Publications by authors named "Vasiliki Christodoulou"

28 Publications

  • Page 1 of 1

Evidence of West Nile virus seropositivity in wild birds on the island of Cyprus.

Comp Immunol Microbiol Infect Dis 2021 Feb 21;74:101592. Epub 2020 Nov 21.

Department of Biological Sciences, University of Cyprus, PO Box 20537, Nicosia 1678, Cyprus. Electronic address:

West Nile Virus (WNV) is an emerging pathogen in Cyprus, with the first human case of infection reported in 2016, and another documented in 2018. A cluster of cases in humans was then reported in 2019. However, little is known regarding which avian species might bring WNV to Cyprus. Here, we investigated seroprevalence of WNV antibodies in migratory and resident birds, captured across Cyprus to assess to what extent human populations might be exposed to WNV. We used Enzyme-Linked Immunosorbent Assay (ELISA) to test for the presence of WNV antibodies in 836 avian blood samples of 44 species captured between 2015 and 2020. A seropositivity rate of 1.3 % was found. The majority of seropositive wild birds belonged to the migratory species Sylvia atricapilla, a common and widespread migrant, implying a high risk of WNV being introduced throughout Cyprus.
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http://dx.doi.org/10.1016/j.cimid.2020.101592DOI Listing
February 2021

Origin of Bluetongue Virus Serotype 8 Outbreak in Cyprus, September 2016.

Viruses 2020 01 14;12(1). Epub 2020 Jan 14.

Pirbright Institute, Woking, Surrey, GU24 0NF, UK.

In September 2016, clinical signs, indicative of bluetongue, were observed in sheep in Cyprus. Bluetongue virus serotype 8 (BTV-8) was detected in sheep, indicating the first incursion of this serotype into Cyprus. Following virus propagation, Nextera XT DNA libraries were sequenced on the MiSeq instrument. Full-genome sequences were obtained for five isolates CYP2016/01-05 and the percent of nucleotide sequence (% nt) identity between them ranged from 99.92% to 99.95%, which corresponded to a few (2-5) amino acid changes. Based on the complete coding sequence, the Israeli ISR2008/13 (98.42-98.45%) was recognised as the closest relative to CYP2016/01-05. However, the phylogenetic reconstruction of CYP2016/01-05 revealed that the possibility of reassortment in several segments: 4, 7, 9 and 10. Based on the available sequencing data, the incursion BTV-8 into Cyprus most likely occurred from the neighbouring countries (e.g., Israel, Lebanon, Syria, or Jordan), where multiple BTV serotypes were co-circulating rather than from Europe (e.g., France) where a single BTV-8 serotype was dominant. Supporting this hypothesis, atmospheric dispersion modelling identified wind-transport events during July-September that could have allowed the introduction of BTV-8 infected midges from Lebanon, Syria or Israel coastlines into the Larnaca region of Cyprus.
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http://dx.doi.org/10.3390/v12010096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019704PMC
January 2020

Leishmania infection in lagomorphs and minks in Greece.

Vet Parasitol Reg Stud Reports 2019 04 27;16:100279. Epub 2019 Feb 27.

Laboratory of Parasitology and Parasitic Diseases, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece. Electronic address:

Greece is an endemic country for human and canine leishmaniosis. Studies about the role of lagomorphs and minks in the epidemiology of the diseases are, so far, limited. The aim of the present study was to investigate the prevalence of Leishmania infection in these animals, in different areas of the country. Samples from 393 domestic and wild rabbits, 90 hares and 200 minks were collected and examined by cytology (spleen imprints) and serology (ELISA), while spleen samples of 116, 56 and 95 of the rabbits, hares and minks, respectively, were examined by a PCR assay targeting the ITS1 region. For every animal examined a form was created, recording information like date, area, animal species, sex, etc. All imprint smears examined were negative, while serology revealed infection in 7.6% (C.I. 5.0-10.3%) rabbits, 6.7% (C.I. 1.5-11.8%) hares and 20% (C.I. 14.5-25.5%) minks. Infection was confirmed by molecular methods in 2.6% (C.I. 0.0-5.5%), 3.6% (C.I. 0.0-8.4%) and 2.1% (C.I. 0.0-5.0%) of the animals, respectively. The statistical analysis showed that minks are most likely to be seropositive and that in rabbits, the breeding method (i.e. homestead reared animals) was associated with infection. Because of the proximity of lagomorphs and minks to humans and dogs it is necessary to further elucidate their role in the epidemiology of leishmaniosis.
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http://dx.doi.org/10.1016/j.vprsr.2019.100279DOI Listing
April 2019

A climate-driven and field data-assimilated population dynamics model of sand flies.

Sci Rep 2019 02 21;9(1):2469. Epub 2019 Feb 21.

Energy, Environment and Water Research Center, The Cyprus Institute, 2121, Aglantzia, Nicosia, Cyprus.

