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.

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.

Comparative Surfaceome Analysis of Clonal Histomonas meleagridis Strains with Different Pathogenicity Reveals Strain-Dependent Profiles.

Histomonas meleagridis, a poultry-specific intestinal protozoan parasite, is histomonosis's etiological agent. Since treatment or prophylaxis options are no longer available in various countries, histomonosis can lead to significant production losses in chickens and mortality in turkeys. The surfaceome of microbial pathogens is a crucial component of host-pathogen interactions. Recent proteome and exoproteome studies on H. meleagridis produced molecular data associated with virulence and in vitro attenuation, yet the information on proteins exposed on the cell surface is currently unknown. Thus, in the present study, we identified 1485 proteins and quantified 22 and 45 upregulated proteins in the virulent and attenuated strains, respectively, by applying cell surface biotinylation in association with high-throughput proteomic analysis. The virulent strain displayed upregulated proteins that could be linked to putative virulence factors involved in the colonization and establishment of infection, with the upregulation of two candidates being confirmed by expression analysis. In the attenuated strain, structural, transport and energy production proteins were upregulated, supporting the protozoan's adaptation to the in vitro environment. These results provide a better understanding of the surface molecules involved in the pathogenesis of histomonosis, while highlighting the pathogen's in vitro adaptation processes.

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.

Clinically Manifesting, Naturally Occurring Fowl Glioma in a Leghorn Chicken in Germany.

Fowl glioma-inducing virus (FGV), a strain of avian leukosis virus (ALV) subgroup A, is the causal agent of fowl glioma characterized by multiple nodular astrocytic growths, gliosis, and lymphocytic encephalitis. Also associated with FGV infection are cases of cerebellar hypoplasia, perineuromas, and nonsuppurative myocarditis. Though fowl glioma has been recognized in several countries, most reports of FGV infection come from Japan. A 9-mo-old brown leghorn from a German farm with nine leghorns was presented to a veterinarian with an impaired general health with torticollis, tremor, and incoordination. Histopathology revealed multifocal nodular astrocytic growths, gliosis, and a lymphoplasmacytic encephalitis. Immunohistochemically, neoplastic astrocytes showed positivity for anti-ALV antibody. FGV was detected in the brain with nested reverse transcription-polymerase chain reaction (RT-PCR) and subsequent sequencing of PCR product. The remaining eight birds were screened for the presence of ALV with real-time RT-PCR. Four leghorns tested positive for exogenous ALV in nested RT-PCR with an identical nucleotide sequence as the leghorn with neurological symptoms. To the authors' knowledge this is the first report of a natural FGV infection in a brown leghorn in Germany with clinical manifestation.

Glioma aviar de manifestación clínica y natural en un pollo Leghorn en Alemania. El virus inductor del glioma del pollo (FGV), una cepa del subgrupo A del virus de la leucosis aviar (ALV), es el agente causal del glioma del pollo caracterizado por crecimientos astrocíticos nodulares múltiples, gliosis y encefalitis linfocítica. También se asocian con la infección por este virus, casos de hipoplasia cerebelar, perineuromas y miocarditis no supurativa. Aunque el glioma aviar se ha reconocido en varios países, la mayoría de los informes de infección por el virus inductor del glioma del pollo provienen de Japón. Un pollo Leghorn marrón de nueve meses de edad proveniente de una granja alemana con nueve aves Leghorns fue remitido a una clínica veterinaria con problemas de salud en general, tortícolis, temblores y falta de coordinación. La histopatología reveló crecimientos astrocíticos nodulares multifocales, gliosis y encefalitis linfoplasmocítica. Inmunohistoquímicamente, los astrocitos neoplásicos mostraron reacción positiva para anticuerpos contra el virus de la leucosis aviar. El virus inductor del glioma del pollo se detectó en el cerebro mediante transcripción reversa y reacción en cadena de la polimerasa anidada (RT-PCR) y con secuenciación posterior del producto de PCR. Las ocho aves restantes se examinaron para detectar la presencia del virus de la leucosis aviar mediante RT-PCR en tiempo real. Cuatro aves Leghorn dieron positivo para virus exógenos de leucosis mediante RT-PCR anidada y con una secuencia de nucleótidos idéntica a la del ave Leghorn con síntomas neurológicos. De acuerdo con el conocimiento de los autores, este es el primer informe de una infección natural por el virus inductor del glioma del pollo en un ave Leghorn marrón en Alemania que presentaba manifestaciones clínicas.

