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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 CD8β+ 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−CD8β− 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−CD8β− 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.

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. Graphic abstract

Recently, outbreaks of adenoviral gizzard erosion (AGE) have been documented in pullets and layers housed free range and in enriched cage systems characterized by increased mortality and a negative impact on egg production. In the present study the pathogenicity of a fowl adenovirus serotype 1 (FAdV-1) field strain as well as the aetiological role of a FAdV-8a strain, both isolated from AGE affected pullets, were investigated in vivo in 20-week-old specific-pathogen-free (SPF) layer-type chickens. Furthermore, the efficacy of a single (week 17) and double (week 14 and 17) application of a live vaccine consisting of an apathogenic FAdV-1 (CELO strain) against challenge with virulent FAdV-1 was investigated. For the first time, AGE was successfully reproduced in adult birds after oral infection of 20-week-old SPF birds with a virulent FAdV-1 field isolate, characterized by pathological changes of the gizzard from 7 days post challenge onwards. In addition, a negative impact of the FAdV-1 infection on the development of the reproductive tract was observed. Thus, confirming the pathogenicity and aetiological role of FAdV-1 in the development of AGE and economic losses due to AGE in layers. In contrast, no pathological changes were observed in birds infected with FAdV-8a. Independent of a single or double application of the live FAdV-1 vaccine strain CELO, no gross pathological changes were observed in gizzards post challenge with the virulent FAdV-1, indicating that complete protection of layers against horizontal induction of AGE was achieved. Nonetheless, virulent FAdV-1 was detected in cloacal swabs and gizzards in both vaccinated groups post challenge determined by the application of an amplification refractory mutation system quantitative PCR used to differentiate between vaccine and challenge strains.

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 CD3e+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.

The avian immune system has been shown to possess a repertoire of cytokines directing T-helper (Th) 1 and Th2 types of immune responses similar to that in mammals. The objective of this study was to establish in situ hybridization (ISH) for the localization of mRNA of selected signal cytokines, chicken interferon-γ (ChIFN-γ), chicken interleukin (ChIL)-4 and ChIL-13 in fixed tissues. RNA probes were generated to hybridize to 488, 318, and 417 bp of the respective target mRNA. Probe concentrations ranging from 100 ng/ml to 400 ng/ml were shown to be suitable to label cells that expressed these cytokines. The specificity of every probe was verified using the respective sense probe. ChIFN-γ, ChIL-4 and ChIL-13 positive cells were observed in the lymphocytic infiltrations of liver and in the periarteriolar lymphatic sheaths of spleen collected from specific-pathogen-free chickens. ISH of these cytokines in a severely inflamed liver due to infiltration with the parasite Histomonas meleagridis revealed the expression of both ChIFN-γ and ChIL-13 mRNA in the mononuclear infiltrates. In conclusion, ChIFN-γ, ChIL-4 and ChIL-13 mRNA were efficiently localized by ISH, which supplies a valid technique to characterize immune responses in fixed tissues.

Histomonosis is a parasitic disease of gallinaceous birds characterized by necrotic lesions in cacum and liver that usually turns fatal in turkeys while it is less severe in chickens. Vaccination using in vitro attenuated Histomonas meleagridis has been experimentally shown to confer protection against histomonosis. The protective mechanisms that underpin the vaccine-induced immune response are not resolved so far. Therefore, the actual study aimed to evaluate the location and quantitative distribution patterns of signature cytokines of type 1 [interferon gamma (IFN-γ)] or type 2 [interleukin (IL)-13] immune responses in vaccinated or infected hosts. An intergroup and interspecies difference in the spatial and temporal distribution patterns of cytokine mRNA positive cells was evident. Quantification of cells showed a significantly decreased percentage of IFN-γ mRNA positive cells at 4 days post-inoculation (DPI) in caeca of turkeys inoculated exclusively with the attenuated or the virulent inocula, compared to control birds. The decrement was followed by a surge of cells expressing mRNA for IFN-γ or IL-13, reaching a peak of increment at 10 DPI. By contrast, turkeys challenged following vaccination showed a slight increment of cecal IFN-γ mRNA positive cells at 4 DPI after which positive cell counts became comparable to control birds. The increase in infected birds was accompanied by an extensive distribution of positively stained cells up to the muscularis layer of cecal tissue whereas the vaccine group maintained an intact mucosal structure. In chickens, the level of changes of positive cells was generally lower compared to turkeys. However, control chickens were found with a higher percentage of IFN-γ mRNA positive cells in cecum compared to their turkey counterparts indicating a higher resistance to histomonosis, similar to the observation in immunized turkeys. In chickens, it could be shown that the changes of cytokine-positive cells were related to variations of mononuclear cells quantified by immunofluorescence. Furthermore, gene expression measured by reverse transcription quantitative real time PCR confirmed variations in organs between the different groups of both bird species. Overall, it can be concluded that a proportionally increased, yet controlled, allocation of IFN-γ mRNA positive cells in caeca hallmarks a protective trait against histomonosis.

