Molecular detection of Leishmania donovani, Leishmania major, and Trypanosoma species in Sergentomyia squamipleuris sand flies from a visceral leishmaniasis focus in Merti sub-County, eastern Kenya

Abstract Background Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remain limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sand fly species responsible for Leishmania transmission in the sub-County and their blood-meal hosts. Methods We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sand flies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (cox1) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Blood-meal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products. Results We sampled 526 sand flies consisting of 8 species, Phlebotomus orientalis (1.52%; n = 8), and 7 Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sand fly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sand fly blood meals. However, we also detected mixed blood meals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood. Conclusions Our findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sand flies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major and L. donovani are known to cause ZCL and VL, respectively. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area.

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Molecular detection of Leishmania donovani, Leishmania major, and Trypanosoma spp. in Sergentomyia squamipleuris sandflies from a visceral leishmaniasis focus in Merti sub-County, eastern Kenya

10.1101/2020.07.29.226191 ◽

2020 ◽

Author(s):

Barrack O. Owino ◽

Jackline Milkah Mwangi ◽

Steve Kiplagat ◽

Hannah Njiriku Mwangi ◽

Johnstone M. Ingonga ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Mus Musculus ◽

Leishmania Donovani ◽

Leishmania Major ◽

Control Measures ◽

Health Concern ◽

Coi Gene ◽

Pcr Analysis ◽

Mechanical Transmission ◽

Reservoir Hosts

AbstractBackgroundVisceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remains limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sandfly species responsible for Leishmania transmission in the sub-County, and their blood-meal hosts.MethodsWe conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sandflies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (COI) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Bloodmeal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products.ResultsWe sampled 526 sandflies consisting of eight species, Phlebotomus orientalis (1.52%; n = 8) and seven Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sandfly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sandfly bloodmeals. However, we also detected mixed bloodmeals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood.ConclusionsOur findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sandflies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major causes ZCL while L. donovani is responsible for VL. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area.

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The Phlebotomus papatasi systemic transcriptional response to trypanosomatid-contaminated blood does not differ from the non-infected blood meal

10.21203/rs.3.rs-46963/v3 ◽

2020 ◽

Author(s):

Megan A. Sloan ◽

Jovana Sadlova ◽

Tereza Lestinova ◽

Mandy J. Sanders ◽

James A. Cotton ◽

...

Keyword(s):

Blood Meal ◽

Leishmania Donovani ◽

Leishmania Major ◽

Transcriptional Response ◽

Sand Fly ◽

Host Responses ◽

Sand Flies ◽

Phlebotomus Papatasi ◽

New Thinking ◽

Contaminated Blood

Abstract Background Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control, require a better understanding of the key step for transmission namely, the establishment of infection inside the fly. Methods In this work we wanted to identify fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, Leishmania donovani and Herpetomonas muscarum: a parasite not transmitted to humans. Results Of these, only L. major is able to successfully establish an infection in P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and were not different from each other. They were also indistinguishable from non-contaminated blood. Conclusions This implies that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.

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The Phlebotomus papatasi systemic transcriptional response to trypanosomatid-contaminated blood does not differ from the non-infected blood meal

10.21203/rs.3.rs-46963/v2 ◽

2020 ◽

Author(s):

Megan A. Sloan ◽

Jovana Sadlova ◽

Tereza Lestinova ◽

Mandy J. Sanders ◽

James A. Cotton ◽

...

Keyword(s):

Blood Meal ◽

Leishmania Donovani ◽

Leishmania Major ◽

Transcriptional Response ◽

Sand Fly ◽

Host Responses ◽

Sand Flies ◽

Phlebotomus Papatasi ◽

New Thinking ◽

Contaminated Blood

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The Phlebotomus papatasi transcriptomic response to trypanosomatid-contaminated blood is robust but non-specific

10.1101/867382 ◽

2019 ◽

Author(s):

Megan A. Sloan ◽

Jovana Sadlova ◽

Tereza Lestinova ◽

Mandy J. Sanders ◽

James A. Cotton ◽

...

