Survival of adult female worms of Onchocerca change in gerbils and hamsters: implications for the development of an in vivo macrofilaricide screening model

doi:10.21203/rs.3.rs-2248793/v1

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Survival of adult female worms of Onchocerca change in gerbils and hamsters: implications for the development of an in vivo macrofilaricide screening model

https://doi.org/10.21203/rs.3.rs-2248793/v1

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published 08 May, 2023

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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 mired by the lack of an appropriate small laboratory animal model to evaluate potential drug candidates in vivo. This study assessed the survival of O. change 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 enzyme-liberated O. change female worms, and sacrificed at various time points to test for survival. Recovered worms 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, 58.6 ± 7.5% female worms were recovered from hamsters, and 20 ± 3.5% from gerbils. Those recovered from gerbils were mostly disintegrated or fragmented, with significantly higher fragmentation observed with enzymatically-liberated worms. FBZ had no significant effect on the number worms recovered, but enhanced embryo degradation in gerbils and reduced worm viability in hamsters. This exploratory study has revealed the gerbil and hamster as permissible rodents to adult female worms of O. change. The hamsters appeared to maintain the worms longer, compared to gerbils.

Onchocerciasis

animal models

macrofilariae

hamster

gerbil

Human onchocerciasis (river blindness) is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus (Etya’alé 2002; O'Neill 1875). Since its discovery in 1874 (O'Neill 1875), this disease has continued to plague people predominantly living in sub-Saharan Africa, maintaining its place as the second leading infectious cause of blindness globally (Boatin and Richards 2006). Immune host responses to antigens of the parasite microfilariae (mf), antigens of the adult worms or the parasite alone, result in various pathologies. These include dermatitis, atrophy of the skin, visual impairment, and blindness (Ward et al. 1988; Wu et al. 2003). The O. volvulus parasite has also been shown to exist in an endosymbiotic relationship with bacteria belonging to the genus Wolbachia (Kozek and Marroquin 1977). These bacteria contribute to the clinical presentation of filarial infections, since bacterial products released from both living and dead worms activate the mammalian innate immune system (Keiser et al. 2002; Landmann et al. 2010). Host responses to Wolbachia antigens have been associated with the development of chronic eye pathology in humans (Saint André et al. 2002; Tamarozzi et al. 2014).

For over 30 years, ivermectin mass drug administration (MDA) has been the principal mode of treatment (Lawrence et al. 2015), with remarkable success in reducing the public health burden of both ocular and dermal manifestations of the disease (APOC 2015). However, ivermectin is only microfilaricidal (Katabarwa et al. 2011) and, induces life-threatening complications in onchocerciasis individuals with concomitant Loa loa infection (Gardon et al. 1997; Metzger and Mordmüller 2014), and possible resistance may have emerged in some areas in Ghana and Cameroon (Umeh et al. 2010). This indicates the drug’s inability to completely break the transmission cycle. As of June 2018, moxidectin at 8 mg/kg body weight oral dose was approved for the treatment of onchocerciasis in patients aged 12 years and older (Keller et al. 2019; Maheu-Giroux and Joseph 2018). Although only microfilaricidal as ivermectin, moxidectin appears to be safer, requiring a single annual dose as opposed to the biannual administration for ivermectin. Ivermectin and moxidectin fall short of achieving the goal of eliminating human onchocerciasis by 2030 (mondiale de la Santé and Organization 2019). It is therefore unlikely that eradication or even stable elimination can be achieved globally without a safe and effective macrofilaricide (Geary and Mackenzie 2011). The potential development of such a macrofilaricide is mired by the lack of an appropriate small animal pre-clinical laboratory model to evaluate drug candidates in vivo (Cupp et al. 2011). O. volvulus does not infect animals other than humans and chimpanzees, and acquiring the large numbers required for routine in vitro screening will be otherwise invasive. Consequently, other closely related Onchocerca species have been used as substitutes for chemotherapeutic research. So far, the African bovine species, O. change has been most frequently used for such purposes, as well as Brugia and Litomosoides species amongst others (Renz et al. 1995; Trees 1992). Apart from being the closest related species to O. volvulus, O. change shares the same vector, Simulium and is common in cattle in Northern Cameroon (Wahl 1996; Wahl et al. 1994; Xie et al. 1994).

