Molecular identification and evolution of protozoa belonging to the <i>Parabasalia</i> group and the <i>genus Blastocystis</i>

My research project is focused in the study two groups of protists: the Parabasalia and Blastocystis, an emerging parasite. The first part is based on the identification and pathogenicity of trichomonads. We confirmed that Pentatrichomonas hominis could be the aetiological agent of gastrointestinal troubles and that the waterborne could be the transmission mode. We confirmed the presence of some parabasalid outside their natural such as lungs and intestinal tract. We found Trichomonas tenax in the lungs, in the feces as well as in the colon, and with Trichomonas vaginalis in the colon of an immunocompetent individual. In the second part, we studied the evolution and systematics of parabasalids using as markers glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin and elongation factor EF-1a. Our data indicate that increasing the number of taxa as well as the addition of new molecular markers greatly improves the robustness of the parabasalid tree and suggest that the ancestral parabasalid cell exhibited a morphologically simple structure. In the third part I have conducted the first investigations onto the molecular epidemiology of human Blastocystis isolates in Italy and Lebanon. In the last part we described a case report of multiple coinfections with Blastocystis isolates in a French patient showing that rural lifestyle and traveling history represent risk factors for transmission and multiple infections with Blastocystis

Molecular phylogeny and evolution of <i>Parabasalia</i> with improved taxon sampling and new protein markers of actin and elongation factor-1α

Background: Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa—in particular determining the root—is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences. Principal Findings: Actin and elongation factor-1a genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported. Conclusions/Significance: We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids

influenza monitoring in sardinia italy identifies h3 subtype in mediterranean wild migratory birds

Introduction: Wild migratory birds are global distributors of pathogens. Sardinia, Italy, is the second largest Island in the Mediterranean and is a land bridge between Europe and Africa. Methodology: We designed a surveillance protocol to investigate wild migratory birds for presence, frequency, and type of avian influenza viruses. We collected over 4,000 avian samples and compared three sampling methods, fecal, cloacal, and tracheal, to determine the most productive for virus identification. To determine frequency of infection, RNA was extracted and RT-PCRs for avian influenza virus genes were run. Positive samples were cultivated for live virus, sub typed and sequenced. Results: Forty-four samples were positive for influenza nucleoprotein gene. We identified two previously unidentified H3 subtype strains and found cloacae to have the highest rate of virus identification and fecal sampling to provide quality RNA and repeatable results for determination of virus presence. Conclusion: Our investigation provides information on the frequency of Mediterranean avian influenza viruses, and validates the initiation of an avian influenza surveillance protocol. Taken together with global avian influenza findings, these results give insight into infectious disease distributions which is important for viral pandemic monitoring and design of preventative measures

Molecular Phylogeny and Evolution of Parabasalia with Improved Taxon Sampling and New Protein Markers of Actin and Elongation Factor-1α

BACKGROUND: Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa-in particular determining the root-is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences. PRINCIPAL FINDINGS: Actin and elongation factor-1α genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported. CONCLUSIONS/SIGNIFICANCE: We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids

Cell Lines for the Development of African Swine Fever Virus Vaccine Candidates: An Update

African swine fever virus (ASFV) is the etiological agent of a highly lethal disease in both domestic and wild pigs. The virus has rapidly spread worldwide and has no available licensed vaccine. An obstacle to the construction of a safe and efficient vaccine is the lack of a suitable cell line for ASFV isolation and propagation. Macrophages are the main targets for ASFV, and they have been widely used to study virus&ndash;host interactions; nevertheless, obtaining these cells is time-consuming and expensive, and they are not ethically suitable for the production of large-scale vaccines. To overcome these issues, different virulent field isolates have been adapted on monkey or human continuous cells lines; however, several culture passages often lead to significant genetic modifications and the loss of immunogenicity of the adapted strain. Thus, several groups have attempted to establish a porcine cell line able to sustain ASFV growth. Preliminary data suggested that some porcine continuous cell lines might be an alternative to primary macrophages for ASFV research and for large-scale vaccine production, although further studies are still needed. In this review, we summarize the research to investigate the most suitable cell line for ASFV isolation and propagation

Cell Lines for the Development of African Swine Fever Virus Vaccine Candidates: An Update

African swine fever virus (ASFV) is the etiological agent of a highly lethal disease in both domestic and wild pigs. The virus has rapidly spread worldwide and has no available licensed vaccine. An obstacle to the construction of a safe and efficient vaccine is the lack of a suitable cell line for ASFV isolation and propagation. Macrophages are the main targets for ASFV, and they have been widely used to study virus–host interactions; nevertheless, obtaining these cells is time-consuming and expensive, and they are not ethically suitable for the production of large-scale vaccines. To overcome these issues, different virulent field isolates have been adapted on monkey or human continuous cells lines; however, several culture passages often lead to significant genetic modifications and the loss of immunogenicity of the adapted strain. Thus, several groups have attempted to establish a porcine cell line able to sustain ASFV growth. Preliminary data suggested that some porcine continuous cell lines might be an alternative to primary macrophages for ASFV research and for large-scale vaccine production, although further studies are still needed. In this review, we summarize the research to investigate the most suitable cell line for ASFV isolation and propagation

Blastocystis, an unrecognized parasite: an overview of pathogenesis and diagnosis.

International audienceBlastocystis sp. is among the few enteric parasites with a prevalence that often exceeds 5% in the general population of industrialized countries and can reach 30-60% in developing countries. This parasite is frequently found in people who are immunocompromised (patients with human immunodeficiency virus/acquired immunodeficiency syndrome or cancer) and a higher risk of Blastocystis sp. infection has been found in people with close animal contact. Such prevalence in the human population and the zoonotic potential naturally raise questions about the impact of these parasites on public health and has increased interest in this area. Recent in vitro and in vivo studies have shed new light on the pathogenic power of this parasite, suggesting that Blastocystis sp. infection is associated with a variety of gastrointestinal disorders, may play a significant role in irritable bowel syndrome, and may be linked with cutaneous lesions (urticaria). Despite recent significant advances in the knowledge of the extensive genetic diversity of this species, the identification of extracellular proteases as virulence factors and the publication of one isolate genome, many aspects of the biology of Blastocystis sp. remain poorly investigated. In this review, we investigate several biological aspects of Blastocystis sp. (diversity and epidemiology, diagnosis tools and pathophysiology). These data pave the way for the following challenges concerning Blastocystis sp. research: deciphering key biological mechanisms and pathways of this parasite and clarification of its clinical impact in humans

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5′-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5′ and 3′ boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5′ trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5′ ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III

Prevalence of Tritrichomonas foetus infections in French catteries.

International audienceRecently, Tritrichomonas foetus, the known etiologic agent of bovine trichomonosis was identified in domestic cats in many countries around the world. In felids, this parasite would be a significant cause of large-bowel diarrhoea. Therefore the aim of the present study was to determine for the first time the prevalence of T. foetus infection in French catteries. In this epidemiological survey, rectal swabs from 140 cats participating in three international shows were tested for the presence of motile parasites by microscopy after culturing. The prevalence of T. foetus infection was 14.3% among cats (20/140) and 15.9% among catteries (18/117). These values were similar to those previously obtained in other European countries. Except for the age, no significant associations were found between the presence of T. foetus and various risk factors of infection such as the size of the cattery, the type of food, or the vicinity of a dog. Internal transcribed region of the ribosomal DNA unit was sequenced from the 20 T. foetus isolates identified in this study. They exhibited 100% identity and are homologous with other sequences of strains isolated from domestic cats in other countries