Sand flies are responsible for the transmission of leishmaniasis, a neglected tropical disease claiming more than 50,000 lives annually. Leishmaniasis is an emerging health risk in tropical and Mediterranean countries as well as temperate regions in North America and Europe. There is an increasing demand for predicting population dynamics and spreading of sand flies to support management and control, yet phenotypic diversity and complex environmental dependence hamper model development. Here, we present the principles for developing predictive species-specific population dynamics models for important disease vectors. Based on these principles, we developed a sand fly population dynamics model with a generic structure where model parameters are inferred using a surveillance dataset collected from Greece and Cyprus. The model incorporates distinct life stages and explicit dependence on a carefully selected set of environmental variables. The model successfully replicates the observations and demonstrates high predictive capacity on the validation dataset from Turkey. The surveillance datasets inform about biological processes, even in the absence of laboratory experiments. Our findings suggest that the methodology can be applied to other vector species to predict abundance, control dispersion, and help to manage the global burden of vector-borne diseases.
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http://dx.doi.org/10.1038/s41598-019-38994-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385250PMC
February 2019

Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

mBio 2018 11 6;9(6). Epub 2018 Nov 6.

Unité de Parasitologiemoléculaire et Signalisation, Institut Pasteur, Paris, France

Protozoan parasites of the genus adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast growth. Together our data draw a complex picture of genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. Protozoan parasites of the genus cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the , , and complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.
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http://dx.doi.org/10.1128/mBio.01399-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222132PMC
November 2018

Phlebotomine sand flies (Diptera: Psychodidae) in the Greek Aegean Islands: ecological approaches.

Parasit Vectors 2018 02 20;11(1):97. Epub 2018 Feb 20.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, School of Medicine, University of Crete, Vassilika Vouton, GR-71003, Heraklion, Greece.

Background: Blood-sucking phlebotomine sand flies are the vectors of the protozoan parasites Leishmania spp. Different Phlebotomus species transmit different Leishmania species causing leishmaniases which are neglected diseases emerging/reemerging in new regions. Thirteen sand fly species, ten belonging to the medically important genus Phlebotomus and three belonging to Sergentomyia are known in Greece. An increasing number of human and dog cases are reported each year from all parts of the country including the Aegean Islands. However, no previous study has been conducted on the sand fly fauna on the islands, except for Rhodes and Samos. The aim of this study was to investigate sand fly species in eleven small Aegean islands; to understand species-specific relationships with environmental and climatic factors and to compare sand fly community parameters among islands. A risk analysis was carried out for each species using climatic and environmental variables.

Results: Nine sand fly species: Phlebotomus neglectus, P. tobbi, P. similis, P. simici, P. perfiliewi, P. alexandri, P. papatasi, Sergentomyia minuta and S. dentata, were collected from the islands studied. Phlebotomus (Adlerius) sp. and Sergentomyia sp. specimens were also collected but not identified to the species level. There was a positive effect of distance from the sea on the abundance of P. neglectus, S. minuta and S. dentata, and a negative effect on the abundance of P. tobbi, P. simici and P. similis. In general, temperature preferences of sand fly populations were between 21 and 29 °C. Nevertheless, there were significant differences in terms of temperature and relative humidity preference ranges among species. The most important species found, P. neglectus, was indisputably the most adapted species in the study area with a very high reaction norm, favoring even the lower temperature and humidity ranges. Overall, the sand fly fauna in the islands was very rich but there were differences in species diversity, as indicated by the values of the Shannon-Wiener index, along with evenness and richness of the sand fly fauna between the islands and altitude ranges in the islands.

Conclusions: The study indicated that the Greek Aegean Islands, however small, maintain a rich sand fly fauna. This includes important vectors of Leishmania spp. representing a risk for parasite transmission to humans and dogs along with the danger of maintaining new Leishmania spp. if introduced to the area.
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http://dx.doi.org/10.1186/s13071-018-2680-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819154PMC
February 2018

Identification of wild-caught phlebotomine sand flies from Crete and Cyprus using DNA barcoding.

Parasit Vectors 2018 02 17;11(1):94. Epub 2018 Feb 17.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, School of Medicine, University of Crete, Vassilika Vouton, P.O. Box 2208, GR-71003, Heraklion, Greece.

Background: Phlebotomine sand flies (Diptera: Psychodidae) are vectors of Leishmania spp., protozoan parasites responsible for a group of neglected diseases called leishmaniases. Two sand fly genera, Phlebotomus and Sergentomyia, contain species that are present in the Mediterranean islands of Crete and Cyprus where the visceral (VL), cutaneous (CL) and canine (CanLei) leishmaniases are a public health concern. The risk of transmission of different Leishmania species can be studied in an area by monitoring their vectors. Sand fly species are traditionally identified using morphological characteristics but minute differences between individuals or populations could be overlooked leading to wrong epidemiological predictions. Molecular identification of these important vectors has become, therefore, an essential tool for research tasks concerning their geographical distribution which directly relates to leishmaniasis control efforts. DNA barcoding is a widely used molecular identification method for cataloguing animal species by sequencing a fragment of the mitochondrial gene encoding cytochrome oxidase I.