Rodents have been in use for in vivo drug or medical research for centuries (Tendongfor et al. 2012; Townson et al. 1988). This has helped the understanding of both the host-parasite relationship and the causes of disease. The Mongolian gerbil (Meriones unguiculatus) has been known to have a uniquely high susceptibility to various parasitic infections, including filariasis (Cho-Ngwa et al. 2019; Shimizu-Suganuma et al. 2000) and is currently used as a model for Brugia and Litomosoides sigmodontis (Mutafchiev et al. 2014; Shimizu-Suganuma et al. 2000). Furthermore, the gerbil was recently shown to harbor adult male worms of O. change with high viability 35 days post-implantation (Cho-Ngwa et al. 2019; Halliday et al. 2014). The Syrian hamster (Mesocricetus auratus) is equally a long standing definitive model of human tapeworms (Taenia solium and T. saginata) (Verster 1971). Different mice species have also been used extensively in the above-mentioned animal model studies (Bianco et al. 1986; Folkard et al. 1997; Townson and Bianco 1982). In this study, we therefore sought to exploit the potential susceptibility of gerbils and hamsters to O. change and to contribute to developing and validating an appropriate Onchocerca small animal model for in vivo macrofilaricide screens.

Ethics statement

The animal protocol (UB-IACUC N^o 002/2017) was approved by the University of Buea Animal Care and Use Committee, following recommendations from the ‘Guide for the Care and Use of Laboratory Animals’, 8th edition by the National Research Council, USA. This in vivo study was reported in accordance with the ARRIVE Guidelines for reporting animal research.

Experimental animals

Male and female Mongolian gerbils and Syrian hamsters were purchased from Charles River (France). These animals were maintained in a conventional animal house at the Biotechnology Unit of the University of Buea until they were at least 8 weeks old. Animals were given food and water ad libidum. Based on Clincalc sample size calculator, at least 5 animals were used per group (except during preliminary stages), and each experiment was repeated at least once. The animals were only handled by trained personnel.

Isolation of O. change adult female worms from cattle skin

Worms were isolated from umbilical cattle skin as previously described (Cho-Ngwa et al. 2010). Fresh pieces of cattle skin with palpable nodules were purchased from the main slaughter house in Douala, washed repeatedly with tap water and rinsed with distilled water. The skin was towel-dried, sterilized with 70% ethanol and allowed to dry in a laminar flow hood. After that, the nodules (pale orange-yellow O. change adult worms) were either mechanically- or enzymatically-liberated.

Mechanical liberation of adult female worms: Using a razor blade, worm masses were excised with care to not hurt the adult worm. The entire mass was subsequently submerged in incomplete culture medium (ICM): RPMI-1640 (SIGMA, USA), supplemented with 25 mM HEPES, 2 g/L sodium bicarbonate, 2 mM L-glutamine, 200 units/ml penicillin, 200 µg/ml streptomycin and 0.25 µg/ml amphotericin B, pH 7.4 in 24-well tissue culture plates. Damaged worms and worms from putrefied nodules were discarded. The plates containing the worms were then incubated at 37°C in humidified air with 5% CO_2, in a HERACELL-150 incubator overnight.

Enzymatic liberation of adult female worms

Using a razor blade, nodules were carefully excised and introduced in 50 ml falcon tubes containing collagenase B (Sigma Aldrich™, Germany). Collagenase B was dissolved in complete culture medium (CCM; ICM plus 5% heat-inactivated fetal bovine serum) at a concentration of 0.5 mg/ml, and sterile-filtered. The tubes containing the worms were incubated at 37°C overnight with gentle agitation. Thereafter, the worms were rinsed at least 7 times with copious amounts of ICM. The worms were allowed to incubate for 15 min at 37°C in humidified air with 5% CO₂, in a HERACELL-150 incubator, and observed microscopically.

