Tetracycline treatment and sex-ratio distortion: a role for Wolbachia in the moulting of filarial nematodes?

Filarial nematodes harbour intracellular bacteria of the genus Wolbachia. These bacteria are thought to be beneficial to the host nematode. Indeed, tetracycline treatments reduce the population of Wolbachia in filarial worms and have detrimental effects on the nematode. Even though various antibiotic-curing experiments have been performed on filariae, the actual role of Wolbachia in the biology of these nematodes is not yet clear. To address this issue, we designed a first experiment on a model filaria (Brugia pahangi), maintained in the gerbil (Meriones unguiculatus). In this experiment, timing of tetracycline treatment was set on the basis of the larval stage of the nematode. This first experiment showed that 2 weeks of treatment started after the L(4)-L(5) moult of males, but before the moult of females, led to significant sex-ratio distortion of the nematodes. We thus hypothesised that tetracycline interferes with the moult in B. pahangi. To test this hypothesis, we designed a second experiment in which antibiotic treatments were started (1). before the moult of both sexes, (2). after the moult of males but before the moult of females, or (3). after the moult of both sexes. Treatment 1 determined a reduction of worm recovery with no sex bias. Treatment 2 led to a male-biased sex-ratio. Treatment 3 had no effect on either worm recovery or sex-ratio. These results thus support the hypothesis that tetracycline treatment interferes with the L(4)-L(5) moult of B. pahangi. The nematodes recovered from the treated and control animals were examined for the presence of Wolbachia using both immunohistochemistry and real-time PCR. In general, nematodes from treated animals showed a dramatic reduction in Wolbachia content. In one group, Wolbachia depletion, as observed at the end of the treatment, was followed by a rebound to 'normal' values 160 days later. Prospects for antifilarial therapy using Wolbachia-targeted tetracycline treatments should thus take into account the possibility of Wolbachia rebound.     

Tetracycline inhibits development of the infective-stage larvae of filarial nematodes in vitro

In recent years, studies have linked tetracycline treatment of filaria-infected animals with reduced adult worm burdens and decreased levels of microfilaremia. These observations are believed to be attributable to clearance of Wolbachia, intracellular rickettsial-like organisms found within filarial tissues. Although maximal worm reductions were observed when treatment was initiated early in infection, it is not known whether tetracycline inhibits development of infective-stage larvae. To address this issue, we studied the effect of tetracycline on three different species of filarial nematodes, Brugia malayi, Brugia pahangi, and Dirofilaria immitis, in a serumfree in vitro system supporting molting to the fourth larval stage. Tetracycline was capable of inhibiting L3 to L4 molting within a dosage range similar to that reported for susceptible rickettsial organisms. However, Wolbachia DNA could still be detected in nematodes from tetracycline-treated cultures. In addition, three other antibiotics with anti-rickettsial and anti-chlamydial activity (chloramphenicol, erythromycin, and ciprofloxacin) failed to inhibit L3 to L4 molting. Although tetracycline is capable of completely blocking molting of infective-stage larvae, it remains possible that this effect is due to pharmacological activities unrelated to its anti-rickettsial functions.     

Ultrastructural evidence of the degenerative events occurring during embryogenesis of the filarial nematode Brugia pahangi after tetracycline treatment

Intracellular bacteria belonging to the genus Wolbachia have been described in filarial nematodes and these microorganisms appear to have evolved an obligatory mutualistic association with their filarial hosts. In fact, antibiotic treatment leads to the clearance of bacteria from worms resulting in a block in embryogenesis and, eventually, death of adult filariae. Currently, the antifilarial action of antibiotic treatment is interpreted as a secondary consequence of the bacteriostatic activity against Wolbachia endosymbionts. Here, we demonstrate by transmission electron microscopy the degenerative events occurring during embryogenesis of Brugia pahangi after tetracycline treatment. After 56 days of treatment the cytoplasm of hypodermal cords was totally void of Wolbachia and numerous vacuoles, residual of cytolitic activity, were observed. In the ovary, the morphology of the oocytes was well conserved 33 days after treatment, but the texture of symbiotic bacteria appeared altered. After 56 days of treatment embryogenesis was dramatically affected and the terminal portion of the ovary appeared totally empty. The authors suggest that the symbiotic bacteria play a direct role in worm metabolism and a long-term bacteriostatic effect may block bacterial activity involved in the active control of cytolysis. As a consequence, the bacteriophorous vacuole is transformed into a digestive vacuole and the whole symbiotic population is disrupted.     

Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia

Wolbachia endosymbiotic bacteria have been shown to be widespread among filarial worms and could thus play some role in the biology of these nematodes. Indeed, tetracycline has been shown to inhibit both the development of adult worms from third-stage larvae and the development of the microfilaraemia in jirds infected with Brugia pahangi. The possibility that these effects are related to the bacteriostatic activity of tetracycline on Wolbachia symbionts should be considered. Here we show that tetracycline treatment is very effective in blocking embryo development in two filarial nematodes, B. pahangi and Dirofilaria immitis. Embryo degeneration was documented by TEM, while the inhibition of the transovarial transmission of Wolbachia was documented by PCR. Phylogenetic analysis on the ssrDNA sequence of the Wolbachia of B. pahangi confirms that the phylogeny of the bacterial endosymbionts is consistent with that of the host worms. The possibility that tetracycline inhibition of embryo development in B. pahangi and D. immitis is determined by cytoplasmic incompatibility is discussed.     

A worm's best friend: recruitment of neutrophils by Wolbachia confounds eosinophil degranulation against the filarial nematode Onchocerca ochengi

Onchocerca ochengi, a filarial parasite of cattle, represents the closest relative of the human pathogen, Onchocerca volvulus. Both species harbour Wolbachia endosymbionts and are remarkable in that adult female worms remain viable but sessile for many years while surrounded by host cells and antibodies. The basis of the symbiosis between filariae and Wolbachia is thought to be metabolic, although a role for Wolbachia in immune evasion has received little attention. Neutrophils are attracted to Wolbachia, but following antibiotic chemotherapy they are replaced by eosinophils that degranulate on the worm cuticle. However, it is unclear whether the eosinophils are involved in parasite killing or if they are attracted secondarily to dying worms. In this study, cattle infected with Onchocerca ochengi received adulticidal regimens of oxytetracycline or melarsomine. In contrast to oxytetracycline, melarsomine did not directly affect Wolbachia viability. Eosinophil degranulation increased significantly only in the oxytetracycline group; whereas nodular gene expression of bovine neutrophilic chemokines was lowest in this group. Moreover, intense eosinophil degranulation was initially associated with worm vitality, not degeneration. Taken together, these data offer strong support for the hypothesis that Wolbachia confers longevity on O. ochengi through a defensive mutualism, which diverts a potentially lethal effector cell response.     

Natural death of adult Onchocerca volvulus and filaricidal effects of doxycycline induce local FOXP3+/CD4+ regulatory T cells and granzyme expression

Immunosuppression in human filarial disease involves regulatory T cells. We hypothesized that natural or worm antigen-induced FOXP3 regulatory T cells could be involved locally, suppressing effector cells via granzymes. Natural and treatment-induced death of worms implies enhanced exposure to worm antigens. Therefore, we examined FOXP3+T cells and granzyme expression in onchocercomas harbouring adult Onchocerca volvulus worms, with respect to worm viability, productivity, the patient's immune status and filaricidal treatment. The immunohistological analysis revealed that dead adult worms were strongly associated with FOXP3+T cells in generalized hyporeactive onchocerciasis. FOXP3+ cells hardly expressed granzymes, but cell contacts with granzyme A+ or B+ cells were frequent. While suramin directly kills most adult worms within 6 months, the Wolbachia depleting antibiotic doxycycline indirectly causes adult worm degeneration within 18 months. Contrary to suramin, depletion of Th1-driving endobacteria most strongly promoted FOXP3+T cells and granzyme-expressing cells. In hyperreactive patients, FOXP3+ cells were less frequent. This is the first demonstration of local FOXP3+Treg cells in human filariasis and their induction by natural worm death and anti-parasitic treatment. We newly report granzyme responses to helminths and their association with immunosuppression. FOXP3+Treg and granzyme+ cells might locally suppress defence against newly acquired worms.     

