Supplementation of broiler diets with high levels of microbial protease and phytase enables partial replacement of commercial soybean meal with raw, full-fat soybean.

A 3 × 3 + 1 factorial, involving three levels of protease (0, 15,000 or 30,000 PROT/kg) and three levels of phytase (1,000, 2,000 or 3,000 FYT/kg), was used to evaluate the effect of replacing commercial soybean meal (SBM) with raw, full-fat soybean (RFSB) at 75 g/kg of diet for broilers. A control diet was used for comparison. Each treatment was replicated six times, with nine birds per replicate. The concentration of trypsin inhibitors (TIs) in the test diets was approximately 10,193.4 TIU/kg. Regardless of enzyme supplementation, feed intake (FI) and body weight gain (BWG) of birds in the control group were superior to those on the test diets. Birds that received the protease-free test diets had reduced FI and BWG, but when supplemented with protease, were similar to the control diet in BWG, FI (except 0-35 days) and feed conversion ratio (FCR). When the test diet was supplemented with elevated levels (extradose) of protease and phytase, the BWG was improved during 0-10 days (p = .05) and 0-24 days (p < .01). Regardless of protease supplementation, the weight of thighs was lower for birds fed the test diets. Birds that received the control diet had smaller weight of pancreas. Increasing the level of phytase supplementation reduced (p < .05) the weight of the pancreas. The apparent ileal digestibility (AID) of CP and AA was higher in birds on the control diets, but this was also improved in test diets by protease supplementation. The activities of trypsin (7%), general proteolytic (11%) and lipase (12%) were slightly increased because of protease supplementation. Mucosal depth and apparent villus surface areas were increased by about 2.9% and 20%, respectively, due to supplementation of elevated level of phytase. It can be concluded that RFSB could partially replace SBM in broiler diets, provided the diets are supplemented with elevated levels of protease and phytase.

Pasture, multi-enzymes, benzoic acid and essential oils positively influence performance, intestinal organ weight and egg quality in free-range laying hens.

1. The objective of this study was to investigate the effect of range type, multi-enzyme applications, and a combination of benzoic acid (BA) and essential oils (EO) on the productive performance, organ weight and egg quality of free-range laying hens. 2. Three hundred laying hens were evaluated for the short-term (6 weeks) and long-term (12 weeks) effects of range type (G = no pasture, P = pasture) and feed additives (T1 = control; T2 = betaglucanase/pectinase/protease; T3 = BA/EO). Body weight, feed intake (FI), feed conversion ratio (FCR), egg production (EP), digestive organ weight, and egg quality (EQ) were evaluated. Data were analysed using SPSS 2.2 in a 2×2×3 factorial arrangement. 3. Hens that ranged on pasture were significantly heavier (2043 g vs. 1996 g; p < 0.001), laid heavier eggs (61.9 g vs. 60.3 g; p < 0.001) and produced darker yolk colour (4.3 vs. 7.0; p < 0.001) compared to hens ranged on gravel. Hens fed T2 were significantly heavier (2050 g) compared to hens fed T1 (2005 g) or T3 (2008 g). Organ weights (gizzard, liver and pancreas) were significantly heavier in hens ranged on pasture (16.8 g/kg BW, 22.3 g/kg BW and 1.89 g/kg BW, respectively) compared to hens ranged on gravel (14.2 g/kg BW, 21.7 g/kg BW and 1.83 g/kg BW, respectively). Over time, body weight (1970-2070 g; p < 0.001) and egg weight (59.5-62.8 g; p < 0.001) increased, FI (123-120 g; p = 0.024) was reduced and FCR (2.36-2.10; p = 0.002) improved 4. In conclusion, hens housed on pasture and fed multi-enzyme supplemented diets had significantly heavier body weight and produced heavier eggs with darker yolk colour. Pasture intake and enzyme supplementation increased digestive organ weight significantly.

Physiological and health-related response of broiler chickens fed diets containing raw, full-fat soya bean meal supplemented with microbial protease.

