Research Note: Evaluation of deoxycholic acid for antihistomonal activity.

Deoxycholic acid (DCA) is a naturally occurring secondary bile acid that originates from intestinal bacterial metabolic conversion of cholate, a primary bile acid. Deoxycholic acid was shown to have antihistomonal properties in vitro, leading to our hypothesis that DCA inclusion within the feed might prevent histomoniasis. Selected dietary concentrations of DCA were evaluated for effects on body weight gain (BWG), lesions, and mortality of turkeys challenged with wild-type Histomonas meleagridis (WTH). Treatments consisted of non-challenged control (NC; basal diet), 0.25% DCA diet + challenge, 0.5% DCA diet + challenge, 1% DCA diet + challenge, and a positive-challenged control (PC; basal diet). All groups were fed a basal starter diet until day 7, at which time DCA diets were administered to the respective groups. On day 14, 2 × 105 WTH cells/turkey were intracloacally administered. H. meleagridis-related lesions were evaluated on day 13 post-challenge. Pre-challenge day 0 to 14 BWG was higher (P ≤ 0.05) in the 0.25% DCA group than in the 1% DCA group. There were no significant differences in pre-challenge day 0 to 14 BWG between any of the other groups. No significant differences in mortalities from histomoniasis occurred in the DCA groups as compared to the PC group. No H. meleagridis lesions or mortalities were observed at any time in the NC group. Presence of H. meleagridis-related liver lesions was higher (P ≤ 0.05) in the 0.5% DCA group as compared to the PC group. Using the same controls and experimental timeline, an additional group was included to evaluate a biliogenic diet formulated with 20% whole egg powder to encourage endogenous bile acid production. The biliogenic diet had no statistical impact on pre-challenge day 0 to 14 BWG, but did not reduce H. meleagridis-related mortalities or lesions after the challenge. Taken together, these data suggest that DCA inclusion within the feed at these concentrations and under these experimental conditions does not prevent histomoniasis.

Research Note: Evaluation of boric acid as a chemoprophylaxis candidate to prevent histomoniasis.

Histomoniasis, caused by the protozoan parasite Histomonas meleagridis, is a disease to which turkeys are especially susceptible. Currently, no chemoprophylaxis compounds are available to mitigate this disease. Boric acid (BA) exhibits antifungal, antiseptic, and antiviral properties and has been used in the treatment of yeast infections. Based on these characteristics, an experiment was conducted to evaluate whether BA might be an efficacious prophylaxis against challenge with wild-type H. meleagridis (WTH). On day-of-hatch, poults were randomly assigned to either a basal control diet or a BA diet (basal diet + 0.2% BA). Groups consisted of a nonchallenged control (NC; basal diet), 0.2% BA + challenge (BA; 0.2% BA diet), and a positive-challenged control (PC; basal diet). On day 21, challenged groups were intracloacally inoculated with 2 × 105 WTH cells/turkey, and lesions were evaluated on day 14 postchallenge. Individual body weights were recorded on day 0, 21, and 35 to calculate the prechallenge and postchallenge body weight gain (BWG). The BA group resulted in lower prechallenge day 0 to day 21 BWG (P = 0.0001) than the NC group. Postchallenge day 21 to day 35, BWG was also lower (P = 0.0503) in the BA group than the PC group. No differences between the BA and PC groups were detected for mortalities associated with histomoniasis. Moreover, liver and cecal lesions were not statistically different between the BA and PC groups. Taken together, these data suggest that BA was not efficacious in the prevention or reduction of histomoniasis disease severity when provided at 0.2% dietary concentration under these experimental conditions.

Research Note: Evaluation of dietary administration of sodium chlorate and sodium nitrate for Histomonas meleagridis prophylaxis in turkeys.

