Partial sequence of the alpha-tubulin gene from Histomonas meleagridis isolates from the United States

Histomonas meleagridis , the causative agent of histomoniasis, is a protozoan parasite classified in the Dientamoebidae (order Tritrichomonadida). The α-tubulin gene of 7 H. meleagridis isolates originating from either domestic chickens or turkeys from the United States was amplified by nested PCR and sequenced. A 91.4-99.8% nucleotide identity was shared among the 7 different sequences, and phylogenetic analysis disclosed that the 7 isolates were divided into at least 3 clades. These sequences had a 91-99% nucleotide identity and a 96-100% amino acid identity compared with 3 H. meleagridis α-tubulin sequences obtained from isolates originating from turkeys in Germany. Further α-tubulin gene analysis from species in the Dientamoebidae will be useful in elucidating the evolutionary relationship of these protozoans.     

First molecular characterisation of hydrogenosomes in the protozoan parasite Histomonas meleagridis

Histomonas meleagridis is a trichomonad species that undergoes a flagellate-to-amoeba transformation during tissue invasion and causes a serious disease in gallinaceous birds (blackhead disease or histomoniasis). Living in the avian cecum, the flagellated form can be grown in vitro in the presence of an ill-defined bacterial flora. Its cytoplasm harbours numerous spherical bodies which structurally resemble hydrogenosomes. To test whether these organelles may be involved in anaerobic metabolism, we undertook the identification of H. meleagridis genes encoding some potentially conserved hydrogenosomal enzymes. The strategy was based on several PCR amplification steps using primers designed from available sequences of the phylogenetically-related human parasite Trichomonas vaginalis. We first obtained a C-terminal sequence of an iron-hydrogenase homologue (Hm_HYD) with typical active site signatures (H-cluster domain). Immunoelectron microscopy with anti-Hm_HYD polyclonal antibodies showed specific gold labelling of electron-dense organelles, thus confirming their hydrogenosomal nature. The whole genes encoding a malic enzyme (Hm_ME) and the alpha-subunit of a succinyl coenzyme A synthetase (Hm_alpha-SCS) were then identified. Short N-terminal presequences for hydrogenosomal targeting were predicted in both proteins. Anti-Hm_ME and anti-Hm_alpha-SCS antisera provided immunofluorescence staining patterns of H. meleagridis cytoplasmic granules similar to those observed with anti-Hm_HYD antiserum or mAb F5.2 known to react with T. vaginalis hydrogenosomes. Hm_ME, Hm_alpha-SCS and Hm_HYD were also detected as reactive bands on immunoblots of proteins from purified hydrogenosomes. Interestingly, anti-Hm_alpha-SCS staining of the cell surface in non-permeabilised parasites suggests a supplementary role for SCS in cytoadherence, as previously demonstrated in T. vaginalis.     

Efficacy of a herbal product against Histomonas meleagridis after experimental infection of turkey poults

Histomoniasis (infectious enterohepatitis, blackhead) is caused by the protozoan parasite Histomonas meleagridis (H. meleagridis). After the ban of all prophylactic and therapeutic drugs in the European Union, histomoniasis is increasingly responsible for considerable economic problems to the poultry industry. The aim of this study was to investigate the effect of a herbal product with extracts from cinnamon, garlic, lemon, and rosemary on H. meleagridis in turkey poults in vivo. For this purpose, 60 two-week-old poults were divided into three groups. Group 1 received the herbal product in the feed six days before infection and in water three days before infection, then in feed and drinking water until the end of the experiment. Groups 2 and 3 were left untreated. At week 3 of age, Groups 1 and 2 were infected intracloacally with H. meleagridis. Three weeks after infection the surviving birds were euthanized and examined for pathological lesions. Mortality was 20% in Group 1 and 50% in Group 2. There were no deaths in Group 3. DNA of histomonads was detected in all examined caeca and livers of the dead birds, but was not detected in any examined organ of the surviving birds of all groups. There was no noticeable difference in the lesion scores of the dead birds between the groups. The surviving birds of all groups did not show lesions post mortem. Since all effective prophylactic and therapeutic drugs against histomoniasis were banned in the EU, under given conditions the investigated herbal product seems to be an effective alternative for the reduction of mortality in turkeys caused by histomoniasis.     

Efficacy of a herbal product against Histomonas meleagridis after experimental infection of turkey poults

Abstract

Histomoniasis (infectious enterohepatitis, blackhead) is caused by the protozoan parasite Histomonas meleagridis (H. meleagridis). After the ban of all prophylactic and therapeutic drugs in the European Union, histomoniasis is increasingly responsible for considerable economic problems to the poultry industry. The aim of this study was to investigate the effect of a herbal product with extracts from cinnamon, garlic, lemon, and rosemary on H. meleagridis in turkey poults in vivo. For this purpose, 60 two-week-old poults were divided into three groups. Group 1 received the herbal product in the feed six days before infection and in water three days before infection, then in feed and drinking water until the end of the experiment. Groups 2 and 3 were left untreated. At week 3 of age, Groups 1 and 2 were infected intracloacally with H. meleagridis. Three weeks after infection the surviving birds were euthanized and examined for pathological lesions. Mortality was 20% in Group 1 and 50% in Group 2. There were no deaths in Group 3. DNA of histomonads was detected in all examined caeca and livers of the dead birds, but was not detected in any examined organ of the surviving birds of all groups. There was no noticeable difference in the lesion scores of the dead birds between the groups. The surviving birds of all groups did not show lesions post mortem. Since all effective prophylactic and therapeutic drugs against histomoniasis were banned in the EU, under given conditions the investigated herbal product seems to be an effective alternative for the reduction of mortality in turkeys caused by histomoniasis.     

