Protective immunity induced by irradiated third-stage larvae of the filaria Acanthocheilonema viteae is directed against challenge third-stage larvae before molting

Jirds (Meriones unguiculatus) were vaccinated with irradiated L3 third-stage larvae (L3) of Acanthocheilonema viteae, and the time required for killing of the challenge L3 was determined. The number of parasites recovered from vaccinated jirds was reduced to about 10% of the control values on the second day after challenge infection and later on. Histological studies revealed an eosinophil-rich infiltrate containing macrophages, neutrophils, and mast cells in the vicinity of the L3 on day 2 after challenge and destruction of the worms by day 4 after challenge. Ultrastructural studies confirmed these data and showed that eosinophils, macrophages, and mast cells were close to the L3 on day 2 after challenge. Flattening of the eosinophils onto the surface of the worms, degranulation of electron-dense material, and rupture of the L3 surface was observed on day 4 after challenge, followed by invasion of the inner of the worms by phagocytic cells. These data show that immune attack against the challenge L3 in vaccinated jirds is initiated between the first and the second day after challenge and that killing occurs around the fourth day after challenge, before the worms undergo their first molt.     

Litomosoides carinii: retardation of worm growth and of migration of challenge infections in jirds (Meriones unguiculatus)

Inbred jirds (Meriones unguiculatus) were divided into three groups; each animal in two of the groups was infected with 30 infective larvae (L3) of Litomosoides carinii. When these infections were patent, the jirds of one of the two infected groups plus those of the third group were injected with 30 L3 L. carinii each. All animals were killed either on day 14 or 24 after the second infection for the recovery, enumeration and measurement of all worms and developing larvae. Challenge larvae were stunted (smaller) and fewer than control larvae. Additionally, fewer challenge larva were recovered on day 14 than on day 24, indicating that migration to the pleural cavity was retarded.     

Induction of lymphatic lesions by Brugia pahangi in jirds with large and small preexisting homologous intraperitoneal infections

The effect of intraperitoneal (i.p.) infections induced by inoculations of 30 or 150 Brugia pahangi third-stage larvae (L3) on the development of infections and lymphatic lesions induced by subsequent homologous subcutaneous (s.c.) inoculations were compared in the present study. Lymphatic lesion severity, as judged by the numbers of lymph thrombi present, and lymphatic lesion scores were significantly reduced in both groups of jirds with existing i.p. infections. The numbers of adult worms that developed, locations of these worms, and the subsequent microfilaremias did not differ significantly between groups. All jirds with i.p. infections developed similar antibody titers to crude somatic adult antigen as measured by ELISA. These levels did not change following s.c. infections. Immediate and delayed footpad swelling responses were also similar in all groups. Results of these experiments support and extend previous studies indicating that i.p. infections of B. pahangi induce a hyporesponsive state in jirds to subsequent s.c. infections without significantly affecting the subsequent parasite burden. This effect appears to be independent of the numbers of L3 inoculated i.p. prior to lymphatic-induced infection. Circulating antibody titers and footpad swelling responses to B. pahangi antigen were not reduced in jirds with the hyporesponsive lymphatic inflammatory response and do not correlate with this condition.     

Growth and development of Haemonchus contortus in jirds, Meriones unguiculatus.

Growth and development of Haemonchus contortus were examined in jirds and were compared to these processes in lambs. Number, sex, size, and stage of development were determined for worms recovered at necropsy at various times postinoculation (PI) from immunosuppressed jirds inoculated with approximately 1,000 exsheathed infective larvae (L3) of H. contortus. In addition, gastric tissue samples from jirds were examined histologically. Parallel studies were done in lambs inoculated with approximately 7,500 L3. Typically, 5-30% of the inoculum established and survived in jirds at reasonably stable numbers to day 14 PI. By day 21 PI, worm numbers in jirds decreased dramatically. Although the parasite was similar in size and development on day 4 PI in jirds and lambs, from day 7 PI on, worms were significantly smaller and less developed in jirds. On histological examination, the parasite was found only in the glandular portion of the stomach of jirds (anatomically similar to its predilection site in the abomasum of lambs), and histological changes were consistent for both host species. Although growth and development of H. contortus are slower and incomplete in jirds, the parasite establishes, grows, and develops (at anatomically comparable sites in both hosts) in this model. Thus, the model appears to provide a useful laboratory host to study H. contortus.     

