Infection dynamics of Salmonella Infantis vary considerably between chicken lines.

ABSTRACTSalmonella (S.) Infantis is the most common serovar in broilers and broiler meat in the European Union. In the field, fast-growing broilers are reported to be more affected than slow-growing and layer birds. The present study investigated the infection dynamics and immunological response of four chicken lines in the course of a S. Infantis infection. Two commercial chicken lines, Ross 308 and Hubbard ISA-JA-757, and two experimental chicken lines, specific pathogen free (SPF) layers and broilers, were infected at 2 days of age. Investigations focused on faecal shedding, bacterial colonization, humoral and cellular immune response. Ross and SPF broilers proved mainly as high shedders followed by Hubbard. SPF layers showed the least shedding. This is in agreement with the caecal colonization; SPF layers harboured significantly less bacteria. Systemic spread of S. Infantis to liver and spleen was highest in Ross broilers compared to the other lines. Spread of infection to in-contact birds, was noticed 5 days post infection in every line. Antibody response occurred in every chicken line from 21 days of age onwards. In contrast to the other chicken lines, significant differences in T cell subsets and monocytes/macrophages were found between infected and negative Hubbard birds at 7 days of age. Uninfected SPF birds had significantly higher immune cell counts than uninfected commercial birds, a fact important for future experimental settings. The results illustrate that the infection dynamics of S. Infantis is influenced by the chicken line resulting in a higher risk of transmission to humans from fast-growing broilers.RESEARCH HIGHLIGHTS Infection dynamics of Salmonella Infantis differs between chicken lines.Layers showed less faecal shedding and caecal colonization compared to broilers.Fast-growing broilers proved more susceptible than slow-growing broilers.

Outbreaks of Streptococcus gallolyticus subsp. pasteurianus in Goslings Characterized by Central Nervous Symptoms.

The present report describes outbreaks of Streptococcus gallolyticus subsp. pasteurianus in young geese flocks in Austria. The flocks, comprising 160-1450 goslings of 2-3 wk of age, experienced increased mortalities The clinical signs were characterized by severe central nervous symptoms, namely leg paddling and torticollis. The postmortem investigation revealed hepatitis, splenitis, and a low amount of liquid fluid in the coelomic cavity. Livers were of fragile texture, with white necrotic areas. The latter were also found in spleens. No macroscopic lesions were seen in brains. Bacteriologic investigation followed by bacterial identification by matrix-assisted laser desorption time-of-flight mass spectrometry and phylogenetic analysis of the partial 16S rRNA region revealed the presence in heart, liver, spleen, and brain of S. gallolyticus subsp. pasteurianus. Histologic investigation revealed multifocal necrosis in liver and spleen samples together with infiltration of mononuclear cells and heterophilic granulocytes. Furthermore, in the lesions, coccoid bacteria could be identified. No histopathologic changes were observed in brain samples from goslings, except in one bird in which accumulation of coccoid bacteria in blood vessels of the brain samples was present. Antibiotic sensitivity tests revealed identical profiles for all strains, which were susceptible to penicillins, cephalosporins, chloramphenicol, imipenem, and tylosin. However, resistance was found against quinolones, aminoglycosides, tetracycline, and trimethoprim-sulfamethoxazole, which are commonly used to treat infections with gram-positive bacteria.

