Brugia pahangi: immunization with early L3 ES alters parasite migration, and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus).

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3 s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3 s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.

Effects of extracorporeal shock wave therapy on desmitis of the accessory ligament of the deep digital flexor tendon in the horse.

OBJECTIVE: To evaluate the effects of extracorporeal shock wave therapy (ESWT) on collagenase-induced lesions in the accessory ligament of the deep digital flexor tendon (ALDDFT) of horses. STUDY DESIGN: Paired, blinded controlled study. ANIMALS: Eight Thoroughbred horses (3 mares, 5 geldings; mean ± SD weight, 464 ± 26 kg, mean age, 8 ± 1.7 years). METHODS: Lesions were created in both ALDDFTs of all horses by injection of 2 × 10(3) IU of collagenase type I. Percent lesion and structure (fiber alignment and echogenicity) were quantified with ultrasonographic imaging 3, 6, and 9 weeks after collagenase injection. After ultrasound examinations, ESWT (1000 shocks at 0.15 mJ/mm2) was applied to 1 ALDDFT in each horse. ALDDFT were harvested 15 weeks after collagenase injection and the microstructure, mRNA levels of collagen types I and III, and collagen and glycosaminoglycan content were evaluated. RESULTS: There were no differences in percent lesion, echogenicity, or fiber alignment between control- and ESWT-treated ligaments at each evaluation time; however, compared with 3-week values, there was a significant increase in percent lesion and echogenicity for EWST treated ligaments at 6 weeks and significant decrease in both variables for treated and control ligaments at 12 weeks. Fiber alignment improved significantly at 9 weeks in controls and at 12 weeks in treated and control ligaments. Collagen type I mRNA levels were significantly higher in the ESWT treatment group compared with the control group 15 weeks after collagenase injection though differences in other mRNA levels, microstructure, and composition were not significant. CONCLUSIONS: Our results do not support an effect of ESWT on collagenase-induced lesions in the equine ALDDFT.

Tissue migration capability of larval and adult Brugia pahangi.

Infection with mosquito-born filarial nematodes occurs when hosts are bitten by a vector carrying the infective third stage larvae (L3) of the parasites. These larvae, deposited on the skin by the feeding mosquito, are presumed to enter the skin via the vector-induced puncture wound. Larvae of Brugia spp. must then migrate from the entry site, penetrate various skin layers, and locate a lymphatic vessel that leads to their lymphatic predilection site. We have recently established an intradermal (ID) infection model using B. pahangi and the Mongolian gerbil, allowing us to investigate the migratory capability ofB. pahangi. Larval and adult parasites recovered from the peritoneal cavities of gerbils were capable of establishing an infection following ID (larvae) or subcutaneous (adult) injection. Third and fourth stage larvae both migrated away from the injection site within hours, although data suggest they localize to different lymphatic tissues at 3 days postinfection (DPI). Immature adult (28 day) B. pahangi also migrated away from their SC inoculation site within 7 DPI. Mature (45 day) adult B. pahangi displayed little migration away from the SC infection site, suggesting tissue migration may be limited to developing stages of the parasite.

Effect of immunostimulatory oligodeoxynucleotides on host responses and the establishment of Brugia pahangi in Mongolian gerbils (Meriones unguiculatus).

Infection of humans with filarial parasites has long been associated with the maintenance of a dominant Th2-type host immune response. This is reflected by increases in interleukin (IL)-4- and IL-5-producing T cells, elevated immunoglobulin (Ig)E and IgG4 levels, and a pronounced eosinophilia. The Mongolian gerbil (Meriones unguiculatus) is permissive for the filarial nematodes Brugia malayi and B. pahangi. As in humans, persistent microfilaremic infections of gerbils with Brugia spp. results in increases in Th2 cytokines such as IL-4 and IL-5. The association of dominant Th2 cytokine profiles with the maintenance of infection suggests that the introduction of Brugia spp. into a strongly Th1-biased environment may adversely affect parasite establishment. Indeed, studies conducted in mice with B. malayi suggest that depleting Th1 effectors such as interferon (IFN)-gamma and nitric oxide results in increased worm recoveries. In the present studies, the Mongolian gerbil was used as a model to investigate the effect of a dominant Th1 cytokine environment on the establishment of B. pahangi. Intraperitoneal (i.p.) administration of immunostimulatory oligodeoxynucleotide (IS ODN) induced the production of IFN-gamma in the peritoneal exudate cells and spleen of gerbils. The presence of IFN-gamma at the time of B. pahangi infection did result in an altered host immune response to B. pahangi. Gerbils that received IS ODN before i.p. B. pahangi infections showed lower levels of the Th2-type cytokines IL-4 and IL-5, compared with animals that received B. pahangi alone (0 + Bp). This alteration in cytokine profile, however, did not alter the establishment or development of B. pahangi in the peritoneal cavity. Furthermore, there was no difference in the granulomatous response of gerbils to soluble adult B. pahangi antigen bound to beads embolized in their lungs, regardless of treatment group, suggesting that IL-4 and IL-5 are not essential contributors to the systemic host inflammatory response to B. pahangi in this model.

