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Parasit Vectors ; 13(1): 580, 2020 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203446


BACKGROUND: Ethiopia is affected by human leishmaniasis caused by several Leishmania species and transmitted by a variety of sand fly vectors of the genus Phlebotomus. The sand fly fauna in Ethiopia is highly diverse and some species are closely related and similar in morphology, resulting in difficulties with species identification that requires deployment of molecular techniques. DNA barcoding entails high costs, requires time and lacks reference sequences for many Ethiopian species. Yet, proper species identification is pivotal for epidemiological surveillance as species differ in their actual involvement in transmission cycles. Recently, protein profiling using MALDI-TOF mass spectrometry has been introduced as a promising technique for sand fly identification. METHODS: In our study, we used an integrative taxonomic approach to identify most of the important sand fly vectors of leishmaniasis in Ethiopia, applying three complementary methods: morphological assessment, sequencing analysis of two genetic markers, and MALDI-TOF MS protein profiling. RESULTS: Although morphological assessment resulted in some inconclusive identifications, both DNA- and protein-based techniques performed well, providing a similar hierarchical clustering pattern for the analyzed species. Both methods generated species-specific sequences or protein patterns for all species except for Phlebotomus pedifer and P. longipes, the two presumed vectors of Leishmania aethiopica, suggesting that they may represent a single species, P. longipes Parrot & Martin. All three approaches also revealed that the collected specimens of Adlerius sp. differ from P. (Adlerius) arabicus, the only species of Adlerius currently reported in Ethiopia, and molecular comparisons indicate that it may represent a yet undescribed new species. CONCLUSIONS: Our study uses three complementary taxonomical methods for species identification of taxonomically challenging and yet medically import Ethiopian sand flies. The generated MALDI-TOF MS protein profiles resulted in unambiguous identifications, hence showing suitability of this technique for sand fly species identification. Furthermore, our results contribute to the still inadequate knowledge of the sand fly fauna of Ethiopia, a country severely burdened with human leishmaniasis.

Parasit Vectors ; 13(1): 467, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917242


BACKGROUND: Phlebotomus pedifer is the vector for Leishmania aethiopica causing cutaneous leishmaniasis (CL) in southwestern Ethiopia. Previous research on the transmission dynamics of CL resulted in recommendations for vector control. In order to target these interventions towards affected areas, a comprehensive understanding of the spatial distribution of P. pedifer at high spatial resolution is required. Therefore, this study determined the environmental predictors that facilitate the distribution of P. pedifer and created a map indicating the areas where conditions are suitable for survival of the vector in southwestern Ethiopia with high spatial resolution. METHODS: Phlebotomus pedifer presence points were collected during two entomological surveys. Climate, vegetation and topographic variables were assembled. Climate variables were interpolated with variables derived from high-resolution digital elevation models to generate topoclimatic layers representing the climate conditions in the highlands. A Maximum Entropy model was run with the presence points, predicting variables and background points, which were selected based on a bias file. RESULTS: Phlebotomus pedifer was the only captured Phlebotomus species in the study area and was collected at altitudes ranging between 1685 and 2892 m. Model projections indicated areas with suitable conditions in a 'belt' surrounding the high mountain peaks. Model performance was high, with train and test AUC values being 0.93 and 0.90, respectively. A multivariate environmental similarity surface (MESS) analysis showed that the model projection was only slightly extrapolated for some of the variables. The mean annual temperature was the environmental variable, which contributed most to the model predictions (60.0%) followed by the seasonality in rainfall (13.2%). Variables representing steep slopes showed very low importance to model predictions. CONCLUSIONS: Our findings indicate that the suitable habitats for P. pedifer correspond well with the altitudes at which CL was reported previously, but the predictions are more widely distributed, in contrast with the description of CL to occur in particular foci. Moreover, we confirm that vector distribution is driven by climate factors, suggesting inclusion of topoclimate in sand fly distribution models. Overall, our model provides a map with a high spatial resolution that can be used to target sand fly control measures in southwestern Ethiopia.