Keyword(s):

Blood Meal ◽

Leishmania Donovani ◽

Leishmania Major ◽

Blood Feeding ◽

Sand Fly ◽

Insect Vector ◽

Sand Flies ◽

Phlebotomus Papatasi ◽

Transcriptomic Response ◽

Gene Expression Response

AbstractLeishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control, require a better understanding of the key step for transmission namely, the establishment of infection inside the fly. In this work we wanted to identify fly transcriptomic signatures associated with infection success or failure. We used next generation sequencing to describe the transcriptome of the sand fly Phlebotomus papatasi when fed with blood alone or with blood containing one of three trypanosomatids: Leishmania major, Leishmania donovani and Herpetomonas muscarum: a parasite not transmitted to humans. Of these, only L. major was able to successfully establish an infection in P. papatasi. However, the transcriptional signatures observed were not specific to success or failure of infection but a generalised response to the blood meal. This implies that sand flies perceive Leishmania as just a feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment.Authors summaryLeishmania are parasites that cause leishmaniasis, a group of serious diseases that affect millions of people, mainly across the subtropics and tropics. They are transmitted to humans by phlebotomine sand flies. However, despite establishment in the insect’s midgut being key to transmission, early infection events inside the insect are still unclear. Here, we study the gene expression response of the insect vector to a Leishmania parasite that is able to establish infection (L. major) one that is unable to do so (L. donovani) as well as one that is not a natural parasite of sand flies (Herpetomonas muscarum). We found that responses following any of the infected blood meals was very similar to uninfected blood meal. However, changes post-blood meal from day 1 to day 9 were dramatic. As a blood feeding insect can accumulate three times its weight in one blood meal, this seems to be the most important physiological change rather than the presence of the parasite. The latter might be just one in a number of microbes the insect encounters. This result will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.

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Entomological Studies in Itaúna, Brazil, an Area With Visceral Leishmaniasis Transmission: Fauna Survey, Natural Leishmania Infection, and Molecular Characterization of the Species Circulating in Phlebotomine Sand Flies (Diptera: Psychodidae)

Journal of Medical Entomology ◽

10.1093/jme/tjz061 ◽

2019 ◽

Vol 56 (5) ◽

pp. 1368-1376 ◽

Cited By ~ 4

Author(s):

Josiane V Lopes ◽

Erika M Michalsky ◽

Nathalia C L Pereira ◽

Adão J V de Paula ◽

Fabiana O Lara-Silva ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Natural Infection ◽

Local Population ◽

Influential Factor ◽

Sand Fly ◽

Health Concern ◽

Leishmania Infection ◽

Lutzomyia Longipalpis ◽

Sand Flies ◽

Phlebotomine Sand Flies

Abstract Among neglected tropical diseases, visceral leishmaniasis (VL) shows great relevance in global terms and is a serious public health concern due to the possibility of severe and lethal forms in humans. In this study, we evaluate entomological factors such as diversity and abundance of phlebotomine sand flies (Diptera:Psychodidae) and the Leishmania species circulating in these species in possible association with VL transmission in the Brazilian town Itaúna. The entomological collections were performed during three consecutive nights, always in the third week of each month, within a period of 12 mo. A total of 1,786 sand fly specimens were collected, from which 20% were collected inside houses. The influence of three local climatic variables (temperature, rainfall, relative humidity) on the population sizes of these insects was evaluated. Temperature was the most influential factor, with a significant positive correlation with the local population size of phlebotomine sand flies collected per month. Lutzomyia longipalpis (Lutz & Neiva, 1912) was the predominant species in the study area. Leishmania DNA was detected in nine out of 133 pools of sand fly females, using nested/PCR, which resulted in a minimal natural infection rate of 2.91%. DNA from Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatida), was detected in Evandromyia cortelezzii (Bréthes, 1923), Ev. evandroi (Costa, Lima & Antunes, 1936), Ev. lenti (Mangabeira, 1938), and Ev. termitophila (Martins, Falcão & Silva, 1964), besides Lu. longipalpis. Our study indicates favorable conditions for VL spreading in Itaúna due to the presence of Lu. longipalpis and Le. infantum-infected phlebotomine sand flies.

