Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annual... more Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annually. Here, we aimed to investigate the likely variations in gene expression of glycopro-tein63 (gp63), heat shock protein 70 (HSP70), histone, arginase, cysteine protease B (CPB), Leishmania homologue of receptors for activated C kinase (LACK), small hydrophilic endoplasmic reticulum-associated protein (SHERP) in metacyclic promastigotes of L. major isolated from Phlebotomus papatasi sand flies and promastigotes excessively cultured in culture medium. The parasites were collected from suspected CL cases in Pasteur Institute of Iran, cultured and inoculated into the female BALB/c mice (2×10 6 promastigotes). Sand flies were trapped in Qom province, fed with the blood of euthanized infected mice and subsequently dissected in order to isolate the midgut including stomodeal valve. The metacyclic promastigotes were isolated from Ph. papatasi (Pro-Ppap) using peanut agglutinin test (PNA), then continuously cultured in RPMI-1640 medium enriched with fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) to reach stationary phase (Pro-Stat). The gene expression was evaluated in both parasitic stages (Pro-Ppap and Pro-Stat) using qRT-PCR. Out results showed a significant increased gene expression at Pro-Ppap stage for gp63 (P = 0.002), SHERP (P = 0.001) and histone (P = 0.026) genes, in comparison with Pro-Stat stage. Noticeably, significant changes were, also, demonstrated in 10 th to 15 th passages [gp63 (P = 0.041), arginase (P = 0.016), LACK (P = 0.025)] and in 5 th to 20 th passage (SHERP) (P = 0.029). In conclusion, the findings of the present study seem to be essential in designing Leishmania studies, in particular regarding host-parasite interaction, immunization and infectivity studies.
Background: Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public ... more 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 difference between Swiss albino (resistant) and BALB/c (susceptible) strains in response to L... more The difference between Swiss albino (resistant) and BALB/c (susceptible) strains in response to L. donovani infection has been linked to inheritable genes, and the GATAq and Lsh group of genes plays a key role in disease transmission. This study sought to investigate effects of gene neutralization through cross breeding and back crossing. SAB mice were generated by cross breeding of BALB/c mice and Swiss Albino mice. This study aimed at investigating the susceptibility, immune response and pathology of SAB mice after L. donovani infection. Serum was collected from all mice groups 4 weeks post infection and levels of IFN-γ and IL-4 cytokine were determined using ELISA. Body and spleen weights were measured, and tissue section stained for histopathology examination. In this study the L. donovani infected swiss mice had significantly low amounts of IL-4 as compared to the SAB mice (P=0.0093). Whereas the IL-4 levels in BALB/c mice were not significantly different to that of SAB mice (P= 0.1507). Conversely infected swiss albino mice had significantly high amounts of IFN-γ compared to SAB (P=0.0216) and BALB/c (P=0.0326) mice. No significant difference in IFN-γ levels were between the groups. No significant difference in body and splenic weights between groups (P>0.05). Pathologically there was observed proliferation of kupffer cells, degenerated hepatocytes and fibrosis in BALB/c and SAB mice. In addition, there was chronic degeneration of structure of spleen of SAB mice characterized by disruption, an indication of severe infection. Results obtained in this study show that SAB mice can be used in studies involving L. donovani parasites. This mouse model can be used in the absence of BALB/c mice owing to their susceptibility to L. donovani. Further studies are needed to be carried out to determine other factors e.g genetic makeup that makes it susceptible to L. donovani parasites.
Additional file 2: Figure S2. Summary matrix of pair-wise patristic distances between the ITS1 se... more Additional file 2: Figure S2. Summary matrix of pair-wise patristic distances between the ITS1 sequences of Trypanosoma spp. identified in this study (red borders) and those registered in the GenBank. The patristic distances between a pair of sequences are represented in the form of a heatmap and their values indicated. The genetic distances were calculated in Geneious Prime (v20.0) following a maximum likelihood phylogenetic analysis implemented in PHYML.
Additional file 1: Figure S1. Detection of Leishmania and Trypanosoma spp. DNA in sand flies by I... more Additional file 1: Figure S1. Detection of Leishmania and Trypanosoma spp. DNA in sand flies by ITS1-PCR. M: 100 bp ladder; 1–4: Sergentomyia squamipleuris sand fly samples; 5 and 6: Leishmania donovani (NLB065) and Leishmania major (Friedlin strain) positive controls; NTC negative control.
BackgroundVisceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public he... more BackgroundVisceral 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 ...
BackgroundVisceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public he... more BackgroundVisceral 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 ...
Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annual... more Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annually. Here, we aimed to investigate the likely variations in gene expression of glycopro-tein63 (gp63), heat shock protein 70 (HSP70), histone, arginase, cysteine protease B (CPB), Leishmania homologue of receptors for activated C kinase (LACK), small hydrophilic endoplasmic reticulum-associated protein (SHERP) in metacyclic promastigotes of L. major isolated from Phlebotomus papatasi sand flies and promastigotes excessively cultured in culture medium. The parasites were collected from suspected CL cases in Pasteur Institute of Iran, cultured and inoculated into the female BALB/c mice (2×10 6 promastigotes). Sand flies were trapped in Qom province, fed with the blood of euthanized infected mice and subsequently dissected in order to isolate the midgut including stomodeal valve. The metacyclic promastigotes were isolated from Ph. papatasi (Pro-Ppap) using peanut agglutinin test (PNA), then continuously cultured in RPMI-1640 medium enriched with fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) to reach stationary phase (Pro-Stat). The gene expression was evaluated in both parasitic stages (Pro-Ppap and Pro-Stat) using qRT-PCR. Out results showed a significant increased gene expression at Pro-Ppap stage for gp63 (P = 0.002), SHERP (P = 0.001) and histone (P = 0.026) genes, in comparison with Pro-Stat stage. Noticeably, significant changes were, also, demonstrated in 10 th to 15 th passages [gp63 (P = 0.041), arginase (P = 0.016), LACK (P = 0.025)] and in 5 th to 20 th passage (SHERP) (P = 0.029). In conclusion, the findings of the present study seem to be essential in designing Leishmania studies, in particular regarding host-parasite interaction, immunization and infectivity studies.
Background: Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public ... more 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 difference between Swiss albino (resistant) and BALB/c (susceptible) strains in response to L... more The difference between Swiss albino (resistant) and BALB/c (susceptible) strains in response to L. donovani infection has been linked to inheritable genes, and the GATAq and Lsh group of genes plays a key role in disease transmission. This study sought to investigate effects of gene neutralization through cross breeding and back crossing. SAB mice were generated by cross breeding of BALB/c mice and Swiss Albino mice. This study aimed at investigating the susceptibility, immune response and pathology of SAB mice after L. donovani infection. Serum was collected from all mice groups 4 weeks post infection and levels of IFN-γ and IL-4 cytokine were determined using ELISA. Body and spleen weights were measured, and tissue section stained for histopathology examination. In this study the L. donovani infected swiss mice had significantly low amounts of IL-4 as compared to the SAB mice (P=0.0093). Whereas the IL-4 levels in BALB/c mice were not significantly different to that of SAB mice (P= 0.1507). Conversely infected swiss albino mice had significantly high amounts of IFN-γ compared to SAB (P=0.0216) and BALB/c (P=0.0326) mice. No significant difference in IFN-γ levels were between the groups. No significant difference in body and splenic weights between groups (P>0.05). Pathologically there was observed proliferation of kupffer cells, degenerated hepatocytes and fibrosis in BALB/c and SAB mice. In addition, there was chronic degeneration of structure of spleen of SAB mice characterized by disruption, an indication of severe infection. Results obtained in this study show that SAB mice can be used in studies involving L. donovani parasites. This mouse model can be used in the absence of BALB/c mice owing to their susceptibility to L. donovani. Further studies are needed to be carried out to determine other factors e.g genetic makeup that makes it susceptible to L. donovani parasites.
Additional file 2: Figure S2. Summary matrix of pair-wise patristic distances between the ITS1 se... more Additional file 2: Figure S2. Summary matrix of pair-wise patristic distances between the ITS1 sequences of Trypanosoma spp. identified in this study (red borders) and those registered in the GenBank. The patristic distances between a pair of sequences are represented in the form of a heatmap and their values indicated. The genetic distances were calculated in Geneious Prime (v20.0) following a maximum likelihood phylogenetic analysis implemented in PHYML.
Additional file 1: Figure S1. Detection of Leishmania and Trypanosoma spp. DNA in sand flies by I... more Additional file 1: Figure S1. Detection of Leishmania and Trypanosoma spp. DNA in sand flies by ITS1-PCR. M: 100 bp ladder; 1–4: Sergentomyia squamipleuris sand fly samples; 5 and 6: Leishmania donovani (NLB065) and Leishmania major (Friedlin strain) positive controls; NTC negative control.
BackgroundVisceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public he... more BackgroundVisceral 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 ...
BackgroundVisceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public he... more BackgroundVisceral 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 ...
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