Cholesterol supplementation improves growth rates of Histomonas meleagridis in vitro.

Research on the energy metabolism of various protozoan parasites showed the essentiality and benefits of cholesterol in the cultivation of these organisms. However, not much is known about the energy metabolism of Histomonas meleagridis, although such information is of high importance to improve cultivation of the parasite for advancements in diagnostics, research and vaccine development. By supplementing a serum enriched cultivation medium with cholesterol, numbers of parasites could be doubled in comparison to unsupplemented negative controls. This effect was demonstrated for two different strains of the parasite, at different levels of in vitro-passages and for histomonads under xenic or monoxenic settings. Supplementing medium free of serum with cholesterol, resulted in significant growth of the parasite over 72 h. However, there were differences in growth behaviour in serum free medium between the different histomonad cultures and continuous passaging of the cultures without serum was not possible. Monitoring the bacterial growth of two different co-cultivated E. coli strains in monoxenic histomonad cultures during these experiments showed that there was no significant impact of cholesterol on the bacteria. Therefore, a direct effect of cholesterol on the parasite itself could be demonstrated. The results of these experiments supply new insights into the metabolism of H. meleagridis and it can be concluded that cholesterol is an important component to enhance parasite growth in vitro.

Critical Review: Future Control of Blackhead Disease (Histomoniasis) in Poultry.

Blackhead disease (histomoniasis) currently has no efficacious drug approved for use in poultry in the United States. Both chickens and turkeys can get the disease, but mortality is most often associated with turkeys. The lack of any approved therapies for blackhead is of concern, especially in the case of valuable turkey breeder candidate flocks. Due to the availability of efficacious drugs for many years, research focused on blackhead was minimal. However, without any drugs or reliable additives, blackhead will continue to be an issue in turkeys and broiler breeder chickens. The American Association of Avian Pathologists annual meeting in San Antonio, Texas, August 6-9, 2016, held a mini-symposium on blackhead. The mini-symposium included university researchers and industry veterinarians discussing blackhead in the United States and Europe including insights on the disease pathogenesis and epidemiology, as well as an update on the current state of blackhead in the United States since the removal of nitarsone from the market in January 2016. This review summarizes the information presented at the mini-symposium and discusses current control measures in an era without efficacious drugs.

Development of a Dry Medium for Isolation of Histomonas meleagridis in the Field.

Blackhead disease is caused by Histomonas meleagridis, an anaerobic protozoan parasite, and results in mortality rates of up to 100% in turkeys and 30% in chickens. Outbreaks of blackhead disease are unpredictable, and the harvesting of H. meleagridis strains from the field would be a great resource for researchers to study its epidemiology. Therefore, the objective of this study was to develop a dry medium that would allow storage at ambient temperatures until needed. Fifty milliliters of horse serum was dried and then mixed with dry medium M199 with Hanks balanced salts (10.6 g), sodium bicarbonate (0.35 g), and rice powder (0.8 g). To test the ability of reconstituted medium to support growth of H. meleagridis, groups of 10 flasks containing 0.2 g of dry medium were stored for 24 hr at 25 and 60 C before testing. Other groups of flasks containing dry medium were stored at 25, 37, and 42 C for 1, 3, or 6 mo. At each test period, the flasks were reconstituted with 10 ml of water, inoculated with 100 000 H. meleagridis cells, and incubated at 40 C for 48 hr. Fresh liquid medium was used as a control. There were no differences in cell counts in medium stored at 25 or 60 C for 24 hr. After 1 mo, cell counts in reconstituted medium were about half that of fresh liquid medium after 48 hr of incubation. But after 3 and 6 mo, the cell counts were not significantly different in all groups (P < 0.05) after 72 hr of incubation. These results show that dried Dwyer medium can be stored at ambient temperatures for extended times and would be an effective tool for obtaining isolates of H. meleagridis from the field.

Long-term in vitro cultivation of Histomonas meleagridis coincides with the dominance of a very distinct phenotype of the parasite exhibiting increased tenacity and improved cell yields.

The majority of research on Histomonas meleagridis was performed in the first half of the last century, especially those on morphological aspects. In the present study identical monoxenic settings for cultures of the same H. meleagridis clonal strain in its virulent low passage and attenuated high passage form enabled a comparative analysis of parasite characteristics. For the first time, it could be shown that long-term in vitro cultivation led to a severe shift in cell morphology, with the occurrence of a very distinct phenotype expressing a flagellated and highly amoebic cell morphology. Furthermore, the attenuated parasites showed better growth rates and a higher tenacity when confronted with adverse conditions. During these experiments up to 100% of the parasites, both virulent and attenuated, assumed a completely rounded morphology elucidated by electron microscopy. The findings indicate that such previously reported cyst-like stages are a defence strategy of H. meleagridis, independent of the passage level in vitro and pathogenicity in vivo. In conclusion, long-term in vitro passaging of H. meleagridis led not only to an attenuation of the parasite, as previously demonstrated, but also to a shift in the parasite's phenotype regarding morphology, growth behaviour and a higher level of tenacity.

