Typhlitis induced by Histomonas meleagridis affects relative but not the absolute Escherichia coli counts and invasion in the gut in turkeys.

Unlike in chickens, dynamics of the gut microbiome in turkeys is limitedly understood and no data were yet published in context of pathological changes following experimental infection. Thus, the impact of Histomonas meleagridis-associated inflammatory changes in the caecal microbiome, especially the Escherichia coli population and their caecal wall invasion in turkeys was investigated. Birds experimentally inoculated with attenuated and/or virulent H. meleagridis and non-inoculated negative controls were divided based on the severity of macroscopic caecal lesions. The high throughput amplicon sequencing of 16SrRNA showed that the species richness and diversity of microbial community significantly decreased in severely affected caeca. The relative abundances of operational taxonomic units belonging to Anaerotignum lactatifermentans, E. coli, and Faecalibacterium prausnitzii were higher and paralleled with a decreased abundances of those belonging to Alistipes putredinis, Streptococcus alactolyticus, Lactobacillus salivarius and Lactobacillus reuteri in birds with the highest lesion scores. Although the relative abundance of E. coli was higher, the absolute count was not affected by the severity of pathological lesions. Immunohistochemistry showed that E. coli was only present in the luminal content of caecum and did not penetrate even severely inflamed and necrotized caecal wall. Overall, it was demonstrated that the fundamental shift in caecal microbiota of turkeys infected with H. meleagridis was attributed to the pathology induced by the parasite, which only led to relative but not absolute changes in E. coli population. Furthermore, E. coli cells did not show tendency to penetrate the caecal tissue even when the intestinal mucosal barriers were severely compromised.

Aerosol is the optimal route of respiratory tract infection to induce pathological lesions of colibacillosis by a lux-tagged avian pathogenic Escherichia coli in chickens.

Pathogenesis of colibacillosis caused by avian pathogenic Escherichia coli (APEC) in poultry is unclear and experimental studies reveal substantial inconsistency. In this study, the impact of three infection routes differing in the site of deposition of inoculum in the respiratory tract, were investigated. Two-weeks-old chickens were infected with a lux-tagged APEC strain via aerosol, intranasally or intratracheally, and sequentially sampled along with uninfected birds. At 1 and 3 days post infection (dpi), liver or spleen to body-weight ratios in all infected groups were significantly higher than in negative control, while at 7 dpi, such differences were significant in both organs in the aerosol-infected group. The infection-strain colonized tracheas and lungs in infected birds at 1 dpi and persisted until 7 dpi. Among infected groups, in lungs, bacterial load at 1 dpi was significantly lower in intranasally-inoculated birds. Histology revealed that, independent of infection route, lesions were mostly seen in the lower respiratory organs (lungs and air sacs) characterized by bronchitis/pneumonia and airsacculitis. Birds infected via aerosol showed the highest mean lesion score in lungs while intranasal application caused the mildest pathological changes, and difference between the two groups was significant at 1 dpi. In spleen, heterophilic infiltrations were prominent in affected birds. Interestingly, tracheas were pathologically unaffected. Altogether, the results demonstrated the importance of infection route, with aerosol being the most suitable to induce pathological lesions of colibacillosis without predisposing factors. Furthermore, the lux-tagged APEC strain was discriminated from native isolates enabling exact differentiation and enumeration.RESEARCH HIGHLIGHTS Lux-tagged APEC strain was used for infection to differentiate from native E. coli.Pathologically, lungs, air sacs and spleen but not trachea were affected.The route of infection strongly impacts the pathological outcome with APEC.The infection with APEC via aerosol caused the most severe lesions in chickens.

Bacterial Infection in Chicken Embryos and Consequences of Yolk Sac Constitution for Embryo Survival.

