Evaluación bibliométrica de la producción científica mundial sobre el virus Toscana: Bibliometric evaluation of the world scientific production on the Toscana virus

Introducción: Los virus de la fiebre de la Mosca de los Arena (familia Bunyaviridae, género Phlebovirus), son un grupo de virus que se encuentra ampliamente distribuidas en la cuenca mediterránea, donde se transmiten por la picadura de flebótomos infectados perteneciente al género Phlebotomus. Objetivo: Evaluar la Producción Científica mundial sobre el Virus Toscana en las bases de datos: Medline/Pubmed, Scopus, Scielo, y Science Citation Index. Metodología: Estudio bibliométrico en cuatro bases de datos, caracterizando la producción científica global sobre el Virus Toscana, se determinó la cantidad, calidad (número de citas) y tipos de estudios desarrollados por cada país, caracterizando los años, la cooperación internacional, el desarrollo, el lugar de publicación, los autores (con índice H). Resultados: En Medline, se encontraron 23 artículos publicados. En Scopus, se encontraron 159 artículos publicados. En Scielo no fueron encontrados artículos, y en SCI fueron encontrados 22 artículos. Discusión: la investigación sobre el TOSV es muy baja, por lo que resultaría importante el fomento de la investigación sobre este virus

Performance of Leishmania PFR1 recombinant antigen in serological diagnosis of asymptomatic canine leishmaniosis by ELISA

Abstract Background Leishmania infantum is a protozoan parasite transmitted by phlebotomine sand flies that causes life-threatening disease in humans and dogs. The dog is the primary reservoir of the parasite and early diagnosis of canine leishmaniosis is crucial at the clinical and epidemiological level. The currently available serological tests for CanL diagnostic show limitations therefore the aim of the present study was to investigate the diagnostic performance of an indirect antibody ELISA based on the Leishmania infantum recombinant antigen PFR1 in asymptomatically infected dogs. One hundred fifty-six dogs including Leishmania-free experimental Beagles and pet dogs from England, Scotland and Leishmania-endemic Murcia in Spain, were tested with the assay. The later were also tested with two commercial L. infantum crude antigen ELISAs (INgezim and Civtest, respectively) and a real-time kinetoplast PCR test. Results Anti-PFR1 antibodies were detected in the four groups of dogs, and the mean log-transformed optical density (OD) values were lowest in Beagles and in dogs from England and highest among dogs from Murcia (p < 0.05). Using the highest OD in beagles as the PFR1 ELISA cut-off point, the estimated seroprevalence was 27% (14-40%) in dogs from Murcia, 4% (0-9%) in dogs from Scotland and 3% (0-8%) in dogs from England (p < 0.05). Seroprevalence in dogs from Murcia according to the INgezim and Civtest ELISAs were 24% (12-37%) and 31% (18-45%), respectively, whilst the prevalence of infection based on PCR in these dogs was 73% (60-86). The percentages of PFR1-positive dogs that tested negative on the INgezim and Civtest ELISAs were 30% and 35%, respectively, and all of them tested positive on the PCR test. Relative to the PCR, the specificity, sensitivity and area under the ROC curve of the PFR1 ELISA were 100%, 36% and 0.74 (0.63-0.86), respectively. Conclusions The ability shown by the PFR1 ELISA to detect infected dogs that go undetected by the crude antigen ELISAs is clinically and epidemiologically useful and PFR1 could be considered a candidate for a multi-antigen-based immunoassay for early detection of L. infantum infected dogs