A 2 × 3 factorial study (protease: 0 or 1,5000 PROT/kg and raw full-fat soya bean meal [RSBM] replacing the commercial SBM at 0, 45 and 75 g/kg of diet) was conducted to examine the performance of broilers. Phytase (2000 FYT/kg) was uniformly added to each diet, each also replicated six times, with eight birds per replicate. Birds were raised in climate-controlled rooms using sawdust as the bedding material and offered starter, grower and finisher diets. Feed intake (FI) and body weight gain (BWG) were reduced (p < .05) due to increasing levels of RSBM, but feed conversion ratio (FCR; 0-35 days) was unaffected. Over the first 24 days, neither RSBM nor protease supplementation affected (p > .05) mortality, footpad dermatitis or intestinal lesions in birds. At day 24, the weight, length, width and strength of tibia bone were reduced in chickens that received an elevated level of RSBM (75 g/kg of diet), but this was not significant at day 35. At day 24 (p < .05) and 35 (p < .01), Ca concentration in the litter was reduced when the RSBM level was increased in the diet, but P content was not affected. On days 24 (p < .05) and 35 (p < .01), the N content in litter was also increased with increase in dietary RSBM. Protease supplementation increased (p < .05) the uric acid concentration in the litter (at day 35), but the reverse was the case for ammonia concentration. Overall, the results of this study indicate that there are no major health-related risks, associated with the replacement of commercial SBM with RSBM (≤25%) in broiler diets.

Performance, nutrient utilization, and energy partitioning in broiler chickens offered high canola meal diets supplemented with multicomponent carbohydrase and mono-component protease.

Two broiler chicken experiments were conducted to investigate the effects of canola meal (CM) replacing soybean meal (SBM) in diets supplemented with carbohydrase and protease on performance and partitioning of energy. First, a 2 × 2 × 2 factorial arrangement of treatments was employed to evaluate: protein meals (CM vs. SBM), carbohydrase (none or 300 mg/kg), protease (none or 200 mg/kg), and their interactions. Each treatment was fed to 6 replicated pens of 16 male broilers (Ross 308) from d 10 to 35. In the second experiment, 32 broiler chicks were used in a 2 × 2 factorial arrangement to investigate CM and carbohydrase effects on energy partitioning. Birds were transferred into 16 closed-circuit calorimeter chambers (4 chambers/diet; 2 birds/chamber) to measure heat production (HP), metabolizable and net energy (NE) by gaseous exchange, and total excreta collection from d 25 to 28. There were no 3-way interactions among experimental factors for any of the performance parameters measured. Birds given CM diets consumed less feed, had lower BW, and exhibited higher FCR compared to the control birds (P < 0.01). Both enzymes, alone or in combination, improved final BW and FCR (P < 0.05). There was an interaction between carbohydrase and protease for FCR over the grower period (P < 0.01), in which the combination of the enzymes resulted in further improvement of FCR. Energy, DM, and crude protein digestibility values were higher in control birds (P < 0.05). There was an interaction of protein meal and carbohydrase for HP, respiratory quotient (P < 0.05), and NE:ME ratio of the diets (P = 0.06). Inclusion of CM without carbohydrase increased HP and decreased NE and NE:ME ratio of the diets (P < 0.05). Carbohydrase decreased HP and increased retained energy (P = 0.06) and NE and NE:ME ratio (P < 0.05). In conclusion, high CM in the diet negatively affects growth performance through reduction in feed consumption, nutrient digestibility, and NE of the diet, which could partly be restored by enzyme supplementation.

Effects of heat treatment on the nutritional value of raw soybean selected for low trypsin inhibitor activity.