Bacterial infections in chicken eggs often cause mortality of embryos and clinical consequences in chicks but the pathological mechanism is unclear. We investigated the pathological changes and bacterial growth kinetics in dead and live embryos following infection with 2 Escherichia coli strains with a different clinical background and with 1 Salmonella Enteritidis strain. In 2 experiments, 12-day-old embryos were infected via the allantoic sac with 100 µl of 1 to 5 × 102 CFU/ml of one of the bacteria. In experiment 1, only dead embryos were sampled until 4 days postinfection (dpi), and surviving embryos were sampled at 5 dpi. In experiment 2, sampling was performed in dead and killed embryos sequentially at 1, 2, 3, and 4 dpi. The bacteria showed varying pathogenicity in embryos. The yolk sacs of dead embryos showed congestion, inflammation, damaged blood vessels, and abnormal endodermal epithelial cells. Such lesions were absent in the yolk sacs of negative control embryos and in those of embryos that survived infection. The livers and hearts of dead embryos showed congestion and lysed erythrocytes with no morphological changes in hepatocytes or myocardial cells. All bacteria multiplied rapidly in the yolks of infected embryos, although this did not predict survival. However, the livers of dead embryos contained significantly higher bacterial loads than the livers of the embryos that survived infection. The results provide evidence that lesions in the yolk sac, which have been neglected to date, coincide with embryonic mortality, underlining the importance of healthy yolk sacs for embryo survival.

Fowl adenovirus (FAdV) fiber-based vaccine against inclusion body hepatitis (IBH) provides type-specific protection guided by humoral immunity and regulation of B and T cell response.

A recombinant fowl adenovirus (FAdV) fiber protein, derived from a FAdV-8a strain, was tested for its efficacy to protect chickens against inclusion body hepatitis (IBH). FAdV-E field isolates belonging to both a homotypic (FAdV-8a) and heterotypic (-8b) serotype were used as challenge. Mechanisms underlying fiber-induced protective immunity were investigated by fiber-based ELISA, virus neutralization assays and flow cytometry of peripheral blood mononuclear cells, monitoring the temporal developments of humoral and cellular responses after vaccination and challenge exposure. Birds were clinically protected from the homologous challenge and showed a significant reduction of viral load in investigated target organs, whereas fiber-based immunity failed to counteract the heterologous serotype infection. These findings were supported in vitro by the strictly type-specific neutralizing activity of fiber immune sera. In protected birds, fiber vaccination prevented a post-challenge drop of peripheral B cells in blood. Furthermore, fiber immunization stimulated CD4+ T lymphocyte proliferation while moderating the CD8α+ T cell response and prevented challenge-induced changes in systemic monocytes/macrophages and γδ+ T cell subpopulations. Both vaccinated and adjuvant-only injected birds experienced a priming of systemic B cells and TCRγδ+ T lymphocytes, which masked possible pre-challenge effects due to the antigen. In conclusion, within FAdV-E, recombinant fiber represents a vaccine candidate to control the adverse effects of homotypic infection by eliciting an effective humoral immunity and regulating B and T cell response, whereas the failure of heterotypic protection suggests a primordial role of humoral immunity for this vaccine.

Spotlight on Histomonosis (blackhead disease): a re-emerging disease in turkeys and chickens.

Histomonosis, or blackhead disease, is a well-known disease in turkeys that can cause high mortality, but outbreaks with lower losses are also observed. The disease is less fatal in chickens but is economically important due to reduced performance and its co-appearance with colibacillosis. The lack of specific prophylactic and therapeutic interventions has led to a re-emergence of the disease in recent years, mainly in turkeys, free-range layers and chicken parent stock.

Cytokine production and phenotype of Histomonas meleagridis-specific T cells in the chicken.

The protozoan parasite Histomonas meleagridis is the causative agent of the re-emerging disease histomonosis of chickens and turkeys. Due to the parasite's extracellular occurrence, a type-2 differentiation of H. meleagridis-specific T cells has been hypothesized. In contrast, a recent study suggested that IFN-γ mRNA+ cells are involved in protection against histomonosis. However, the phenotype and cytokine production profile of H. meleagridis-specific T cells still awaits elucidation. In this work, clonal cultures of a virulent monoxenic strain of H. meleagridis were used for infecting chickens to detect IFN-γ protein and IL-13 mRNA by intracellular cytokine staining and PrimeFlow™ RNA Assays, respectively, in CD4+ and CD8ß+ T cells. Infection was confirmed by characteristic pathological changes in the cecum corresponding with H. meleagridis detection by immunohistochemistry and H. meleagridis-specific antibodies in serum. In splenocytes stimulated either with H. meleagridis antigen or PMA/ionomycin, IFN-γ-producing CD4+ T cells from infected chickens increased in comparison to cells from non-infected birds 2 weeks and 5 weeks post-infection. Additionally, an increase of IFN-γ-producing CD4-CD8ß- cells upon H. meleagridis antigen and PMA/ionomycin stimulation was detected. Contrariwise, frequencies of IL-13 mRNA-expressing cells were low even after PMA/ionomycin stimulation and mainly had a CD4-CD8ß- phenotype. No clear increase of IL-13+ cells related to H. meleagridis infection could be found. In summary, these data suggest that H. meleagridis infection induces a type-1 differentiation of CD4+ T cells but also of non-CD4+ cells. This phenotype could include γδ T cells, which will be addressed in future studies.

