Francisco Ponce-Gordo

Genetic heterogeneity in internal transcribed spacer genes of Balantidium coli (Litostomatea, Ciliophora).

The species Balantidium coli is the only ciliate that parasitizes humans. It has been described in other primates, and it has been proposed that the species B. suis from pigs and B. struthionis from ostriches are synonyms of B. coli. Previous genetic analysis of pig and ostrich Balantidium isolates found a genetic polymorphism in the ITS region but its taxonomic relevance was not established. We have extended the genetic analysis to Balantidium isolates of pig, gorilla, human and ostrich origin. We have PCR-amplified and sequenced the ITS region of individual Balantidium cells. The predicted ITS secondary structures of the sequences obtained were transferred by homology modelling to the sequences of other Trichostomatia ciliates (Isotricha, Troglodytella, Lacrymaria and Spathidium) and compared to determine the importance of the differences in the primary sequences. The results show that the ITS2 secondary structure of the species considered follows the general pattern of other ciliates, although with some deviations. There are at least two main types of ITS sequence variants in B. coli which could be present in the same cell and they are common to the mammal and avian hosts studied. These data do not support B. suis and B. struthionis as distinct species.

High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain.

Echinococcus granulosus is the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated with E. granulosus sensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography of E. granulosus s. s. G1 in part of its main distribution range in Europe using 8274 bp of mtDNA; (ii) compare the results with those derived from previously used shorter mtDNA sequences and highlight the major differences. We sequenced a total of 91 E. granulosus s. s. G1 isolates from six different intermediate host species, including humans. The isolates originated from seven countries representing primarily Turkey, Italy and Spain. Few samples were also from Albania, Greece, Romania and from a patient originating from Algeria, but diagnosed in Finland. The analysed 91 sequences were divided into 83 haplotypes, revealing complex phylogeography and high genetic variation of E. granulosus s. s. G1 in Europe, particularly in the high-diversity domestication centre of western Asia. Comparisons with shorter mtDNA datasets revealed that 8274 bp sequences provided significantly higher phylogenetic resolution and thus more power to reveal the genetic relations between different haplotypes.

Genetic diversity and phylogeography of highly zoonotic Echinococcus granulosus genotype G1 in the Americas (Argentina, Brazil, Chile and Mexico) based on 8279 bp of mtDNA

Echinococcus granulosus is a taeniid cestode and the etiological agent of an infectious zoonotic disease known as cystic echinococcosis (CE) or hydatid disease. CE is a serious public health concern in many parts of the world, including the Americas, where it is highly endemic in many regions. Echinococcus granulosus displays high intraspecific genetic variability and is divided into multiple genotypes (G1-G8, G10) with differences in their biology and etiology. Of these, genotype G1 is responsible for the majority of human and livestock infections and has the broadest host spectrum. However, despite the high significance to the public and livestock health, the data on genetic variability and regional genetic differences of genotype G1 in America are scarce. The aim of this study was to evaluate the genetic variability and phylogeography of G1 in several countries in America by sequencing a large portion of the mitochondrial genome. We analysed 8279bp of mtDNA for 52 E. granulosus G1 samples from sheep, cattle and pigs collected in Argentina, Brazil, Chile and Mexico, covering majority of countries in the Americas where G1 has been reported. The phylogenetic network revealed 29 haplotypes and a high haplotype diversity (Hd=0.903). The absence of phylogeographic segregation between different regions in America suggests the importance of animal transportation in shaping the genetic structure of E. granulosus G1. In addition, our study revealed many highly divergent haplotypes, indicating a long and complex evolutionary history of E. granulosus G1 in the Americas.

New mitogenome and nuclear evidence on the phylogeny and taxonomy of the highly zoonotic tapeworm Echinococcus granulosus sensu stricto

Cystic echinococcosis, a zoonotic disease caused by Echinococcus granulosus sensu lato (s. l.), is a significant global public health concern. Echinococcus granulosus s. l. is currently divided into numerous genotypes (G1-G8 and G10) of which G1-G3 are the most frequently implicated genotypes in human infections. Although it has been suggested that G1-G3 could be regarded as a distinct species E. granulosus sensu stricto (s. s.), the evidence to support this is inconclusive. Most importantly, data from nuclear DNA that provide means to investigate the exchange of genetic material between G1-G3 is lacking as none of the published nuclear DNA studies have explicitly included G2 or G3. Moreover, the commonly used relatively short mtDNA sequences, including the complete cox1 gene, have not allowed unequivocal differentiation of genotypes G1-G3. Therefore, significantly longer mtDNA sequences are required to distinguish these genotypes with confidence. The main aim of this study was to evaluate the phylogenetic relations and taxonomy of genotypes G1-G3 using sequences of nearly complete mitogenomes (11,443bp) and three nuclear loci (2984bp). A total of 23 G1-G3 samples were analysed, originating from 5 intermediate host species in 10 countries. The mtDNA data demonstrate that genotypes G1 and G3 are distinct mitochondrial genotypes (separated by 37 mutations), whereas G2 is not a separate genotype or even a monophyletic cluster, but belongs to G3. Nuclear data revealed no genetic separation of G1 and G3, suggesting that these genotypes form a single species due to ongoing gene flow. We conclude that: (a) in the taxonomic sense, genotypes G1 and G3 can be treated as a single species E. granulosus s. s.; (b) genotypes G1 and G3 should be regarded as distinct genotypes only in the context of mitochondrial data; (c) we recommend excluding G2 from the genotype list.

