In vitro haemagglutination and attenuation of microfilarial motility by haemolymph from individual blackflies (Simulium ornatum) infected with Onchocerca lienalis

Two in vitro tests were used to investigate the effect of Onchocerca lienalis Stiles infection on the haemolymph of Simulium ornatum Meigen. The first of these examined the effect of infected haemolymph on the motility of fresh O. lienalis or Brugia pahangi Buckley & Edeson microfilariae. Incubation of haemolymph from individual flies with fresh microfilariae was performed in the wells of Terasaki micro-tissue culture plates. Motility of both species of parasite was found to be significantly attenuated when compared to worms incubated in control haemolymph groups. The second assay was that of agglutination of cat erythrocytes in the presence of haemolymph from individual flies, also performed in Terasaki plates. This test demonstrated significant increases in the rates of haemagglutination in the haemolymph of O. lienalis infected blackflies. The titre appeared to increase during the initial 5 days of infection up to a level of 1/32+, but then fell between day 5 and 7 to a maximum level of 1/2. The proportion of flies exhibiting haemagglutination also rose following infection. Despite the apparent absence of melanization and encapsulation, simuliids may have at least two humoral haemolymph components available to them for parasite regulation; a fast-acting factor responsible for rapid parasite death, and more specific agglutinins, possibly lectins. The role of the latter in defence is as yet unclear.     

Angiostrongylus cantonensis: in vitro cultivation from the first-stage to infective third-stage larvae

The first-stage larvae of Angiostrongylus cantonensis were cultured in various media at 27 degrees C. The most suitable medium for the development was Chernin's balanced salt solution supplemented with 10% L-15, 10% tryptose phosphate broth, 20% fetal calf serum, and 26 mM sodium bicarbonate. Addition of sodium bicarbonate to the medium facilitated early development of the first-stage larvae. When the first-stage larvae were cultured in the medium under 5% CO2 in air, the worms developed gradually to become quiescent and showed the C shape. Thereafter, the larvae developed to the second stage, retaining their first sheath. About 23 days later, the larvae began to develop to the third stage, being enclosed within the sheaths of the first and second molts. Under these conditions, the larvae developed uniformly and 82% of the larvae reached the third stage 50 days later. About 70% of the third-stage larvae discarded their two sheaths, showing almost the same size as those obtained in vivo. When these exsheathed larvae were inoculated into rats, they developed into adult worms and deposited numerous first-stage larvae.     

Immunity to Onchocerca lienalis microfilariae in mice. II. Effects of sensitization with a range of heterologous species

The model of Onchocerca lienalis microfilariae (mff) injected into CBA and T.O. strains of mice has been used to examine immunity to skin-dwelling microfilariae following exposure to a range of species of helminths. Mice which had received a primary infection with O. cervicalis mff were significantly resistant to challenge with O. lienalis mff (58% reduction relative to challenge controls). Immunization with the uterine contents (eggs and mff) of O. lienalis, O. gutturosa or O. volvulus conferred equivalent levels of protection against challenge with O. lienalis mff (66 to 75%). Similar results were obtained with immunizations in mice that employed either fresh or freeze-killed eggs of O. gutturosa. Significant reductions in the recoveries of O. lienalis mff were also demonstrated following the intraperitoneal implantation of adult male worms of O. gutturosa (30 to 52%), the adults of either sex of Dipetalonema viteae (60%), or after infection with Trichinella spiralis (27 to 81%). Infections with the trematode, Schistosoma mansoni, had a negligible effect on mff recoveries. It is concluded that partial resistance in mice to Onchocerca mff may be stimulated by factors, yet to be determined, that are neither stage nor species-specific.     

The morphogenesis of Ascaris suum to the infective third-stage larvae within the egg.

Studies of the morphology of Ascaris suum larvae developing in the egg during embryonation in vitro at room temperature showed that 2 molts take place within the egg. The first larval stage (L1) appeared in the egg after 17-22 days of cultivation, the first molt to the second larval stage (L2) took place from day 22 to day 27, and the second molt to the third larval stage (L3) started on day 27 and continued during the 60-day observation period. Infectivity of the eggs was studied by oral egg inoculation in mice and showed that the L3 are the infective stage for mice. Molting to the L3 stage occurs gradually over a period of 2-6 wk, and it is recommended to have an additional maturation period so the infectivity of an egg batch may reach maximum level.     