Implanting isolated female worms into gerbils and hamsters

Animals were sedated with ketamine/xylazine at 80/5 mg/kg and 100/5 mg/kg body weight for gerbils and hamsters, respectively. The abdominal area of each animal was shaved and sterilized with 95% ethanol. Using a size 23 sterile surgical blade (Everich, China), a small incision (~ 1 cm) was made on the skin and peritoneal membrane of each animal. Implanting one female worm at a time, at least 3 and at most 15 worms were blotted on absorbent paper (to decrease any serum carryover from CCM) and implanted carefully in the peritoneum of the respective animals. Using a sterile nylon monofilament non-absorbable suture needle (Medtrue Enterprise, Nigeria), interrupted stitches were made. The peritoneal membrane was stitched separately from the skin, to avoid worm displacement from the peritoneum. To mimic what occurs in natural infections, some implantations were done subcutaneously in hamsters, sedated as indicated above. The skin at the nape of the neck was sterilized, shaved, cut opened and worms implanted, and the area stitched (as described above). The stitched area (abdominal or nape of the neck) was sterilized with 95% ethanol; animals were placed in cages containing freshly sterilized beddings and monitored daily. Daily monitoring involved continuous ethanol sterilization of the sutured area until complete drying/healing of wound.

Drug administration

Animals implanted with adult worms were numbered, and received either flubendazole (FBZ-cat# 34091; Sigma-Aldrich, Germany) subcutaneously (sc) at a dose of 20 mg/kg body weight once daily (SID, for gerbils) or twice daily (BID, for hamsters) or vehicle (0.5% HEC (hydroxyethyl cellulose), 0.1% Tween 80, and 99.4% water). Animals were treated for 7 days beginning 3 days after adult worm implantation.

Recovery of O. change adult female worms from gerbils and hamsters

Animals were sacrificed via cervical dislocation between 7- and 60-days post-implantation of O. change adult female worms, and each peritoneum was washed with ICM (the animals were coded during sacrifice and assaying, and only decoded during analyses). Using an inverted microscope, peritoneal wash was analyzed for the presence of O. change mf. The skin and peritoneal membrane at the site of implantation were chopped and incubated in ICM at 37°C for 4 hours, including the shaved and properly chopped earlobes of each animal. Incubation was done in 6-well tissue culture plates. After incubation, the medium was observed (Nikon Eclipse TS100) for the presence of O. ochengi mf in 5 separate portions of 100 µL each carefully spread on petri dishes. However, realising that mf were not consistent in the skin, mf detection in the skin was discontinued. Efforts were made to ensure that the gerbils and hamsters were sacrificed about the same time on the endpoint date, as the time when they were implanted.

Eventually, only the peritoneum of each animal was cut wide open and thoroughly observed for the presence of male and/or female adult worms. Male and female nodules obtained or carefully excised from the animal were placed in collagenase B solution at a concentration of 5 mg/ml and incubated for at least 2 hours at 37°C with gentle agitation.

Recovered female worms/nodules were incubated in 15 ml falcon tubes with each tube corresponding to one animal. After incubation, all female worms were thoroughly washed with copious amounts of ICM, and each worm introduced into a well containing fresh 1 ml CCM. Both male and female worms recovered were observed under an inverted microscope (Nikon Eclipse TS100) and their motility scores, presence of uterine mf, periplasmic streaming, and general health state recorded. The collagenase media from which worms were retrieved, including the media used for washing the female worms, were also checked for mf under the microscope (Nikon Eclipse TS100).

Assessing the viability of worms recovered from gerbils and hamsters

Worm viability was assessed based on 2 main methods; estimation (visual and optical density) of percentage inhibition of formazan (blue color) formation (MTT assay) or the embryogram.

