Morphology does not allow differentiating the species of the Phlebotomus perniciosus complex: Molecular characterization and investigation of their natural infection by Leishmania infantum in Morocco.

Morphological and DNA-based complemented approaches were applied for characterization of sympatric populations of Phlebotomus longicuspis and Phlebotomus perniciosus in Morocco. Both sand fly species are generally recorded in sympatry in North Africa but on few occasions have been molecularly characterized. The diagnostic confusion of these species has led to errors in their geographical distribution and probably, in the assignment of their role in the transmission of L. infantum. Sand flies were caught inside households in El Borouj, central Morocco, in 2014-2015. For female sand flies, detection of L. infantum natural infection and blood meal identification were carried out. According to morphological identification, Phlebotomus longicuspis s.l. (34.7%) was the second most abundant Phlebotomus species after P. sergenti, followed by atypical Phlebotomus perniciosus (7.1%); 11.6% of the male specimens of P. longicuspis s.l. were identified as P. longicuspis LCx according to the number of coxite setae. The density of Larroussius species was very high (31 Larroussius/light trap/night) in the peripheral neighbourhood of Oulad Bouchair (p = 0.001) where the first case of cutaneous leishmaniasis due to Leishmania infantum was detected in 2017. Phylogenetic trees based on three independent genes highlighted three well-supported clusters within P. perniciosus complex that could be interpreted as corresponding to P. perniciosus, P. longicuspis s.s. and an undescribed species, all coexisting in sympatry. Some females with typical morphology of P. longicuspis were genetically homologous to P. perniciosus. The taxa cannot be differentiated by morphological methods but characterized by a distinctive genetic lineage for which the synapomorphic characters are described. Leishmania infantum was detected in females of all clusters with a low parasite load. Population genetics will help to assess the threat of the geographical spread of L. infantum in Morocco by determining the density, abundance and vector role of the species of the P. perniciosus complex identified correctly.

Understanding the factors that determine the emergence of anthroponotic cutaneous leishmaniasis due to Leishmania tropica in Morocco: Density and mitochondrial lineage of Phlebotomus sergenti in endemic and free areas of leishmaniasis.

Anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica is spreading to new areas in Morocco. Exposure to the vector, Phlebotomus sergenti, is the only proven risk factor. Our objective was to compare the densities and genetic characteristics of P. sergenti populations in two nearby localities in Morocco, one in an ACL endemic area (El Borouj) and another in a nonendemic area (Sidi Hajjaj). P. sergenti density was significantly higher in the endemic area than in the nonendemic town (p = 0.032). A different predominant P. sergenti mitochondrial lineage was evidenced in each one of the two localities, and for the first time, the P. sergenti lineage acting as a vector of L. tropica has been identified. Bioclimatic differences were detected between both localities. In conclusion we found differences in both the density and the mitochondrial lineage of P. sergenti populations that may explain the different epidemiological situation. Given that the density of P. sergenti in the locality without ACL cases seems sufficient to allow transmission, the main factor that would justify its nonendemic character could be the absence of P. sergenti Lineage IV, which seems to prefer warmer and drier climates.

Intra and peridomiciliary comparison of density, sex ratio and gonotrophic stage of Phlebotomus sergenti in an active anthroponotic cutaneous leishmaniasis focus in Morocco.

Cutaneous leishmaniasis due to Leishmania tropica represents a major public health problem due to its ability to spread into non-endemic areas by means of its vectors, and the associated dramatic psychosocial impact. The objective of this work was to compare the intra and extradomiciliary density, sex ratio and gonotrophic stage of sand flies from a recent active focus in Morocco. This field study is based on the need to optimize the effectiveness of control programs. Two different capture methods, CDC light traps and sticky traps, were used at two different times of the year, corresponding with the peaks of sand fly abundance. 7,815 sand flies were captured and classified into 13 species belonging to genera Sergentomyia (50.8%) and Phlebotomus (49.2%). Phlebotomus sergenti was the most abundant and frequent species of the genus Phlebotomus both inside (49.3%) and outside houses (52.1%) and it showed the highest density in extradomiciliary captures in June. The proportion of blood-fed females was similar indoors and outdoors (21.5% and 26.3%, respectively). Females in the three gonotrophic stages were found in 26% houses and this was significantly associated with some factors related to housing conditions. Therefore, P. sergenti seems well adapted to both indoors and outdoors biotopes where these females coexist with males. These findings suggest that the adoption of additional measures could benefit the strategy of the Moroccan health authorities, currently consisting of indoor insecticide spraying, given that transmission may also occur outdoors.

Seasonal dynamics of phlebotomine sand flies and autochthonous transmission of Leishmania infantum in high-altitude ecosystems in southern Spain.