Results: DNA barcoding was used to identify individuals of five sand fly species (Phlebotomus papatasi, P. similis, P. killicki, Sergentomyia minuta, S. dentata) circulating in the islands of Crete and Cyprus during the years 2011-2014. Phlebotomus papatasi is a known vector of zoonotic CL in the Middle East and it is found in both islands. Phlebotomus similis is the suspected vector of Leishmania tropica in Greece causing anthroponotic CL. Phlebotomus killicki was collected in Cyprus for the first time. Sergentomyia minuta, found to present intraspecific diversity, is discussed for its potential as a Leishmania vector. Molecular identification was consistent with the morphological identification. It successfully identified males and females, which is difficult when using only morphological characters. A phylogenetic tree was constructed based on the barcodes acquired, representing their genetic relationships along with other species from the area studied. All individuals identified were clustered according to their species and subgenus.

Conclusions: Molecular identification of sand flies via DNA barcoding can accurately identify these medically important insects assisting traditional morphological tools, thus helping to assess their implication in Leishmania transmission.
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http://dx.doi.org/10.1186/s13071-018-2676-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816364PMC
February 2018

Canine tick-borne pathogens in Cyprus and a unique canine case of multiple co-infections.

Ticks Tick Borne Dis 2017 03 26;8(3):341-346. Epub 2016 Dec 26.

Diagnostic Laboratories, Langford Vets, School of Veterinary Sciences, University of Bristol, Langford, UK. Electronic address:

Canine tick-borne pathogens such as Ehrlichia canis and Hepatozoon canis are widespread in the Mediterranean basin but have never been reported or investigated in Cyprus. We describe herein the presence of canine tick-borne pathogens in three dogs with clinical signs compatible with vector-borne diseases from Paphos area of Cyprus. Molecular and phylogenetic analysis revealed the presence of E. canis, Anaplasma platys, H. canis, Babesia vogeli and Mycoplasma haemocanis in Cyprus. One dog co-infected with E. canis, H. canis, B. vogeli and M. haemocanis is, to the best of our knowledge, the first report of this multiple co-infection in dogs. The tick-borne pathogens reported in the current study should be considered in the differential diagnoses in dogs exposed to ticks in Cyprus.
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http://dx.doi.org/10.1016/j.ttbdis.2016.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315763PMC
March 2017

DNA sequencing confirms PCR-RFLP identification of wild caught Larroussius sand flies from Crete and Cyprus.

Acta Trop 2016 Dec 5;164:314-320. Epub 2016 Sep 5.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, School of Medicine, University of Crete, Vassilika Vouton, P.O. Box 2208, GR-71003, Heraklion, Greece. Electronic address:

Many Phlebotomine sand fly species (Diptera, Psychodidae) are vectors of the protozoan parasite Leishmania causing a group of diseases called the leishmaniases. The subgenus Larroussius includes sand fly vectors found in South East Mediterranean Basin responsible for Visceral (VL) and Cutaneous human leishmaniasis (CL). It is important to monitor these medically important insects in order to safely predict possible Leishmania transmission cycles. Leishmania infantum is endemic in the islands of Crete and Cyprus with increasing VL cases in humans and dogs and in Cyprus the newly introduced Leishmania donovani causes both VL and CL in humans. The morphological identification of the females of the subgenus Larroussius often presents difficulties. Morphology and COI PCR - RFLP were used to identify wild caught Larroussius sand flies belonging to Phlebotomus tobbi, P. perfiliewi, and P. neglectus species from Crete and Cyprus. The identification results were further confirmed by sequencing (DNA barcoding) and Bayesian phylogenetic analysis. COI PCR - RFLP, when correctly optimized and with respect to geographical origin, can serve as an initial patterning identification tool when large sand fly numbers need to be identified. It could accurately assign Larroussius females and males to their taxa overcoming the difficulties of morphological identification. Finally, DNA barcoding will contribute to a molecular identification database to be used for in-depth species studies.
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http://dx.doi.org/10.1016/j.actatropica.2016.09.003DOI Listing
December 2016

Seroprevalence of Sandfly-Borne Phleboviruses Belonging to Three Serocomplexes (Sandfly fever Naples, Sandfly fever Sicilian and Salehabad) in Dogs from Greece and Cyprus Using Neutralization Test.

PLoS Negl Trop Dis 2016 Oct 26;10(10):e0005063. Epub 2016 Oct 26.

UMR "Emergence des Pathologies Virales" (EPV: Aix-Marseille Univ - IRD 190 - Inserm 1207 - EHESP), Marseille, France.

Phleboviruses transmitted by sandflies are endemic in the Mediterranean area. The last decade has witnessed the description of an accumulating number of novel viruses. Although, the risk of exposure of vertebrates is globally assessed, detailed geographic knowledge is poor even in Greece and Cyprus where sandfly fever has been recognized for a long time and repeatedly. A total of 1,250 dogs from mainland Greece and Greek archipelago on one hand and 422 dogs from Cyprus on the other hand have been sampled and tested for neutralising antibodies against Toscana virus (TOSV), Sandfly fever Sicilian virus (SFSV), Arbia virus, and Adana virus i.e. four viruses belonging to the 3 sandfly-borne serocomplexes known to circulate actively in the Mediterranean area. Our results showed that (i) SFSV is highly prevalent with 71.9% (50.7-84.9% depending on the region) in Greece and 60.2% (40.0-72.6%) in Cyprus; (ii) TOSV ranked second with 4.4% (0-15.4%) in Greece and 8.4% (0-11.4%) in Cyprus; (iii) Salehabad viruses (Arbia and Adana) displayed also substantial prevalence rates in both countries with values ranging from 0-22.6% depending on the region and on the virus strain used in the test. These results demonstrate that circulation of viruses transmitted by sand flies can be estimated qualitatively using dog sera. As reported in other regions of the Mediterranean, these results indicate that it is time to shift these viruses from the "neglected" status to the "priority" status in order to stimulate studies aiming at defining and quantifying their medical and veterinary importance and possible public health impact. Specifically, viruses belonging to the Sandfly fever Sicilian complex should be given careful consideration. This calls for implementation of direct and indirect diagnosis in National reference centers and in hospital microbiology laboratories and systematic testing of unelucidated febrile illness and central and peripheral nervous system febrile manifestations.
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http://dx.doi.org/10.1371/journal.pntd.0005063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081206PMC
October 2016

