Efficacy of a spot-on formulation containing moxidectin 2.5%/imidacloprid 10% for the treatment of Cercopithifilaria spp. and Onchocerca lupi microfilariae in naturally infected dogs from Portugal.

BACKGROUND: Onchocerca lupi and Cercopithifilaria spp. are vector-borne filarioids of dogs, which harbour skin microfilariae (mfs), the former being of zoonotic concern. Proper treatment studies using compounds with microfilaricidal activity have not been performed. Therefore, this study aimed to assess the efficacy of a commercially available spot-on formulation containing moxidectin 2.5%/imidacloprid 10% for the treatment of O. lupi or Cercopithifilaria spp. skin-dwelling mfs in naturally infected dogs. METHODS: Privately owned dogs (n = 393) from southern Portugal were sampled via skin biopsies to identify and count mfs in 20 µl of skin sediment. A total of 22 mfs-positive dogs were allocated to treatment group (n = 11; G1) or left untreated as a control (n = 11; G2). As a pilot investigation to test the treatment efficacy, five dogs assigned to G1 were treated four times at monthly intervals with moxidectin 2.5%/imidacloprid 10% spot-on formulation on SDs 0, 28 (± 2), 56 (± 2), and 84 (± 2). Based on the negative results for both O. lupi and/or Cercopithifilaria spp. mfs of dogs in the pilot study from SD28 onwards, the remaining six dogs in G1 were treated at SD0 and assessed only at SD28. RESULTS: Of the 393 animals sampled, 78 (19.8%) scored positive for skin-dwelling mfs. At the pilot investigation, a mean number of 19.6 mfs for O. lupi was recorded among five infected dogs whereas no mfs were detected at SD28. At SD0, the mean number of Cercopithifilaria spp. larvae was 12.6 for G1 and 8.7 for G2. The mean number of mfs for G2 was 20.09. CONCLUSIONS: Results herein obtained suggest that a single treatment with moxidectin 2.5%/imidacloprid 10% spot-on formulation is efficacious against skin-dwelling mfs in dogs. The microfilaricidal effect of moxidectin could also be useful in reducing the risk of O. lupi infection for humans.

The central adaptor molecule TRIF influences L. sigmodontis worm development.

Worldwide approximately 68 million people are infected with lymphatic filariasis (Lf), provoked by Wuchereria bancrofti, Brugia malayi and Brugia timori. This disease can lead to massive swelling of the limbs (elephantiasis) and disfigurement of the male genitalia (hydrocele). Filarial induced immune regulation is characterised by dominant type 2 helper T cell and regulatory immune responses. In vitro studies have provided evidence that signalling via Toll-like receptor-mediated pathways is triggered by filarial associated factors. Nevertheless, until now, less is known about the role of the adapter molecule TRIF during in vivo infections. Here, we used the rodent-specific nematode Litomosoides sigmodontis to investigate the role of TLR signalling and the corresponding downstream adapter and regulatory molecules TRIF, MyD88, IRF1 and IRF3 during an ongoing infection in semi-susceptible C57BL/6 mice. Interestingly, lack of the central adapter molecule TRIF led to higher worm burden and reduced overall absolute cell numbers in the thoracic cavity (the site of infection) 30 days post-infection. In addition, frequencies of macrophages and lymphocytes in the TC were increased in infected TRIF-/- C57BL/6 mice, whereas frequencies of eosinophils, CD4+ and CD8+ T cells were reduced. Nevertheless, cytokine levels and regulatory T cell populations remained comparable between TRIF-deficient and wildtype C57BL/6 mice upon 30 days of L. sigmodontis infection. In summary, this study revealed a crucial role of the adapter molecule TRIF on worm recovery and immune cell recruitment into the site of infection 30 days upon L. sigmodontis infection in C57BL/6 mice.

Myeloid cell recruitment versus local proliferation differentiates susceptibility from resistance to filarial infection.

