Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the Leishmania mexicana Β-TUBULIN locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A. We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania, resulting in loss of this gene from the genome during the evolution of these parasites.

• Keywords
• Leishmania, catalase, dixeny, evolution, virulence,
• MeSH
• Virulence Factors genetics   metabolism   MeSH
• Catalase genetics   metabolism   MeSH
• Cells, Cultured MeSH
• Leishmania mexicana genetics   growth & development   pathogenicity   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Protozoan Proteins genetics   MeSH
• Psychodidae parasitology   MeSH
• Life Cycle Stages genetics   MeSH
• Teschovirus genetics   MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Virulence Factors MeSH
• Catalase MeSH
• Protozoan Proteins MeSH

BACKGROUND: Simulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC-MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC-MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase. CONCLUSIONS/SIGNIFICANCE: This study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.

• MeSH
• Child MeSH
• Adult MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Insect Vectors physiology   MeSH
• Immunoglobulin G blood   MeSH
• Immunoglobulin M blood   MeSH
• Insect Bites and Stings epidemiology   immunology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Onchocerciasis MeSH
• Child, Preschool MeSH
• Aged MeSH
• Simuliidae physiology   MeSH
• Saliva * MeSH
• Animals MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Ghana MeSH
• Sudan MeSH
• Names of Substances
• Immunoglobulin G MeSH
• Immunoglobulin M MeSH

BACKGROUND: Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius. CONCLUSIONS/SIGNIFICANCE: We have successfully optimized the ICT, now also suitable to be used with whole canine blood. The test is able to reflect the seasonal fluctuation in dog exposure and showed a good detectability in a field population of naturally exposed dogs, particularly in areas with a high seroprevalence of bitten dogs. Furthermore, our study showed the existence of favorable cross-reactions with other sand fly vectors thereby expanding its use in the field.

• MeSH
• Insect Vectors parasitology   physiology   MeSH
• Immunoassay methods   MeSH
• Leishmania infantum physiology   MeSH
• Leishmaniasis blood   diagnosis   parasitology   veterinary   MeSH
• Mice, Inbred BALB C MeSH
• Dog Diseases blood   diagnosis   parasitology   MeSH
• Phlebotomus parasitology   physiology   MeSH
• Dogs MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host-vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. METHODS: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. RESULTS: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. CONCLUSIONS: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas.

• Keywords
• Canine leishmaniosis, Longitudinal study, Markers of exposure, North-east Spain, Phlebotomus perniciosus, Saliva proteins,
• MeSH
• Endemic Diseases veterinary   MeSH
• Insect Vectors parasitology   MeSH
• Immunity, Humoral MeSH
• Immunoglobulin G analysis   MeSH
• Leishmania infantum isolation & purification   MeSH
• Leishmaniasis blood   parasitology   veterinary   MeSH
• Longitudinal Studies MeSH
• Dog Diseases diagnosis   immunology   parasitology   MeSH
• Phlebotomus immunology   MeSH
• Antibodies, Protozoan blood   MeSH
• Antibodies blood   MeSH
• Dogs immunology   parasitology   MeSH
• Seasons MeSH
• Salivary Proteins and Peptides immunology   MeSH
• Salivary Glands chemistry   parasitology   MeSH
• Saliva immunology   microbiology   parasitology   MeSH
• Animals MeSH
• Check Tag
• Dogs immunology   parasitology   MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Spain epidemiology   MeSH
• Names of Substances
• Immunoglobulin G MeSH
• Antibodies, Protozoan MeSH
• Antibodies MeSH
• Salivary Proteins and Peptides MeSH

BACKGROUND: Canine leishmaniasis (CanL) is a zoonotic disease, caused by Leishmania infantum and transmitted by Phlebotomus perniciosus in the Mediterranean basin. Previously, an ELISA based on the P. perniciosus salivary protein SP03B was proposed as a valid tool to screen for canine exposure to sand fly bites across regions endemic for CanL. Although this approach is useful in laboratory settings, a practical tool for immediate application in the field is needed. In this study we propose the rSP03B sero-strip, the first immunochromatographic test (ICT) in the field of vector exposure able to rapidly screen dogs living in endemic areas for the presence of P. perniciosus and to aid in the evaluation of vector control programs. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was prepared using the bacterially expressed recombinant protein rSP03B as antigen. For test optimization, pre-immune sera from non-bitten laboratory-bred Beagles were used as negative controls. In order to validate the test, sera from laboratory-bred Beagles experimentally exposed to P. perniciosus bites were used as positive controls. Additionally, all samples were tested by ELISA using whole salivary gland homogenate (SGH) and the rSP03B protein as antigen. An almost perfect degree of agreement was found between the ICT and the SGH-ELISA. Furthermore, the newly proposed rSP03B sero-strip showed a sensitivity of 100% and a specificity of 86.79%. CONCLUSIONS/SIGNIFICANCE: We developed a simple and rapid ICT based on the P. perniciosus rSP03B salivary protein, able to replace the standard ELISA used in previous studies. Our rSP03B sero-strip showed to be highly sensitive and specific in the detection of antibodies (IgG) against P. perniciosus saliva. In the future, this test can be employed during large-scale epidemiological studies of CanL in the Mediterranean area to evaluate the efficacy of vector control programs.

• MeSH
• Time Factors MeSH
• Chromatography, Affinity veterinary   MeSH
• Enzyme-Linked Immunosorbent Assay methods   MeSH
• Insect Vectors MeSH
• Insect Proteins MeSH
• Insect Bites and Stings immunology   veterinary   MeSH
• Leishmania infantum MeSH
• Dog Diseases diagnosis   parasitology   MeSH
• Phlebotomus immunology   MeSH
• Dogs MeSH
• Reagent Strips MeSH
• Sensitivity and Specificity MeSH
• Serologic Tests veterinary   MeSH
• Zoonoses MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Insect Proteins MeSH
• Reagent Strips MeSH

BACKGROUND: Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania's ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. METHODOLOGY/PRINCIPAL FINDINGS: The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. CONCLUSIONS/SIGNIFICANCE: L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana's general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.

• MeSH
• Insect Vectors parasitology   MeSH
• Leishmania mexicana genetics   pathogenicity   MeSH
• Leishmaniasis, Cutaneous parasitology   MeSH
• Macrophages parasitology   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• GTP-Binding Proteins genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Psychodidae parasitology   MeSH
• Virulence MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• GTP-Binding Proteins MeSH
• Protozoan Proteins MeSH

BACKGROUND: Leishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties. METHODS AND FINDINGS: In this review, we focus on 3 main aspects of sand fly salivary molecules: (1) structure and composition of salivary glands, including the properties of salivary molecules related to hemostasis and blood feeding, (2) immunomodulatory properties of salivary molecules and the diverse impacts of these molecules on leishmaniasis, ranging from disease exacerbation to vaccine development, and (3) use of salivary molecules for field applications, including monitoring host exposure to sand flies and the risk of Leishmania transmission. Studies showed interesting differences between salivary proteins of Phlebotomus and Lutzomyia species, however, no data were ever published on salivary proteins of Sergentomyia species. CONCLUSIONS: In the last 15 years, numerous studies have characterized sand fly salivary proteins and, in parallel, have addressed the impact of such molecules on the biology of the host-sand fly-parasite interaction. The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas.

• MeSH
• Leishmania immunology   MeSH
• Psychodidae parasitology   physiology   MeSH
• Salivary Proteins and Peptides immunology   metabolism   MeSH
• Saliva immunology   parasitology   MeSH
• Feeding Behavior * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Salivary Proteins and Peptides MeSH