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.

Complete genomes of the eukaryotic poultry parasite Histomonas meleagridis: linking sequence analysis with virulence / attenuation.

BACKGROUND: Histomonas meleagridis is a protozoan parasite and the causative agent of histomonosis, an important poultry disease whose significance is underlined by the absence of any treatment and prophylaxis. The recent successful in vitro attenuation of the parasite urges questions about the underlying mechanisms. RESULTS: Whole genome sequence data from a virulent and an attenuated strain originating from the same parental lineage of H. meleagridis were recruited using Oxford Nanopore Technology (ONT) and Illumina platforms, which were combined to generate megabase-sized contigs with high base-level accuracy. Inspecting the genomes for differences identified two substantial deletions within a coding sequence of the attenuated strain. Additionally, one single nucleotide polymorphism (SNP) and indel targeting coding sequences caused the formation of premature stop codons, which resulted in the truncation of two genes in the attenuated strain. Furthermore, the genome of H. meleagridis was used for characterizing protein classes of clinical relevance for parasitic protists. The comparative analysis with the genomes of Trichomonas vaginalis, Tritrichomonas foetus and Entamoeba histolytica identified ~ 2700 lineage-specific gene losses and 9 gene family expansions in the H. meleagridis lineage. CONCLUSIONS: Taken as a whole, the obtained data provide the first hints to understand the molecular basis of attenuation in H. meleagridis and constitute a genomics platform for future research on this important poultry pathogen.

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.

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.

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.

Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens.

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1-35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.

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.

Interplay between Histomonas meleagridis and Bacteria: Mutualistic or Predator-Prey?

Histomonas meleagridis is an extracellular protozoan parasite and the aetiological agent of histomonosis, an important poultry disease whose impact is greatly accentuated by inaccessibility of any treatment. A special feature of the parasite is its intricate interplay with bacteria in vitro and in vivo, the focus of this article.

Membrane associated proteins of two Trichomonas gallinae clones vary with the virulence.

Oropharyngeal avian trichomonosis is mainly caused by Trichomonas gallinae, a protozoan parasite that affects the upper digestive tract of birds. Lesions of the disease are characterized by severe inflammation which may result in fatality by starvation. Two genotypes of T. gallinae were found to be widely distributed in different bird species all over the world. Differences in the host distribution and association with lesions of both genotypes have been reported. However, so far no distinct virulence factors of this parasite have been described and studies might suffer from possible co-infections of different genotypes. Therefore, in this paper, we analyzed the virulence capacity of seven clones of the parasite, established by micromanipulation, representing the two most frequent genotypes. Clones of both genotypes caused the maximum score of virulence at day 3 post-inoculation in LMH cells, although significant higher cytopathogenic score was found in ITS-OBT-Tg-1 genotype clones at days 1 and 2, as compared to clones with ITS-OBT-Tg-2. By using one representative clone of each genotype, a comparative proteomic analysis of the membrane proteins enriched fraction has been carried out by a label free approach (Data available via ProteomeXchange: PXD013115). The analysis resulted in 302 proteins of varying abundance. In the clone with the highest initial virulence, proteins related to cell adhesion, such as an immuno-dominant variable surface antigen, a GP63-like protein, an armadillo/beta-catenin-like repeat protein were found more abundant. Additionally, Ras superfamily proteins and calmodulins were more abundant, which might be related to an increased activity in the cytoskeleton re-organization. On the contrary, in the clone with the lowest initial virulence, larger numbers of the identified proteins were related to the carbohydrate metabolism. The results of the present work deliver substantial differences between both clones that could be related to feeding processes and morphological changes, similarly to the closely related pathogen Trichomonas vaginalis.

PCR and serology confirm the infection of turkey hens and their resilience to histomonosis in mixed flocks following high mortalities in toms.

