Genetically different clonal isolates of Trichomonas gallinae, obtained from the same bird, can vary in their drug susceptibility, an in vitro evidence.

Trichomonas gallinae is a flagellated protozoon which parasitizes in the upper digestive tract of different birds, especially columbiformes (doves and pigeons) and falconiformes. The parasite is also a common inhabitant of the crop of psittacine birds and is frequently detected in budgerigars. The lesions associated with T. gallinae infection of the upper digestive tract range from mild inflammation of the mucosa to large caseous lesions that block the lumen of the oesophagus. Nitroimidazoles are considered to be the drugs of choice for the treatment of trichomonosis. However, only a few studies report the existence of resistant strains of T. gallinae to these drugs. Thus, in the present investigation cloned cultures of T. gallinae obtained from budgerigars and pigeons were analysed for the first time for their in vitro susceptibilities against four 5´-nitroimidazole derivates, including metronidazole, dimetridazole, ronidazole and ornidazole. Significantly different minimal lethal concentrations (MLCs) were observed for them against all four drugs. The lowest MLCs revealed the Trichomonas isolates obtained from two budgerigars, ranging from 2.0 ± 0.3 to 3.0 ± 0.7 µg/ml for metronidazole and dimetridazole, and from 2.0 ± 0.6 to 6.7 ± 1.7 µg/ml for ornidazole and ronidazole. Contrary to this, the highest MLCs were recorded for one Trichomonas isolate obtained from a pigeon, ranging from 83.3 ± 6.7 (for dimetridazole and ronidazole) to 103.3 ± 3.3 µg/ml (for metronidazole and ornidazole). The data obtained for the resistance testing were further compared with already available genetic data of the small subunit rRNA gene sequences and ITS-1, 5.8S rRNA and ITS-2 sequences, indicating a certain correlation between in vitro results and strain relationships.

Molecular analysis of clonal trichomonad isolates indicate the existence of heterogenic species present in different birds and within the same host.

Trichomonas gallinae is a flagellated protozoon and the etiological agent of avian trichomoniasis. Despite its importance, especially in columbiformes and falconiformes, only a few molecular studies have yet been performed in order to investigate the degree of genetic diversity and cross-transmissibility between different isolates of this parasite. To address these questions 63 clonal cultures of Trichomonas spp. isolates were established by successful isolation of single trichomonads from a mixture of micro-organisms obtained from 17 birds belonging to five different species. All birds were from Austria with the exception of one bird which originated from the Czech Republic. The sequence of the complete genomic region spanning the two ribosomal RNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was determined for all 63 isolates. In addition, in order to compare the results obtained with the ITS1-5.8S-ITS2 region the sequence of the 18S rRNA gene was determined from a subset of isolates. Unrooted phylogenetic trees inferred by distance, parsimony, and likelihood methods suggest the existence of at least three clusters within the T. gallinae species complex, two groups being closely related to the human pathogens, Trichomonas vaginalis and Trichomonas tenax. Furthermore, for the first time two different trichomonad sequence types isolated at the same time from a single bird could be detected in the crops of two pigeons.

Experiments to produce cysts in cultures of Histomonas meleagridis--the agent of histomonosis in poultry.

Experiments were done with cultured trophozoite stages of different clonal strains (Histomonas meleagridis/Turkey/Austria/2922-C6/04 and H. meleagridis/Chicken/Hungary/5009-C2/05) of H. meleagridis in order to induce a cyst formation as it is known in other intestinal parasites. It was shown that the best multiplication of H. meleagridis occurred at 40 degrees C in a full medium 199, when fetal calf serum and rice starch had been added. Under these conditions, numerous amoebic stages (8-12 microm in diameter) without and a few with flagellum were seen showing regular reproduction rates. When the conditions of culture were experimentally changed-and thus became worse-by decreasing the temperature, by deprivation of the medium from fetal calf serum and/or rice starch, and by changing the osmolarity, the pH, or the MgCl(2) concentration, many of the amoebic stages (containing starch granules) were destroyed, and several had obtained a spherical shape. If the culture conditions became even worse, smaller spherical stages occurred, which had only diameters of 4-7 microm and which appeared more condensed. Both spherical stages did not contain starch granules. All the previously seen stages disappeared constantly. Since a similar decrease of the optimal living conditions also occurs when intestinal or cloacal feces are deposited outside from the bird's body, the results obtained here may underline the interpretation that some of the formerly amoebic stages are able to become large spherical stages and later small spherical stages. The large spherical stage would be some type of precysts while the smaller ones would represent true cysts.

Light and transmission electron microscopic studies on the encystation of Histomonas meleagridis.

