Echinococcus granulosus: biological comparison of cattle isolates from endemic regions of Argentina and Spain / Echinococcus granulosus: comparación biológica de aislados de bovinos de regiones endémicas de Argentina y España

In the present study we have compared cattle isolates of Echinococcus granulosus from Argentina and Spain. The aim was to compare and determine if there exist phenotypic and genetic differences within E. granulosus cattle isolates between an endemic area of Spain (where the disease is mainly restricted to a sheep-dog cycle) and an endemic area of Argentina (where cattle are the most abundant intermediate hosts). The Spanish samples were previously identified as G1 genotype. The Argentinean samples were also identified as G1, but some variants were found for the cytochrome c oxidase-1 (CO1) and NADH dehydrogenase-1 (ND1) mitochondrial genes. When comparing the cyst features and the morphology of the larval rostellar hooks in both regions, some differences were found. The morphometric analyses of the larval rostellar hooks showed the existence of two distinct clearly separated groups (one corresponding to the Argentinean samples and the other to the Spanish ones). In conclusion, there are some genetic and phenotypic differences within E. granulosus cattle isolates from Argentina and Spain. Probably these differences, more important from an epidemiological point of view, are related to different steps in the disease control in both countries. Further studies involving other epidemiological, morphometric and molecular data, including other types of livestock, would contribute to clarify and expand the present work.

El objetivo del presente trabajo fue determinar si existen diferencias fenotípicas y genéticas entre los aislados de Echinococcus granulosus de origen bovino provenientes de dos regiones geográficas donde la hidatidosis es endémica, una de España (donde predomina el ciclo perro-oveja) y una de Argentina (donde el bovino es el hospedador intermediario más importante). Las muestras españolas fueron previamente identificadas como pertenecientes al genotipo G1. Las muestras argentinas también correspondían al genotipo G1, pero entre ellas se registraron algunas microvariantes de los genes mitocondriales citocromo c oxidasa-1 (CO1) y NADH deshidrogenasa- 1 (ND1). La comparación de las características de los quistes y de la morfología de los ganchos rostelares del metacestode mostró ciertas diferencias. En conclusión, existen algunas diferencias genéticas y fenotípicas entre los aislados de E. granulosus de Argentina y España. Probablemente estas diferencias, más importantes desde el punto de vista epidemiológico, podrían estar relacionadas con diferentes etapas en los programas de control de la enfermedad en los dos países. Estudios adicionales que involucren datos epidemiológicos, morfométricos y moleculares provenientes de otros tipos de ganado contribuirán a clarificar y ampliar la información aportada por este trabajo.

Genetic identification and host range of the Spanish strains of Echinococcus granulosus.

Three strains of Echinococcus granulosus have been previously identified in Spain (namely 'sheep', 'horse' and 'pig'), but these Spanish strains have not been properly genotyped yet. The aim of the present research was to identify the genotype to which they correspond to. Cyst isolates were obtained from different host species, and the strain to which each belonged was established by analysis of its random amplified polymorphic DNA (RAPD) banding patterns. These results were compared to those obtained with the analysis of two mitochondrial fragment sequences (cytochrome oxidase 1 (CO1) and NADH dehydrogenase 1 (ND1)) from each isolate. The Spanish 'sheep' strain corresponded with the genotype 1 (G1) of the parasite, infecting Spanish sheep, cattle, goat, pig, wild boar and human; the Spanish 'horse' strain corresponded with the genotype 4 (G4), only infecting Spanish horses; and the Spanish 'pig' strain corresponded with the genotype 7 (G7), infecting Spanish goat, pig and wild boar. Goat, pig and wild boar can be infected by two genotypes, G1 and G7. This circumstance, and especially the possibility of sylvatic intermediate hosts serving as reservoirs of the G1 genotype of the parasite, must be taken into consideration by authorities in order to develop and evaluate effective anti-hydatidosis programmes.

The effect of solubilization on the oral bioavailability of three benzimidazole carbamate drugs.