Molecular characterization of Histomonas meleagridis and other parabasalids in the United States using the 5.8S, ITS-1, and ITS-2 rRNA regions

Extracted DNA from 28 Histomonas meleagridis -infected avian tissue samples from multiple hosts and geographic locations was analyzed for variation in the 5.8S rRNA and the flanking internal transcribed spacer regions (ITS 1 and ITS 2). Samples were amplified by polymerase chain reaction, sequenced, and compared with known sequences from GenBank accessions of H. meleagridis and other related protozoa. The analyses revealed significant genetic variation within H. meleagridis sequences and suggested the possibility of multiple genotypes within the samples or a possible misdiagnosis. Related protozoa found in some samples were mostly identified as Tetratrichomonas spp. However, 1 sample had a 93% identity to Simplicimonas similis , a newly described organism, suggesting the possibility of a new pathogen in poultry. A phylogenetic tree analyzing the 5.8S and flanking ITS regions was inconclusive and we were unable to resolve all H. meleagridis into a single grouping. In contrast, a tree constructed only on the 5.8S rRNA grouped all but 1 H. meleagridis sample into 1 clade, including GenBank accessions submitted from Europe. This suggests that the 5.8S region alone is more reliable in identifying H. meleagridis than are the combined 5.8S and flanking ITS regions. There was no correlation between genotypes and host species or geographic location, suggesting that H. meleagridis moves freely between multiple avian species in the sampled regions.     

Identification of transposable elements which activate gene expression in Pseudomonas cepacia.

This study demonstrated that transposable elements in Pseudomonas cepacia could be inserted upstream of a poorly expressed gene and increase its expression more than 30-fold. Five elements, TnPc1, IS402, IS403, IS404, and IS405, were isolated by their ability to increase expression of the beta-lactamase gene of the broad-host-range plasmid pRP1. Increased expression resulted only from insertion of these elements, suggesting that insertional activation is an important means of elevating gene expression in this organism. Four of the elements inserted between a PstI site within the beta-lactamase gene and a BamHI site located 375 base pairs upstream of its promoter. The element IS403 inserted distal to the BamHI site within the coding region for the gene tnpR, suggesting that insertional activation can act over greater than expected distances. In addition, the element IS402 activated the beta-lactamase genes carried on plasmids pRP1 and pMR5 (temperature-sensitive pRP1) equally well in opposite orientations, demonstrating that insertional activation by this element occurs independent of its orientation.     

Identification of Vibrio harveyi using PCR amplification of the toxR gene

AIMS: The aim of this study was to develop an effective method for the identification of Vibrio harveyi based on using the toxR gene as a taxonomic marker. METHODS AND RESULTS: Primers for the toxR gene were designed for specificity to V. harveyi, and incorporated in a polymerase chain reaction (PCR). The results of the PCR, which took <5 h from DNA extraction to amplification, revealed positive amplification of the toxR gene fragment in 20 V. harveyi isolates including type strains, whereas DNA from 23 other Vibrionaceae type strains and 13 Vibrio parahaemolyticus strains were negative. The detection limit of the PCR was 4.0 x 10(3) cells ml(-1). In addition, the technique enabled the recognition of V. harveyi from diseased fish. CONCLUSIONS: The PCR was specific and sensitive, enabling the identification of V. harveyi within 5 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR allowed the rapid and sensitive detection of V. harveyi.     

Qualitative and quantitative histochemical changes in the caecum and liver of turkeys infected with Histomonas meleagridis.

Changes in the amount and distribution of acid and alkaline phosphatase, non-specific esterase, glycogen, lipid and acid mucopolysaccharide in the caecal wall and liver of turkey poults infected with Histomonas meleagridis have been studied histochemically. A microdensitometer was used to measure changes in activity and distribution of acid phosphatase and non-specific esterase in the caecal mucosa. During the course of the infection there is a marked reduction in activity and distribution of acid phosphatase and non-specific esterase but little change in the amounts and distribution of alkaline phosphatase, glycogen, lipid and acid mucopolysaccharide in the wall of the main part of the caecum. Similar, but smaller, changes occurred in the wall of the neck region of the caecum. In the liver most changes occurred in the immediate vicinity of the parasites. Initially, there was a reduction in the amount of glycogen in the parasitic lesions but later in the infection there was a marked loss of glycogen in all regions of the liver. Changes in the caecum are apparently brought about by the parasite prior to and after invasion of the caecal tissues; changes in the liver occur after it has been invaded.     

Pilot study on the efficacy of paromomycin as a histomonostatic feed additive in turkey poults experimentally infected with Histomonas meleagridis

Paromomycin is an aminoglycoside antibiotic with activity against protozoa. Currently, paromomycin is registered for food producing animal species. In a pilot study we evaluated the efficacy of different doses of paromomycin in the feed against Histomonas meleagridis in experimentally challenged turkey poults. Groups consisting of 30 birds each were given feed with 100, 200 and 400 ppm paromomycin, respectively, starting on day 1 through to day 42. One group of 30 birds was left untreated. On day 21 all birds were infected intracloacally with H. meleagridis. Additionally, 10 birds were kept as a non-infected and non-treated control group. Before the challenge there was no significant difference between untreated and treated groups with regards to feed consumption and feed conversion rate. After the challenge, mortality was 80% in the infected non-treated birds. In the treated groups the mortality rate was 73.3%, 43.3% and 20%, respectively. No histomonal DNA was found in caeca and livers of the surviving birds. In addition, higher doses of paromomycin (200 and 400 ppm) led to reduced counts of Clostridium perfringens in the droppings. In conclusion, a prophylactic application of paromomycin as a feed additive was effective against the challenge with H. meleagridis under experimental conditions.