Ascorbic acid potentiates the Giardia duodenalis growth inhibitory activity of pure Terminalia ferdinandiana Exell compounds.

Giardiasis, one of the most common causes of diarrhoeal disease, is caused by gastrointestinal protozoal parasites of the genus Giardia. Metronidazole is the most commonly used drug to treat giardiasis. However, metronidazole resistance is increasingly common, making the development of new anti-giardial drugs a high priority. A panel of 11 compounds previously identified in T. ferdinandiana fruit extracts were investigated for the ability to inhibit G. duodenalis proliferation. Eight of the 11 compounds inhibited the growth of all three G. duodenalis strains. 2,3-Dihydroxyphenyl B-D-glucopyranosiduronic acid (DPGA) was the most potent anti-giardial compound, with IC50 values as low as 126 µM (38 µg/mL). Notably, DPGA inhibited a metronidazole-resistant G. duodenalis strain with similar activity as determined for the metronidazole-sensitive strains. Furthermore, the activity of DPGA was greatly potentiated when it was tested in combination with ascorbic acid, to approximately 17 µM (5 µg/mL) for the metronidazole-sensitive G. duodenalis strains and 40 µM (12 mg/mL) for the resistant strain. The T. ferdinandiana tannins (gallic acid and chebulic acid) were moderate inhibitors of G. duodenalis growth when tested in combination with ascorbic acid, although they had only low levels of activity when tested alone. All of the tested compounds (and their combinations with ascorbic acid) displayed low toxic effects and all compounds are conformed to Lipinski's rules of 5 with few violations, indicating their potential as drug leads and chemotherapies for the treatment and prevention of giardiasis.

[CHARACTERISTICS OF PARASITIC PROTOZOA METABOLISM AND POSSIBILITIES OF ANTIPROTOZOA DRUG DEVELOPMENT (REVIEW)].

One of the most poorly studied areas of protozoology is metabolic processes of parasitic protozoa. Study of the biochemistry of parasites required for the development of effective chemotherapy of protozoal diseases. Some amitochondrial parasites of humans, such as Giardia intestinalis, Entamoeba histolytica, Trichomonas sp., living in an environment with low oxygen content, have specialized cellular organelles-hydrogenosomes (like mitochondria provide cells with simple energy). The study of the functioning of these organelles allows us to consider them as targets for the development of аntiprotozoal drugs. The target for chemotherapy in the treatment of trypanosomiasis can be processes related to the characteristics of the glycolytic pathway or a decrease in the level of energy substrate, such as glucose. This leads to a rapid decrease in ATP levels in the cell of the parasite, an overall loss of mobility and disappearance of trypanosomes from the bloodstream of the infected host. Also, glucose transporters located in the membrane of the parasite can be targets for drugs.

Synergistic effects of fenbendazole and metronidazole against Giardia muris in Swiss mice naturally infected.

In this study were proposed different protocols for the treatment of mice naturally infected with Giardia muris. Male Swiss mice were divided into seven groups, with five animals each, in a blind, controlled, randomized by drawing lots and once-repeated experiment. Parasite detection and cure control were performed using the Faust method and search by trophozoites in the intestinal mucosa. Clinical parameters (weight, water and feed consumption, elimination of excreta, aspect of the fur and feces) were also evaluated. All animals were treated with metronidazole (M), fenbendazole (F), and probiotics (P), administered intragastrically, during 7 days. M1, FM1, and F1 groups were treated 1×/day; M3, FM3, and PM3 groups 3×/day; and ST (control group) received only water. After the 5th and 7th days of treatment, the animals in FM1/FM3 and PM3/M3 groups presented, respectively, negative results and remained negative in the following 10 days. Animals in F1 group consumed less water (p = 0.00010) compared with FM1/FM3/PM3. The animals in M1 group compared with FM3/M3, F1 compared with M3, and ST compared with FM1/FM3/M3/PM3 consumed a larger amount of feed (p = 0.00001). The animals in F1 group compared with FM3/M1/M3/PM3, FM1 compared with FM3, and ST compared with FM3/M1/M3/PM3 eliminated lower volume of excreta (p = 0.00001). The results show that the association between F and M potentiates the effects, indicating a synergistic action of these two drugs, and FM1 is the best protocol due to early negativity in the animals, lower concentrations of the drugs, lower risk of toxicity and stress, and less alterations in clinical parameters.