Histomoniasis is currently a re-emerging disease of major significance for many commercial turkey and broiler breeder production companies because of the unavailability of drugs or vaccines. The protozoa Histomonas meleagridis (HM) requires the presence of enteric microflora to promote the disease. The objectives of this research note were to evaluate the effect of dietary administration of sodium chlorate (SC) and sodium nitrate (SN) in vitro and in vivo for HM prophylaxis in poults. A total of 128 day-of-hatch female poults obtained from a commercial hatchery were wing-tagged and randomly assigned into 1 of 4 experimental groups: negative control (NC), positive control, dietary inclusion of SC (3,200 ppm) and SN (500 ppm). Poults from groups SC and SN started on their respective diets on day 12. All groups, except the NC, were challenged with 2 × 105 HM on day 19. Controls were fed a basal diet, identical to the treatment diets but not supplemented with SC or SN. Body weight gain (BWG) was determined weekly, starting on day 1 until day 28, and postchallenge morbidity and mortality were recorded. On day 28 of age, all surviving poults were lesion scored for hepatic and cecal lesions. Ceca and distal ileum were collected on day 28 for bacterial recovery on selective media for total aerobic, lactic acid bacteria, or gram-negative bacteria. The addition of SC and SN in the in vitro growth of HM greatly reduced the growth of the protozoa after 20 h of incubation when compared with the control nontreated group (P < 0.05). However, dietary supplementation of SC and SN had no effect against HM in vivo, as was demonstrated by BWG, the severity of lesions in the liver and ceca or bacterial recovery of treated poults when compared with the positive control group.

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.

Histomonosis in poultry: previous and current strategies for prevention and therapy.

Histomonosis is a parasitic disease of poultry with worldwide prevalence. The disease can cause morbidity and mortality in chicken and turkey flocks entailing severe economic losses. In the first half of the last century, there was a high demand to control histomonosis as the turkey industry was severely affected by the disease. Consequently, numerous chemical compounds were tested for their efficacy against Histomonas meleagridis with varying outcomes, that are summarized and specified in this review. At the same time, preliminary attempts to protect birds with cultured histomonads indicated the possibility of vaccination. Several years ago antihistomonal drugs were banned in countries with tight regulations on pharmaceuticals in order to comply with the demand of consumer protection. As a consequence, outbreaks of histomonosis in poultry flocks increased and the disease became endemic again. New approaches to prevent and treat histomonosis are, therefore, needed and recently performed studies focused on various areas to combat the disease, from alternative chemotherapeutic substances to plant-derived compounds until vaccination, altogether reviewed here. Considering existing regulations and the overall outcome of experimental studies, it can be concluded that vaccination is very promising, despite the fact that various challenges need to be addressed until the first ever developed vaccine based upon live flagellates in human or bird medicine can be marketed.

Antiprotozoal Effects of the Tomato Tetrasaccharide Glycoalkaloid Tomatine and the Aglycone Tomatidine on Mucosal Trichomonads.

The present study investigated the inhibitory effects of the commercial tetrasaccharide tomato glycoalkaloid tomatine and the aglycone tomatidine on three mucosal pathogenic protozoa that are reported to infect humans, cattle, and cats, respectively: Trichomonas vaginalis strain G3, Tritrichomonas foetus strain D1, and Tritrichomonas foetus strain C1. A preliminary screen showed that tomatine at 100 µM concentration completely inhibited the growth of all three trichomonads. In contrast, the inhibition of all three pathogens by tomatidine was much lower, suggesting the involvement of the lycotetraose carbohydrate side chain in the mechanism of inhibition. Midpoints of concentration-response sigmoid plots of tomatine on the three strains correspond to IC50 values, the concentration that inhibits 50% of growth of the pathogenic protozoa. The concentration data were used to calculate the IC50 values for G3, D1, and C1 of 7.9, 1.9, and 2.2 µM, respectively. The results show an approximately 4-fold variation from the lowest to the highest value (lowest activity). Although the inhibition by tomatine was not as effective as that of the medicinal drug metronidazole, the relatively low IC50 values for both T. vaginalis and T. foetus indicated tomatine as a possible natural alternative therapeutic for trichomoniasis in humans and food-producing (cattle and pigs) and domestic (cats) animals. Because tomatine has the potential to serve as a new antiprotozoan functional (medical) food, the distribution of this glycoalkaloid in tomatoes and suggestions for further research are discussed.

Antihistomonal effects of artemisinin and Artemisia annua extracts in vitro could not be confirmed by in vivo experiments in turkeys and chickens.