MTT/Formazan assay

The viability of female worms was assessed based on visual estimation of the percentage inhibition of formazan (blue color) formation. This was done by incubating the nodules in 500 µl of 0.5 mg/ml MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] for 30 minutes, removing the worms, blotting with absorbent paper and taking electronic photographs against a white background. Viability scores ranged from 0% (no blue coloration seen), 25% inhibition of formazan, 50% inhibition of formazan, 75% inhibition of formazan, 90% inhibition of formazan to 100% (entire worm appeared blue) (see Fig. 1).

To further investigate the viability of female worms, each worm was weighed (mg), the formazan dissolved in DMSO (by incubating the worms at 37°C in 200 µl of DMSO for 30 minutes), and the optical density measured at 595 nm (OD₅₉₅) using a microplate reader (Molecular Devices, USA). The final value was obtained by dividing each OD₅₉₅ reading by the weight of the worm (mg).

The embryogram

Worms were crushed in 1x PBS, spread on petri dishes (100 µL portions) and viewed under an inverted microscope (Nikon Eclipse TS100). Using already existing reference graphic developmental embryonic stages (Schulz-Key 1988), different stages of the embryo observed were counted and recorded. At any given point, the aim was to assay the embryo in terms of percentages (number of a given stage present for every 100 embryos counted).

Statistical analyses

Data were analyzed using GraphPad Prism 5.0 software and statistical analyses performed using Mann-Whitney U or Kruskal-Wallis tests and Dunn’s post-test. Where applicable, the Chi-squared test, as well as the one-way ANOVA and Bonferroni post-test were used. Data presented shows mean ± SEM. Data were considered statistically significant when p < 0.05.

The Mongolian gerbil can support a short-term host environment for O. change adult female worms

Figure 2 Recovery of O. ochengi female worms from gerbils. a) Mechanically-liberated female worms before implantation. b) Enzymatically-liberated worms before implantation. c) Onchocermata (black arrow) showing vascularization (arrow head) on day 7 in gerbils. d) Disintegrated adult females after enzyme (collagenase) digestion on day 30 post-implantation (4x magnification).

Following implantation of adult female O. change worms, female nodules with thick and hardened (calcified) walls were recovered from the peritoneum of infected gerbils by day 7 (Fig. 2c). Oftentimes, nodules were attached to body fat around the stomach and large intestines. Newly formed blood vessels were also observed around nodules by day 7 (Fig. 2c). Repeated occurrences of female worm aggregates within a nodule were observed, which could only be separated after enzymatic digestion. Generally, worms from nodules excised from gerbils 20 days post-implantation and enzymatically liberated were found to be fragmented (Fig. 2d). This indicated decreased viability, and killing of implanted adult females over time (Fig. 3a-c). For intact (non-fragmented) worms, an average worm recovery of 20 ± 3.5% was recorded 26 days post-implantation (Fig. 3a). Female worms recovered from 17 of 18 gerbils analyzed for female worm survival on day 26, contained uterine mf. Interestingly, O. change mf were observed in the skin and peritoneal membrane as early as day 10. By day 42, motile mf were recovered in the earlobes of 50% of infected gerbils.

No significant difference was observed in the number and viability of female worms recovered (Fig. 4a-c) from the control and FBZ-treated groups. Oftentimes, male worms (which were probably embedded in female masses at time of implant) were equally recovered unaffected, except in case of one FBZ-treated gerbil in which male worms were either dead or far less viable, compared to those from control animals. There was a general tendency of more female worms from the control group to contain motile uterine mf, compared to those recovered from FBZ-treated gerbils (Fig. 4d). This was indicative of the effect of FBZ on embryogenesis, hence, the embryogram was used to further analyze recovered worms (Table 1). From the embryogram (Table 1), there were more degenerating embryos (95.18%) and fewer normal embryos (4.82%) in the FBZ-treated group, compared with the control group (90.21% and 9.79%, respectively). However, this was not significantly different.

