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.

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.

Aerosol vaccination of chicken pullets with irradiated avian pathogenic Escherichia coli induces a local immunostimulatory effect.

The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1ß and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1ß was elevated. Changes were observed in macrophages and TCR-γδ+ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4+ cells in spleen and a rise of Bu-1+ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ+CD8α+ T cells in the lung and IFN-γ+TCR-γδ+ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.

A novel genotype of avian hepatitis E virus identified in chickens and common pheasants (Phasianus colchicus), extending its host range.

In 2019, outbreaks of hepatitis-splenomegaly syndrome (HSS) were observed in six commercial layer chicken flocks, belonging to three different Polish farms, and characterized by increased mortality, hemorrhagic hepatitis with attached blood clots on the liver surface, and splenomegaly. Diseased flocks were initially investigated for the presence of avian hepatitis E virus (aHEV) - the etiological agent of HSS - by conventional reverse transcriptase polymerase chain reaction, which revealed aHEV sequences clustering separately from all known aHEV genotypes. Additionally, an aHEV genome was identified for the first time in common pheasants, from a flock in France, using Next Generation Sequencing. This genome clustered together with the Polish aHEVs here investigated. Complete genome aHEV sequences from the HSS outbreaks confirmed the divergent cluster, with a shared nucleotide sequence identity of 79.6-83.2% with other aHEVs, which we propose to comprise a novel aHEV genotype - genotype 7. Histology and immunohistochemistry investigations in the liver and spleen established an association between aHEV and the observed lesions in the affected birds, consolidating the knowledge on the pathogenesis of aHEV, which is still largely unknown. Thus, the present investigation extends the natural host range and genotypes of aHEV and strengthens knowledge on the pathogenesis of HSS.

Local cellular immune response plays a key role in protecting chickens against hepatitis-hydropericardium syndrome (HHS) by vaccination with a recombinant fowl adenovirus (FAdV) chimeric fiber protein.

Fowl adenovirus (FAdV)-induced diseases hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) have been affecting the poultry industry with increasing severity in the last two decades. Recently, a subunit vaccine based on a chimeric fiber protein with epitopes from different fowl adenovirus serotypes (named crecFib-4/11) has been shown to confer simultaneous protection against both HHS and IBH. However, the underlying immune mechanisms in chickens are still enigmatic, especially because of frequently absent neutralizing response despite high levels of protection. In this study, we investigated the kinetics of the humoral and cellular immune responses in specific pathogen-free chickens after vaccination with crecFib-4/11 and/or challenge with a HHS-causing strain, on a systemic level, as well as locally in target and lymphoid organs. The humoral response was assessed via enzyme-linked immunosorbent assay (ELISA) and virus neutralization test in serum, while the cellular immune response was determined by phenotyping using flow cytometry. Although vaccination induced serum antibodies, as confirmed by ELISA, such antibodies exhibited no pre-challenge neutralizing activity against FAdV-4. Nevertheless, immunized birds experienced a significant B cell increase in the liver upon challenge, remaining high throughout the experiment. Furthermore, vaccination stimulated the proliferation of cytotoxic T lymphocytes, with earlier circulation in the blood compared to the challenge control and subsequent increase in liver and spleen. Overall, these findings imply that protection of chickens from HHS after crecFib-4/11 vaccination relies on a prominent local immune response in the target organs, instead of circulating neutralizing antibodies.

Experimental reproduction of histomonosis caused by Histomonas meleagridis genotype 2 in turkeys can be prevented by oral vaccination of day-old birds with a monoxenic genotype 1 vaccine candidate.

