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58 • "PROTIST—2016
Periodontal disease is one of the most important health concerns for companion animals. Previous studies have demonstrated that at least half of all dogs will have some form of the disease within their lifetime which, without early intervention, can lead to painful periodontal ligament destruction, alveolar bone loss, and eventual loss of teeth. The recent focus of research into canine periodontitis has been the identification and characterisation of the bacterial communities present. However, other microorganisms are known to inhabit the oral cavity and could also influence the disease process. Our recent research has identified two oral protists that can inhabit the canine oral periodontium. Trichomonas sp. and Entamoeba gingivalis were detected using PCR and next generation sequencing and had an overall prevalence of56.52 % (52/92) and 4.34 % (4/92) respectively in UK dogs. Both were statistically associated to animals with periodontal disease indicating their potential involvement in the periodontal disease process. Further work has led to the development of a quantitative PCR assay to measure protist abundance. The qPCR assay has been utilised on plaque samples, collected over a 60 week period from individual teeth of miniature schnauzer dogs (n =52), to investigate longitudinal changes in abundance of both Trichomonas sp. and E. gingivalis as dogs progress from mild gingivitis to early stage periodontitis (<25 % attachment loss). These findings provide the first conclusive evidence for the presence of canine oral protozoa in dog plaque and suggest a possible role for protozoa in the periodontal disease process.
NAKED AMOEBAE OF UKRAINIAN POLIS-SYAFAUNA Patsyuk M.K.
Zhytomyr Ivan Franko State University kostivna@ukr.net
At present, the naked amoebae of the Kyiv, Zhytomyr and Volyn Polissya fauna are represented by 47 species, which belong to 5 classes, 11 orders, 15 families and 20 species.
According to our research the most distributed in the Ukrainian Polissya waters are: Saccamoeba stagnicola Page, 1974, Korotnevella stella Schaeffer, 1926, Vexillifera sp., Vannella (cf) lata Page, 1988, Cochliopodium sp.(1), Mayorella cantabrigiensis Page, 1983, Thecamoeba striata Penard, 1890, Vahlkampfia sp.(1), Vahlkampfia sp.(2). The such species as Amoeba proteus Leidy, 1878, Polychaos sp., Saccamoeba wakulla Bovee, 1972, Rhizamoeba sp. (2), Cochliopodium sp. (2), Pellita digitata (Greef, 1866) Smirnov et Kudryavtsev, 2004, Mayorella
penardi Page, 1972, Thecamoeba sphaeronucleolus Greef, 1891, Penardia mutabilis Cash, 1904 (have been registered by us in the Volyn Polissya waters), Willaertia sp. and Acanthamoeba sp. (have been registered by us in the Zhytomyr Polissya waters) are less distributed. Among the above listed species 3, which belong to genera Vahlkampfia Chatton et Lalung-Bonnaire, 1912 and Acanthamoeba Vol-konsky, 1931 are parasitic. The 14 naked amoebae morphotypes are registered in the different water-bodies: polytactic, monotactic, flamellian, lens-like, striate, rugose, lingulate, lanceolate, fan-shaped, in the different waters mayorellian, dactylopodial, acanthopodial, branched and eruptive. The amoebae with polytactic and acanthopodial morphotypes might be mentioned as the least distributed.
EFFECT OF THE SPECTRUM OF AVAILABLE NITROGEN SOURCES ON PROROCENT-RUM MINIMUM MORPHOLOGY AND PHYSIOLOGY
Pechkovskaya S.A.12, Matantseva O.V.2, Filatova N.A.2, Telesh I.V.3
1 - Department of microbiology, St. Petersburg State University, Russia
2 - Institute of Cytology RAS, St. Petersburg, Russia
3 - Zoological Institute RAS, St. Petersburg, Russia juliya_94@inbox.ru
Dinoflagellates are the prosperous group of aquatic eukaryotes. The ongoing eutrophication of coastal areas provides a competitive advantage to these organisms due to their ability to assimilate nitrogen from a variety of dissolved organic and inorganic sources. The analysis of genomic and transcriptomic databases revealed the presence of transporters and enzymes involved in uptake and assimilation of basic nitrogenous compounds present in seawater in dinoflagellates. In laboratory experiments with the culture of dinoflagellates Prorocentrum minimum, we investigated how the spectrum of available nitrogen sources influences morphological and physiological parameters of cells. We showed that addition of nitrate, ammonium, urea, glycine and their combinations to the culture growing on nitrate causes various physiological cell responses. The incorporation of H3-uridine by dinoflagellate cells revealed an increase in the RNA synthesis rate after the addition of supplementary nitrogen sources to the culture. Remarkably, the extent to which RNA synthesis was enhanced differed depending on the available nitrogen sources. For example, the largest increase in the level of RNA synthesis was achieved in response to addition of the ammonium/urea combination. The analysis of
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the natural fluorescence of cellular photosynthetic pigments demonstrated that the observed increase in transcription was not directly linked to their concentration in cells. Moreover, this analysis revealed heterogeneity in the pigment fluorescence among distinct cells within the model culture. Funded in part by RFBR, project 15-29-02706.