Leishmaniasis is a vector-borne disease transmitted by sand flies. A dozen species have been involved in the transmission of Leishmania infantum in the Mediterranean region. Climate change may alter sand fly distribution at particular altitudes and latitudes. The objective of this study was to interrogate the existence of stable populations of sand flies in high-altitude ecosystems and evaluate if these populations are enough to support autochthonous transmission of leishmaniasis. These altitudinal conditions can be found in Sierra Nevada (southern Spain). Therefore, we have determined the sand fly population dynamics in different biotopes located at elevations above 1,300 m a.s.l. and searched for evidence of leishmaniasis transmission. Five collecting sites above 1,300 m a.s.l. containing large livestock concentrations were selected. Sand flies were caught using CDC light traps from May to November, annually from 2008 to 2013, and these were morphologically identified. Association between sand fly density or presence and temperature/humidity was estimated by linear and logistic regression, respectively. Leishmania infantum detection in female sand flies was performed by PCR. Diagnosis of canine leishmaniasis (CanL) was carried out by indirect immunofluorescence and PCR. A total of 2,973 specimens of 5 sand fly species were collected from June to October. Phlebotomus perniciosus was the most frequent (100%), abundant (80.1%) and densest species (9.8 sand flies/trap). The minimum temperature on the day of capture was the most important variable factor for sand fly presence and P. perniciosus density. An increase in altitude showed a negative effect over the sand fly diversity and activity period, driving changes in seasonal dynamics similar to those reported by latitudinal changes. CanL prevalence was 23%, a similar rate to previous surveys carried out on randomly selected dogs from towns in southern Spain. A successful host-vector-pathogen network was found at this altitude characterised by 9.9% L. infantum infection rate in non-blood fed P. perniciosus and Phlebotomus ariasi females and high CanL prevalence that entails an increase in the leishmaniasis risk area driven by sand fly colonization.

Risk factors for the expansion of cutaneous leishmaniasis by Leishmania tropica: Possible implications for control programmes.

Cutaneous leishmaniasis (CL) caused by Leishmania tropica is emerging in new areas, initially as outbreaks and then establishing endemic foci. There is little evidence of the risk factors and effectiveness of existing control measures, what limits our ability to generalize in different epidemiological settings. The disease is described as anthroponotic; however, zoonotic outbreaks have been reported in some countries. Our aim was to identify risk factors in a recently reported endemic focus in Morocco in order to design more effective control programmes. A case-control study was conducted from September 2014 to October 2015 for epidemiological data collection from families with and without CL cases. Sandflies were captured and L. tropica infection determined. The presence of potential animal reservoirs was evaluated. 71 CL cases (44 diagnosed between 2013 and 2015) and 137 healthy people were surveyed. The average age of the new cases was 33.1 ± 22.3 years, and 69.0% were women. Phlebotomus sergenti was the most abundant species with a density of 4.27 sandflies/trap/night and differences between houses with and without CL cases were detected (p-value = 0.014). Overall, 2.7% female P. sergenti and 3.0% dogs were positive for L. tropica. Human, cat, rabbit and bird blood was detected in blood-fed P. sergenti females. 45% people used preventive measures that were not translated into a reduction in the individual risk of acquiring CL. Exposure to P. sergenti was the only risk factor found, and the reduction in its density could be achieved through the improvement of water wells management, organic fertilizers' disposal and dogs control. The lack of effectiveness of indoor residual spraying and treated nets are attributable to poor compliance and misuse of them. In addition, result optimization of the awareness campaigns on the public is possible by involving patients with CL to explain their own experience.

Anisakis simplex s.l. parasitization in mackerel (Scomber japonicus) caught in the North of Morocco--prevalence and analysis of risk factors.

Consumption of mackerel (Scomber japonicus) is very widespread in Morocco, where its production is an important economic pillar. We investigated the prevalence of infection by Anisakis spp. in mackerel caught in Moroccan waters, analyzing infection risk factors. The prevalence was generally higher in fish from the Atlantic (67.9%) than from the Mediterranean (57.0%), but they did not differ in the mean abundance, intensity, or prevalence of muscle parasitization. A. pegreffii was the predominant species (82.6% of larvae) followed by the hybrid A. simplex s.s./A. pegreffii (16.3%). Only one L3 of A. simplex s.s. was found in a specimen of S. japonicus from the Atlantic. Mackerel infection was associated with total fish weight, gonad weight, catch area, and catch season. However, muscle infection was associated solely with total weight and parasite loads. The consumption of lower-weight mackerel may be a good prophylactic measure against human anisakiasis.

Genetic variability of Anisakis simplex s.s. parasitizing European hake (Merluccius merluccius) in the Little Sole Bank area in the Northeast Atlantic.

In this study, we researched the presence of anisakids in specimens of Merluccius merluccius caught in the area of Little Sole Bank, in the Northeast Atlantic, and found that 100% of the European hake examined were infected and showed high average values of abundance (976.88) and intensity (976.88). The larvae were identified in morphological terms as morphotype type I and in molecular terms as Anisakis simplex s.s via polymerase chain reaction (PCR) restriction fragment length polymorphism of the rDNA. The genetic variability of the A. simplex s.s population in the North Atlantic is notable, with at least two ribosomal and three mitochondrial haplotypes which are different from the specimen used as control, reflecting the diversity of this species, an aspect which has scarcely been studied to date. The cox-2 gene appears to be an interesting candidate for generating new genetic markers which can be applied to differentiate between A. simplex s.s and Anisakis pegreffii. We detected 11 fixed differences in this gene, and it also offers the advantage of being easily amplified by PCR. The high prevalence of infection by A. simplex s.s and the extremely high average intensity and abundance values can have significant repercussions on public health, especially among populations which regularly eat insufficiently cooked or raw fish and have a certain genetic predisposition; the genetic variability of the parasite could be another factor to take into account.