Parasit Vectors ; 13(1): 276, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487217


BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

PLoS Negl Trop Dis ; 14(3): e0007947, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196501


BACKGROUND: Cutaneous leishmaniasis (CL) is a major public health concern in Ethiopia. However, knowledge about the complex zoonotic transmission cycle is limited, hampering implementation of control strategies. We explored the feeding behavior and activity of the vector (Phlebotomus pedifer) and studied the role of livestock in CL transmission in southwestern Ethiopia. METHODS: Blood meal origins of engorged sand flies were determined by sequencing host DNA. A host choice experiment was performed to assess the feeding preference of P. pedifer when humans and hyraxes are equally accessible. Ear and nose biopsies from livestock were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches and CDC light traps to determine at which time and where P. pedifer is mostly active. PRINCIPAL FINDINGS: A total of 180 P. pedifer sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source indoors (65.9%, p < 0.001), while no significant differences were determined outdoors and in caves. In caves, hyraxes were represented in blood meals equally as humans (45.5% and 42.4%, respectively), but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes (p = 0.009). Only a single goat nose biopsy from 412 animal samples was found with Leishmania RNA. We found that P. pedifer is predominantly endophagic (p = 0.003), but occurs both indoors and outdoors. A substantial number of sand flies was active in the early evening, which increased over time reaching its maximum around midnight. CONCLUSION: In contrast to earlier suggestions of exclusive zoonotic Leishmania transmission, we propose that there is also human-to-human transmission of CL in southwestern Ethiopia. Livestock does not play a role in CL transmission and combined indoor and outdoor vector control measures at night are required for efficient vector control.

Reservatórios de Doenças/parasitologia , Comportamento Alimentar , Leishmania/isolamento & purificação , Gado/parasitologia , Phlebotomus/fisiologia , Phlebotomus/parasitologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Transmissão de Doença Infecciosa , Etiópia , Feminino , Humanos , Leishmaniose Cutânea/transmissão , Masculino , Pessoa de Meia-Idade , Adulto Jovem
PLoS Negl Trop Dis ; 13(8): e0007667, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425506


BACKGROUND: Ochollo is a village in southern Ethiopia burdened with cutaneous leishmaniasis (CL), where Phlebotomus pedifer is the only vector for Leishmania aethiopica and hyraxes are confirmed reservoir hosts. A detailed description of the different players of transmission, and the ecology and seasonality of the vector needs to be established in order to accomplish efficient control programs. METHODS AND FINDINGS: Between March 2017 and February 2018, a monthly sandfly collection was carried out in different habitats and records of temperature and humidity were taken. Rodents and hyraxes were trapped in the dry and wet season. All samples were screened for Leishmania kinetoplast DNA (kDNA). Positive samples were further processed for determination of the Leishmania species and the species of the sandfly/small mammal that was found infected. Additionally, the species of 400 sandfly specimens from different habitats and seasons was identified. 17,190 Sergentomyia and Phlebotomus sandflies were caught and showed an overall kDNA prevalence of 2.6%, all were L. aethiopica infections only found in P. pedifer. The overall sandfly and P. pedifer abundance peaked in the dry season and was negatively correlated with the %RH. The kDNA prevalence varied over the months and was negatively correlated with the temperature. Total sandfly abundance did not differ between the sampled habitats, but P. pedifer was the distinct predominant species only in caves. Moreover, significantly more infected sandflies were found in caves. Only 1/192 rodents were kDNA positive, while 20.0% (5/25) of Heterohyrax brucei were found infected. CONCLUSIONS: This study suggests that caves may be a source of multiplication of the infection. If an outdoor control program would be considered, it would be useful to focus on caves in the wet season, when the sandfly abundance is lowest. The captured rodent species appear not important for transmission and the contribution of hyraxes in transmission should be further investigated.

DNA de Protozoário/análise , Reservatórios de Doenças , Vetores de Doenças , Procaviídeos/parasitologia , Leishmania/genética , Leishmaniose Cutânea/epidemiologia , Psychodidae/parasitologia , Animais , DNA de Protozoário/genética , Transmissão de Doença Infecciosa , Etiópia/epidemiologia , Feminino , Humanos , Umidade , Leishmaniose Cutânea/transmissão , Masculino , Carga Parasitária , Prevalência , Psychodidae/crescimento & desenvolvimento , Estações do Ano , Temperatura