1. Two broiler experimentss and a layer experiments were conducted on Kunitz trypsin inhibitor (Kti) soybeans (SB) of low trypsin inhibitor (TI) activity to determine their nutritive value when included as mash in least-cost poultry diets. 2. Experiment 1 compared chick performance on the Kti or raw SB using a commercial full-fat SB meal (FFSBM) and a solvent extracted SB meal (SBM) as controls during a 20 d experimental period. Broiler experiment 2 compared Kti and raw SB, non-steamed, or steam-pelleted with and without DL-methionine supplementation added to every treatment containing 170 g SB/kg. For each broiler experiment the levels of each SB were 70, 120 and 170 g/kg with the control birds fed only 170 g SB/kg. 3. The layer experiment, compared steam-pelleted Kti and raw SB against a non-steamed Kti and raw SB each fed at two levels (70 and 110 g/kg) x 30 replicates from 29 weeks of age for 19 weeks in a completely randomised design. Production parameters were measured when diets were formulated to contain minimum required specifications and calculated apparent metabolisable energy (AME). At the completion of each trial, 2 broiler birds from each cage and 5 layer birds per treatment were killed, weighed, and their liver and pancreas weighed. 4. Both broiler experiments indicated that production parameters on the Kti SB treatments were significantly lower (P<0.05) than on the two commercial control SB treatments. However, the Kti treatments were superior to the raw SB treatments. 5. Pancreas weight increased with increasing inclusion of both raw and Kti SB, suggesting that a TI was causing the depression in performance. The AME of the Kti SB was similar to that of commercial FFSB meal. After steam conditioning, the raw SB meal AME value of 9.5 MJ/kg dry matter (DM) was improved to 14.1 MJ/kg DM by reduced TI activity, but this AME improvement with TI activity reduction, plus the supplementation with DL-methionine on birds fed the raw SB had no effect (P>0.05) on any parameter evaluated in experiment 2. 6. The layer experiment showed that hens on the Kti SB treatments had significantly greater live weight gain (LWG), egg weight and daily egg mass than birds given raw SB. A reduced food intake (FI) was observed in the Kti treatments but egg mass was generally similar to that on the FFSB control diet, indicating that Kti SB supported excellent egg production at an inclusion of 110 g/kg. The depressed performance observed for broiler chicks suggest that younger birds are more susceptible to the effects of SB TI.

Cloning of a pyruvate phosphate dikinase from Trypanosoma cruzi.

We have cloned and characterised a gene that encodes a putative pyruvate phosphate dikinase (PPDK) from Trypanosoma cruzi, an enzyme that catalyses the reversible conversion of phosphoenolpyruvate to pyruvate. PPDK is absent in mammalian cells, but has been found in a wide variety of other organisms, including plants and bacteria. In T. cruzi, two genes (PPDK1 and PPDK2) are present in a tandem array localised on a 1 Mbp chromosome. Northern and Western blot analyses indicates that PPDK is expressed as a 100-kDa protein in epimastigote, amastigote and trypomastigote forms. PPDK1 and PPDK2 encode an identical protein of 100.8 kDa with a C-terminal extension ending with the sequence AKL, a signal for glycosomal import. Both T. cruzi and T. brucei enzymes possess a 23-residue insertion, that is absent in other PPDKs. A three-dimensional alignment with the crystal structure of the enzyme from Clostridium symbiosum predicts that this insertion is located on the surface of the nucleotide-binding domain. Phylogenetic studies indicate that bacterial and protist PPDKs cluster as a separate group from those of plants. The evolutionary implications and possible role of this enzyme in T. cruzi is discussed.

Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein.

The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypanosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the addition of myristate and palmitate at its amino terminal segment and both modifications are required for calcium-modulated flagellar membrane association. FCaBP has four sequence motifs for potential calcium binding, and comparison to other calcium-acyl switch proteins, such as recoverin, suggested that only two of these sites are functional. Because it is not possible to predict with certainty the calcium binding affinity or selectivity based on motif analysis alone, we determined the quantitative calcium binding activity of FCaBP by direct ligand binding using the flow dialysis method. The results demonstrated the presence of two calcium binding sites in the full length FCaBP and in a mutant (FCaBPdelta12) lacking the amino terminal pair of sites. FCaBPdelta12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBPdelta34), did not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from other myristoyl switch proteins such as recoverin. The results add to a growing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the apparently novel mechanism by which FCaBP undergoes calcium modulated flagellar membrane association and its relation to calcium signal transduction.