Ultrastructural changes in the third-stage, infective larvae of ruminant nematodes treated with sainfoin (Onobrychis viciifolia) extract

Plants rich in condensed tannins are an alternative to chemical anthelmintics to control gastrointestinal nematodes (GINs) in ruminants. Previous functional studies have shown that sainfoin extracts affect the two forms of infective larvae (L3), ensheathed and exsheathed. However, the mechanisms of action remain unknown. The aim of this study was thus to compare ultrastructural changes in ensheathed and exsheathed L3 of two GIN species after in vitro contact with sainfoin extracts using transmission electron microscopy. The main changes identified were an alteration of the hypodermis, the presence of numerous vesicles in the cytoplasm and degeneration and/or death of muscular and intestinal cells. The changes suggested similar and nonspecies-specific lesions in the two nematode species. Comparison of the modifications found in the ensheathed vs. exsheathed L3s revealed different locations of the main cellular changes depending on the larval form. It is hypothesized that these spatial differences in lesions are mainly influenced by the presence of the sheath which favors contact between the active compounds and either the cuticle or the digestive tract. Overall, our observations suggest that the functional changes observed in the biology of GIN L3s after contact with sainfoin extracts are mediated through a direct mode of action, i.e. different interactions between the bioactive plant metabolites and the nematode structure depending on the route of contact.     

Serum-stimulated feeding in vitro by third-stage infective larvae of the canine hookworm Ancylostoma caninum

Developmentally arrested nonfeeding infective larvae of hookworms resume development after entry into the host, presumably in response to a signal encountered during invasion. Logically, an initial step in the resumption of development might be the resumption of feeding. An in vitro assay for feeding is described for the third-stage larvae of the canine hookworm Ancylostoma caninum. Populations of larvae incubated under hostlike conditions in the presence of 10% canine serum resume feeding within 6 hr, as evidenced by the uptake of fluorescein-labeled bovine serum albumin. Feeding is dependent on the presence of canine serum, and peaks by 24 hr incubation. Maximal feeding levels occur at temperatures above 34 C with a gas phase of 5% CO2/95% air, whereas culture medium and pH are unimportant for feeding. Serum concentrations between 0.1% and 1.0% (v/v) initiate feeding, and the response peaks at approximately 8.0% serum. Serum triggers feeding within 6 hr and is not required for feeding to continue once initiated. The saturation effect and the trigger phenomenon suggest that the initiation of feeding is a receptor-mediated response.     

Infective larvae of five Onchocerca species from experimentally infected Simulium species in an area of zoonotic onchocerciasis in Japan

Microfilariae of five Onchocerca species, O. dewittei japonica (the causative agent of zoonotic onchocerciasis in Oita, Kyushu, Japan) from wild boar (Sus scrofa), O. skrjabini and O. eberhardi from sika deer (Cenus nippon), O. tienalis from cattle, and an as yet unnamed Onchocerca sp. from wild boar, were injected intrathoracically into newly-emerged black flies of several species from Oita to search the potential vector(s) of these parasites and identify their infective larvae. Development of O. dewittei japonica microfilariae to the infective larvae occurred in Simulium aokii, S. arakowae, S. bidentatum, S. japonicum, S. quinquestriatum, and S. rufibasis while development of infective larvae of O. skrjabini, O. eberhardi, and the unnamed Onchocerca sp. was observed in S. aokii, S. arakawae, and S. bidentatum. Development of O. lienalis microfilaria to infective larvae occurred in S. arakawae. Based on the morphology of infective larvae obtained, we proposed a key of identification of Onchocerca infective larvae found in Oita. We also reconsider the identification of three types of infective larvae previously recovered from Simulium species captured at cattle sheds: the large type I larvae that may be an undescribed species; the small type III identified as O. lienalis may include O. skrjabini too; the intermediary type II that may be O. gutturosa, or O. dewittei japonica, or the unnamed Onchocerca sp. of wild boar.     

Chemokinetic behavior of the infective third-stage larvae of Strongyloides ratti on a sodium chloride gradient.

