Histomonas wenrichi n. sp. (Mastigophora: Mastigamoebidae), a Nonpathogenic Parasite of Gallinaceous Birds

SYNOPSIS. A nonpathogenic histomonad, morphologically identical to that found in pheasants by Wenrich in 1943, is described as Histomonas wenrichi n. sp. Its dimensions are about 1.5 × those of H. meleagridis. It has 4 flagella instead of the 1 or 2 that characterize the latter species. It does not multiply in the host tissues nor produce visible responses to its presence there, and it does not exist as the clear "tissue form" that is common in active H. meleagridis infections.
Histomonas wenrichi n. sp. can be transmitted to turkeys, chickens, and pheasants by either rectal inoculation or by feeding embryonated eggs of Heterakis gallinarum grown in birds harboring the nonpathogenic histomonad in the ceca. Usually fewer than half the female heterakids so grown produce eggs capable of transmitting this histomonad, and a total of 200 to several thousand eggs from worms recovered from Histomonas -infected birds may be required to produce one Histomonas infection.
Histomonas wenrichi n. sp. has been propagated in more than 2000 chickens and turkeys over an 8-year period, during which time it has been transmitted by 18 generations of Heterakis. It has frequently been grown in birds in which H. meleagridis was also present, and upon reisolation it maintained all of its original characteristics. It has not yet been cultivated in vitro.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Prevalence and pathology of the nematode Heterakis gallinarum, the trematode Paratanaisia bragai, and the protozoan Histomonas meleagridis in the turkey, Meleagris gallopavo

The prevalence of infection and associated pathology induced by two helminth and one protozoan species infecting Brazilian turkeys are reported. The intestinal nematode Heterakis gallinarum appeared with a prevalence of 70% in the infected birds, without gross lesions when not associated to the protozoan Histomonas meleagridis. Histological findings in the ceca were represented by the presence of H. gallinarum worms, intense chronic diffuse inflammatory processes with mononuclear and polymorphonuclear (heterophils) leucocyte infiltrations. The prevalence of the protozoan H. meleagridis associated to H. gallinarum was of 2.5% and microscopic examination revealed a severe inflammatory process in the liver and cecum with the presence of small clear areas with round eosinophilic parasites. Gross lesions were absent in turkeys infected with the renal digenetic trematode Paratanaisia bragai; the parasite was prevalent in 20% of the cases and cross-sections of the kidneys showed a remarkable distension of the collecting ducts with several worms in the lumen. The walls of the ducts presented a discrete heterophilic infiltrate among mononuclear cells.     

The wild turkey as a host for Heterakis gallinarum and Histomonas meleagridis.

Freshly embryonated eggs of Heterakis gallinarum gathered from naturally infected domestic turkeys and chickens developed the first 4 weeks essentially as well in young wild turkeys as in domestic poults, but then became progressively retarded and failed in most birds to result in females with fertile eggs. There was no significant difference in the prevalence or progress of infections with Histomonas meleagridis in the two kinds of turkeys, both of which differed from chickens only in that the latter had neither liver involvement nor mortality. In a second test, heterakids hatched from eggs stored 5-6 months at 4 C (comparable to overwintering) sustained very heavy losses in all birds, with greatly accelerated liberations of H. meleagridis, Few worms reached maturity and still fewer produced fertile eggs. In turkeys, and especially in wild turkeys, replacement of infective stages was so poor, that these birds were of no importance in contaminating the soil.     

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.     

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.     

Two sites of intracellular localization of rhodaminyl-phalloidin in hepatocytes

The uptake of a fluorescent phallotoxin (tetramethylrhodaminyl-phalloidin) into rat hepatocytes has been studied. The experiments were performed in vitro, using freshly isolated hepatocyte suspensions or monolayers of hepatocytes cultured for up to 5 days, as well as in vivo, by investigating cryostat sections of a liver from an animal injected with the labelled toxin. In vitro, in freshly isolated hepatocytes, a staining of actin was observed. On the contrary, if the hepatocytes were cultured, only fluorescent endocytotic vesicles were found accumulated around the nucleus, and remaining in the cells unchanged for several days. In vivo, both fluorescent patterns were observed, often in one and the same cell. The endocytotic vesicles of rhodaminylphalloidin looked very similar to those obtained with fluoresceinyl-concanavalin A. navalin A. We conclude that in all systems the fluorescent phallotoxin enters the hepatocytes by endocytosis. However, in the freshly isolated cells the endocytotic vesicles apparently undergo some kind of processing with release of the toxin and subsequent staining of cellular actin, while in cultured hepatocytes the endocytotic vesicles persist unprocessed.     

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.     

Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD can be induced in salmon by exposure to freshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic differences between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions. Immunoblot analysis using polyclonal antisera, revealed differences in the antigen profiles of two cultured isolates of Neoparamoeba sp. when they were grown on agar versus in liquid medium. However, the antigen profiles of the two isolates were very similar when they were grown under the same culture conditions. Comparison of these antigen profiles with a preparation from parasites freshly isolated from infected gills revealed a very limited number of shared antigens. In addition monoclonal antibodies (mAbs) raised against surface antigens of cultured parasites were used in an indirect immunofluorescence assay to assess the expression of specific surface antigens of Neoparamoeba sp. after various periods in culture. Significant changes in antigen expression of freshly isolated parasites were observed after 15 days of in vitro culture. The use of mAb demonstrated progressive exposure/expression of individual antigens on the surface of the freshly isolated parasites during the period in culture.     