Reporte de caso­Brotes de Streptococcus gallolyticus subsp. pasteurianus en gansitos caracterizados por síntomas nerviosos centrales. El presente informe describe brotes de Streptococcus gallolyticus subsp. pasteurianus en parvadas de gansos jóvenes en Austria. Las bandadas, que comprendían entre 160 a 1450 gansos de 2 a 3 semanas de edad, experimentaron aumento de la mortalidad. Los signos clínicos se caracterizaron por síntomas severos del sistema nervioso central, incluyendo, movimientos de pataleo y tortícolis. La investigación post mórtem reveló hepatitis, esplenitis y la presencia de líquido en la cavidad celómica en poca cantidad. Los hígados presentaron textura frágil, con áreas necróticas blancas. Estos últimos también se encontraron en bazos. No se observaron lesiones macroscópicas en el cerebro. La investigación bacteriológica seguida de la identificación bacteriana mediante espectrometría de masas MALDI-TOF y el análisis filogenético de la región parcial de ARNr 16S revelaron la presencia en el corazón, el hígado, el bazo y en el cerebro de S. gallolyticus subsp. pasteurianus. La investigación histológica reveló necrosis multifocal en muestras de hígado y bazo junto con infiltración de células mononucleares y granulocitos heterófilos. Además, en las lesiones se pudieron identificar bacterias de morfología cocoide. No se observaron cambios histopatológicos en muestras de cerebro de los gansitos, excepto en un ave en la que se observó acumulación de bacterias cocoides en los vasos sanguíneos de las muestras de cerebro. Las pruebas de sensibilidad a los antibióticos revelaron perfiles idénticos para todas las cepas, que eran susceptibles a penicilinas, cefalosporinas, cloranfenicol, imipenem y tilosina. Sin embargo, se encontró resistencia contra quinolonas, aminoglucósidos, tetraciclina y trimetoprim-sulfametoxazol, que se usan comúnmente para tratar infecciones con bacterias grampositivas.

Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and racing pigeons (Columba livia): genetic and morphometric characterisation of clonal cultures.

Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur.

Histomonosis in poultry: previous and current strategies for prevention and therapy.

Histomonosis is a parasitic disease of poultry with worldwide prevalence. The disease can cause morbidity and mortality in chicken and turkey flocks entailing severe economic losses. In the first half of the last century, there was a high demand to control histomonosis as the turkey industry was severely affected by the disease. Consequently, numerous chemical compounds were tested for their efficacy against Histomonas meleagridis with varying outcomes, that are summarized and specified in this review. At the same time, preliminary attempts to protect birds with cultured histomonads indicated the possibility of vaccination. Several years ago antihistomonal drugs were banned in countries with tight regulations on pharmaceuticals in order to comply with the demand of consumer protection. As a consequence, outbreaks of histomonosis in poultry flocks increased and the disease became endemic again. New approaches to prevent and treat histomonosis are, therefore, needed and recently performed studies focused on various areas to combat the disease, from alternative chemotherapeutic substances to plant-derived compounds until vaccination, altogether reviewed here. Considering existing regulations and the overall outcome of experimental studies, it can be concluded that vaccination is very promising, despite the fact that various challenges need to be addressed until the first ever developed vaccine based upon live flagellates in human or bird medicine can be marketed.

An in vitro attenuated strain of Histomonas meleagridis provides cross-protective immunity in turkeys against heterologous virulent isolates.

In the current study, cross-protective immunity induced by a well-defined clonal strain of Histomonas meleagridis, attenuated by prolonged in vitro cultivation against different clonal heterologous isolates of the same parasite was investigated. For this purpose, 86 turkey poults were assigned to groups consisting of 9-10 birds. Birds of four groups were vaccinated on their 1st day of life followed by re-vaccination on their 14th day of life when the remaining turkeys were left untreated. The challenge was performed using four strains of H. meleagridis that were isolated from chickens or turkeys from different outbreaks of histomonosis in Europe and three of them showed diversities in their genome. Hence, every strain used for the challenge was applied to a group of vaccinated and a group of non-vaccinated birds while birds of the negative control group were sham inoculated. Non-vaccinated birds suffered from severe histomonosis due to the challenge with fatalities reaching from 5 to 10 turkeys per group. Vaccinated birds did not contract clinical signs of the disease following challenge and the increase in weight was unaffected compared to birds of the negative control group. A significant difference in lesion scores was recorded between vaccinated and non-vaccinated groups, with very few instances of liver involvement in the former groups. Livers of vaccinated birds that were without recordable macroscopic lesions were also found negative by immunohistochemical investigation. According to the data obtained, the present study demonstrates, for the first time, the cross-protective capability of a tentative vaccine strain of H. meleagridis attenuated in vitro against heterologous virulent isolates of different origin.