Both TH2-dependent helminth killing and suppression of the TH2 effector response have been attributed to macrophages (MΦ) activated by IL-4 (M(IL-4)). To investigate how M(IL-4) contribute to diverse infection outcomes, the MΦ compartment of susceptible BALB/c mice and more resistant C57BL/6 mice was profiled during infection of the pleural cavity with the filarial nematode, Litomosoides sigmodontis. C57BL/6 mice exhibited a profoundly expanded resident MΦ (resMΦ) population, which was gradually replenished from the bone marrow in an age-dependent manner. Infection status did not alter the bone-marrow derived contribution to the resMΦ population, confirming local proliferation as the driver of resMΦ expansion. Significantly less resMΦ expansion was observed in the susceptible BALB/c strain, which instead exhibited an influx of monocytes that assumed an immunosuppressive PD-L2+ phenotype. Inhibition of monocyte recruitment enhanced nematode killing. Thus, the balance of monocytic vs. resident M(IL-4) numbers varies between inbred mouse strains and impacts infection outcome.

Twelve-month longitudinal parasitological assessment of lymphatic filariasis-positive individuals: impact of a biannual treatment with ivermectin and albendazole.

OBJECTIVE: Mass drug administration (MDA) for the control of lymphatic filariasis (LF), in Ghana, started in the year 2000. While this had great success in many implementation units, there remain areas with persistent transmission, after more than 10 years of treatment. A closer examination of the parasite populations could help understand the reasons for persistent infections and formulate appropriate strategies to control LF in these areas of persistent transmission. MATERIALS AND METHODS: In a longitudinal study, we assessed the prevalence of microfilaraemia (mf) in two communities with 12 years of MDA in Ghana. In baseline surveys 6 months after the National MDA in 2014, 370 consenting individuals were tested for antigenaemia using immunochromatographic test (ICT) cards and had their mf count determined through night blood surveys. 48 ICT positives, of whom, 17 were positive for mf, were treated with 400 µg/kg ivermectin + 400 mg albendazole and subsequently followed for parasitological assessment at 3-month intervals for 1 year. This overlapped with the National MDA in 2015. RESULTS: There was a 68% parasite clearance 3 months after treatment. The pre-treatment mf count differed significantly from the post-treatment mf counts at 3 months (P = 0.0023), 6 months (P = 0.0051), 9 months (P = 0.0113) and 12 months (P = 0.0008). CONCLUSION: In these settings with persistent LF transmission, twice-yearly treatment may help accelerate LF elimination. Further large-scale evaluations are required to ascertain these findings.

Migratory phase of Litomosoides sigmodontis filarial infective larvae is associated with pathology and transient increase of S100A9 expressing neutrophils in the lung.

Filarial infections are tropical diseases caused by nematodes of the Onchocercidae family such as Mansonella perstans. The infective larvae (L3) are transmitted into the skin of vertebrate hosts by blood-feeding vectors. Many filarial species settle in the serous cavities including M. perstans in humans and L. sigmodontis, a well-established model of filariasis in mice. L. sigmodontis L3 migrate to the pleural cavity where they moult into L4 around day 9 and into male and female adult worms around day 30. Little is known of the early phase of the parasite life cycle, after the L3 is inoculated in the dermis by the vector and enters the afferent lymphatic vessels and before the moulting processes in the pleural cavity. Here we reveal a pulmonary phase associated with lung damage characterized by haemorrhages and granulomas suggesting L3 reach the lung via pulmonary capillaries and damage the endothelium and parenchyma by crossing them to enter the pleural cavity. This study also provides evidence for a transient inflammation in the lung characterized by a very early recruitment of neutrophils associated with high expression levels of S100A8 and S100A9 proteins.

Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis.

Litomosoides sigmodontis: a jird urine metabolome study.

