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.     

Brugia malayi: stage-specific expression of carbohydrates containing N-acetyl-D-glucosamine on the sheathed surfaces of microfilariae

Microfilariae, infective larvae, and adult worms of Brugia malayi were incubated with a panel of seven lectins in order to study the expression of surface carbohydrates. Infective larvae and adult worms did not bind any of the lectins utilized. Microfilariae, on the other hand, bound wheat germ agglutinin. The binding of this lectin was saturable and specific, and attributed to the presence of N-acetyl-D-glucosamine. In addition, microfilariae derived in vitro bound concanavalin A, indicating the presence of glucose and/or mannose on this stage of the parasite. The fact that similar concanavalin A binding was not seen on microfilariae recovered directly from the infected host implies that there is masking or loss of parasite surface antigens as microfilariae mature in vivo.     

Studies with Brugia pahangi. I. Parasitological observations on primary infections of cats (Felis catus)

The large majority of cats given a single inoculation of third stage larvae of Brugia pahangi became microfilaraemic. Some cats had microfilariae in their blood 53 or 54 days after infection and most had become positive before 72 days after infection. In the majority of cats microfilarial counts remained very steady between 2 and 10 microfilariae per mm³ for long periods. At autopsy 10·7% of the infective larvae injected were recovered as adult worms. The recovery of adult worms was directly related to the number of larvae injected. The microfilarial level did not increase significantly with an increase in the number of adult worms.     

Brugia pahangi: stage-specific antigens on microfilariae detected by serum from infected jirds or by monoclonal antibodies

Experiments were carried out to determine whether there are stage-specific antigens on microfilariae of Brugia pahangi, using sera from Mongolian jirds infected with B. pahangi and monoclonal antibodies against microfilariae of B. pahangi. These studies showed that microfilariae have both stage-specific and nonspecific antigens. The nonspecific antigens were also present on adult worms and on infective larvae. Among monoclonal antibodies, 6 out of 14 clones produced antibodies against the microfilarial stage-specific antigens, and 8 clones produced antibodies against nonspecific antigens. These monoclonal antibodies could not distinguish between adults, microfilariae, or infective larvae of B. malayi and B. pahangi.     

Setaria cervi: immunoprophylactic potential of glutathione-S-transferase against filarial parasite Brugia malayi

Glutathione-S-transferase (GST) has been detected in the adult female Setaria cervi, a bovine filarial parasite. The role of S. cervi GST antigen in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell mediated reaction as well as in situ inoculation of filarial parasites within a microchamber in Mastomys. The immune sera from glutathione-S-transferase immunized Mastomys promoted the adherence of peritoneal exudate cells to B. malayi microfilariae and infective larvae in vitro inducing 80.7 and 77.6% cytotoxicity, respectively in 72 h. In the microchambers implanted in the immunized Mastomys host cells could migrate and adhere to the microfilariae and infective larvae and induced 77.8 and 75% cytotoxicity to B. malayi microfilariae and infective larvae in 72 h, respectively. These results suggest that native GST from S. cervi is effective in inducing protection against heterologous B. malayi filarial parasite and thus has potential in immunoprophylaxis.     

Chemical mutagenesis of the parasitic nematode Strongyloides ratti to isolate ivermectin resistant mutants

We describe a strategy for the mutagenesis of the free-living adult generation of Strongyloides ratti and selection of worms carrying new mutations in the subsequent F2 generation of infective larvae. We demonstrate that this strategy is successful via the selection of infective larvae that are resistant to the anthelmintic ivermectin at a concentration of 10 ng/ml. The majority of these larvae were unable to give rise to patent infections when used to infect parasite naive rats, implying that the majority of the ivermectin resistance mutations confer pleiotropic defects on parasitic, but not on free-living, development.     

Suppression of parasitemia in rodent filariasis (Litomosoides carinii) by immunization with BCG and microfilaria. I. Intracutaneous inoculation of BCG

After intracutaneous inoculation of BCG and challenge by subcutaneous injection of infective larvae of Litomosoides carinii, the parasitaemia of the filarial infection in cotton rats remains significantly lower when BCG and larvae are applied in the region of the same popliteal and ileal lymph nodes. However, when the infective larvae are directed to other regional lymph nodes (Ln cubitales and axillares), the depression of microfilaraemia is missed. The worm load (recovery rate) and the expulsion of microfilariae by the adult worms are not influenced by the BCG inoculation. Obviously BCG stimulates the lymphatic tissue unspecifically, and the infective larvae produce the first antigen contact, which is boostered by the microfilariae at the onset of patency. When the intracutaneous BCG inoculation is combined with specific antigen stimulation by simultaneous injection of blood microfilariae in the region of the same lymph nodes, the microfilaraemia of the challenge infection disappears completely or remains extremely low.     

A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds

A 43 kiloDaltan (kDa) antigen fraction (CFA₂-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory–secretory products. Further analysis was done on the immunoprophylactic potential of CFA₂-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA₂-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA₂-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA₂-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA₂-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.     