Optimum inclusion of field peas, faba beans, chick peas and sweet lupins in poultry diets. I. Chemical composition and layer experiments.

1. Experiments were undertaken to determine the chemical composition and apparent metabolisable energy (AME) of field peas, faba beans, sweet lupins and chick peas and the production of hens when each was included in nutritionally similar diets at 250 g/kg in 2 experiments. 2. Amino acid composition, crude protein and AME agreed well with previously published measurements. Detailed analysis of the non-starch polysaccharides (NSPs) showed that sweet lupins were much higher than the other grain legumes in the soluble NSPs and that the NSPs were particularly high in arabinose. The condensed tannin content was highest in field peas followed by faba beans. 3. In the 1st layer experiment over 40 weeks, hen-day egg production was lowest on the faba bean-based diet and egg weight and egg mass were also lowest. Relative viscosity of digesta in the small intestine of hens fed on the sweet lupin-based diet was highest, followed by that of hens fed on field peas. Enlargement of the pancreas was observed in hens consuming chick peas. 4. In experiment 2, in which only sweet lupins and faba beans were used, steam or cold pelleting showed few effects, nor did dehulling of faba beans but egg weight was lower when diets were steam pelleted. Daily food intake was 5.7 g/bird lower on the steam than cold pelleted diets and food conversion ratio tended to be improved (P=0.082). 5. It was concluded that field peas could support good production at 250 g/kg of layer diet. Although chick peas and sweet lupins supported good performance, there was concern about the increased weight of the pancreas and high gut viscosity respectively. Faba beans showed similar hen-d egg production in the 2nd experiment to that of sweet lupins but egg weight tended to be about 0.8 g lower than when on the sweet lupin-based diets.

Optimum inclusion of field peas, faba beans, chick peas and sweet lupins in poultry diets. II. Broiler experiments.

1. Three experiments were undertaken to determine the optimum inclusion rates of field peas, faba beans, chick peas and sweet lupins in broiler starter and finisher diets in amounts up to 360 g/kg. 2. In experiment A chickens in cages grown to 21 d on diets with field peas and faba beans gave better growth rate and feed efficiency than those with sweet lupins and chick peas. Growth rate and Food conversion ratio (FCR) improved with increasing amounts of faba beans in the diet while for chick peas growth rate and FCR declined. Digesta viscosity and excreta stickiness scores were much higher on diets with sweet lupins. Steam pelleting improved growth rate and FCR on all diets. 3. In experiment B birds were in cages and grown from 21 to 42 d. There were no differences between grain legumes (when combined for all inclusions) for growth rate, food intake or FCR. Viscosity was again much higher on the sweet lupin-based diets while the pancreas was significantly enlarged on the diets with chick peas, as observed previously in chickens grown to 21 d. Steam pelleting of diets gave a consistent and positive response for weight gain and FCR. 4. Experiment C was carried out in pens each holding 60 birds under semi-commercial conditions and grown to 42 d on starter and finisher diets with the same grain legumes as used previously but each at 2 rates of inclusion similar to those in commercial practice. Field peas at 200 to 300 g/kg and chick peas at 150 to 220 g/kg gave inferior growth to faba beans (150 to 180 g/kg) and sweet lupins (120 g/kg). 5. The results of these experiments allowed tentative recommendations to be made to industry for inclusion rates of these cultivars of the 4 grain legumes. These were: field peas 300 g/kg; faba beans 200 g/kg, chick peas 100 g/kg and sweet lupins <100 g/kg. Wet droppings and high gut viscosity were serious problems with sweet lupins although these were not so obvious in experiment C.

Homologues of the 24-kDa flagellar Ca(2+)-binding protein gene of Trypanosoma cruzi are present in other members of the Trypanosomatidae family.