The study deals with the pleomorphic zooflagellate Histomonas meleagridis, which was cultivated under different stress conditions to induce a possible encystation. In the present paper, the morphological changes were analyzed by light and electron microscopy. The determination of the proliferation under different adverse conditions led to conclusions on the tenacity of the flagellate. H. meleagridis parasitizes in the intestinal tract of galliform birds and may cause enormous losses in poultry farming. For the development of new therapy approaches, clarification of the transmission pathways will be helpful. Different clonal cultures of H. meleagridis established by micromanipulation and exposed to media lacking different ingredients, inappropriate temperatures, and/or distinct reagents were investigated. Lowering of temperature was proven to have adverse effects on the survival of H. meleagridis. The flagellate could not survive in a frozen medium, and survival in a temperature of 4 degrees C lasted no longer than 23 h. An addition of sodium chloride induced an increased proliferation; pH values between 2 and 8 set limits for the survival of the parasite in different ways. H. meleagridis was able to survive under high acidic conditions for only 1 h. The major amount of cells, which could be discovered in the controls, measured 8-12 microm appeared amoebic (stage 1) and were filled with enclosures of rice starch. A rounding of most cells was noted 4 h at 4 degrees C after incubation in minimal essential medium in the absence of rice starch and fetal calf serum. A higher osmolarity of the medium, which was initiated by the addition of sodium chloride or magnesium chloride, did not induce an encystation process. After addition of hypochlorite base and cultivating at pH values between 7 and 8, spherical stages without a flagellum were formed (stage 2) measuring about 8-12 microm in diameter. Their interior consisted of a central and a peripheral region when studied by transmission electron microscopy. This aspect was due to the location of the glycogen granules. The central zone was described as totally filled with the carbohydrates, which made totally invisible the other organelles. The solidity of the amorphous layer below the cell membrane seemed to hinder the invasion of the glycogen granules. The amorphous layer below the cell membrane made it apparently possible that the cell might survive under adverse conditions-at least for a short time. This special structure might enable H. meleagridis to proceed a fast transmission and to infect many birds in a rather short time, which was shown in the past by several studies. Double-membraned cells, which were guessed to be cyst-like structures of the parasite, were also detected (stage 3). The size of these cells, however, was much smaller than that of the amoebic stages or the above-described spherical forms of H. meleagridis. Furthermore, the small cells were characterized by other granula structures. These findings might be interpreted that the small stages are possibly long-term (true) cysts and that the spherical stages with the amorphous layer beneath the cell membrane might be short-term cysts. Both, however, should be able to survive situations outside of a body and thus might be transmitted from feces to another animal.

Axenization and optimization of in vitro growth of clonal cultures of Tetratrichomonas gallinarum and Trichomonas gallinae.

A rapid and simple procedure was established to obtain clonal axenic cultures of Tetratrichomonas gallinarum and Trichomonas gallinae and to optimize their in vitro growth conditions. Medium 199 was used for axenization of two genetically different clones of T. gallinarum and T. gallinae. Six different media were used to optimize the growth behaviour of axenically grown parasites: Medium 199, TYM, TYI-S-33, Hollander fluid (HF), Trichomonas vaginalis (TV) and modified TV media. The highest cell yields for both axenic clones of T. gallinarum were obtained in modified TV medium without antibiotics. The maximum numbers of trophozoites of T. gallinae were obtained in an optimized HF medium. This study demonstrated that axenic cultures for T. gallinarum and T. gallinae could be obtained avoiding the migration technique through a V-tube. Following axenization and optimization, both clones of T. gallinarum and T. gallinae could be propagated both aerobically and anaerobically.

Antiprotozoal activities determined in vitro and in vivo of certain plant extracts against Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp.

A total of 43 plant substances provided as raw material and different kinds of extracts (aqueous, ethanol, and heptane) from 18 different organic wastes obtained from the food/feed industry were investigated for their in vitro activities against clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum, and Blastocystis sp. Ethanolic extracts of thyme, saw palmetto, grape seed, and pumpkin fruit proved to be most efficacious. Thus, these extracts were further tested in vivo in turkeys experimentally infected with H. meleagridis by administrating the substances to the birds through the drinking water. Even though a delayed mortality was noticed in some birds medicated with the extracts of thyme, grape seed, and pumpkin fruit, all birds died or had to be euthanized the latest within 5 weeks post infection--with the exception of one bird which was probably never infected with histomonads--due to a severe typhlohepatitis indicative for histomonosis. In addition, none of the substances were able to prevent the spreading of H. meleagridis from infected to in-contact birds. Thus, these studies clearly demonstrate that in vitro studies are of limited value to assess the efficacy of plant substances against histomonosis.