Fowl aviadenovirus serotype 1 confirmed as the aetiological agent of gizzard erosions in replacement pullets and layer flocks in Great Britain by laboratory and in vivo studies.

An investigation into the aetiology and pathogenesis of adenoviral gizzard erosion has been conducted following three natural outbreaks affecting one flock of 6-week-old replacement pullets and two consecutive placements of free range layers at the age of 21 and 23 weeks. Affected flocks showed increased mortality (0.12-0.30% per week), and gizzard lesions were consistent with fowl aviadenovirus (FAdV) involvement. To substantiate the initial findings, a selection of archived formalin-fixed paraffin-embedded gizzard samples from another 12 pullet and layer flocks, for which macroscopic and histopathological diagnosis of the disease were recorded in Great Britain during the period 2009-2016, were also investigated. In situ hybridization (ISH), virology and/or PCR confirmed the presence of FAdV species-A, serotype-1 (FAdV-A, FAdV-1) DNA in gizzard samples of all 15 cases investigated. Co-infections with additional FAdV serotypes including FAdV-8a were detected by serology and/or virology in two of the pullet flocks. However, species-specific in situ hybridization revealed that pathological changes of affected gizzards were only associated with the detection of FAdV-A. A subsequent in vivo study infecting 21-day-old SPF pullets with FAdV-1 or FAdV-8a strains isolated from the 6-week-old replacement pullets revealed characteristic pathomorphological changes only in the gizzards from birds infected with FAdV-1. While infection with FAdV-8a was confirmed by virology and serology, infected SPF birds did not develop pathomorphological changes. Therefore, the aetiological involvement of the isolated FAdV-8a in the development of adenoviral gizzard erosion in commercial pullets has been ruled out.

The outcome of experimentally induced inclusion body hepatitis (IBH) by fowl aviadenoviruses (FAdVs) is crucially influenced by the genetic background of the host.

In the present study, inclusion body hepatitis (IBH) was experimentally induced by oral inoculation of two groups of specific pathogen-free (SPF) broilers and two groups of SPF layers at day-old with either a fowl aviadenovirus (FAdV)-D or a FAdV-E strain. A substantial variation in the degree of susceptibility was observed with mortalities of 100 and 96% in the FAdV-E and D infected SPF broiler groups, respectively, whereas in the groups of infected SPF layers mortalities of only 20 and 8% were noticed. Significant changes in clinical chemistry analytes of all infected birds together with histopathological lesions indicated impairment of liver and pancreas integrity and functions. Furthermore, significantly lower blood glucose concentrations were recorded at peak of infection in both inoculated SPF broiler groups, in comparison to the control group, corresponding to a hypoglycaemic status. High viral loads were determined in liver and pancreas of SPF broilers already at 4 days post-infection (dpi), in comparison to SPF layers, indicating a somewhat faster viral replication in the target organs. Overall, highest values were noticed in the pancreas of SPF broilers independent of the virus used for infection. The actual study provides new insights into the pathogenesis of IBH, a disease evolving to a metabolic disorder, to which SPF broilers were highly susceptible. Hence, this is the first study to report a significant higher susceptibility of SPF broiler chickens to experimentally induced IBH in direct comparison to SPF layers.

The 60S ribosomal protein L13 is the most preferable reference gene to investigate gene expression in selected organs from turkeys and chickens, in context of different infection models.