Toxoplasmosis in female high school students, pregnant women and ruminants in Cyprus.

Trans R Soc Trop Med Hyg 2016 06;110(6):359-66

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece

Background: The protozoan parasite Toxoplasma gondii is important to human and animal health worldwide. This is the first study of prevalence of infection with T. gondii and associated risk factors in human populations and small ruminants in Cyprus.

Methods: A random sample of 18 schools out of 46 participated: 1056 girls aged 16 to 18 years completed a questionnaire and were serologically tested for Toxoplasma between 2008 and 2011 (response rate 30%). In addition, infection with T. gondii laboratory results of 23 076 pregnant women tested between 2009 and 2014 were obtained from hospital records. Finally, 163 (out of 3123) farms were randomly sampled and blood samples from 515 sheep and 581 goats were obtained.

Results: Estimated seropositivity prevalence in female students was 6.5% (95% CI 4.3 to 8.7%) and 18% (95% CI 17 to 19%) in pregnant women. Overall, 40.1% of the ruminants tested were seropositive (95% CI 37.2% to 43.0%). Seropositivity differed according to geographical region in all three groups.

Conclusions: Further studies are needed to investigate the differences between regions that lead to differing prevalence levels and patterns between ruminants and humans so that health education policies can be developed to help prevent infection and reduce environmental contamination.
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http://dx.doi.org/10.1093/trstmh/trw038DOI Listing
June 2016

Geographical Distribution of MDR1 Expression in Leishmania Isolates, from Greece and Cyprus, Measured by the Rhodamine-123 Efflux Potential of the Isolates, Using Flow Cytometry.

Am J Trop Med Hyg 2016 05 21;94(5):987-92. Epub 2016 Mar 21.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece; Veterinary Services of Cyprus, Nicosia, Cyprus; Veterinary Services of Lasithi prefecture, Crete, Greece; Laboratory of Flow Cytometry, Faculty of Medicine, University of Crete, Crete, Greece

Leishmaniasis, a neglected vector-borne disease caused by the protozoan parasite Leishmania, is encountered in 98 countries causing serious concerns to public health. The most alarming is the development of parasite drug resistance, a phenomenon increasingly encountered in the field rendering chemotherapy ineffective. Although resistance to drugs is a complex phenomenon, the rate of efflux of the fluorescent dye Rhodamine-123 from the parasite body, using flow cytometry, is an indication of the isolate's ability to efflux the drug, thus avoiding death. The rate of efflux measured 275 Leishmania strains, isolated from patients and dogs from Greece and Cyprus, was measured and mapped to study the geographical distribution of the multidrug resistance (MDR) gene expression as an indication of the drug resistance of the parasite. The map showed that out of the seven prefectures, where dogs presented high efflux rates, five also had patients with high efflux rates. In one, out of the 59 prefectures studied, the highest number of isolates with efflux slope α > 1, in both human and dog isolates, was found; a fact which may suggest that spread of drug resistance is taking place. The virulence of the Leishmania strains, assessed after infecting human macrophages of the THP-1 cell line, fluctuated from 1% to 59.3% with only 2.5% of the isolates showing infectivity > 50%. The most virulent strains were isolated from Attica and Crete.
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http://dx.doi.org/10.4269/ajtmh.15-0658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856631PMC
May 2016

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

PLoS Negl Trop Dis 2016 Feb 22;10(2):e0004458. Epub 2016 Feb 22.

Veterinary Faculty, Mehmet Akif Ersoy University, Burdur, Turkey.

Background: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported.

Methods/principal Findings: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am.

Conclusions: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.
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http://dx.doi.org/10.1371/journal.pntd.0004458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4762948PMC
February 2016

Detection of viable Mycobacterium avium subspecies paratuberculosis in powdered infant formula by phage-PCR and confirmed by culture.

Int J Food Microbiol 2016 Jan 21;216:91-4. Epub 2015 Sep 21.

Division of Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, United Kingdom.