Antigeny ze slin flebotomů a protilátková odpověd pobodaných hostitelů

Leishmanióza je onemocnění, které se vyskytuje především v rozvojových zemích Afriky, Asie a Latinské Ameriky a jehož význam je velmi podceňován. Zároveň je toto onemocnění přítomno také severní Ameriky a Evropy a kvůli globálnímu oteplování se šíří spolu se svými rezervoáry a přenašeči - flebotomy do vyšších nadmořských výšek a oblastí severnějších a jižnějších zeměpisných šířek. V této dizertační práci proto shrnujeme současné informace o šíření leishmaniózy a jejích přenašečů a rezervoárů a v Evropě. Riziko přenosu leishmaniózy je úzce spjato s pravděpodobností kontaktu s flebotomy a protilátková odpověď založená na imunoglobulinech typu G (IgG) proti jejich slinám byla prokázána jako vhodný nástroj pro zjištění poštípání napadených hostitelů. Sliny flebotomů ovšem mají několik nevýhod pro tuto metodu - jedná se o směs mnoha látek s různými chemickými a antigenními vlastnostmi a jejich získání přímo z flebotomů je velmi pracné a nákladně. Proto jsme se zaměřili na přípravu rekombinantních proteinů, které budou zastupovat nejsilnější slinné antigeny a nahradí tak slinné žlázy jako základ screeningových metod. Zvolili jsme dva druhy evropských flebotomů - Phlebotomus (P.) papatasi a Phlebotomus (P.) perniciosus, a identifikovali jsme nejvíce antigenní proteiny v jejich slinných žlázách, které jsme...Leishmaniases are neglected diseases occurring mainly in developing countries of Africa, Asia and Latin America. However, these diseases are also present in Europe and North America and due to climate changes and human activities they spread to higher latitudes and altitudes. In these theses, we review the current information about the spread of leishmaniases, its vectors and reservoirs in Europe. The risk of Leishmania transmission is closely connected with the host-vector contact. Recently, immunoglobulin G (IgG) antibody response to sandfly saliva has been proven as a reliable marker of the host exposure. However, sandfly saliva is a complex mixture of components with different chemical and antigenic properties and it is laborious and expensive to acquire. Therefore, we have focused on preparation of major salivary antigens in the form of recombinant proteins that would be capable to replace the saliva in immunological screenings. We choose two European vectors, Phlebotomus (P.) papatasi and Phlebotomus (P.) perniciosus and identified their major salivary antigens by western blotting and mass spectrometry. We expressed these proteins in the bacterial system and test their antigenicity using ELISA and western blotting with sera of mice and dogs bitten by these sand fly species. The most promising...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Comparative Evolution of Sand Fly Salivary Protein Families and Implications for Biomarkers of Vector Exposure and Salivary Vaccine Candidates

Sand fly salivary proteins that produce a specific antibody response in humans and animal reservoirs have been shown to be promising biomarkers of sand fly exposure. Furthermore, immunity to sand fly salivary proteins were shown to protect rodents and non-human primates against Leishmania infection. We are missing critical information regarding the divergence amongst sand fly salivary proteins from different sand fly vectors, a knowledge that will support the search of broad or specific salivary biomarkers of vector exposure and those for vaccines components against leishmaniasis. Here, we compare the molecular evolution of the salivary protein families in New World and Old World sand flies from 14 different sand fly vectors. We found that the protein families unique to OW sand flies are more conserved than those unique to NW sand flies regarding both sequence polymorphisms and copy number variation. In addition, the protein families unique to OW sand flies do not display as many conserved cysteine residues as the one unique to the NW group (28.5% in OW vs. 62.5% in NW). Moreover, the expression of specific protein families is restricted to the salivary glands of unique sand fly taxon. For instance, the ParSP15 family is unique to the Larroussius subgenus whereas phospholipase A2 is only expressed in member of Larroussius and Adlerius subgenera. The SP2.5-like family is only expressed in members of the Phlebotomus and Paraphlebotomus subgenera. The sequences shared between OW and NW sand flies have diverged at similar rates (38.7 and 45.3% amino acid divergence, respectively), yet differences in gene copy number were evident across protein families and sand fly species. Overall, this comparative analysis sheds light on the different modes of sand fly salivary protein family divergence. Also, it informs which protein families are unique and conserved within taxon for the choice of taxon-specific biomarkers of vector exposure, as well as those families more conserved across taxa to be used as pan-specific vaccines for leishmaniasis

Recombinant antigens from Phlebotomus perniciosus saliva as markers of canine exposure to visceral leishmaniases vector

BACKGROUND: Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs. METHODOLOGY/PRINCIPAL FINDINGS: The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate. CONCLUSIONS/SIGNIFICANCE: Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral leishmaniases for vector exposure and for estimating the risk of L. infantum transmission in dogs.This research has been funded by Instituto de Salud Carlos III ( PS09/02075 and PI1202021); Xunta de Galicia (10 PXIB 918 273PR) Fundación Mutua Madrileña. Centro de Investigacion Biomedica en Red Fisiopatología de la Obesidad y Nutrición is an ISCIII iniciative (CB06/03). CC is funded by CIBER Fisiopatología Obesidad y Nutrición (CB06/03), LLS is funded by the University Professional Development Program (FPU) of the Spanish Ministry of Education, SB-F by Xunta de Galicia. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.S