Five different Artemisia annua-derived materials (i.e. dry leaves, pure artemisinin, and hexane, dichloromethane or methanol extracts of leaves) were screened for their in vitro activities against six clonal cultures of Histomonas meleagridis. Except for the methanol extract, all tested materials displayed in vitro activity against all tested protozoal clones. Neither the dry plant material, extracts nor artemisinin showed any antibacterial activity against the xenic bacteria accompanying the six H. meleagridis clones at concentration levels identical to the antihistomonal setting. The dichloromethane extract of dry leaves (Ext-DCM) (minimal lethal concentration=1.0 mg/ml) and artemisinin (half-maximal inhibitory concentration=1.295 mg/ml) had the most promising antihistomonal properties and were therefore subsequently tested in a standardized experimental infection model in both turkeys and chickens infected with clonal H. meleagridis. There were no differences between treatment groups, where all infected turkeys showed severe clinical histomonosis and demonstrated severe typhlohepatitis typical for histomonosis. Consistent with the infection model used, the infected chickens did not show any adverse clinical signs but contracted severe lesions in their caeca 7 and 10 days post infection (d.p.i.), liver lesions were absent to mild after 7 d.p.i. and progressed to severe lesions at 10 d.p.i.; thus no differences between treatment groups were observed. In conclusion, neither artemisinin nor Ext-DCM was able to prevent experimental histomonosis in turkeys and chickens at the given concentrations, which is contrary to the antihistomonal effect noticed in vitro even though the same clonal culture was used. The results of this study therefore clearly demonstrate the importance of defined in vivo experimentation in order to assess and verify in vitro results.

Factors influencing the activity of tiamulin against Histomonas meleagridis in vitro.

In this study, we investigated the activity of tiamulin fumerate against three strains of Histomonas meleagridis in vitro under different conditions. Tiamulin reduced histomonal growth of all three strains at concentrations of 20 ppm and higher. Cultures in phosphate-buffered saline-based medium were more susceptible than cultures in traditional Dwyers medium. When the cultures were inoculated with higher numbers of histomonads, the activity of tiamulin was reduced. Bacteria present in the cultures were resistant against tiamulin.

Partial sequence of the beta-tubulin of Histomonas meleagridis and the activity of benzimidazoles against H. meleagridis in vitro.

The protozoan parasite Histomonas meleagridis is a member of the family Monocercomonadidae in the class Trichomonada. Due to food safety concerns, currently no prophylactic or therapeutic drug against the parasite is licensed in the European Union. Benzimidazoles are antiparasitic drugs, and some of them are licensed for use in food-producing animals. Benzimidazoles act on beta-tubulin, and the beta-tubulin sequence allows predictions about the efficacy of benzimidazoles. In this study, we sequenced and analyzed a part of the beta-tubulin gene of five H. meleagridis strains and of Dientamoeba fragilis. In each Histomonas strain, three to five different sequences were found. No clustering of sequences from the same strain was recognizable. A phylogenetic tree based on the amino acid sequences of trichomonal beta-tubulin genes placed the histomonal sequences on a branch with D. fragilis, separate from Monocercomonas sp. and Tritrichomonas foetus. All histomonal amino acid sequences predicted a susceptibility to benzimidazoles. However, when we tested the efficacy of five benzimidazoles, namely, albendazole, fenbendazole, flubendazole, mebendazole, and nocodazole, on H. meleagridis in vitro, all tested drugs showed no efficacy, even though the concentrations tested were higher than the concentrations found to be effective against Trichomonas vaginalis and T. foetus by other investigators.

Antiprotozoal activities determined in vitro and in vivo of certain plant extracts against Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp.

A total of 43 plant substances provided as raw material and different kinds of extracts (aqueous, ethanol, and heptane) from 18 different organic wastes obtained from the food/feed industry were investigated for their in vitro activities against clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum, and Blastocystis sp. Ethanolic extracts of thyme, saw palmetto, grape seed, and pumpkin fruit proved to be most efficacious. Thus, these extracts were further tested in vivo in turkeys experimentally infected with H. meleagridis by administrating the substances to the birds through the drinking water. Even though a delayed mortality was noticed in some birds medicated with the extracts of thyme, grape seed, and pumpkin fruit, all birds died or had to be euthanized the latest within 5 weeks post infection--with the exception of one bird which was probably never infected with histomonads--due to a severe typhlohepatitis indicative for histomonosis. In addition, none of the substances were able to prevent the spreading of H. meleagridis from infected to in-contact birds. Thus, these studies clearly demonstrate that in vitro studies are of limited value to assess the efficacy of plant substances against histomonosis.