A single strain of Tetratrichomonas gallinarum causes fatal typhlohepatitis in red-legged partridges (Alectoris rufa) to be distinguished from histomonosis.

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.

Identification of a new reovirus causing substantial losses in broiler production in France, despite routine vaccination of breeders.

Numerous cases of tenosynovitis appeared in France causing high morbidity in free-range and standard broilers. The main clinical findings were lameness, stunting and non-uniform bodyweights. Although the natural mortality was low, the economic losses due to birds that had to be removed from the flock prematurely, downgrading of carcases and lower average weights at slaughter were substantial. Postmortem examinations, bacteriological, virological and serological examination confirmed the aetiology of avian orthoreovirus (ARV)-induced tenosynovitis. The isolated ARVs were analysed serologically and genetically. Sequencing of σC RT-PCR products and phylogenetic analysis revealed a new type of ARV. The virus was not neutralised in serum neutralisation test using monovalent sera from vaccinated chickens. Together with the flock data, epidemiology of these recent reovirus outbreaks in France was reconstructed. It is concluded that these reovirus isolates differ serologically and genetically from the well described reovirus isolates used in commercial vaccines which were not capable of preventing the disease. The outbreaks resulted in substantial losses in broilers from vaccinated breeders.

Riemerella anatipestifer outbreaks in commercial goose flocks and identification of isolates by MALDI-TOF mass spectrometry.

Several outbreaks of Riemerella anatipestifer in commercial geese occurred within a short time period. A serious disease was recognized in the affected birds, mainly characterized by depression and severe neurologic disturbances. The morbidity ranged from 20 to 30% and the mortality from 5 to 20%. Generally, the clinical signs started at the age of 8 to 10 days. Post-mortem examination revealed fibrinous pericarditis, perihepatitis and airsacculitis in all birds. Some of the birds also had synovitis of the tibio-tarsal joints and oedematous swelling of the subcutaneous tissues around these joints and metatarsus. Histology revealed a characteristic severe inflammation with heterophilic granulocytes in different organs. Bacteriological investigation was made from several organs and R. anatipestifer could be isolated from all birds investigated. The identification of these clinical isolates, done for the first time by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, confirmed the aetiology. Sequence analysis showed 100% similarity between the clinical isolates, indicating a common source of infection.

Vaccination against histomonosis prevents a drop in egg production in layers following challenge.

The effect of attenuated Histomonas meleagridis on pullets was investigated and the protection of vaccinated adult laying hens against a severe challenge was studied in the same experimental setting. Four groups of 25 pullets were set up at 18 weeks of life and birds in two groups were vaccinated with in vitro-attenuated H. meleagridis. Chickens in two groups (vaccinated and non-vaccinated) were challenged 5 weeks later with virulent histomonads, while the remaining groups were retained until termination of the study 11 weeks post vaccination. Vaccination of pullets did not have any impact on their subsequent performance. Egg production of non-vaccinated but challenged birds dropped significantly (P ≤ 0.05) between 2 and 4 weeks post challenge (p.c.) to 58.7%, compared with 90% in control chickens. At 4 weeks p.c., the drop in egg production in vaccinated and challenged birds was significantly lower (P=0.02) than in non-protected layers. Pathological changes were found only in challenged birds 2 and 6 weeks p.c. Several non-vaccinated birds showed severe lesions in the caeca with sporadic involvement of the liver and atrophy of the reproductive tract. Vaccination prior to challenge reduced the incidence of pathological findings. For the first time, vaccination of pullets with in vitro-attenuated histomonads could be shown to be an effective and safe prophylactic tool to prevent a severe drop in egg production of commercial layers following experimental infection.

Antihistomonal effects of artemisinin and Artemisia annua extracts in vitro could not be confirmed by in vivo experiments in turkeys and chickens.