Histomonosis (syn. blackhead disease) is caused by the protozoan parasite Histomonas meleagridis and can result in high mortality in turkey flocks, a situation driven by the limitation of prophylactic and therapeutic interventions. Multi-locus sequence typing confirmed the existence of two genotypes, with the vast majority of reported histomonosis outbreaks being caused by genotype 1 in contrast to only a few detections of genotype 2. For the first time, genotype 2 of H. meleagridis was successfully isolated from an outbreak of histomonosis in a flock of 5-week-old turkeys and a clonal culture was established. Using this culture, an experimental infection was performed in naïve turkeys. The animal trial reflected the observations from the field outbreak and coincided with a previously reported case of histomonosis caused by genotype 2, albeit no mortality was observed in the infected birds whereas 17.1% mortality was noticed in the field outbreak from appearance of disease until slaughter. Post mortem investigations demonstrated that lesions were restricted to the caeca in the field outbreak and the experimental trial. In parallel with the experimental reproduction of pathological changes, an oral vaccination of day-old turkeys with a monoxenic genotype 1 vaccine was carried out to determine efficacy against a genotype 2 challenge. Successful vaccine uptake was characterized by the presence of the vaccine in the caeca determined by qPCR and immunohistochemistry (IHC). Excretion of the vaccine strain was confirmed prior challenge, with the majority of birds developing antibodies. The new monoxenic vaccine was able to minimize lesions in the caeca demonstrating heterologous protection. No parasites were detected in the liver by IHC in any of the vaccinated birds, compared to non-vaccinated animals. However, in 6 out of 17 birds of the vaccinated group a positive signal was obtained by real time PCR from liver samples with 2 positives being typeable by conventional PCR as genotype 2. Overall, H. meleagridis genotype 2 infection was successfully reproduced. Experimental vaccination with a genetically distantly related genotype 1 was able to reduce lesions, supporting protection by a recently developed vaccine candidate as an efficacious prophylactic strategy.

A novel Chaphamaparvovirus is the etiological agent of hepatitis outbreaks in pheasants (Phasianus colchicus) characterized by high mortality.

In the present study, we report the occurrence of several outbreaks of hepatitis in flocks of young pheasants in France, between 2017 and 2021. The disease was characterized by prostration, apathy and a median cumulative mortality of 12%, with the birds presenting multifocal to coalescing necrotizing hepatitis on necropsy. Severe extensive areas of degeneration and necrosis were observed in the liver, with degenerative hepatocytes presenting large amphophilic to acidophilic intranuclear inclusion bodies. Transmission electron microscopy examination of liver samples showed the presence of parvovirus-like virions of 21-24 nm, a finding already reported decades ago. Further investigations by Next Generation Sequencing and PCR revealed the complete genome of a novel species of parvovirus, here designated Phasianus chaphamaparvovirus 1 (PhChPV-1), that belongs to the new genus Chaphamaparvovirus in the Hamaparvovirinae subfamily. In situ hybridization and real-time PCR confirmed the etiology of the outbreaks, demonstrating the viral genome in the lesions. The findings establish the etiology of a pathology first described in pheasants 50 years ago and pave the way for a targeted protection strategy.

Production of interferon gamma and interleukin 17A in chicken T-cell subpopulations hallmarks the stimulation with live, irradiated and killed avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis with different clinical manifestations. The disease is associated with compromised animal welfare and results in substantial economic losses in poultry production worldwide. So far, immunological mechanisms of protection against colibacillosis are not comprehensively resolved. Therefore, the present study aimed to use an ex vivo model applying chicken mononuclear cells stimulated by live and inactivated APEC. For this purpose, an 8-color flow cytometry panel was set up to target viable chicken immune cells including CD45+, CD8α+, CD4+, TCR-γδ+, Bu-1+ cells and monocytes/macrophages along with the cytokines interferon gamma (IFN-γ) or interleukin 17A (IL-17A). The 8-color flow cytometry panel was applied to investigate the effect of live and two different types of inactivated APEC (formalin-killed APEC and irradiated APEC) on the cellular immune response. For that, mononuclear cells from spleen, lung and blood of 10-week-old specific pathogen-free layer birds were isolated and stimulated with live, irradiated or killed APEC. Intracellular cytokine staining and RT-qPCR assays were applied for the detection of IFN-γ and IL-17A protein level, as well as at mRNA level for spleenocytes. Ex vivo stimulation of isolated splenocytes, lung and peripheral blood mononuclear cells (PBMCs) from chickens with live, irradiated or killed APEC showed an increasing number of IFN-γ and IL-17A producing cells at protein and mRNA level. Phenotyping of the cells from blood and organs revealed that IFN-γ and IL-17A were mainly produced by CD8α+, TCR-γδ+ T cells as well as CD4+ T cells following stimulation with APEC. Expression level of cytokines were very similar following stimulation with live and irradiated APEC but lower when killed APEC were applied. Consequently, in the present study, an ex vivo model using mononuclear cells of chickens was applied to investigate the cellular immune response against APEC. The results suggest the relevance of IFN-γ and IL-17A production in different immune cells following APEC infection in chickens which needs to be further investigated in APEC primed birds.

