacterium avium subspecies (ssp.) avium was identified. This study gives an overview of morphological changes and infectious diseases from one location of the European flyway population. It contributes to future health studies on Common Eiders in the Baltic and Wadden Seas by providing baseline information to compare with other areas or circumstances.

Infectious bronchitis (IB) is the most important Coronavirus infection in poultry. The IB virus (IBV) spreads rapidly in the affected flocks, and although airborne-spread is the principle route of transmission, contact also plays an important epidemiological role. This Gammacoronavirus infects susceptible chickens at all age groups. It affects not only the respiratory tract, but may also affect the urogenital and possibly to some extent the intestinal tract, depending on the strain characteristics. Besides strict biosecurity measures, vaccination is the most important strategy to control clinical disease. Vaccines, mainly live attenuated vaccines, have been used for almost 80 years. The high mutation and recombination rates and the frequent emergence of new virus variants in association with poor cross-protection, provide a continuous pressure to develop new vaccine candidates and strategies. Therefore, IB is a disease chickens will continue to be challenged with, and eradiation seems to be unrealistic at this point. The availability of advanced molecular techniques is currently opening up new opportunities to develop new generation vaccines and respond to newly emerging strains more quickly compared to traditional approaches. It is important that these new vaccine candidates stimulate the necessary immune mechanisms to provide protection at the main virus entrance site, the respiratory tract. Therefore, they should induce mucosal immunity including humoral as well as cell mediated reactions. The practicability for mass vaccination has to be ensured to be able to use these new vaccines in poultry production.

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.

Abstract Background Campylobacter jejuni is considered as a chicken commensal. The gut microbiota and the immune status of the host may affect its colonization. Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens, which allows secondary pathogens to invade or exacerbates their pathogenesis. To investigate the effect of IBDV-induced immunosuppression on the pathogenesis of C. jejuni, broiler chickens were inoculated with a very virulent (vv) strain of IBDV at 14 days post hatch followed by C. jejuni inoculation at 7 (Experiment A) or 9 (Experiment B) days post virus (IBDV) inoculation. Results vvIBDV-infection led to a depression in caecal lamina propria B lymphocytes and the anti-C. jejuni-antibody response starting at 14 days post C. jejuni inoculation (pbi). The C. jejuni-colonization pattern was comparable between mono-inoculated groups of both experiments, but it varied for vvIBDV + C. jejuni co-inoculated groups. In Experiment A significant higher numbers of colony forming units (CFU) of C. jejuni were detected in the caecum of co-inoculated birds compared to C. jejuni-mono-inoculated birds in the early phase after C. jejuni-inoculation. In Experiment B the clearance phase was affected in the co-inoculated group with significantly higher CFU at 21 days pbi compared to the mono-inoculated group (P

Respiratory pathogens are a health threat for poultry. Co-infections lead to the exacerbation of clinical symptoms and lesions. Mycoplasma gallisepticum (M. gallispeticum) and Avian Metapneumovirus (AMPV) are two avian respiratory pathogens that co-circulate worldwide. The knowledge about the host–pathogen interaction of M. gallispeticum and AMPV in the chicken respiratory tract is limited. We aimed to investigate how co-infections affect the pathogenesis of the respiratory disease and whether the order of invading pathogens leads to changes in host–pathogen interaction. We used chicken tracheal organ cultures (TOC) to investigate pathogen invasion and replication, lesion development, and selected innate immune responses, such as interferon (IFN) α, inducible nitric oxide synthase (iNOS) and IFNλ mRNA expression levels. We performed mono-inoculations (AMPV or M. gallispeticum) or dual-inoculations in two orders with a 24-h interval between the first and second pathogen. Dual-inoculations compared to mono-inoculations resulted in more severe host reactions. Pre-infection with AMPV followed by M. gallispeticum resulted in prolonged viral replication, more significant innate immune responses, and lesions (p < 0.05). AMPV as the secondary pathogen impaired the bacterial attachment process. Consequently, the M. gallispeticum replication was delayed, the innate immune response was less pronounced, and lesions appeared later. Our results suggest a competing process in co-infections and offer new insights in disease processes.

