Question of agent of camel balantidiosis solved: Molecular identity, taxonomic solution and epidemiological considerations.

Domestic camels (Camelus bactrianus, the Bactrian camel; and Camelus dromedarius, the dromedary) are pseudo-ruminant herbivores kept as livestock in rural, inhospitable regions (cold deserts and dry steppes of Asia, arid to semi-arid regions of Africa, western and central Asia). Their close contact with humans makes them a potential reservoir for zoonotic parasite infections, as has been suggested for human balantidiasis. However, there is confusion about the ciliate species that infects camels: Infundibulorium cameli was originally described in dromedaries, but this name has almost never been used and most authors identified their findings as Balantioides coli and, to a lesser extent, Buxtonella sulcata, a cattle ciliate. To clarify the taxonomic status of the parasite and the corresponding zoonotic significance for camels, we performed morphological characterization of cysts and genetic analysis (SSU-rDNA and ITS markers) of B. coli-like isolates from Bactrian camels from Bulgaria and from dromedaries from Spain and the United Arab Emirates. Our results indicate that the camel ciliate is not B. coli, nor is it B. sulcata, but is a different species that should be placed in the same genus as the latter. Thus, camels are not a reservoir for human balantidiasis. Although the correct genus name would be Infundibulorium according to the principle of priority, this would lead to confusion since this name has almost fallen into disuse since its initial description, but Buxtonella is almost universally used by researchers and veterinarians for the cattle ciliate. We therefore propose to apply the reversal of precedence and use Buxtonella as the valid genus name. Consequently, we propose Buxtonella cameli n.comb. as the name for the camel ciliate.

Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and racing pigeons (Columba livia): genetic and morphometric characterisation of clonal cultures.

Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur.

Morphological and molecular identification of Tetratrichomonas flagellates from the giant anteater (Myrmecophaga tridactyla).

A tetratrichomonad flagellate found in the diarrhoeic faeces of a 5 years-old male giant anteater (Myrmecophaga tridactyla) was characterised by morphological and genetic analysis. This protozoan presents four anterior flagella of unequal length and a recurrent flagellum attached to the undulating membrane without a free end portion, and a broad axostyle projection. Numerous vacuoles of different sizes containing bacteria and digestion products were found. The complete sequence of the DNA coding for the 16S rRNA-ITS1-5.8S rRNA-ITS2 region was also obtained in order to compare this isolate with other tetratrichomonad species. The sequence obtained was identical to others previously obtained by other researchers from bovines and turtles (Geochelone sp.). It is not easily explainable how the same organism could be found in such different hosts and locations; however these results indicate that some tetratrichomonad species could have a wide host range and could survive in a wide range of environmental conditions.

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.

Tentative identification of the species of Balantidium from ostriches (Struthio camelus) as Balantidium coli-like by analysis of polymorphic DNA.

The characteristics of Balantidium from ostriches (Struthio camelus) are similar to those of Balantidium coli; however, the species Balantidium struthionis was proposed on the basis of the host species (ostriches) and the shape of the macronucleus (with a deep depression in one side). In the present work, we have performed morphological and genetic comparisons between isolates of Balantidium from ostriches and B. coli from pigs to determine the specific status of B. struthionis. The morphological characteristics of the trophozoites of Balantidium from ostriches were reviewed in 100 trophozoites from two isolates. The macronucleus' shape of the ostrich Balantidium was highly variable, thus the use of this criterion for diagnostic purposes is not reliable. Besides, very few trophozoites showed a deep depression in their macronucleus and almost all the trophozoites conform to the description of B. coli. The complete sequence of the DNA coding for the 18s-rRNA-ITS1-5.8s-rRNA-ITS2 regions were obtained by PCR from five pig and five ostrich isolates. The sequences corresponding to the 18s and 5.8s-rRNA genes were identical for the ostrich and pig isolates. Two clearly different genotypes were found in the analysis of the ITS1 and ITS2 regions of the pig isolates; the genotype A was identified in all isolates, while the genotype B was found in only two of them. Their sequences show clear differences from that published corresponding to a B. coli gorilla isolate, which we will consider as a different genotype, C. In our opinion, these different B. coli genotypes reflect the genetic variability of this organism, but further studies would be necessary to determine if it could have practical importance. The polymorphism of the ITS regions have been also found in the ostrich isolates. The same genotypes A and B have been identified, although not as mixed infections. The morphological characteristics and the genetic results suggest that the species name B. struthionis is a synonym of B. coli; however, until experimental infections are carried out to determine if the parasite is transmissible between pigs and ostriches, it would be preferable to tentatively designate it as B. coli-like.

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.

Entamoeba struthionis n.sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus).

In the present work we identify the species of Entamoeba from ostriches (Struthio camelus). The complete sequence of the small subunit ribosomal RNA gene from this organism has been compared with those published for other species of the genus and clear differences have been found. These results confirm previous data which showed differences on parasite morphology and class of host with the other Entamoeba species. Taking all these data together, it can be concluded that the organism from ostriches is a new species whose proposed name is Entamoeba struthionis n.sp. This species probably infects rheas (Rhea americana), but genetic analysis of isolates from this host should be performed to confirm morphological data. Also, comparison of gene sequences with data from other authors on cysts recovered from human stool samples showed the possibility that this amoeba may affect humans. Further studies are needed to determine the risk of transmission of this new species to humans.

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.

Parasites from farmed ostriches (Struthio camelus) and rheas (Rhea americana) in Europe.

During a 4-year-period, more than 500 ostriches and several rheas, all born in European countries and raised in Spain and Portugal, have been analyzed for the presence of ectoparasites and endoparasites. A total of 29 parasite species have been found, most of them of the gastrointestinal tract. Some of the helminth species found may represent spureous parasitosis, as only the eggs (of an ascarid and a trematode) were found in some samples. From the organisms identified, the ectoparasites (lice-Struthiolipeurus rheae, S. nandu; mites-Dermoglyphus pachycnemis, Gabucinia bicaudata), helminths (cestoda-Houttuynia struthionis- and nematoda-Libyostrongylus sp., Codiostomum struthionis-) and the ciliate Balantidium struthionis are known as ratite specific parasites. Capillaria eggs and larvae were also found; there are no previous records of this parasite from ostriches, and the data available do not allow to do a temptative specific diagnosis. Among protozoa, most of the species now found are described for the first time in ratites. They include organisms also found in other birds (Trichomonas gallinae, Tetratrichomonas gallinarum, Chilomastix gallinarum, Spironucleus meleagridis and Pleuromonas jaculans), and organisms whose specific status cannot be established until further analysis are performed (Cryptosporidium sp., Eimeria sp. and/or Isospora sp., Entamoeba sp. of the one-nucleate and of the eight-nucleate mature cyst groups, Endolimax sp., Iodamoeba sp., Monocercomonas sp., Retortamonas sp., Giardia sp., Blastocystis sp. and euglenids).

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: 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.