Transmission electron microscopic studies of stages of Histomonas meleagridis from clonal cultures.

Histomonas meleagridis is a 10-20 microm-sized flagellated protozoan, causing histomoniasis in gallinaceous birds. Different strains of H. meleagridis from different origins were used to establish clonal cultures, which can be traced back to a single cell. Cells from these clonal cultures were examined by transmission electron microscopy. Results gave detailed insights in the ultrastructure showing a single flagellum, a band of microtubules remnants of an axostyle or of a costa, respectively pelta, hydrogenosomes, nucleus, spindle apparatus, and other organelles of the trophozoites of 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.

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.

PCR for the identification and differentiation of Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis spp.

In the present investigation PCR assays were developed for the rapid detection and differentiation of two poultry flagellates: Histomonas meleagridis and Tetratrichomonas gallinarum as well as the protozoan microorganism: Blastocystis spp. The nucleotide sequences of the small subunit ribosomal RNAs were used for primer construction obtaining fragments which vary in size for each microorganism. The established PCRs were able to detect DNA obtained from one microorganism of T. gallinarum and Blastocystis spp. propagated in vitro, proving the high analytical sensitivity of the method. DNA isolated from 10 protozoa was sufficient to detect H. meleagridis. To assess specificity, each PCR assay was performed with DNA from either H. meleagridis and/or T. gallinarum and/or Blastocystis spp. as well as with DNA from several other protozoan parasites (Eimeria tenella, Toxoplasma gondii, Cryptosporidia spp., Trichomonas gallinae, Entamoeba invadens, Entamoeba ranarum), fungi (Aspergillus fumigatus, Candida albicans), bacteria (Staphylococcae, Streptococcae, E. coli, Clostridium perfringens, Camplyobacter jejuni, Proteus) and viruses (fowl adenovirus serotype 4, avian reovirus) as well as livers and caecal samples from turkeys and specified pathogen free (spf) chickens. No cross-reactions with any of these samples were observed with the primer sets for the detection of H. meleagridis and Blastocystis spp. The primers designed for the identification of T. gallinarum yielded a PCR product with DNA of Trichomonas gallinae that had the identical size as the amplicon obtained with DNA from T. gallinarum. However, no PCR products resulted from any of the other samples tested with these primers. Liver and caecal samples from turkeys and chickens from flocks with outbreaks of histomonosis also named as "histomoniasis" originating from geographically distinct regions were investigated with the established PCRs. This is also the first report about the detection of the nucleic acid of H. meleagridis, T. gallinarum and Blastocystis spp. nucleic acid in the livers and/or caeca of laying hens and turkeys obtained from field outbreaks. Hence, the established PCR assays proved to be a rapid and sensitive diagnostic tool for the direct detection and differentiation of H. meleagridis, T. gallinarum and Blastocystis spp. nucleic acid in organ samples of infected turkeys and chickens regardless of the geographic origin.

Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. established through micromanipulation.

Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. were established for the first time. Single microbes were successfully isolated from a mixture of micro-organisms obtained from caecal contents of turkeys, using a micromanipulation approach. The cloned parasites were propagated in vitro and maintained through continuous passages multiplying to high numbers. Identification of the protists was done by morphological investigation identifying various forms of each parasite. PCR and partial sequencing of the small subunit rRNA were used to confirm clonality and to determine the relationship of the cloned parasites with known protozoan parasites. The clonal cultures established by this technique will be useful to gain more insight into the biological repertoire of the organisms. In addition, refined infection experiments in different poultry species can now be performed to elucidate the pathological pathways of the respective protozoa.

Rapid transmission of the protozoan parasite Histomonas meleagridis in turkeys and specific pathogen free chickens following cloacal infection with a mono-eukaryotic culture.

In the present investigation, the pathogenicity and transmission of a mono-eukaryotic culture of Histomonas meleagridis for commercial turkeys and specific pathogen free (SPF) chickens is described for the first time. Two separate trials with the same kind of experimental design were performed, one with commercial turkeys and one with SPF chickens. In each experiment, two different groups were included, which were housed in separate rooms. The first group contained four control birds, whereas the second group consisted of 10 infected and four in-contact birds. The birds were infected via the cloaca at 14 days of age with 380,000 cells of a mono-eukaryotic culture of H. meleagridis consisting of a cloned isolate (Turkey/Austria/2922-66/04). Reisolation of the parasite from turkeys and chickens under experimental conditions was performed for the first time. The infected birds started to excrete the parasite as soon as 2 days post infection. Rapid spread of the parasite to in-contact turkeys and chickens was noticed, based on reisolation of live parasites. Reisolation of the pathogen was impossible from two of the four in-contact SPF chickens at any time, whereas all of the infected turkeys were found positive. Intermittent shedding of the parasite was noticed in infected turkeys and SPF chickens, but the phenomenon was much more severe in the SPF chickens as these birds survived the infection. All of the infected and in-contact turkeys died between days 11 and 14 post infection, whereas no death was recorded in the SPF chickens, which were killed 6 weeks after the infection. Typical lesions were recorded in the caeca and livers of the infected turkeys. In addition, a heavy destruction of the bursa of Fabricius was seen in all of the infected and one of the in-contact turkeys. Altogether, the present investigations are of importance for an understanding of the pathogenicity and transmission of H. meleagridis in poultry.

Sacbrood virus of the honeybee (Apis mellifera): rapid identification and phylogenetic analysis using reverse transcription-PCR.

Sacbrood virus (SBV) infects larvae of the honeybee (Apis mellifera), resulting in failure to pupate and death. Until now, identification of viruses in honeybee infections has been based on traditional methods such as electron microscopy, immunodiffusion, and enzyme-linked immunosorbent assay. Culture cannot be used because no honeybee cell lines are available. These techniques are low in sensitivity and specificity. However, the complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a reverse transcription-PCR (RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. RT-PCR was used to target five different areas of the SBV genome using infected honeybees and larvae originating from geographically distinct regions. The RT-PCR assay proved to be a rapid, specific, and sensitive diagnostic tool for the direct detection of SBV nucleic acid in samples of infected honeybees and brood regardless of geographic origin. The amplification products were sequenced, and phylogenetic analysis suggested the existence of at least three distinct genotypes of SBV.