The Enrichment of Histomonas meleagridis and Its Pathogen-Specific Protein Analysis: A First Step to Shed Light on Its Virulence.

Since the discovery of Histomonas meleagridis in 1893, the necessity of isolating pure H. meleagridis has been highlighted over the years in the battle against histomonosis. Insights into the molecular characteristics of this protozoon open possibilities to proper treatment. Axenization of H. meleagridis in vitro cultures cocultured with bacteria has been unsuccessful. Numerous unsuccessful attempts at culturing H. meleagridis axenically have reinforced the assumption that the protozoa had an obligate relationship with certain bacteria originating from the host ceca. Within these perspectives, we enriched H. meleagridis cells from a mono-eukaryotic culture copropagated with host cecal bacteria by flow cytometry. The enrichment of histomonads was confirmed through transmission electron microscopy and two-dimensional gel electrophoresis. For the first time several protein spots were successfully identified. The majority of spots were annotated as cytoskeletal proteins. Actin microfilaments are known to be a key player in cell spreading, cell adhesion, phagocytosis, signal transduction, and several other processes. Together with the identification of superoxide dismutase, the information generated from protein analysis of H. meleagridis may serve as a very first step toward understanding its pathogenesis and virulence.

Gut microeukaryotes during anorexia nervosa: a case report.

BACKGROUND: Few studies have focused on eukaryote community in the human gut. Here, the diversity of microeukaryotes in the gut microbiota of an anorexic patient was investigated using molecular and culture approaches. CASE PRESENTATION: A 21-year-old Caucasian woman was admitted in an intensive care unit for severe malnutrition in anorexia nervosa. One stool specimen was collected from the anorexic patient, culture and polymerase chain reaction-based explorations yielded a restricted diversity of fungi but four microeukaryotes Tetratrichomonas sp., Aspergillus ruber, Penicillium solitum and Cladosporium bruhnei previously undescribed in the human gut. CONCLUSIONS: Establishing microeukaryote repertoire in gut microbiota contributes to the understanding of its role in human health.

Syrian hamsters (Mesocricetus auratus) with simultaneous intestinal Giardia sp., Spironucleus sp., and trichomonad infections.

A commercial facility producing hamsters with a history of infection by dwarf tapeworm (Hymenolepis nana) submitted 15 animals for necropsy and postmortem parasitological and microscopic examination. No tapeworms were detected grossly or microscopically. Fecal examination including gastrointestinal mucosal smears demonstrated mixed intestinal bacteria and low numbers of Giardia sp. Histologic examination of small intestine demonstrated filling of the small intestinal crypts by large numbers of 7-9 µm × 3 µm, rod to crescent or teardrop-shaped flagellates consistent with Spironucleus sp. These organisms had two 1-µm, basophilic, oval nuclei and multiple superficial flagella-like structures. Much larger 10-15 µm × 8-10 µm, oval to pear-shaped organisms were also present in lower numbers and usually located with the crypts. These larger flagellates had multiple flagella and a basophilic rod-shaped nucleus. The larger flagellates included Giardia sp., which had an intimate interface with the surface of the mucosal epithelium, bilaterally symmetry, and binucleation. Lower numbers of trichomonads were also present and were distinguished by an undulating surface membrane and a single nucleus. The mucosa was hyperplastic and moderately inflamed. Although the tapeworm infection was resolved, diagnosis of multiple intestinal flagellates by fecal examination is complicated by the varying sensitivity and diagnostic accuracy of different types of fecal analysis for different flagellate types. Key differences in the morphology and location of the different types of flagellates as observed by histology of intestinal tissues provide important additional diagnostic information to distinguish trichomonads, Spironucleus sp., and Giardia sp.

Establishing mono-eukaryotic Histomonas meleagridis cultures from in vivo infection contaminated with Tetratrichomonas gallinarum and Blastocystis spp.