The anthelmintic activity of a novel organic arsenical, R7/45, upon Brugia pahangi in Meriones unguiculatus

The new organic arsenical R7/45 is a rapidly acting and very potent anthelmintic against adult Brugia pahangi in jirds. Against adult worms implanted into the peritoneal cavity 5 subcutaneous (SC) injections at 2.5 mg/kg of R7/45 killed 100% of adult worms. A single dose SC of 20 mg/kg was 100% effective and 10 mg/kg 76% effective against adult worms. When jirds were autopsied at different times after treatment at 20 mg/kg SC 89% of worms were dead within three days. R7/45 was not active when given by stomach intubation. Pretreatment of jirds with R7/45 had no effect on adult worms subsequently implanted into jirds. R7/45 was highly active against third and fourth stage larvae of B. pahangi in jirds.     

Acquired immunity in rats against Angiostrongylus cantonensis infection

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).     

Induction of host resistance to Brugia pahangi in jirds (Meriones unguiculatus) protected by chemoprophylaxis

Jirds were given a chemoprophylactic inoculation of flubendazole (FMBZ) and then five injections of infective larvae of Brugia pahangi whilst still protected by the FMBZ. When the drug was thought to be non-effective the jirds (and controls) were given a challenge infection of B. pahangi larvae. By comparison with control jirds the treated-infected-challenged jirds had 40% fewer adult worms. The control treated-challenged jirds contained mostly sterile female worms showing that they were still partially protected by FMBZ but worms numbers were not significantly reduced as compared with untreated controls.     

Oral transmission of Brugia pahangi and Dipetalonema viteae to adult and neonatal jirds

Sullivan J. J. and Chernin E. 1976. Oral transmission of Brugia pahangi and Dipetalonema viteae to adult and neonatal jirds. International Journal for Parasitology6: 75–78. Confirming previous studies, anaesthetized adult jirds became infected after oral doses of 100 infective larvae of B. pahangi; 4 of 5 jirds became microfilaria-positive and all 5 harbored adult worms. Among 7 unanaesthetized adult jirds similarly exposed, none developed microfilaraemia although 5 each harbored a few adult worms. In these unanaesthetized jirds, presumably, rapid passage of the inoculum through the mouth permitted fewer larvae to penetrate the mucosa, and the rest were probably killed in the stomach. Unanaesthetized 4-day old jirds proved highly and equally susceptible to oral or subcutaneous infection with B. pahangi as indicated by microfilaraemias and large worm-burdens. Direct and indirect evidence suggest that the baby's stomach and small intestine are not inimical to swallowed larvae, thus accounting for the relatively numerous mature worms in the peritoneal cavity. Third-stage larvae of D. viteae, readily infective subcutaneously, succeeded relatively infrequently in maturing when given orally to anaesthetized adult or to unanaesthetized baby jirds. Consistent oral infectivity may thus be a feature of filariae more closely related to B. pahangi.     

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.     

Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia

Wolbachia endosymbiotic bacteria have been shown to be widespread among filarial worms and could thus play some role in the biology of these nematodes. Indeed, tetracycline has been shown to inhibit both the development of adult worms from third-stage larvae and the development of the microfilaraemia in jirds infected with Brugia pahangi. The possibility that these effects are related to the bacteriostatic activity of tetracycline on Wolbachia symbionts should be considered. Here we show that tetracycline treatment is very effective in blocking embryo development in two filarial nematodes, B. pahangi and Dirofilaria immitis. Embryo degeneration was documented by TEM, while the inhibition of the transovarial transmission of Wolbachia was documented by PCR. Phylogenetic analysis on the ssrDNA sequence of the Wolbachia of B. pahangi confirms that the phylogeny of the bacterial endosymbionts is consistent with that of the host worms. The possibility that tetracycline inhibition of embryo development in B. pahangi and D. immitis is determined by cytoplasmic incompatibility is discussed.     

