Efficacy of oryzalin and associated histological changes in Cryptosporidium-infected neonatal rats.

This paper reports the anti-cryptosporidial effects of, and concomitant amelioration of the histological changes in the gut of neonatal rats with intestinal cryptosporidiosis treated with the dinitroaniline, oryzalin. The ED50 was determined to be 7 mg/kg using twice daily doses administered for 3 consecutive days. A maximum inhibition of 85.5% was achieved at 25 mg/kg and this inhibition remained constant despite increasing the oryzalin dose to 200 mg/kg. Cryptosporidiosis significantly decreased the intestinal villus/crypt (VC) ratio by approximately 50% (duodenum = 2.3, jejunum = 2.5 and ileum = 1.7) when compared to uninfected untreated controls (duodenum = 4.3, jejunum = 5.9 and ileum = 4.5). Treatment with oryzalin doubled the VC ratio in the duodenum, jejunum and ileum following doses of 5 mg, 50 mg and 200 mg/kg respectively. Oryzalin concentrations in the small intestine contents and plasma were determined, using HPLC, at 0.5, 1 and 2 h after dosing. The much greater dose required to return VC ratios to normal in the ileum (200 mg/kg) compared to the duodenum (6.25 mg/kg) appeared to reflect the decreased concentration of the drug in the distal small intestine. Concentrations of oryzalin equivalent to the in vitro IC50 were maintained for 2 h in the first half of the small intestine following a single dose of 100 mg/kg.

Plasmodium: assessment of the antimalarial potential of trifluralin and related compounds using a rat model of malaria, Rattus norvegicus.

A rodent model of malaria, Plasmodium berghei was used to assess the antimalarial potential of dinitroaniline herbicides. Trifluralin, pendimethalin, oryzalin, and benfluralin were all active against P. berghei in vitro at, or close to, submicromolar concentrations, with a rank order of potency similar to that against other protozoa. The dinitroanilines did not elicit a cytotoxic effect against a mammalian cell line at concentrations 100-fold higher than those for activity against P. berghei. Neither trifluralin nor oryzalin exhibited any antimalarial activity in vivo after oral administration at the maximum dose tolerated by the host. In a pharmacokinetic study, it was found that the lack of in vivo antimalarial activity was due to poor absorption. Other DNs which have better absorption characteristics than either trifluralin or oryzalin may offer more scope for antimalarial activity in vivo.

Expression of Giardia duodenalis beta-tubulin as a soluble protein in Escherichia coli.

The beta-tubulin gene of the parasitic protozoan Giardia duodenalis has been expressed for the first time using a novel and direct method. The protein was expressed in both soluble and insoluble forms in an Escherichia coli-based expression system. The level of expression was found to be affected by several variables including the incubation temperature, length of time for which expression was carried out, and the E. coli culture volume. The protein expression system contributed no additional amino acids to the final fusion protein and the polyhistidine fusion sequence was easily removed from the beta-tubulin protein using a specific enterokinase enzyme. The expression system also provided a means of preparing a soluble protein and purifying it by a relatively straightforward affinity chromatography method to give a very high level of protein purity. This makes the protein suitable for a number of applications for characterization including beta-tubulin antibody assays, alpha-/beta-tubulin-binding regions, and beta-tubulin folding intermediates.

Towards the eradication of hookworm in an isolated Australian community.

Hookworm (Ancylostoma duodenale) and other enteric parasites such as Giardia and Hymenolepis are common in Aboriginal communities in northem Australia, and their presence is associated with iron deficiency, anaemia, and failure to thrive. We report the outcome of a sustained, community-based control programme that used regular albendazole in one isolated community. Whereas hookworm has been effectively controlled by the programme, no sustained effect on the prevalence of Giardia and Hymenolepis was seen; the control of these parasites will depend on improvements in health education. This programme might serve as a model for community-based or population-based drug treatment programmes elsewhere.

The binding and distribution of albendazole and its principal metabolites in Giardia duodenalis.