Table 1

Effect of FBZ on embryogenesis in *O. change* female worms recovered from FBZ-treated gerbils. Embryogram showing the normal and degraded events of different *O. change* embryonic stages as percents.
Embryo stage	Recovery (%)
	Embryo state (normal or degenerating)	Control (vehicle-treated)	FBZ-treated (20 mpk, SID, sc, 7 days)
Large morula	Nor	0.93	0.64
	Deg	33.10	24.76
	Sub-total	34.03	25.40
Horseshoe	Nor	0.23	0
	Deg	5.59	0.96
	Sub-total	5.83	0.96
Pretzel	Nor	1.17	0
	Deg	12.82	36.66
	Sub-total	13.99	36.66
Mf	Nor	7.46	4.18
	Deg	38.69	32.80
	Sub-total	46.15	36.98
Total	Nor	9.79	4.82
Total	Deg	90.21	95.18
Nor = normal morphology; Deg = degenerating morphology; Total = sum of embryo with either normal (Nor) or degenerating (Deg) morphology

The Syrian hamster harbors more O. change adult females and for over 30 days

Intact female worms with thin-walled nodules were recovered from hamsters 7 days post-implantation (Fig. 5a). Similar to gerbils, nodules were often attached to body fat around the stomach and large intestines. Although not as prominent as observed in gerbils (Fig. 2d), newly formed blood vessels were observed around nodules by day 7 (Fig. 5a). It was repeatedly observed that female worms aggregated to form nodules and these aggregates could be separated after enzyme (collagenase) digestion or with the use of forceps. The average recovery of viable adult females was 58.6 ± 7.5% on day 26 (Fig. 6a and b). Generally, female masses excised from hamsters were viable (Fig. 5b), with periplasmic movement, motile uterine mf, and visible motile displacement of some females, extending till day 60 post-implantation.

Worms implanted subcutaneously disintegrated by day 15 hence this route of implantation was discontinued.

The hamster could be considered a better host to female worms compared to gerbils, based on the recovery of viable and sometimes partially motile female worms by day 60 post-implantation. In addition, tests with FBZ indicated similar results to what obtained with female worms from gerbils. Both recovery (Fig. 7a) and viability of female (Fig. 7b) worms had no significant difference between the control and FBZ-treated groups. However, OD values from the MTT showed significantly reduced worm viability upon FBZ treatment compared to the control group (Fig. 7c). No worm from either group (control or FBZ) had motile uterine mf.

The method of adult worm isolation only affects worm recovery in gerbils

The gerbils and hamsters were implanted with either mechanically- or enzymatically-liberated worms. In an effort to prevent possible wounding of worms that could occur during mechanical liberation, it was postulated that implanting enzymatically-liberated female worms would lead to better recovery. Paradoxically, worm recovery from gerbils with mechanically-liberated worms was significantly higher (p = 0.0128), compared to those recovered from gerbils with enzymatically-liberated worms (Table 2). Moreover, mf were only recovered from animals implanted with mechanically-liberated worms. Interestingly, worms which were mechanically-liberated before implantation formed more calcified nodules in gerbils, compared to those resulting from enzymatically-liberated worms.

Conversely, no significant difference was observed with worm recovery from hamsters, irrespective of the type of worm (enzymatically or mechanically-liberated) implanted (Table 2).