Five different Artemisia annua-derived materials (i.e. dry leaves, pure artemisinin, and hexane, dichloromethane or methanol extracts of leaves) were screened for their in vitro activities against six clonal cultures of Histomonas meleagridis. Except for the methanol extract, all tested materials displayed in vitro activity against all tested protozoal clones. Neither the dry plant material, extracts nor artemisinin showed any antibacterial activity against the xenic bacteria accompanying the six H. meleagridis clones at concentration levels identical to the antihistomonal setting. The dichloromethane extract of dry leaves (Ext-DCM) (minimal lethal concentration=1.0 mg/ml) and artemisinin (half-maximal inhibitory concentration=1.295 mg/ml) had the most promising antihistomonal properties and were therefore subsequently tested in a standardized experimental infection model in both turkeys and chickens infected with clonal H. meleagridis. There were no differences between treatment groups, where all infected turkeys showed severe clinical histomonosis and demonstrated severe typhlohepatitis typical for histomonosis. Consistent with the infection model used, the infected chickens did not show any adverse clinical signs but contracted severe lesions in their caeca 7 and 10 days post infection (d.p.i.), liver lesions were absent to mild after 7 d.p.i. and progressed to severe lesions at 10 d.p.i.; thus no differences between treatment groups were observed. In conclusion, neither artemisinin nor Ext-DCM was able to prevent experimental histomonosis in turkeys and chickens at the given concentrations, which is contrary to the antihistomonal effect noticed in vitro even though the same clonal culture was used. The results of this study therefore clearly demonstrate the importance of defined in vivo experimentation in order to assess and verify in vitro results.

Seroprevalence of Histomonas meleagridis in pullets and laying hens determined by ELISA.

Serum samples from 1120 layers from 56 flocks and 400 pullets from 20 flocks were tested by an indirect sandwich ELISA to investigate the prevalence of antibodies to Histomonas meleagridis in chickens kept in alternative husbandry systems. The overall prevalence of antibodies to H meleagridis in layers was 37.3 per cent, and positive birds were identified in 50 flocks. This was significantly higher than in pullets, where only 8.3 per cent of the birds tested positive. Optical density (OD) values obtained from pullet sera were much lower than the OD values from layers; however, positive birds were detected in half of the pullet flocks. In particular, all birds from an organic pullet flock were found to be positive, with high OD values. Overall, the highest prevalence of positive sera was obtained from birds kept in free-range flocks. Attempts to reisolate live histomonads from birds in 18 layer flocks were unsuccessful.

Progression of histomonosis in commercial chickens following experimental infection with an in vitro propagated clonal culture of Histomonas meleagridis.

Four commercial strains of chickens, namely, ISA brown leghorn (ISA), TETRA-SL brown (TETRA-SL), Lohmann brown (LB), and Lohmann LSL (LSL), were infected with a well-defined clonal culture of Histomonas meleagridis (H. meleagridis/Turkey/Austria/2922-C6/04) to investigate their susceptibility to histomonosis. Each group included 16 chickens, which were housed under the same conditions in separate pens. All chickens were infected with 10(4) histomonads orally and intracloacally at 14 days of age. No mortality or clinical signs were observed during the experiment in all birds. Three birds of each chicken strain were euthanatized on days 4, 7, 10, 14, and 21 postinfection. Incidence of histomonosis on the basis of cecal lesions was found to be 64.00% in TETRA-SL, 62.50% in LB, 53.12% in LSL, and 43.75% in ISA chickens. Fewer lesions were noticed in livers than in ceca, with an incidence of 15.62% in TETRA-SL, 9.37% in LB, and 3.12% in ISA chickens. No liver lesions were found in the LSL chickens. Statistical analysis revealed that there was no significant difference (P > 0.05) in susceptibility to experimental H. meleagridis infection based on cecal and liver involvement. Polymerase chain reaction (PCR) and immunohistochemistry were found to be reliable tools to confirm the presence of histomonads and changes in the ceca. However, some negative PCR results were recorded from the livers despite the presence of macroscopic lesions. Additionally, DNA of H. meleagridis was detected by PCR in a few of the lungs, but immunohistochemistry was negative. Nucleic acid of the protozoan parasite was not detected in samples from kidney, brain, spleen, or bursa of Fabricius. Altogether, the high susceptibility of commercial chicken lines to histomonosis could be demonstrated and characterized by severe lesions in the ceca and insignificant involvement of the liver, approaching a maximum on days 7-14 postinfection.