Studies with Brugia pahangi 12. The activity of levamisole.

The effects of levamisole on adults, third stage infective larvae, and microfilariae of Brugia pahangi were studied in in vitro culture and in vivo against developing stages in the vector mosquito and in infected cats. In vitro the drug was effective only at dose levels much higher than can be tolerated by mammals. It was active against the developmental stages of the worm in the vector Aedes aegypti. The drug was strongly microfilaricidal in cats but less effective against adult worms.     

Studies with Brugia pahangi 17. The anthelmintic effects of diethylcarbamazine.

Diethylcarbamazine (DEC) was active in vitro against infective larvae and microfilariae of Brugia pahangi but only at high concentrations. When fed to mosquitoes which were infected with B. pahangi it had little or no activity. In jirds it was inactive against B. pahangi microfilariae and adults when administered at 300 mg/kg for 5 days either by the intraperitoneal or oral route. In cats given 25 or 50 mg DEC/kg intraperitoneally on 3 or 5 occasions it was not microfilaricidal, but most of the adult worms died within 30 days of the end of treatment. Although most microfilariae disappeared from the blood of cats immediately (i.e., within an hour) after treatment, they reappeared within a few hours in the same numbers. Microfilarial levels were reduced after treatment but there was no precipitate decline as occurs in human B. malayi patients.     

Dissociation of the protective immune response in the mouse to Strongyloides ratti

The generation of protective immunity by various stages in the life-cycle of Strongyloides ratti and the phases against which resistance is directed has been examined in murine strongyloidiasis. Mice were exposed to natural, complete infections, were treated with thiabendazole (which largely resembles the natural infection), were treated with cambendazole (which restricts infection to the larval stage), or infected directly by oral transfer of adult worms. Mice that were infected with infective larvae alone did not become resistant to infective larvae or the complete infection but were resistant to adult worms implanted directly into the gut. Mice exposed to adult worms alone were resistant to natural infections and adults worms implanted directly but were not resistant to infective larvae. On the other hand, mice that had received prior natural infections showed evidence of resistance to infective larvae, adult worms, and natural, complete infections. It is concluded that there is immunological cross-reactivity between infective larvae and adult worms but that under certain circumstances the infective larvae are able to evade the host's protective immune response.     

Development of the plerocercoid I of Ophiotaenia europaea in reptiles

The existence of a two-host life-cycle in ophiotaeniid proteocephalideans was tested experimentally using Ophiotaenia europaea as a model. Three species of reptiles, Natrix natrix, Natrix tessellata and Lacerta viridis, were fed with experimentally infected copepods containing a large number of infective plerocercoids I. A few plerocercoids, most of which were dead, corresponding morphologically to the plerocercoid II developmental stage of O. europaea, were found encysted in the intestinal wall of N. natrix (8 days p.i.), N. tessellata (5 and 150 days p.i.) and L. viridis (40 days p.i.), while no plerocercoids or adult worms were recovered from their intestines. The results indicate that the infective plerocercoid I of O. europaea cannot undergo further development when ingested directly by the final host (a reptile), and that environmental temperature stimuli cannot initiate a reverse plerocercoid migration to the gut followed by strobilization.     

Acknowledgment

The uptake and incorporation in vitro of various nucleic acid precursors by microfilariae, third-stage infective larvae, 10-day-old juveniles and adult worms of Brugia pahangi were investigated using scintillation counting and autoradiographic techniques. A significant uptake of uracil and of purines, including adenine, hypoxanthine, and guanine was demonstrated in this study. No evidence was obtained for the uptake and incorporation of thymine, cytosine, orotate, formate, folate or p-aminobenzoic acid by either micro- or macrofilariae of B. pahangi.     

Suppression of the parasitemia in rodent filariasis (Litomosoides carinii) by immunization with BCG and microfilaria. II. Intravenous BCG application

By intravenous (i.v.) inoculation of living tuberculosis bacteria (BCG) non-specific resistance to microfilariae of Litomosoides carinii (Filarioidea) is induced in cotton rats. This is only possible using the preparation "Immune-BCG Pasteur F" (suspended germs), but not with "Vaccin-BCG pour scarifications" (lyophilized tuberculosis bacteria). After inoculation of Immune-BCG, followed by a challenge infection by 60 infective larvae 6 weeks later, a patent infection develops. However, the level of microfilaraemia is constantly lower than in the control. After challenge infection 12 weeks later, this effect has disappeared. Immune-BCG has no influence on the worm load or the output of microfilariae by the adult worms. If i.v. inoculation of Immune-BCG is combined with a subcutaneous injection of specific antigen--living embryos from the uteri of adult worms--the BCG-activated immune system undergoes specific sensitization. Upon challenge infection 6 weeks later, the microfilaraemia is completely suppressed, but the worm load and production of microfilariae by the adult female worms are normal. If Immune-BCG is injected i.v. 3 days before intraperitoneal injection of freeze-killed microfilariae, there is still constantly reduced microfilaraemia when challenge infection follows 12 weeks later. Obviously, the effect of this relatively weak antigen may be increased by BCG stimulation.     