The neglected tropical disease onchocerciasis affects more than 35 million people worldwide with over 95% in Africa. Disease infection initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious L3 larvae. New treatments and diagnostics are required to eradicate this parasitic disease. Herein, we describe that a previously discovered biomarker for onchocerciasis, N-acetyltyramine-O-glucuronide (NATOG) is also present in urine samples of jirds infected with the onchocerciasis model nematode Litomosoides sigmodontis. Increased NATOG values paralleled a progressing infection and demonstrated that quantification of NATOG in this rodent model can be utilized to track its infectivity. Moreover, our findings suggest how NATOG monitoring may be used for evaluating potential drug candidates.

Development of patent Litomosoides sigmodontis infections in semi-susceptible C57BL/6 mice in the absence of adaptive immune responses.

BACKGROUND: One of the most advantageous research aspects of the murine model of filariasis, Litomosoides sigmodontis, is the availability of mouse strains with varying susceptibility to the nematode infection. In C57BL/6 mice, L. sigmodontis worms are largely eliminated in this strain by day 40 post-infection and never produce their offspring, microfilariae (Mf). This provides a unique opportunity to decipher potential immune pathways that are required by filariae to achieve a successful infection. In this study we tracked worm development and patency, the production of microfilariae and thus the transmission life-stage, in Rag2IL-2Rγ(-/-) mice which are deficient in T, B and NK cell populations. FINDINGS: Although worm burden was comparable between wildtype (WT) and Rag2IL-2Rγ(-/-) mice on d30, by day 72 post-infection, parasites in Rag2IL-2Rγ(-/-) mice were still in abundance, freely motile and all mice presented high quantities of Mf both at the site of infection, the thoracic cavity (TC), and in peripheral blood. Levels of cytokine (IL-4, IL-6, TNFα) and chemokine (MIP-2, RANTES, Eotaxin) parameters were generally low in the TC of infected Rag2IL-2Rγ(-/-)mice at both time-points. The frequency of neutrophils however was higher in Rag2IL-2Rγ(-/-)mice whereas eosinophils and macrophage populations, including alternatively activated macrophages, were elevated in WT controls. CONCLUSION: Our data highlight that adaptive immune responses prevent the development of patent L. sigmodontis infections in semi-susceptible C57BL/6 mice and suggest that induction of such responses may offer a strategy to prevent transmission of human filariasis.

Vector competence of Aedes aegypti mosquitoes for filarial nematodes is affected by age and nutrient limitation.

Mosquitoes are one of the most important vectors of human disease. The ability of mosquitoes to transmit disease is dependent on the age structure of the population, as mosquitoes must survive long enough for the parasites to complete their development and infect another human. Age could have additional effects due to mortality rates and vector competence changing as mosquitoes senesce, but these are comparatively poorly understood. We have investigated these factors using the mosquito Aedes aegypti and the filarial nematode Brugia malayi. Rather than observing any effects of immune senescence, we found that older mosquitoes were more resistant, but this only occurred if they had previously been maintained on a nutrient-poor diet of fructose. Constant blood feeding reversed this decline in vector competence, meaning that the number of parasites remained relatively unchanged as mosquitoes aged. Old females that had been maintained on fructose also experienced a sharp spike in mortality after an infected blood meal ("refeeding syndrome") and few survived long enough for the parasite to develop. Again, this effect was prevented by frequent blood meals. Our results indicate that old mosquitoes may be inefficient vectors due to low vector competence and high mortality, but that frequent blood meals can prevent these effects of age.

Comparative analysis of the secretome from a model filarial nematode (Litomosoides sigmodontis) reveals maximal diversity in gravid female parasites.