Recently we demonstrated that co-infection with Avibacterium paragallinarum and Gallibacterium anatis leads to increased severity of clinical signs of infectious coryza in birds. The present study examined the interaction of these two pathogens in chickens by evaluation of histologic lesions in sinus infraorbitalis and nasal turbinates, applying a defined scoring scheme ranging from 0 to 3. Furthermore, for the first time, an in situ hybridization (ISH) technique was applied to detect A. paragallinarum in tissues. The samples were received from vaccinated and nonvaccinated birds that were infected with A. paragallinarum and/or G. anatis. Vaccinated birds were mostly devoid of any histopathologic lesions except a few birds with lesion score 1 at 7 and 14 days postinfection (dpi). Likewise, nonvaccinated birds infected with G. anatis only did not present microscopic changes in the sinus infraorbitalis, except in a single bird at 7 dpi. Interestingly, median lesion scores caused by G. anatis infection were significantly higher in the nasal turbinates of infected birds than in negative control at 7 and 14 dpi. The most prominent histologic changes were recorded from sinus infraorbitalis and nasal turbinates of nonvaccinated birds that were infected either with A. paragallinarum only or together with G. anatis. ISH demonstrated positive signals for A. paragallinarum in exudates present in the lumen or attached to the epithelial layer of investigated tissues. Such signals were mainly detected in tissues from birds with the highest histopathologic lesion scores.

Typhlohepatitis was observed in a flock of 2500 red-legged partridges in Great Britain, characterized by the sudden deaths of 15 birds within 2 days. Necropsy of five dead birds revealed severe lesions in the caeca with thickened caecal walls, a reddened lining and bloody contents. The livers contained multiple miliary lesions and similar pathological changes were found in the spleens of some birds. Microscopic examination of intestinal contents showed the occurrence of coccidial oocysts in two partridges. Different methods for the detection of bacteria from liver and intestine samples were conducted without positive results. Histopathological examination revealed the presence of protozoan parasites in the caecum, liver and spleen of the affected birds. In situ hybridization (ISH) for the detection of trichomonads resulted in positive findings and polymerase chain reaction (PCR) confirmed the presence of Tetratrichomonas gallinarum in the lesions. Additionally, archived tissues of red-legged partridges from different flocks suffering from severe typhlohepatitis in Great Britain in 2008 and 2009 were re-investigated by ISH and PCR. Beside the sporadic occurrence of histomonosis, in most of the cases trichomonads were detected by ISH in the caecum and liver of affected birds. Furthermore, dissemination of the flagellate into the lung and bursa of Fabricius could be demonstrated. Analyses of T. gallinarum DNA obtained from the different cases resulted in homologous nucleotide sequences. Altogether, the results demonstrate the circulation of a virulent strain of T. gallinarum in reared red-legged partridges.

Gizzard erosion in broilers due to an infection with virulent fowl adenovirus serotype 1 (FAdV-1) is an emerging disease. Although experimental studies were performed, a possible prevention strategy was not reported so far. The present study was set up to determine (i) a possible influence of birds' age at time of inoculation on the pathogenicity of a European FAdV-1 field strain (PA7127), (ii) the virulence of a apathogenic FAdV-1 strain (CELO), and (iii) its capability to protect SPF broilers from adenoviral gizzard erosion caused by the field virus. Oral infection of birds with PA7127 at 1-, 10- and 21-days of life, resulted in reduced weight gain compared to non-infected birds, with significance for birds infected at day-old. Independent of the birds' age at time of inoculation, clinical signs appearing approximately one week after challenge coincided with gizzard lesions. Birds infected exclusively with CELO at the first day of life did not show any clinical signs or pathological changes in the gizzard, confirming the apathogenicity of this European FAdV-1. A similar result was obtained for birds orally infected at the first day of life with CELO and challenged three weeks later with the pathogenic PA7127 strain. Therefore, complete protection of adenoviral gizzard erosion in broilers by vaccination of day-old birds could be demonstrated for the first time, although virus excretion was detected post challenge. Establishment of an amplification refractory mutation system quantitative PCR (ARMS-qPCR) facilitated the identification of the FAdV-1 strain and presence of challenges virus was confirmed in one sample.

In the present study the effects of dietary gizzard stimulation on the development and severity of adenoviral gizzard erosion were investigated. For this purpose, specific pathogen-free broilers were divided into six groups, investigating the influence of an oat-containing diet with higher fibre content, a whole wheat-containing diet and a control diet of nearly identical composition, but containing ground wheat. For each feed administered, one group of birds was experimentally infected on the 10th day of age by the oral route with virulent fowl adenovirus serotype 1 (FAdV-1), recently proven to induce gizzard erosions, while the respective negative control groups remained uninfected. Experimental feed was administered from 2 days post-infection onwards. No significant differences on gizzard health or in weight gain could be detected between uninfected control groups or between FAdV-1 infected groups that received different experimental feed. However, independent of the supplied diet, a significantly reduced weight gain was noted from 7 days post-infection onwards in FAdV-1 infected broilers compared to uninfected birds that received the same diet. Macroscopically, discolouration and erosion of the koilin layer and inflammation of the gizzard mucosa were observed in all FAdV-1 infected groups. Histologically, necrosis, degeneration of gizzard epithelial cells and multiple basophilic intranuclear inclusion bodies were observed. In summary, after experimental infection with FAdV-1 development of gizzard erosion in chickens was not influenced by the feeding regimes investigated. Therefore, it is unlikely that dietary gizzard stimulation influences the outcome of adenoviral gizzard erosion in vertically infected 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.