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INVESTIGATION OF LEISHMANIASIS VECTORS IN EL-KADOBA VILLAGE, WHITE NILE STATE, SUDAN

International Journal of Research -GRANTHAALAYAH ◽

10.29121/granthaalayah.v5.i1.2017.1938 ◽

2017 ◽

Vol 5 (1) ◽

pp. 430-441

Author(s):

Belal Abdallah A. Adam ◽

Moawia Mukhtar Hassan ◽

Osman Mohammed Abd Elnour ◽

Ahmed Hamid Awadallah

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Morphological Characters ◽

Sand Fly ◽

Leishmania Infection ◽

Sand Flies ◽

Kala Azar ◽

Ecological Aspects ◽

Binocular Microscope ◽

White Nile

Visceral leishmaniasis (VL; kala-azar) is one of the most important parasitic tropical diseases in Sudan and the Sudan is considered to be one of the most important foci in the world. The visceral leishmaniasis has been described in Sudan since the beginning of the twentieth century. In Sudan, VL is caused by Leishmania donovani complex: MON 18, MON 30 and MON 82 zymodemes and The proven vector is Phlebotomus (Larroussius) orientalis, in this study a survey was carried out to identify the principal vector of VL based on Leishmania infection, morphological characters and to determine some ecological aspects of the sand flies prevalent in the area . Ten species of sand flies were recorded, three Phlebotomus species and seven Sergentomyia species of these sandflies, P. rodhaini was collected only from Acacia seyal/Balanities aegyptiaca at the Island whereas S. hunti was collected from the Acacia nilotica forest only of the total collections, P. orientalis representing 3.80% (248 specimens) of the total collection and P. papatasi were 3.11% of the collection. Sand flies identification was done under a binocular microscope at 40x (magnification). The main features used for sand flies identification were the sperm theca of the female, the termination of the male and the pharyngeal and the ciboria toothed structures of both sexes. According to results of this study we recommended the following: more studies are needed in the future to determine the transmission season, and infection rates of Leishmania parasites in human and the animal host in this area. Annually Entomological surveys must be done to determine density of Sand fly Vectors and Encourage the use of personal protection tools (ITNs), repellents, and improving of houses to avoid bite of sand fly.

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Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

10.1101/2020.11.18.388553 ◽

2020 ◽

Author(s):

Subir Karmakar ◽

Nevien Ismail ◽

Fabiano Oliveira ◽

James Oristian ◽

Wen Wei Zhang ◽

...

Keyword(s):

Immune Response ◽

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Leishmania Major ◽

Protective Efficacy ◽

Sand Fly ◽

Endemic Region ◽

Cytokines And Chemokines ◽

Human Leishmaniasis ◽

Strong Candidate

AbstractVisceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen−/−). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen−/− against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen−/− did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen−/−-immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen−/− immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-γ and TNF-α, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen−/− which showed equal protection as laboratory-grade LmCen−/− parasites in hamsters. Importantly, GLP-grade LmCen−/− parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen−/− parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

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Second Blood Meal by Female Lutzomyia longipalpis: Enhancement by Oviposition and Its Effects on Digestion, Longevity, and Leishmania Infection

BioMed Research International ◽

10.1155/2018/2472508 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 5

Author(s):

C. S. Moraes ◽

K. Aguiar-Martins ◽

S. G. Costa ◽

P. A. Bates ◽

R. J. Dillon ◽

...

Keyword(s):

Blood Meal ◽

Molecular Mechanisms ◽

Sand Fly ◽

Leishmania Infection ◽

Lutzomyia Longipalpis ◽

Sand Flies ◽

Gonotrophic Cycle ◽

Trypsin Activity ◽

The Impact ◽

Blood Meals

Lutzomyia longipalpis is the main vector of visceral leishmaniasis (VL) in America. Physiological and molecular mechanisms of Leishmania infection in sand flies have been studied during the first gonotrophic cycle. There are few studies about these interactions during the second gonotrophic cycle mainly because of the difficulties maintaining sand flies through sequential feeds. Here we standardized conditions to perform the second blood feed efficiently, and our results show that oviposition is an essential factor for the success of multiple feeds. We evaluated the impact of the second blood meal on longevity, protein digestion, trypsin activity, and Leishmania mexicana development within L. longipalpis gut. Mortality of blood-fed females increases after second blood meal as compared to sugar-fed females. Trypsin activity was lower during the second gonotrophic cycle. However, no difference in protein intake was observed between blood meals. There was no difference in the population size of Leishmania in the gut after both blood meals. In this work, we presented an optimized protocol for obtaining sufficient numbers of sand fly females fed on a second blood meal, and we described some physiological and parasitological aspects of the second gonotrophic cycle which might influence the vectorial competence of sand flies.

