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Onchocerciasis is a devastating skin and eye disease that afflicts about 21 million people, most of whom live in sub-Saharan Africa. Its control with the microfilaricidal drug ivermectin is limited, thus necessitating the development of preclinical animal models to aid in the discovery of a macrofilaricide. Previously, we found that Onchocerca ochengi (the closest relative of the human O. volvulus) worm masses survive better in hamsters than in gerbils. The aim of this study was to compare the survival of O. ochengi adult male worms and their susceptibility to flubendazole (FBZ, a macrofilaricide) in gerbils and hamsters. The animals were intraperitoneally implanted with O. ochengi male worms, treated with FBZ, and sacrificed 35 days post-implantation. Unlike gerbils which had some worms moving freely in the peritoneum and some in newly formed nodules (neo-nodules), all the worms in the hamsters were found in neo-nodules. FBZ significantly decreased worm burden, motility, and viability in gerbils whereas it had no significant effect in hamsters. These results highlight a major difference in how O. ochengi adult male worms are sustained and affected by FBZ in gerbils compared to hamsters. Understanding the difference between these two models is important in the development of effective macrofilaricides for onchocerciasis. [ABSTRACT FROM AUTHOR]

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases caused by filarial nematodes that utilize insect vectors for transmission to their human hosts. Current control strategies are based on annual or biannual mass drug administration (MDA) of the drugs Ivermectin or Ivermectin plus Albendazole, respectively. These drug regimens kill the first-stage larvae of filarial worms (i.e., microfilariae) and interrupt the transmission of infections. MDA programs for these microfilaricidal drugs must be given over the lifetime of the filarial adult worms, which can reach 15 years in the case of Onchocerca volvulus. This is problematic because of suboptimal responses to ivermectin in various endemic regions and inefficient reduction of transmission even after decades of MDA. There is an urgent need for the development of novel alternative treatments to support the 2030 elimination goals of onchocerciasis and lymphatic filariasis. One successful approach has been to target Wolbachia, obligatory endosymbiotic bacteria on which filarial worms are dependent for their survival and reproduction within the human host. A 4–6-week antibiotic therapy with doxycycline, for example, resulted in the loss of Wolbachia that subsequently led to extensive apoptosis of somatic cells, germline, embryos, and microfilariae, as well as inhibition of fourth-stage larval development. However, this long-course regimen has limited use in MDA programs. As an alternative approach to the use of bacteriostatic antibiotics, in this study, we focused on autophagy-inducing compounds, which we hypothesized could disturb various pathways involved in the interdependency between Wolbachia and filarial worms. We demonstrated that several such compounds, including Niclosamide, an FDA-approved drug, Niclosamide ethanolamine (NEN), and Rottlerin, a natural product derived from Kamala trees, significantly reduced the levels of Wolbachia in vitro. Moreover, when these compounds were used in vivo to treat Brugia pahangi-infected gerbils, Niclosamide and NEN significantly decreased adult worm survival, reduced the release of microfilariae, and decreased embryonic development depending on the regimen and dose used. All three drugs given orally significantly reduced Wolbachia loads and induced an increase in levels of lysosome-associated membrane protein in worms from treated animals, suggesting that Niclosamide, NEN, and Rottlerin were effective in causing drug-induced autophagy in these filarial worms. These repurposed drugs provide a new avenue for the clearance of adult worms in filarial infections. [ABSTRACT FROM AUTHOR]

Background: Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease. Methodology/Principal findings: Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay. Conclusions/Significance: Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays. Author summary: Onchocerca volvulus is a nematode parasite that is the causative agent of river blindness in Africa, the Americas and Yemen and is transmitted by blackflies of the genus Simulium. Efforts to reduce and eliminate this neglected tropical disease focus on mass drug administration (MDA) with ivermectin and require reliable and accurate diagnostic assays to monitor the progress of once or twice-yearly treatments. One important measure of the impact of MDA is to screen pools of vector flies collected in endemic areas for the presence of L3-stage (infective) larvae. Since dissection of individual flies and microscopic examination to search for larvae in the flies is very time-consuming and results in many false negative and false positive results (due to morphologically similar larvae of closely related animal parasites), DNA-based PCR assays have proven extremely useful. An existing PCR-ELISA method based on amplification and detection of the O150 repeat found in the parasites has proven very useful over the last 25 years. An updated real-time qPCR-based method using a mitochondrial DNA target (OvND5) has now been developed that is more sensitive, more species specific, less-time consuming and less costly than the original O150 PCR-ELISA assay. This assay is ready for field evaluation. [ABSTRACT FROM AUTHOR]