Trophozoites of the protozoan parasite Giardia duodenalis were exposed to various albendazole concentrations for 4 h, washed, fixed and incubated with antibodies raised against albendazole and its two major metabolites albendazole sulphoxide and albendazole sulphone. Tubulin antibodies were also used. A peroxidase- or FITC-conjugated secondary antibody was used to detect the primary antibody with transmission electron microscopy or confocal laser scanning microscopy, respectively. Albendazole, a benzimidazole compound, was detected in the mid-dorsal region of trophozoites, albendazole sulphoxide in the posterior-dorsal region and albendazole sulphone in clusters above the median bodies. Tubulin was recognised in the ventral disk. This is the first indication that G. duodenalis may be capable of metabolising albendazole and the potential path of the metabolised drug traced within the trophozoite. Fluorescence measurements revealed that albendazole sulphoxide binding decreased and albendazole sulphone binding increased with exposure of the trophozoites to increasing albendazole concentration. This indicates that if albendazole was being metabolised by trophozoites, it occurred to a greater extent following exposure to higher albendazole concentrations.

A comparison of the effects of a benzimidazole and the dinitroanilines against Leishmania infantum.

Leishmania infantum promastigotes and amastigotes were axenically cultured and exposed to the known tubulin binding compounds, the dinitroanilines, trifluralin, benfluralin, pendimethalin, oryzalin and the precursor of the dinitroanilines, chloralin, as well as isomers of chloralin and trifluralin and to the benzimidazole, albendazole. Drug induced inhibition was observed using [3H]thymidine uptake compared with untreated controls. In vitro analysis demonstrated a significant difference in the activity of five of the seven dinitroanilines between both life cycle stages of L. infantum. The amastigotes were 20-times more sensitive to chloralin and its isomer than to the dinitroanilines whereas the promastigotes were similar in sensitivity to the dinitroanilines and to chloralin and its isomer. This interesting finding suggests that the dinitroaniline precursors may have different target sites in the amastigotes to those within the promastigotes. Additionally, both chloralin and its isomer, and to a lesser extent benfluralin, caused a substantial stimulation of thymidine incorporation (up to 50%) at low concentrations. Dose response analysis suggests that the dinitroanilines may have more than one mode of action against L. infantum amastigotes and promastigotes. The inhibitory effects of the dinitroanilines against L. infantum vary from previous findings using the dinitroanilines against other Leishmania spp. The 348 base pair DNA sequence coding for beta-tubulin from amino acid residues 132 to 248 was obtained for L. infantum and used to compare the in vivo efficacy of albendazole with predicted activity based on beta-tubulin sequences of known benzimidazole sensitive protozoa. The use of beta-tubulin sequence as a predictive model of benzimidazole activity is discussed with particular reference to L. infantum.

A comparison of the effects of two dinitroanilines against Cryptosporidium parvum in vitro and in vivo in neonatal mice and rats.

The effects of two dinitroanilines, oryzalin and trifluralin, were compared against Cryptosporidium parvum, in vitro using HCT-8 cells and in vivo using neonatal Swiss ARC mice and Wistar neonatal rats. In vitro, oryzalin and trifluralin exhibited IC(50) values (concentration necessary to cause a 50% inhibition) of 750 and 800 nM, respectively. A viability assay showed that neither compound produced a cytotoxic effect on the host cells at concentrations as high as 1 microM. The in vivo component of this study consisted of inoculation of neonatal mice and neonatal rats with 10(5) viable oocysts of C. parvum per animal and the subsequent treatment of this infection with trifluralin and oryzalin administered via gastric intubation. At doses of 100 mg kg(-1) body weight administered twice daily for 3 consecutive days, trifluralin had no statistically significant effect on the number of oocysts recovered from the gut of either rats or mice compared with controls, whereas at the same concentration, oryzalin caused 90 and 79% inhibition of oocysts recovered from mice and rats, respectively.

Assessment of drugs against Cryptosporidium parvum using a simple in vitro screening method.

A rapid semi-quantitative screening method was devised for assessing the anticryptosporidial and cytotoxic effects of putative chemotherapeutic compounds. The method is suitable as an initial rapid screening procedure from which compounds demonstrating anticryptosporidial activity can be identified for further analysis. It has the advantages of speed, low cost and concurrent assessment of anticryptosporidial and cytotoxic effects and allows accurate determination of minimum lethal concentrations. Of the 71 compounds screened, six completely inhibited cryptosporidial growth at 1 microM (monensin, salinomycin, alborixin, lasalocid, trifluralin and nicarbazin) and a further eight showed significant anticryptosporidial activity at 1 or 20 microM (halquinol, bleomycin, suramin, mitomycin, doxycycline hydrochloride, toltrazuril, chloroquine phosphate and teniposide). Twelve compounds were found to have some degree of cytotoxicity at 1 microM and a further 12 at 20 microM.

PCR-based DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer.