Table 2

The adult worm isolation method significantly affects worm recovery in gerbils but not hamsters. Average percent recovery of female worms from gerbils and hamsters, following the respective worm isolation method
Animal	Duration between worm implantation and worm recovery (days)	Average % recovery of female worms from animals implanted with either enzymatically- or mechanically- liberated worms		Level of significance based Mann-Whitney U test
Animal		enzymatically-liberated	mechanically-liberated	Level of significance based Mann-Whitney U test
Gerbils	35	0 ± 0	26.7 ± 4.6	p = 0.0128
Hamsters	35	23.2 ± 5.1	25.7 ± 10	p = 0.8590

The lack of a convenient small animal-Onchocerca model for macrofilaricide drug screens has been a major challenge in onchocerciasis drug discovery. The only short-term female worm model currently reported is the severe combined immune-deficient (SCID) mice which can harbor females of O. change, forming onchocermata analyzed on day 7 post-implantation (Halliday et al. 2014). This timing is too short to monitor slow acting drug action. In addition, mice are not considered sizeable enough to harbor a high numbers of the rather large female worms in a single experiment. The aim of this study was therefore first to assess the survival of O. change adult female worms in the more sizeable gerbils and hamsters over time, given their susceptibility to O. change male worms, and other parasitic infections (Hardin et al. 1992; Verster 1971), in order to guide the development of better in vivo drug screening models. Both rodents were permissible to the adult female worms of O. change. However, the female worms were generally viable in the gerbils before day 30, after which most became fragmented. On the other hand, the hamsters were susceptible beyond day 30, with 100% viable females recovered in some cases, howbeit with very low recovery rates.

The prolonged susceptibility of hamsters to the adult female worms could be attributable to immunological hyporesponsiveness, a quality reported to be displayed by many rodents and primates permissive to a given filarial parasite (Babayan et al. 2012). This state of tolerance is believed to significantly facilitate the survival of these large extracellular parasites for several years in their respective hosts. Specifically, hamsters infected with Acanthocheilonema viteae have been shown to display reduced responsiveness to T-cell mitogen (Weiss 1978), even lower than that of their natural hosts; gerbils. This aspect has also been observed in gerbils infected with B. pahangi (Portaro et al. 1976; Shimizu-Suganuma et al. 2000). This hyporesponsiveness is not observed in non-permissive and/or short-term hosts. Furthermore, the endosymbiont Wolbachia has been implicated in the protection of the bovine O. change from lethal immune mechanisms such as eosinophil infiltration and degranulation (MacDonald et al. 2004; Nfon et al. 2006). This could explain why the prolonged stay of adult female worms in the gerbil result in disintegrated worms, which may sometimes release unwanted antigens (Babayan et al. 2012; Ravindran 2001).

Unlike with adult female worms, studies in our laboratory have shown that gerbils can tolerate O. change mf for as long as 50 days. This could imply that different stages of the parasite provoke different immune responses. The significant difference observed between the recovery of enzymatically-liberated and mechanically-liberated worms from gerbils, with the former recording more disintegration, could be attributed to worm exposure to physiological proteases, in the absence of the nodule coat/tissue. The nodular tissue which is present in the mechanically-liberated worms may contain some primitive immune-survival surface elements encouraging the formation of more calcified nodules, serving as a protection barrier against host enzymatic degradation and immune complexes.

FBZ, the only known highly effective Onchocerca macrofilaricide, was chosen to validate the implanted gerbils and hamsters at a dose of 20 mg/kg once daily for 7 days. Female worm recovery appeared not to be affected by FBZ. The OD values obtained from analyzing MTT/Formazan in worms however showed that FBZ significantly reduced female worm viability in hamsters. Apparently this effect was not yet strong enough to be captured by the visual color scoring. Thus increasing the dose of FBZ could have led to a more evident reduction in worm viability. The few recovered male worms (which were probably hidden within the nodular masses during implantation) were not affected by FBZ. However, existing studies show FBZ as very potent against O. change male worms when exclusively male worms are implanted in gerbils (Cho-Ngwa et al. 2019). Acute exposure to benzimidazoles (including FBZ) has been said to have few noticeable effects on many nematodes, even at very high concentrations owing to their mode of action, i.e. disrupting the tubulin-microtubule equilibrium in cells, leading to cessation of nutrient transport and eventual cell death. These effects take time to become evident. Hence, it is possible that the male worms are protected in the presence of female worms. Furthermore, FBZ used in human trials proved to be slow-acting (5 weeks after initiation of treatment and 2 months post last treatment) (Geary et al. 2019), and the female worms are unable to survive long enough in these rodents for any observable drug effect. However, further analysis via the embryogram indicated more disintegrated embryos from FBZ-treated gerbils, compared to control ones. Given the slow-acting nature of FBZ, and the fact that female worms naturally deteriorate in artificial hosts, it could be suggested that a combination of higher non-toxic doses, earlier recovery time points, and the embryogram, will be the most likely way to assay O. change female worm viability. It is therefore essential for a macrofilaricide capable of killing the adult worms within 30 days to be found. Amocarzine was shown to be a partially effective macrofilaricide in the Americas (Poltera et al. 1991), but showed no additional advantage when used in combination with ivermectin in Africa (Awadzi et al. 1997).