Filarial nematodes (superfamily Filarioidea) are responsible for an annual global health burden of ∼6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans. No vaccine exists for the major filarial diseases, lymphatic filariasis and onchocerciasis; in part because research on protective immunity against filariae has been constrained by the inability of the human-parasitic species to complete their lifecycles in laboratory mice. However, the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction. Here, we provide a comprehensive analysis of excretory-secretory products from L. sigmodontis across five lifecycle stages and identifications of host proteins associated with first-stage larvae (microfilariae) in the blood. Applying intensity-based quantification, we determined the abundance of 302 unique excretory-secretory proteins, of which 64.6% were present in quantifiable amounts only from gravid adult female nematodes. This lifecycle stage, together with immature microfilariae, released four proteins that have not previously been evaluated as vaccine candidates: a predicted 28.5 kDa filaria-specific protein, a zonadhesin and SCO-spondin-like protein, a vitellogenin, and a protein containing six metridin-like ShK toxin domains. Female nematodes also released two proteins derived from the obligate Wolbachia symbiont. Notably, excretory-secretory products from all parasite stages contained several uncharacterized members of the transthyretin-like protein family. Furthermore, biotin labeling revealed that redox proteins and enzymes involved in purinergic signaling were enriched on the adult nematode cuticle. Comparison of the L. sigmodontis adult secretome with that of the human-infective filarial nematode Brugia malayi (reported previously in three independent published studies) identified differences that suggest a considerable underlying diversity of potential immunomodulators. The molecules identified in L. sigmodontis excretory-secretory products show promise not only for vaccination against filarial infections, but for the amelioration of allergy and autoimmune diseases.

Eotaxin-1 is involved in parasite clearance during chronic filarial infection.

Eosinophil migration as key feature of helminth infection is increased during infection with filarial nematodes. In a mouse model of filariasis, we investigated the role of the eosinophil-attracting chemokine Eotaxin-1 on disease outcome. BALB/c and Eotaxin-1(-/-) mice were infected with the rodent filaria Litomosoides sigmodontis, and parasitic parameters, cellular migration to the site of infection, and cellular responsiveness were investigated. We found increased parasite survival but unaffected eosinophil migration to the site of infection in Eotaxin-1(-/-) mice. Expression of CD80 and CD86 was reduced on eosinophils from Eotaxin-1(-/-) mice after in vitro TLR2 stimulation and exposure to filarial antigen, respectively, suggesting a potential reduced activation state of eosinophils in Eotaxin-1 deficient mice. We further demonstrated that macrophages from Eotaxin-1(-/-) mice produce decreased amounts of IL-6 in vitro, a cytokine found to be associated with parasite containment, suggesting possible mechanisms by which Eotaxin-1 regulates activation of inflammatory cells and thus parasite survival.

Mosquito-parasite interactions can shape filariasis transmission dynamics and impact elimination programs.

The relationship between mosquito vectors and lymphatic filariasis (LF) parasites can result in a range of transmission outcomes. Anophelines are generally characterized as poor vectors due to an inability to support development at low densities. However, it is important to understand the potential for transmission in natural vectors to maximize the success of elimination efforts. Primary vectors in Papua New Guinea (n = 1209) were dissected following exposure to microfilaremic blood (range 8-233 mf/20 µl). We examined density dependent and species-specific parasite prevalence, intensity and yield, barriers to parasite development as well as impacts on mosquito survival. We observed strikingly different parasite prevalence and yield among closely related species. Prevalence of infective stage larvae (L3s) ranged from 4.2% to 23.7% in An. punctulatus, 24.5% to 68.6% in An. farauti s.s. and 61.9% to 100% in An. hinesorum at low and high density exposures, respectively. Injection experiments revealed the greatest barrier to parasite development involved passage from the midgut into the hemocoel. The ratio of L3 to ingested mf at low densities was higher in An. hinesorum (yield = 1.0) and An. farauti s.s. (yield = 0.5) than has been reported in other anopheline vectors. There was a negative relationship between mosquito survival and bloodmeal mf density. In An. farauti s.s., increased parasite yield and survival at low densities suggest greater competence at low microfilaremias. In Papua New Guinea the likelihood of transmission will be strongly influenced by vector composition and changes in the mf reservoir as a result of elimination efforts. Global elimination efforts will be strengthened by the knowledge of transmission potential in the context of current control measures.