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Response to thermal and infection stresses in an American vector of visceral leishmaniasis

10.1101/2020.11.23.373100 ◽

2020 ◽

Author(s):

Kelsilandia A. Martins ◽

Caroline S. Morais ◽

Susan J. Broughton ◽

Claudio R. Lazzari ◽

Paul A. Bates ◽

...

Keyword(s):

Heat Shock ◽

Body Temperature ◽

Visceral Leishmaniasis ◽

Heat Shock Proteins ◽

Blood Meal ◽

Blood Feeding ◽

The Body ◽

Sand Fly ◽

Sand Flies ◽

Shock Proteins

AbstractThe phlebotomine sand fly Lutzomyia longipalpis is the primary insect vector of visceral leishmaniasis in the Americas. For ectothermic organisms such as sand flies, the ambient temperature is a critical factor influencing all aspects of their life. However, the impact of temperature has been ignored in previous investigations of stress-induced responses by the vector, such as taking a blood meal or during Leishmania infection. Therefore, this study explored the interaction of Lu. longipalpis with temperature by evaluating sand fly behaviour across a thermal gradient after sugar or blood-feeding, and infection with Leishmania mexicana. Thermographic recordings of sand fly females fed on mice were analysed, and the gene expression of heat shock proteins HSP70 and HSP90(83) was evaluated when insects were exposed to extreme temperatures or infected. The results showed that 72h after blood ingestion females of Lu. longipalpis became less active and preferred relatively low temperatures. However, at later stages of blood digestion females increased their activity and remained at higher temperatures prior to taking a second blood meal; this behaviour seems to be correlated with the evolution of their oocysts and voracity for a second blood meal. No changes in the temperature preferences of female sand flies were recorded in the presence of a gut infection by Le. mexicana, indicating that this parasite has not triggered behavioural immunity in Lu. longipalpis. Real-time imaging showed that the body temperature of female flies feeding on mice increased to the same temperature as the host within a few seconds after landing. The body temperature of females remained around 35 ± 0.5 °C until the end of blood-feeding, revealing a lack of thermoregulatory behaviour. Analysis of expression of heat shock proteins revealed insects increased expression of HSP90(83) when exposed to higher temperatures, such as during blood feeding. Our findings suggest that Lu. longipalpis interacts with the environmental temperature by using its behaviour to avoid temperature-related physiological damage during the gonotrophic cycle. However, the expression of certain heat shock proteins might be triggered to mitigate against thermal stress in situations where a behavioural response is not the best option.

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The Phlebotomus Papatasi Transcriptomic Response to Trypanosomatid-contaminated Blood is Robust but non-specific

10.21203/rs.3.rs-46963/v1 ◽

2020 ◽

Author(s):

Megan A. Sloan ◽

Jovana Sadlova ◽

Tereza Lestinova ◽

Mandy J. Sanders ◽

James A. Cotton ◽

...

Keyword(s):

Blood Meal ◽

Leishmania Donovani ◽

Leishmania Major ◽

Sand Fly ◽

Phlebotomus Papatasi ◽

Transcriptomic Response ◽

Infected Blood ◽

New Thinking ◽

Contaminated Blood ◽

Generation Sequencing

Abstract Background Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control, require a better understanding of the key step for transmission namely, the establishment of infection inside the fly.Methods In this work we wanted to identify fly transcriptomic signatures associated with infected blood meal with non-infected blood meal as our baseline. We used next generation sequencing to describe the transcriptome of the sand fly Phlebotomus papatasi when fed with blood alone or with blood containing one of three trypanosomatids: Leishmania major, Leishmania donovani and Herpetomonas muscarum: a parasite not transmitted to humans.Results Of these, only L. major was able to successfully establish an infection in P. papatasi. However, the transcriptional signatures observed were not specific to success or failure of infection but a generalised response to the blood meal.Conclusions This implies that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. This result will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.

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