A full-length cDNA encoding the 24-kDa flagellar Ca(2+)-binding protein (FCaBP) of the Dm28c clone of Trypanosoma cruzi was cloned and characterized. Comparison of the deduced amino acid sequence with those of the FCaBPs of other T. cruzi strains revealed greater than 97% sequence conservation. FCaBP-like genes are found in Trypanosoma conorhini, Trypanosoma freitasi, Trypanosoma lewisi, Herpetomonas megaseliae, Leptomonas seymouri, and Phytomonas serpens, but not in Crithidia deanei, Leishmania amazonensis, or Endotrypanum schaudinni: Among various T. cruzi strains, FCaBP genes are located on chromosomes of different size, although all strains possess multiple FCaBP genes organized as tandemly arranged gene families. Northern and Western blot analyses revealed that FCaBP mRNAs are produced in all organisms possessing FCaBP-hybridizing sequences, indicating that expression of FCaBP or an FCaBP-like protein is common to a number of trypanosomatid species.

Digestion of 14C-labelled condensed tannins from Desmodium intortum in sheep and goats.

An experiment was conducted to investigate the metabolism of condensed tannin (CT) in sheep and goats offered a mixture of Digitaria decumbens (700 g/kg) and Desmodium intortum (300 g/kg) hay. Radioactive 14CO2 was used to label CT in young growing desmodium plants, [14C]CT was extracted, purified and infused intraruminally, and the metabolism of [14C]CT was followed in the rumen and lower digestive tract of both species. Digestion of DM, organic matter (OM), cell-wall constituents (CWC), N and the efficiency of rumen microbial synthesis were determined using a continuous intraruminal infusion of 51Cr EDTA, YbCl3 and Na235SO4. The measurements taken for sheep and goats respectively were: intake, 21 and 30 g/kg0.9 per d; digestibilities (g/g) of DM, 0.566 and 0.505; OM 0.578 and 0.508; neutral-detergent fibre, 0.584 and 0.532; and acid-detergent fibre, 0.535 and 0.435. None of these measurements was significantly different (P > 0.05) between animal species. There was an apparent net gain in lignin across the rumen and whole intestinal tract for both animal species (19 and 29% for sheep and goats respectively). There were no significant differences between sheep and goats (P > 0.05) detected for any measurements of N excretion and utilization. The overall efficiency of N digestion and utilization was also similar between species. The routes of CT metabolism were compared for both colorimetric estimates (butanol-HCl) of dietary CT (DCT) and the specific radioactivity of [14C]CT in digesta (abomasum) and excreta (urine and faeces) of both sheep and goats. [14C]CT showed total losses of 57 and 56% in sheep and goats respectively whilst losses of DCT of 71 and 70% were detected with butanol-HCl in sheep and goats respectively. The apparent losses of DCT across the rumen of sheep and goats were 12 and 9% whilst higher losses (49 and 42% for sheep and goats respectively) were observed for [14C]CT. Losses of DCT in the lower intestinal tract accounted for 69 and 71% of the total CT leaving the abomasum. By comparison, only 40 and 35% of [14C]CT was lost during intestinal passage in sheep and goats respectively. It was concluded that the infused free [14C]CT interacted with DCT and entered the protein and fibre-bound DCT pools. The loss of DCT during passage through the intestines was considered to be a consequence of either absorption of free CT or the degradation products of CT. It was assumed that free CT arose in the lower gastrointestinal tract from protein-CT and fibre-CT dissociation to be digested and/or absorbed. The higher recoveries of [14C]CT in faeces (32 and 35%) compared with DCT (27 and 26%) for sheep and goats respectively) were associated with the excretion of [14C] degradation products or conjugates which were not reactive to butanol-HCl. It was concluded that both methods (butanol-HCl and labelling CT with 14C) detected a substantial disappearance of CT (free, protein, and fibre-bound) during metabolism in the gastrointestinal tract in sheep and goats.

The effects of condensed tannins from Desmodium intortum and Calliandra calothyrsus on protein and carbohydrate digestion in sheep and goats.