Surveys from different parts of the world have reported that viable Mycobacterium avium subsp. paratuberculosis (MAP) can be cultured from approximately 2% of samples of retail pasteurised milk samples. Pasteurised milk is used for the production of powdered infant formula (PIF) and therefore there is a concern that MAP may also be present in these products. Several studies have previously reported the detection of MAP in PIF using PCR-based assays. However, culture-based surveys of PIF have not detected viable MAP. Here we describe a phage amplification assay coupled with PCR (page-PCR) that can rapidly detect viable MAP in PIF. The results of a small survey showed that the phage-PCR assay detected viable MAP in 13% (4/32) of PIF samples. Culture detected viable MAP in 9% (3/32) PIF samples, all of which were also phage-PCR positive. Direct IS900 PCR detected MAP DNA in 22% (7/32) of PIF samples. The presence of viable MAP in PIF indicates that MAP either survived PIF manufacturing or that post-production contamination occurred. Irrespective of the route of MAP contamination, the presence of viable MAP in PIF is a potential public health concern.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2015.09.011DOI Listing
January 2016

Erratum to: Frequency of MDR 1-related p-gp overexpression in Greek Leishmania isolates.

Parasitol Res 2015 Sep;114(9):3565

Center of Anatomy, Institute II, Laboratory for Medical and Molecular Parasitology, Medical School, University of Cologne, Cologne, Germany,

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http://dx.doi.org/10.1007/s00436-015-4572-2DOI Listing
September 2015

A cluster of four cases of cutaneous leishmaniasis by Leishmania donovani in Cyprus: a case series.

J Med Case Rep 2014 Oct 24;8:354. Epub 2014 Oct 24.

Archbishop Makarios Hospital, Department of Paediatrics, 6 Korytsas St,, Strovolos, 1474 Nicosia, Cyprus.

Introduction: Leishmaniasis is endemic in more than 95 countries and is the only tropical/subtropical vector-borne disease that has been endemic in Southern Europe for decades. To the best of our knowledge, this is the first case of cutaneous leishmaniasis by Leishmania donovani in a child and the first cluster with adult cases reported in Europe.

Case Presentation: We describe a familial cluster of four cutaneous leishmaniasis cases among Greek Cypriots caused by L. donovani in a Paphos village, in Cyprus. A 6-year-old boy (Case number 1) had a persistent lesion in the left angle of his upper lip, a 60-year-old woman (Case number 2) presented with a 2 cm-diameter glabella lesion on her forehead, a 60-year-old man (Case number 3) developed a lesion on his moustache area and a 40-year-old woman (Case number 4) had a lesion on her neck. In Case number 3 the lesion was self-cured; the other cases recovered after surgical resection followed by liposomal amphotericin B (Case numbers 1 and 4) or thermotherapy and liposomal amphotericin B (Case number 2).

Conclusions: This familial cluster of cutaneous leishmaniasis, due to the anthroponotic L. donovani, shows that the sand fly species responsible for transmitting this parasite species is found in the area around the three neighbouring houses involved. The factors favourable for the survival, spread and contact of the vector with people could be assessed in this area for the establishment of preventative measures to safeguard public health.
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http://dx.doi.org/10.1186/1752-1947-8-354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4220636PMC
October 2014

The use of spatial analysis to estimate the prevalence of canine leishmaniasis in Greece and Cyprus to predict its future variation and relate it to human disease.

Am J Trop Med Hyg 2014 Aug 23;91(2):336-41. Epub 2014 Jun 23.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece; Veterinary Services of Cyprus, Nicosia, Cyprus

Climatic, environmental, and demographic changes favor the emergence of neglected vector-borne diseases like leishmaniasis, which is spreading through dogs, the principle host of the protozoan Leishmania infantum. Surveillance of the disease in dogs is important, because the number of infected animals in an area determines the local risk of human infection. However, dog epidemiological studies are costly. Our aim was to evaluate the Emerging Diseases in a Changing European Environment (EDEN) veterinary questionnaire as a cost-effective tool in providing reliable, spatially explicit indicators of canine leishmaniasis prevalence. For this purpose, the data from the questionnaire were compared with data from two epidemiological studies on leishmaniasis carried out in Greece and Cyprus at the same time using statistical methods and spatial statistics. Although the questionnaire data cannot provide a quantitative measure of leishmaniasis in an area, it indicates the dynamic of the disease; information is obtained in a short period of time at low cost.
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http://dx.doi.org/10.4269/ajtmh.13-0459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125258PMC
August 2014

Frequency of MDR1-related p-gp overexpression in Greek Leishmania isolates.

Parasitol Res 2014 Mar 7;113(3):1225-32. Epub 2014 Feb 7.

Center of Anatomy, Institute II, Laboratory for Medical and Molecular Parasitology, Medical School, University of Cologne, Cologne, Germany,

In this work, we investigated Greek Leishmania isolates (n = 70) for their individual MDR1-gene-related p-gp (belonging to the ABC-B subfamily of permeases) expression levels by means of flow cytometric analysis of Rhodamine 123 extrusion kinetics. Of all used isolates, 5.71% express this drug-extruding ABC-transporter at alarming levels and are distributed widely over the country. Some 33% of all examined isolates originated on the island of Crete though none of the strains showed vastly elevated p-gp extrusion activity, indicating a reasonable implementation of anti-leishmanial compounds in this part of the country. Compared to isolates obtained from canine tissue, human Leishmania isolates were superior both in size and in subcellular differentiation in flow cytometry. Furthermore, a specific t test confirmed verapamil hydrochloride to be a highly potent p-gp reversal agent with p < 0.0001. In a second test series, the loading of Leishmania with Rhodamine 123 was moreover reduced when occurring under influence of verapamil hydrochloride, a known p-gp reversal agent, indicating an ATP-dependant influx of the fluorescent dye and therewith the drug itself. In a final, third experiment series, it was shown that Sb(V) does not act upon the promastigote form of Leishmania.
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http://dx.doi.org/10.1007/s00436-014-3761-8DOI Listing
March 2014

Will the introduction of Leishmania tropica MON-58, in the island of Crete, lead to the settlement and spread of this rare zymodeme?