Immunity to Litomosoides carinii in Mastomys natalensis. II. Effects of chemotherapeutically abbreviated and postpatent primary infections on challenges with various stages of the parasite

Naive Mastomys natalensis, Litomosoides carinii-infected M. natalensis at a postpatent stage of the infection and L. carinii-infected M. natalensis treated chemotherapeutically with furazolidone (FUR), FUR and diethylcarbamazine (FUR/DEC) or amoscanate (AMOS) were challenged by either injection or implantation of 40 third stage larvae (L3, s.c.), 40 fourth stage larvae (L4, 16 days old, i.p.), 20 male and 20 female preadult worms (36 days old, i.p.), 12 adult female worms (i.p.) or 6 X 10(6) microfilariae/kg (i.v.). Microfilaraemia in animals challenged at a postpatent stage (independent of the kind of challenge), was either totally suppressed or at least greatly reduced. Necropsy of L3-challenged animals showed that neither the length of the worms nor their content of morphologically intact, intrauterine stages was affected. Infected, treated animals challenged with developing stages (L3, L4 and preadult worms) showed reduced levels of microfilaraemia (by up to 75%). Dissection of AMOS-treated, L3-challenged animals showed that both the developmental rate and the fertility of the worms were affected. Microfilaraemia was also reduced after implantation of adult worms into treated animals. This was independent of the interval between treatment and challenge (44-150 days) except in animals challenged 10 days after AMOS-treatment, which showed no difference from naive controls. However, infected, treated M. natalensis, cotton rats and gerbils did not develop immunity against intravenously injected blood microfilariae.     

Delayed macrofilaricidal activity of diethylcarbamazine against Brugia pahangi in Mongolian jirds

The macrofilaricidal activity of diethylcarbamazine (DEC) was confirmed in jirds infected with Brugia pahangi. Seventy jirds were inoculated subcutaneously with 100 infective larvae. At 20 weeks post-infection, the microfilaraemic jirds were divided into two groups, untreated and treated. For the treated group, 200 mg kg(-1) of DEC was injected intraperitoneally for 5 consecutive days. One, 4, 8, 12, 16 and 27 weeks after the final treatment, 4-7 jirds in each group were sacrificed to measure adult worm burdens. The number of adult worms recovered from treated jirds was comparable to controls at earlier necropsy (1 and 4 weeks post-treatment). However, at late necropsy (8 weeks and later) the recovery rate of adult worms in treated jirds was significantly lower than that in untreated controls, indicating an adultcidal effect of DEC. The present study demonstrates that DEC requires 8 weeks to kill B. pahangi adult worms in jirds and that the Mongolian jird is a useful model for screening antifilarial activity.     

Differential tissular distribution of Litomosoides sigmodontis microfilariae between microfilaremic and amicrofilaremic mice following experimental infection

Filariases are caused by onchocercid nematodes that are transmitted by arthropod vectors. More than 180 million people are infected worldwide. Mass drug administration has been set up in many endemic areas to control the parasite burden. Although very successful in limiting microfilarial load, transmission has not been completely interrupted in such areas. A proportion of infected patients with lymphatic filariasis or loiasis are known to be amicrofilaremic, as they do not present microfilariae in their bloodstream despite the presence of adult worms. A mirror status also exists in CBA/Ca mice infected with Litomosoides sigmodontis, the well-established model of filariasis. Using this model, the goal of this study was to determine if the kinetics of blood clearance of microfilariae differed between amicrofilaremic CBA/Ca mice and microfilaremic BALB/c mice. For this purpose, a qPCR approach was devised to detect microfilariae in different tissues, after a controlled inoculation of microfilariae. We showed that the rapid clearance of microfilariae from the pleural cavity or from the bloodstream of CBA/Ca mice was associated with a massive accumulation of first stage larvae in the lungs, liver and spleen.     

The ability of Aedes aegypti mosquitoes to survive and transmit infective larvae of Brugia pahangi over successive blood meals

The mortality of Aedes aegypti mosquitoes increased; immediately following a blood meal containing microfilariae of Brugia pahangi, when infective larvae began to migrate out of the flight muscles and when infective larvae were lost from the mosquitoes during a blood meal. When infective mosquitoes took a second blood meal 86.2% of the infective larvae escaped from their bodies. However, only 50.3% escaped when mosquitoes fed through a thin layer of cotton. Infective larvae in the abdomen of the mosquitoes stood the least chance of escaping from the insects. When infective mosquitoes were offered a third blood meal four days later, the proportion of infective larvae in the head and labium had risen from 56.6% in the control group to 66.0% and 69.4% in the two test groups. At this third feed 54.7% and 75.7% of the infective larvae were lost from mosquitoes with a low and medium pre-feeding worm burden respectively. This suggests that the escape of infective larvae from mosquitoes with only a few worms is less efficient than from mosquitoes with a medium worm burden.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.