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Abstract Background Wolbachia (wBm) is an obligate endosymbiotic bacterium of Brugia malayi, a parasitic filarial nematode of humans and one of the causative agents of lymphatic filariasis. There is a pressing need for new drugs against filarial parasites, such as B. malayi. As wBm is required for B. malayi development and fertility, targeting wBm is a promising approach. However, the lifecycle of neither B. malayi nor wBm can be maintained in vitro. To facilitate selection of potential drug targets we computationally ranked the wBm genome based on confidence that a particular gene is essential for the survival of the bacterium. Results wBm protein sequences were aligned using BLAST to the Database of Essential Genes (DEG) version 5.2, a collection of 5,260 experimentally identified essential genes in 15 bacterial strains. A confidence score, the Multiple Hit Score (MHS), was developed to predict each wBm gene's essentiality based on the top alignments to essential genes in each bacterial strain. This method was validated using a jackknife methodology to test the ability to recover known essential genes in a control genome. A second estimation of essentiality, the Gene Conservation Score (GCS), was calculated on the basis of phyletic conservation of genes across Wolbachia's parent order Rickettsiales. Clusters of orthologous genes were predicted within the 27 currently available complete genomes. Druggability of wBm proteins was predicted by alignment to a database of protein targets of known compounds. Conclusion Ranking wBm genes by either MHS or GCS predicts and prioritizes potentially essential genes. Comparison of the MHS to GCS produces quadrants representing four types of predictions: those with high confidence of essentiality by both methods (245 genes), those highly conserved across Rickettsiales (299 genes), those similar to distant essential genes (8 genes), and those with low confidence of essentiality (253 genes). These data facilitate selection of wBm genes for entry into drug design pipelines.

