Orientation of Onchocerca lienalis stiles (Filarioidea: Onchocercidae) microfilariae to black fly saliva.

Black flies (Simulium spp.) are intermediate hosts and vectors of parasitic nematodes belonging to the genus Onchocerca (Filarioidea Onchocercidae). Infection and subsequent transmission of infective third-stage larvae occur at the vertebrate host-skin interface. Experimental evidence presented here demonstrates that Onchocerca lienalis Stiles microfilariae orient to one or more components (microfilarial orientation factor [s]; MOF) in black fly saliva. MOFs may serve as a means for microfilariae to find and infect black flies during the act of blood-feeding. Directed movement through the host's skin to the bite site is necessary because Onchocerca spp. microfilariae do not circulate in the blood. The substance directing microfilarial orientation appears to be a salivary protein, but it is not the Simulium vittatum Zetterstedt erythema protein (SVEP) described from New World Simulium spp. These results support earlier field observations that associated increased numbers of cutaneous microfilariae with black fly feeding and indicate that a fundamental molecular mechanism linked to vector saliva may be key for the maintenance of the life cycle of Onchocerca spp. Salivary molecules that induce orientation of microfilariae to the bite site are potential targets for use in transmission-blocking vaccines to uncouple this primary vector infection step.

Thrombostasin: purification, molecular cloning and expression of a novel anti-thrombin protein from horn fly saliva.

Thrombostasin (TS), a novel protein found in the saliva of Haematobia irritans (horn fly), was purified by high-performance liquid chromatography from the saliva of field-collected insects. This protein, which inhibits thrombin, accounts for anti-clotting activity in horn fly saliva [J. Med. Entomol. 37 (2000) 416] and is the first purified anti-hemostatic factor described from the Stomoxyinae, a large group of blood-feeding insects that are major pests of livestock world-wide. The purified TS had an apparent molecular weight of 16.7 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed two isoelectric groups with isoelectric points (pIs) of approximately 4.6 and 4.8. Mass spectroscopy analysis, however, resulted in at least three major isoforms that range in mass from 9213 to 9274 Da. A 243-bp coding sequence was obtained from cDNA by using a degenerate primer deduced from the N-terminal sequence of the purified TS. The conceptual translation of the 243-bp sequence showed that the 81-amino-acid peptide, whose first 30 amino acids match those of the N-terminal sequence, had a predicted mass of 9213 Da with pI 4.14. A full-length TS cDNA was generated by rapid amplification of cDNA ends of the 5' and sequential polymerase chain reaction (PCR) amplification. It contained a 5'-end 12-bp segment preceding the putative ATG start codon, followed by a 54-bp sequence corresponding to a secretory signal and an additional 228-bp coding sequence preceding residues revealed by N-terminal sequencing of purified TS. The fidelity of the PCR-generated TS sequence was confirmed in genomic DNA and by biological activity of recombinant TS produced in a baculovirus expression system. Database comparisons revealed no homology between TS and other known molecules. Because of the paucity of other anti-hemostatic factors in horn fly saliva, TS may play a critical role in maintenance of the ectoparasitic lifestyle of horn flies.

Polymorphism of the thrombostasin gene in the horn fly (Haematobia irritans) revealed in a cDNA library and in genomic DNA.

Thrombostasin (TS) is a newly described thrombin-inhibiting protein isolated from the saliva of the horn fly (Haematobia irritans), a blood-sucking ectoparasite of cattle. This report provides a detailed characterization of the TS gene and the first analysis of the allelic complexity of a gene for an anti-hemostatic protein from a blood-feeding insect. Multiple point mutations at fixed positions in the TS gene were identified in a cDNA library prepared from mRNA isolated from horn fly salivary glands. When translated, the variant mRNAs would specify five biochemically active peptides that differ in molecular weight, isoelectric point and predicted secondary structure. Allelic variation with the same mutation pattern was revealed in the genomes of individual flies collected in the field and sampled from a long-standing laboratory colony. Approximately 60% of flies examined carried heterozygous alleles, including five additional alleles not found in the cDNA library. Comparative analysis of the allelic mutations and the predicted effects on secondary structures of the active proteins produced suggest that the TS gene may be undergoing evolutionary selection.