The movements of the infective third-stage larvae (L3) of a rodent parasitic nematode Strongyloides ratti were examined on a sodium chloride (NaCl) gradient set up on agarose plates. The movements of larvae were followed by observing their tracks on the surface of the agarose. The direction of movement depended on the NaCl concentration at the point of their initial placement on the gradient. Larvae placed at between 230 and 370 mM NaCl tended to migrate towards areas of lower concentration. On the other hand, when placed at concentrations less than 20 mM NaCl, larvae tended to migrate initially towards higher concentrations but did not linger in areas where the concentration was over approximately 80 mM NaCl. It seems that S. ratti L3, tested in vitro, prefer regions with a concentration of NaCl below 80 mM NaCl. Two typical chemokinetic behaviors are seen; a unidirectional avoidance movement when initially placed in unfavorable environmental conditions and a random dispersal movement when placed within an area of favorable conditions. Track patterns were straight in the avoidance movement but included multiple changes of direction and loops in the dispersal movement. This study introduces an assay system suitable for studying chemokinetic behavior of larvae of Strongyloides ratti.     

Studies of the growth-regulating effects of ivermectin on larval Onchocerca lienalis in vitro

At concentrations of 0.1-100 ng/ml ivermectin inhibited L3-L4 molting by Onchocerca lienalis in vitro. The degree of inhibition was dose-dependent with a significant effect apparent at 0.1 ng/ml and complete inhibition occurring at 100 ng/ml. The ED50 for molt inhibition was 0.19 ng/ml. Molt-inhibiting levels of the drug were not acutely toxic to the worms. In the presence of 10 ng/ml, a concentration giving 95% molt inhibition, motility at day 7 postinoculation was 71% of that seen in nontreated controls. A more pronounced effect on motility was apparent in larvae under long-term cultivation in the presence of ivermectin. Kinetic studies indicated that the majority of the larvae respond irreversibly to the drug within the first 2 hr of exposure. Twenty-four hours of exposure were required for a maximal response. The inhibitory effects of ivermectin were less pronounced if larvae were allowed to develop under normal culture conditions for 24 or more hours prior to the initiation of drug treatment.     

CD4+-dependent immunity to Onchocerca volvulus third-stage larvae in humans and the mouse vaccination model: common ground and distinctions

Onchocerciasis is a major filarial disease and is the second most common cause of infectious blindness in the world. Disease development after infection with Onchocerca volvulus varies widely and is determined by the host's immune response to the parasite. Vector control and administration of ivermectin has reduced infection and disease rates significantly. However, limitations of these programmes, including ivermectin's selective activity on microfilariae, the need for 10-15 years of annual treatments, logistical obstacles and the potential emergence of drug-resistant strains demand alternative strategies. A vaccine that targets O. volvulus infective third-stage larvae (L3) could provide an additional tool to guarantee successful elimination of infection with O. volvulus. An essential step in the development of immunoprophylactic procedures and reagents is the identification of host immune responses toward antigens of O. volvulus L3 and L3 developing to the fourth-stage larvae that are associated with protection against these stages of the parasite. This review summarises the recent advancements in understanding the immune mechanisms in particular the CD4(+) responses to L3 stages in humans and in the mouse vaccination model. Comparison between the two uncovered common immunological elements in naturally exposed humans and mice vaccinated with radiation attenuated L3 or recombinant O. volvulus antigens, as well as significant differences. These studies promisingly suggest that the O. volvulus mouse model is a very useful adjunct to the studying of natural infection in humans and could provide us with the tools to identify the target molecules and the effector immune correlates of protection in humans responsible for attrition of L3 stages. Since some of these antigens may exist in other nematodes, any insight gained into the mechanisms of vaccine-induced anti-O. volvulus L3 protective immunity in both humans and mice could be applicable to the development of vaccines against other nematode infections.     

In vitro activity of levamisole on the infective larvae, microfilariae and adult worms of Breinlia Sergenti

In vitro activity of levamisole on the infective larvae, microfilariae and adult worms of Breinlia sergenti. International Journal for Parasitology4: 207–210. Levamisole shows in vitro activity against the infective larvae, microfilariae and adult worms of Breinlia sergenti. The polygraph studies using the adult worms indicate that levamisole causes an increase in the muscle tone; this action being dose related. The adult worms are more sensitive to the drug than the infective larvae and microfilariae. In vitro, levamisole is more potent compared with diethylcarbamazine against all the three stages of B. sergenti.     

Quantitative studies on the eggs of Simulium (Simulium) ornatum Meigen and Simulium (Wilhelmia) equinum L. in a chalk stream in southern England

Abstract.

