Onchocerca ochengi male worms implanted in SCID mice and Gerbil: Relationship between microfilaridermia status of cows, nodular worm viability and fertility and worm survival in the rodents.

BACKGROUND: Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. METHODS: Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. RESULTS: Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf-. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf- cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf- cows, respectively. Female worms from nodules of mf- cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. CONCLUSION: Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents.

In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening.

BACKGROUND: Mansonellosis arises from infections with threadlike filarial nematodes in millions of individuals, especially in sub-Saharan Africa. Since infections present no overt clinical symptoms but attenuate immune responses that might lead to increased susceptibility and worsened disease course of concomitant infections, it is truly a neglected tropical disease. Nevertheless, only few studies focus on identifying suitable safe drugs for its control and little is known about the requirements for in vitro maintenance of the Mansonella perstans transmission stage. This study, therefore, evaluated the survival of M. perstans microfilariae (mf) using in vitro conditions that have been shown to promote survival of Loa loa, a closely related filarial nematode. Furthermore, the in vitro microfilaricidal effect of 15 agents was assessed on this helminth. METHODS: The ability of two basic culture media; Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and a monkey kidney epithelial cell line (LLC-MK2) to support the survival of M. perstans microfilariae was investigated. Subsequently, 6 anti-helminthics, 5 anti-malarials, 1 anti-microbacterial, 2 trypanocidals and 1 anti-cancer agent were tested in vitro against mf. The suitability of the culture media as well as the effect of the anti-infective agents on mf survival was assessed by scoring their motility. RESULTS: FBS supplement and additional LLC-MK2 cells significantly improved the survival of mf in DMEM and RPMI-1640 culture. In detail, RPMI-1640 supplemented with 10% FBS and LLC-MK2 cells sustained the maintenance of mf for at least 20 days (100.00 ±â€¯0.00% survival). In co-cultures with LLC-MK2 cells without serum, M. perstans mf were maintained in DMEM and RPMI-1640 medium with a motility above 99% by day 5. Mefloquine displayed the highest microfilaricidal effect in vitro followed by artesunate. CONCLUSION: Both RPMI and DMEM in the presence of LLC-MK2 cells are suitable for the maintenance of M. perstans mf in vitro. In absence of the feeder cells, the addition of 10% FBS to RPMI-1640 medium improved the parasite survival rate and motility. The microfilaricidal activity of mefloquine and artesunate on M. perstans mf was documented for the first time in this study and can therefore be considered as reference for further screening of agents against this parasite stage.

Advances in preclinical platforms of Loa loa for filarial neglected tropical disease drug and diagnostics research.

The tropical disease, loiasis, caused by the filarial parasite, Loa, has gained prominence in global public health as a cause of excess mortality and a barrier to the elimination of the related prioritized neglected tropical diseases (NTDs), lymphatic filariasis and onchocerciasis, within Central Africa. There are no effective drug cures or vaccines available to treat loiasis safely. Here we review recent advances in loiasis preclinical platform technologies, including novel in vitro culturing systems, animal models and innovations in experimental infections of the L. loa vector, Chrysops, that have facilitated access to all L. loa filarial life-cycle stages. We detail applications of these new model systems in anti-filarial drug screening, diagnostic development, immunology, and pathophysiology research. Finally, we provide an overview of how loiasis preclinical platforms may be further utilized in translational medicine applications to support the development of much needed new interventions against filarial NTDs.

Dataset on in vitro maintenance of Mansonella perstans microfilariae and drug testing.

Endemic communities of Mansonella perstans infections have been neglected since associated pathology remains undefined. Consequently, improvements in drug therapy have also been ignored despite a large number of infected individuals in areas of Cameroon. Thus, we established an in vitro system to culture M. perstans microfilariae (Mf); the transmission stage of infection. In short, we compared the ability of two renowned culture media (Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640)) to sustain Mf in culture. Media were supplemented with 10% fetal bovine serum (FBS) and monkey kidney epithelial cells (LLC-MK2) were used as feeder cells. As readout we assessed Mf survival and motility using a standardised microscopy assessment strategy. Moreover, this in vitro culture system was used to test susceptibility levels of microfilariae to different chemotherapeutic agents. Parasite motility was scored daily using a graded system and analysed using the average motility and area under the motility curve of M. perstans Mf. These datasets were analysed and discussed in detail in the related article entitled: "In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening" [1].

Effect of flubendazole on developing stages of Loa loa in vitro and in vivo: a new approach for screening filaricidal agents.

BACKGROUND: Loiasis, an often-neglected tropical disease, is a threat to the success of lymphatic filariasis and onchocerciasis elimination programmes in rainforest areas of the central and western Africa. Its control and even its elimination might be possible through the use of a safe macrofilaricide, a prophylactic drug, or perhaps a vaccine. This present study evaluated the effect of flubendazole (FLBZ) on the development of Loa loa L3 in vitro and in vivo. METHODS: Infective stages of L. loa were isolated and co-cultured in Dulbecco's Modified Eagle's Medium in the presence of monkey kidney epithelial cells (LLC-MK2) feeder cells. FLBZ and its principal metabolites, reduced flubendazole (RFLBZ) and hydrolyzed flubendazole (HFLBZ), were screened in vitro at concentrations 0.05, 0.1, 0.5, 1 and 10 µg/ml. The viability of the parasites was assessed microscopically daily for 15 days. For in vivo study, a total of 48 CcR3 KO mice were infected subcutaneously with 200 L. loa L3 and treated with 10 mg/kg FLBZ once daily for 5 consecutive days. Twenty-four animals were used as control and received L3 and vehicle. They were dissected at 5, 10, 15 and 20 days post-treatment for worm recovery. RESULTS: The motility of L3 larvae in vitro was reduced from the second day of incubation with drugs at in vivo plasma concentration levels, with a strong correlation found between reduced motility and increased drug concentration (Spearman's rho = -0.9, P < 0.0001). Except for HFLBZ (0.05 µg/ml and 0.01 µg/ml), all concentrations of FLBZ, HFLBZ and RFLBZ interrupted the moulting of L. loa infective larvae to L4. In vivo, regardless of the experimental group, there was a decrease in parasite recovery with time. However, at each time point this reduction was more pronounced in the group of animals treated with FLBZ compared to equivalent control. Parasites were recovered from the flubendazole-treated groups only on day 5 post-inoculation at an average rate of 2.1%, a value significantly lower (Mann-Whitney U-test, U = 28, P = 0.0156) than the average of 31.1% recovered from the control group. CONCLUSIONS: This study reveals the ability of flubendazole to inhibit the development of L. loa L3 both in vitro and in vivo, and in addition validates the importance of in vitro and animal models of L. loa as tools for the development of drugs against loiasis.