High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Entomological surveillance and spatiotemporal risk assessment of sand fly-borne diseases in Cyprus.

Visceral and cutaneous leishmaniases are important public health concerns in Cyprus. Although the diseases, historically prevalent on the island, were nearly eradicated by 1996, an increase in frequency and geographical spread has recently been recorded. Upward trends in leishmaniasis prevalence have largely been attributed to environmental changes that amplify the abundance and activity of its vector, the phlebotomine sand flies. Here, we performed an extensive field study across the island to map the sand fly fauna and compared the presence and distribution of the species found with historical records. We mapped the habitat preferences of Phlebotomus papatasi and P. tobbi, two medically important species, and predicted the seasonal abundance of P. papatasi at unprecedented spatiotemporal resolution using a climate-sensitive population dynamics model driven by high-resolution meteorological forecasting. Our compendium holds a record of 18 species and the locations of a subset, including those of potential public and veterinary health concern. We confirmed that P. papatasi is widespread, especially in densely urbanized areas, and predicted that its abundance uniformly peaks across the island at the end of summer. We identified potential hotspots of P. papatasi activity even after this peak. Our results form a foundation to inform public health planning and contribute to the development of effective, efficient, and environmentally sensitive strategies to control sand fly populations and prevent sand fly-borne diseases.

First detection of WNV RNA presence in field-collected mosquitoes in Cyprus.

West Nile virus (WNV) infections have increased over recent years to the extent that WNV has become one of the most widespread arboviruses in the world, with potential consequences for both human and animal health. While much is known about WNV and the vectors that transmit it from their primary hosts across continental Europe, little is known about the epidemiology of the disease on the island of Cyprus. In this study, the aim was to investigate the prevalence of WNV infection in potential mosquito vectors for the first time in the Republic of Cyprus, using WNV surveillance of mosquitoes. Mosquitoes were collected in 2019, during which an outbreak in humans had occurred, and sampled mosquitoes were then examined for WNV infection by testing them for the presence of WNV RNA. Of 126 mosquito pools tested, one pool, containing Culex pipiens mosquitoes sampled from the Nicosia district, was found to be positive for the presence of WNV RNA. The positive pool found in this study represents the first demonstration of WNV in mosquitoes in Cyprus and confirms that human cases in Cyprus are likely the result of transmission via local Culex mosquitoes.

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

West Nile Virus (WNV)1 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)2 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.

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

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.

Leishmania infection in lagomorphs and minks in Greece.

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.

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

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.

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

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania 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 in vitro 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 Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania 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.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania 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 L. donovani, L. major, and L. tropica 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.

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

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.

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

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.

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

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.

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.

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

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.

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

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.

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.

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.

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

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.

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

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.

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.