Epidemic of cutaneous fowlpox in a naïve population of chickens and turkeys in Austria: Detailed phylogenetic analysis indicates co-evolution of fowlpox virus with reticuloendotheliosis virus.

Cutaneous fowlpox is a disease of chickens and turkeys caused by the fowlpox virus (FWPV), characterized by the development of proliferative lesions and scabs on unfeathered areas. FWPVs regularly carry an integrated, active copy of the reticuloendotheliosis virus (REV), and it has been hypothesized that such FWPVs are more problematic in the field. Extensive outbreaks are usually observed in tropical and sub-tropical climates, where biting insects are more difficult to control. Here, we report an epidemic of 65 cutaneous fowlpox cases in Austria in layer chickens (91% of the cases) and broiler breeders and turkeys, all of them unvaccinated against the disease, from October 2018 to February 2020. The field data revealed appearance in flocks of different sizes ranging from less than 5000 birds up to more than 20,000 animals, with the majority raised indoors in a barn system. The clinical presentation was characterized by typical epithelial lesions on the head of the affected birds, with an average decrease of 6% in egg production and an average weekly mortality of 1.2% being observed in the flocks. A real-time multiplex polymerase chain reaction (PCR) confirmed the presence of FWPV-REV DNA, not only in the lesions but also in the environmental dust from the poultry houses. The integration of the REV provirus into the FWPV genome was confirmed by PCR, and revealed different FWPV genome populations carrying either the REV long terminal repeats (LTRs) or the full-length REV genome, reiterating the instability of the inserted REV. Two selected samples were fully sequenced by next generation sequencing (NGS), and the whole genome phylogenetic analysis revealed a regional clustering of the FWPV genomes. The extensive nature of these outbreaks in host populations naïve for the virus is a remarkable feature of the present report, highlighting new challenges associated with FWPV infections that need to be considered.

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.

Vaccination against the Protozoan Parasite Histomonas meleagridis Primes the Activation of Toll-like Receptors in Turkeys and Chickens Determined by a Set of Newly Developed Multiplex RT-qPCRs.

Histomonosis in turkeys and chickens is caused by the extracellular parasite Histomonas meleagridis, but the outcome of the disease varies depending on the host species. So far, studies on the immune response against histomonosis focus mainly on different traits of the adaptive immune system. Activation of toll like receptors (TLR) leads to the interplay between cells of innate and adaptive immunity with consequences on B and T cell clonal expansion. Therefore, the present investigation focused on the interaction of virulent and/or attenuated histomonads with the innate immune system of turkeys and chickens at 4, 10, 21 days post inoculation. The expression of TLRs (TLR1A, 1B, 2A, 2B, 3, 4, 5, 6(Tu), 7, 13(Tu) and 21(Ch)) and pro-inflammatory cytokines (IL1ß and IL6) were analysed in caecum and spleen samples by RT-qPCR. Most frequent significant changes in expression levels of TLRs were observed in the caecum following infection with virulent parasites, an effect noticed to a lower degree in tissue samples from birds vaccinated with attenuated parasites. TLR1B, 2B and 4 showed a continuous up-regulation in the caecum of both species during infection or vaccination, followed by challenge with virulent parasites. Vaccinated birds of both species showed a significant earlier change in TLR expression following challenge than birds kept non-vaccinated but challenged. Expression of TLRs and pro-inflammatory cytokines were associated with severe inflammation of diseased birds in the local organ caecum. In the spleen, changes in TLRs and pro-inflammatory cytokines were less prominent and mainly observed in turkey samples. In conclusion, a detailed comparison of TLRs and pro-inflammatory cytokines of the innate immune system following inoculation with attenuated and/or virulent H. meleagridis of two avian host species provides an insight into regulative mechanisms of TLRs in the development of protection and limitation of the disease.