Acta Trop 2014 Apr 24;132:125-30. Epub 2014 Jan 24.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece. Electronic address:

The rare zymodeme, Leishmania tropica MON-58, was isolated from a young Afghan refugee with a facial cutaneous lesion who had come to live in Crete early 2008. The same zymodeme variant was isolated from a local dog that had never travelled outside the island, with symptoms of visceral leishmaniasis, which stayed in the area where the patient worked during the summer months. This is the first record of L. tropica in a host, other than human, in Greece and another example of introduction of a vector borne pathogen in a focus where local vector/s can sustain it, with the risk of initiation of new transmission cycle/s.
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http://dx.doi.org/10.1016/j.actatropica.2014.01.003DOI Listing
April 2014

Genetic diversity and structure in Leishmania infantum populations from southeastern Europe revealed by microsatellite analysis.

Parasit Vectors 2013 Dec 5;6:342. Epub 2013 Dec 5.

Laboratory of Molecular Parasitology, Hellenic Pasteur Institute, 127 Vasilissis Sofias Avenus, 11521, Athens, Greece.

Background: The dynamic re-emergence of visceral leishmaniasis (VL) in south Europe and the northward shift to Leishmania-free European countries are well-documented. However, the epidemiology of VL due to Leishmania infantum in southeastern (SE) Europe and the Balkans is inadequately examined. Herein, we aim to re-evaluate and compare the population structure of L. infantum in SE and southwestern (SW) Europe.

Methods: Leishmania strains collected from humans and canines in Turkey, Cyprus, Bulgaria, Greece, Albania and Croatia, were characterized by the K26-PCR assay and multilocus enzyme electrophoresis (MLEE). Genetic diversity was assessed by multilocus microsatellite typing (MLMT) and MLM Types were analyzed by model- and distance- based algorithms to infer the population structure of 128 L. infantum strains.

Results: L. infantum MON-1 was found predominant in SE Europe, whilst 16.8% of strains were MON-98. Distinct genetic populations revealed clear differentiation between SE and SW European strains. Interestingly, Cypriot canine isolates were genetically isolated and formed a monophyletic group, suggesting the constitution of a clonal MON-1 population circulating among dogs. In contrast, two highly heterogeneous populations enclosed all MON-1 and MON-98 strains from the other SE European countries. Structure sub-clustering, phylogenetic and Splitstree analysis also revealed two distinct Croatian subpopulations. A mosaic of evolutionary effects resulted in consecutive sub-structuring, which indicated substantial differentiation and gene flow among strains of both zymodemes.

Conclusions: This is the first population genetic study of L. infantum in SE Europe and the Balkans. Our findings demonstrate the differentiation between SE and SW European strains; revealing the partition of Croatian strains between these populations and the genetic isolation of Cypriot strains. This mirrors the geographic position of Croatia located in central Europe and the natural isolation of the island of Cyprus. We have analysed the largest number of MON-98 strains so far. Our results indicate extensive gene flow, recombination and no differentiation between MON-1 and MON-98 zymodemes. No correlation either to host specificity or place and year of strain isolation was identified. Our findings may be associated with intensive host migration and common eco-epidemiological characteristics in these countries and give valuable insight into the dynamics of VL.
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http://dx.doi.org/10.1186/1756-3305-6-342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029556PMC
December 2013

Leishmaniases in Greece.

Am J Trop Med Hyg 2013 Nov 23;89(5):906-15. Epub 2013 Sep 23.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece; Veterinary Services of Cyprus, Nicosia, Cyprus; University Montpellier 1, Faculty of Medicine, Laboratory of Parasitology-Mycology, Hospital University Centre (CHRU) of Montpellier and French National Reference Centre for Leishmaniases, CNRS 5290 - IRD 224 - University Montpellier 1&2 (UMR "MIVEGEC").

Abstract. During the last 35 years, visceral leishmaniasis has spread in Greece with autochthonous human cases appearing in 41 of the 54 prefectures. The occurrence of the disease was mapped and related to dog seropositivity, environmental and geospatial risk factors. Average dog seropositivity was 22.1% and positive animals were found in 43 of 54 prefectures. Factors like: altitude, presence of water bodies, land use, wind speed, mean land surface temperature, mean relative humidity, and mean annual rainfall were found to affect dog seropositivity. Cases of cutaneous leishmaniasis, caused by Leishmania tropica are also increasing. Phlebotomus similis believed to be the potential vector of L. tropica in Greece, was found in areas where the disease is widespread but also where cases have never been reported implying a danger of introduction of this anthroponotic parasite to new regions.
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http://dx.doi.org/10.4269/ajtmh.13-0070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820334PMC
November 2013

Drug resistance in natural isolates of Leishmania donovani s.l. promastigotes is dependent of Pgp170 expression.