Safety of avirulent histomonads to be used as a vaccine determined in turkeys and chickens.

In the present work, chickens and turkeys were infected with virulent or attenuated Histomonas meleagridis to investigate and compare the effect of both isolates on birds. Thereby, histomonads of a clonal culture were propagated in vitro either for a short period of time (21 passages) to preserve virulence or for 295 passages to achieve attenuation. On the first day of life birds of each species were infected with either virulent or attenuated parasites. Throughout the experiment, all birds were examined daily for clinical signs attributable to the infection. Furthermore, the excretion of viable parasites was determined after in vitro reisolation from cloacal swabs. For the investigation of pathological changes of organs a defined number of infected birds were killed on d 4, 7, 10, 14, and 21 postinfection (PI) and necropsy was performed. By this routine, changes in livers and ceca were classified by a scoring system to evaluate the severity of lesions. Samples of cecum, liver, and lung were generated and screened for the presence of parasites by PCR and immunohistochemistry. Turkeys infected with virulent histomonads showed first clinical manifestation of histomonosis on d 10 PI, whereas the remaining birds did not express clinical signs. Positive reisolations of virulent and attenuated histomonads were obtained intermittently from individual chickens and turkeys from d 2 PI until the end of the experiment. Both species of birds displayed lesions in the ceca and the liver following infection with virulent parasites, whereas no changes occurred in birds inoculated with attenuated histomonads. The PCR revealed the dissemination of virulent histomonads in ceca, livers, and lungs of some chickens and turkeys in contrast to attenuated parasites, which were exclusively found in cecal samples. The attenuated isolate of H. meleagridis did not induce clinical signs or pathological changes and offers high safety after infection of chickens and turkeys. Therefore, the in vitro attenuation and the use of avirulent histomonads represent a viable tool for vaccination against histomonosis.

Experimental infection of turkeys and chickens with a clonal strain of Tetratrichomonas gallinarum induces a latent infection in the absence of clinical signs and lesions.

The pathogenicity of a mono-eukaryotic culture of Tetratrichomonas gallinarum in specific pathogen free (SPF) chickens and turkeys was studied. Two experiments of identical design were performed: the first with SPF chickens and the second with commercial turkeys. Each experiment included three groups. Groups 1 and 2 each contained 12 infected and three in-contact birds. The birds in these groups were infected on the first day of life, either cloacally (group 1) or orally (group 2). Group 3 consisted of four control birds. Re-isolation of the parasite from cloacal swabs was performed to verify the excretion of T. gallinarum. The infected birds excreted trichomonads from the second day post-infection. Spread of the flagellate from infected to in-contact birds was detected after 5 days post-infection (dpi), based on the re-isolation of the protozoa. No clinical signs or deaths were recorded in chickens or turkeys. Three birds were killed at 4, 8, 14 and 21dpi and various tissues were collected for pathological examination. No gross lesions were noted. Protozoal DNA was demonstrated in the oesophagus, duodenum, jejunum, caecum, liver, lung, bursa of Fabricius and brain by polymerase chain reaction and in-situ hybridization. No antibodies were detected in the serum of infected birds by enzyme linked immunosorbent assay. Microscopical changes were only present in the caecum, where there was sloughing of the epithelium associated with the presence of numerous flagellates on the epithelial surface, within the crypts of Lieberkühn and in the lamina propria. These changes were found in caecal samples from infected and in-contact birds. These studies have demonstrated the rapid transmission of T. gallinarum between both turkeys and chickens and the establishment of a latent infection in both species.

Outbreak of cutaneous form of poxvirus on a commercial turkey farm caused by the species fowlpox.