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.     

Possible direct effect of diethylcarbamazine on the infective larvae of Brugia pahangi

The direct action of diethylcarbamazine (DEC) on the infective larvae of Brugia pahangi was studied. The larvae were cultured in RPMI 1640 supplemented with foetal bovine serum and antibiotics for 22 days. Most of the larvae remained alive for 8 days, but survival rate of larvae decreased rapidly from day 10 onwards. The larvae did not grow in the culture system. The addition of DEC did not affect the morbidity of the larvae and no difference was observed in the morphological characteristics between the larvae cultured in the presence or absence of DEC. The infective larvae were cultured in vitro for 5 days in the presence or absence of DEC, and inoculated into jirds. The animals were necropsied at intervals, and developing larvae and adult worms were recovered. When the larvae were cultured without DEC and then inoculated subcutaneously into jirds, 29.8% of the inoculum was recovered 3-15 days, and 25% 19-22 weeks, post-inoculation. However, when the larvae were exposed to DEC in vitro and inoculated into jirds, the rate of recovery was reduced to 25% 3-15 days post-inoculation and 2% after 19-22 weeks. When the control larvae cultured in vitro were inoculated intraperitoneally into jirds, 41.3% of inoculum was recovered 3-15 days, and 42.8% 19-22 weeks, post-inoculation. Again the corresponding value for larvae exposed to DEC in vitro was reduced to 19.8% 3-15 days, and 8% 19-22 weeks, post-inoculation. It was observed that the larvae exposed to DEC in vitro were retarded in their development in jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of surrogate rodent hosts for larval Onchocerca lienalis and induction of protective immunity in a model system.

The objectives of this project were to screen a variety of inbred rodent species and strains to determine their usefulness as surrogate hosts for the study of the early larval development of Onchocerca lienalis and then to use a selected model to study the induction of protective immunity. In the primary screen, 6 strains of mice, 5 strains of rats, jirds, and multimammate rats were tested. Animals were infected with fresh O. lienalis by subcutaneous implantation of third-stage larvae (L3) contained in diffusion chambers covered with 5.0-microns pore-size membranes. After 7 days the chambers were recovered, and larval viability and growth were assessed. Approximately one-half of inoculated larvae were recovered alive regardless of the host tested. Larvae were implanted in CBA/J and DBA/2J mice in chambers covered with membranes that prevented host cells from entering; survival and growth rates of the larvae were not altered by the absence of cells from the chambers. Cryopreserved larvae were implanted in chambers with 5.0-microns pore-size membranes in CBA/J and DBA/2J mice and Wistar Furth rats for 3-28 days. No statistically significant difference was seen in the larval recoveries on days 3-28 in all 3 hosts. Statistically significant increases in length were seen in the 3 strains from day 3 to day 14, after which growth appeared to cease. Molting from L3 to fourth-stage larvae was observed in all 3 hosts beginning on day 3, with most larvae completing the molt by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brugia pahangi: granulomatous lesion development in jirds following single and multiple infections

The development of adult worm burdens and microfilaremias were determined in jirds which received 2, 3, or 4 subcutaneous inoculations of 50 Brugia pahangi infective larvae. Parasite burdens in multiply inoculated jirds were compared to those in four different groups of jirds which received single inoculations of 50 infective larvae. One of each of these singly inoculated groups was infected on the same day that one of the inoculations was given to the multiply infected jirds. Thus, the duration of the infections in the four groups of jirds receiving one inoculation was 54, 118, 189, and 254 days. The development of lymphatic lesions and granulomatous hypersensitivity to B. pahangi antigen was assessed in all jirds at necropsy. The percentage recoveries of adult worms and their locations did not differ in the singly inoculated jirds with infections of different durations. A protective resistance to reinfection, as measured by adult worm recovery in multiply infected jirds, did not occur. The lymphatic lesion scores and numbers of intralymphatic thrombi was greatest in singly inoculated jirds examined 54 days after infection. Pulmonary granuloma areas around adult filarial antigen coated beads embolized in the lungs of jirds 3 days prior to necropsy were also greatest in singly inoculated jirds examined 54 days after infection. Using criteria of lesion scores and lymph thrombi numbers to assess lymphatic lesion severity, a decrease in lesion severity as well as pulmonary granuloma size around antigen coupled beads was seen by 118 days after infection in singly inoculated jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apodemus sylvaticus, a new host for Acanthocheilonema viteae (Nematoda: filarioidea)

