Maria Antoniou

Leishmania donovani leishmaniasis in Cyprus.

1recently discussed the emergence of Leishmania donovani in Cyprus. We would like to provide some additional data regarding possible vector species that might have caused the disease outbreak on this island. The presence of L donovani was reported in the eastern Mediterranean basin about 10 years ago in the vicinity of Kassab, northern Syria, when an isolate taken from Phlebotomus tobbi—a suspected vector of canine leishmaniasis caused by Leishmania infantum at the time of the infected sandfl y’s discovery—was typed as belonging to a zymodeme close to L donovani MON-3. 2

The role of indigenous phlebotomine sandflies and mammals in the spreading of leishmaniasis agents in the Mediterranean region

An updated view of the establishment and spread of the leishmaniases in Europe is presented, mostly with respect to newly emerging and re-emerging foci and the incrimination of neglected as well as new reservoir hosts. At the same time, a concept of specific versus permissive vectors reassesses the potential role of various sandfly species in Leishmania transmission and considers the risk of introduction of exotic Leishmania species in Europe. The leishmaniases are dynamic diseases and the circumstances of transmission are continually changing in relation to environmental, demographic and human behavioural factors. Changes in the habitat of the natural hosts and vectors, immunosuppressive conditions (like infection with human immunodeficiency virus (HIV) or organ transplantation-associated therapies in humans) and the consequences of war, all contribute to the transformation of the epidemiology of leishmaniasis. Such changes should be considered when studying the spread of the disease throughout Europe for targeted control measures to safeguard public health.

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

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.

Leishmaniases and the Cyprus Paradox

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.

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).

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.

Rats as indicators of the presence and dispersal of six zoonotic microbial agents in Cyprus, an island ecosystem: a seroepidemiological study.

A total of 622 rats (402 Rattus norvegicus and 220 R. rattus frugivorus) were collected in 51 different areas in Cyprus during 2000-2003 and used as indicators of the presence and dispersal of six zoonotic microbial agents. IgG antibodies against Rickettsia typhi (241/496, 48.6%), R. conorii (209/500, 41.8%), Toxoplasma sp. (138/494, 27.9%), Coxiella burnetti (63/494, 12.8%), Bartonella henselae (52/494, 10.5%) and Leishmania infantum (36/494, 7.3%) were detected by indirect immunofluorescence test. There was variation in the association between the seropositivity of the six microbial agents and other factors. Rat species affected R. typhi and R. conorii seropositivity, the prefecture where the rats were caught affected R. typhi, C. burnetii, B. henselae, T. gondii and L. infantum, the sampling season impacted on R. typhi, R. conorii, T. gondii and L. infantum, and the flea species affected R. typhi, R. conorii and B. henselae. These results were analysed using geographical information system (GIS) technology and the seropositivity in rats against the pathogens tested appeared to follow the occurrence of these pathogens in humans. This suggests that rats could be used as disease sentinels and, together with GIS technology, they could be a useful tool for the identification of endemic foci and high-risk areas for each pathogen.

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.

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 μg (high dose) or 100 μg (low dose) DNA prime (day 0) and 1 × 108 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-γ 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-γ 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/Treg 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.

Identification of phlebotomine sand flies (Diptera: Psychodidae) by matrix-assisted laser desorption/ionization time of flight mass spectrometry

BackgroundPhlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens. To elucidate their role as vectors, proper species identification is crucial. Since traditional morphological determination is based on minute and often dubious characteristics on their head and genitalia, which require certain expertise and may be damaged in the field-collected material, there is a demand for rapid, simple and cost-effective molecular approaches.MethodsSix laboratory-reared colonies of phlebotomine sand flies belonging to five species and four subgenera (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius) were used to evaluate the discriminatory power of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Various storage conditions and treatments, including the homogenization in either distilled water or given concentrations of formic acid, were tested on samples of both sexes.ResultsSpecimens of all five analysed sand fly species produced informative, reproducible and species-specific protein spectra that enabled their conclusive species identification. The method also distinguished between two P. sergenti colonies originating from different geographical localities. Protein profiles within a species were similar for specimens of both sexes. Tested conditions of specimen storage and sample preparation give ground to a standard protocol that is generally applicable on analyzed sand fly specimens.ConclusionsSpecies identification of sand flies by MALDI-TOF MS is feasible and represents a novel promising tool to improve biological and epidemiological studies on these medically important insects.

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.