Anti-giardial therapeutic potential of dichloromethane extracts of Zingiber officinale and Curcuma longa in vitro and in vivo.

Giardiosis is one of the common parasitic diarrhoea in humans, especially in children, worldwide. Many drugs are used for its treatment, but there is evidence of drug resistance, insufficient efficacy and unpleasant side effects. Natural products are good candidates for discovering more effective anti-giardial compounds. This study evaluated the activity of extracts of Zingiber officinale (ginger) and Curcuma longa (curcumin) against Giardia lamblia in vitro and in vivo. Giardia cyst suspension was prepared from children faecal specimens. For the in vitro experiment, 1, 10 and 50 mg/mL dichloromethane extracts of ginger and curcumin separately were incubated with Giardia cysts for 5, 10, 30 and 60 min. The viability was distinguished by 0.1 % eosin and a haemocytometer. For the in vivo experiments, Balb/c mice were infected with Giardia cyst suspension containing 10,000 cysts/mL. Infected mice were administered 10 and 20 mg kg(-1) day(-1) ginger and curcumin extracts separately for 7 days post-infection. The effectiveness of the extracts was evaluated by faecal cyst and intestinal trophozoite counts and histopathological examination of the small intestine. In vitro ginger extract had a higher significant effect on cyst viability than curcumin, in a dose- and time-dependent manner. In vivo ginger (more effective) and curcumin extracts significantly treated infected mice, and this was evidenced by the faecal cyst and intestinal trophozoite counts reduction, in addition to evident improvement of intestinal mucosal damages induced by Giardia infection. Z. officinale and C. longa extracts may represent effective and natural therapeutic alternatives with low side effects and without drug resistance in the treatment of giardiosis.

Ethanol and isopropanol in concentrations present in hand sanitizers sharply reduce excystation of Giardia and Entamoeba and eliminate oral infectivity of Giardia cysts in gerbils.

Enteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls of Giardia and Entamoeba have a single layer composed of fibrils of ß-1,3-linked GalNAc and ß-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts of G. duodenalis and 90 to 100% of cysts of Entamoeba invadens (a nonpathogenic model for E. histolytica), as shown by nuclear labeling with propidium iodide and failure to excyst in vitro. Alcohols with or without drying collapsed the cyst walls of Giardia but did not collapse the cyst walls of Entamoeba. To validate the in vitro results, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000 G. duodenalis cysts, while a commercial hand sanitizer (Purell) killed E. invadens cysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission of Giardia and Entamoeba.

Albendazole and its derivative JVG9 induce encystation on Giardia intestinalis trophozoites.

In the present study, we evaluated the effect of an albendazole (ABZ) derivative JVG9 on cultured Giardia intestinalis. To assess the JVG9 effects, we evaluated the tubulin cytoskeleton by confocal microscopy, and we found that the characteristic staining was modified. The scanning electron microscopy images revealed extremely damaged trophozoites and cyst-like cells. The confocal images revealed that this drug triggered the expression of cyst wall protein 1 and encystation. We also found that at low doses, AL triggered the encystation process too.

Performic acid for advanced wastewater disinfection.

The disinfection efficiency of performic acid (PFA) against various microbial contaminants has been studied in municipal secondary effluent. The study demonstrated that PFA provides rapid, efficient and safe disinfection, degrading both bacteria and viruses even at low doses. The resistance order starting from the most resistant microorganism is as follows: MS2-coliphages > DNA-coliphages > enterococci and Escherichia coli. PFA is also efficient in the elimination of Salmonella spp., Clostridium perfringens spores and Giardia cysts. The results showed that a PFA dose as low as 0.5-1 mg L(-1) with contact time of 10 min was efficient in achieving and maintaining for 72 h the disinfection level required for unrestricted agricultural water reuse (≤3 log units for faecal coliforms). However, the optimal dose will depend on the quality of wastewater. Regarding the formation of by-products during disinfection with PFA, very low amounts of hydrogen peroxide and organic per-acids were observed; active oxygen was not detected. The amounts of adsorbable organically bound halogens (AOX) compounds formed were significantly lower compared to the amounts generated during chlorine disinfection. This chlorine-free solution enables compliance with microbiological criteria for various water reuse applications and is already on the market for advanced disinfection.