The effect of solubilization by complexation with povidone on the oral bioavailability of three anthelmintic benzimidazole carbamate drugs: mebendazole (MBZ), albendazole (ABZ) and ricobendazole (RBZ), was studied in mice. The following in vitro characteristics of the initial raw materials and the drug-povidone complexes were evaluated: melting point (MP); mean dissolution time (MDT); solubility constants (Cs) in n-octanol, acid (pH 1.2) and neutral (pH 7.4) aqueous media; apparent partition coefficients (P) and capacity factors (k'W) determined by HPLC. The following in vivo parameters were also evaluated: AUC(0-infinity), C(max), T(max) and MRT. The possible relationship between in vitro characteristics and in vivo parameters was explored and it was found that an increase in solubility, especially in acidic medium, leads to an increase in AUC and C(max) and a decrease in T(max). Therefore, dissolution seems to be the absorption limiting step for these drugs. For the in vivo parameters related to the amount of absorbed drug (AUC and C(max)), the best correlation was obtained with the in vitro characteristics related to solubility which are Cs, MP and MDT. On the other hand, there were good linear correlations between T(max) which is an in vivo parameter related to the rate of drug absorption, and the lipophilia/hydrophilia (logP and log k'W) relation-parameters.

Effect of two formulations of benzimidazole carbamates on the viability of cysts of Echinococcus granulosus in vivo.

Two different preparations, solution and suspension, of three benzimidazole carbamate drugs, mebendazole, albendazole and ricobendazole, were compared by analyzing their in vivo activity against Echinococcus granulosus cysts in a mouse model. Polyvinylpyrrolidone was used for the elaboration of drug solutions and these formulations manifested better results in terms of reduction of number of viable hydatid cysts in mice than the reference drug suspensions. The effect was more prominent on mebendazole-treated mice, at doses of 25-50 mg/kg. There was a correlation between ED50 and pharmacokinetical parameters of AUC0-infinity and Cmax, showing that a significant improvement on solubility affects the in vivo activity of these drugs.

Further molecular discrimination of Spanish strains of Echinococcus granulosus.

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.

Observations on the Echinococcus granulosus horse strain in Spain.

The Spanish sheep and horse strains of Echinococcus granulosus possess several differential characteristics in their metacestode stage. Cysts from sheep vary widely in size and fertility, but they usually have a thin cyst wall and, when fertile, a whitish hydatid sand formed by brood capsules and protoscoleces. Two types of infections have been observed in horses: one resembling that of sheep, caused by small, non-fertile cysts with a thin wall, and a second type caused by medium to large, always fertile cysts with a thick wall. In this latter case, hydatid sand is always dark brown in color and formed mainly by brood capsules (with almost no free protoscoleces) and abundant calcareous corpuscles. These characteristics of the fertile equine cysts, which were identified in other studies by genetic, biochemical, immunological and physiological criteria as belonging to the horse strain, have not been previously described nor observed in cysts from other host species. It is considered that the horse strain possesses a strong intermediate host specificity.

Echinococcus granulosus: characterization of the Spanish strains using in vitro vesicular development.

Differences in in vitro vesicular development (microcyst formation) in three Spanish strains of Echinococcus granulosus (sheep-cattle, horse and pig-goat) are reported. Microcyst formation occurred in 19-37 days (sheep strain), 9-18 days (pig strain) and 35-47 days (horse strain). Comparing these results with those from human samples (microcyst formation in 24-38 days), it is possible to consider the sheep strain as the most likely source of human infections in Spain.

Differentiation of Spanish strains of Echinococcus granulosus using larval rostellar hook morphometry.

Two multivariate statistical procedures (Principal Component Analysis and Discriminant Function Analysis) were used to analyse morphometric data from larval hooks of Spanish samples of Echinococcus granulosus previously characterized in physiological, biochemical and genetic studies. The 5 physical variables considered could be reduced to 2 functional variables ("length of hooks" and "number of hooks"). The number of hooks was found not to be a reliable character to differentiate between strains of this parasite. Three Spanish strains (sheep-cattle, pig, and horse strains) were identified; these results are compatible with those obtained using other techniques. We consider larval hook morphology as a valid criterion for identifying E. granulosus strains in Spain, with potential use for epidemiological studies.