Susceptibility of Apodemus sylvaticus and A. agrarius to infection with Acanthocheilonema viteae was compared with that of hamsters and jirds. Microfilaremia in A. sylvaticus was first noted on day 52 post-infection (p.i.) and lasted during the course of the study (up to day 150 p.i.). Maximum microfilaremic levels (female worm basis) of A. sylvaticus [mean +/- S.D. (n) = 690 +/- 1288(6)] were considerably higher than those of hamsters [16 +/- 18(6)] and jirds [51 +/- 25(5)]. Adult worm recovery in A. sylvaticus ranged from 2 to 40% of the number of infective larvae inoculated. Worm development in A. sylvaticus resembled that in hamsters and jirds. In contrast, microfilaremia was not detected in, nor adult worms recovered from A. agrarius throughout the study.     

Effect of flubendazole on the number of first-stage larvae of Angiostrongylus cantonensis released in the faeces of treated rats

The effect of flubendazole orally administered at 10 mg/kg/day for 5 consecutive days (the 11th, 20th or 40th post-infection) on the number of first-stage larvae (L1) of Angiostrongylus cantonensis released in the faeces of rats each infected with 40 third-stage larvae was determined. Faecal examination for 5 months, the period from medication to dissection of rats, showed that L1 release ceased in all the rats of medicated groups by about 1 week after the termination of dosing and resumed 1-2 months later in 86% of the rats which were dissected at the end of experiments with the recovery of adult worms of both sexes. Throughout the period of 5 months, about 2-4 x 10(4) L1/gram of fresh faeces was recorded in non-medicated control groups. There was a 38-79% reduction in adult worms at the dissection. Microscopic examination of the uteri of the remaining adult worms and lung tissues of rats confirmed no normal egg production in the adult worms from rats of medicated groups, except the rats with the resumption of faecal L1 release.     

Angiostrongylus costaricensis: culture of third-stage larvae to young adults in a defined medium

The third-stage larvae of Angiostrongylus costaricensis were successfully cultured to young adults in a chemically defined medium. The most suitable medium for the development was Waymouth's medium among eight defined media examined. Twenty-eight days after cultivation in this medium, 77% of the larvae developed to young adults, although these worms gradually died thereafter. When Waymouth's medium was supplemented with mouse red blood cells, these young adult worms continued their development. The mean body lengths of the worms cultivated in Waymouth's medium supplemented with RBCs were significantly larger than those of the worms in the medium without RBCs on Days 14 and 21 after cultivation. Addition of RBCs was essential for their further development. At 28 days after cultivation, the maximum body length of the worms was 2.1 mm for males and 3.3 mm for females. Additions of serum, yeast extract lactalbumin hydrolysate, and growth factors to Waymouth's medium did not provide any additional benefits for worm development.     

In vitro cultivation of Dipetalonema viteae third-stage larvae: effect of the gas phase

The effect of the gas phase on the in vitro growth and development of Dipetalonema viteae (Nematoda: Filarioidea) third-stage larvae obtained from the tick vector and 3 day infections of jirds was examined. Measurements of the oxygen (pO2) and carbon dioxide (pCO2) tensions and the pH in the medium were made for each gas phase. In cultures gassed with 5% carbon dioxide in nitrogen the pO2 was between 32 and 50 mm Hg, the pCO2 ranged from 25 to 40 mm Hg and the pH was between 7.2 and 7.4. This gas phase resulted in the best growth and development of third-stage larvae to the fourth-stage. Survival and development of larvae were decreased in cultures with oxygen tensions less than 20 mm Hg and greater than 50 mm Hg.     

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.