Brugia pahangi: comparative susceptibility of the Mongolian jird, Meriones unguiculatus, and the PD4 inbred hamster, Mesocricetus auratus

The susceptibility of Mongolian jirds, Meriones unguiculatus, and PD4 hamsters, Mesocricetus auratus, to Brugia pahangi was compared based on the percentage adult worm recoveries, mean microfilaremia levels, and adult worm lengths. Fourteen male jirds and seventeen male PD4 hamsters were each inoculated subcutaneously in the left inguinal region with 90-100 L3 of B. pahangi and necropsied 130-150 days after inoculation. There were no significant differences between jirds and hamsters in mean adult worm recoveries (24.7 vs 25.4%) and prepatent periods (69.9 vs 77 days after inoculation). In hamsters, 85% of recovered worms were found in the heart and lungs and 15% were found in genital lymphatic vessels. In jirds, distribution of recovered worms was 66% in genital lymphatics, 23% in the heart and lungs, 8% in the peritoneal cavity, and 3% in lymphatic vessels in other sites. The mean microfilaremia level in jirds (16.5/20 microliter) was significantly higher than in hamsters (8.7/20 microliter. Female worms in the genital lymphatics of jirds were significantly longer than female worms in the genital lymphatics of PD4 hamsters (33.5 vs 27.3 mm). Lengths of worms in other locations were similar between the two species.     

Real-time PCR for quantification of the bacterial endosymbionts (Wolbachia) of filarial nematodes

Filarial nematodes harbour intracellular symbiotic bacteria belonging to the genus Wolbachia. Wolbachia is thought to play an important role in the biology of the nematode. Moreover, Wolbachia appears to be involved in the immunopathogenesis of filariasis and in the onset of the side-effects of antifilarial therapy. Investigations in these research areas require reliable methods to quantify Wolbachia both in nematodes and in vertebrate tissues. To this purpose, we designed a quantitative real-time PCR targeted on the ftsZ gene of the Wolbachia of Brugia pahangi, a model filarial species maintained in gerbils. The method was applied to quantify Wolbachia in Brugia pahangi, from animals with or without tetracycline treatment. Our results show that tetracycline treatment leads to dramatic reduction or clearance of Wolbachia from the nematode. Results obtained from different replicates were reproducible and the method appeared very sensitive compared to other PCR protocols for Wolbachia detection. Real-time PCR is thus an appropriate method for investigations on the biological role of Wolbachia and on the implication of these bacteria in the pathogenesis of filariasis. With slight modifications of the primers and probe, the protocol we have developed could be applied in studies of the human pathogen Brugia malayi and on the model filarial species Litomosoides sigmodontis.     

Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs

There is still a pressing need for effective adulticide treatment for human and animal filarial infections. Like many filarial nematodes, Dirofilaria immitis, the causative agent of canine heartworm disease, harbours the bacterial endosymbiont Wolbachia, which has been shown to be essential for worm development, fecundity and survival. Here the authors report the effect of different treatment regimens in dogs experimentally infected with adult D. immitis on microfilariemia, antigenemia, worm recovery and Wolbachia content. Treatment with ivermectin (IVM; 6 microg/kg per os weekly) combined with doxycycline (DOXY; 10 mg/kg/day orally from Weeks 0-6, 10-12, 16-18, 22-26 and 28-34) resulted in a significantly faster decrease of circulating microfilariae and higher adulticidal activity compared with either IVM or DOXY alone. Quantitative PCR analysis of ftsZ (Wolbachia DNA) and 18S rDNA (nematode DNA) absolute copy numbers showed significant decreases in Wolbachia content compared with controls in worms recovered from DOXY-treated dogs that were not, however, associated with worm death. Worms from IVM/DOXY-treated dogs, on the other hand, had Wolbachia/nematode DNA ratios similar to those of control worms, suggesting a loss of both Wolbachia and nematode DNA as indicated by absolute copy number values. Histology and transmission electron microscopy of worms recovered from the IVM/DOXY combination group showed complete loss of uterine content in females and immunohistochemistry for Wolbachia was negative. Results indicate that the combination of these two drugs causes adult worm death. This could have important implications for control of human and animal filarial infections.     