Conventional diagnosis of filarial infections is based on morphological identification of microfilariae using light microscopy and requires considerable expertise, is time-consuming, and can be subjective. Loop-mediated isothermal amplification (LAMP) has advantages over microscopy or PCR because of its operational simplicity, rapidity and versatility of readout options. LAMP assays represent a major step forward in improved filarial diagnostic tools suitable for low resource settings and field applicability. The study goal was to retrospectively evaluate the performance and suitability of the O-150, RF4, and Mp419 LAMP assays for diagnosing Onchocerca volvulus , Loa loa and Mansonella perstans infections, respectively, in humans and vectors under experimental and natural field conditions. Surveys were conducted in four health districts of Cameroon using skin snip and thick blood film methods to detect skin ( O. volvulus ) and blood ( L. loa and M. perstans ) dwelling microfilaria in humans. Engorged vectors ( Simulium spp ., Chrysops spp ., and Culicoides spp.) were evaluated by LAMP. Dissected, wild-caught vectors were also analyzed. LAMP showed a prevalence of 40.4% ( O. volvulus ), 17.8% ( L. loa ) and 36.6% ( M. perstans ) versus 20.6% ( O. volvulus ), 17.4% ( L. loa ) and 33.8% ( M. perstans ) with microscopy. Simulium spp. were dissected for microscopy and pooled for LAMP. The O-150 LAMP assay infection rate was 4.3% versus 4.1% by microscopy. Chrysops spp. were dissected and analyzed individually in the LAMP assay. The RF4 LAMP assay infection rate was 23.5% versus 3.3% with microscopy. The RF4 LAMP assay also detected parasites in Chrysops spp. fed on low microfilaremic volunteers. The Mp419 LAMP assay infection rate was 0.2% for C. milnei and 0.04% for C. grahamii , while three other species were LAMP-negative. The sensitivity, species specificity, rapidity and ease of its use of these filarial LAMP assays, and validation of their performance in the field support use as alternatives to microscopy as diagnostic and surveillance tools in global health programs aimed to eliminate onchocerciasis., Competing Interests: Conflict of interest ZL and CC are employees of New England Biolabs, manufacturer of LAMP reagents described in the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Filarial diseases, including lymphatic filariasis and onchocerciasis, are considered among the most devastating of all tropical diseases, affecting over 86 million people worldwide. To control and more rapidly eliminate onchocerciasis requires treatments that target the adult stage of the parasite. Drug discovery efforts are challenged by the lack of preclinical animal models using the human-pathogenic filariae, requiring the use of surrogate parasites for Onchocerca volvulus for both ex vivo and in vivo evaluation. Herein, we describe a platform utilizing phenotypic ex vivo assays consisting of the free-living nematode Caenorhabditis elegans, microfilariae and adult filariae of the bovine filariae Onchocerca lienalis and Onchocerca gutturosa, respectively, as well as microfilariae and adult filariae of the feline filariae Brugia pahangi, the rodent filariae Litomosoides sigmodontis and the human-pathogenic filariae Brugia malayi to assess activity across various surrogate parasites. Utilization of those surrogate nematodes for phenotypic ex vivo assays in order to assess activity across various parasites led to the successful establishment of a screening cascade and identification of multiple compounds with potential macrofilaricidal activity and desirable physicochemical, MW = 200-400 and low lipophilicity, logP <4, and pharmacokinetic properties, rat and human liver S9 stability of ≥70% remaining at 60 min, and AUC exposures above 3 μM h. This platform demonstrated the successful establishment of a screening cascade which resulted in the discovery of potential novel macrofilaricidal compounds for futher drug discovery lead optimization efforts. This screening cascade identified two distinct chemical series wherein one compound produced a significant 68% reduction of adult Litomosoides sigmodontis in the mouse model. Successful demonstration of efficacy prompted lead optimization medicinal chemistry efforts for this novel series., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The impact of large scale Mass Drug Adminstration (MDA) of ivermectin on active onchocerciasis transmission by Simulium damnosum, which transmits the parasite O. volvulus is of great importance for onchocerciasis control programmes. We investigated in the Mbam river system area, the impact of MDA of ivermectin on entomological indices and also verify if there are river system factors that could have favoured the transmission of onchocerciasis in this area and contribute to the persistence of disease. We compared three independent techniques to detect Onchocerca larvae in blackflies and also analyzed the river system within 9 months post-MDA of ivermectin., Method: Simulium flies were captured before and after 1, 3, 6 and 9months of ivermectin-MDA. The biting rate was determined and 41% of the flies dissected while the rest were grouped into pools of 100 flies for DNA extraction. The extracted DNA was then subjected to O-150 LAMP and real-time PCR for the detection of infection by Onchocerca species using pool screening. The river system was analysed and the water discharge compared between rainy and dry seasons., Principal Findings: We used human landing collection method (previously called human bait) to collect 22,274 adult female Simulium flies from Mbam River System. Of this number, 9,134 were dissected while 129 pools constituted for molecular screening. Overall biting and parous rates of 1113 flies/man/day and 24.7%, respectively, were observed. All diagnostic techniques detected similar rates of O. volvulus infection (P = 0.9252) and infectivity (P = 0.4825) at all monitoring time points. Onchocerca ochengi larvae were only detected in 2 of the 129 pools. Analysis of the river drainage revealed two hydroelectric dams constructed on the tributaries of the Mbam river were the key contributing factor to the high-water discharge during both rainy and dry seasons., Conclusion: Results from fly dissection (Microscopy), real-time PCR and LAMP revealed the same trends pre- and post-MDA. The infection rate with animal Onchocerca sp was exceptionally low. The dense river system generate important breeding sites that govern the abundance of Simulium during both dry and rainy seasons., Competing Interests: The authors have declared that no competing interests exist.

Background: The mass drug administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that has, in turn, led to reduction of infection levels in Chrysops vectors. Accurate parasite detection is essential for assessing loiasis transmission as it provides a potential alternative or indirect strategy for addressing the problem of co-endemic loiasis and lymphatic filariasis through the Onchocerciasis Elimination Programme and it further reflects the true magnitude of the loiasis problem as excess human mortality has been reported to be associated with the disease. Although microscopy is the gold standard for detecting the infection, the sensitivity of this method is compromised when the intensity of infection is low. The loop-mediated isothermal amplification (LAMP) assay of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. The aim of this study was to validate the Loa loa LAMP assay for the detection of infected Chrysops spp. under experimental and natural field conditions., Methods: Two sets of 18 flies were fed on volunteers with either a low (< 10 mf/ml) or high (> 30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed using LAMP for the detection of L. loa infection. In addition, a total of 9270 flies were collected from the north-west, east, and south-west regions (SW 1 and 2) of Cameroon using sweep nets and subjected to microscopy (7841 flies) and LAMP (1291 flies plus 138 nulliparous flies) analyses., Results: The LAMP assay successfully detected parasites in Chrysops fed on volunteers with both low and high microfilariaemic loads. Field validation and surveillance studies revealed LAMP-based infection rates ranging from 0.5 to 31.6%, with the lowest levels in SW 2 and the highest infection rates in SW 1. The LAMP assay detected significantly higher infection rates than microscopy in four of the five study sites., Conclusion: This study demonstrated the potential of LAMP as a simple surveillance tool. It was found to be more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.