SUMMARY The necessity to easily establish Histomonas meleagridis cultures has been underlined extensively by many researchers in order to gain more insights in the biology of H. meleagridis. In addition the occurrence of different protozoa in the caeca of birds impedes, however, the isolation and propagation of H. meleagridis from field outbreaks. Therefore, in a kinetic study using transmission electron microscopy the deleterious effects of adventitious protozoa including Tetratrichomonas gallinarum and Blastocystis spp. on cultured H. meleagridis were examined. To overcome this issue, an easy and successful approach to establish the mono-eukaryotic H. meleagridis culture free of other host's protozoa is proposed. At 10 days post infection, liver lesions of H. meleagridis-infected birds were isolated and inoculated into culture media pre-incubated with caecal bacteria. After 48 h of incubation, presence of H. meleagridis in the cultures was confirmed through morphological evaluation. Additionally, TEM examination and analysis by PCR amplification of the small subunit rRNA gene could exclude the co-cultivation of T. gallinarum and Blastocystis spp. Furthermore, after successful propagation and maintenance of the cultured H. meleagridis, its pathogenicity was affirmed in an infection experiment in turkeys.

A genomic analysis of Histomonas meleagridis through sequencing of a cDNA library.

Histomonas meleagridis, a flagellated protozoan of the Order Trichomonadida, is the causative agent of blackhead disease in gallinaceous birds. Few genes have been identified in this organism; thus, little is known regarding the molecular basis for its metabolism, virulence, and antigenicity. To identify new genes, a cDNA library derived from a lab strain of H. meleagridis was sequenced and annotated. Data obtained from these experiments identified 3,425 H. meleagridis genes. Analysis of the data allowed the identification of 81 genes coding for putative hydrogenosomal proteins and was used to determine the codon usage frequency. Sequence information also identified bacteria that are cultured with H. meleagridis. Future analysis of these data should provide valuable molecular insights into H. meleagridis and provide the platform for molecular studies aimed at understanding the pathogenesis of blackhead disease.

Escherichia coli strongly supports the growth of Histomonas meleagridis, in a monoxenic culture, without influence on its pathogenicity.

Based on clonal cultures of Histomonas meleagridis, monoxenic cultures have, to our knowledge for the first time, been established in a liquid medium. The faecal flora was exchanged for defined bacterial strains by selective destruction of the initial bacteria with a variety of antibiotics, keeping the flagellate alive. The growth of the protozoan parasite was found to depend on the bacteria, especially on their energy metabolism. Escherichia coli was found to strongly support the growth of the parasite, whereas Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa were less efficient. Confocal laser microscopy showed that H. meleagridis could take up green fluorescent protein-tagged E. coli DH5α, suggesting that bacteria serve as a food supply for the protozoa. By exchanging the bacterial flora for E. coli strain DH5α in H. meleagridis cultures that underwent continuous in vitro passages, it was possible to show that the in vivo attenuation process was independent of the bacteria. Furthermore, the gut flora in infected turkeys had no negative effect on the protozoan's virulence. Consequently, attenuation depends not on the bacteria in the culture but on the in vitro passages. Finally, the experiments provided evidence that the infection of turkeys with H. meleagridis enabled infection of the liver with E. coli.

Persistence of Histomonas meleagridis in or on materials used in poultry houses.

Histomonas meleagridis is the causative agent of blackhead disease or histomonosis in turkeys, and previous research suggests that this parasite survives poorly outside of hosts except within heterakid nematodes. However, we investigated the viability of H. meleagridis in or on several artificially contaminated materials kept at ambient room temperature (22 +/- 2 C) to mimic the situation in the field. The protozoan survived for up to 1 hr on wood, rubber, and metal; up to 3 hr on egg-tray cartons, egg shells, and bricks; up to 6 hr on straw, turkey feathers, and feed; and up to 9 hr in nonchlorinated tap water and fecal matter. Therefore, contaminated water, fresh fecal matter, or both could play a role in transmission of the parasite within and among poultry houses rather than other materials tested in this study.

Axenization and optimization of in vitro growth of clonal cultures of Tetratrichomonas gallinarum and Trichomonas gallinae.

A rapid and simple procedure was established to obtain clonal axenic cultures of Tetratrichomonas gallinarum and Trichomonas gallinae and to optimize their in vitro growth conditions. Medium 199 was used for axenization of two genetically different clones of T. gallinarum and T. gallinae. Six different media were used to optimize the growth behaviour of axenically grown parasites: Medium 199, TYM, TYI-S-33, Hollander fluid (HF), Trichomonas vaginalis (TV) and modified TV media. The highest cell yields for both axenic clones of T. gallinarum were obtained in modified TV medium without antibiotics. The maximum numbers of trophozoites of T. gallinae were obtained in an optimized HF medium. This study demonstrated that axenic cultures for T. gallinarum and T. gallinae could be obtained avoiding the migration technique through a V-tube. Following axenization and optimization, both clones of T. gallinarum and T. gallinae could be propagated both aerobically and anaerobically.

