The poultry red mite,Dermanyssus gallinae,a potential vector of Erysipelothrix rhusiopathiae causing erysipelas in hens

Erysipelas is a bacterial disease caused by Erysipelothrix rhusiopathiae, which may infect swine as well as several other species of mammals and birds, including domestic fowl. In poultry, erysipelas may cause sudden high mortality due to septicemia. This communication describes the first isolation of E. rhusiopathiae from the haematophagous poultry red mite, Dermanyssus gallinae DeGeer (Acari: Dermanyssidae), that was collected on three farms where hen erysipelas was diagnosed. The bacteria were isolated from the integument as well as from the interior of the mites. Serotypes 1a and 1b of E. rhusiopathiae found in the mites corresponded with those isolated from the diseased birds. These findings imply that D. gallinae is a potential vector of E. rhusiopathiae. The current lack of effective measures to control D. gallinae causes recurring mite problems in poultry facilities once afflicted by this parasite. Consequently, mites containing E. rhusiopathiae may act as reservoir hosts of this bacterium, allowing it to persist in the poultry house between flock cycles as a source of infection for the replacement pullets. The zoonotic potentials of both E. rhusiopathiae and D. gallinae should also be considered.     

Detection of Salmonella sp. in Dermanyssus gallinae using an FTA filter-based polymerase chain reaction

Salmonella spp. bacteria are responsible for some of the most important zoonoses worldwide. Because Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) has been recently reported to be an experimental vector of Salmonella Enteritidis, it would be of benefit to evaluate the presence of this bacterium in mites. A molecular detection tool associating a simple filter-based DNA preparation with a specific 16S rDNA Salmonella sp. polymerase chain reaction (PCR) amplification was described. The limit of detection with this method was 2 x 10(4) bacteria per mite. To adapt this technique for large-scale studies, two sizes of mite pools were tested and a preliminary investigation was carried out on mites from 16 currently or previously contaminated farms. Mites sampled from one farm of each type were positive for Salmonella, suggesting that Dermanyssus could act as a reservoir between flocks. In further investigations, it will be necessary to carry out a large-scale study to assess the role of D. gallinae in the epidemiology of avian salmonellosis.     

Analysis of the Antigenic Relationships Among Trichomonas,Histomonas, Dientamoeba and Entamoeba III. Immunoelectrophoresis Technics*

Antigens prepared from Trichomonas gallinae, Histomonas meleagridis, Dientamoeba fragilis, Entamoeba invodens and Entamoeba histolytica were separated by electrophoresis in agar gels and reacted with antisera prepared in rabbits against each of the 5 species. The most numerous and strongest precipitin lines were obtained from reactions between the homologous antigens and antisera. Direct and cross-absorption reaction methods were employed with each antiserum and the various antigens to ascertain quantitatively the immunologic relationships among the several organisms. Trichomonas shared many common antigens with Histomonas, fewer with Dientamoeba and none with either species of Entamoeba. Histomonas was more closely related antigenically to Dientamoeba than to Trichomonas. The histomonad had only a few weakly cross-reacting antigens in common with the 2 Entamoeba species. Dientamoeba shared the most common antigens with Histomonas, fewer with Trichomonas and the fewest with Entamoeba. Somewhat stronger cross-reactivity was obtained with anti-Dientamoeba serum and E. invadens than between this immune serum and E. histolytica. The 2 species of Entamoeba shared the largest number of common antigens with each other, and to a much lesser extent both species cross reacted with Dientamoeba. Anti-Entamoeba sera had only a few weak cross-reacting precipitins with Histomonas. No antigenic relationship was found between either species of Entamoeba and Trichomonas.     

Characterization of nucleoside triphosphate diphosphohydrolase activity in Trichomonas gallinae and the influence of penicillin and streptomycin in extracellular nucleotide hydrolysis

Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides.     

Histamine release factor from Dermanyssus gallinae (De Geer): characterization and in vitro assessment as a protective antigen

A cDNA encoding a 174-amino-acid orthologue of a tick histamine release factor (HRF) was identified from the haematophagous poultry red mite Dermanyssus gallinae. The predicted D. gallinae HRF protein (Dg-HRF-1) sequence is highly conserved with the tick HRFs (identity 52-54%) and to a lesser degree with translationally controlled tumour proteins (TCTP) from mammals and other invertebrates (range 38-47%). Phylogenetically, Dg-HRF-1 partitions with the tick HRF clade suggesting a shared linage and potentially similar function(s). A recombinant Dg-HRF-1 protein (rDg-HRF-1) was produced and shown to induce degranulation of rat peritoneal mast cells in vitro, confirming conservation of the histamine-releasing function in D. gallinae. Polyclonal antibodies were generated in rabbits and hens to rDg-HRF-1. Western blotting demonstrated that native Dg-HRF is a soluble protein and immunohistochemical staining of mite sections revealed that the distribution of Dg-HRF, although ubiquitous, is more common in mite reproductive, digestive and synganglion tissues. A survey of hens housed continuously in a mite-infested commercial poultry unit failed to identify IgY specific for recombinant or native Dg-HRF, indicating that Dg-HRF is not exposed to the host during infestation/feeding and may therefore have potential as a vaccine using the concealed antigen approach. To test the protective capability of rDg-HRF-1, fresh heparinised chicken blood was enriched with yolk-derived anti-Dg-HRF IgY antibodies and fed to semi-starved mites using an in vitro feeding system. A statistically significant increase in mortality was shown (P = 0.004) in mites fed with anti-Dg-HRF IgY after just one blood meal. The work presented here demonstrates, to our knowledge for the first time, the feasibility of vaccinating hens with recombinant D. gallinae antigens to control mite infestation and the potential of rDg-HRF-1 as a vaccine antigen.     

