Co-Circulation of Phleboviruses and Leishmania Parasites in Sand Flies from a Single Site in Italy Monitored between 2017 and 2020

Sand flies transmit Leishmania infantum, which is responsible for causing leishmaniasis, as well as many phleboviruses, including the human pathogenic Toscana virus. We screened sand flies collected from a single site between 2017 and 2020 for the presence of both phleboviruses and Leishmania. The sand flies were sampled with attractive carbon dioxide traps and CDC light traps between May and October. We collected more than 50,000 sand flies; 2826 were identified at the species level as Phlebotomus perfiliewi (98%) or Phlebotomus perniciosus (2%). A total of 16,789 sand flies were tested in 355 pools, and phleboviruses were found in 61 pools (6 Toscana virus positive pools, 2 Corfou virus positive pools, 42 Fermo virus positive pools, and 7 Ponticelli virus positive pools, and 4 unidentified phlebovirus positive pools). Leishmania was found in 75 pools and both microorganisms were detected in 16 pools. We isolated nine phleboviruses from another 2960 sand flies (five Ponticelli viruses and for Fermo viruses), not tested for Leishmania; the complete genome of a Fermo virus isolate was sequenced. The simultaneous detection in space and time of the Fermo virus and L. infantum is evidence that supports the co-circulation of both microorganisms in the same location and partial overlap of their cycles. A detailed characterization of the epidemiology of these microorganisms will support measures to limit their transmission.

Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis

Background The sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin. Dogs are the main reservoir host of this disease. The main objective of this study was to determine, under both laboratory and field conditions, if dogs infected with L. infantum, were more attractive to female P. perniciosus than uninfected dogs. Methodology/Principal findings We carried out a series of host choice experiments and found that infected dogs were significantly more attractive to P. perniciosus than uninfected dogs in the laboratory as well as in the field. Significantly more P. perniciosus fed on infected dogs than on uninfected dogs. However, the fecundity of P. perniciosus fed on infected dogs was adversely impacted compared to uninfected dogs by lowering the number of laid eggs. Phlebotomus perfiliewi, the second most abundant sand fly species in the field site and a competent vector of L. infantum had similar trends of attractivity as P. perniciosus toward infected dogs under field conditions. Conclusions The results strongly suggest that L. infantum causes physiological changes in the reservoir host which lead to the host becoming more attractive to both male and female P. perniciosus. These changes are likely to improve the chance of successful transmission because of increased contact with infected hosts and therefore, infected dogs should be particularly targeted in the control of zoonotic visceral leishmaniasis in North Africa.

Insecticide Susceptibility Status of Wild Population of Phlebotomus kandelakii and Phlebotomus perfiliewi transcaucasicus Collected from Visceral Leishmaniasis Endemic Foci in Northwestern Iran

Background: Phlebotomus kandelakii and Phlebotomus perfiliewi transcaucasicus sand flies are the vectors of vis­ceral leishmaniasis in Iran. The aim of this study was to evaluate the susceptibility of Ph. kandelakii and Ph. perfil­iewi trans­caucasicus, collected from an endemic focus of visceral leishmaniasis (VL) in the northwest of Iran, to different select­ed insecticides. Methods: Sand flies were collected from the villages of Meshkinshahr and Germi Counties using light traps and aspira­tors from May to October 2019. The sand flies were identified as Ph. kandelakii and Ph. perfiliewi transcauca­sicus us­ing standard identification keys. Susceptibility test was carried out against DDT (4%), Malathion (5%), Propoxur (0.1%) and Lambda-cyhalothrin (0.05%) according to the WHO standard method. MedCalc statistical software was employed to calculate LT50 and LT90 and to compare the chances of sand flies surviving the exposure to the studied in­secticides. Results: A total of 1,278 female specimens were used for the susceptibility tests. Out of which 1,063 samples were used for the test and 215 for the control group. The estimated LT50 values for DDT (4%), Malathion (5%), Propoxur (0.1%), and Lambda-cyhalothrin (0.05%) for Ph. kandelakii were 15.1, 13.4, 15.4 and 5.8 minutes respectively, and for Ph. per­filiewi transcaucasicus were 11.9, 15.6, 15.9 and 5.8 minutes respectively. Conclusion: This susceptibility studies revealed different LT50 values for different insecticides efficient against Ph. kandelakii and Ph. perfiliewi transcaucasicus. The regular monitoring for the resistance of Ph. kandelakii and Ph. per­filiewi transcaucasicus sand flies seems necessary in diseases control programs in this area.