Programmed cell death in Giardia.

Programmed cell death (PCD) has been observed in many unicellular eukaryotes; however, in very few cases have the pathways been described. Recently the early divergent amitochondrial eukaryote Giardia has been included in this group. In this paper we investigate the processes of PCD in Giardia. We performed a bioinformatics survey of Giardia genomes to identify genes associated with PCD alongside traditional methods for studying apoptosis and autophagy. Analysis of Giardia genomes failed to highlight any genes involved in apoptotic-like PCD; however, we were able to induce apoptotic-like morphological changes in response to oxidative stress (H2O2) and drugs (metronidazole). In addition we did not detect caspase activity in induced cells. Interestingly, we did observe changes resembling autophagy when cells were starved (staining with MDC) and genome analysis revealed some key genes associated with autophagy such as TOR, ATG1 and ATG 16. In organisms such as Trichomonas vaginalis, Entamoeba histolytica and Blastocystis similar observations have been made but no genes have been identified. We propose that Giardia possess a pathway of autophagy and a form of apoptosis very different from the classical known mechanism; this may represent an early form of programmed cell death.

Synthesis of benzologues of Nitazoxanide and Tizoxanide: a comparative study of their in vitro broad-spectrum antiprotozoal activity.

We have synthesized two new benzologues of Nitazoxanide (NIT) and Tizoxanide (TIZ), using a short synthetic route. Both compounds were tested in vitro against six protozoa (Giardia intestinalis, Trichomonas vaginalis, Entamoeba histolytica, Plasmodium berghei, Leishmania mexicana and Trypanosoma cruzi). Compound 1 (benzologue of NIT) showed broad antiprotozoal effect against all parasites tested, showing IC(50)'s<5 µM. This compound was five-times more active than NIT, and 18-times more potent than metronidazole against G. intestinalis. It was 10-times more active than pentamidine against L. mexicana, and it was sevenfold more potent than benznidazole versus T. cruzi. This compound could be considered as a new broad spectrum antiprotozoal agent.

An image-based Pathogen Box screen identifies new compounds with anti-Giardia activity and highlights the importance of assay choice in phenotypic drug discovery.

Giardia duodenalis, the most prevalent human intestinal parasite causes the disease, giardiasis. On an annual basis G. duodenalis infects ~1 billion people, of which ~280 million develop symptomatic disease. Giardiasis can be severe and chronic, causing malnutrition, stunted growth and poor cognitive development in children. Current treatment options rely on drugs with declining efficacy and side-effects. To improve the health and well-being of millions of people world-wide, new anti-Giardia drugs with different modes of action to currently used drugs are required. The Medicines for Malaria Venture's Pathogen Box, a collection of bio-active compounds specifically chosen to stimulate infectious disease drug discovery, represents an opportunity for the discovery of new anti-Giardia agents. While the anti-Giardia activity of Pathogen Box compounds has been reported, this work failed to identify known anti-Giardia controls within the compound set. It also reported the activity of compounds previously screened and shown to be inactive by others, suggesting data may be inaccurate. Given these concerns the anti-Giardia activity of Pathogen Box compounds was re-assessed in the current study. Data from this work identified thirteen compounds with anti-Giardia IC50 values ≤2 µM. Five of these compounds were reference compounds (marketed drugs with known anti-microbial activity), or analogues of compounds with previously described anti-Giardia activity. However, eight, including MMV676358 and MMV028694, which demonstrated potent sub-µM IC50s against assemblage A, B and metronidazole resistant parasites (0.3 µM and 0.9 µM respectively), may represent new leads for future drug development. Interestingly, only four of these compounds were identified in the previously reported Pathogen Box screen highlighting the importance of assay selection and design when assessing compounds for activity against infectious agents.

Comparative Analysis of Antigiardial Potential of Heat Inactivated and Probiotic Protein of Probiotic Lactobacillus rhamnosus GG in Murine Giardiasis.