Molecular investigations of cytochrome c oxidase subunit I (COI) and the internal transcribed spacer (ITS) in the poultry red mite, Dermanyssus gallinae, in northern Europe and implications for its transmission between laying poultry farms

Samples of Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) from more than 49 Norwegian and Swedish laying poultry farms, and additional samples collected from Scottish, Finnish, Danish and Dutch layer farms, were compared genetically. Analysis of partial mitochondrial gene cytochrome c oxidase subunit I (COI) sequences of mites from Norway and Sweden revealed 32 haplotypes. Only single haplotypes were found on most farms, which suggests that infections are recycled within farms and that transmission routes are few. Both Norwegian and Swedish isolates were found in the two major haplogroups, but no haplotypes were shared between Norway and Sweden, indicating little or no recent exchange of mites between these countries. There appears to be no link between haplotypes and geographical location as identical haplotypes were found in both the northern and southern Swedish locations, and haplotypes were scattered in locations between these extremes. The current data suggest that wild birds in Sweden are not a reservoir for D. gallinae infection of layer farms as their mites were genetically distinct from D. gallinae of farm layer birds. Transmission of the poultry red mite in Scandinavia is thus likely to depend on synantropic factors such as the exchange of contaminated material or infested birds between farms or facilities.     

Metabolic Changes in Trichomonas gallinae Resulting from Growth in Various Carbohydrates*†

SYNOPSIS. The influence of the type of growth carbohydrate on the subsequent metabolic activity of Trichomonas gallinae was investigated. Washed suspensions of cells collected from CPL-glucose, CPL-maltose, CPL-galactose, and CPL-glucose-maltose media were examined in the Warburg respirometer for their ability to utilize glucose, maltose, and galactose. Comparisons of the metabolic parameters of substrate consumption, changes in glycogen content, and CO₂ and H₂ production were made. The pattern of utilization of the sugars, both qualitatively and quantitatively, depended upon the type of carbohydrate in the CPL medium used to culture the cells and upon the time of exposure of the cells to a particular sugar in the medium.     

An Improved Method for the Isolation of Highly Polymerized Native Deoxyribonucleic Acid from Certain Protozoa*

A relatively simple phenol extraction method, with EDTA as the nuclease inhibitor, is described for the isolation of purified, highly polymerized native DNA from Trichomonas vaginalis, Trichomonas gallinae, and Tritrichomonas foetus; it is applicable also to Tetrahymena pyriformis. RNase Tl, RNase A (Worthington's R), pronase, and α-amylase digestions constitute important steps in obtaining satisfactory yields of DNA. High degree of polymerization of the isolation product was estimated by hyperchromicity at O.D.₂₆₀ after DNase treatment and by CsCl gradient analysis. The double-stranded condition of the DNA samples was estimated by the latter method and by denaturation with NaOH, and the molecular weight by sucrose gradient analysis. Purity of the samples was determined spectrophotometrically and by chemical analyses for protein and glycogen. DNA percent recovery was estimated by the diphenylamine reaction.     

Analysis of the Antigenic Relationships Among Trichomonas,Histomonas, Dientamoeba,and Entamoeba.* II. Gel Diffusion Methods

SYNOPSIS. Antisera were developed in rabbits against Trichomonas gallinae, Histomonas meleagridis, Dientamoeba fragilis, Entamoeba invadens, and Entamoeba histolytica. In reactions between these antisera and antigens prepared from each of the 5 species the most numerous and strongest precipitin lines appeared on gel diffusion agarose plates between the homologous antigens and antisera. Anti-Trichomonas serum cross-reacted most strongly with Histomonas, somewhat less with Dientamoeba, but gave no lines with the 2 species of Entamoeba. Anti-Histomonas serum cross-reacted strongly with both Trichomonas and Dientamoeba, and weakly with E. invadens and E. histolytica. Dientamoeba antiserum gave many precipitin lines with Histomonas, fewer with Trichomonas, and fewest with the 2 species of Entamoeba. Stronger reactions were noted between anti-Dientamoeba serum and E. invadens than between this serum and E. histolytica. Immune sera prepared against the 2 species of Entamoeba gave the most numerous precipitin lines in intrageneric cross-reactions, but the reaction between either of these antisera and Histomonas was weak. Somewhat stronger reactions were observed between the 2 anti-Entamoeba sera and Dientamoeba. Trichomonas failed to react with either of the anti-Entamoeba sera.