Isolation and Molecular Typing of Leishmania infantum from Phlebotomus perfiliewi in a Re-Emerging Focus of Leishmaniasis, Northeastern Italy

Visceral leishmaniasis (VL) caused by Leishmania (L.) infantum is a public health threat in the Emilia-Romagna region, northeastern Italy, but its epidemiology has not been fully elucidated in this area. The objective of this study was to characterize Leishmania infection in sand flies collected in a re-emerging focus of VL in the Bologna province. During the summer of 2016, 6114 sand flies were collected, identified, and tested for Leishmania detection. Of the identified sand flies, 96.5% were Phlebotomus (P.) perfiliewi and 3.5% were P. perniciosus. Detected parasites were characterized by biomolecular methods (multilocus microsatellite typing and characterization of repetitive region on chromosome 31), and quantified by real-time PCR. The prevalence of Leishmania infection in individually-tested P. perfiliewi sand flies varied from 6% to 10% with an increasing trend during the season. Promastigotes of L. infantum were isolated by dissection in one P. perfiliewi female; the isolated strain (Lein-pw) were closely related to Leishmania parasites from VL cases in northeastern Italy, but differed from strains isolated in dogs from the same area. Our findings strongly support the vector status of P. perfiliewi for human VL in the study area.

Isolation of a Trypanosome Related to Trypanosoma theileri (Kinetoplastea: Trypanosomatidae) from Phlebotomus perfiliewi (Diptera: Psychodidae)

The Trypanosoma theileri group includes several trypanosome species hardly distinguishable due to the lack of discriminating morphological characters. Trypanosomes belonging to this group have been isolated from different bovine, ovine, and cervids in Europe, Africa, Asia, and Americas. The principal vectors of the T. theileri group are considered tabanid flies; however, T. melophagium is transmitted exclusively by sheep keds. In 2016, 128 sand flies out of 2,728 trapped in Valsamoggia municipality, Italy, were individually dissected and an unknown trypanosome strain, named TrPhp1, was isolated from a female of the sand fly Phlebotomus perfiliewi. Sequence analysis placed this trypanosome in the T. theileri group with very high homology to other trypanosomes detected in European cervids. This is the first report of the T. theileri group isolation from a sand fly, and the possible role of this insect group in the trypanosome transmission cycle is discussed. Within the T. theileri group, the phylogenetic analysis distinguished several lineages, which, unfortunately, do not correspond with their host specificity and their taxonomic status remains ambiguous.

Inventaire des phlébotomes dans le foyer leishmanien de Draa El Mizan, Kabylie

Les leishmanioses, maladies à transmission vectorielle liée au genre Phlebotomus, constituent un problème de santé publique en Algérie, essentiellement dans leur forme cutanée. Dans la région de Draa El Mizan (Kabylie), coexistent depuis long-temps laleishmaniose viscérale, et la forme cutanée liée àLeishmania infantum.Durant l’été 2013, une enquête entomologique a été menée à Draa El-Mizan, dans le but de réaliser un inventairefaunistique des phlébotomes à l’aide de pièges lumineux type CDC.Sur un total de 22.998 spécimens récoltés, un échantillon représentatif de 1500 individus a fait l’objet d’une identificationmorphologique par microscope optique.Le genre Phlebotomus est prédominant avec 97,41% des individus représentant 5 taxons où prévalent Phlebotomus perfiliewi (4,63%) et Phlebotomus perniciosus (19,4%). Le sex-ratio M/F global est de 1,39, mais en faveur des individus femelles pour l’espèce Phlebotomus perfiliewi (sex-ratio M/F=0,82).A Draa El Mizan, les conditions climatiques associées aux activités humaines de type pastorales entretiennent la prolifération du genre Phlebotomus inféodé à L. Infan-tum. Par ailleurs, l’utilisation des pièges lumineux type CDC a montré son intérêt dans la surveillance entomologique des maladies vectorielles.

Detection ofLeishmania Infantumin red foxes (Vulpes vulpes) in Central Greece

SUMMARYThis is the first record ofLeishmaniadetection 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 forLeishmania infantumandLeishmania tropicaby molecular methods (polymerase chain reaction (PCR) and restriction fragment length polymorphism) and serology (indirect immunofluorescent antibody test; when blood samples were available).Leishmania infantumDNA 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 forLeishmaniain areas where the sandfly vectors are found. In the prefectures of Larisa and Magnisia, adjacent to Fthiotida,Phlebotomus perfiliewi and Phlebotomus tobbi(known vectors ofL. infantum) have been reported.