In vivo effect of herbal products against Histomonas meleagridis in turkeys.

Histomoniasis is a serious disease in poultry. All chemotherapeutics with known efficacy against its causative agent, Histomonas meleagridis, have been banned from use as prophylactic or therapeutic use in production animals. In a search for possible alternatives, the in vivo effects of the herbal products Enteroguard and Protophyt were examined. Two-week-old turkeys allocated into 13 groups of 18 birds were either sham inoculated (negative control group) or were inoculated with 100, 3162 or 200 000 histomonads per bird. Control groups (no feed additives, dimetridazole, or Histostat-50) were included in the study. No morbidity or mortality was observed in the negative control group or in the groups inoculated with 100 histomonads per bird. Mortality was 100% in the groups inoculated with 200 000 histomonads per bird and either untreated (positive control group) or receiving Protophyt SP, Protophyt SP and Protophyt B, Enteroguard, or Histostat-50. Mortality was 17% in the dimetridazole-treated group. In the groups inoculated with 3162 histomonads per bird, mortality was 100% for the positive control group and the group receiving Enteroguard, and was 94% in the group receiving Protophyt SP. In the present study, Enteroguard or Protophyt was not found to be effective against histomoniasis.

Ectobiotic spirochetes of flagellates from the termite Mastotermes darwiniensis: attachment and cyst formation.

The association of the gut flagellates Mixotricha paradoxa and Deltotrichonympha sp. from the termite Mastotermes darwiniensis with ectobiotic spirochetes and bacterial rods is investigated with light and electron microscopy. Treatment with different chemicals disturbing molecular interactions and use of the freeze-fracture and freeze-etch technique show that hydrophobic interactions and integral membrane proteins seem to be involved in the firm attachment at the contact sites. Application of antibiotics reduces the number of ectobionts and leads to a disintegration of the cortical attachment systems. As a result Mixotricha becomes spherical and immotile. In both flagellates the antibiotics have a further effect: they lead to a transformation of some of the spirochetes into cystic bodies. Cyst formation of ectobiotic spirochetes is here reported for the first time. Starvation has a similar but less dramatic influence than antibiotics. The cysts contain protoplasmic cylinders in the periphery and sometimes larger central bodies. Production of dormant cystic forms may be a survival mechanism under hostile conditions.

Effect of coated plant extracts on Histomonas meleagridis and growth of bacteria in vitro.

Three coated plant extracts (RepaXol and two experimental formulations) were tested for their minimal lethal concentration for histomonads in vitro and the effect of those substances on the bacterial growth in the histomonadal culture. After 48 hr, RepaXol and experimental formulation B were lethal to histomonads at a concentration of 1.25 microl/ml. Experimental formulation C was lethal at a concentration of 2.5 microl/ml. All products also decreased the growth of bacteria at concentrations inhibiting the growth of histomonads.

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.

In vitro effect of essential oils from Cinnamomum aromaticum, Citrus limon and Allium sativum on two intestinal flagellates of poultry, Tetratrichomonas gallinarum and Histomonas meleagridis.

Essential oils may be effective preventive or curative treatments against several flagelated poultry parasites and may become primordial either to organic farms, or as more drugs are bannished. The anti-flagellate activity of essential oils obtained from fresh leaves of Cinnamomum aromaticum, Citrus limon pericarps and Allium sativum bulbs was investigated in vitro on Tetratrichomonas gallinarum and Histomonas meleagridis. On T. gallinarum, the minimal lethal concentration (MLC) at 24 hours was 0.25 microliter/ml for C. aromaticum oil, and 0.125 microliter/ml for C. limon and A. sativum oils. On H. meleagridis, MLC was 0.5 microliter/ml for C. aromaticum oil and 1 microliter/ml for C. limon and A. sativum oils at 24 and 48 hours. Moreover, no synergistic effects were evidenced in vitro. The essential oil constituents, based on their GC retention times have been also identified. The major component is trans-cinnamaldehyde (79%) for C. aromaticum; limonene for C. limon (71%) and diallyl tri- and disulfide (79%) for A. sativum. Even if concentration and protocol adaptations are required for successful in vivo treatments, it appears that these oils may be useful as chemotherapeutic agents against several poultry parasites.