Glycolysis and gluconeogenesis are, in part, driven by the interconversion of 3- and 2-phosphoglycerate (3-PG and 2-PG) which is performed by phosphoglycerate mutases (PGAMs) which can be cofactor dependant (dPGAM) or cofactor independent (iPGAM). The African trypanosome, Trypanosoma brucei, possesses the iPGAM form which is thought to play an important role in glycolysis. Here, we report on the use of RNA interference to down-regulate the T. brucei iPGAM in procyclic form T. brucei and evaluation of the resulting phenotype. We first demonstrated biochemically that depletion of the steady state levels of iPGM mRNA correlates with a marked reduction of enzyme activity. We further show that iPGAM is required for cell growth in procyclic T. brucei.

Crisis events can produce waves of shock, fear, and disbelief throughout a community. The university campus is a community and no stranger to crisis events. Incidents such as natural disasters, bus accidents, fire, and death on campus have demonstrated this fact. Communities, including the university, are becoming more aware of the need to develop plans to respond to these crisis events. Communities that are prepared to respond to such events may moderate the impact that the crisis has on a community. This article discusses the issues confronted by a small liberal arts university's faculty and staff as they worked together to develop and sustain a crisis response team.

The resistance of the human parasite Brugia malayi to the antiparasitic activity of cyclosporin A (CsA) may arise from the presence of cyclophilins with relatively low affinity for the drug. The structure of the complex of B. malayi cyclophilin (BmCYP-1) and CsA, with eight independent copies in the asymmetric unit, has been determined at a resolution of 2.7 A. The low affinity of BmCYP-1 for CsA arises from incomplete preorganization of the binding site so that the formation of a hydrogen bond between His132 of BmCYP-1 and N-methylleucine 9 of CsA is associated with a shift in the backbone of approximately 1 A in this region.

Cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase activity and bind the immunosuppressive agent cyclosporin A (CsA). Brugia malayi is a filarial nematode parasite of humans, for which a cyclophilin-like domain was identified at the N-terminal of a protein containing 843 amino acid residues. There are two differences in sequence in the highly conserved CsA binding site: A histidine and a lysine replace a tryptophan and an alanine, respectively. The crystal structure of this domain has been determined by the molecular replacement method and refined to an R-factor of 16.9% at 2.15 A resolution. The overall structure is similar to other cyclophilins; however, major differences occur in two loops. Comparison of the CsA binding site of this domain with members of the cyclophilin family shows significant structural differences, which can account for the reduced sensitivity of the Brugia malayi protein to inhibition by CsA.

Cyclosporin A (CsA) was initially developed as an immunosuppressive drug. In the past several years, it has been shown to possess antiparasite activity independent of the immune system. It is not known how the drug exerts these antiparasite effects, or why it is stage and/or species specific. The answers may lie in the enzymatic function of cyclophilins. The cyclophilins are a growing family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPiase) activity and bid CsA to varying degrees. PPiases have been shown to play a role in the folding of many essential proteins. Antony Page, Sanjay Kumar and Clotilde Carlow here review parasite cyclophilins and their association with CsA. The possible biological function of parasite cyclophilins and their potential role in future drug discovery are also discussed.

The cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosporin A (CsA) to varying degrees. We have isolated a cDNA clone encoding a novel cyclophilin from the human filarial parasite Brugia malayi. This gene possesses an N-terminal domain homologous to cyclophilins from diverse phyla (49-60% amino acid sequence identity) and a hydrophilic C-terminal domain. The cyclophilin domain was overexpressed in Escherichia coli and found to possess peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 7.9 x 10(6) M-1 s-1. A histidine residue in lieu of tryptophan in the highly conserved CsA-binding site suggests that B. malayi cyclophilin is more closely related to the cyclophilin-like proteins described recently from natural killer (NK) cells, plants, and the 40 kDa cyclophilins from mammals. In accordance with the histidine-containing CsA-binding domain, the B. malayi enzyme was relatively insensitive to inhibition by CsA, since an IC50 value of 860 nM (compared to 19 nM for human cyclophilin A) was determined.