Immunization with L. sigmodontis microfilariae reduces peripheral microfilaraemia after challenge infection by inhibition of filarial embryogenesis.

BACKGROUND: Lymphatic filariasis and onchocerciasis are two chronic diseases mediated by parasitic filarial worms causing long term disability and massive socioeconomic problems. Filariae are transmitted by blood-feeding mosquitoes that take up the first stage larvae from an infected host and deliver it after maturation into infective stage to a new host. After closure of vector control programs, disease control relies mainly on mass drug administration with drugs that are primarily effective against first stage larvae and require many years of annual/biannual administration. Therefore, there is an urgent need for alternative treatment ways, i.e. other effective drugs or vaccines. METHODOLOGY/PRINCIPAL FINDINGS: Using the Litomosoides sigmodontis murine model of filariasis we demonstrate that immunization with microfilariae together with the adjuvant alum prevents mice from developing high microfilaraemia after challenge infection. Immunization achieved 70% to 100% protection in the peripheral blood and in the pleural space and furthermore strongly reduced the microfilarial load in mice that remained microfilaraemic. Protection was associated with the impairment of intrauterine filarial embryogenesis and with local and systemic microfilarial-specific host IgG, as well as IFN-γ secretion by host cells from the site of infection. Furthermore immunization significantly reduced adult worm burden. CONCLUSIONS/SIGNIFICANCE: Our results present a tool to understand the immunological basis of vaccine induced protection in order to develop a microfilariae-based vaccine that reduces adult worm burden and prevents microfilaraemia, a powerful weapon to stop transmission of filariasis.

On a Cercopithifilaria sp. transmitted by Rhipicephalus sanguineus: a neglected, but widespread filarioid of dogs.

BACKGROUND: This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. RESULTS: The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9% and 10.5% by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6% and 45.5% of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7% to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2% to 16.7%) was higher than that detected by PCR (from 0% to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. CONCLUSIONS: Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i.e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs.

Filarial worms reduce Plasmodium infectivity in mosquitoes.

BACKGROUND: Co-occurrence of malaria and filarial worm parasites has been reported, but little is known about the interaction between filarial worm and malaria parasites with the same Anopheles vector. Herein, we present data evaluating the interaction between Wuchereria bancrofti and Anopheles punctulatus in Papua New Guinea (PNG). Our field studies in PNG demonstrated that An. punctulatus utilizes the melanization immune response as a natural mechanism of filarial worm resistance against invading W. bancrofti microfilariae. We then conducted laboratory studies utilizing the mosquitoes Armigeres subalbatus and Aedes aegypti and the parasites Brugia malayi, Brugia pahangi, Dirofilaria immitis, and Plasmodium gallinaceum to evaluate the hypothesis that immune activation and/or development by filarial worms negatively impact Plasmodium development in co-infected mosquitoes. Ar. subalbatus used in this study are natural vectors of P. gallinaceum and B. pahangi and they are naturally refractory to B. malayi (melanization-based refractoriness). METHODOLOGY/PRINCIPAL FINDINGS: Mosquitoes were dissected and Plasmodium development was analyzed six days after blood feeding on either P. gallinaceum alone or after taking a bloodmeal containing both P. gallinaceum and B. malayi or a bloodmeal containing both P. gallinaceum and B. pahangi. There was a significant reduction in the prevalence and mean intensity of Plasmodium infections in two species of mosquito that had dual infections as compared to those mosquitoes that were infected with Plasmodium alone, and was independent of whether the mosquito had a melanization immune response to the filarial worm or not. However, there was no reduction in Plasmodium development when filarial worms were present in the bloodmeal (D. immitis) but midgut penetration was absent, suggesting that factors associated with penetration of the midgut by filarial worms likely are responsible for the observed reduction in malaria parasite infections. CONCLUSIONS/SIGNIFICANCE: These results could have an impact on vector infection and transmission dynamics in areas where Anopheles transmit both parasites, i.e., the elimination of filarial worms in a co-endemic locale could enhance malaria transmission.