In vitro activity of therapeutic drugs against Histomonas meleagridis (Smith, 1895).

Histomoniasis or blackhead is a life-threatening disease of turkeys that is caused by a flagellated protozoan, Histomonas meleagridis. The development of an assay to measure the sensitivity of drugs traditionally used against this parasite, as reputed to be effective against other protozoan parasites, is described. The in vitro minimum lethal concentrations (MLC), time for drug efficacy, and parasite viability after removal of residual drugs were determined. Three of the 10 tested drugs, fenbendazole, albendazole, and sulfadiazine, were found to be ineffective against H. meleagridis. Nifursol, the only compound still authorized as a feed additive in Europe, is an inhibiting agent but is not lethal in vitro. Roxarsone, an arsenical derivate similar to nitarsone (the only authorized drug in United States), is effective at high concentration (200 microg/mL) after a long exposure (48 h). The lethal activity of dimetridazole, metronidazole, ronidazole, tinidazole, and furazolidone in vitro was demonstrated. Dimetridazole (MLC = 25 microg/mL after 6 h of exposure), metronidazole (MLC = 50 microg/mL after 24 h), and furazolidone (MLC = 50 microg/mL after 24 h) are rapidly effective at low concentrations. These results confirm the effectiveness of dimetridazole, a drug that has been used in the treatment and prevention of blackhead. In May 2002 this compound was removed as feed additive in Europe.

Unusual diversity in alpha-amanitin sensitivity of RNA polymerases in trichomonads.

Previous studies in the parasitic protist Trichomonas vaginalis have revealed that protein coding genes are transcribed by an alpha-amanitin-resistant RNA polymerase (RNAP) II. To investigate whether this unusual property is a general characteristic of trichomonads, we addressed the physiology of RNA synthesis in lysolecithin-permeabilized cells. Unlike in T. vaginalis, RNAP II in Tritrichomonas foetus was highly sensitive to the inhibitor alpha-amanitin. On the other hand, RNAP III, identified by its sensitivity to the specific inhibitor tagetitoxin, was found to be resistant to alpha-amanitin in Tritrichomonas foetus, but showed a typical intermediate sensitivity in T. vaginalis. Extension of this study to an additional seven trichomonad species confirmed this genera specific pattern of alpha-amanitin sensitivity and highlighted an unusual diversity in RNAPs among trichomonads, a closely related group of unicellular eukaryotes.

The effectiveness of benzimidazole derivatives for the treatment and prevention of histomonosis (blackhead) in turkeys.

The benzimidazole derivatives, albendazole and fenbendazole were evaluated for their effectiveness in the treatment and prevention of histomonosis (blackhead) in turkeys. Histomonosis was produced in 5 week-old birds by placing them on broiler breeder litter known to be contaminated with Heterakis gallinae ova and the protozoan Histomonas meleagridis. In the first trial, at the onset of confirmed clinical disease, birds were treated orally with metronidazole, a compound known to be effective against Histomonas. Those receiving metronidazole had significantly greater mean body weight gains during the treatment period and the 2 weeks following treatment than untreated controls. Treated birds also had significantly lower caecal and liver lesion scores. These findings served to validate the method of disease reproduction and establish its suitability for testing the benzimidazoles. Similar trials were conducted to determine the therapeutic value of albendazole at 100.0 mg/kg of body weight and fenbendazole at 10.0 mg/kg body weight, administered orally twice a day for 5 consecutive days. Under these conditions, both drugs were found to be ineffective as treatments. A final trial was conducted to assess the prophylactic value of albendazole and fenbendazole administration. At the time of placement on contaminated litter, birds were medicated as previously described with the exception that treatment was continued for 14 consecutive days, the approximate incubation period for histomonosis. The trial was terminated on the 16th day. In the case of both albendazole and fenbendazole, treatment was associated with a significant increase in mean body weight gain and lower caecal and liver lesion scores. It is believed that the observed prophylactic effect may be attributed to the destruction of the transport vector e.g., Heterakis larvae, or to direct killing of the flagellated form of Histomonas which is normally found in the caecal lumen and is considered to be more sensitive to chemotherapeutic agents than the amoeboid form found in tissues.