Atypical Manifestation of Cutaneous Fowlpox in Broiler Chickens Associated with High Condemnation at a Processing Plant.

The present case is an unusual report of cutaneous fowlpox with an atypical appearance and incidence in broilers. Gross skin lesions were noticed in 41-day-old commercial broilers during the veterinary inspection at a processing plant in the north of Iran. The skin lesions were only observed on feathered skin areas of the broilers and remained unnoticed until slaughter. Round, nodular or coalescent, elongated, reddish-brown proliferative lesions were mainly located on the back, thighs, and proximal areas of the neck of broilers. Nonfeathered skin, including the wattle, comb, eyelids, and legs, were not affected. This condition incurred high losses due to a 5.3% condemnation and trimming of carcasses. Cutaneous lesions were sampled for histopathology and molecular virology for further investigations. Histopathology revealed multifocal necrotic dermatitis with epidermal eosinophilic cytoplasmic inclusion bodies in the skin lesions. Molecular investigations confirmed the presence of fowlpox virus (FWPV) in the proliferative lesions, with further investigations identifying two FWPV genome populations, one carrying a portion of the reticuloendotheliosis virus (REV) and the other a nearly complete REV provirus. Furthermore, the 4b core protein gene-based molecular analysis clustered the field virus into clade A of the genus Avipoxvirus.

Reporte de caso- Manifestación atípica de viruela aviar cutánea en pollos de engorde asociada con altas de decomisos en una planta de procesamiento. El presente caso es un informe inusual de viruela aviar cutánea con apariencia e incidencia atípicas en pollos de engorde. Se observaron lesiones severas cutáneas en pollos de engorde comerciales de 41 días durante la inspección veterinaria en una planta de procesamiento en el norte de Irán. Las lesiones cutáneas solo se observaron en las áreas de piel emplumada de los pollos de engorde y pasaron desapercibidas hasta el procesamiento. Las lesiones proliferativas redondas, nodulares o coalescentes, alargadas, de color marrón rojizo se localizaron principalmente en el dorso, los muslos y en las áreas proximales del cuello de los pollos de engorde. La piel sin plumas, incluidos las barbillas, la cresta, los párpados y las piernas, no se vio afectada. Esta condición generó grandes pérdidas debido a un 5.3% de decomisos y recorte de canales. Se tomaron muestras de las lesiones cutáneas para histopatología y virología molecular para investigaciones diagnósticas. La histopatología reveló dermatitis necrótica multifocal con cuerpos de inclusión citoplasmáticos eosinófilos epidérmicos en las lesiones cutáneas. Las investigaciones moleculares confirmaron la presencia del virus de la viruela aviar (FWPV) en las lesiones proliferativas, con investigaciones adicionales que identificaron dos poblaciones del genoma del virus de la viruela aviar, una que portaba una porción del virus de la reticuloendoteliosis (REV) y la otra con un provirus del virus de la reticuloendoteliosis casi completo. Además, el análisis molecular basado en el gene de la proteína del núcleo 4b agrupó el virus de campo en el clado A del género Avipoxvirus.

An Outbreak of Pullorum Disease in a Young Layer Parent Flock in Austria Presented with Central Nervous System Signs.

The present report describes an outbreak of Pullorum disease in a young layer parent stock in Austria. The flock, which comprised 14,220 Lohmann brown layer chickens, experienced high mortality from the first week of life, reaching a total of 1905 chickens in the fifth week, when the flock was depopulated. Clinical signs included uneven size of the chicks, pasty vents, apathy, and diminished water and feed intake, with some birds presenting central nervous system signs such as tremors and torticollis. The postmortem investigation of 43 birds, of ages 1 to 4 weeks, revealed retained yolk sacs filled with caseous exudate, purulent airsacculitis, hepatitis with whitish pinpoint coalescing necrotic foci, splenitis with splenomegaly, hemorrhagic-mucoid enteritis in the small intestine, fibrinous typhlitis, nephromegaly, and urate deposits in the ureters and cloaca. Inflammation and/or necrosis were identified in liver, spleen, kidney, small intestine, and heart by histopathology. However, no histopathologic lesions were observed in the brain. Salmonella enterica was isolated from heart, liver, spleen, and brain in pure culture. Group-specific serotyping determined the presence of group D, with S. enterica subspecies enterica serovar Gallinarum being confirmed based on the Kauffmann-White scheme. A duplex PCR further identified S. enterica subspecies enterica serovar Gallinarum biovar Pullorum as the responsible agent for the outbreak. Subsequently, the grandparent flocks, from which the affected flock originated, were tested and found to be negative for Salmonella Pullorum, with no other progenies from the same grandparents developing disease. Although the source of the pathogen could not be identified, such findings highlight the importance of "old" pathogens such as Salmonella Pullorum causing sudden high mortality in chicks, even in a highly controlled environment.