Horn fly (Diptera: Muscidae) saliva targets thrombin action in hemostasis.

The horn fly, Hematobia irritans (L.), is an important pest of livestock because the adult stage of both sexes are aggressive blood-feeders. Remarkably, even though horn fly adults feed recurrently on their hosts as ectoparasites, these flies lack the ADP-responsive antiplatelet aggregation and vasodilatory antihemostatic systems described for other blood-feeding Diptera. Horn fly salivary gland extracts do interfere with the normal coagulation process as demonstrated by the recalcification time assay. Using this as a baseline, the effects of saliva on recalcification time, activated partial thromboplastin time, prothrombin time, and thrombin time were measured to determine which arm(s) of the coagulation cascade might be impacted. Factor-deficient plasma assays also were used to measure possible perturbations in clotting. Gland-free saliva delayed the recalcification time as well as the activated partial thromboplastin time, prothrombin time, and thrombin time. Saliva also further delayed clotting times of plasmas deficient in factor V, factor VIII, and factor XIII, indicating that other factors in the coagulation cascade were inhibited. Although horn fly saliva did not alter the ability of deficient plasma reconstituted with factor X to clot, it did inhibit deficient plasma reconstituted with factor II (thrombin). Antithrombin activity in saliva was confirmed by its ability to interfere with thrombin hydrolysis of fibrinogen, its normal substrate, and by its inhibition of thrombin action on a chromagenic substrate that mimics the hydrolytic site of fibrinogen. Thus, horn fly saliva contains a factor that specifically targets thrombin, a key component in the coagulation cascade. While the biochemical mechanisms of inhibition may vary, this antihemostatic characteristic is shared with other zoophilic Diptera such as black flies, Simulium spp., and tsetse, Glossina morsitans morsitans Westwood, that feed on ungulates.

Blood-feeding strategy of Haematobia irritans (Diptera: Muscidae).

The economic impact on livestock production by Haematobia irritans (L.) is estimated to approach $1 billion per year in North America. However, there is little information regarding the blood-feeding strategy used by these insects. Information presented here shows that horn fly saliva interferes with the normal coagulation response as measured by the recalcification time assay. The relative anticoagulant activity on a per-gland basis was more than or equal to that reported for Simulium vittatum Zetterstedt, a common hematophagous black fly that also feeds on cattle. However, unlike S. vittatum, H. irritans salivary factors do not inhibit platelet aggregation using apyrase and have no detectable vasodilative activity. In this regard, the horn fly is strikingly different from blood-feeding species in the lower Diptera and shows a much more limited repertoire of antihemostatic factors.

Analyses of cDNA and recombinant protein for a potent vasoactive protein in saliva of a blood-feeding black fly, Simulium vittatum.

A cDNA was cloned from the salivary glands of a blood-feeding black fly Simulium vittatum. The encoded protein has been given the name Simulium vittatum erythema protein or SVEP, because of its ability to increase blood perfusion in skin capillaries, resulting in the well-characterized erythema of black fly bites. The full-length cDNA contains 548 base pairs which encode 152 amino acid residues of the nascent protein. Post-translational processing produces a mature, secreted protein of 133 residues with a molecular mass of 15.4 kDa. Recombinant SVEP (rSVEP) was produced in a baculovirus expression system and purified by a one-step reversed-phase HPLC procedure. Analyses of physical properties and biological potency demonstrated fidelity of rSVEP to the native protein. Recombinant SVEP relaxed rabbit aorta preparations when preconstricted with 2 micromol l-1 phenylephrine or 25 mmol l-1 K+ but not with 60 mmol l-1 K+. Further, the rSVEP-induced relaxation response of phenylephrine-constricted aorta was inhibited by glibenclamide (10 micromol l-1), suggesting that at least part of its action to relax smooth muscle may result from the opening of ATP-dependent K+ channels. SVEP is a novel salivary-gland-derived vasoactive protein that may be essential for blood feeding by black flies and could potentially enhance transmission of filarial parasites.

Black fly (Diptera:Simuliidae) salivary secretions: importance in vector competence and disease.

