Histomonas meleagridis after One Thousand in vitro Passages

SYNOPSIS. During a 7-year period Histomonas meleagridis survived 1000 passages in Medium 199 fortified with serum and antibiotic-killed bacteria. The histomonads were originally isolated from a chicken's cecal dropping, and the bacteria were cultivated from the cecal contents of a normal turkey. In many respects, the histomonads remained unchanged during cultivation, but in some other respects they changed considerably, perhaps irreversibly.
Morphologically, the histomonads propagated in vitro showed only slight changes. When returned to birds, they resumed the structure characteristic of lumen-dwelling individuals of this species. However, they had long since lost their ability to produce disease in either chickens or turkeys, and organisms of the tissue-dwelling type were rarely seen. The long-cultivated histomonads are actually as nonpathogenic as Histomonas wenrichi , but they have none of the distinguishing morphologic characteristics of the latter.
As has been reported elsewhere, H. meleagridis long maintained in the tissue culture medium has also lost much of its ability to immunize either chickens or turkeys against infection with virulent strains of this parasite. Also lost in the process of adaptation to its restricted medium has been the ability to multiply satisfactorily in vitro with the complement of bacteria normal to the ceca of the birds. However, in some instances, histomonads which have become adapted to in vitro cultivation are still able to live in birds with this diversified flora.
Activity of the histomonads cultivated in vitro differed but little from that of H. meleagridis freshly isolated from birds, when both were viewed under the same conditions. Histomonads from each of the above sources multiplied most satisfactorily in their accustomed habitat, probably because of a difference in nutritional requirements.     

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.     

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.     

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.     

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.     

Lightmicroscopic Observations on Structure and Division of Histomonas meleagridis (Smith)*

SYNOPSIS. Culture forms and lumen-dwelling phases of the ameboflagellate Histomonas meleagridis, which are structurally indistinguishable from each other, have a single flagellum. Their well-developed pelta is connected to the anterior segment of the broad, spatulate axostylar capitulum, applied to the left-ventral surface of the nearly spheroid or somewhat ellipsoid or ovoid nucleus. The capitulum narrows into a very slender axostylar trunk that tapers to a fine point and does not project beyond the body surface. The parabasal apparatus consists of a V-shaped parabasal body and a large parabasal filament. A new flagellum appears early during division and soon approaches its full length. The 2 flagella persist thruout division and each becomes the locomotory organelle of a daughter histomonad. The arms of the parental parabasal body appear to separate, each going to 1 of the daughter mastigont systems; some parabasal material is lost early in division. The 2nd arm is regenerated in each daughter parabasal body. The large parabasal filament seems not to be retained in the parental mastigont system, and new filaments are seen at both poles before 2 daughter nuclei are formed. The old axostyle degenerates from the anterior toward the posterior end; at the same time lamellar primordia of the daughter pelta-axostyle complexes appear in the separating mastigont systems that are connected by an extranuclear spindle during the entire division process. The structure and taxonomic status of H. meleagridis are discussed in the light of this and previous studies.