Preliminary results on the effect of tetracycline on the embryogenesis and symbiotic bacteria (Wolbachia) of Dirofilaria immitis. An update and discussion.

The distribution and phylogeny of Wolbachia in filarial species suggests that these endosymbiotic bacteria may be important in the biology of their filarial hosts. An experiment to falsify this hypothesis would be to treat filarial worms with antibiotics which are active against intracellular bacteria. Indeed, it has already been shown that tetracycline treatment inhibits development in a model filarial species (Brugia pahangi) at different stages of the life cycle, in both mosquito and mammalian hosts. Here we discuss these previous data and present new results on the effect of tetracycline on the embryogenesis of the canine filaria Dirofilaria immitis.     

Dipetalonema viteae and Brugia pahangi transplant infections in gerbils for use in antifilarial screening

Transplanted infections of Dipetalonema viteae and Brugia pahangi have been evaluated as tools for experimental chemotherapy. Attempts were made to establish these filariae in similar pharmacokinetic sites within the same host, so that direct comparisons of in vivo drug susceptibilities could be made. Unfortunately, it was not possible to establish B. pahangi in the subcutaneous tissues, the preferred site of D. viteae. Therefore, intraperitoneal B. pahangi and subcutaneously implanted D. viteae in gerbils were used for the study. D. viteae infections were significantly enhanced by concomitant infections with B. pahangi, while B. pahangi infection rates were unaffected by the presence of D. viteae. Experiments with amoscanate, CGP6140 and Mel W demonstrated the importance of employing both B. pahangi and D. viteae for antifilarial discovery work and the fundamental effect of parasite location on drug efficacy. D. viteae rapidly migrate from the peritoneal cavity of gerbils following implantation; twenty one hours after infection 73% of transplanted worms were found in the subcutaneous tissues. It was shown that the migration response could be used as a stringent parameter for demonstrating antifilarial activity. D. viteae were exposed to antifilarial drugs for 24 hours in vitro, washed and implanted into the peritoneal cavity of gerbils. At autopsy, 5 days later, 10(-8)M ivermectin and milbemycin D had prevented migration; CGP6140, amoscanate, suramin, flubendazole and furapyrimidone were also detected at less than 10(-6)M using this parameter. In all cases the migration response was more sensitive to drugs than parasite kill. Ivermectin's ability to inhibit worm migration through the tissues is discussed, with respect to the role of itinerant males in the reproductive cycle of Onchocerca volvulus.     

Macrofilaricidal activity of tetracycline against the filarial nematode Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship

Filarial nematodes are important and widespread parasites of animals and humans. We have been using the African bovine parasite Onchocerca ochengi as a chemotherapeutic model for O. volvulus, the causal organism of 'river blindness' in humans, for which there is no safe and effective drug lethal to adult worms. Here we report that the antibiotic, oxytetracycline is macrofilaricidal against O. ochengi. In a controlled trial in Cameroon, all adult worms (as well as microfilariae) were killed, and O. ochengi intradermal nodules resolved, by nine months' post-treatment in cattle treated intermittently for six months. Adult worms removed from concurrent controls remained fully viable and reproductively active. By serial electron-microscopic examination, the macrofilaricidal effects were related to the elimination of intracellular micro-organisms, initially abundant. Analysis of a fragment of the 16S rRNA gene from the O. ochengi micro-organisms confirmed them to be Wolbachia organisms of the order Rickettsiales, and showed that the sequence differed in only one nucleotide in 858 from the homologous sequence of the Wolbachia organisms of O. volvulus. These data are, to our knowledge, the first to show that antibiotic therapy can be lethal to adult filariae. They suggest that tetracycline therapy is likely to be macrofilaricidal against O. volvulus infections in humans and, since similar Wolbachia organisms occur in a number of other filarial nematodes, against those infections too. In that the elimination of Wolbachia preceded the resolution of the filarial infections, they suggest that in O. ochengi at least, the Wolbachia organisms play an essential role in the biology and metabolism of the filarial worm.     

