Synthesis and in-vitro antifungal activity of 6-trifluoromethyipyrazolo[3,4-6d]pyrimidin-4(5H)-thiones

Twelve 6-trifluoromethylpyrazolo[3,4-d]pyrimidin-4(5H)-thiones were prepared by the reaction between 4-thiocarbamoyl-5-aminopyrazoles and trifluoroacetic anhydride. They were tested in vitro for antifungal activity against a series of phytopathogenic fungi of different taxonomic classes. The EC50 and MIC values of four compounds were comparable or inferior to those of reference commercial fungicides in controlling Sclerotinia minor, Corticium solani and Phytium ultimum.     

Long term improvement in the treatment of canine leishmaniosis using an antimony liposomal formulation

Pharmacokinetic and clinical effectiveness of liposome-encapsulated N-methylglucamine antimoniate (LMA) was performed in dogs suffering from experimental leishmaniosis. LMA was compared with N-methylglucamine antimoniate (MGA), the same drug in its free form. Sb plasma concentrations for LMA were always higher than those for MGA. Mean residence time (MRT), half-life time (t(1/2)) and clearance (Cl) showed that Sb was eliminated slower after liposome administration. The high volume of distribution (Vd) obtained with LMA suggests that Sb could achieve therapeutic concentrations in parasite-infected tissues. Average plasma concentration at steady state (Css(ave)) shows that Sb body concentrations after LMA treatment (9.8 mg/kg Sb, each 24h) would be effective in Leishmania infantum canine infection. Comparing LMA with MGA in a 1-year follow-up we observed no relapses for LMA and total protein and gammaglobulin concentrations were within normal range, while for MGA both began to rise 3 months after treatment. Use of antimonial liposomal formulations may restore effectiveness to an existing drug and reduce toxicity.     

Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.     

Identification of optimal regions for phylogenetic studies on VP1 gene of foot-and-mouth disease virus: analysis of types A and O Argentinean viruses

An analysis of the informative content of sequence stretches on the foot-and-mouth disease virus (FMDV) VPI gene was applied to two important viral serotypes: A and O. Several sequence regions were identified to allow the reconstruction of phylogenetic trees equivalent to those derived from the whole VPI gene. The optimal informative regions for sequence windows of 150 to 250 nt were predicted between positions 250 and 550 of the gene. The sequences spanning the 250 nt of the 3' end (positions 400 to 650), extensively used for FMDV phylogenetic analyses, showed a lower informative content. In spite of this, the use of sequences from this region allowed the derivation of phylogenetic trees for type A and type O FMDVs which showed topologies similar to those previously reported for the whole VP1 gene. When the sequences determined for viruses isolated in Argentina, between 1990 and 1993, were included in these analyses, the results obtained revealed features of the circulation of type A and type O viruses in the field, in the months that preceded the eradication of the disease in this country. Type A viruses were closely related to an Argentinean vaccine strain, and defined an independent cluster within this serotype. Among the type O viruses analysed, two groups were distinguished; one was closely related to the South American vaccine strains, while the other was grouped with viruses of the O3 subtype. In addition, a detailed phylogeny for type A FMDV is presented.     

An in vitro study of hematopoietic progenitor cells from peripheral blood of rabbits

The purpose of this study was to isolate and cultivate a subpopulation of pluripotent stem cells (PSCs) from the peripheral blood of rabbits, which are frequently used in veterinary research as an animal model. Pluripotent stem cells, as described in human beings, are fibroblast-like cells that exhibit a CD34 marker, specific from other hematopoietic stem cells. Commonly used human commercial media has been researched for culturing rabbit PSCs. These findings allow us to contemplate the direct application of this simple and standardized methodology in several areas of study, such as of the pharmacological effect of many drugs on hematopoietic cells, veterinary practice, and even the study of new strategies in cellular therapy for some human diseases.     

Evaluation of Natural Chemical Compounds Against Root-lesion and Root-knot Nematodes and Side-effects on the Infectivity of Arbuscular Mycorrhizal Fungi

The survival of two species of plant parasitic nematodes: the root-lesion nematode Pratylenchus brachyurus, and the root-knot nematode Meloidogyne javanica, was evaluated in saturated atmospheres of 12 natural chemical compounds. The infectivity of two isolates of arbuscular mycorrhizal fungi: Glomus mosseae and Glomus intraradices, under identical experimental conditions, was also determined. All the compounds tested exerted a highly significant control against M. javanica and among them, benzaldehyde, salicilaldehyde, borneol, p-anisaldehyde and cinnamaldehyde caused a mortality rate above 50% over P. brachyurus. The infectivity of G. intraradices was inhibited by cinnamaldehyde, salicilaldehyde, thymol, carvacrol, p-anisaldehyde, and benzaldehyde, while only cinnamaldehyde and thymol significantly inhibited mycorrhizal colonization by G. mosseae.     

Multispecies and multiple anthelmintic resistance on cattle nematodes in a farm in Argentina: the beginning of high resistance?

There are very few resistance records on cattle nematodes. South American successive records have been increasing rapidly since the year 2000. In Argentina, increasing dissatisfaction based on the exclusive use of macrocyclic lactones has prompted the use of benzimidazoles in the Pampean region. The studied farm is located in the Argentina humid Pampas and had apparently poor results after anthelmintic treatments. Evaluation of resistance was firstly based on faecal egg reduction after treatment in November 2001 and June 2002 and was complemented with worm counts obtained by necropsy in June 2002. The study reports that the reduction of faecal egg excretion after benzimidazole (reduction 31-79%) or ivermectin treatments (76-97%) in November 2001 was insufficient and these poor results were confirmed in June 2002 (benzimidazoles (65-89%) and ivermectin (47-77%)). Several methods for evaluating faecal egg count reduction were performed and yielded different results. A new method based on the estimated faecal egg counts (corrected from the initial faecal egg counts and animal type using a general linear model) was used and gave higher flexibility in the interpretation of putative resistance to anthelmintics. From necropsy results, it could be concluded that Cooperia oncophora was resistant to avermectins and benzimidazoles and that Cooperia punctata, Ostertagia ostertagi and Haemonchus placei were resistant to benzimidazoles. This case of multispecies and multidrug resistance is probably not unique and could reflect the emergence of resistances in Argentina. This emergence is probably due to the intensive use of anthelmintics, the absence of refugia, and the frequent circulation of infected cattle.     

SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis

Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.