When blood-feeding, black flies introduce secretions into the feeding lesion that act in a coordinated manner on the 3 arms of the vertebrate hemostatic system (platelet aggregation, coagulation, and vasoconstriction). Apyrase activity inhibits platelet aggregation and is ubiquitous in the saliva of black flies, although activity per gland varies by species and has a positive association with anthropophagy. Anticoagulants target components in the final common pathway of the coagulation cascade, including factors V, Xa, and II (thrombin). The antithrombin salivary protein may exert a redundant effect by inhibiting the role of thrombin in platelet aggregation. Antithrombin presence and activity also varies among black fly species, and exhibits a positive correlation with zoophagy. Vasodilation of capillaries to increase blood supply to the feeding wound appears to be an important requirement for Simulium spp., because substantial erythema-inducing activity, has been demonstrated in salivary glands of all New World species examined. Salivary glands of Simulium ochraceum (Walker), a highly anthropophilic vector of Onchocerca volvulus (Leuckhart), contain greater vasodilator activity than several other species, including S. metallicum Bellardi, a secondary zoophagic vector of human onchocerciasis. Simulium vittatum Zetterstedt saliva affects immune cell responses and cytokine production. The ability of the saliva to modulate components of the host immune system provides an opportunity for enhancing transmission of pathogens during bloodfeeding. Thus, the likely possibility that effective pathogen transmission relies on vector saliva may complement present efforts aimed at target epitopes of O. volvulus or identify additional molecules to be investigated as part of a "river blindness" vaccine cocktail. Components in saliva also may enhance the transmission of other microbial agents either by a cofeeding process similar to that observed in ixodid ticks or through rupture of the labrum during escape of Onchocerca infective stage larvae. In a few instances, saliva of some Simulium spp. also has been associated with extensive tissue and organ pathology, including hemorrhagic shock and death. Pathologic signs associated with this syndrome indicate an enhanced antihemostatic activity in saliva.

Cellular hemolymph response of Simulium vittatum (Diptera:Simuliidae) to intrathoracic injection of Onchocerca lienalis (Filarioidea:Onchocercidae) microfilariae.

Hemolymph cellular changes in Simulium vittatum Zetterstedt in response to intrathoracic injection with Onchocerca lienalis Stiles were characterized by increased numbers of tissue fragments that contained cells embedded within a noncellular matrix. Cell numbers within the matrix fragments increased both for sham and microfilariae-injected files, indicating a blastogenic response to injection alone. Morphological characteristics of fixed, Giemsa-stained cells within these tissue fragments were most similar to those previously described for prohemocytes. The total population of freely circulating hemocytes collected 24 h after injection also responded to injection alone. Differential cell counts showed a complex pattern of changes that was influenced strongly by increased numbers of prohemocytes at 24 h in microfilariae-injected flies. Fat body fragments collected in hemocoel perfusates at 24 h were fewer when flies were maintained at 27 degrees C than at 21 degrees C. More fat body fragments were collected from microfilariae-injected flies than from control and sham-injected flies held at 27 degrees C. Injection of 1.25-5 micrograms of lipopolysaccharide per fly did not elicit similar hemolymph-associated matrix tissue and fat body changes, indicating that S. vittatum response to microfilaria infection and bacteria infection are likely to involve different mechanisms.

Novel anticoagulant from salivary glands of Simulium vittatum (Diptera: Simuliidae) inhibits activity of coagulation factor V.

Anticoagulant activity was detected in fractions of a reversed phase-high-performance liquid chromatography (RP-HPLC) of salivary gland lysate from Simulium vittatum Zetterstedt. Using a plasma recalcification time assay, these fractions did not inhibit factor Xa or thrombin. HPLC-purified fractions showing the anticoagulant property were pooled and examined using the activated partial thromboplastin time test conducted on normal plasma and plasmas deficient in factors V, VIII, IX, XI, and XII. The anticoagulant prolonged the clotting time of all the plasmas, except plasma deficient in factor V. The detection of antifactor V activity, together with other anticoagulants reported from Simulium spp. indicates a feeding strategy that targets enzymes in the terminal portion of the coagulation cascade.

Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status.

Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum, a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 +/- 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 +/- 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 +/- 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S. (Ps.) ochraceum (24.8 +/- 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum, a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S. (N.) exiguum (10.9 +/- 0.6 mU) than in Simulium (Notolepria) gonzalezi (5.9 +/- 1.9 mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O.volvulus.