BACKGROUND: Histomonosis, caused by the protozoan parasite Histomonas meleagridis, is a severe disease especially in turkeys where it can cause high mortalities. Recently, outbreaks were described in which turkey hens showed no clinical signs despite high mortalities in toms, from which they were separated only by a wire fence. The present study investigated three similar outbreaks of histomonosis whereby in two of them only a few hens were being affected and none in the third. Hens from all flocks were kept until end of production and slaughtered as scheduled. However, in all three cases, the disease progressed in toms reaching nearly 100% within two weeks. METHODS: Following diagnosis of the disease, tissue samples were obtained from toms and hens at necropsy. Environmental dust, cloacal swabs and blood were taken on three successive farm visits within compartments of hens and toms and tested by real-time PCR or ELISA. The DNA from a total of 18 samples positive for H. meleagridis was further subjected to conventional PCR utilizing the 18S rRNA primers and sequenced for phylogenetic analysis. RESULTS: All tissue samples and some cloacal swabs were tested positive. Dust samples confirmed the presence of H. meleagridis DNA that spread within entire houses up to 6 weeks after the first clinical signs of histomonosis. Sequence analysis of the 18S rRNA locus demonstrated the presence of the same strain in birds of both sexes within each of the turkey houses. Investigation of serum samples two weeks post-initial diagnosis and prior to euthanasia resulted in antibody detection in 73% of toms and 70% of hens. Until the end of the investigation the number of positive hens per farm increased up to 100% with mean OD-values approaching those noticed in toms prior to euthanasia. CONCLUSIONS: For the first time it could be demonstrated that turkey hens kept in the same house as toms became infected during fatal outbreaks in toms. This highlights the value of different diagnostics methods in order to trace the parasite in connection with the host response. The strange phenomenon that only single hens succumb to the diseases despite being infected requires further investigations.

Detection of Histomonas meleagridis DNA in dust samples obtained from apparently healthy meat turkey flocks without effect on performance.

Environmental dust samples obtained from 65 turkey flocks in France, of which six suffered from histomonosis whereas the rest remained apparent healthy until the end of production, were tested for the presence of Histomonas meleagridis DNA by recently developed real-time PCR based on the 18S rRNA locus. In order to determine the genotype of detected histomonads, positive samples were further subjected to conventional 18S PCR and sequencing. Additionally, production data of all tested flocks, such as average daily gain, feed conversion ratio and production index, were statistically evaluated and compared to see the effect of positive dust samples in apparently healthy flocks. Histomonad DNA was detected in the dust obtained from all six clinically affected and, surprisingly, in nine apparent healthy flocks. Sequencing of the 18S rRNA gene resulted in only one DNA sample homologous to H. meleagridis whereas 11 others revealed the presence of several other flagellates. Average daily gain and production index were negatively affected in flocks with clinical histomonosis, resulting in significant difference in comparison with the data obtained from clinically healthy flocks independent of the presence of histomonad DNA in the dust. Overall, there was no significant difference following statistical analysis of production parameters between the two last mentioned groups of tested flocks. Altogether, this is the first investigation demonstrating the presence of H. meleagridis DNA in environmental dust samples obtained from clinically unaffected turkey flocks. However, this finding could not be correlated with impact on production based on analysis and comparison of selected production data. RESEARCH HIGHLIGHTS Environmental dust obtained from clinically healthy turkey flocks, in addition to dust from flocks affected by histomonosis, was found positive for the presence of Histomonas meleagridis DNA. Histomonas-positive dust samples in clinically unaffected flocks did not have a negative effect on production parameters. The results demonstrate a wider spread of H. meleagridis DNA in flocks of commercial meat turkeys than previously thought.

Molecular characterization of Histomonas meleagridis exoproteome with emphasis on protease secretion and parasite-bacteria interaction.

The exoproteome of parasitic protists constitutes extracellular proteins that play a fundamental role in host-parasite interactions. Lytic factors, especially secreted proteases, are capable of modulating tissue invasion, thereby aggravating host susceptibility. Despite the important role of exoproteins during infection, the exoproteomic data on Histomonas meleagridis are non-existent. The present study employed traditional 1D-in-gel-zymography (1D-IGZ) and micro-LC-ESI-MS/MS (shotgun proteomics), to investigate H. meleagridis exoproteomes, obtained from a clonal virulent and an attenuated strain. Both strains were maintained as mono-eukaryotic monoxenic cultures with Escherichia coli. We demonstrated active in vitro secretion kinetics of proteases by both parasite strains, with a widespread proteolytic activity ranging from 17 kDa to 120 kDa. Based on protease inhibitor susceptibility assay, the majority of proteases present in both exoproteomes belonged to the family of cysteine proteases and showed stronger activity in the exoproteome of a virulent H. meleagridis. Shotgun proteomics, aided by customized database search, identified 176 proteins including actin, potential moonlighting glycolytic enzymes, lytic molecules such as pore-forming proteins (PFPs) and proteases like cathepsin-L like cysteine protease. To quantify the exoproteomic differences between the virulent and the attenuated H. meleagridis cultures, a sequential window acquisition of all theoretical spectra mass spectrometric (SWATH-MS) approach was applied. Surprisingly, results showed most of the exoproteomic differences to be of bacterial origin, especially targeting metabolism and locomotion. By deciphering such molecular signatures, novel insights into a complex in vitro protozoan- bacteria relationship were elucidated.