The present report documents the occurrence of a poxvirus infection in commercial meat turkeys. The affected farm had six flocks, with a total of 11,680 birds at different ages; birds from two of these flocks were affected. The clinical picture was characterized by severe epithelial lesions and proliferations on the head and neck regions as reported for the cutaneous form of poxvirus infection. Except for these lesions, no adverse clinical signs or gross pathologic lesions were observed. Only a low number of birds was affected (n = 20) and no increase of mortality could be seen. Bacteriologic investigations from the lesions revealed multiresistant Staphylococcus aureus. Eosinophilic inclusions (Bollinger bodies) in histologic examinations in the cytoplasm of keratinocytes were noticeable. Typical pox virions were demonstrated by electron microscopy, and poxvirus was isolated on the chorioallantoic membrane of specific-pathogen-free chicken eggs. Further identification of the poxvirus species was carried out by PCR and sequencing, revealing an infection with the species fowlpox. Layers in vicinity of the turkey farm that also were affected by fowlpox were considered as potential source of infection. Although it is assumed that avian poxviruses are strongly species specific, the present case report reinforces the changing picture of poxvirus infections in turkeys. Furthermore, it supports the assumption of previous data that fowlpox virus has to be seen as recently emerging pathogen in turkeys.

Oral vaccination of 1-day-old turkeys with in vitro attenuated Histomonas meleagridis protects against histomonosis and has no negative effect on performance.

One-day-old turkey poults were vaccinated against histomonosis (syn. histomoniasis) via the oral route by application of in vitro attenuated Histomonas meleagridis. Subsequently, two different groups composed of 14 birds each were challenged cloacally with highly virulent histomonads after 2 or 4 weeks. Two additional groups of non-vaccinated birds were infected with the challenge inoculum at the same time points. In addition, a group of 19 birds, of which 14 were vaccinated but not challenged, were kept for clinical and serological examinations. Non-vaccinated and non-challenged birds (n=10) represented the negative control group. All non-vaccinated but infected birds and 10 out of 14 vaccinated turkeys challenged 2 weeks post vaccination (w.p.v.) contracted severe histomonosis. Turkeys challenged 4 w.p.v. and all remaining birds used in this experiment did not show any pathognomonic clinical signs. In addition, no adverse effect regarding the weight gain could be observed in birds that were vaccinated but not challenged. The excretion of attenuated and virulent live histomonads was observed very infrequently by re-isolation, but transmission to in-contact birds was very efficient. Presence of antibodies was first noticed 3 w.p.v. and antibody levels remained above the cut-off value until termination of the experiment at 16 w.p.v. The present experiment demonstrates for the first time the potential efficacy of in vitro attenuated histomonads used as an orally applied vaccine to 1-day-old turkeys for protection against fatal histomonosis without affecting performance.

Oral infection of turkeys with in vitro-cultured Histomonas meleagridis results in high mortality.

In the present study a well-defined clonal culture of Histomonas meleagridis was used to investigate whether turkeys can be infected orally with in vitro-propagated parasites in the absence of any vector. Therefore, two subsequent experiments were arranged to study the morbidity and mortality of a virulent strain of histomonads in turkeys following crop instillation of protozoa and a feed restriction for 5 h. Soon after infection, the parasites triggered total morbidity and mortality in a group of 14 turkeys, of which 10 birds were orally infected and the others were kept as in-contact birds. Administration of the same number of histomonads into either the cloaca or the crop of individually housed birds was investigated subsequently, to avoid any bird-to-bird transmission. All eight cloacally infected turkeys and six out of eight orally infected turkeys contracted severe histomonosis, with only two birds surviving the infection expressing no clinical signs. During pathological investigations, all birds showed pathognomonic lesions in the livers and caeca indicative for histomonosis, except the two turkeys that survived the infection. The results of the experiments verify for the first time the effective oral infection of 1-day-old turkeys with clonal in vitro-cultured H. meleagridis.

A virulent mono-eukaryotic culture of Histomonas meleagridis is capable of inducing fatal histomonosis in different aged turkeys of both sexes, regardless of the infective dose.

