L.M. MacDonald

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.

The development of a real-time quantitative-PCR method for characterisation of a Cryptosporidium parvum in vitro culturing system and assessment of drug efficacy.

A reliable and sensitive quantitative-PCR (Q-PCR) method using an in vitro culturing system for Cryptosporidium parvum has been developed and characterised. This method allows standardisation of an in vitro culturing system and its development for quantitative assessment using PCR. This system was assessed against an established counting method which is widely used to enumerate parasites, particularly following exposure to antiparasitic compounds. There are several sources of variability inherent in in vitro culturing systems which could result in an inaccurate final amount of DNA being detected per culture well. These can be summarised as cumulative effects due to variability in the in vitro system, and the DNA extraction and quantification method. Analysis of the variability in this in vitro culturing system using Q-PCR indicates that it offers higher sensitivity and specificity when compared with counting methods as well as providing a vast improvement in sample throughput and efficiency. Further to this, we have also determined a method for calculating the inhibitory concentration of anticryptosporidial compounds and present a comparison of this method with a counting method and published data. We conclude that this method of quantification could be used as a substitute for haemocytometer methods, particularly, and also antibody-based techniques.

Characterization of factors favoring the expression of soluble protozoan tubulin proteins in Escherichia coli

The alpha- and beta-tubulin genes of the parasitic protozoa Giardia duodenalis, Cryptosporidium parvum, and Encephalitozoon intestinalis have been overexpressed in soluble form using Escherichia coli-based expression systems. Several expression systems were compared in terms of the amount of soluble protein produced with different fusion partners, strains of E. coli BL21, and expression temperatures. The cleavability of the fusion partner was also assessed in terms of post-expression applications of the recombinant protein. The maltose-binding protein (MBP) and glutathione S-transferase (GST) fusion partners produced the highest expression levels for all six proteins without the formation of inclusion bodies. The expression system also provided a means of purifying the soluble protein using affinity and anion-exchange chromatography while minimizing protein losses. The yield and purity were therefore very high for both the MBP and GST systems. The tubulin monomers were demonstrated to be assembly-competent using a standard dimerization assay and also retained full antigenicity with monoclonal antibodies. This study presents several methods which are suitable for producing soluble tubulin monomers and, thus, circumventing the formation of inclusion bodies which necessitates re-folding of the tubulin.

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.

The application of quantitative-PCR for high throughput screening of novel compounds against cryptosporidium parvum In Vitro and their subsequent IC50

A quantitative-PCR (Q-PCR) method that uses an in vitro culturing system for Cryptosporidium parvum has been developed. This sensitive method allows standardization of an in vitro culturing system and its development for quantitative assessment using PCR. This chapter presents a study in which this system was assessed against an established counting method which is widely used to enumerate parasites, particularly following exposure to anti-parasitic compounds. There are several sources of variability inherent in, in vitro culturing systems which could result in an inaccurate final amount of DNA being detected per culture well. These can be summarized as cumulative effects because of variability in the in vitro system and the DNA extraction and quantification method. Analysis of the variability in this in vitro culturing system using Q-PCR indicates that it is a consistent and reliable system which offers higher sensitivity and specificity when compared with counting methods as well as providing a vast improvement in sample.

An Examination of the Activity of the Dinitroanilines on Cryptosporidium Parvum Using In Vitro, In Vivo and Target Expression Methods

More than 200 compounds have been tested for activity against Cryptosporidium parvum, both in vitro and in vivo, there is still no effective treatment. Previous studies have revealed the anticryptosporidial effect of the tubulin specific herbicides, the dinitroanilines. The in vitro activities of two members of this class of compounds, oryzalin and trifluralin have been demonstrated against Cryptosporidium. Recent studies exposed IC50 values for oryzalin and trifluralin against Cryptosporidium of 750 and 800 nM, respectively. This chapter presents a study, the aim of which on-going study is to examine tubulin as an effective target both in vivo and ex vivo.