• 1 The egg masses of Simulium ornatum, S.equinum and S.vernum are described and information is given on numbers of eggs per egg mass and the size and number of batches laid by individual females.
• 2 The oviposition sites were studied in relation to water velocity, wind speed and direction.
• 3 The incubation periods at different river temperatures and the effects of desiccation on percentage hatch and incubation times were investigated.
• 4 The numbers of eggs laid were assessed for the winter generations of S. ornatum and S.equinum. The relevance of these to the overall production estimates is discussed

     

Skin penetration by ensheathed third-stage infective larvae of Necator americanus, and the host's immune response to larval antigens.

In vitro experiments were conducted to assess skin penetration by ensheathed third-stage infective larvae (L3) of Necator americanus. The fact that only a small proportion of larval sheaths was recoverable from the outer skin surface suggested that some larvae penetrate mouse skin without undergoing exsheathment. Penetration by ensheathed larvae was confirmed visually using a novel fluorescein isothiocyanate (FITC)-labelling technique in which viable ensheathed larvae were fluoresceinated, applied onto intact mouse skin, and their progress monitored in frozen skin sections. This direct observation that the L2-derived sheath can present antigens to the host's immune system was also monitored by immunoassay to provide confirmatory information regarding skin penetration by ensheathed larvae. Sera from humans infected with Necator americanus were shown to react in ELISA against antigens stripped by detergent (cetyltrimethylammonium bromide) from the sheath surface, and with antigens contained in L3-exsheathing fluid. These data suggest that the host's immune response, as a result of antigenic stimulation by the cast sheath and exsheathing fluid, could in fact be diverted away from the potentially vulnerable L3 stage.     

Acquired hookworm immunity in the golden hamster (Mesocricetus auratus) elicited by living Necator americanus third-stage infective larvae

The aim of the study is to demonstrate and understand the acquired immunity in golden hamsters (Mesocricetus auratus) elicited by primary Necator americanus infective third-stage larvae (L3) infection. Hamsters infected with 150 L3 for 1, 2, 3, 6 and 10 weeks, were challenged with the same number of L3 and sacrificed 25 days post challenge. The primarily infected hamsters exhibited 99-100% protection against subsequent L3 challenge compared to un-infected naive hamsters. The acquired immunity was developed as early as 1 week post L3 infection and lasted up to 10 weeks. Similar protective immunity was obtained in hamsters infected with N. americanus L3 and then treated orally with a single of 100mg/kg albendazole, followed by challenge with N. americanus L3 4 and 8 weeks post-treatment. The infected hamsters exhibited a rise in IgG antibodies against L3 and juvenile adult worm antigens. Histological examination showed that challenging L3 were trapped in the skin of primarily infected hamsters and surrounded or infiltrated by different inflammatory cells. The trapped L3 were damaged and dead followed by the formation of granulomas encasing dead worms. The results demonstrate that hamsters primarily infected with N. americanus L3 develop acquired immunity against re-infection.     

Temporal changes in the level of infestation of Simulium ornatum Meigen (complex) (Simuliidae: Diptera) larvae by the endosymbiotic fungus Harpella melusinae Lichtwardt (Harpellales: Trichomycetes)

The fungus Harpella melusinae (Harpellales: Trichomycetes) is obligately associated with the midguts of larval Simuliidae (Diptera). The level of infestation of a population of Simulium ornatum by H. melusinae was monitored at a stream in Hampshire, England. Significant temporal changes in the level of infestation were recorded during monthly and weekly collections; a twenty-fold increase being recorded over a nine-day period. Possible mechanisms by which these changes occur are discussed.     

Efficiency of Simulium sanctipauli as a vector of Onchocerca volvulus in the forest zone of Ghana

The role of Simulium sanctipauli Vajime & Dunbar (Diptera: Simuliidae) as a vector of Onchocerca volvulus (Leuckart) (Spirurida: Onchocercidae) in the forest zone of central Ghana was studied in the Upper Denkyira district, where onchocerciasis is hyperendemic. Simulium sanctipauli was found to be a highly efficient vector, with a mean of 377 infective (L3) larvae in the heads of 1000 parous and 122 in the heads of 1000 biting flies. The overall infection rate of 44% of the parous flies with L1, L2 and L3 stages of O. volvulus (identity confirmed by polymerase chain reaction) demonstrates marked anthropophily. Female flies dispersed over a wide area and can transmit onchocerciasis up to at least 10 km away from their breeding sites. Annual community-directed treatments with ivermectin did not have a noticeable effect on the infection rates and parasitic loads of fly populations, which were as high 2 months after as 3 months before the distribution of ivermectin. This failure can be attributed to poor coverage, with treatment taken by only 24.4% of the population of the six study villages.