The present study was designed to envisage the antigiardial efficacy of killed probiotic and probiotic protein (PP) of Lactobacillus rhamnosus GG in murine giardiasis. Experimentally, it was observed that animal administered either with probiotic protein emulsified with adjuvant (PP(E) + Giardia) or killed probiotic (killed probiotic (i/p) + Giardia) had significantly reduced Giardia cycle with respect to observed severity and duration of giardiasis compared with Giardia-infected mice. Further, it was found that animals belonging to PP(E) + Giardia and killed probiotic (i/p) + Giardia had significantly high levels of antigiardial IgA antibody and nitric oxide both in serum and in intestinal fluid compared with Giardia-infected and counter control mice. Histopathologyically, also animals belonging to PP(E) + Giardia and killed probiotic (i/p) + Giardia animals had intact mucosal epithelium lining, basal crypts, and normal villi along with increased goblet cells compared with severe microvillus atrophy, vacuolated epithelial cells, and ileitis in Giardia-infected mice. This is the first-ever study to demonstrate that prior administration of either killed probiotics or probiotic protein of effective probiotic reduced both the severity and the duration of giardiasis mainly by modulating the gut microbiome and morphology along with mucosal immunity, but animals belonging to PP(E) + Giardia had better response than killed probiotic (i/p) + Giardia suggesting that probiotic components do have adjuvant potential and may be used as the vaccine candidate for gastrointestinal diseases.

Anti-Giardia Drug Discovery: Current Status and Gut Feelings.

Giardia parasites are ubiquitous protozoans of global importance that impact a wide range of animals including humans. They are the most common enteric pathogen of cats and dogs in developed countries and infect ∼1 billion people worldwide. While Giardia infections can be asymptomatic, they often result in severe and chronic diseases. There is also mounting evidence that they are linked to postinfection disorders. Despite growing evidence of the widespread morbidity associated with Giardia infections, current treatment options are limited to compound classes with broad antimicrobial activity. Frontline anti-Giardia drugs are also associated with increasing drug resistance and treatment failures. To improve the health and well-being of millions, new selective anti-Giardia drugs are needed alongside improved health education initiatives. Here we discuss current treatment options together with recent advances and gaps in drug discovery. We also propose criteria to guide the discovery of new anti-Giardia compounds.

Insights about the structure of farnesyl diphosphate synthase (FPPS) and the activity of bisphosphonates on the proliferation and ultrastructure of Leishmania and Giardia.

BACKGROUND: The enzyme farnesyl diphosphate synthase (FPPS) is positioned in the intersection of different sterol biosynthesis pathways such as those producing isoprenoids, dolichols and ergosterol. FPPS is ubiquitous in eukaryotes and is inhibited by nitrogen-containing bisphosphonates (N-BP). N-BP activity and the mechanisms of cell death as well as damage to the ultrastructure due to N-BP has not yet been investigated in Leishmania infantum and Giardia. Thus, we evaluated the effect of N-BP on cell viability and ultrastructure and then performed structural modelling and phylogenetic analysis on the FPPS enzymes of Leishmania and Giardia. METHODS: We performed multiple sequence alignment with MAFFT, phylogenetic analysis with MEGA7, and 3D structural modelling for FPPS with Modeller 9.18 and on I-Tasser server. We performed concentration curves with N-BP in Leishmania promastigotes and Giardia trophozoites to estimate the IC50via the MTS/PMS viability method. The ultrastructure was evaluated by transmission electron microscopy, and the mechanism of cell death by flow cytometry. RESULTS: The nitrogen-containing bisphosphonate risedronate had stronger anti-proliferative activity in Leishmania compared to other N-BPs with an IC50 of 13.8 µM, followed by ibandronate and alendronate with IC50 values of 85.1 µM and 112.2 µM, respectively. The effect of N-BPs was much lower on trophozoites of Giardia than Leishmania (IC50 of 311 µM for risedronate). Giardia treated with N-BP displayed concentric membranes around the nucleus and nuclear pyknosis. Leishmania had mitochondrial swelling, myelin figures, double membranes, and plasma membrane blebbing. The same population labelled with annexin-V and 7-AAD had a loss of membrane potential (TMRE), indicative of apoptosis. Multiple sequence alignments and structural alignments of FPPS proteins showed that Giardia and Leishmania FPPS display low amino acid identity but possess the conserved aspartate-rich motifs. CONCLUSIONS: Giardia and Leishmania FPPS enzymes are phylogenetically distant but display conserved protein signatures. The N-BPs effect on FPPS was more pronounced in Leishmania than Giardia. This might be due to general differences in metabolism and differences in the FPPS catalytic site.