Evaluation of reference genes for expression studies in chickens and turkeys is very much limited and unavailable for various infectious models. In this study, eight candidate reference genes HMBS, HPRT1, TBP, VIM, TFRC, RPLP0, RPL13 and RPS7 were evaluated by five different algorithms (GeNorm, NormFinder, BestKeeper©, delta CT, RefFinder) to assess their stability. In order to analyze a broad variation of tissues, spleen, liver, caecum and caecal tonsil of different aged specific pathogen free (SPF) layer chickens and commercial turkeys, uninfected or infected with the extracellular pathogen Histomonas meleagridis, were included. For tissue samples from SPF chickens RPL13 and TBP were found to be the most stable reference genes. Further testing of RPL13 and TBP in the same organs of uninfected and infected SPF broiler chickens with the intracellular pathogen fowl aviadenovirus confirmed this finding. In tissue samples from turkeys, a stable expression of RPL13 and TFRC genes was noticed. Overall, the determined reference genes should be considered whenever gene expression studies in spleen, liver, caecum and caecal tonsil of chickens and turkeys are performed.

Selected clinical chemistry analytes correlate with the pathogenesis of inclusion body hepatitis experimentally induced by fowl aviadenoviruses.

In the present study, clinical chemistry was applied to assess the pathogenesis and progression of experimentally induced inclusion body hepatitis (IBH). For this, five fowl aviadenovirus (FAdV) strains from recent IBH field outbreaks were used to orally inoculate different groups of day-old specific pathogen-free chickens, which were weighed, sampled and examined during necropsy by sequential killing. Mortalities of 50% and 30% were recorded in two groups between 6 and 9 days post-infection (dpi), along with a decreased weight of 23% and 20%, respectively, compared to the control group. Macroscopical changes were seen in the liver and kidney between 6 and 10 dpi, with no lesions being observed in the other organs. Histological lesions were observed in the liver and pancreas during the same period. Plasma was collected from killed birds of each group at each time point and the following clinical chemistry analytes were investigated: aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), bile acids, total protein, albumin, uric acid and lipase. Plasma protein profile, AST and GLDH, together with bile acids values paralleled the macroscopical and histopathological lesions in the liver, while plasma lipase activity levels coincided with lesions observed in pancreas. In agreement with the histology and clinical chemistry, viral load in the target organs, liver and pancreas, was highest at 7 dpi. Thus, clinical chemistry was found to be a valuable tool in evaluating and monitoring the progression of IBH in experimentally infected birds, providing a deeper knowledge of the underlying pathophysiological mechanisms of a FAdV infection in chickens.

Aberrant Clinical Appearance and Pathomorphology Noticed During an Outbreak of Histomonosis Indicates a Different Pathogenesis of Histomonas meleagridis Genotype 2.

The present case report describes a remarkable feature of Histomonas meleagridis characterized by aberrant clinical appearance and pathomorphologic lesions, which were mainly confined to the ceca, noticed during a field outbreak of histomonosis. In a flock of meat turkey toms, sudden death was noticed at the end of week 5 in the absence of specific clinical signs. Instead of the well-known sulfur-colored feces, some caseous cores were found in the litter. Mortality up to 17% per week was noticed in the first 2 wk of observation, after which it declined to approximately 1% per week. In the 10th week of life roughly 31% of the birds had died before the remaining birds were killed to preclude further economic losses due to insufficient growth or continuing mortality. Necropsy of affected birds on the farm and during a more detailed investigation of 15 birds prior to the killing of the flock revealed severe lesions in the ceca characterized by thickened cecal walls filled with necrotic and caseous material. Additionally, some ruptured and necrotic ceca were noticed together with localized peritonitis. Despite such severe typhlitis, only one of the sectioned birds showed pathomorphologic changes in the liver. Test tube flotation from collected fecal samples revealed only sporadic occurrence of coccidial oocysts and no nematodes. However, the presence of H. meleagridis was confirmed by PCR and/or immunohistochemistry, with specific antibodies against the parasite in a majority of the investigated ceca and in four liver samples. Remarkably, genetic characterization revealed H. meleagridis genotype 2, about which no detailed investigations have been reported so far. Although PCR detected a concurrent presence of Tetratrichomonas gallinarum, an involvement in the lesions could be excluded based upon histologic investigations. Finally, infection with Escherichia coli and Gallibacterium anatis was demonstrated by bacteriologic smears of internal organs, most likely a secondary infection. Altogether, the results demonstrate an aberrant clinical appearance and pathomorphology in turkeys suffering from histomonosis. Pathomorphologic changes were characterized by severe inflammation of the ceca with minimal liver involvement, indicating a different pathogenesis of H. meleagridis genotype 2.