In vitro development of cyathostomin larvae from the third stage larvae to the fourth stage: morphologic characterization, effects of refrigeration, and species-specific patterns.

A mixed population of equine cyathostomin (Nematoda, Strongyloidea) infective third stage larvae (L3) was cultured in vitro using a cell-free medium. Some L3 were cultured immediately after Baermann collection from fecal cultures, while others were kept in water at 4 degrees C for 7 days before initiating the in vitro cultures. Cultures were examined daily for viability. At days 2, 7, 14 and 21 larvae were collected for identification of developmental stage and morphological changes, using both light and scanning electron microscopy. Larvae were classified as early L3 (EL3), developing L3 (DL3), late L3 (LL3) and fourth stage larvae (L4) on the basis of morphological features. Viability remained high throughout the entire study period in cultures of both non-refrigerated (84.7%) and refrigerated (77.4%) larvae. However, viability of the non-refrigerated was significantly greater from 7 through 21 days of culture. Significant differences were also observed in the percentage of DL3 between the non-refrigerated and refrigerated larval cultures by day 7. The highest percentage of DL3 larvae (22.5%) was reached at the end of study in those larvae that were not previously refrigerated. The data suggests that prior refrigeration decreases viability and slows L3 development. At day 21 LL3 larvae were only a small percentage of the DL3: 6.9 and 5% in non-refrigerated and refrigerated cultures, respectively. Few of these larvae freed themselves from the L3 cuticle and moulted to L4 stage. Characteristics of individual species in vitro developmental patterns were determined by the molecular identification of individual larvae in pools of larvae randomly collected at days 0 and 21. Seven species (Coronocyclus coronatus, Cylicostephanus goldi, Cylicostephanus longibursatus, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus ashworthi, Petrovinema poculatum) were identified in the day 0 pool. The greatest tendency to develop in vitro was shown by the genus Cylicostephanus with the species C. goldi and C. longibursatus that developed to the LL3-L4 stages. C. nassatus, C. ashworthi and C. coronatus did not progress in their development beyond the EL3 stage, while no apparent signs of development were registered for C. catinatum.

Brugia pahangi: in vivo tissue migration of early L3 alters gene expression.

Events occurring during early filarial nematode migrations are central to parasite establishment but rarely studied. Brugia pahangi larvae injected intradermal (ID) into the hind limb of the gerbil (Meriones unguiculatus) can be recovered from the popliteal lymph node (POP) at 3 days post-infection (DPI). They have been designated migrating larvae (IDL3). Alternatively, L3 recovered at 3DPI from the peritoneal cavity (IPL3) do not migrate. Subtracted cDNA libraries using IDL3 and IPL3 revealed distinct gene profiles between IDL3 and IPL3. Troponin-c was significantly upregulated in IDL3, while Cathepsin L was significantly increased in IPL3. Differences in mRNA levels were also observed with these and other genes between IDL3, IPL3 and L3 isolated from mosquitoes (VL3). These data suggest that migratory activity, exposure to potentially different host environments and/or host location may be important external factors in influencing larval gene expression.

Inflammatory responses to migrating Brugia pahangi third-stage larvae.

Despite being central to parasite establishment and subsequent host pathological and immunologic responses, host-parasite interactions during early third-stage filarial larva (L3) migration are poorly understood. These studies aimed to define early tissue migration of Brugia pahangi L3 in the gerbil (Meriones unguiculatus) and measure host cellular responses during this period. Gerbils were intradermally inoculated in the hind limb with 100 B. pahangi L3, and necropsies were performed at various times. At 3 h, most L3 (96.3%) were recovered from tissues associated with the infection site, with marked L3 migration occurring by 24 h. Larvae were dispersed throughout the lymphatics at 7 days postinfection (dpi), and at 28 dpi, most parasites were recovered from the spermatic cord lymphatics. Parasites were identified histologically at all time points. Inflammatory cells, primarily neutrophils, were frequently observed around larvae in the dermis and muscle near the injection site at 3 h and 24 h. Levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha mRNA peaked at 3 h in all tissues, with IL-6 levels also high in the spleen at 28 dpi. Levels of IL-4 mRNA were elevated in all tissues at 28 dpi. These observations demonstrate that L3 migrate quickly through various tissues and into lymph nodes in a predictable pattern. Migrating L3 induce an early acute inflammatory response that is modulated as parasites establish in the lymphatics. Polarization of the host response towards a dominant Th2-like profile is present at 7 dpi and is well established by 28 dpi in this permissive host.

Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus.

Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.

Brugia pahangi and Wolbachia: the kinetics of bacteria elimination, worm viability, and host responses following tetracycline treatment.

Wolbachia spp., first reported from filariae nearly 30 years ago, have been suggested to contribute to the pathogenesis associated with human filarial infection. Tetracycline has been used to cure filariae of Wolbachia, as a novel means of chemotherapeutic treatment for both ocular and lymphatic filariasis. Tetracycline treatment of L4 or adult Brugia pahangi in vivo resulted in Wolbachia clearance. Less tetracycline was required to clear Wolbachia when treatment began at the L4 stage, compared with adults. Female worms died earlier than male worms when tetracycline was administered at the L4 stage. In all cases, Wolbachia clearance was closely associated with worm death. Worm recoveries decreased following the L4-L5 molt, suggesting tetracycline does not interrupt molting in this model system. Despite worm death and the assumed release of both bacterial- and worm-derived molecules, differences in inflammatory cell population and T cell cytokine mRNA profiles were negligible between tetracycline-treated and non-treated B. pahangi infected gerbils. These data suggest the contribution of Wolbachia to the in vivo induction of the gerbil immune response to B. pahangi may be small.

Removal of Wolbachia from Brugia pahangi is closely linked to worm death and fecundity but does not result in altered lymphatic lesion formation in Mongolian gerbils (Meriones unguiculatus).

Approximately 30 years ago, researchers reported intracellular bacteria in filarial nematodes. These bacteria are relatives of the arthropod symbiont Wolbachia and occur in many filarial nematodes, including Brugia pahangi and Brugia malayi. Wolbachia bacteria have been implicated in a variety of roles, including filaria development and fecundity and the pathogenesis of lymphatic lesions associated with filarial infections. However, the role of the bacteria in worm biology or filarial disease is still not clear. The present experiments support previous data showing that tetracycline eliminates or reduces Wolbachia bacteria in B. pahangi in vivo. The elimination of Wolbachia was closely linked to a reduction in female fecundity and the viability of both sexes, suggesting that the killing of Wolbachia is detrimental to B. pahangi. The gerbils treated with tetracycline showed reduced levels of interleukin-4 (IL-4) and IL-5 mRNA in renal lymph nodes and spleens compared with the levels in B. pahangi-infected gerbils not treated with tetracycline. However, similar findings were noted in B. pahangi-infected gerbils treated with ivermectin, suggesting that the loss of circulating microfilariae, not the reduction of Wolbachia bacteria, was associated with the altered cytokine profile. Despite the change in T-cell cytokines, there was no difference in the sizes of renal lymph nodes isolated from gerbils in each treatment group. Furthermore, the numbers, sizes, or cellular compositions of granulomas examined in the lymphatics or renal lymph nodes did not differ with treatment. These data suggest that Wolbachia may not play a primary role in the formation of lymphatic lesions in gerbils chronically infected with B. pahangi.

Infection outcome and cytokine gene expression in Brugia pahangi- infected gerbils (Meriones unguiculatus) sensitized with Brucella abortus.

Filarial infections have been associated with the development of a strongly polarized Th2 host immune response and a severe impairment of mitogen-driven proliferation and type 1 cytokine production in mice and humans. The role of this polarization in the development of the broad spectra of clinical manifestations of lymphatic filariasis is still unknown. Recently, data gathered from humans as well as from immunocompromised mouse models suggest that filariasis elicits a complex host immune response involving both Th1 and Th2 components. However, responses of a similar nature have not been reported in immunologically intact permissive models of Brugia infection. Brucella abortus-killed S19 was inoculated into the Brugia-permissive gerbil host to induce gamma interferon (IFN-gamma) production. Gerbils were then infected with B. pahangi, and the effect of the polarized Th1 responses on worm establishment and host cellular response was measured. Animals infected with both B. abortus and B. pahangi showed increased IFN-gamma and interleukin-10 (IL-10) and decreased IL-4 and IL-5 mRNA levels compared with those in animals infected with B. pahangi alone. These data suggest that the prior sensitization with B. abortus may induce a down regulation of the Th2 response associated with Brugia infection. This reduced Th2 response was associated with a reduced eosinophilia and an increased neutrophilia in the peritoneal exudate cells. The changes in cytokine and cellular environment did not inhibit the establishment of B. pahangi intraperitoneally. The data presented here suggest a complex relationship between the host immune response and parasite establishment and survival that cannot be simply ascribed to the Th1/Th2 paradigm.