THE HIGH COMPLEXITY AND DYNAMIC EVOLUTION OF THE RAS SUPERFAMILY OF GTPASES IN NAEGLERIA Petrzelkova R.1, Herman E.K.2, Dacks J.B.2, Elias M.1
1 - University of Ostrava, Faculty ofScience, Department ofBiology and Ecology, Ostrava, Czech Republic
2 - Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada losssina@gmail.com
Ras superfamily GTPases constitute a vast group of proteins involved in many eukaryote-specific processes. The last eukaryotic common ancestor appears to have possessed at least several tens of Ras superfamily genes, but gene duplications and losses in different eukaryotic lineages have modified this ancestral set such that substantially different gene complements may be present in different eukaryote groups. One extreme are taxa harbouring an extensively expanded Ras superfamily complement, as is the case of a free-living amoeboflagellate Nae-gleria gruberi (Heterolobosea). Recently, genome sequences of three strains of Naegleria fowleri, a causative agent of primary amebic meningoencephalitis (PAM), became available for analysis. In order to assess the differences between the two species and the three strains, we identified and annotated the Ras superfamily genes in the newly sequenced N. fowleri genomes and reannotated the respective gene complement in the previously published N. gruberi genome. The sets of Ras superfamily genes turned out to differ substantially between the two species, as N. gruberi harbours over 350 genes, whereas N. fowleri exhibits a much less expanded set with "only" over 200 genes. In contrast, little, if any, differences were found for the three N. fowleri strains. Phylogenetic analyses revealed both species-specific duplications and losses as the factors responsible for the different gene numbers in the two species. The evolution of the Ras superfamily in the genus Naegleria is thus surprisingly dynamic and points to a hidden level of differentiation in cellular physiology of different Naegleria species.
EXPRESSION AND PURIFICATION OF A PNEUMOCYSTIS JIROVECII SYNTHETIC RECOMBINANT ANTIGEN AND APPLICATION IN THE DEVELOPMENT OF A SEROLOGICAL RAPID DIAGNOSTIC TEST Pinto Mafalda12, Cardoso Fernando2, Gomes Ines1,3, Pereira Eulália4, Peixoto Miguel4, Franco Ricardo1, Matos Olga2
1 - UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa (UNL), Portugal
2 - Medical Parasitology Unit, Group ofOpportunistic Protozoa/HIV and Other Protozoa, GHTM, Instituto de Higiene e Medicina Tropical, UNL, Portugal
3 - Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal
4 - UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciencias, Universidade do Porto, Portugal
omatos@ihmt.unl.pt
Pneumocystis pneumonia (PcP) is an infectious disease caused by Pneumocystis jirovecii, an atypical fungus. PcP remains a major cause of respiratory illness among immunosuppressed patients. Current PcP diagnosis is based on the detection of P. jirovecii in respiratory specimens, obtained by invasive methods such as bronchoalveolar lavage, followed by cytochemical staining, immunofluorescent staining with monoclonal antibodies (IF/Mab) or PCR. Therefore, the possibility of an early diagnostic method allowing the use of biological specimens obtained non-invasively, is highly desirable. Rapid diagnostic tests (RDTs) using gold nanoparticles (AuNPs) allow a more sensitive, fast and cheap diagnosis, to be used in developing countries. The goal of this work is to develop an immunochromatographic RDT for the detection of P. jirovecii in non-invasive specimens like serum. In this test, spherical AuNPs are conjugated with a multi-epitope synthetic recombinant antigen (msr) which will allow the detection of circulating anti-P. jirovecii antibodies in sera. In order to obtain the highest amount of pure antigen, the expression vector pLATE 31, which contains the coding sequence for the MSG g antigen, was isolated and cloned in E. coli XJb (DE3). Extraction and purification through affinity chromatography with immobilized metallic ions, ELISA, SDS-PAGE and Western-Blot, were performed with the objective to obtain the maximum quantity of antigen and determine its purity. The antigen was then used to form bionanoconjugates with AuNPs, previously