Pathogenesis of Gallibacterium anatis in a natural infection model fulfils Koch's postulates: 1. Folliculitis and drop in egg production are the predominant effects in specific pathogen free layers.

Pathogenicity of Gallibacterium anatis was studied in specific pathogen free layers in a controlled environment, applying the intranasal route for experimental infection. At 30 weeks, 37 hens were infected with 0.4 ml of 1.53 × 10(8) colony-forming units/ml suspension of G. anatis strain 07990 whereas equal numbers of hens were left uninfected for control. Following experimental infection, clinical signs and the number and weight of the eggs were recorded daily until 5 weeks post infection. Three birds from each group were killed at 3, 7, 10, 28 and 38 days post infection (d.p.i.) for necropsy and sampling for bacteriological and histopathological examinations. Additionally, necropsy examination was performed on all remaining birds at 38 d.p.i. G. anatis infection was found to have an immediate and severe effect on egg production, showing early and persistent colonization in respiratory and reproductive organs as well as in the gut of infected layers. In birds killed at various time points, G. anatis infection caused focal necrosis in the liver (1/37), folliculitis (2/37), pericarditis (3/37), haemorrhagic follicles (2/37), ruptured follicles (20/37), yolk in the body cavity (2/37) and egg peritonitis (1/37). The inflammation of the ovaries could be further confirmed by histopathological examination. Recovery of G. anatis from yolk at 10 d.p.i. indicates the potential of vertical transmission. Altogether, lesions reflect typical findings of G. anatis infection reported in natural cases. Thus, for the first time, lesions and the consecutive disease caused by G. anatis infection have been reproduced experimentally in a natural infection model.

Pathogenesis of Gallibacterium anatis in a natural infection model fulfils Koch's postulates: 2. Epididymitis and decreased semen quality are the predominant effects in specific pathogen free cockerels.

Pathogenesis of Gallibacterium anatis was investigated in specific pathogen free cockerels. Birds aged 35 weeks were infected intranasally with G. anatis whereas negative controls were left uninfected. Following infection, necropsy, bacteriological and histopathological investigations were performed in birds killed at 3, 7, 10, 28 and 38 days post infection (d.p.i.). Additionally, semen samples were collected twice a week until 5 weeks post infection for quality assessment. No clinical signs and gross pathological lesions were seen throughout the experiment. Bacteriological investigation revealed that G. anatis colonized the upper respiratory tract at 3 d.p.i. and could be isolated from testis and epididymis at 7 d.p.i. onwards. Bacterial persistence was found in the respiratory tract, gut and testis until the termination of the study at 38 d.p.i. Furthermore, G. anatis was isolated from semen arguing for the possibility of vertical transmission. Histopathological examination showed infiltration of mononuclear cells in epididymal tissue, indicating an inflammation. Density, total motility, progressive motility and membrane integrity of sperms were significantly decreased in infected birds as compared with control chickens. Along with these findings, an increase in spermatozoa with morphological defects was observed at different time points. In conclusion, the present study provides novel data on the impact of a G. anatis infection in cockerels in a natural infection model, thus helping to elucidate bacterial distribution, pathological lesions as well as influences on semen quality.

Subclinical circulation of avian hepatitis E virus within a multiple-age rearing and broiler breeder farm indicates persistence and vertical transmission of the virus.

In a prospective longitudinal study, a broiler breeder flock and its progeny were monitored for the presence of avian hepatitis E virus (HEV) RNA and antibodies. The flock was part of a multiple-age farm where the presence of avian HEV with clinical signs (increased mortality and decreased egg production) was demonstrated in several previous production cycles. Samples were taken twice at the rearing site and several times at the production site from broiler breeders including cockerels and day-old chicks. The samples were investigated by conventional and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and histological methods. At all time points, samples from the hens were positive for avian HEV RNA. The birds did not show any clinical signs, even though histopathological lesions of non-specific aetiology in the liver and spleen could be demonstrated. A significant increase in the number of positive birds and viral load was seen in week 45, in accordance with an increase in antibody titres. In comparison, cockerels investigated in week 62 tested negative by RT-PCR and ELISA. Avian HEV RNA was also detected in day-old chicks hatched from eggs laid in week 25, indicating vertical transmission. All partial helicase and capsid sequences retrieved within this study clustered together and were identical to previous sequences obtained from the same multiple-age farm. In conclusion, avian HEV persisted on the farm over years and circulated between the rearing and the production sites without causing any clinical signs although high viral loads in the adult hens were observed.