Campylobacter (C.) is the most common food-borne zoonosis in humans, which mainly manifests with watery to bloody diarrhoea. While C. jejuni is responsible for most cases of infection, C. coli is less frequently encountered. The object of the study was to prove the clinical impact of mono- and co-colonisation of C. coli and C. jejuni on weaned piglets in an infection model and to investigate the impact on transepithelial transport processes in the jejunum and caecum. At an age of eight weeks, eight pigs were infected with C. coli (ST-5777), 10 pigs with C. jejuni (ST-122), eight pigs with both strains, and 11 piglets served as control. During the four-week observation period, no clinical signs were observed. During dissection, both strains could be isolated from the jejunum and the caecum, but no alteration of the tissue could be determined histopathologically. Mono-infection with C. jejuni showed an impact on transepithelial ion transport processes of the caecum. An increase in the short circuit current (Isc) was observed in the Ussing chamber resulting from carbachol- and forskolin-mediated Cl− secretion. Therefore, we speculate that caecal colonisation of C. jejuni might affect the transport mechanisms of the intestinal mucosa without detectable inflammatory reaction.

Abstract 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.

The 2009 pandemic influenza A virus (IAV) H1N1 strain (H1N1pdm09) has widely spread and is circulating in humans and swine together with other human and avian IAVs. This fact raises the concern that reassortment between H1N1pdm09 and co-circulating viruses might lead to an increase of H1N1pdm09 pathogenicity in different susceptible host species. Herein, we explored the potential of different NS segments to enhance the replication dynamics, pathogenicity and host range of H1N1pdm09 strain A/Giessen/06/09 (Gi-wt). The NS segments were derived from (i) human H1N1- and H3N2 IAVs, (ii) highly pathogenic- (H5- or H7-subtypes) or (iii) low pathogenic avian influenza viruses (H7- or H9-subtypes). A significant increase of growth kinetics in A549 (human lung epithelia) and NPTr (porcine tracheal epithelia) cells was only noticed in vitro for the reassortant Gi-NS-PR8 carrying the NS segment of the 1918-descendent A/Puerto Rico/8/34 (PR8-wt, H1N1), whereas all other reassortants showed either reduced or comparable replication efficiencies. Analysis using ex vivo tracheal organ cultures of turkeys (TOC-Tu), a species susceptible to IAV H1N1 infection, demonstrated increased replication of Gi-NS-PR8 compared to Gi-wt. Also, Gi-NS-PR8 induced a markedly higher expression of immunoregulatory and pro-inflammatory cytokines, chemokines and interferon-stimulated genes in A549 cells, THP-1-derived macrophages (dHTP) and TOC-Tu. In vivo, Gi-NS-PR8 induced an earlier onset of mortality than Gi-wt in mice, whereas, 6-week-old chickens were found to be resistant to both viruses. These data suggest that the specific characteristics of the PR8 NS segments can impact on replication, virus induced cellular immune responses and pathogenicity of the H1N1pdm09 in different avian and mammalian host species.

Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive poultry disease. IBD virus (IBDV) is the causative agent, which may lead to high morbidity and mortality rates in susceptible birds. IBDV-pathogenesis studies have focused mainly on primary lymphoid organs. It is not known if IBDV infection may modify the development of the gut associated lymphoid tissues (GALT) as well as the microbiota composition. The aim of the present study was to investigate the effects of IBDV-infection on the bursa of Fabricius (BF), caecal tonsils (CT) and caecum, and to determine the effects on the gut microbiota composition in the caecum. Commercial broiler chickens were inoculated with a very virulent (vv) strain of IBDV at 14 (Experiment 2) or 15 (Experiment 1) days post hatch (dph). Virus replication, lesion development, immune parameters including numbers of T and B lymphocytes, macrophages, as well as the gut microbiota composition were compared between groups. Rapid IBDV-replication was detected in the BF, CT and caecum. It was accompanied by histological lesions including an infiltration of heterophils. In addition a significant reduction in the total mucosal thickness of the caecum was observed in vvIBDV-infected birds compared to virus-free controls (P < 0.05). vvIBDV infection also led to an increase in T lymphocyte numbers and macrophages, as well as a decrease in the number of B lymphocytes in the lamina propria of the caecum, and in the caecal tonsils. Illumina sequencing analysis indicated that vvIBDV infection also induced changes in the abundance of Clostridium XIVa and Faecalibacterium over time. Overall, our results suggested that vvIBDV infection had a significant impact on the GALT and led to a modulation of gut microbiota composition, which may lead to a higher susceptibility of affected birds for pathogens invading through the gut.