A factorial experiment was conducted to study the effects of condensed tannins (CT) from the tropical legumes Desmodium intortum and Calliandra calothyrsus on the digestion and utilization of protein and carbohydrate in sheep and goats. CT-free Centrosema pubescens was also fed for comparison with the CT legumes, and each legume was included (300 g/kg DM) in a basal diet of pangola grass (Digitaria decumbens). Pangola grass alone was used as a control diet. There were no significant (P > 0.05) differences between sheep and goats for the efficiency of digestion of N (0.574, SE 0.013), organic matter (OM; 0.519, SE 0.010), neutral-detergent fibre (NDF; 0.524, SE 0.011) and acid-detergent fibre (ADF; 0.407, SE 0.016). Diets containing desmodium and calliandra were digested less well in the rumen (64 and 62% of total OM digested) when compared with the pangola and centrosema diets (74 and 73% of total OM digested in rumen). There was an apparent net gain of 30% in ADF across the digestive tract of sheep and goats given calliandra, and this gain was ascribed to the formation of 'artifact' fibre as a result of fibre-tannin interaction. Overall, inclusion of legume at 300 g/kg in the diet significantly increased (P < 0.05) the concentration of acetic acid and decreased butyric acid concentration in the rumen fluid of sheep and goats. Significantly higher proportions of dietary N apparently reached the abomasum of animals given the diets containing desmodium (50%) and calliandra (56%) when compared with animals given the centrosema and pangola diets (35%). Sheep and goats given the CT diets also had higher excretions of faecal N. This increment of faecal N (14%) did not affect post-rumen N digestion (P > 0.05) since animals given CT diets absorbed more N (19%) per kg total OM digested than those given the control diets. It was concluded that whilst the low levels of CT provided in desmodium (1.0%) and calliandra (2.3%) diets protected dietary protein from degradation in the rumen, there were no overall beneficial or detrimental effects of CT in these diets for sheep or goats. A method was developed to categorize CT into fractions representative of their forms (free, protein-bound, and fibre-bound) during the digestion process. A quantitative model of CT metabolism during passage through the digestive tract was developed from the measured exchanges of CT between free, protein-bound and fibre-bound pools in the rumen and lower digestive tract. CT interchange mainly occurred in the reticulo-rumen of both animal species. Desmodium and calliandra free CT showed net losses of 68 and 78% in the rumen respectively and 57 and 68% of the fibre-bound CT was lost in the same site for sheep and goats respectively. However, protein-bound CT increased across the rumen by 73 and 56% for both animal species. Post-rumen losses of the total CT abomasal flow were 86 and 83% (free CT) for sheep and goats respectively, 70 and 66% (protein-bound CT), whilst 28% loss of fibre-bound CT occurred in sheep and goats respectively.

Experimental chemotherapy with combinations of ergosterol biosynthesis inhibitors in murine models of Chagas' disease.