Histomonas meleagridis (Protozoa: Trichomonadidae): analysis of growth requirements in vitro.

Histomonads grew rapidly in Dwyer's medium, consisting of medium 199, chick embryo extract, serum, and rice powder, reaching a population size of about 5 x 10(5) in 3-4 days, followed by a rapid decline. Substitution of other cell culture media (L-15, MEM, or RPMI) for M199 was also satisfactory, except for Waymouth's medium, which produced a lower and later peak of growth. Omission of serum or rice rendered media unsuitable for growth. Bacteriological culture media did not support growth of histomonads. Media that included glucose were unsuitable because the pH of the cultures dropped to about 4. The effect of glucose on pH was due to the action of bacteria. Oxygen inhibited growth of histomonads. There was no growth when culture tubes were not capped tightly, regardless of the medium used. Histomonads grew well with rice flour, cornstarch, oat flour, rye flour, and buckwheat flour. Barley and blue corn meal were less satisfactory. It appeared that the requirements for growth of the lumen phase Histomonas meleagridis included a suitable physiological saline, serum (of any source), and a starch source (grain flour). Anaerobic conditions and a pH near neutral were best. Histomonads separated into pure cultures by flow cytometry would not grow without the inclusion of an unspecified species of bacteria.

Identification and molecular characterization of numerous Histomonas meleagridis proteins using a cDNA library.

SUMMARYHistomonas meleagridis is a protozoan parasite of various galliform birds causing a type of enterohepatitis termed histomonosis or 'blackhead disease'. Due to the ban of chemotherapeutic substances and an increase in free-range poultry production, histomonosis is currently a re-emerging disease. So far limited molecular knowledge is available. In the present work, mRNAs coding for antigenic proteins of H. meleagridis were identified. For this purpose, a cDNA expression library was constructed from a mono-eukaryotic culture of H. meleagridis. The library was screened with polyclonal rabbit serum raised against purified H. meleagridis trophozoites. Polyclonal rabbit serum specifically recognized the same major H. meleagridis antigens as chicken and turkey sera originating from animal trials, but displayed a significantly lower bacteria-dependent background signal. After 2 rounds of screening, a total of 95 positive clones were sequenced. Bioinformatics analyses were performed on nucleotide and deduced amino acid sequences, identifying 37 unique clones. Based on the homology to other protozoan parasites, mostly Trichomonas vaginalis, the clones were grouped according to functional aspects: structural proteins, possible surface proteins, oxygen reducing proteins, ribosomal proteins, protein kinases and various other intracellular proteins.

Light and transmission electron microscopic studies on trophozoites and cyst-like stages of Histomonas meleagridis from cultures.

The present study deals with Berlin strains of Histomonas meleagridis, the specimens of which were cultivated in Dwyer's medium. The light and electron microscopic examination revealed that the cultivated trophozoite stages (reaching about 10 mum in size) appeared more or less spherical, although their surface (covered by a single membrane) showed amoeba-like waves. All stages were uni-nucleated and reproduced by binary fission with an extranuclear spindle apparatus. Some trophozoites appeared ovoid and possessed a single flagellum with a typical microtubular 9 x 2 + 2 arrangement. Furthermore, the latter were characterized by an inner row of typical microtubules (remnant of an axostyle) and a Golgi apparatus (both adjacent to the nucleus), multivesicular structures, hydrogenosomes, and many food vacuoles containing either starch grains or bacteria. Their cytoplasm was densely filled with glycogen granules and ribosomes. Similar stages were also documented in the caeca and cloaca of chicken when being inoculated (via cloaca) with such culture stages. In addition to these typical trophozoites, the cultures contained a low number of 10-mum-sized spherical cyst-like stages with a surrounding amorphous layer. The cytoplasm of some of these cyst-like stages-when studied by electron microscopy-appeared with two membranes or had formed an amorphic, cyst-wall-like layer at their surface, apparently corresponding to their light microscopical appearance. Such stages might be involved in transmission from one host to another and probably have been missed before in microscopical examinations of infected poultry.

Transmission electron microscopic studies of stages of Histomonas meleagridis from clonal cultures.

Histomonas meleagridis is a 10-20 microm-sized flagellated protozoan, causing histomoniasis in gallinaceous birds. Different strains of H. meleagridis from different origins were used to establish clonal cultures, which can be traced back to a single cell. Cells from these clonal cultures were examined by transmission electron microscopy. Results gave detailed insights in the ultrastructure showing a single flagellum, a band of microtubules remnants of an axostyle or of a costa, respectively pelta, hydrogenosomes, nucleus, spindle apparatus, and other organelles of the trophozoites of H. meleagridis.