Filarial parasites develop faster and reproduce earlier in response to host immune effectors that determine filarial life expectancy.

Humans and other mammals mount vigorous immune assaults against helminth parasites, yet there are intriguing reports that the immune response can enhance rather than impair parasite development. It has been hypothesized that helminths, like many free-living organisms, should optimize their development and reproduction in response to cues predicting future life expectancy. However, immune-dependent development by helminth parasites has so far eluded such evolutionary explanation. By manipulating various arms of the immune response of experimental hosts, we show that filarial nematodes, the parasites responsible for debilitating diseases in humans like river blindness and elephantiasis, accelerate their development in response to the IL-5 driven eosinophilia they encounter when infecting a host. Consequently they produce microfilariae, their transmission stages, earlier and in greater numbers. Eosinophilia is a primary host determinant of filarial life expectancy, operating both at larval and at late adult stages in anatomically and temporally separate locations, and is implicated in vaccine-mediated protection. Filarial nematodes are therefore able to adjust their reproductive schedules in response to an environmental predictor of their probability of survival, as proposed by evolutionary theory, thereby mitigating the effects of the immune attack to which helminths are most susceptible. Enhancing protective immunity against filarial nematodes, for example through vaccination, may be less effective at reducing transmission than would be expected and may, at worst, lead to increased transmission and, hence, pathology.

Mosquito infection responses to developing filarial worms.

Human lymphatic filariasis is a mosquito-vectored disease caused by the nematode parasites Wuchereria bancrofti, Brugia malayi and Brugia timori. These are relatively large roundworms that can cause considerable damage in compatible mosquito vectors. In order to assess how mosquitoes respond to infection in compatible mosquito-filarial worm associations, microarray analysis was used to evaluate transcriptome changes in Aedes aegypti at various times during B. malayi development. Changes in transcript abundance in response to the different stages of B. malayi infection were diverse. At the early stages of midgut and thoracic muscle cell penetration, a greater number of genes were repressed compared to those that were induced (20 vs. 8). The non-feeding, intracellular first-stage larvae elicited few differences, with 4 transcripts showing an increased and 9 a decreased abundance relative to controls. Several cecropin transcripts increased in abundance after parasites molted to second-stage larvae. However, the greatest number of transcripts changed in abundance after larvae molted to third-stage larvae and migrated to the head and proboscis (120 induced, 38 repressed), including a large number of putative, immunity-related genes (approximately 13% of genes with predicted functions). To test whether the innate immune system of mosquitoes was capable of modulating permissiveness to the parasite, we activated the Toll and Imd pathway controlled rel family transcription factors Rel1 and Rel2 (by RNA interference knockdown of the pathway's negative regulators Cactus and Caspar) during the early stages of infection with B. malayi. The activation of either of these immune signaling pathways, or knockdown of the Toll pathway, did not affect B. malayi in Ae. aegypti. The possibility of LF parasites evading mosquito immune responses during successful development is discussed.

Screening of different classes of proteases in microfilarial and adult stages of Setaria cervi.

Many of the filarial proteases involved in critical physiological functions are expressed in stage-specific manner and belong to various mechanistic classes. Setaria cervi, a bovine filarial parasite express different classes of proteases. This parasite shows strong antigenic cross-reactivity with human filarial parasites Wuchereria bancrofti and Brugia malayi. Somatic extracts of S. cervi microfilariae (mf) and adult stages as well as their excretory-secretory (ES) products were screened for the presence of different classes of proteases using general (casein, bovine hemoglobin) and class specific substrates. Detergent-soluble extracts of male and female worms were also screened. Significant enzyme activity was detected in ES products both at pH 5.0 and 7.0 with casein. Cathepsin B-like activity was found to be much higher in membrane-bound extract than in the crude-soluble extract. However, it was also found to be actively secreted by both mf and adult worms. Cathepsin D-like activity assayed at pH 3.0 was very low both in somatic extract as well as in ES products. Collagenase activity at neutral pH showed higher levels, both in somatic extract and ES products. Cathepsin L-like activity was detected only in crude-soluble extract but was below detectable limit in ES products. Leucine aminopeptidase activity was significant both in crude-soluble extract and ES products. This study, thus, might be helpful for a better understanding of host-parasite interaction and identification of appropriate virulence factors that may be targeted as vaccine and/or drug targets against lymphatic filariasis.

