The current status of phlebotomine sand flies in Albania and incrimination of <i>Phlebotomus neglectus</i> (Diptera, Psychodidae) as the main vector of <i>Leishmania infantum</i> - Fig 2

<p><b>Sand fly collection sites (A and B).</b> Map showing: (A) 2006 sampling sites targeting foci of recent VL transmission and <i>Culicoides</i> midges monitoring in the frame of a bluetongue-disease surveillance program (see text for the sampling frame design); (B) 2011 sampling sites targeting foci of recent VL transmission.</p

Albanian <i>Leishmania</i> isolates identified from 1988 through 2012 by MultiLocus Enzyme Electrophoresis (MLEE) or PCR-restriction fragments length polymorphism of ribosomal internal transcribed spacer-1 (ITS-1 PCR-RFLP).

<p>Albanian <i>Leishmania</i> isolates identified from 1988 through 2012 by MultiLocus Enzyme Electrophoresis (MLEE) or PCR-restriction fragments length polymorphism of ribosomal internal transcribed spacer-1 (ITS-1 PCR-RFLP).</p

Spatial distribution of sand fly sampling sites and human leishmaniasis cases in the study area for the period 2001–2014.

<p>Spatial distribution of sand fly sampling sites and human leishmaniasis cases in the study area for the period 2001–2014.</p

Prevalence and sand fly species by county and district collected in the 2006 <i>Culicoides</i>-monitoring study in southernmost counties of Albania (Study 2).

<p>Prevalence and sand fly species by county and district collected in the 2006 <i>Culicoides</i>-monitoring study in southernmost counties of Albania (Study 2).</p

Molecular detection and characterization of <i>Leishmania</i> from wild-caught sand flies in Albania.

<p><b>(A)</b> Nested-PCR targeting a <i>Leishmania</i> sp. small-subunit ribosomal DNA sequence. Lane C₁-: uninfected reared sand fly DNA; lane C₂-: PCR Master Mix with no DNA; lanes 2, 4, 5, 7, 8, 10, 11: positive <i>P</i>. <i>neglectus</i> pools; lane 13: positive <i>P</i>. <i>tobbi</i> pool; lanes 1, 3, 6, 9, 12, 14: negative <i>P</i>. <i>perfiliewi</i> pools; lane C₁+: <i>L</i>. <i>infantum</i> promastigotes DNA; C₂+: <i>L</i>. <i>infantum</i> promastigotes DNA mixed with uninfected reared sand fly DNA; lane M: 100 base pair ladder (Promega). <b>(B)</b> ITS-1 n-PCR-RFLP for <i>Leishmania</i> species characterization. Lane M: 100 base pair ladder (Promega); lane 1: <i>Leishmania</i> isolate from Albanian <i>P</i>. <i>neglectus</i> (IMJN/AL/2011/MJN2-ISS3056); lane 2: <i>L</i>. <i>infantum</i> (human isolate from Albania, MHOM/AL/2006/ISS2840); lane 3: <i>L</i>. <i>infantum</i> (dog isolate from Albania, MCAN/AL/2006/ISS2841); lane 4: WHO reference strain for <i>L</i>. <i>infantum</i> (MHOM/TN/1980/IPT-1).</p

Significant non-zero CCHFV migration rates worldwide.

<p>Rates supported by a BF of >3 are highlighted: the relative strength of the support is indicated by the colour of the lines (from dark red = weak to light red = strong). Dotted lines indicate non-significant linkages. The map was reconstructed using SPREAD (see Methods). The numbers indicate the mean estimated year in which the virus entered the area. </p

The current status of phlebotomine sand flies in Albania and incrimination of <i>Phlebotomus neglectus</i> (Diptera, Psychodidae) as the main vector of <i>Leishmania infantum</i>

<div><p>The incidence of visceral leishmaniasis (VL) in Albania is higher than in other countries of southern Europe, however the role of local sand fly species in the transmission of <i>Leishmania infantum</i> was not addressed conclusively. In 2006, a country-wide collection of sand flies performed in 14 sites selected based on recent occurrence of VL cases showed that <i>Phlebotomus neglectus</i> was by far the most prevalent species (95.6%). Furthermore, 15% of pools made from 422 <i>P</i>. <i>neglectus</i> females tested positive for <i>Leishmania</i> sp. genomic DNA. In the same year, <i>Culicoides</i> trapping was performed for bluetongue disease surveillance in 91 sites of southern Albania, targeting livestock farms regardless recent occurrence of VL in the surveyed areas. In 35 sites where sand flies were collected along with midges, <i>Phlebotomus perfiliewi</i> was the most prevalent among the <i>Phlebotomus</i> species identified, however search for leishmanial DNA in females of this species was unsuccessful. In 2011, sand flies were trapped in 4 sites of north Albania characterized by high VL incidence, and females were dissected to search for <i>Leishmania</i> infections. Both <i>P</i>. <i>neglectus</i> and <i>P</i>. <i>tobbi</i> were collected at high densities. Two positive specimens were detected from a sample of 64 <i>P</i>. <i>neglectus</i> trapped in one site (3.1%). Parasites were successfully cultured from one specimen and characterized as belonging to <i>Leishmania infantum</i> zymodeme MON-1, the only zymodeme so far identified as the agent of human and canine leishmaniasis in the country. Altogether our studies indicate that <i>P</i>. <i>neglectus</i> is the main leishmaniasis vector in Albania.</p></div

Sand fly species diversity and cumulative relative abundance recorded during three entomological studies carried out in Albania in 2006 and 2011.

<p>Sand fly species diversity and cumulative relative abundance recorded during three entomological studies carried out in Albania in 2006 and 2011.</p

The current status of phlebotomine sand flies in Albania and incrimination of <i>Phlebotomus neglectus</i> (Diptera, Psychodidae) as the main vector of <i>Leishmania infantum</i> - Fig 2

<p><b>Sand fly collection sites (A and B).</b> Map showing: (A) 2006 sampling sites targeting foci of recent VL transmission and <i>Culicoides</i> midges monitoring in the frame of a bluetongue-disease surveillance program (see text for the sampling frame design); (B) 2011 sampling sites targeting foci of recent VL transmission.</p

The maximum clade credibility (MCC) tree of CCHFV S gene sequences.

<p>The branches are coloured on the basis of the most probable location of the descendent nodes (A=Africa, AL=Albania, ASC=Central Asia, BU=Bulgaria, CH=China, G=Greece, KO=Kosovo, MO=Middle East, PA=Pakistan, T=Turkey). The numbers on the internal nodes indicate significant posterior probabilities (pp>0.8), and the scale at the bottom of the tree represents the number of years before the last sampling time (2010). The main geographical clades (genotypes) have been highlighted.</p