Morphological aspects of Monocercomonas sp. and investigation on probable pseudocysts occurrence.

Monocercomonas sp. is a flagellate protozoan found in the large intestine of snakes and in insects. Light microscopy revealed the measurements of morphological features of the trophozoites. Scanning electron microscopy showed in detail the emergence of the three anterior flagella, the recurrent flagellum, the axostyle, and the absence of undulating membrane. In addition, we described spherical forms which are probably pseudocysts. The investigation on the occurrence of this process was carried out through the incubation of Monocercomonas sp. trophozoites in several stressful conditions, such as pH change, nutrient depletion and different temperatures. Results revealed high pseudocyst formation at acidic pH values (4.0, 5.0, and 6.0), in absence of serum and in incubation at 37 degrees C. The occurrence of these pseudocystic forms in trichomonads life cycle is under investigation. This study describes the external structure of Monocercomonas sp., as demonstrated by light and scanning electron microscopy. Moreover, to our knowledge, this is the first time that formation of probable pseudocysts is shown in Monocercomonas sp., contributing to the research field on termite protozoa biology.

Improved culture of Histomonas meleagridis in a modification of Dwyer medium.

Dwyer medium is the most frequently employed culture medium for Histomonas meleagridis. Both for subculturing and for resuscitation of H. meleagridis from storage in liquid nitrogen, modified Dwyer medium with an increased rice powder concentration (0.8%) and no chicken embryo extract proved superior to Dwyer standard medium, with threefold (10(6.3) vs. 10(5.8) histomonads/ml) to 10-fold (10(6.7) vs. 10(5.8) histomonads/ml) higher concentrations of parasites after resuscitation or subculturing, respectively.

Blackhead disease (histomoniasis) in poultry: a critical review.

After its discovery in 1893 in Rhode Island, blackhead disease was reported across the continent and soon in many other countries. It decimated the turkey industry in New England and followed production like a faithful shadow. Blackhead disease causes high mortality in turkeys, sometimes approaching 100% of a flock. In chickens, the mortality may be 10%-20% with high morbidity, although many outbreaks pass unnoticed. Early workers identified Histomonas meleagridis, a protozoan related to Entamoeba histolytica, Giardia lamblia, and Trichomonas, as the causative agent. Like many other parasites, its life cycle is complex, involving as an intermediate host, the common cecal worm Heterakis gallinarum. The necessity for bacteria for Histomonas to become virulent in the turkey and chicken, notably Escherichia coli, Bacillus subtilis, and Clostridium spp., was discovered by research in gnotobiotic birds. Changes in management brought the disease under control, although it remained the first cause of mortality in turkeys until modern antihistomonal products were developed after WWII. The ban of nitroimidazole products in the United States and Europe was followed by an upsurge in reported cases in turkeys and chickens. Immunization is not an option for prevention, as birds do not reliably become resistant to reinfection after suffering a primary exposure. Recent research demonstrated that histomoniasis could spread rapidly through a flock of turkeys by direct contact, probably involving the phenomenon of cloacal drinking. Direct transmission was not demonstrated for chickens, stressing dependence on H. gallinarum as the source of infection. The lack of suitable treatment drugs or vaccines emphasizes the importance of prevention by worm control and management.

Failure to establish infection with Tetratrichomonas sp. in the reproductive tracts of heifers and bulls.

Experimental infection of the reproductive tracts of heifers and bulls with Tetratrichomonas sp. isolated from preputial smegma of virgin bulls was attempted. Nine heifers and four bulls were challenged by inoculation of 7 x 10(6) Tetratrichomonas sp. into the vaginal lumen and preputial cavity, respectively. Vaginal mucus and preputial smegma samples were collected and cultured for Tetratrichomonas sp. Heifers were slaughtered in groups of three at 2, 9 and 21 days after inoculation. Two heifers and two bulls infected with Tritrichomonas foetus and two uninfected heifers were used as controls for the model infection. Tetratrichomonas sp. were only isolated in vaginal mucus of 7/9 inoculated heifers at 6h post-inoculation, and genital secretions taken at slaughter time from vagina, uterus and oviduct were cultural negative. Bulls challenged with Tetratrichomonas sp. remained cultural negative. Since Tetratrichomonas sp. survived only a few hours in the female genitalia and did not survive in the male genitalia after experimental challenge, Tetratrichomonas sp. did not colonize the genital tract. These were likely trichomonads from the digestive tract. Collection of clean samples without fecal contamination from the reproductive tract is proposed as a measure to avoid Tetratrichomonas sp. transitory genital infection.