Effect of immune reconstitution on resistance to Brugia pahangi in congenitally athymic nude mice

The dichotomy of resistance to Brugia pahangi (Nematoda: Filarioidea) between nonsusceptible, euthymic C3H/HeN mice, heterozygotic for the "nu" gene (+/nu), and susceptible, congenitally-athymic "nude" (nu/nu) C3H/HeN mice, suggests that resistance is thymus-dependent. To test this hypothesis, the effect of syngeneic neonatal thymus grafts and neonatal thymus cell suspensions on recovery of worms at day 40 PI, and responses to Concanavalin A (Con A) were examined in reconstituted nudes. Nude recipients of a thymus graft 7 or 14 wk before subcutaneous inoculation with 50 infective larvae (L3) yielded no worms and responded strongly to Con A. Serum from these mice reacted in two lines of identity with serum from similarly-infected heterozygotes by double radial immunodiffusion against an adult worm saline extract. Nude recipients of a thymus 2 days or 3 wk before inoculation harbored an average of three or two worms, respectively. Intravenous injection of nude recipients with 10(7) or 10(8) neonatal thymus cells seven weeks before inoculation was less effective in conferring resistance to B. pahangi and responsiveness to Con A. Complete resistance to B. pahangi could be adoptively transferred to nude mice by 10(8) spleen cells obtained from infection-primed heterozygotes and injected intravenously on the day of larval inoculation. The same numbers of worms were significantly reduced. less effective when injected 3 wk before inoculation, although numbers of worms were significantly reduced. Passive transfer of primed heterozygote serum, containing high titers of antibodies to adult worm and larval antigens, failed to protect nude recipients against a larval inoculum in the absence of cellular reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Obligatory symbiotic Wolbachia endobacteria are absent from Loa loa

BACKGROUND: Many filarial nematodes harbour Wolbachia endobacteria. These endobacteria are transmitted vertically from one generation to the next. In several filarial species that have been studied to date they are obligatory symbionts of their hosts. Elimination of the endobacteria by antibiotics interrupts the embryogenesis and hence the production of microfilariae. The medical implication of this being that the use of doxycycline for the treatment of human onchocerciasis and bancroftian filariasis leads to elimination of the Wolbachia and hence sterilisation of the female worms. Wolbachia play a role in the immunopathology of patients and may contribute to side effects seen after antifilarial chemotherapy. In several studies Wolbachia were not observed in Loa loa. Since these results have been doubted, and because of the medical significance, several independent methods were applied to search for Wolbachia in L. loa. METHODS: Loa loa and Onchocerca volvulus were studied by electron microscopy, histology with silver staining, and immunohistology using antibodies against WSP, Wolbachia aspartate aminotransferase, and heat shock protein 60. The results achieved with L. loa and O. volvulus were compared. Searching for Wolbachia, genes were amplified by PCR coding for the bacterial 16S rDNA, the FTSZ cell division protein, and WSP. RESULTS: No Wolbachia endobacteria were discovered by immunohistology in 13 male and 14 female L. loa worms and in numerous L. loa microfilariae. In contrast, endobacteria were found in large numbers in O. volvulus and 14 other filaria species. No intracellular bacteria were seen in electron micrographs of oocytes and young morulae of L. loa in contrast to O. volvulus. In agreement with these results, Wolbachia DNA was not detected by PCR in three male and six female L. loa worms and in two microfilariae samples of L. loa. CONCLUSIONS: Loa loa do not harbour obligatory symbiotic Wolbachia endobacteria in essential numbers to enable their efficient vertical transmission or to play a role in production of microfilariae. Exclusively, the filariae cause the immunopathology of loiasis is patients and the adverse side effects after antifilarial chemotherapy. Doxycycline cannot be used to cure loiais but it probably does not represent a risk for L. loa patients when administered to patients with co-infections of onchocerciasis.     