Analysis of migration success of Onchocera lienalis microfilariae in the haemocoel of Simulium vittatum.

Migration success (i.e. the proportion of worms that reach the thorax) of Onchocerca lienalis microfilariae (mf) in the haemocoel of Simulium vittatum was studied by inoculating mf into the posterior abdomen, and recording their distribution in the blackfly body at predetermined time points. Mf arrive into the thorax by active locomotion rather than by drifting in haemolymph currents. Migration into the thorax was completed by 12 h post inoculation (pi) but was not continuous throughout this period. Migration proceeded in two phases; the first occurred 0-2 h pi and the second at 6-12 h pi. Overall, migration success 12-24 h pi was only 36%, indicating that a substantial number of mf failed to reach the thorax, either because they were eliminated by the fly's defensive response or because they remained in the abdomen. Migration success was density independent. Mf that arrive into the thorax within 2 h pi did not differ in their migration potential from mf that remained in the abdomen at this time. In flies where more mf migrated successfully there was lower mf loss, indicating that migration success was linked to mf loss. Moreover, the proportion of mf in the thorax was not correlated with mf loss, suggesting that mf loss affected the number of mf that migrated successfully, rather than the reverse causal relationship.

Anticoagulant activity in salivary gland extracts of black flies (Diptera: Simuliidae).

Anticoagulant activity was determined in salivary gland extracts from four species of black flies, i.e., Simulium vittatum Zetterstedt, Simulium argus Williston, Simulium metallicum Bellardi, and Simulium ochraceum Walker. Inhibition of coagulation factor Xa occurred among all four, whereas thrombin inhibition was detected in S. argus and S. vittatum only. Both bovine and human alpha-thrombins were inhibited with the highest activity occurring with S. argus salivary gland extracts. Factor Xa inhibition was highest in S. ochraceum, an anthropophilic species and vector of Onchocerca volvulus, and lowest in S. vittatum, a primiparous autogenous species that is also zoophilic. Total soluble salivary gland extract protein also varied among the four species with the highest concentration measured in S. ochraceum and the lowest in S. vittatum. A positive correlation was observed between the amount of soluble protein and percentage of inhibition of factor Xa for the four species.

Onchocerca lienalis: rapid clearance of microfilariae within the black fly, Simulium vittatum.

A rapid decrease of about a third of the number of Onchocerca lienalis microfilariae (mf) parenterally inoculated into Simulium vittatum black flies occurred within 5 hr postinoculation (pi). The change of mf counts over time was modeled by a segmented linear regression. During 2 hr pi the slope was -3.5 mf/hr (P < or = 0.001) and between 2 and 24 hr pi the slope was -0.1 mf/hr. Although significantly different from the former slope (P < 0.001), the latter was not significantly different from zero (P > 0.2). The decrease could not be attributed to excretion of mf. Microfilariae (especially those heat-killed prior to inoculation) in intermediate stages of destruction were observed in flies dissected 5 hr pi but not immediately after injection. No short- or long-term (24 hr pi) effects of the injection procedure alone on mf survival were evident. A constant proportion of mf was eliminated regardless of dose within a range of 5 to 100 mf/fly during 24 hr pi. However, a second injection of 50 mf/fly 2.5 hr following an injection of the same dose resulted in a significantly lower proportion of mf eliminated. These results suggest that the availability of an active factor(s) in the fly was reduced 2.5 hr after the first inoculation. The change in the availability of this factor(s) may partly explain the change in clearance rate occurring 2 hr pi. Soluble factor(s), rather than a sequence of cellular responses, seems to be involved in the rapid clearance because it occurred in freshly killed flies at a similar rate to that observed in live flies. The hypothesis that mf differ in their innate susceptibility to rapid clearance was rejected as mf that were recovered 2 hr pi and reinoculated into other flies were eliminated faster than unexposed controls. It is concluded that the rapid clearance of mf represents an as yet undescribed immune response to macroparasites of the fly host.

Salivary apyrase in African and New World vectors of Plasmodium species and its relationship to malaria transmission.