The Na+/H+ antiporter in rat alveolar type II cells and its role in stimulated surfactant secretion

We used the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+/H+ exchange in freshly isolated rat alveolar type II cells and alveolar type II cells in primary culture. The intracellular pH (pHi) of freshly isolated alveolar type II cells was 7.36 +/- 0.05 (n = 3). When freshly isolated alveolar type II cells were acid loaded with nigericin in sodium-free buffer, the pHi dropped to 6.59 +/- 0.04 and remained low in sodium-free buffer. When acid-loaded cells were subsequently incubated with NaCl, pHi increased in a dose-dependent manner. Amiloride (0.1 mM) inhibited the sodium-induced increase in pHi. When the acid-loaded cells were resuspended in an unbuffered choline chloride solution, the cells secreted H+ in a sodium-dependent and amiloride-inhibitable manner. Alveolar type II cell monolayers, which were cultured for 22 h on glass coverslips and then loaded with BCECF, had a resting pHi of 7.48 +/- 0.05 (n = 4). Nigericin acidified these cultured cells in the absence of sodium and NaCl increased the pHi of these acid loaded cells as observed in freshly isolated cells. Secretagogues of pulmonary surfactant, 12-O-tetradecanoylphorbol 13-acetate (TPA) and terbutaline, did not change pHi. Inhibition of the Na+/H+ antiporter by the addition of amiloride to a Na+ containing medium or the substitution of choline for Na+ did not inhibit stimulated phosphatidylcholine secretion. We conclude that pHi regulation in rat alveolar type II cells is in part mediated by an amiloride-sensitive Na+/H+ antiporter, but this system appears not to be involved in TPA- or terbutaline-induced pulmonary surfactant secretion in primary culture.     

PACAP is an islet neuropeptide which contributes to glucose-stimulated insulin secretion

Pituitary adenylate cyclase activating peptide (PACAP) is a ubiquitously distributed neuropeptide which also is localized to pancreatic islets and stimulates insulin secretion. We examined whether endogenous PACAP within the islets might contribute to glucose-stimulated insulin secretion by immunoneutralizing endogenous PACAP. Immunocytochemistry showed that PACAP immunoreactivity is expressed in nerve terminals within freshly isolated rat islets, but not in islets that had been cultured for 48 h. In contrast, islet endocrine cells did not display PACAP immunoreactivity. Addition of either of two specific PACAP antisera markedly inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from freshly isolated rat islets, whereas a control rabbit serum did not affect glucose-stimulated insulin secretion. In contrast, the PACAP antisera had no effect on glucose-stimulated insulin secretion in cultured islets. Based on these results we therefore suggest that PACAP is an islet neuropeptide which is required for the normal insulinotropic action of glucose.     

Lichen protein affinity towards walls of cultured and freshly isolated phycobionts and its relationship to cell wall cytochemistry

Abstract The binding capacity of protein fractions ( M _r > 8000) from five lichens, Hypocenomyce scalaris, Hypogymnia physodes, Ramalina fastigiata, Umbilicaria pustulata and Xanthoria parietina was tested towards their freshly isolated phycobionts and towards ten cultures of phycobionts, five of which were from the above-mentioned lichens. The protein fractions from H. scalaris, R. fastigiata, U. pustulata and X. parietina bind intensely to a cultured Pseudotrebouxia from H. physodes and a cultured Trebouxia from X. parietina , but not to the other cultured algae, and not to the freshly isolated algae. A protein fraction between M _r 3500 and 8000 from H. physodes shows a faint binding to its own cultured algae. The nature of the binding and its relevance in connection with the mutual recognition of alga and fungus are briefly discussed.     

Histomoniasis and reticuloendotheliosis in a wild turkey (Meleagris gallopavo) in North Carolina

A moribund wild turkey (Meleagris gallopavo) died shortly after it was discovered in Martin County, North Carolina (USA). The 4.3-kg female turkey appeared in good condition with no visible external lesions or evidence of injury. There were 2- to 5-mm yellow-white plaques on the mucosal surfaces of the oral cavity and mid-esophagus. The liver had large, multifocal, irregular pale areas on cut and uncut surfaces. The spleen contained multifocal, pale, hard, nodules. Microscopic changes in the liver consisted of large multifocal coalescing areas of necrosis. Occasional spherical 10 to 15 microns in diameter organisms consistent with Histomonas meleagridis were present in the necrotic areas. Viable hepatic parenchyma contained multifocal infiltrations of numerous mononuclear cells interpreted as neoplastic cells resembling lymphoblasts and plasma cells. Similar neoplastic cell infiltrates, consistent with the lymphoproliferative disease reticuloendotheliosis, were present in spleen, lung, and esophageal and oral mucosa. Reticuloendotheliosis virus, subtype 2, was isolated from samples of liver and spleen.