PLoS One 2013 11;8(6):e65467. Epub 2013 Jun 11.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece.

Resistance of pathogens to drugs is a growing concern regarding many diseases. Parasites like Leishmania, Plasmodium and Entamoeba histolytica; and neoplastic cells, present the multidrug-resistant phenotype rendering chemotherapy ineffective. The acquired resistance of Leishmania to antimony has generated intense research on the mechanisms involved but the question has not yet been resolved. To test the hypothesis that drug efflux in Leishmania, as measured by flow cytometry using the fluorescent dye Rhodamine-123, is largely dependent on the number of efflux pumps an isolate can express, the amount of Pgp 170 molecules was assessed in ten field isolates (5 "resistant" and 5 "susceptible") using: Western Blotting, Confocal and Transmission Electron Microscopy, and proteomics. Their survival after exposure to three antileishmanial drugs, in vitro, was evaluated and clinical data were compared to the in vitro results. All isolates were resistant to Glucantime but susceptible to Miltefosine, whilst Amphotericin B was more effective on the "susceptible" isolates. The MDR gene, expressing the transmembrane efflux pump Pgp 170, appears to play a key role in the phenomenon of drug resistance. When "susceptible" versus "resistant" parasites were compared, it was shown that the higher the number of Pgp 170 molecules the higher the Rhodamine-123 efflux from the parasite body and, when exposed to the drug, the number of efflux pumps increased. However, the rate of this increase was not linear and it is possible that there is a maximum number of Pgp 170 molecules an isolate can express. Nevertheless, the phenomenon is a complex one and other factors and proteins are involved in which the HSP-70 group proteins, detected in the "resistant" isolates, may play a significant role.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065467PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679129PMC
January 2014

Re-emergence of visceral and cutaneous leishmaniasis in the Greek Island of Crete.

Vector Borne Zoonotic Dis 2012 Mar 4;12(3):214-22. Epub 2012 Jan 4.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece.

Leishmaniases are vector-borne diseases transmitted by phlebotomine sand flies. Three species of Leishmania are found in the Mediterranean basin: Leishmania infantum, the most common species responsible for both visceral (VL) and cutaneous leishmaniasis (CL); Leishmania major, found in North Africa and Middle East causing CL; Leishmania tropica with a limited presence in Europe, causing CL. During the last 25 years, Crete has become an endemic zone for L. infantum with a high number of infected dogs and an increasing number of human cases every year; in the last 4 years, the incidence has reached an average of seven VL patients per year in a population of 600,000. At the same time, CL has re-emerged in Crete due to L. tropica, with an average of three CL cases per year in the last 4 years. Isolates were typed as L. infantum MON-1 and MON-98 and L. tropica MON-300, a zymodeme not reported before. Both VL and CL have spread to the whole of the island during the last 25 years, primarily in semi-urban and urban areas with altitudes of 0-50 m. The prevailing Phlebotomus species were Phlebotomus neglectus (proven vector of L. infantum) and Phlebotomus similis (suspected vector of L. tropica).
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http://dx.doi.org/10.1089/vbz.2011.0004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3300062PMC
March 2012

Leishmania infantum and Toxoplasma gondii: Mixed infection of macrophages in vitro and in vivo.

Exp Parasitol 2011 Jul 24;128(3):279-84. Epub 2011 Feb 24.

Laboratory of Clinical Bacteriology Parasitology Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece.

Although macrophages have a microbicidal role in the immune system they themselves can be infected by pathogens. Often a simultaneous infection by more than one microbe may occur in a single cell. This is the first report of coinfection of macrophages with Toxoplasma gondii and Leishmania infantum, in vitro and in vivo. L. infantum does not cause severe disease in mice but T. gondii, RH strain, is lethal. Cell culture studies using THP-1 macrophages dually infected in vitro revealed that 4.3% harbored both parasites 24h after infection. When mice were infected with both parasites on the same day 7.3% of the infected cells carried both parasites 7 days later. Yet, if mice were first infected with L. infantum and then with Toxoplasma (5 days post-infection) 18.7% of the macrophages hosted either parasite but concomitant infection could not be found and mice, already harboring L. infantum, survived Toxoplasma's lethal effect.
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http://dx.doi.org/10.1016/j.exppara.2011.02.022DOI Listing
July 2011

Leishmaniases and the Cyprus paradox.

Am J Trop Med Hyg 2010 Mar;82(3):441-8

Veterinary Services of Cyprus, Nicosia, Cyprus.

