Microfilarial periodicity in Mastomys natalensis

The microfilarial level in the peripheral blood of Mastomys natalensis infected with the filarial parasites, Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae was monitored at two-hour intervals for 24 hours. The microfilariae of B. pahangi and B. malayi exhibited nocturnal and diurnal subperiodicity, respectively; no such periodicity was seen in L. carinii and D. viteae infections. The level of B. pahangi and D. viteae microfilariae in peripheral blood was significantly increased when the host was anaesthetized with diethylether or pentothal sodium. Ether-induced anaesthesia had no effect on the level of B. malayi microfilariae but pentothal was most effective. The peripheral blood count of L. carinii microfilariae tended to decrease in the anaesthetized animals but the reduction was not statistically significant.     

Filaricidal efficacy of anthelmintically active cyclodepsipeptides

PF 1022A, a novel anthelmintically active cyclodepsipeptide, and Bay 44-4400, a semisynthetic derivative of PF 1022A were tested for filaricidal efficacy in Mastomys coucha infected with Litomosoides sigmodontis, Acanthocheilonema viteae and Brugia malayi. The parent compound PF 1022A showed limited anti-filarial efficacy in L. sigmodontis and B. malayi infected animals. Oral doses of 5 x 100 mg/kg on consecutive days caused only a temporary decrease of microfilariaemia levels. By contrast, Bay 44-4400 was highly effective against microfilariae of all three species in single oral, subcutaneous and cutaneously applied (spot on) doses. Minimum effective doses (MED, reducing parasitaemia density by > or =95%) determined 3 and 7 days after treatment were 3.125-6.25 and 6.25-12.5mg/kg, respectively. Using the spot on formulation, doses of 6.25mg/kg (L. sigmodontis), 12.5mg/kg (A. viteae) and 25mg/kg (B. malayi) were required to cause reductions of microfilaraemia levels by > or =95% until day 56. Adulticidal effects, determined as minimum curative doses (MCD, eliminating adult parasites within 56 days by >95%) after single dose treatment were limited to A. viteae (MCD, 100mg/kg independent of the route of administration). Repeated oral treatment (100mg/kg on 5 consecutive days) killed all adult L. sigmodontis but did not affect B. malayi. However, single doses of 6.25 and 25mg/kg resulted in severe pathological alterations of intrauterine stages of L. sigmodontis and B. malayi, respectively. These alterations may be responsible for long-lasting reductions of microfilaraemia even when curative effects could not be achieved.     

Immunity to Litomosoides carinii in Mastomys natalensis. I. Effect of immunization with microfilariae and existing primary infections on the parasitaemia after microfilariae injection and challenge infection

Subcutaneous injections of intrauterine stages of Litomosoides carinii into Mastomys natalensis induced strong immunity to i.v. injected blood microfilariae. Immunity, developed after boostering with an i.p. and an i.v. injection of microfilariae, did not totally suppress the parasitaemia of a challenge infection but reduced significantly the microfilaraemia level. No effect was found on number and size of the worms of the challenge infection, the number of microfilariae or the number of leucocytes in the pleural cavity. Delayed type hypersensitivity reactions in challenged animals were similar to those in non-immunized, infected controls. Sera of immunized animals agglutinated microfilariae and mediated cell attachment to microfilariae. Challenge infections did not change this until the end of the fourth week post infection but sera taken 32 days after challenge and later failed to induce such reactions. Challenge infections performed 120 or 240 days after a primary infection did not increase the parasitaemia of recipients. Dissections carried out 130 days after the challenge showed that (a) the developmental rate of the challenge infection was reduced by about 50%; (b) the size of the challenge parasites was reduced; and (c) that these worms produced significantly less embryonic stages in comparison to worms of primary infections, of which about 90% were abnormal.     

Separation of viable microfilariae free of blood cells on Percoll gradients

A consistent and reproducible method is described for isolating pure populations of microfilariae of Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae, free of cells, from blood, by density gradient centrifugation on Percoll in 0.25 M sucrose. The recovery of the microfilariae was 85 to 97%.     

Studies of potential filaricides: Part 14--Activity of 1-iso-butoxycarbonyl-4-methylpiperazine against experimental filariasis

The synthesis and filaricidal activity of 1-iso-butoxycarbonyl-4-methylpiperazine against Litomosoides carinii in Sigmodon hispidus and Dipetalonema viteae in Mastomys natalensis is reported. At an intraperitoneal or oral dose of 3 mg/kg given for 6 days, the compound removed 91% of the circulating microfilariae but had no effect on adult L. carinii. However, it killed all microfilariae and adults of D. viteae at a subcutaneous dose of 50 mg/kg given for 6 days. The compound also possessed chemoprophylactic activity against the larvae of L. carinii and D. viteae at a dose of 30 and 50 mg/kg respectively.     

