The Wolbachia Symbiont: Here, There and Everywhere.

Wolbachia symbionts, first observed in the 1920s, are now known to be present in about 30-70% of tested arthropod species, in about half of tested filarial nematodes (including the majority of human filarial nematodes), and some plant-parasitic nematodes. In arthropods, they are generally viewed as parasites while in nematodes they appear to be mutualists although this demarcation is not absolute. Their presence in arthropods generally leads to reproductive anomalies, while in nematodes, they are generally required for worm development and reproduction. In mosquitos, Wolbachia inhibit RNA viral infections, leading to populational reductions in human RNA virus pathogens, whereas in filarial nematodes, their requirement for worm fertility and survival has been channeled into their use as drug targets for filariasis control. While much more research on these ubiquitous symbionts is needed, they are viewed as playing significant roles in biological processes, ranging from arthropod speciation to human health.

In Silico Identification of Novel Biomarkers and Development of New Rapid Diagnostic Tests for the Filarial Parasites Mansonella perstans and Mansonella ozzardi.

Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.

Crystal structure of the cyclophilin-like domain from the parasitic nematode Brugia malayi.

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.

Protective immunity to Brugia malayi larvae in BALB/c mice: potential of this model for the identification of protective antigens.

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.

Anti-idiotypic antibodies function as a surrogate surface epitope of Brugia malayi infective larvae.

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.

Resistance to Onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous Onchocerca species.

Embryonic stages of various Onchocerca species have been used to stimulate resistance in CBA mice to challenge injections with the microfilariae of Onchocerca lienalis. Comparable levels of resistance to challenge (29-37% reductions) were conferred by living, freeze-killed, or sonicated organisms administered with Freunds' Complete Adjuvant (FCA). Antigens extracted in saline, or with the detergent sodium deoxycholate, were also protective. Adjuvants enhanced the protective effect, particularly FCA (78% reduction), Freunds' Incomplete Adjuvant (74% reduction), aluminum hydroxide (70% reduction) and Bordetella pertussis (70% reduction). Detergent extracts prepared from intact embryos with n-octyl glucoside also stimulated significant levels of protection against microfilarial challenge when given with FCA (37-45% reductions). Levels of resistance induced by immunizations with intact organisms were greatest following subcutaneous (s.c.) injection over the neck or by intramuscular inoculation. Soluble extracts were also particially effective given by s.c. inguinal or intraperitoneal injection. A time-interval of greater than 3 weeks between the completion of immunization and challenge was required for the expression of immunity. Cross-protection against challenge with O. lienalis microfilariae was also afforded to mice by immunization with intact embryos or detergent extracts of Onchocerca gutturosa (45 and 34% reductions), Onchocerca gibsoni (66 and 47% reductions) or Onchocerca volvulus (58 and 41% reductions). It is concluded that the embryonic stages of both human and animal parasites provide a source of cross-protective antigens of value in studies on resistance to Onchocerca microfilariae in experimental hosts.

An analysis of the humoral immune response of dogs following vaccination with irradiated infective larvae of Dirofilaria immitis.

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.

Transfer of immunity to the microfilariae of Onchocerca lienalis in 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 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.

Parasite-specific immune responses to Onchocerca lienalis microfilariae in normal and immunodeficient 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.

Parasite cyclophilins and antiparasite activity of cyclosporin A.

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.

Further studies on the resistance to Onchocerca microfilariae in CBA mice.

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.

Crystal structure of the complex of brugia malayi cyclophilin and cyclosporin A.

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.

Molecular characterization of a cyclosporin A-insensitive cyclophilin from the parasitic nematode Brugia malayi.

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.