An Alliance of Gel-Based and Gel-Free Proteomic Techniques Displays Substantial Insight Into the Proteome of a Virulent and an Attenuated Histomonas meleagridis Strain.

The unicellular protozoan Histomonas meleagridis is notorious for being the causative agent of histomonosis, which can cause high mortality in turkeys and substantial production losses in chickens. The complete absence of commercially available curative strategies against the disease renders the devising of novel approaches a necessity. A fundamental step toward this objective is to understand the flagellate's virulence and attenuation mechanisms. For this purpose we have previously conducted a comparative proteomic analysis of an in vitro cultivated virulent and attenuated histomonad parasite using two-dimensional electrophoresis and MALDI-TOF/TOF. The current work aimed to substantially extend the knowledge of the flagellate's proteome by applying 2D-DIGE and sequential window acquisition of all theoretical mass spectra (SWATH) MS as tools on the two well-defined strains. In the gel-based experiments, 49 identified protein spots were found to be differentially expressed, of which 37 belonged to the in vitro cultivated virulent strain and 12 to the attenuated one. The most frequently identified proteins in the virulent strain take part in cytoskeleton formation, carbohydrate metabolism and adaptation to stress. However, post-translationally modified or truncated ubiquitous cellular proteins such as actin and GAPDH were identified as upregulated in multiple gel positions. This indicated their contribution to processes not related to cytoskeleton and carbohydrate metabolism, such as fibronectin or plasminogen binding. Proteins involved in cell division and cytoskeleton organization were frequently observed in the attenuated strain. The findings of the gel-based studies were supplemented by the gel-free SWATH MS analysis, which identified and quantified 42 significantly differentially regulated proteins. In this case proteins with peptidase activity, metabolic proteins and actin-regulating proteins were the most frequent findings in the virulent strain, while proteins involved in hydrogenosomal carbohydrate metabolism dominated the results in the attenuated one.

Unravelling the differences: comparative proteomic analysis of a clonal virulent and an attenuated Histomonas meleagridis strain.

The current study focused on Histomonas meleagridis, a unicellular protozoan, responsible for histomonosis in poultry. Recently, the occurrence of the disease increased due to the ban of effective chemotherapeutic drugs. Basic questions regarding the molecular biology, virulence mechanisms or even life cycle of the flagellate are still puzzling. In order to address some of these issues, we conducted a comparative proteomic analysis of a virulent and an attenuated H. meleagridis strain traced back to a single cell and propagated in vitro as monoxenic mono-eukaryotic cultures. Using two-dimensional electrophoresis (2-DE) for proteome visualization with computational 2-DE gel image and statistical analysis, upregulated proteins in either of the two H. meleagridis strains were detected. Statistical analysis fulfilling two criteria (≥threefold upregulation and P < 0.05) revealed 119 differentially expressed protein spots out of which 62 spots were noticed in gels with proteins from the virulent and 57 spots in gels with proteins from the attenuated culture. Mass spectrometric analysis of 32 protein spots upregulated in gels of the virulent strain identified 17 as H. meleagridis-specific. The identification revealed that these spots belonged to eight different proteins, with the majority related to cellular stress management. Two ubiquitous cellular proteins, actin and enolase, were upregulated in multiple gel positions in this strain, indicating either post-translational modification or truncation, or even both. Additionally, a known virulence factor named legumain cysteine peptidase was also detected. In contrast to this, mass spectrometric analysis of 49 protein spots, upregulated in gels of the attenuated strain, singled out 32 spots as specific for the flagellate. These spots were shown to correspond to 24 different proteins that reflect the increased metabolism, in vitro adaptation of the parasite, and amoeboid morphology. In addition to H. meleagridis proteins, the analysis identified differential expression of Escherichia coli DH5α proteins that could have been influenced by the co-cultivated H. meleagridis strain, indicating a reciprocal interaction of these two organisms during monoxenic cultivation.