Detection of antibodies in Brugia pahangi-infected cats by counter immunoelectrophoresis, indirect fluorescent antibody test and enzyme-linked immunosorbent assay

In cats infected with Brugia pahangi, antibodies first appeared against the larvae (L3), then against the adults (L5) and the microfilariae (mf). Homologous antigens were better than antigens prepared from heterologous species (Dirofilaria immitis, Dipetalonema viteae, Litomosoides carinii and Onchocerca gutturosa) in detecting antibodies to B. pahangi in the infected cats by indirect fluorescent antibody test (IFAT). Metabolic products of L5, but not L3 or mf, of B. pahangi were antigenic and were used in the enzyme-linked immunosorbent assay (ELISA) for detection of antibodies. Using various homologous antigens, IFAT was found to be more sensitive than counter immunoelectrophoresis and ELISA in the detection of antibodies in the infected cats. The best antigen was cryosections of L3, with a positivity rate of 81%. However, using L3, L5 and mf antigens in IFAT, a total positivity of 97% was obtained.     

Reaginic and homocytotropic IgG antibodies in Schistosoma mansoni infected Mastomys natalensis: time courses in untreated infections and after chemoprophylactic and chemotherapeutic treatment

Mastomys natalensis were infected percutaneously with 250 cercariae of Schistosoma mansoni and treated effectively with 5 X 50 mg amoscanate/kg body-weight 12-16 (I), 28-32 (II), 56-60 (III) and 101-105 days p.i. (IV) respectively. Levels of reaginic antibodies (RAb) and homocytotropic IgG antibodies (GAb) were assessed by passive cutaneous anaphylaxis tests (PCA) and compared with those in infected, untreated animals. In untreated animals RAb were first detected in the 6th week p.i. Maximum titres around 1:80 were reached after 100 days. High levels persisted until the end of the experiment 185 days p.i. RAb did not develop after treatment I. After treatment II only low PCA titres occurred. After treatment III the time course was similar to the controls but levels were reduced. Treatment IV did not interfere with the occurrence of specific RAb. In the case of GAb, independent of any treatment, after a first peak of PCA titres in the 5th week either a negative pattern or only very low titres were found in week 7. In untreated infected Mastomys GAb levels increased thereafter to maximum titres of about 1:80 at day 70. Later than 100 days p.i. relatively constant titres were observed. The second rise of antibody levels did not occur after early treatment. After treatment II only low transient titres developed. Animals treated in the early patency (III) showed reduced PCA titres and were negative at the end of the observation period. Treatment given 101 to 105 days p.i. rather enhanced the development of GAb.     

Studies on acquired resistance of the cotton rat against microfilariae of Litomosoides carinii. 2. Injection of microfilariae during prepatency

Cotton rats infected by infective third-stage larvae of Litomosoides carinii were treated at increasing time intervals by a threefold injection of living homologous microfilariae (mf) during the prepatent period. Starting with the first treatment 3, 4 or 5 weeks p.i. seven animals remained completely and two almost mf-negative (1 or 2 mf/mm3 each only once) until 16 weeks p.i. Starting 6, 7 or 8 weeks p.i. six animals developed a normal level of parasitaemia between 42 and 436 mf/mm3, two animals developed a continuous level of 1-2 mf/mm3. The number of fertile adult worms shedding great numbers of microfilariae in the pleural cavity was equal in all animals. However, in mf-negative animals the lung capillary blood showed, in the geometric mean, only 0.6% of the mf-concentration seen in mf-positive animals. The hypothesis is proposed that microfilariae accumulating primarily in the lung capillaries absorb all aggressive components specifically reacting with microfilarial antigens, i.e. neutralize the immune response against them to enable the development of the parasitaemia in the peripheral blood.     

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.     

Sharing of antigens among filarial species revealed by antibody dependent cell-mediated reactions

Antisera raised in albino rats against microfilariae ofLitomosoides carinii, Brugia pahangi, Brugia malayi and sera fromBancroftian elephantiasis patients promoted rat neutrophil-mediated adherence and cytotoxicity to the microfilariae. Pre-treatment of the immune sera, with microfilarial antigen at a final concentration of 5 and 25μg per ml blocked cellular adherence and cytotoxicity to the microfilariae indicating the presence of cross-reactive antibodies. The heterologous immune sera were effective in eliminating the circulatingLitomosoides carinii microfilariae inMastomys natalensis. Contribution No. 766 from Hindustan CIBA-GEIGY Research Centre.     

Brugia malayi: intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10(6) microfilariae. Ferrets (Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 +/- 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3-4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10(6) microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (greater than or equal to 1:200) and microfilarial agglutination titers (greater than or equal to 1:40) rapidly cleared injected microfilariae (less than 24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Occurrence of microfilariae in various hollow organs and in the peritoneal cavity after treatment of Litomosoides carinii infected Mastomys natalensis with diethylcarbamazine and haloxon

Treatment of Litomosoides carinii infected Mastomys natalensis with diethylcarbamazine (DEC: 500 mg/kg p.o.) was followed by increased occurrence of microfilariae in the bronchi of the host after 40 min and lasting at least until 6 h after treatment. After 4 h, increased levels of larvae were observed in the gut. Only a few microfilariae occurred in the bladder and sputum. Accumulations of microfilariae were found furthermore in the Lnn. hepaticae whereas no changes were observed in the inguinal or jejunal and lung and pleura associated lymph nodes. Increased numbers of microfilariae were found in the peritoneal cavity only after 8 and continuing until at least 48 h after treatment. In contrast, after haloxon treatment (100 mg/kg p.o.) an accumulation of microfilariae was found in the peritoneal cavity only, following a time course similar to that after DEC.     