Echinococcus granulosus in Spain: strain differentiation by SDS-PAGE of somatic and excretory/secretory proteins.

A comparison was made, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of excretory/secretory (ES)-crude and immunopurified (with the corresponding anti-host serum) hydatid fluids-and somatic (S)-protoscoleces-proteins, from several ovine, equine, swine, bovine and human Echinococcus granulosus Spanish isolates. Likewise, the host influence on parasitic ES protein expression was studied, comparing purified hydatid fluids from ovine and equine cysts obtained from natural hosts and in RNMI mice. Purified hydatid fluids patterns, under reducing conditions, yielded the most precise differentiation of Spanish strains of E. granulosus into three groups (ovine-bovine-human, equine and swine), the finding of a characteristic 82 kDa band in equine isolates, and an unusual arrangement of bands between 50 and 6 kDa in swine samples. In addition, differences were found amongst crude and purified hydatid fluids, especially in bovine and swine isolates. The total protein patterns of protoscoleces were most complex, and therefore could not be used for strain differentiation. Finally, the purified hydatid fluids from cysts developed in natural and experimental hosts showed similar protein patterns, suggesting the lack of host influence, under our experimental conditions, on the expression of parasitic ES proteins.

Echinococcus granulosus: genomic and isoenzymatic study of Spanish strains isolated from different intermediate hosts.

The phenomenon on intraspecific variation in Echinococcus granulosus is already documented in Spain, where unilocular hydatidosis is an endemic disease. The first speciation studies, focused at a genomic level, showed the existence of three different strains: ovine-bovine-human, equine and swine-caprine. In the present study, the genomic identification, by random amplified polymorphic DNA technique (RAPD) of a larger number of Spanish E. granulosus isolates, using five different primers, showed the maintenance of these groups. Thus, some of these strains may not be infective for man. These conclusions were supported by a phenotypic characterization of the same isolates by zymodeme technique, showing the five isoenzyme systems used that Spanish E. granulosus strains can also be distinguished at a phenotypic level by isoenzymatic patterns. Both techniques (RAPD and zymodemes) were used for statistical analysis and for the construction of two dendrograms, which were slightly different. In addition, some intrastrain variation was detected with both techniques, a phenomenon that is directly related to the different speciation theories proposed for E. granulosus strains. The epidemiological implications of the results are discussed in the text.

Echinococcus granulosus: observations on strobilar development in in vitro monophasic culture.

The in vitro cultivation technique of Echinococcus granulosus protoscoleces usually states the necessity of a biphasic medium with a solid protein substrate for strobilar development to take place; otherwise, in a monophasic medium, protoscoleces follow a vesicular development. However, in some monophasic cultures, the development of several strobilate individuals (in different quantities and stages of development, depending on the culture) were observed. The only known difference from cultures made previously and since, where the development was vesicular, was the batch of foetal calf serum used in the constitution of the liquid medium, and this is presumed to be the cause of this unexpected strobilar development.

Comparative genetic analysis of Swiss and Spanish isolates of Echinococcus granulosus by southern hybridization and Random Amplified Polymorphic DNA technique.

Swiss and Spanish isolates of Echinococcus granulosus were compared using different molecular biological techniques: Genomic DNAs isolated from parasites originating from various intermediate hosts were subjected to Southern hybridization with different probes, the same source of DNA was used for DNA amplification using the Random Amplified Polymorphic DNA (RAPD) technique. With both methods the various isolates (metacestodes) of E. granulosus exhibited characteristic banding patterns which allowed us to assign them to the following groups of homologous profiles: (a) isolates of horse and donkey origin from Spain and Switzerland; (b) isolates of cattle origin from Switzerland; (c) isolates of sheep, cattle and human origin from Spain; (d) isolates of pig origin from Spain and Switzerland and of goat origin from Spain. By RAPD (Southern hybridization not examined) two isolates of human origin from Switzerland were showing banding patterns distinct from groups (a-d). The results provide further evidence that the morphological and biological differences of several strains of E. granulosus are also detectable on the genetic level using molecular biological techniques.