Infection of poultry with low pathogenic avian influenza viruses (LPAIV) is often associated with mild respiratory symptoms but may also lead to loss in egg production in laying birds. In vivo susceptibility of the reproductive tract for LPAIV infection was reported for turkeys and chickens, but virus-interaction with epithelial cells of the oviduct and possible stimulation of the local antiviral immune responses have not been characterized. In this study, we wanted to investigate the suitability of magnum organ cultures (MOC) as an in vitro model to study virus-host interactions. We compared the susceptibility of duck (Du), chicken (Ch), and turkey (Tu) MOC for three different influenza A viruses (IAV). Overall, the course of infection and the antiviral immune response varied between strains as well as host cell origin, but MOC gave reproducible results for all investigated parameters within each species. While pandemic (p) H1N1 and H9N2 efficiently replicated in MOC-Ch and MOC-Tu, MOC-Du were significantly less susceptible to infection as indicated by a reduced replication level for both viruses (p < 0.05). Overall, virus replication levels did not correlate with interferonα (IFNα) mRNA-expression levels in neither species. H9N2-infection led to a significant upregulation of interferonλ (IFNλ) mRNA expression in MOC of all species compared to the non-infected controls (p < 0.05), while a correlation with replication levels was only seen for MOC-Tu. pH1N1-infection induced only significant upregulation of IFNλ mRNA expression in MOC-Tu at 48 hours post infection (p < 0.05), but the expression pattern did not correlate with replication levels. Our results show that MOC are a suitable model to study IAV-interaction with the mucosal surface of the avian reproductive tract. The data suggest that the reproductive tract may play a role in the pathobiology of IAV in poultry.

ABSTRACT The human diarrheal pathogens Campylobacter jejuni and Campylobacter coli interfere with host innate immune signaling by different means, and their flagellins, FlaA and FlaB, have a low intrinsic property to activate the innate immune receptor Toll-like receptor 5 (TLR5). We have investigated here the hypothesis that the unusual secreted, flagellin-like molecule FlaC present in C. jejuni, C. coli, and other Campylobacterales might activate cells via TLR5 and interact with TLR5. FlaC shows striking sequence identity in its D1 domains to TLR5-activating flagellins of other bacteria, such as Salmonella, but not to nonstimulating Campylobacter flagellins. We overexpressed and purified FlaC and tested its immunostimulatory properties on cells of human and chicken origin. Treatment of cells with highly purified FlaC resulted in p38 activation. FlaC directly interacted with TLR5. Preincubation with FlaC decreased the responsiveness of chicken and human macrophage-like cells toward the bacterial TLR4 agonist lipopolysaccharide (LPS), suggesting that FlaC mediates cross-tolerance. C. jejuniflaC mutants induced an increase of cell responses in comparison to those of the wild type, which was suppressed by genetic complementation. Supplementing excess purified FlaC likewise reduced the cellular response to C. jejuni. In vivo, the administration of ultrapure FlaC led to a decrease in cecal interleukin 1β (IL-1β) expression and a significant change of the cecal microbiota in chickens. We propose that Campylobacter spp. have evolved a novel type of secreted immunostimulatory flagellin-like effector in order to specifically modulate host responses, for example toward other pattern recognition receptor (PRR) ligands, such as LPS. IMPORTANCE Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins.

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.

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants.

BACKGROUND: Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses. METHODOLOGY/PRINCIPAL FINDINGS: To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses. CONCLUSIONS/SIGNIFICANCE: Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.