Identifikace, exprese a charakterizace antigenů slinných žláz Phlebotomus orientalis

Flebotomové (Diptera: Psychodidae) jsou přenašeči leishmanií (Kinetoplastida: Trypanosomatidae), medicínsky a veterinárně významných parazitů způsobujících leishmaniózu. Tito prvoci jsou inokulováni společně s obsahem slinných žláz do hostitele během sání krve. Slinné proteiny hrají důležitou roli v počáteční fázi vývoje parazita v hostiteli, ale jsou zásadní i pro samice flebotomů a jejich sání krve. Bylo prokázáno, že proteiny ze slin flebotomů jsou mimo jiné schopny hydrolýzy ATP a ADP, vazby biogenních aminů a dalších funkcí, díky nimž dochází k inhibici hemostatických reakcí a modulaci reakcí imunitního systému, což ve svém důsledku umožňuje úspěšné nasátí krve. V minulosti bylo složení slin popsáno u dvanácti druhů flebotomů a my přidali další - Phlebotomus orientalis, který je hlavním přenašečem Leishmania donovani, původce lidské viscerální leishmaniózy ve východoafrických zemích. Z jeho slinných žláz jsme popsali třináct proteinových rodin, což odpovídá dříve publikovaným studiím z ostatních druhů flebotomů, a také jsme identifikovali hlavní antigeny pro dva druhy hostitelů - myši a psy. U opakovaně poštípaných hostitelů dochází k tvorbě specifických IgG protilátek proti slinných proteinům flebotomů. Antigeny ze slinných žláz (nebo jejich varianty připravené v rekombinantní formě) lze...Sand flies (Diptera: Psychodidae) are vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae), medically and veterinary important parasites causing leishmaniasis. These protozoans are inoculated into the host during blood-feeding together with the vector saliva. Salivary components are crucial for sand fly female blood-sucking but also play a major role in the initial stage of parasite development. It was demonstrated that this rich mixture of proteins performs different functions after its injection into the host like hydrolyzing ATP and ADP, binding biogenic amines, and others which inhibits the haemostatic reaction and modulate the immune system thus enables the successful feeding. So far, cDNA salivary transcriptomes have been constructed for twelve sand fly species and we added another one - Phlebotomus orientalis, the main vector of Leishmania donovani, a causative agent of human visceral leishmaniasis in East African countries. We described members of thirteen protein families which corresponded with previously published salivary cDNA libraries of other sand fly species and showed the main salivary antigens using sera of experimentally bitten mice and naturally exposed dogs. Specific IgG antibody response is induced against sand fly salivary proteins in repeatedly bitten hosts....Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Recombinant proteins and synthetic peptides as tools for studies on antibody response against Phlebotomus orientalis