In Cyprus, leishmaniasis has been considered exclusively a veterinary problem. It was prevalent before 1945, and until its recent reemergence, it was nearly eradicated by 1996 as a consequence of the destruction of reservoir hosts and vectors. A survey carried out to provide an unbiased estimate of current transmission rates in dogs and humans showed a 9-fold increase in dog seroprevalence (reaching 14.9%) compared with 10 years ago. However, no human cases caused by Leishmania infantum were detected, although L. donovani cases were reported recently. The 62 strains isolated from dogs were typed as L. infantum MON-1 (98.4%), which is the predominating zymodeme in the Mediterranean region, and MON-98 (1.6%). The Phlebotomus species P. tobbi (vector of L. infantum in Cyprus), P. galilaeus, and P. papatasi were the predominant species captured. Two transmission cycles seem to run in parallel in Cyprus: in dogs with L. infantum and in humans with L. donovani.
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http://dx.doi.org/10.4269/ajtmh.2010.09-0282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829906PMC
March 2010

Increasing incidence of zoonotic visceral leishmaniasis on Crete, Greece.

Emerg Infect Dis 2009 Jun;15(6):932-4

University of Crete, Heraklion, Greece.

To determine whether the incidence of canine leishmaniasis has increased on Crete, Greece, we fitted infection models to serodiagnostic records of 8,848 dog samples for 1990-2006. Models predicted that seroprevalence has increased 2.4% (95% confidence interval 1.61%-3.51%) per year and that incidence has increased 2.2- to 3.8-fold over this 17-year period.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727332PMC
http://dx.doi.org/10.3201/eid1506.071666DOI Listing
June 2009

Selection of appropriate serological tests to measure the incidence of natural Leishmania infantum infection during DNA/MVA prime/boost canine vaccine trials.

Vet Parasitol 2009 Jun 31;162(3-4):207-13. Epub 2009 Mar 31.

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.

In response to the increasing need for field trials of experimental DNA vaccines against zoonotic visceral leishmaniasis in dogs, our aim was to validate the use of ELISA protocols which will be suitable for detection of natural infection in vaccinated dogs. We have previously demonstrated that DNA/modified vaccinia virus Ankara (MVA) vaccine expressing tryparedoxin peroxidase (TRYP) induced high titres of TRYP antigen-specific IgG in immunized dogs. Here we report our findings that seroconversion to an unrelated diagnostic antigen rK39 did not occur in vaccinated dogs, and that responses to crude Leishmania infantum promastigote antigen (CLA) were weak and short-lived. This is in contrast to strong responses to both antigens shown in naturally infected dogs. To select an appropriate serological test for measurement of infection incidence, we also tested longitudinal samples from an immunologically well-characterized cohort of naturally infected dogs. The sensitivity of CLA ELISA was superior to that of rK39 in early stage infection (from 2 months before, to 2 months after the first detection of infection by PCR or parasitological culture), and more sensitive than rK39 in cross-sectional sampling (81.0% vs 61.9%). We conclude that CLA ELISA will provide sensitive estimates of L. infantum infection incidence in DNA/MVA vaccinated dogs, though optimal testing would include rK39, or a similar recombinant antigen, to improve overall specificity.
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http://dx.doi.org/10.1016/j.vetpar.2009.03.037DOI Listing
June 2009

A prime/boost DNA/Modified vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safe and immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis.

Vaccine 2009 Feb 16;27(7):1080-6. Epub 2008 Dec 16.

Populations and Disease Research Group, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.

Previous studies demonstrated safety, immunogenicity and efficacy of DNA/modified vaccinia virus Ankara (MVA) prime/boost vaccines expressing tryparedoxin peroxidase (TRYP) and Leishmania homologue of the mammalian receptor for activated C kinase (LACK) against Leishmania major challenge in mice, which was consistent with results from TRYP protein/adjuvant combinations in non-human primates. This study aimed to conduct safety and immunogenicity trials of these DNA/MVA vaccines in dogs, the natural reservoir host of Leishmania infantum, followed-up for 4 months post-vaccination. In a cohort of 22 uninfected outbred dogs, blinded randomised administration of 1000 microg (high dose) or 100 microg (low dose) DNA prime (day 0) and 1x10(8)pfu MVA boost (day 28) was shown to be safe and showed no clinical side effects. High dose DNA/MVA vaccinated TRYP dogs produced statistically higher mean levels of the type-1 pro-inflammatory cytokine IFN-gamma than controls in whole blood assays (WBA) stimulated with the recombinant vaccine antigen TRYP, up to the final sampling at day 126, and in the absence of challenge with Leishmania. TRYP vaccinated dogs also demonstrated significantly higher TRYP-specific total IgG and IgG2 subtype titres than in controls, and positive in vivo intradermal reactions at day 156 in the absence of natural infection, observed in 6/8 TRYP vaccinated dogs. No significant increases in IFN-gamma in LACK-stimulated WBA, or in LACK-specific IgG levels, were detected in LACK vaccinated dogs compared to controls, and only 2/9 LACK vaccinated dogs demonstrated DTH responses at day 156. In all groups, IgG1 subclass responses and antigen-specific stimulation of IL-10 were similar to controls demonstrating an absence of Th2/T(reg) response, as expected in the absence of in vivo restimulation or natural/experimental challenge with Leishmania. These collective results indicate significant antigen-specific type-1 responses and in vivo memory phase cellular immune responses, consistent with superior potential for protective vaccine immunogenicity of DNA/MVA TRYP over LACK.
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http://dx.doi.org/10.1016/j.vaccine.2008.11.094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2663027PMC
February 2009
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