The potential for the non-coding intergenic rDNA spacer (IGS) to DNA fingerprint Giardia duodenalis isolates was investigated. Conserved PCR primers, specific for the flanking large and small rDNA genes, were used to amplify the IGS from 52 in vitro-cultured Giardia isolates. Four distinct IGS-PCR size groups (1.35-1.6 kb) were observed, which correlated closely with the major genetic assemblages established previously for the same isolates using isoenzyme analysis. IGS-PCR size groups A (1.42 kb) C (1.4 kb) and D (1.35 kb) corresponded to isoenzyme assemblage A, and IGS-PCR group B (1.6 kb) to isoenzyme assemblage B. Amplified products from IGS-PCR size groups A and B, which contained 50/52 isolates, were subsequently digested with 8 different restriction enzymes and their profiles compared. Analysis separated isolates within each IGS-PCR size group into 2 distinct clusters which correlated almost exactly with the same genetic groups established previously using isoenzyme electrophoresis. Within each cluster, both methods exhibited a similar capacity to distinguish between Giardia genotypes although they established different genetic relationships between individual isolates. Much of the variability associated with the IGS was attributed to isolates harbouring multiple IGS-sequence types. Restriction analysis of IGS-PCR products amplified from cloned and parent lines of a human isolate BAH 39, which contains multiple IGS variants, showed that trophozoite populations are homogeneous with respect to the types of IGS-variants they maintain. Furthermore, in vitro culture of the cloned isolate BAH39c9 over a 6-year period also failed to reveal variation in IGS-PCR digestion profiles. These results suggest that IGS-PCR RFLP profiles are inherently stable. IGS-PCR analysis was successfully applied to 11 Giardia cyst samples highlighting the potential for this approach to genotype Giardia isolates without the need for in vitro culture.

Efficacy of albendazole against Giardia and hookworm in a remote Aboriginal community in the north of Western Australia.

The parasitological, clinical efficacy and tolerability of albendazole in the treatment for both giardiasis and hookworm infection in a remote Aboriginal population was investigated. Albendazole at a dose rate of 400 mg daily for 5 days was highly effective in reducing hookworm egg numbers and both Giardia antigen and cysts. The 36.6% prevalence of Giardia prior to treatment fell to 12% between days 6 and 9, 15% for days 10-17 and rose to 28% between days 18 and 30. Tolerability and clinical efficacy were excellent. The effect of albendazole on hookworm was longer lasting than that on Giardia, reducing percent infection from over 76-2% on days 6-9 and zero by day 18-30 despite conditions highly conducive to rapid re-infection. We conclude that albendazole is highly efficacious against both parasites when used as described but that long term community benefit may require additional education programmes to avoid re-infection with Giardia although treatment strategies would seem appropriate for hookworm.

Effects of tiletamine/zolazepam premedication on propofol anaesthesia in dogs.

The cardiovascular and pulmonary effects of tiletamine/zolazepam, propofol and tiletamine/zolazepam plus propofol were studied in five mongrel dogs. A cannula inserted into a raised carotid artery was used to measure mean arterial pressure (MAP) and heart rate continuously and to collect arterial blood for the determination of pH, PO2, PCO2, bicarbonate and base balance. Respiratory frequency and rectal temperature were also recorded. In the two propofol groups premedication had no significant effect on the time to rejection of an endotracheal tube and the return to sternal recumbency. The MAP and heart rate increased after tiletamine/zolazepam alone and after tiletamine/zolazepam plus propofol, although propofol alone reduced MAP and transiently increased heart rate. Respiratory frequency decreased transiently in both propofol groups in association with a significant increase in PaCO2 and decrease in PaO2. The most notable change was the hypoxaemia in the tiletamine/zolazepam plus propofol group in which the PaO2 was reduced. In all the dogs given tiletamine/zolazepam alone undesirable side effects were observed, effects which also occurred during the recovery of the dogs given tiletamine/zolazepam plus propofol.

Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality.