Sliny flebotomů obsahují proteiny a peptidy, které mají důležitou roli při sání krve. Některé z těchto proteinů jsou zároveň antigenní a v opakovaně poštípaném hostiteli se proti nim vytvářejí protilátky. Pomocí imunoblotu s psími séry jsme identifikovali antigenní proteiny slinných žláz Phlebotomus orientalis, který je hlavním přenašečem viscerální leishmaniózy v Súdánu a Etiopii. Pět nejnadějnějších proteinů bylo připraveno v rekombinantní formě v bakteriích E. coli a použito jako antigen v ELISA testech se séry domácích zvířat z Etiopie a myší z chovu, které byly vystaveny poštípání P. orientalis. Jako pozitivní kontrola byl použit homogenát slinných žláz (SGH). Nejlepší antigenní vlastnosti prokázaly dva rekombinantní proteiny Yellow-related protein PorSP24 a ParSP25-like protein PorSP65. Protilátková odpověď proti těmto proteinům vysoce korelovala s odpovědí proti SGH, zejména v testech s ovčími a psími séry. V testech s psími séry však byla u obou antigenů detekována také nespecifická vazba protilátek. Dále bylo zjištěno, že v testech s SGH a rekombinantními proteiny slinných žláz P. orientalis nedochází ke zkříženým reakcím se séry myší poštípaných P. papatasi a Sergentomyia schwetzi. Z obou výše zmíněných slinných proteinů byly následně navrženy a syntetizovány peptidy, které by měly být...Sand fly saliva contains proteins and peptides that have an important role in bloodfeeding. Some of those proteins are antigenic and repeated sand fly bites result in a specific antibody response of the bitten host. Antigenic salivary proteins of Phlebotomus orientalis, main vector of visceral leishmaniasis in Sudan and Ethiopia, were identified using immunoblot with dog sera. The 5 most promising antigens were expressed in an E. coli bacterial system. Subsequently, these proteins were tested in ELISA with sera of domestic animals from Ethiopia naturally exposed to P. orientalis, and with sera of mice bitten experimentally by this sand fly species. Salivary gland homogenate (SGH) was used as the positive control. The best antigenic properties were detected in two recombinant proteins, Yellow-related protein PorSP24 and ParSP25-like protein PorSP65, especially in tests with sheep and dog sera. However, nonspecific binding of dog sera was also detected using both antigens. In addition, we proved that sera of mice repeatedly bitten by P. papatasi and Sergentomyia schwetzi do not crossreact with SGH and the tested recombinant proteins of P. orientalis. In a second part of this thesis we designed peptides representing epitopes recognized by specific anti-saliva antibodies. Two peptides were derived from...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Charakterizace a antigenní vlastnosti yellow-related proteinů ze slinných žláz flebotomů

3 ABSTRAKT Yellow-related proteiny (YRP) tvoří početnou rodinu proteinů, jejíž členové byli identifikováni v transkriptomech slinných žláz všech dosud studovaných druhů flebotomů. Tato rodina je zařazena do skupiny tzv. MRJP/Yellow proteinů, které se vyskytují u hmyzu i některých dalších organismů. Přestože tyto proteiny sdílejí podobnou terciární strukturu tvořenou šestiramenným beta-propelerem s centrálně umístěným tunelem, liší se ve svých funkcích. Struktura a amin-vázající vlastnosti YRP flebotomů byly popsány pouze na základě jediné studie u druhu Lutzomyia longipalpis. V naší práci jsme namodelovali struktury celkem 32 YRP ze slinných žláz 13 druhů flebotomů. Zatímco celkový tvar a struktura všech proteinů byla obdobná, jednotlivé YRP se mezi druhy i v rámci jednoho druhu odlišovaly zejména v povrchovém náboji, vlastnostech tunelu a v aminokyselinovém složení vazebného místa pro biogenní aminy. Tyto modifikace ukazují na rozdílné funkce jednotlivých proteinů, jež byly experimentálně potvrzeny ve druhé části našeho projektu. Zaměřili jsme se na vazebné vlastnosti YRP druhů Phlebotomus perniciosus a P. orientalis, kteří jsou důležitými přenašeči prvoků rodu Leishmania. Síla vazby pro serotonin, histamin a katecholaminy se lišila jak mezi druhy, tak v rámci obou proteinů každého druhu. Přes tyto...2 ABSTRACT Yellow-related proteins (YRPs) form an abundant protein family, whose members were found in salivary gland transcriptomes of all sand fly species studied up to date. This protein family belongs to the group of MRJP/Yellow proteins occurring in insects and some other organisms. Though sharing similar folding as a six-bladed β-propeller comprising central tunnel, MRJP/Yellow proteins adopted different functions. The structure and biogenic amine-binding property described for the sand fly salivary YRPs was based on a single study conducted on Lutzomyia longipalpis. In the present work, we have modelled the structures of 32 salivary YRPs belonging to 13 different sand fly species. We have shown the general structural similarity of these proteins along with both inter- and intra-specific differences in surface charge, tunnel parameters and in amino acids composition of the amine-binding motif. These modifications indicated divergence in function of individual YRPs, which was experimentally verified in the second project focused on identification of the amine-binding properties of YRPs in two important vectors of Leishmania; Phlebotomus perniciosus and P. orientalis. In each species, two YRPs differ in affinities for biogenic amines serotonin, histamine and catecholamines. However, in both sand fly...Katedra parazitologieDepartment of ParasitologyFaculty of SciencePřírodovědecká fakult