Immunologic Analysis by Gel Diffusion of Effects of Prolonged Cultivation on Histomonas meleagridis (Smith)*

SYNOPSIS. Antigens were prepared from each of 4 lines of Histomonas meleagridis: Hm-L1, a strain highly virulent for both turkeys and chickens; Hm-L1 /C12, Hm-L1 /C24, Hm-L1 /C52, 3 avirulent substrains derived from Hm-L1 after 12, 24, 52 weeks of in vitro cultivation, respectively. Hm-L1 strain and the 3 substrains were maintained in liquid nitrogen. Antisera were developed in rabbits against Hm-L1 and Hm-L1 /C24 parasites. Both antisera were reacted on gel diffusion plates with homologous and heterologous antigens. Two groups of precipitin lines and/or bands designated arbitrarily as A and B, were observed on the slides. Analysis of these bands revealed the common antigenic composition of the 4 histomonads with respect to some of the group A and group B antigens. The concentrations and numbers of precipitin lines in both groups increased, however, with the length of cultivation. These antigenic differences are discussed in the light of their possible relationship to pathogenicity.     

Infectivity of Histomonas meleagridis of Cecal and Liver Origins Compared

SYNOPSIS. Turkey poults were inoculated rectally with 100, 1000, 10,000, or 100,000 Histomonas meleagridis from the ceca of a group of experimentally infected turkeys. Other poults were given the same numbers of histomonads from an infected liver from the same group of source birds, Comparisons of the incidence of infection, liver involvement, mortality, and average survival time following these inoculations showed that organisms of cecal origin were about 100 times more effective in producing histomoniasis than were organisms of liver origin. It is suggested that this difference in infectivity resulted from heavy losses of histomonads of liver origin that were due to various selective processes.     

Effect of Bile on Infectivity of Histomonas meleagridis of Liver Origin

SYNOPSIS After 30 min exposure to fresh turkey bile at 99 F, 100,000 Histomonas meleagridis of turkey liver origin failed to infect any of 20 poults inoculated rectally. Only one of 20 poults similarly inoculated with histomonads exposed to fresh turkey bile for only 5 min at 99 F became infected. Fifteen of 20 poults became infected following rectal inoculation with 100,000 H. meleagridis of liver origin held in physiologic saline for 30 min at 99 F. All infected birds developed liver lesions and died. Altho H. meleagridis was very abundant in the livers of all potential donor poults and all infected recipient poults, histomonads were never found in the gall bladders of any of these birds. In some such birds, a few apparently degenerating histomonads were detected in the duodenum near the entrance of the bile ducts. Histomonads of liver origin are regarded as of little or no consequence in the transmission of this protozoon.     

Effect of dimetridazole on transmission of Histomonas meleagridis by Heterakis gallinarum

The administration of an antihistomonal drug, dimetridazole, at a dose of 0.08% in feed, controlled experimental infections with Histomonas meleagridis in chickens. The treated birds developed no lesions and the duration of infection with H. meleagridis was reduced. This drug regimen, however, did not always prevent incorporation of H. meleagridis into eggs of Heterakis gallinarium; heterakid eggs pooled from medicated chickens in which H. meleagridis had never been detected transmitted the protozoan to 1 of 10 turkeys fed the eggs. Thus, therapeutic treatment of chickens with dimetridazole may reduce, but not eliminate, transmission of H. meleagridis by eggs of H. gallinarum from medicated birds.     

Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.     

In vitro Cultivation of Histomonas meleagridis Free of Demonstrable Bacteria

Histomonas meleagridis was successfully cultured without demonstrable bacteria in a modified tissue culture medium enriched by fresh hamster liver and metal ions.     

Isolation of Histomonas meleagridis from Embryonated Eggs of Heterakis gallinarum*

SYNOPSIS. Histomonas meleagridis was isolated from eggs of Heterakis gallinarum by culturing artificially hatched eggs in a modified DeVolt's alkaline serum medium. The presence of the protozoon in the cultures was established by microscopic examination and confirmed by producing histomonosis in turkeys by intracecal inoculation with quantities of the cultures.     

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.