In this study, the expression of an Onchocerca volvulus Ov33 homolog was demonstrated in Dirofilaria immitis. Rabbit antiserum raised against a recombinant fusion protein of O. volvulus, MBP/OvD 5B (Ov33), was found to react with a 31-33 kDa glycoprotein (DiT33) of adult worms of D. immitis. An antibody response to MBP/OvD 5B was observed in dogs, as early as 11 weeks post infection with infective larvae of D. immitis, and in dogs with occult infection. Cats both experimentally and naturally infected with D. immitis also reacted strongly with the recombinant antigen. In contrast, sera from dogs receiving chemically-abbreviated infection or from animals harbouring a variety of other helminths failed to react. These data suggest that antibody responses generated by DiT33 may have potential in immunodiagnosis of heartworm infection in cats and dogs.

In this study, dogs were immunized with irradiated L3 larvae of Dirofilaria immitis. Following challenge with non-irradiated L3, vaccinated dogs had an average of 71% fewer adult worms compared to non-vaccinated animals. A comparative analysis of eosinophil and antibody responses of these two groups of dogs is presented. Vaccinated dogs preferentially recognized several larval (14, 20, 30, 34, 39 kDa), adult worm (20 kDa) and microfilarial (36, 38, 71, 84 kDa) antigens. To characterize these antigens, the extent of glycosylation was assessed. The data suggest that an earlier response to these antigens may be important in the protection induced in dogs by administration of irradiated L3 of D. immitis.

Anti-idiotypic (AB2) antibodies were generated in rabbits following immunization with a murine IgM monoclonal antibody (AB1) recognizing a surface determinant of Brugia malayi infective stage larvae. AB2 specifically inhibited the binding of AB1 to B. malayi larvae. Furthermore, AB2 had the ability to mimic the original antigen since mice immunized with AB2 possessed serum antibodies (AB3) specific for the B. malayi surface determinant. The presence of anti-surface antibodies (AB3 and AB1) induced either by AB2 immunization or by administration of AB1, did not alter the outcome of an intraperitoneal infection of B. malayi larvae in BABL/c mice when compared to untreated animals. AB3 antibodies like AB1, were IgM, thus indicating an isotype restricted response to the B. malayi epitope. There were no detectable cell mediated responses to the surface determinant in mice immunized with AB2, assessed by lymphocyte blastogenesis or IL3 production in vitro in response to the idiotope as presented by living larvae. The lack of cellular responses and/or the previously demonstrated rapid shedding of the epitope may explain the inability of AB1 or AB2 to protect mice against larval challenge in this study.

We analyzed the antigenicity and stability of the surface of skin microfilariae (mf) of Onchocerca cervicalis, a horse parasite. These mf express antigens on their surface that are cross-reactive with the cattle parasite O. lienalis and with the human parasite O. volvulus. The surface of living O. cervicalis mf was radioiodinated using Iodogen and the labeled components were solubilized in buffers containing sodium dodecyl sulphate (SDS), or extracted with the milder detergent octyl-beta-D-glucopyranoside (OGP). Electrophoresis of this material showed seven prominent bands, one of which (14 kDa) was specifically precipitated by antisera from rabbits immunized with mf from either O. cervicalis, O. lienalis, or O. volvulus, and by human sera obtained from infected individuals in Chiapas, Mexico. Other components were precipitated by either the rabbit or the human sera. In addition, antisera from mice immunized with O. cervicalis mf bound specifically to the surface of freeze-thawed uterine O. lienalis and O. volvulus mf as detected by immunofluorescence. This fluorescence was lost from the surface of O. cervicalis mf in a temperature-dependent fashion. Live mf incubated on ice with mouse anti-mf antisera and secondary FITC-GAM, showed uniform surface fluorescence. When these mf were incubated at 37 degrees C, but not at 0 degrees C, the fluorescent pattern changed with time. First, small non-fluorescent patches arose, followed by an increasingly wide belt devoid of fluorescence, and finally, no visible fluorescence. These changes in the mf surface suggest potential mechanisms for immune evasion by filarial parasites.