Metronidazole-sensitive organisms in children with severe acute malnutrition: an evaluation of the indication for empiric metronidazole treatment.

OBJECTIVES: Children with severe acute malnutrition (SAM) are treated with empiric amoxicillin or penicillin and gentamicin because of the high risk of severe infections. Experts have suggested, based on available evidence, adding metronidazole to cover anaerobic bacteraemia and diarrhoea caused by Giardia duodenalis or Clostridium difficile. The objective of this study was to assess the importance of these infections in children with SAM. METHODS: Children from 6 months to 15 years with SAM were enrolled and followed clinically. Aerobic and, when patient weight permitted, anaerobic blood cultures were done using Bactec® system, and isolates identified with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Stool samples were tested for C. difficile, G. duodenalis and Entamoeba histolytica by PCR. RESULTS: A total of 334 children were enrolled and 174 out of 331 (53%) for which data on this was available had diarrhoea. Of 273 patients tested by blood culture, 11 had bacteraemia (4.0%, 95% CI 2.3-7.1%) but none with strict anaerobic bacteria (0/153, 95% CI 0-2.4%). There was no difference in the prevalence of C. difficile between children with (5/128, 4%) and without (7/87, 8%) diarrhoea (OR 0.47, 95% CI 0.14-1.53), and no difference in the prevalence of Giardia between these groups (78/138, 60% vs. 46/87, 53%; OR 1.34, 95% CI 0.77-2.32). Children with C. difficile had higher mortality than those without this infection (3/11, 27%, vs. 7/186, 4%; OR 43, 95% CI 3.9-483). CONCLUSION: Our results do not provide support for empiric metronidazole to cover for anaerobic bacteraemia. Trials evaluating the effect of empiric treatment and its effect on G. duodenalis and C. difficile are warranted.

Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases).

Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.

Encystation and expression of cyst antigens by Giardia lamblia in vitro.

The cyst form of Giardia lamblia is responsible for transmission of giardiasis, a common waterborne intestinal disease. In these studies, encystation of Giardia lamblia in vitro was demonstrated by morphologic, immunologic, and biochemical criteria. In the suckling mouse model, the jejunum was shown to be a major site of encystation of the parasite. Small intestinal factors were therefore tested as stimuli of encystation. An antiserum that reacted with cysts, but not with cultured trophozoites was raised in rabbits and used as a sensitive probe for differentiation in vitro. Cultured trophozoites that were exposed to bile salts showed a more than 20-fold increase in the number of oval, refractile cells that reacted strongly with anticyst antibodies, and in the expression of major cyst antigens. Exposure to primary bile salts resulted in higher levels of encystation than exposure to secondary bile salts. These studies will aid in understanding the differentiation of an important protozoan pathogen.

Theoretical considerations and modeling of chemical inactivation of microorganisms: inactivation of Giardia Cysts by free chlorine.

Chemical inactivation of microorganisms is a common process widely employed in many fields such as in treatment of water, preservation in food industry and antimicrobial treatments in healthcare. For economy of applications and efficiency of treatment establishment the minimum dosage of breakpoint in the chemical application becomes essential. Even though experimental investigations have been extensive, theoretical understanding of such processes are demanding. Commonly employed theoretical analyses for the inactivation of microorganisms and depletion of chemicals include kinetics expressing the rates of depletion of chemical and microorganisms. The terms chemical demand (x) and specific disinfectant demand (alpha) are often used in theoretical modeling of inactivation. The value of specific disinfectant demand (alpha) has always been assumed to be a constant in these models. Intracellular concentration built up within the cells of the microorganisms during inactivation could lead to possible weakening effects of microorganisms thereby requiring lower doses as disinfection proceeds makes the assumption of constant alpha inaccurate. Model equations are formulated based on these observations co-relating the parameters alpha and x with a progressive inactivation (N/N(0)). The chemical concentration (C) is also presented in terms of the inactivation time (t) and the survival ratio (N/N(0)) for given pH and temperature conditions. The model is examined using experimentally verified Ct data of Giardia Cysts/chlorine system. The respective values of x for different survival ratios were evaluated from the data using MatLab software. Proposed model correlating for the disinfectant demand (x) with the survival ratio (N/N(0)) fits satisfactorily with those evaluated from data. The rate constants for different pH and temperature conditions are evaluated which showed compatibility with the Arrhenius model. The dependence of frequency factors with pH indicated compatibility with accepted models. The Ct values regenerated with the kinetic data shows a very accurate fit with published data.

