Natural Clerodendrum-derived tick repellent: learning from Nepali culture

Ticks attaching to ear canals of humans and animals are the cause of otoacariasis, common in rural areas of Nepal. The plant Clerodendrum viscosum is used in multiple indigenous systems of medicine by ethnic communities in the Indo-Nepali-Malaysian region. Visiting the Chitwan National Park, we learned that in indigenous medicine, flower extract of C. viscosum is utilized to treat digestive disorders and extracts from leaves as tick repellent to prevent ticks from invading or to remove them from the ear canal. The objective of our study was to provide support to indigenous medicine by characterizing the in vivo effect of leave extracts on ticks under laboratory conditions and its phytochemical composition. We collected plant parts of C. viscosum (leaves and flowers) and mango (Mangifera indica) leaves at the Chitwan National Park, previously associated with repellent activity to characterize their effect on Ixodes ricinus ticks by in vivo bioassays. A Q-ToF high-resolution analysis (HPLC-ESI-QToF) was conducted to elucidate phenolic compounds with potential repellent activity. Clerodendrum viscosum and M. indica leaf extracts had the highest tick repellent efficacy (%E = 80–100%) with significant differences when compared to C. viscosum flowers extracts (%E = 20–60%) and phosphate-buffered saline. Phytochemicals with tick repellent function as caffeic acid, fumaric acid and p-coumaric acid glucoside were identified in C. viscosum leaf extracts by HPLC-ESI-QToF, but not in non-repellent flower extracts. These results support the Nepali indigenous medicine application of C. viscosum leaf extracts to repel ticks. Additional research is needed for the development of natural and green repellent formulations to reduce the risks associated with ticks resistant to acaricides.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.Peer reviewe

Toxoplasma gondii Tetravalent Chimeric Proteins as Novel Antigens for Detection of Specific Immunoglobulin G in Sera of Small Ruminants

The detection of Toxoplasma gondii infection in small ruminants has important significance for public health and veterinary medicine. This study, for the first time, describes the reactivity of four tetravalent chimeric proteins (AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, AMA1-SAG2-GRA1-ROP1, and SAG2-GRA1-ROP1-GRA2) containing immunodominant regions from the AMA1 (apical membrane antigen 1), SAG2 (surface antigen 2), GRA1 (dense granule antigen 1), GRA2 (dense granule antigen 2), and ROP1 (rhoptry antigen 1) with specific IgG antibodies from the sera of small ruminants with the use of an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISA based on a Toxoplasma lysate antigen (TLA). All chimeric proteins were characterized by high specificity (between 96.39% to 100%), whereas the sensitivity of the IgG ELISAs was variable (between 78.49% and 96.77%). The highest sensitivity was observed in the IgG ELISA test based on the AMA1-SAG2-GRA1-ROP1. These data demonstrate that this chimeric protein can be a promising serodiagnostic tool for T. gondii infection in small ruminants

Borrelia burgdorferi BmpA-BBK32 and BmpA-BBA64: New Recombinant Chimeric Proteins with Potential Diagnostic Value

Currently, the diagnosis of Lyme disease is based mostly on two-tiered serologic testing. In the new generation of immunoenzymatic assays, antigens comprise whole-cell lysates of members of the Borrelia burgdorferi sensu lato (s.l.) species complex, with the addition of selected recombinant proteins. Due to the high diversity of members of the B. burgdorferi s.l. genospecies and the low degree of conservation among the amino acid sequences of their proteins, serodiagnostic methods currently in use are not sufficient for the correct diagnosis of borreliosis. Two divalent chimeric proteins (BmpA-BBK32 and BmpA-BBA64) were expressed in Escherichia coli. Following purification by one-step metal-affinity chromatography, preparations were obtained containing milligram levels of chimeric protein exhibiting electrophoretic purity in excess of 98%. Reactivity of the new chimeric proteins with specific human IgG antibodies was preliminarily determined by Western blot. For this purpose, 20 negative sera and 20 positive sera was used. The new chimeric proteins were highly reactive with IgG antibodies contained in the serum of patients suffering from borreliosis. Moreover, no immunoreactivity of chimeric proteins was observed with antibodies in the sera of healthy people. These promising results suggest that new chimeric proteins have the potential to discriminate between positive and negative sera

Vaccines

Peer reviewe

Epitope mapping of BmpA and BBK32 Borrelia burgdorferi sensu stricto antigens for the design of chimeric proteins with potential diagnostic value

Lyme disease is a tick-borne zoonosis caused by Gram-negative bacteria belonging to the Borrelia burgdorferi sensu lato (s.l.) group. In this study, IgM- and IgG-specific linear epitopes of two B. burgdorferi sensu stricto (s.s.) antigens BmpA and BBK32 were mapped using a polypeptide array. Subsequently, two chimeric proteins BmpA-BBK32-M and BmpA-BBK32-G were designed to validate the construction of chimeras using the identified epitopes for the detection of IgM and IgG, respectively, by ELISA. IgG-ELISA based on the BmpA-BBK32-G antigen showed 71% sensitivity and 95% specificity, whereas a slightly lower diagnostic utility was obtained for IgM-ELISA based on BmpA-BBK32-M, where the sensitivity was also 71% but the specificity decreased to 89%. The reactivity of chimeric proteins with nondedicated antibodies was much lower. These results suggest that the identified epitopes may be useful in the design of new forms of antigens to increase the effectiveness of Lyme disease serodiagnosis. It has also been proven that appropriate selection of epitopes enables the construction of chimeric proteins exhibiting reactivity with a specific antibody isotype.This study was supported by the additional budget of the grant IJC2020-042710 by the Ministerio de Ciencia, Innovación y Universidades Spain. Marinela Contreras is funded by the same grant.Peer reviewe

The first study on the usefulness of recombinant tetravalent chimeric proteins containing fragments of SAG2, GRA1, ROP1 and AMA1 antigens in the detection of specific anti-Toxoplasma gondii antibodies in mouse and human sera.

This study presents an evaluation of four tetravalent recombinant chimeric proteins containing fragments of the Toxoplasma gondii antigens, SAG2, GRA1, ROP1 and AMA1, as potential replacements of a the soluble, whole-cell tachyzoite lysate (TLA) used in serological assays. Recombinant chimeric proteins (SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1) obtained by genetic engineering were tested for their reactivity with specific IgM and IgG antibodies from sera of experimentally infected mice and humans with T. gondii infection using an enzyme-linked immunosorbent assay (ELISA). In total 192 serum samples from patients with acquired T. gondii infection and 137 sera from seronegative individuals were examined. The reactivity of chimeric antigens with antibodies generated during T. gondii invasion was measured and compared to the results obtained in assays based on whole-cell Toxoplasma antigen. Chimeric proteins proved effective in differentiation between T. gondii-infected and uninfected individuals (100% sensitivity and specificity in the IgG ELISAs) which shows their potential usefulness as a replacements for TLA in standardized commercial tests for the serodiagnosis of toxoplasmosis. In addition, the chimeric proteins were tested for use in avidity determination. Obtained results were comparable to those of the corresponding commercial assays, suggesting the utility of these proteins for avidity assessment. Furthermore, this study demonstrated that the AMA1-SAG2-GRA1-ROP1 chimeric protein has the potential to distinguish specific antibodies from serum samples of individuals with the early and chronic phase of T. gondii infection