The salivary gland activities of apyrase, an enzyme that prevents platelet aggregation by eliminating ADP, were compared among five members of the Anopheles gambiae species complex and An. albimanus. Within the An. gambiae group, An. quadriannulatus exhibited the lowest amount of enzyme activity at all pH levels measured. Apyrase activity could be separated into three groups at pH 7.5 and 8.0. The two most anthropophilic species (An. gambiae and An. arabiensis) exhibited higher activity at pH 9.0. Anopheles merus and An. melas, both saltwater taxa, and An. albimanus, a New World species, exhibited peak apyrase activity at pH 8.0. When the effects of divalent cations (Ca++, Mg++) on enzyme activity were compared at pH 8.5, apyrase activity in the presence of Mg++ could be separated into three levels. Anopheles gambiae and An. quadriannulatus exhibited reduced activity in the presence of Mg++. Anopheles arabiensis, An. merus, and An. melas displayed the highest relative levels of activity. Anopheles albimanus, with a Mg:Ca ratio of 0.80, was most similar to An. arabiensis. These biochemical differences suggest that different isoenzymes of apyrase have developed within the genus Anopheles.

Vasodilative activity in black fly salivary glands.

Salivary gland extracts of several Simulium spp. were shown to contain vasodilative activity as measured by the rapid and persistent induction of erythema in response to intradermal injection into rabbit skin. Total salivary gland activities were approximately equal for S. vittatum, S. metallicum, S. bivittatum, and S. argus (titers of 0.03-0.02 pairs of gland). Total gland activity in the highly anthropophilic species S. ochraceum, however, was an order of magnitude greater, with erythema produced by as little as 0.002 pairs of glands. Tests for physical stability of the activities from two species (S. vittatum and S. ochraceum) indicated that the vasodilators were proteinaceous and heat stable. A two-step, reversed-phase high-performance liquid chromatography (HPLC) procedure was developed that isolated both activities with similar elution patterns. Homogeneity of the purified protein from S. vittatum was confirmed by capillary gel electrophoresis. Electrospray ionization mass spectroscopy of the S. vittatum protein detected a mass of 15,351 daltons. Similarity in elution times of the proteins from a TSK HPLC column predict some structural similarities between the S. vittatum and S. ochraceum vasodilator proteins.

Modulation of murine immunological responses by salivary gland extract of Simulium vittatum (Diptera: Simuliidae).

The influence of Simulium vittatum Zetterstedt salivary gland extract on several immunological mechanisms was investigated in murine model hosts (laboratory mice). These mechanisms included the expression of major histocompatibility complex class II cell surface molecules, the in vitro mitogen responsiveness of lymphoid cells, and the antibody responses to heterologous foreign antigens (sheep erythrocytes). Experiments were designed to determine the influence of salivary gland extract following in vivo inoculation or in vitro inclusion in cell culture. In vivo inoculation of salivary gland extract reduced the percentage of Ia+ cells in spleen cell populations, although this difference was ameliorated by a 2 d in vitro culture period, regardless of whether salivary gland extract was included in culture. Salivary gland extract had no effect on Ia expression by cells derived from regional lymph nodes or the skin (epidermis). In vivo inoculation with salivary gland extract did not affect the responsiveness of splenic lymphocytes to mitogens, whereas in vitro exposure to salivary gland extract reduced both T and B cell mitogenesis. Finally, antibody responses to sheep erythrocytes were enhanced if salivary gland extract was included as a coinoculant, although this was expressed only at the systemic level regardless of the route of antigen delivery. In light of these results, immunomodulatory functions of black fly saliva are postulated; they are operative at different levels on different subcompartments of the immune system, possibly via cytokine modulation.

Antibody responses of BALB/c mice to salivary antigens of hematophagous black flies (Diptera: Simuliidae).