The tolerance of these rodents to adult worm stages of the closest in phylogeny to O. volvulus is a promising finding. Although FBZ had marginal effect on worm and embryo viability, using imaging techniques to fully ascertain the health of the female worm and identifying blood or urine biomarkers to monitor the most effective implantation/treatment timeline may improve these models.

This exploratory study has revealed the Mongolian gerbil and the Syrian hamster as potential small animal models to validate the effects of candidate drugs on adult O. change female worms. The hamster was found to host adult female worms much longer than gerbils, with viable worms recovered from hamsters up to 60 days post-implantation. Further optimization of these models to support this exploratory study could result in the long-awaited small animal female worm macrofilaricide model for onchocerciasis, to validate candidate drugs for effect on worm burden and/or reproduction of worms.

Competing interests

The authors declare that they have no competing interest.

Authors' contributions

Rene Bilingwe Ayiseh performed the experiments, analyzed the results, and drafted the manuscript.

Glory Enjong Mbah co-performed the experiments, analyzed the results, and drafted the manuscript.

Faustin Pascal Tsague Manfo contributed in developing the methodology and reviewed the manuscript.

Tessy-Koko Kulu and Fabrice Ngoh Njotu assisted in performing the experiments and acquiring data, and reviewed the manuscript.

Elvis Monya, Emmanuel Menang Ndi and Irene Memeh Tumanjongprovided laboratory samples, contributed in developing the methodology, and reviewed the manuscript.

Evans Ngandung Mainsah was involved in developing the methodology and in project administration.

Judy Sakanariand Sara Lustigmanwere involved in conception, funding and reviewing the manuscript.

Fidelis Cho-Ngwa did the conception, sourced for funding, designed and supervised the experiments, and reviewed the manuscript.

Funding

This study was supported by grants from the Bill and Melinda Gates Foundation (www.gatesfoundation.org. Grant numbers OPP1098475 and OPP1017584) to Fidelis Cho-Ngwa, Judy Sakanari, and Sara Lustigman. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgements

We thank Mr. Gamua Stanley and the staff of the Biotechnology Unit, Faculty of Science, University of Buea, Cameroon, for their technical support.

Conflicts of interest/Competing interests

I declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Availability of data and material

Data can be made available upon request but not material as it was all used in the study.

Ethics approval

Not applicable for humans as they were not involved in this study. The animal protocol (UB-IACUC N^o 002/2017) was approved by the University of Buea Animal Care and Use Committee

Consent to participate

Not applicable

Consent for publication

Not applicable

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Survival of adult female worms of Onchocerca change in gerbils and hamsters: implications for the development of an in vivo macrofilaricide screening model

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Abstract

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Introduction

Methods

Ethics statement

Experimental animals

Implanting isolated female worms into gerbils and hamsters

Drug administration

Assessing the viability of worms recovered from gerbils and hamsters

Statistical analyses

Results

The method of adult worm isolation only affects worm recovery in gerbils

Discussion

Conclusion

Declarations

Competing interests

References

Additional Declarations

Status:

Journal Publication

Version 1