Protective immune responses against the infective larvae of Brugia malayi have been demonstrated in BALB/c mice. Various factors governing resistance to reinfection have been examined to provide baseline data for use of this model in studies of immunoprophylaxis. Parasites that established following a primary infection survived for approximately 10 days, following which numbers declined rapidly to a low level. Resistance was evidenced by a more rapid clearance of secondary infection parasites. The degree of immunity expressed was not related to the route of administration of the initial infection (subcutaneous, intravenous, intramuscular, or intraperitoneal). However, both the level of resistance and the rapidity of its expression were dependent on dose, with as few as 2 larvae stimulating measurable immunity. Sensitization with living male or female adult worms, fourth stage larvae or microfilariae of B. malayi, or infective larvae of B. pahangi conferred substantial resistance to larval challenge. Significant levels of immunity were also induced by dead B. malayi larvae (46%) and their aqueous extracts (76%), but not with the corresponding insoluble fraction. We suggest that this experimental system is ideally suited to aid in the identification of putative protective antigens in brugian filariasis.

The model of Onchocerca lienalis microfilariae (mf) in CBA mice has been employed to examine the immunological mechanisms underlying the destruction of skin-dwelling mf in immunised hosts. Spleen cells from highly resistant donors transferred to naive, syngeneic recipients conferred significant protection against mf challenge, with up to 78% reductions in parasite recoveries. Levels of resistance afforded by adoptive transfers of immune cells were dose dependent and only significant above a threshold of 4 x 10(6) splenocytes. Mixed T and B lymphocytes elicited resistance in recipients, although neither cell population was protective alone. Immune serum evoked resistance to mf challenge by passive transfer, but only when collected shortly after booster immunisations of the donors. Xenogeneic sera from immunised rabbits or calves failed to confer protection to recipient mice. It is concluded that resistance to O. lienalis mf in mice is likely to be primarily governed by a thymus-dependent humoral response.

Various factors governing resistance to the microfilariae (mf) of Onchocerca lienalis in mice have been examined to provide baseline data for use of this model in immunological studies. The survival of mf during a primary infection followed a similar course in the skin from most anatomical regions of the body. Mice were highly resistant to secondary infections, manifested by parasite densities over the body that were reduced by 83-100% compared with controls. Recoveries of mf from the ears, used in later experiments, were a representative measure of parasite survival in other skin sites. The resistance to challenge induced by a primary infection was not dependent on the route of administration (intravenous, intraperitoneal, subcutaneous and intramuscular) of the latter and was apparently systemic. Primary infections of various durations that were chemically-abbreviated conferred maximum protection when of 15 days or longer (95-97%) and substantial resistance when of only 7 days (84%). Similar levels of protection were demonstrated in mice that were sensitized with single or multiply-divided mf doses, or challenged in a similar manner. Primary infections containing as few as 20 mf induced almost the same degree of protection (80%) as 50 to 10,000 mf (88-97%). Apparently, resistance to re-exposure with O. lienalis mf is mediated by a highly effective mechanism(s) in CBA mice.

The model of Onchocerca lienalis microfilariae (mf) in CBA mice has been employed to examine the immunological mechanisms underlying the destruction of skin-dwelling mf in onchocerciasis. Comparative studies among immunologically intact (CBA/H) or deficient (CBA/N, T-cell-deprived) syngeneic animals demonstrated that levels of mf of a primary infection were reduced most rapidly in fully immunocompetent mice. Significant reductions in recoveries of a secondary infection were evident in CBA/H (80%) and CBA/N (44%) mice, but not in T-cell-deprived animals. The establishment of primary and secondary infections was apparently not influenced by complement, as judged by C3 depletion with Cobra Venom Factor. Eosinophilia was demonstrated to varying degrees in all infected animals; similar levels occurred in CBA/H and CBA/N mice which were greatly elevated after mf challenge. In contrast, the eosinophil response of T-cell-deprived mice was weak and not potentiated during secondary infection. Type I immediate hypersensitivity responses to soluble mf antigen (mf-Ag) were mounted by all groups, but significantly less strongly in T-cell-deprived mice. Type IV delayed responses were generally weak, although CBA/N mice reacted strongly in the early phase of primary infections. During the first 2 weeks of infection CBA/H and T-cell-deprived mice mounted rapid IgM responses to mf-Ag. Subsequently, levels of IgG2a, IgG2b and IgG1 increased in all mice. There was a potentiated IgG2a, IgG2b and IgG1 response in all groups following challenge, with levels of IgG1 highest in CBA/H mice. IgE responses were also detected by passive cutaneous anaphylaxis during primary and secondary infections. Peak levels of parasite-specific antibodies coincided with the timing of mf clearance.