Reporte de caso­Brote de pulorosis en una parvada de reproductores de postura jóvenes en Austria que presentó signos del sistema nervioso central. El presente reporte describe un brote de pulorosis en un lote de reproductoras de postura jóvenes en Austria. La parvada, que comprendió 14,220 gallinas de postura Lohmann, experimentó alta mortalidad desde la primera semana de vida, alcanzando un total de 1905 gallinas en la quinta semana, cuando la parvada se despobló. Los signos clínicos incluyeron tamaño desigual de pollito, empastamiento de la cloaca, apatía y disminución del consumo de agua y alimento, y algunas aves presentaron signos del sistema nervioso central como temblores y tortícolis. La investigación post mórtem de 43 aves, de 1 a 4 semanas de edad, reveló sacos vitelinos retenidos llenos de exudado caseoso, aerosaculitis purulenta, hepatitis con focos necróticos coalescentes blanquecinos, esplenitis con esplenomegalia, enteritis hemorrágica-mucoide en el intestino delgado, tiflitis fibrinosa, nefromegalia y depósitos de uratos en los uréteres y cloaca. Se identificaron inflamación y/o necrosis en hígado, bazo, riñón, intestino delgado y corazón mediante histopatología. Sin embargo, no se observaron lesiones histopatológicas en el cerebro. Se aisló Salmonella enterica de corazón, hígado, bazo y cerebro en cultivo puro. La serotipificación específica de grupo determinó la presencia del grupo D, con S entérica subespecie enterica serovar Gallinarum que se confirmó según el esquema de Kauffmann-White. Un método dúplex de PCR identificó S. enterica subspecie enterica serovar Pullorum como el agente responsable del brote. Posteriormente, las parvadas de abuelas, de las que se originó la parvada afectada, fueron analizadas y resultaron negativas para Salmonella Pullorum, sin que ninguna otra progenie de los mismos abuelos desarrollara la enfermedad. Aunque no se pudo identificar la fuente del patógeno, tales hallazgos resaltan la importancia de patógenos "viejos" como Salmonella Pullorum que causan una alta mortalidad repentina en los pollitos, incluso en un ambiente altamente controlado.

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.

Excretion of Histomonas meleagridis following experimental co-infection of distinct chicken lines with Heterakis gallinarum and Ascaridia galli.

BACKGROUND: Histomonosis is a severe re-emerging disease of poultry caused by Histomonas meleagridis, a protozoan parasite which survives in the environment via the cecal worm Heterakis gallinarum. Following infection, the parasites reside in the ceca and are excreted via host feces. In the present work, male birds of conventional broiler (Ross 308, R), layer (Lohmann Brown Plus, LB) and a dual-purpose (Lohmann Dual, LD) chicken line were infected with 250 embryonated eggs of Ascaridia galli and Heterakis gallinarum, respectively, with the latter nematode harboring Histomonas meleagridis, to investigate a co-infection of nematodes with the protozoan parasite in different host lines. METHODS: In weekly intervals, from 2 to 9 weeks post infection (wpi), individual fecal samples (n = 234) from the chickens were collected to quantify the excretion of H. meleagridis by real-time PCR and to determine the number of nematode eggs per gram (EPG) in order to elucidate excretion dynamics of the flagellate and the nematodes. This was further investigated by indirect detection using plasma samples of the birds to detect antibodies specific for H. meleagridis and worms by ELISA. The infection with H. meleagridis was confirmed by histopathology and immunohistochemistry to detect the flagellate in the cecum of representing birds. RESULTS: The excretion of H. meleagridis could already be observed from the 2nd wpi in some birds and increased to 100% in the last week of the experiment in all groups independent of the genetic line. This increase could be confirmed by ELISA, even though the number of excreted H. meleagridis per bird was generally low. Overall, histomonads were detected in 60% to 78% of birds with temporary differences between the different genetic lines, which also showed variations in the EPG and worm burden of both nematodes. CONCLUSIONS: The infection with H. gallinarum eggs contaminated with H. meleagridis led to a permanent excretion of the flagellate in host feces. Differences in the excretion of H. meleagridis in the feces of genetically different host lines occurred intermittently. The excretion of the protozoan or its vector H. gallinarum was mostly exclusive, showing a negative interaction between the two parasites in the same host.