The filarial endosymbiont Wolbachia sp. is absent from Setaria equina

Wolbachia sp. was first reported in filarial nematodes over 25 yr ago. Today, much research is focused on the role of these bacteria in filarial worm biology. The filarial symbionts are closely related to arthropod symbionts, which are known to modify host reproduction and biology through various mechanisms. Similarly, it has been suggested that Wolbachia sp. is essential for long-term survival and reproduction of filariae. We report that Wolbachia sp. 16S rDNA was not found in the equine filarial nematode Setaria equina, using either polymerase chain reaction (PCR) or DNA hybridization. In addition, ultrastructural analysis of adult worms did not reveal the presence of Wolbachia sp. in hypodermal cords or reproductive tissues. These data suggest that like Onchocerca flexuosa and Acanthocheilonema vitae, S. equina may not be dependent on Wolbachia sp. for survival.     

Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria

Unlike in many other helminth infections, neutrophilic granulocytes are major cellular components in the hosts immune response against filarial worms. The pathways that drive the immune response involving neutrophils are unclear. This study shows that Wolbachia endobacteria (detectable by polyclonal antibodies against endobacterial heat shock protein 60 and catalase and by polymerase chain reaction being sensitive to doxycycline treatment) are direct and indirect sources of signals accounting for neutrophil accumulation around adult Onchocerca volvulus filariae. Worm nodules from untreated onchocerciasis patients displayed a strong neutrophil infiltrate adjacent to the live adult worms. In contrast, in patients treated with doxycycline to eliminate the endobacteria from O. volvulus and to render the worms sterile, the neutrophil accumulation around live adult filariae was drastically reduced. Neutrophils were absent in worm nodules from the deer filaria Onchocerca flexuosa, a species which does not contain endobacteria. Extracts of O. volvulus extirpated from untreated patients showed neutrophil chemotactic activity and in addition, induced strong TNF-alpha and IL-8 production in human monocytes, in contrast to filarial extracts obtained after doxycycline treatment. Thus, neutrophil chemotaxis and activation are induced directly by endobacterial products and also indirectly via chemokine induction by monocytes. These results show that the neutrophil response is a characteristic of endobacteria-containing filariae.     

Eosinophils contribute to killing of adult Onchocerca ochengi within onchocercomata following elimination of Wolbachia

Many filarial nematodes, including Onchocerca volvulus (the cause of human 'River Blindness'), have a mutually dependent relationship with Wolbachia bacteria. There has been much interest in Wolbachia as a chemotherapeutic target, since there are no macrofilaricidal drugs (i.e., lethal to adult worms) of low toxicity. Using the bovine parasite O. ochengi, we previously demonstrated that combined intensive and intermittent (COM) oxytetracycline treatment induces a sustained depletion of Wolbachia and is macrofilaricidal, whereas a short intensive regimen (SIR) is non-macrofilaricidal. To understand how targeting Wolbachia with oxytetracycline can lead to worm death, O. ochengi nodules (onchocercomata) were sequentially excised from cattle administered COM or SIR therapy, and cell infiltrates were microscopically quantified. Pre-treatment, worms were surrounded by neutrophils, with eosinophils rare or absent. At 8-12weeks after either regimen, eosinophils increased around worms and were observed degranulating on the cuticle. However, with the SIR treatment, neutrophils returned to predominance by 48weeks, while in the COM group, eosinophilia persisted. These observations suggest that accumulation of degranulating eosinophils over a prolonged period is a cause rather than an effect of parasite death, and the macrofilaricidal mechanism of antibiotics may relate to facilitation of eosinophil infiltration around worms by ablation of Wolbachia-mediated neutrophilia.     

