Detection of Leishmania Infantum in red foxes (Vulpes vulpes) in Central Greece.

This is the first record of Leishmania detection in foxes in Greece. Spleen, lymph nodes, bone marrow and blood samples were collected from 47 red foxes (Vulpes vulpes) found dead or captured, narcotized and freed after bleeding, from November 2009 to 2011, in Fthiotida prefecture, central Greece. This is an endemic for canine leishmaniasis area with several human visceral leishmaniasis cases. The samples were tested for Leishmania infantum and Leishmania tropica by molecular methods (polymerase chain reaction (PCR) and restriction fragment length polymorphism) and serology (indirect immunofluorescent antibody test; when blood samples were available). Leishmania infantum DNA was detected in 28 animals (59·5%). PCR positivity was related to animal age, sex, weight, characteristics of the area trapped, presence of leishmaniasis symptoms and presence of endo- and ecto-parasites. The results were related to dog seropositivity obtained earlier in the area. The findings support the hypothesis that this wild canid may serve as a reservoir for Leishmania in areas where the sandfly vectors are found. In the prefectures of Larisa and Magnisia, adjacent to Fthiotida, Phlebotomus perfiliewi and Phlebotomus tobbi (known vectors of L. infantum) have been reported.

The proliferation potential of promastigotes of the main Leishmania species of the old world in NNN culture medium prepared using blood of four different mammals.

The efficacy of the in vitro cultivation of promastigotes of four Leishmania spp. was tested in the biphasic Novy-MacNeal-Nicolle (NNN) medium prepared using blood from different animals (horse, donkey, goat and sheep). The aim was to test which NNN preparation gave the best yield in the shortest time for different parasite species, in order to obtain a large crop of promastigotes for experimental work and for antigen preparation. Promastigotes of Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania major, the four main parasite species occurring in the old world, were defrosted from -80 °C and placed, at equal numbers, in the 4 different NNN preparations. At the end of the 7th day, the NNN medium using horse blood produced the greatest number of promastigotes for all Leishmania spp. tested, whilst goat blood proved the poorest medium, providing culture results only for L. infantum. This finding may be explained by the fact that Leishmania is a nicotinamide adenine dinucleotide (NAD) auxotroph and horse erythrocytes support NAD-dependent microorganisms.

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

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.

Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples.

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.

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.

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.

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.

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.

Serological investigation for West Nile virus, Anaplasma ovis and Leishmania infantum in Greek cattle.

This study investigates the seroprevalence of Greek adult cattle against West Nile virus, Anaplasma ovis and Lehismania infantum. In total, 156 serum samples were examined, drawn from cattle between 2-4 years old. All the examined cattle originated from slaughterhouses of 4 prefectures in Northern Greece (Thessaloniki, Pella, Chalkidiki, Kilkis), in 2 of which (Thessaloniki, Pella) human cases of West Nile virus had been recorded some months before. Thirty out of the 156 (18.6%) samples have tested positive for West Nile virus and fifty-five (35.9%) samples for Anaplasma ovis. All the examined samples tested negative for Leishmania infantum. The prefectures with positive samples against West Nile virus also showed human cases of West Nile virus infections. This should raise questions whether cattle could become markers for West Nile virus activity in high risk areas.

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.

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.

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

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.

Leishmaniases in Greece.

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.

Immunogenicity and diagnostic potential of synthetic antigenic cell surface glycans of Leishmania.

Detection and quantification of pathogen-derived antigenic structures is a key method for the initial diagnosis and follow-up of various infectious diseases. Complex parasitic diseases such as leishmaniasis require highly sensitive and specific tests prior to treatment with potentially toxic drugs. To investigate the diagnostic potential of cell surface glycans found on Leishmania parasites, we identified diagnostically relevant glycan epitopes and used synthetic glycan microarrays to screen sera from infected humans and dogs. On the basis of the screening results, we selected a tetrasaccharide to generate anti-glycan antibodies. The corresponding tetrasaccharide-carrier protein conjugate was immunogenic in mice, and sera obtained from immunized mice specifically detected the Leishmania parasite. These results demonstrate how synthetic glycan arrays, in combination with immunological methods, help to identify promising carbohydrate antigens for pathogen detection.

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

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).