Trichomonads in birds--a review.

Members of the family Trichomonadidae, mainly Trichomonas gallinae and Tetratrichomonas gallinarum, represent important parasites in birds with worldwide presence, since being reported in the 19th century. Especially Columbiformes, Falconiformes and Strigiformes can be severely affected by trichomonads, whereas the majority of infections in Galliformes and Anatiformes are subclinical although severe infections are occasionally reported. With the recent appearance of deadly infections in wild Passeriformes the protozoan parasite T. gallinae obtained greater attention which will be addressed in this review. Although light microscopy remains the method of choice to confirm the presence of trichomonads molecular studies were introduced in recent years, in order to characterize the parasites and to establish relationships between isolates. Isolation of trichomonads is a prerequisite for detailed in vitro and in vivo studies and different media are reported to obtain suitable material. The limited information about virulence factors will be reviewed in context with the pathogenicity of trichomonads which varies greatly, indicating certain strain heterogeneity of the parasites. Options for treatment characterized by the leading role of imidazoles whose activity is sometimes hampered by resistant parasites remains a challenge for the future. Introducing more standardized genetic studies and investigations concentrating on the host-pathogen interaction should be helpful to elucidate virulence factors which might lead to new concepts of treatment.

A comprehensive study of colisepticaemia progression in layer chickens applying novel tools elucidates pathogenesis and transmission of Escherichia coli into eggs.

Colisepticaemia caused by avian pathogenic Escherichia coli (APEC) is a challenging disease due to its high economic importance in poultry, dubious pathogenesis and potential link with zoonosis and food safety. The existing in vitro studies can't define hallmark traits of APEC isolates, suggesting a paradigm shift towards host response to understand pathogenesis. This study investigated the comprehensive pathological and microbial progression of colisepticaemia, and transmission of E. coli into eggs using novel tools. In total 48 hens were allocated into three groups and were inoculated intratracheally with ilux2-E. coli PA14/17480/5-/ovary (bioluminescent strain), E. coli PA14/17480/5-/ovary or phosphate buffered saline. Infection with both strains led to typical clinical signs and lesions of colibacillosis as in field outbreaks. Based on lung histopathology, colisepticaemia progression was divided into four disease stages as: stage I (1-3 days post infection (dpi)), stage II (6 dpi), stage III (9 dpi) and stage IV (16 dpi) that were histologically characterized by predominance of heterophils, mixed cells, pyogranuloma, and convalescence, respectively. As disease progressed, bacterial colonization in host organs also decreased, revealed by the quantification of bacterial bioluminescence, bacteriology, and quantitative immunohistochemistry. Furthermore, immunofluorescence, immunohistochemistry, and bacteria re-isolation showed that E. coli colonized the reproductive tract of infected hens and reached to egg yolk and albumen. In conclusion, the study provides novel insights into the pathogenesis of colisepticemia by characterizing microbial and pathological changes at different disease stages, and of the bacteria transmission to table eggs, which have serious consequences on poultry health and food safety.

Quantity of virulent fowl adenovirus serotype 1 correlates with clinical signs, macroscopical and pathohistological lesions in gizzards following experimental induction of gizzard erosion in broilers.

In the present study day-old specific-pathogen-free (SPF) and commercial broilers with maternally derived fowl adenovirus serotype 1 (FAdV-1) antibodies were orally infected with a European "pathogenic" FAdV-1, isolated from broilers showing signs of gizzard erosion. During the experiment, broilers were observed and weighed daily up to 17 days post infection (dpi). Clinically, both infected groups showed significant decrease of weight compared to respective negative control groups. Birds were examined by necropsy at 3, 7, 10, 14 and 17 dpi. Pathological changes in the gizzards were noticed in both experimentally infected groups from 7 dpi onwards. Macroscopically, erosion of the koilin layer and inflammation or ulceration of the gizzard mucosa were observed. Histologically, presence of FAdV-1 in intranuclear inclusion bodies of degenerated glandular epithelial cells was demonstrated by in-situ hybridization and inflammatory cell infiltration of the lamina propria, submucosa and muscle layer was detected. Tissue samples were investigated by a recently developed real-time PCR and the viral DNA load was calculated from gizzard, liver, spleen and cloacal swabs with the highest amounts of FAdV-1 DNA found in the gizzard. For the first time, successful reproduction of clinical signs in broilers as well as pathological lesions in the gizzard were achieved with a European FAdV-1 isolate displaying some genetic differences to so far reported virulent FAdV-1 from Japan. Furthermore, highest viral load in gizzards could be linked with macroscopical and histological lesions. Therefore, the conducted analyses provide important insights into the pathogenesis of adenoviral gizzard erosion.