Cloned Histomonas meleagridis passaged in vitro resulted in reduced pathogenicity and is capable of protecting turkeys from histomonosis.

Histomonas meleagridis is a flagellated protozoan parasite and the aetiological agent of histomonosis (histomoniasis or blackhead disease), a severe disease in poultry with very limited options for treatment. In the present investigation an inactivated vaccine, based on destroyed parasites and administered by intramuscular injection, failed to induce an efficient protection against a severe challenge. By cloacal infection of 14-day-old turkeys with cloned protozoa passaged in vitro for 95, 215 or 295 times, respectively, severe attenuation could be demonstrated as none of the infected birds died. Following infection with one of the higher passages, the birds resisted the challenge with virulent parasites. Using this approach no mortality due to histomonosis could be recorded in all of the 42 birds subjected to vaccination, either through direct infection or kept as in-contact birds, whereas all of the control animals died. Pathological lesions in the liver and caeca were only noticed in a few birds. The absence of parasitic DNA in the liver of vaccinated birds confirmed the effect of vaccination. For the first time vaccination of turkeys is reported to induce a solid protection against a severe challenge, both based on a clonal culture of Histomonas meleagridis.

Poxvirus infection in a great tit (Parus major).

Four great tits (Parus major) with nodular lesions suggestive of poxvirus infections were observed in a garden in Vienna, Austria. One of the birds was submitted for examination. Because of its poor condition, the bird had to be euthanatized and was subsequently necropsied. An avipoxvirus infection was confirmed by histopathology, electron microscopy, and polymerase chain reaction and sequencing. This is the second report of naturally occurring poxvirus infection in great tits and the first of its kind in central Europe.

Differences in the in vitro susceptibility of mono-eukaryotic cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp. to natural organic compounds.

Currently, all pharmaceuticals for the treatment or prophylaxis of blackhead disease (histomonosis) caused by the flagellate Histomonas meleagridis are banned from the market. Consequently, great interest exists on the finding of alternative drugs for the abatement of histomonosis. In this study, carvacrol, Cassia oil, an essential oil (EO) mixture containing thyme and rosemary EO and a Quillaja saponaria saponin were examined using in vitro assays for antiprotozoal and antibacterial activity testing established against cloned xenic cultures of different isolates of Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp. Whereas similar minimal lethal concentrations (MLCs) of five Histomonas isolates were obtained for both carvacrol and the EO mixture as well as for the saponin, significantly different MLCs were observed for them with Cassia oil, ranging from 0.25 up to 0.50 microl/ml. Testing the Blastocystis isolates, different MLCs were obtained for all substances, whereas the Tetratrichomonas gallinarum isolates showed identical susceptibilities. The effects are independent of the bacteria, underlining the need of well-defined protozoan cultures for these investigations.

Development of a multiplex RT-PCR for the simultaneous detection of three viruses of the honeybee (Apis mellifera L.): acute bee paralysis virus, Black queen cell virus and Sacbrood virus.

A single-step multiple-target (multiplex) reverse transcription-PCR (RT-PCR) was developed for the simultaneous detection and differentiation of three economically important viruses of the honeybee Apis mellifera L.: Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV) and Sacbrood virus (SBV). Three compatible sets of primers, specific for each virus, were designed in conserved regions of the viral genomes for use in a one-step (single tube) RT-PCR assay. The individual RT-PCR assays and the combined multiplex assay were optimized for highest sensitivity and specificity. The multiplex RT-PCR assay was tested on field samples collected from Austrian honeybee colonies. All three viruses were detected, and their identity was confirmed by sequencing of the PCR products. The described multiplex RT-PCR proved to be an accurate tool for rapid simultaneous detection of ABPV, BQCV and SBV directly in honeybee specimens.

Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples: comparison with isolation and biochemical characterization.

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.

Phylogenetic analysis of acute bee paralysis virus strains.

Reverse transcription-PCR assays have been established for a quick, sensitive, and specific diagnosis of acute bee paralysis virus (ABPV), a common virus of the honeybee (Apis mellifera), directly from clinical samples. A 3,071-nucleotide fragment of the ABPV genome, which includes the entire capsid polyprotein gene, was amplified from Austrian, German, Polish, and Hungarian ABPV samples and sequenced, and the sequences were compared. The alignment of a smaller fragment with ABPV sequences from the United States and the United Kingdom revealed nucleotide identity rates between 89 and 96%, respectively. Phylogenetic trees which display the molecular relationship between the viruses of different geographic origin were constructed.