The humoral antibody responses to salivary antigens of Simulium vittatum Zetterstedt were investigated in a BALB/c mouse laboratory model. Production of antisera was stimulated by intraperitoneal immunization with salivary gland extract or by feeding flies directly on depilated mice. Antibody responses in these two groups of mice were compared by western blotting, thus characterizing "true" salivary immunogens present in salivary gland extract. Immunized mice developed IgG, IgM, and IgE antibodies which recognized several salivary gland components, ranging in molecular weight between 26 and 67 kDa. Sera from bitten mice recognized fewer antigens, indicating that some components of the salivary gland extract were poorly immunogenic or absent from the saliva secreted during blood feeding. Antisera raised against S. vittatum also were used to identify cross-reactive immunogens and allergens in salivary gland extracts from other New World simuliids (Simulium argus Williston, S. metallicum Bellardi, and S. ochraceum Walker). SDS-PAGE protein profiles indicated a high degree of similarity between salivary gland extract of S. vittatum and S. argus, and several cross-reacting antigens were identified by western blotting. In contrast, protein profiles of S. ochraceum and S. metallicum differed from the former species, both qualitatively and quantitatively. Antisera demonstrated a low degree of cross-reactivity against salivary gland extract of S. ochraceum, whereas no cross-reactivity was detected against S. metallicum. These observations were confirmed using a monoclonal antibody raised against S. vittatum salivary gland extract (designated SVSG.1.F10), which showed cross-reactivity against S. argus but failed to recognize salivary gland components of either S. ochraceum or S. metallicum.

Ivermectin: reduction in prevalence and infection intensity of Onchocerca volvulus following biannual treatments in five Guatemalan communities.

Residents of five hyperendemic communities located in the central focus of onchocerciasis in Guatemala were treated with ivermectin (Mectizan) or placebo every six months for 30 months. The effects of treatment on prevalence and the intensity of skin infection (microfilarial skin density [MFD]) were evaluated. Significant and persistent reductions in both of these indices were achieved by coverage of 80.7% of the eligible populations. The highest proportionate reductions in both indicators of infection occurred after the first treatment, followed by more gradual decreases through the fourth treatment. In one community in which the mean coverage was 92.7%, prevalence decreased from 74.0% at pretreatment to 34.9% after four treatments, while the MFD decreased from 7.8 to 2.0; reductions of 52.8% and 74.3% from pretreatment values, respectively. In every ivermectin-treated community except one, in which drug acceptance was low, the mean community MFD values were reduced to the level associated with low infectiousness for the vector, Simulium ochraceum. Moreover, the category of MFD associated with high vector infectiousness was reduced at least ten-fold over the pretreatment level. One community had low participation during the first two treatments (32.8% and 22.7% of those eligible). This increased to 55.2% at the third treatment because of implementation of an educational program describing both the disease and the beneficial effects of ivermectin and because skin biopsies and nodulectomies were not performed. Secondary reaction rates for all communities were 29.5%, 9.9%, 10.3%, 8.2%, and 7.1% for the first through fifth treatments, respectively. Pruritus was the most common (34.0%) secondary reaction, followed by facial edema (31.8%). All reactions were classified as mild to moderate. Recommendations for mass distribution of ivermectin in Guatemala are given.

The effects of repetitive community-wide ivermectin treatment on transmission of Onchocerca volvulus in Guatemala.

The effects of biannual ivermectin treatment at the community level on transmission of Onchocerca volvulus during the dry season were measured over a 30-month period in Guatemala. In the Los Tarrales Transmission Zone, an area encompassing three villages, significant changes occurred in both the prevalence and quantity of infection in the Simulium ochraceum vector population. These included a 76% reduction in females with infective stage larvae (L3S) and an 80% reduction in number of L3S per 1,000 parous flies. Significant reductions in both the mean infective biting density (IBD) and mean transmission potential (TP) also occurred. In Santa Emilia, the prevalence of infection with L3S in S. ochraceum was significantly reduced by 77% from the baseline value. The number of O. volvulus L3S per 1,000 parous flies was also reduced by 92%. Changes in both the IBD and TP were substantial but not significant due to the high degree of variance in the occurrence of O. volvulus L3S in the vector population. This was due, in part, to the movement of infected migrant workers into the finca (coffee farm). In Los Andes, four recurrent treatments successfully blocked transmission of infective stage larvae. Prevalence (flies with all stages of developing larvae) in the vector population was reduced by 89% over the two-year period; yearly reductions in both the IBD and TP were also highly significant, ultimately ending in zero values. This finding is particularly striking since prior to treatment, Los Andes exhibited the highest IBD of the three study locations and the second highest TP.