We report the effects of ketoconazole and the bistriazole ICI 195,739 acting alone or in combination with the allylamine terbinafine (Lamisil) on murine models of Chagas' disease. Mice infected with 10(5) Trypanosoma (Schizotrypanum) cruzi blood trypomastigotes and treated orally with 30 mg of ketoconazole per kg of body weight per day for 7 days, starting at 24 h postinoculation, had 100% survival after 35 days, while controls (untreated) or animals that received 15 mg of ketoconazole or 100 mg of terbinafine per kg/day by the same route had 0% survival after the same period of time. However, all mice receiving the combination of 15 mg of ketoconazole plus 100 mg of terbinafine per kg/day survived for 35 days after infection; it was shown that the survival of the animals treated with this combination was statistically greater than that obtained with either drug acting alone and was indistinguishable from that observed with the high doses of ketoconazole, indicating a synergistic action of the drugs in vivo. However, most animals that survived after the 7-day treatments were not cured, as indicated by a delayed but persistent parasitemia. When the treatment was extended to 14 days, 100% survival was obtained 10 weeks after inoculation for mice treated with 30 mg of ketoconazole per kg/day and the combination of 15 mg of ketoconazole per kg/day plus 100 mg of terbinafine per kg/day, while two-thirds of the mice treated with 15 mg of ketoconazole per kg/day alone were alive after the 14-day treatment; controls or animals that received 100 mg of terbinafine per kg/day did not survive after 25 days. Parasitemia in all surviving mice was negative after 55 days but parasitological cure, as assessed by subinoculation of organs in naive animals, was predominant only in animals that received the combined drug treatment. We also investigated the bistriazole ICI 195,739 and found, as reported previously, that just 1 mg of the compound per kg/day administered orally for 5 days was enough to protect most mice from death 30 days after inoculation, but no parasitological cures were observed. However, in the protocol used in the present study, the protective activity of ICI 195,739 at suboptimal doses (0.5 mg/kg/day) could be enhanced when it was used in combination with terbinafine at doses of the allylamine that by themselves induced no significant protection. Survival of the mice was inversely correlated with the levels of parasitemia in all cases. Extension of the treatment period with the triazole to 15 days at 1 mg/kg/day afforded definitive protection against death, with parasitological cure being achieved in 50% of mice at 10 weeks postinoculation, but no enhancement of its activity at suboptimal doses was observed when it was used in combination with terbinafine during this extended observation period. Taken together, these results supports the proposition that ketoconazole used in combination with terbinafine could be useful in the treatment of humans with Chagas' disease because it can promote parasitological cure without the need to resort to the use of high levels of the azole, which is known to interfere with hepatic function and steroid synthesis in the host. They also support the conclusions of previous in vitro studies which suggested that the triazole ICI 195,739 blocks the proliferation of T. cruzi by a mechanism which differs from those of classical ergosterol biosynthesis inhibitors.

Mevinolin (lovastatin) potentiates the antiproliferative effects of ketoconazole and terbinafine against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies.

We have studied the antiproliferative effects of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, on the protozoan parasite Trypanosoma (Schizotrypanum) cruzi and its ability to potentiate the action of specific ergosterol biosynthesis inhibitors, such as ketoconazole and terbinafine, both in vitro and in vivo. Against the epimastigote form in vitro, mevinolin produced a dose-dependent reduction of the growth rate up to 25 microM, but at 50 and 75 microM, complete growth arrest and cell lysis took place after 144 and 96 h, respectively. A systematic study of the effects of mevinolin combined with ketoconazole and terbinafine, which act at different points in the ergosterol biosynthesis pathway, on the proliferation of epimastigotes indicated a synergic action, as shown by concave isobolograms and fractional inhibitory concentration indexes ranging from 0.17 to 0.54. Analysis of the sterol composition and de novo sterol synthesis in control and treated cells by thin-layer and gas-liquid chromatographies showed that the antiproliferative effects of the drug alone and in combination were correlated with the depletion of the endogenous ergosterol pool and particularly with a critical (exogenous) cholesterol/endogenous 4-desmethyl sterol ratio in the cells. When we studied the effects of mevinolin on the amastigote form proliferating inside Vero cells in vitro, only very modest effects on the parasites were observed up to 0.75 microM; above this concentration, significant deleterious effects on the host cells were found. However, when the same concentration of the drug was combined with ketoconazole, it was able to reduce by a factor of 10 the concentration of the azole required to eradicate the parasite (from 10 to 1 nM), again indicating a synergic action. On the other hand, a combination of mevinolin and terbinafine had only additive effects on amastigotes, but a ternary combination of mevinolin, ketoconazole, and terbinafine was again clearly synergistic. In vivo studies with a murine model of Chagas' disease showed that mevinolin can also potentiate the therapeutic effects of ketoconazole in this system; combined treatment with the two drugs at doses that alone offered only limited protection against the parasite was able to essentially eliminate circulating parasites and produce complete protection against death. These results confirm the synergic action against the proliferative stages of T. cruzi both in vitro and in vivo and in vivo of combined ergosterol biosynthesis inhibitors that act at different points in the pathway and suggest that mevinolin combined with azoles, such as ketoconazole, can be used in the treatment of human Chagas' disease.