Comparative utilization of pyruvate by Brugia pahangi, Dipetalonema viteae, and Litomosoides carinii

The metabolism of pyruvate by the adult filarial parasites Brugia pahangi, Dipetalonema viteae, and Litomosoides carinii has been compared. Istopic carbon-balance studies indicate the presence of significant pyruvate dehydrogenase activity in L. carinii but little or no activity in either B. pahangi or D. viteae. In all 3 helminths, the quantities of pyruvate that were completely oxidized to CO2 and water were very small. The activities of some of the tricarboxylic acid cycle enzymes of B. pahangi also were determined. In particular, a relatively low level of isocitrate dehydrogenase was noted in the mitochondria of B. pahangi. It is suggested that the tricarboxylic acid energy generating pathway is of doubtful importance as an energy yielding pathway in any of these parasites.     

Antibody responses of Wuchereria bancrofti patients to recombinant Brugia pahangi superoxide dismutase

Lymphatic filarial parasite Brugia malayi contains significant amount of Cu/Zn superoxide dismutase (SOD) activity in the extract of different life stages and in the excretory-secretory product of adults. In the present study recombinant SOD from B. pahangi has been used to see the antibody response in Wuchereria bancrofti infected patients. The recombinant SOD from B. pahangi reacted specifically with W. bancrofti infected sera in ELISA and immunoblotting. The reactivity of IgM subclass was more as compared to IgG subclass both in the asymptomatic microfilaraemic and symptomatic amicrofilaraemic when tested by ELISA. Serum from other helminthic infection was very low and found to be insignificant. The antibody response to rec SOD was directly proportional to the number of microfilariae in infected patients. The circulating filarial SOD was detected in filarial patients using polyclonal antibodies raised against recombinant Cu/Zn SOD in rabbits. The apparent molecular masses as determined by immunoblotting were 29 and 22 kDa. The specificity of recombinant SOD could be explored for its use in immunodiagnosis of lymphatic filariasis.     

Brugia malayi and Brugia pahangi: transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti

Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L2, and L3 larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L3 larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L3's recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microfilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microfilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microfilariae to develop to the L3 stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microfilariae also failed to develop to L3 in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 ng/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microfilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical and ultrastructural studies on Dipetalonema viteae (Filarioidea)

The antigenic properties of adult male and female of Dipetalonema viteae were studied by immunocytochemistry. Using antisera of the rodents Meriones unguiculatus and Mastomys natalensis infected with D. viteae, the binding of antibodies to sections of filariae embedded in Epon was assayed by the peroxidase-antiperoxidase (PAP) technique and by electron microscopy. The optimal staining intensity was obtained with an antiserum dilution of 1:5000. Control sera were obtained from sex and age matched uninfected animals. Female D. viteae showed maximal antigen-antibody reactions within the uterus: in the inner uterus wall, in the "nutrient channels" between the maturing eggs and the differentiating microfilariae, on the eggshells, in the cuticula of microfilariae and in the spermatheca on the cell membrane of the mature spermatozoa. Male filariae showed binding of antibodies in the vesicula seminalis: in the nucleus and the nuclear membrane of primary spermatocytes and on maturing spermatids. Less pronounced antigen-antibody reactions in the cuticula, muscle and intestine were observed in both sexes. The PAP-technique offers significant improvements in comparison with other techniques, e.g., immunofluorescence, used to detect antigens on filariae: the PAP-technique has an increased sensitivity with a concomitant reduction in nonspecific background and can be used for both light and electron microscopy; moreover, PAP-treated tissues can be stored indefinitely at room temperature.     

The carbohydrate metabolism of Brugia pahangi microfilariae.

Evidence is presented that the microfilariae of Litomosoides carinii, Dipetalonema viteae and Brugia pahangi have an aerobic requirement for motility, but possibly not for survival. In addition, the data suggest that in an in vitro anaerobic environment, B. pahangi microfilariae ferment glucose only as far as lactate. In an aerobic environment, however, the data are consistent with a portion of glucose being dissimilated via a one step oxidative decarboxylation of pyruvate formed from glycolysis to acetate and CO2. In addition, a low level of complete oxidation, possibly via a tricarboxylic acid cycle pathway, may be occurring. Finally, if B. pahangi microfilariae are immobilized with levamisole in an aerobic atmosphere, the drug appears to alter the aerobic glucose metabolism of the parasite both qualitatively and quantitatively. A decreased glucose utilization occurs, together with a shift to a more nearly homolactate fermentation. It is suggested that the effects of levamisole on the metabolism of the microfilariid are secondary to the observed paralysis.     

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.