Assessing pathogenicity of Gallibacterium anatis in a natural infection model: the respiratory and reproductive tracts of chickens are targets for bacterial colonization.

Two separate bird trials were performed to establish a reliable route of infection for Gallibacterium anatis in chickens, comparing intranasal (i.n.) and intravenous (i.v.) applications. Additionally, three mutually divergent isolates from three geographical locations, as shown by MALDI-TOF-MS and partial rpoB gene sequence analysis, were compared. In the first trial, birds were infected with one of the selected isolates by the i.v. or i.n. route. Subsequently, birds were killed 3, 12 and 24 h post infection following i.v. infection while at 3, 7 and 10 days post infection (dpi) in the case of i.n. infection along with birds of the control group. As a result, i.n. infection showed prominent and consistent bacterial tissue distribution in different organs persisting until 10 dpi, which was a striking contrast to the i.v. infection route. Likewise, histopathology revealed mild to severe tracheal lesions following i.n. infection. The second trial was set up to confirm both the achieved results and the robustness of i.n. infection but with an extended observation period, until 28 dpi In agreement with the preceding trial, identical results for bacteriological and histopathological examinations were obtained with persistency of bacteria until 28 dpi Comparing the three different isolates from Mexico, China and Austria, the Mexican isolate showed a somewhat higher pathogenicity than the other strains. Consequently, pathogenesis of G. anatis strains was studied in chickens elucidating i.n. infection as the most reliable route characterized by a long-lasting bacteraemia, targeting the respiratory and reproductive tract.

1H-NMR based-metabolomics reveals alterations in the metabolite profiles of chickens infected with ascarids and concurrent histomonosis infection.

BACKGROUND: Gut infections of chickens caused by Ascaridia galli and Heterakis gallinarum are associated with impaired host performance, particularly in high-performing genotypes. Heterakis gallinarum is also a vector of Histomonas meleagridis that is often co-involved with ascarid infections. Here, we provide a first insight into the alteration of the chicken plasma and liver metabolome as a result of gastrointestinal nematode infections with concomitant histomonosis. 1H nuclear magnetic resonance (1H-NMR) based-metabolomics coupled with a bioinformatics analysis was applied to explore the variation in the metabolite profiles of the liver (N = 105) and plasma samples from chickens (N = 108) experimentally infected with A. galli and H. gallinarum (+H. meleagridis). This was compared with uninfected chickens at different weeks post-infection (wpi 2, 4, 6, 10, 14, 18) representing different developmental stages of the worms. RESULTS: A total of 31 and 54 metabolites were quantified in plasma and aqueous liver extracts, respectively. Statistical analysis showed no significant differences (P > 0.05) in any of the 54 identified liver metabolites between infected and uninfected hens. In contrast, 20 plasma metabolites including, amino acids, sugars, and organic acids showed significantly elevated concentrations in the infected hens (P < 0.05). Alterations of plasma metabolites occurred particularly in wpi 2, 6 and 10, covering the pre-patent period of worm infections. Plasma metabolites with the highest variation at these time points included glutamate, succinate, trimethylamine-N-oxide, myo-inositol, and acetate. Differential pathway analysis suggested that infection induced changes in (1) phenylalanine, tyrosine, and tryptophan metabolism, (2) alanine, aspartate and glutamate metabolism; and 3) arginine and proline metabolism (Pathway impact > 0.1 with FDR adjusted P-value < 0.05). CONCLUSION: In conclusion, 1H-NMR based-metabolomics revealed significant alterations in the plasma metabolome of high performing chickens infected with gut pathogens-A. galli and H. gallinarum. The alterations suggested upregulation of key metabolic pathways mainly during the patency of infections. This approach extends our understanding of host interactions with gastrointestinal nematodes at the metabolic level.