Onchocerciasis, the second leading infectious cause of blindness, afflicts approximately 21 million people globally. Its control is limited to the use of the microfilaricidal drugs, ivermectin and moxidectin. Both drugs are unable to kill the adult worms which can survive for up to 15 years in patients, justifying the urgent need for potent and novel macrofilaricides that kill adult worms. The development of such drugs has been hindered by the lack of an appropriate small laboratory animal model to evaluate potential drug candidates in vivo. This study assessed the survival of O. ochengi female worms and their embryos over time in two laboratory rodents: gerbils and hamsters and tested using "proof-of-concept" studies, whether known macrofilaricidal drugs can kill these worms. Animals were surgically implanted with mechanical or collagenase-liberated O. ochengi worm masses, and necropsied at various time points to test for survival. Recovered worm masses were assessed for viability by biochemical analysis (MTT/formazan assay) or fecundity (embryogram). Flubendazole (FBZ) administered at 20 mg/kg body weight was used to validate both rodent models. By day 26 post-implantation of 15 worm masses, a median of 7.00 (4.00–10.00) was recovered from hamsters, and 2.50 (2.00–4.00) from gerbils. Worm masses recovered from gerbils were mostly disintegrated or fragmented, with significantly higher fragmentation observed with collagenase-liberated worm masses. FBZ had no significant effect on the number of worm masses recovered, but enhanced embryo degradation in gerbils and reduced worm mass viability in hamsters. This exploratory study has revealed the gerbil and hamster as permissible rodents to adult female worms of O. ochengi. The hamsters appeared to maintain the worms longer, compared to gerbils. [ABSTRACT FROM AUTHOR]

Background: Onchocerciasis (river blindness) caused by the filarial worm Onchocerca volvulus is a neglected tropical disease that affects the skin and eyes of humans. Mass drug administration with ivermectin (IVM) to control the disease often suffers from severe adverse events in individuals co-injected with high loads of Loa loa microfilariae (mf). Thus loiasis animal models for counter-screening of compounds effective against onchocerciasis are needed, as are the corresponding onchocerciasis screening models. The repertoire of such models is highly limiting. Therefore, this study was aimed at developing and validating mf immunocompetent small animal models to increase tools for onchocerciasis drug discovery. Methodology/Principal findings: O. ochengi mf from cattle skin and L. loa mf from human blood were used to infect BALB/c mice and Mongolian gerbils, and IVM was used for model validation. O. ochengi mf were given subcutaneously to both rodents while L. loa mf were administered intravenously to mice and intraperitoneally to gerbils. IVM was given orally. In an 8-day model of O. ochengi mf in BALB/c mice, treatment with IVM depleted all mf in the mice, unlike the controls. Also, in a 2.5-day model of L. loa mf in BALB/c, IVM significantly reduced mf in treated mice compared to the untreated. Furthermore, the gerbils were very susceptible to O. ochengi mf and IVM eradicated all mf in the treated animals. In the peritoneal L. loa mf gerbil model, IVM reduced mf motility in treated animals compared to the controls. In a 30-day gerbil co-injection model, IVM treatment cleared all O. ochengi mf and reduced motility of L. loa mf. Both mf survived for up to 50 days in a gerbil co-injection model. Conclusions/Significance: We have developed two immunocompetent small animal models for onchocerciasis and loiasis that can be used for microfilaricide discovery and to counter-screen onchocerciasis macrofilarides Author summary: Onchocerciasis (river blindness) caused by the filarial worm Onchocerca volvulus is a severe skin disease and affects the eyes leading to vision loss. Drugs that are effective against O. volvulus should be ineffective against another filarial worm Loa loa, as severe adverse events has been reported in treated co-infected individuals. Thus, animal models are needed to screen for compounds effective against onchocerciasis but not against the Loa loa microfilariae (mf). O. ochengi mf from cattle skin and L. loa mf from human blood were used to infect BALB/c mice and Mongolian gerbils, and ivermectin (IVM) was used for model validation. O. ochengi mf survived both animals and treatment with IVM significantly reduced the number of mf. In a BALB/c L. loa mf model, IVM significantly reduced mf in treated mice compared to the untreated. L. loa mf survived in peritoneum of gerbils. A gerbil co-injection model (with both O. ochengi and L. loa mf) was developed and both mf survived for up to 50 days in this model. In conclusion, we have developed two immunocompetent small animal models for onchocerciasis and loiasis that can be used for microfilaricide discovery. [ABSTRACT FROM AUTHOR]

Onchocerciasis and lymphatic filariasis are neglected tropical diseases caused by infection with filarial worms. Annual or biannual mass drug administration with microfilaricidal drugs that kill the microfilarial stages of the parasites has helped reduce infection rates and thus prevent transmission of both infections. However, success depends on high population coverage that is maintained for the duration of the adult worm's lifespan. Given that these filarial worms can live up to 14 years in their human hosts, a macrofilaricidal drug would vastly accelerate elimination efforts. Here, we have evaluated the repurposed drug pyrvinium pamoate as well as newly synthesized analogs of pyrvinium for their efficacy against filarial worms in vitro and in vivo. We found that pyrvinium pamoate, tetrahydropyrvinium and one of the analogs were highly potent in inhibiting worms in in vitro whole-worm screening assays, and that all three compounds reduced female worm fecundity and inhibited embryogenesis in the Brugia pahangi-gerbil in vivo model of infection. [ABSTRACT FROM AUTHOR]