Light microscopic morphometrics, ultrastructure, and molecular phylogeny of the putative pycnotrichid Ciliate, Buxtonella sulcata.

The ciliate, Buxtonella sulcata, was isolated from a bull cow near Tišnov, Czech Republic, and fixed for light (LM), scanning electron (SEM) and transmission electron microscopic (TEM) study. Presented here are the basic morphometrics from LM study, and the fine-structure of both somatic and vestibular ciliary, and other structures. While many morphological features are similar to ciliates belonging to the order Vestibuliferida, some differences have been discovered, and are presented here. Especially emphasized are the microtubular and fibrilar components of the basic kinetid structures for both somatic and vestibular regions of these protists. Also observed in both TEM and SEM samples were enigmatic membrane bulges at the base of many somatic cilia. These ciliates are seen to have abundant endocytoplasmic bacteria, as seen in LM and TEM. This evaluation of the ultrastructural morphology of B. sulcata from cattle is accompanied by detailed determination of its small subunit rRNA (SSU rRNA) gene sequence and also of internal transcribed spacers (ITS1-5.8rRNA-ITS2). All of these data will contribute to unravel the phylogenetic relationships of medically and veterinary important intestinal ciliates.

New insights into the molecular phylogeny of Balantidium (Ciliophora, Vetibuliferida) based on the analysis of new sequences of species from fish hosts

We obtained sequences of the small subunit ribosomal RNA (rRNA) for two new isolates of Balantidium from fishes, Balantidium polyvacuolum and Balantidium ctenopharingodoni. This is the first introduction of molecular data of Balantidium species from fish hosts in the phylogenetic analyses of the ciliate subclass Trichostomatia. Despite the fact that these species share morphological characteristics common to other species of Balantidium, the phylogenetic analysis of their sequences has shown that they are to be placed in a different branch closely related to the so-called Australian clade. Thus, our results indicate that the genus Balantidium is polyphyletic and possibly should be represented by two different genera; however, the analysis of more species from other poikilothermic hosts (amphibians, reptiles) should be made before a revised taxonomical proposal could be made.

Development of a specific polymerase chain reaction assay for the detection of Basidiobolus

The etiology of chronic diarrhea is complex in humans and animals. It is always necessary to evaluate a list of differential diagnosis, including bacteria, protozoa and fungi. Basidiobolomycosis is a fungal disease reported sporadically worldwide, mainly caused by B. ranarum, a frequent organism found in soil or in the intestine and skin of lizards and frogs. It is an opportunistic pathogen that causes infections characterized by granulomatous lesions in the subcutaneous tissues as well as in the intestinal wall in humans and animals. In this work we have developed a PCR technique to differentiate Basidiobolus from other causes of intestinal disease in dogs and humans. To test the specificity of the PCR assay we included closely related organisms, common intestinal microbiota and pathogenic organisms, such as Aspergillus, Candida, Cryptosporidium, Escherichia, Giardia, Mucor, Proteus, Rhizopus and Salmonella. Pythium insidiosum, which cause clinically similar disease in dogs but require a different treatment. Only Basidiobolus was positive to the PCR assay.

Morphology of Nyctotheroides hubeiensis Li et al. 1998 from Frog Hosts with Molecular Phylogenetic Study of Clevelandellid Ciliates (Armophorea, Clevelandellida)

The morphology of Nyctotheroides hubeiensis (Acta Hydrobiol. Sin. 1998, 22(suppl.):187), collected from the rectum of Phelophylax nigromaculatus, is presented in this paper based on detailed morphological information and molecular data. Our phylogenetic analysis showed that N. hubeiensis fell into the Nyctotheroides clade, which was strongly supported as monophyletic and clustered as basal to the genera Nyctotherus and Clevelandella. Also, the monophyly of the Order Clevelandellida and the affinity of parasitic nyctotherids and free‐living metopids were indicated in our work. The origin of clevelandellid ciliates as well as their possible evolutionary history was also discussed here; however, the analysis of more species from other vertebrate hosts (fish, reptiles) should be made before a well‐supported conclusion can be drawn.

Global phylogeography and genetic diversity of the zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1

Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682 bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n = 222), G1 (n = 212) and human G1 samples (n = 41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.

The benefits of analysing complete mitochondrial genomes: Deep insights into the phylogeny and population structure of Echinococcus granulosus sensu lato genotypes G6 and G7

Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the species complex Echinococcus granulosus sensu lato. Within this complex, genotypes G6 and G7 have been frequently associated with human CE worldwide. Previous studies exploring the genetic variability and phylogeography of genotypes G6 and G7 have been based on relatively short mtDNA sequences, and the resolution of these studies has often been low. Moreover, using short sequences, the distinction between G6 and G7 has in some cases remained challenging. The aim here was to sequence complete mitochondrial genomes (mitogenomes) to obtain deeper insight into the genetic diversity, phylogeny and population structure of genotypes G6 and G7. We sequenced complete mitogenomes of 94 samples collected from 15 different countries worldwide. The results demonstrated that (i) genotypes G6 and G7 can be clearly distinguished when mitogenome sequences are used; (ii) G7 is represented by two major haplogroups, G7a and G7b, the latter being specific to islands of Corsica and Sardinia; (iii) intensive animal trade, but also geographical isolation, have likely had the largest impact on shaping the genetic structure and distribution of genotypes G6 and G7. In addition, we found phylogenetically highly divergent haplotype from Mongolia (Gmon), which had a higher affinity to G6.