Histomonosis (syn. histomoniasis) is a parasitic disease which affects predominately turkeys but also other avian species. Concurrent with the ban of therapeutic and prophylactic substances, the disease, caused by the flagellated protozoon Histomonas meleagridis, is more frequently reported. Due to somewhat diverse results reported in the past, a well-characterized culture was used in the present study to investigate the possible influence of certain parameters on the outcome of the disease. For this study, turkeys were infected with different doses of the mono-eukaryotic culture Histomonas meleagridis/Turkey/Austria/2922-C6/04 using birds of both sexes at various ages. All study groups consisted of 14 birds, of which 10 birds were directly infected via the cloacal route and four birds were kept as in-contact birds. This scheme was used to investigate the pathogenicity of the cloned isolate in 1-day-old and 14-day-old turkeys. In 8-week-old turkeys, only eight birds out of 12 were infected. When 1-day-old and 8-week-old turkeys were infected with 10(4) histomonads per bird, all turkeys died between 11 and 21 days postinfection or had to be euthanatized due to their poor condition. In a group of 14 poults, infective doses of either 10 histomonads (100 histomonads among 10 birds) or 10(3) histomonads per bird had hardly any influence on the first notification of clinical signs. However, even though the onset of clinical signs and mortality was delayed with the lower dose, none of the birds survived the infection. As a consequence, no differences were noticed between male and female turkeys using the mono-eukaryotic culture of Histomonas meleagrigis/Turkey/Austria/2922-C6/04 in the current experimental setting.

In-situ hybridization for the detection and identification of Histomonas meleagridis in tissues.

For use in an in-situ hybridization method, three probes (HM, TR and BL) were designed to hybridize, respectively, with (1) Histomonas meleagridis, (2) Tetratrichomonas gallinarum, and (3) a broad range of micro-organisms, including Blastocystis spp. Mono-eukaryotic cultures were used to test the specificity of the three oligonucleotides and to optimize the hybridization procedure before applying the probes to archived samples of various tissues and to a culture of Trichomonas gallinae. Specific detection of H. meleagridis was possible with the HM probe, but the other two probes were less specific. The TR probe detected members of the Trichomonadidae (Tetr. gallinarum and Tr. gallinae). Positive signals from a great variety of microorganisms, including fungi and protozoa from different animal species, were obtained with the BL probe. However, neither H. meleagridis nor the two members of the Trichomonadidae mentioned above were detected with this probe, allowing the exclusion of these parasites. Use of the three probes makes possible the accurate detection of H. meleagridis and its distinction from other micro-organisms in tissue samples.

Broad dissemination of Histomonas meleagridis determined by the detection of nucleic acid in different organs after experimental infection of turkeys and specified pathogen-free chickens using a mono-eukaryotic culture of the parasite.

Histomonas meleagridis, a flagellated protozoan parasite, is the causative agent of histomonosis (syn. histomoniasis, blackhead) in turkeys and chickens. The organs primarily affected by the parasite are the caeca and the liver. Until now, only few reports exist in which the parasite has been diagnosed in tissues other than those mentioned above. Hence, the aim of this study was to perform a systematic investigation of various organs of turkeys and specified pathogen-free chickens following an experimental infection with a mono-eukaryotic culture of Histomonas meleagridis in order to determine the dissemination of the flagellate in infected birds. Molecular methods like PCR and in situ hybridization were used for this purpose. For the first time, the DNA of the parasite could be detected in 13 different organs of infected turkeys by PCR including the proventriculus, duodenum, jejunum, caeca, pancreas, bursa of Fabricius, liver, kidney, spleen, heart, lung, thymus and the brain. Most of these findings were further confirmed by in situ hybridization. In contrast to the turkeys that all died shortly after the infection, all of the chickens survived without displaying any clinical symptoms. Even at necropsy, only mild pathological changes were observed in the caeca. Nevertheless, the parasite could also be detected in various organs of these birds, namely the caeca, bursa of Fabricius, kidney, heart and the brain.