Effect of vinegar on the viability of Giardia duodenalis cysts.

The inactivation of Giardia duodenalis cysts by vinegar was investigated. Experiments were carried out in 100 ml volume of vinegar (acetic acid 4%), undiluted or diluted in distilled water in ratios of 1:1, 1:15.6, and 1:62.5 (vol/vol), which were inoculated with 5x10(5) cysts obtained from human feces. Experiments were performed at room temperature (21+/-1 degrees C) and at 4 degrees C. After contact times of 1.5 min, 10, 30, and 60 min, the cysts were recovered from the treatment fluid and subjected to an in vitro excystation assay to determine their viability. The relative viability, which was calculated in relation to controls (maximum excystation percentage), was significantly affected (p<0.1) by the vinegar concentration, contact time, and temperature. At 21+/-1 degrees C, no cysts remained viable after being treated with undiluted vinegar for 60 min, while the treatment with 1:1, 1:15.6, and 1:62.5 vinegar-water mixtures decreased the relative viability to 1.8%, 19.4%, and 56.4%, respectively. The relative viability after corresponding treatments at 4 degrees C also decreased, but 23.6% to 48.8% remained viable after 60 min, and thus complete inactivation was not obtained with any treatment at that temperature.

Treatment of naturally occurring, asymptomatic Giardia sp. in dogs with Drontal Plus flavour tablets.

The administration of three consecutive daily doses of the recommended 1x dose of Drontal Plus flavour tablets (Bayer) was examined for its effect on Giardia sp. cyst-shedding in 7 treated and 7 untreated random-source dogs. Dogs were treated on study days 0, 1 and 2. Cysts were quantified using direct immunofluorescent labelling on days -7, -5, -3 and -2, and daily from day 1 through 11. Three treated dogs never shed cysts again during the study, one shed again only on day 4, and the remaining three dogs started to shed again on days 8, 9 and 11. The mean numbers of cysts per gramme in the faeces of the treated dogs were significantly reduced (t-tests using log(10)(counts)) on days 1 and 2 (geometric means: controls = 447,000; treated = 1,050; p = 0.004) and days 3 to 8 (geometric means: controls = 23,400; treated 5.0; p < 0.001). Four controls that had been consistently positive, changed to negative status on day 11, and thus, on the final day of the trial, there were only three positive control and three positive treated dogs. Three consecutive days of treatment with Drontal Plus flavour tablets halted Giardia sp. cyst shedding by dogs. But starting six days post third treatment, some of the dogs started shedding cysts again. Since the prepatent period of Giardia sp. can be as short as 4 days, shedding of Giardia sp. cysts 6 days after treatment could be caused by a reinfection.

Synergistic effect of febantel and pyrantel embonate in elimination of Giardia in a gerbil model.

The objectives of the study were to determine the optimal dose of febantel, pyrantel embonate and a combination of febantel/pyrantel embonate required to effectively treat Giardia in a gerbil model and to determine if there is a synergistic effect with the two drugs. SPF gerbils were infected by oral inoculation with 105 Giardia duodenalis trophozoites (day 0). On days 5 to 7, animals (n = 6) were treated once daily via oral gavage with febantel, pyrantel embonate, febantel and pyrantel embonate, metronidazole or placebo. Gerbils were euthanised 24 hours after last treatment and duodenal trophozoites were enumerated on a haemocytometer to obtain a concentration of trophozoites/ cm of gut. Febantel alone, effectively eliminated Giardia trophozoites at 160 and 80 mg/kg. Pyrantel embonate did not eliminate Giardia from the animals but significantly reduced parasite counts at all dosages. Febantel combined with pyrantel embonate effectively eliminated Giardia trophozoites at 160, 80 and 40 mg/kg. Metronidazole did not eliminate Giardia trophozoites from the gut. All placebo-treated animals were heavily infected with Giardia trophozoites. It can be concluded that febantel is more effective in elimination of Giardia infections when combined with pyrantel embonate compared to the agents used alone.