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.

Comparative investigation of IFN-γ-producing T cells in chickens and turkeys following vaccination and infection with the extracellular parasite Histomonas meleagridis.

The re-emerging disease histomonosis is caused by the protozoan parasite Histomonas meleagridis that affects chickens and turkeys. Previously, protection by vaccination with in vitro attenuated H. meleagridis has been demonstrated and an involvement of T cells, potentially by IFN-γ production, was hypothesized. However, comparative studies between chickens and turkeys on H. meleagridis-specific T cells were not conducted yet. This work investigated IFN-γ production within CD4+, CD8α+ and TCRγδ+ (chicken) or CD3ε+CD4-CD8α- (turkey) T cells of spleen and liver from vaccinated and/or infected birds using clonal cultures of a monoxenic H. meleagridis strain. In infected chickens, re-stimulated splenocytes showed a significant increase of IFN-γ+CD4+ T cells. Contrariwise, significant increments of IFN-γ-producing cells within all major T-cell subsets of the spleen and liver were found for vaccinated/infected turkeys. This indicates that the vaccine in turkeys causes more intense systemic immune responses whereas in chickens protection might be mainly driven by local immunity.

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.

Co-infection of Chicken Layers With Histomonas meleagridis and Avian Pathogenic Escherichia coli Is Associated With Dysbiosis, Cecal Colonization and Translocation of the Bacteria From the Gut Lumen.

Histomonosis in chickens often appears together with colibacillosis in the field. Thus, we have experimentally investigated consequences of the co-infection of birds with Histomonas meleagridis and avian pathogenic Escherichia coli (APEC) on the pathology, host microbiota and bacterial translocation from the gut. Commercial chicken layers were infected via oral and cloacal routes with lux-tagged APEC with or without H. meleagridis whereas negative controls were left uninfected. Except one bird, which died due to colibacillosis, no clinical signs were recorded in birds infected with bioluminescence lux gene tagged E. coli. In co-infected birds, depression and ruffled feathers were observed in 4 birds and average body weight gain significantly decreased. Typhlitis caused by H. meleagridis was present only in co-infected birds, which also had pronounced microscopic lesions in systemic organs such as liver, heart and spleen. The 16S rRNA gene amplicon sequencing showed that in co-infected birds, corresponding to the severity of cecal lesions, microbial species richness and diversity in caeca greatly decreased and the abundance of the Escherichia group, Helicobacter and Bacteroides was relatively higher with a reduction of commensals. Most of the shared Amplicon Sequencing Variants between cecum and blood in co-infected birds belonged to Pseudomonas, Staphylococcus, and members of Enterobacteriaceae while those assigned as Lactobacillus and members of Ruminococcaceae and Lachnospiraceae were found mainly in negative controls. In infected birds, E. coli in the cecal lumen penetrated into deeper layers, a phenomenon noticed with higher incidence in the dead and co-infected birds. Furthermore, numbers of lux-tagged E. coli in caeca were significantly higher at every sampling date in co-infected birds. Altogether, infection of layers with H. meleagridis and E. coli resulted in more severe pathological changes, dramatic shift in the cecal mucosa-associated microbiota, higher tissue colonization of pathogenic bacteria such as avian pathogenic E. coli in the gut and increased penetration of E. coli from the cecal lumen toward peritoneum. This study provides novel insights into the parasite-bacteria interaction in vivo highlighting the role of H. meleagridis to support E. coli in the pathogenesis of colibacillosis in chickens.

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.