Wolbachia endosymbiotic bacteria of Brugia malayi mediate macrophage tolerance to TLR- and CD40-specific stimuli in a MyD88/TLR2-dependent manner

Lymphatic filarial nematodes are able to down-regulate parasite-specific and nonspecific responses of lymphocytes and APC. Lymphatic filariae are reliant on Wolbachia endosymbiotic bacteria for development and survival. We tested the hypothesis that repeated exposure to Wolbachia endosymbionts would drive macrophage tolerance in vitro and in vivo. We pre-exposed murine peritoneal-elicited macrophages to soluble extracts of Brugia malayi female worms (BMFE) before restimulating with BMFE or TLR agonists. BMFE tolerized macrophages (in terms of IFN-beta, IL-1beta, IL-6, IL-12p40, and TNF-alpha inflammatory cytokine production) in a dose-dependent manner toward self, LPS, MyD88-dependent TLR2 or TLR9 ligands (peptidoglycan, triacyl lipopeptide, CpG DNA) and the MyD88-independent/TRIF-dependent TLR3 ligand, polyinosinic-polycytidylic acid. This was accompanied with down-regulation in surface expression of TLR4 and up-regulation of CD14, CD40, and TLR2. BMFE tolerance extended to CD40 activation in vitro and systemic inflammation following lethal challenge in an in vivo model of endotoxin shock. The mechanism of BMFE-mediated macrophage tolerance was dependent on MyD88 and TLR2 but not TLR4. Evidence that desensitization was driven by Wolbachia-specific ligands was determined by use of extracts from Wolbachia-depleted B. malayi, aposymbiotic filarial species, and a cell line stably infected with Wolbachia pipientis. Our data promote a role for Wolbachia in contributing toward the dysregulated and tolerized immunological phenotype that accompanies the majority of human filarial infections.     

The anthelmintic activity of a novel organic arsenical, R7/45, upon Brugia pahangi in Meriones unguiculatus

The new organic arsenical R7/45 is a rapidly acting and very potent anthelmintic against adult Brugia pahangi in jirds. Against adult worms implanted into the peritoneal cavity 5 subcutaneous (SC) injections at 2.5 mg/kg of R7/45 killed 100% of adult worms. A single dose SC of 20 mg/kg was 100% effective and 10 mg/kg 76% effective against adult worms. When jirds were autopsied at different times after treatment at 20 mg/kg SC 89% of worms were dead within three days. R7/45 was not active when given by stomach intubation. Pretreatment of jirds with R7/45 had no effect on adult worms subsequently implanted into jirds. R7/45 was highly active against third and fourth stage larvae of B. pahangi in jirds.     

Altered adult worm location in young male jirds infected with Brugia pahangi

Male jirds (Meriones unguiculatus) were inoculated subcutaneously with 100 Brugia pahangi L3 each at 2, 6, 10, and 15 wk of age to compare their susceptibility and pathologic reactivity to infection. Adult worm recoveries (mean +/- SD) ranged from 24.1 +/- 15.1 to 36.4 +/- 13.9 at 60 days postinfection. No significant difference in susceptibility was measured among the 4 age groups. Jirds infected at 2 wk of age had significantly fewer (alpha less than or equal to 0.025) testicular and intralymphatic worms than all other age groups. Numbers of intralymphatic thrombi were significantly lower (alpha less than or equal to 0.01) in jirds infected at 2 wk of age. Lymphatic lesion severity, expressed as the number of intralymphatic thrombi per intralymphatic worm, was similar between age groups. These data indicate no differences in susceptibility or lymphatic lesion formation following B. pahangi infection in 2-wk-old male jirds, despite altered adult worm location.