Seasonal and diurnal biting activities and zoonotic filarial infections of two Simulium species (Diptera: Simuliidae) in northern Thailand.

Seasonal and daily biting activity patterns, and natural filarial infections of adult black flies attracted to human bait were investigated at Ban Pang Faen, a rural area in Chiang Mai Province in northern Thailand. Collections were carried out twice a month from 0600 to 18-00 hours from January 2005 to February 2006. Among ten Simulium species collected, S. nodosum and S. asakoae were predominant occupying 57.3% and 37.2% of the total 16,553 females, respectively. These two predominant species showed different patterns in seasonal abundance: majority of S. nodosum (86.7%) were collected in hot season (from mid February to mid May), while most of S. asakoae (74.5%) were collected in rainy season (from mid May to mid October). For the daily biting activity, S. nodosum had two patterns: the main one was unimodal with a peak from 17-00 to 18-00, and the other was bimodal and had the major peak from 1600 to 18-00 and the minor one from 07-00 to 09-00. The pattern of S. asakoae was mostly unimodal with a peak from 06-00 to 10-00. The filarial larvae found in S. nodosum and S. asakoae were morphologically different from each other. The short and thick infective larvae found in S. asakoae differed from all known filarial larvae; it is suggested that they might be a bird parasite, Splendidofilariinae or Lemdaninae. The infection of the mammophilic S. nodosum with large Onchocerca type infective larvae was confirmed in this area. Natural filarial infections were found in each month (except December) in either S. nodosum or S. asakoae or in both. Monthly infection rates with all stages of larvae were 0.6-5.0% for S. nodosum, and 1.0-4.0% for S. asakoae. It is suggested that people in this village are exposed to the risk of infection with zoonotic filariae throughout the year.

Pleural cellular reaction to the filarial infection Litomosoides sigmodontis is determined by the moulting process, the worm alteration, and the host strain.

The filarial nematode Litomosoides sigmodontis model was used to decipher the complex in vivo relationships between filariae, granulomas and leukocytes in the host's pleural cavity. The study was performed from D5 p.i.: to D47 p.i. in resistant C57BL/6 mice, to D74 p.i. in susceptible BALB/c mice, and to D420 p.i. in permissive jirds. We showed that, during the first month, leukocytes only clustered as granulomas around shed cuticles (exuviae) and with eosinophils as the major constituents. In addition, carbohydrates residues became abundant on exuviae only, suggesting a glycan-dependent mechanism of eosinophil attachment. Neutrophils were absent from the pleural cavity of all rodents and from the murine granulomas, but they made up 25% of the granuloma cell population in jirds. After the first month of infection granulomas formed around developed adult worms and morphological evidence suggested that leukocytes preferentially clustered around altered, but still motile, worms. No carbohydrates were detected on these worms and neutrophils were abundant in those granulomas. Finally, a rare third type of granuloma was observed in the resistant mice only; they contained young newly moulted adult worms; typically these granulomas were attached to the lateral lines of the worm via eosinophils; this feature correlated with the persistence of carbohydrate residues on the worms' lateral lines. Neutrophils were always in low proportion in all granulomas from resistant mice, suggesting difference in their adhesive properties in these mice. In vitro neutrophil recruitment in resistant mice was similar to that observed in susceptible mice although they expressed less cell surface CD11b.