A polymerase chain reaction-based method for genotyping Giardia duodenalis isolates using a polymorphic region near the 5' end of the small subunit ribosomal (SSU) RNA gene is described. Analysis was performed using Giardia cysts purified directly from feces. Isolates were collected from humans and dogs living in isolated Aboriginal communities where Giardia infections are highly endemic. This is the first report of the genetic characterization of Giardia from dogs and humans living in the same locality. Comparison of the SSU-rRNA sequences from 13 human and 9 dog isolates revealed 4 different genetic groups. Groups 1 and 2 contained all of the human isolates, whereas groups 3 and 4 consisted entirely of Giardia samples recovered from dogs. One dog sample contained templates from both groups 2 and 3. These results suggest that zoonotic transmission of Giardia infections between humans and dogs does not occur frequently in these communities. The dog-associated SSU-rRNA sequences have not been reported before, suggesting a new G. duodenalis subgroup. A genetic basis for the differences observed between the groups was supported by sequence analysis of 9 in vitro cultured isolates that were placed into the same genetic groups established by enzyme electrophoresis.

Failure of pyrantel in treatment of human hookworm infections (Ancylostoma duodenale) in the Kimberley region of north west Australia.

A survey of 108 individuals from a coastal Aboriginal community in north Western Australia revealed that two species of gastrointestinal protozoan parasites (Giardia duodenalis--39.8%, Entamoeba coli--40.7%) and five gastrointestinal helminths (Hymenolepis nana--54.6%, Hookworm [Ancylostoma duodenale]--30.6%, Enterobius vermicularis--6.5%, Trichuris trichiura--2.8%, Strongyloides stercoralis 1.9%) were present. A total of 29 individuals infected with hookworm were offered treatment with either pyrantel pamoate at a single dose rate of 10 mg/kg body weight or albendazole (single 400 mg dose). Seven days after treatment stool samples were examined. Pyrantel had no significant effect against hookworm. In contrast, albendazole cleared hookworm infections completely and reduced the prevalence of Giardia. The former result suggests that locally A. duodenale is resistant to pyrantel and despite its relatively low cost and wide availability, should not be considered a drug of choice at this dose rate in the treatment of hookworm infections (A. duodenale) in endemic regions.

Cyclosporine a immunosuppression in sheep with response enhancement by concomitant ketoconazole.

1. The pharmacokinetics of a single dose of Cyclosporine A (CsA) administered to sheep by intravenous (i.v.) route were examined. 2. Concomitant administration of ketoconazole was found to increase the area under the blood CsA concentration-time curve (AUC) and was effective when administered by the oral or intraperitoneal route. 3. The effects of CsA and ketoconazole on the immune system of sheep were also assessed. 4. A single dose of CsA 5 mg/kg resulted in abrogation of in vitro lymphocyte function manifest at 24 h after injection of CsA. Normal responsiveness recovered in 48-72 h. Numbers of T lymphocytes in peripheral blood were elevated transiently at 48 h although no other significant alteration in lymphocyte subsets was observed with this treatment. 5. Concomitant ketoconazole administration enhanced the CsA-induced suppression of in vitro lymphocyte responses. Blood levels of CsA (AUC values to 24 h) were significantly elevated with concomitant ketoconazole administration and depression of lymphocyte responses to mitogens were also significantly enhanced. An increase in the proportion of T4 positive cells in the blood was observed at 48 h and at 7 days after administration of CsA with ketoconazole. 6. These findings indicate that CsA effectively abrogates immunocompetence in the sheep and this immunosuppressive effect is enhanced by concomitant administration of ketoconazole.

Giardia duodenalis: exposure to metronidazole inhibits competitive interactions between isolates of the parasite in vitro.

The competitive interactions of genetically distinct isolates of Giardia duodenalis with different growth rates were studied in vitro. Electrophoretic analysis of mixed cultures showed that competition between 2 cloned isolates occurs under normal in vitro culture conditions, with faster-growing isolates outcompeting those with slower growth rates. The addition of sublethal concentrations of metronidazole to clonal mixtures in vitro prevented the competitive exclusion, which was seen in normal culture. This apparently occurred because the drug reduced the growth rate of the faster-growing but not the slower-growing clone.

A comparison of two methods for assessing drug sensitivity in Giardia duodenalis.

This study compared two published methods for assessing the in-vitro drug sensitivity of Giardia duodenalis: the 3H-thymidine incorporation assay (BOREHAM et al. 1984) which radiometrically measures nucleic acid synthesis; and inhibition of adherence (MELONI et al. 1990). Giardia trophozoites were exposed to a range of concentrations of metronidazole or albendazole for 24 hours and their viability measured by both assays in order to determine the degree of correlation between the two methods of measuring viability. Due to the different modes of action of metronidazole and albendazole on Giardia, measuring the inhibition of adherence appears to be a more accurate indicator of trophozoite viability than measuring 3H-thymidine incorporation.