Acanthocheilonema viteae: vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens

Jirds (Meriones unguiculatus) were immunized with irradiated (35 krad) stage-3 larvae (L3) of Acanthocheilonema viteae. The induced resistance against homologous challenge infection and the antibody response of the animals were studied. Immunization with 3, 2, or 1 dose of 50 irradiated L3 induced approximately 90% resistance. Immunization with a single dose of only 5 irradiated L3 resulted in 60.8% protection while immunization with a single dose of 25 L3 induced 94.1% protection. The protection induced with 3 doses of 50 irradiated L3 did not decrease significantly during a period of 6 months. Sera of a proportion, but not all resistant jirds, contained antibodies against the surface of vector derived L3 as defined by IFAT. No surface antigens of microfilariae or adult worms were recognized by the sera. Vaccinated animals had antibody responses against antigens in the inner organs of L3 and in the cuticle and reproductive organs of adult worms as shown by IFAT. Immunoblotting with SDS-PAGE-separated L3 antigens and L3-CSN revealed that all sera contained antibodies against two exported antigens of 205 and 68 kDa, and against a nonexported antigen of 18 kDa. The 205-kDa antigen easily degraded into fragments of 165, 140, 125, and 105 kDa which were recognized by resistant jird sera. Various antigens of adult worms, but relatively few antigens of microfilariae, were also recognized. To test the relevance of exported antigens of L3 to resistance, jirds were immunized with L3-CSN together with a mild adjuvant. This immunization induced 67.7% resistance against challenge infection and sera of the immunized animals recognized the 205- and 68-kDa antigens of L3.     

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.     

Attempted vaccination of jirds (Meriones unguiculatus) against Brugia pahangi with radiation attenuated infective larvae

Jirds were vaccinated by three to five subcutaneous (SC) injections of infective larvae of Brugia pahangi which had been irradiated at 25, 45 or 90 krads from a 60Co source. They were challenged either SC or intraperitoneally. Vaccination with four doses of 50 larvae irradiated with 25 krads produced 49.3% resistance to IP challenge worms and 39.8% against SC challenge worms. Five doses of larvae irradiated with 45 krads produced 62% resistance to SC challenge. Three doses of larvae irradiated with 90 krads produced 74.9% resistance to SC challenge and five doses produced 76.2% resistance. The reasons why irradiated larvae produce resistance whereas normal larvae do not are discussed.     

Dirofilaria immitis: fate and immunogenicity of irradiated infective stage larvae in beagles

Experiments were conducted on the fate of irradiated infective larvae of Dirofilaria immitis in dogs, and on the effect of these infections on a challenge dose of nonirradiated larvae administered at a later date. Six dogs were inoculated with 200 to 296 irradiated larvae; in no case was a patent infection established. No living worm was recovered beyond 66 days. Eight dogs inoculated with 200 to 2401 irradiated larvae over varying periods of time were exposed 57 to 190 days after the final inoculation of irradiated larvae, to a challenge infection of 200 to 250 nonirradiated (normal) larvae. The results showed that the number of worms which developed to maturity in these dogs was sharply reduced compared to that in the 5 controls (dogs inoculated with normal larvae only). The most striking effect was seen in “vaccinated” dogs which were challenged 3 months or more after the final administration of irradiated larvae.     

Development of Taenia asiatica cysticerci to infective stage and adult stage in Mongolian gerbils

The development of metacestodes and adult worms of Taenia asiatica in Mongolian gerbils (Meriones unguiculatus) were observed. Cysticerci were recovered from gerbils subcutaneously injected with hatched oncospheres. The recovery rate ranged from 0.1 to 3.2%. No cysticerci were recovered from the orally inoculated gerbils. The infectivity of the cysticerci recovered at 48 weeks post-infection was evaluated. Tapeworms were recovered on day 14 post-infection from the small intestine of 5 of 11 gerbils, with a recovery rate of 27% (6 worms recovered/22 worms inoculated). Three and four adult worms were recovered from two human volunteers who ingested five cysticerci after 4 months post-infection. In worms recovered from gerbils, segmentation and genital primordia in the posterior proglottids and hooklets in the residual rostellum were observed. The results indicate that gerbils can serve as an alternative intermediate host and that partial development of the adult worm stage occurs in gerbils.     

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.     

Alterations in the longevity and fecundity of adult Trichinella pseudospiralis related to method of isolation of infective larvae

The infectivity of Trichinella pseudospiralis infective larvae was reduced significantly following exposure to low pH or a combination of 1% pepsin at low pH compared to that for larvae isolated in phosphate-buffered saline (PBS) at pH 7.0. Reduction of host gastric pH by administration to mice of sodium bicarbonate solution in PBS was accompanied by an increase in the infectivity of larvae isolated in 1% pepsin/HCl (P/HCl) compared to that for worms inoculated into hosts given PBS alone. Fewer adult worms developing from larvae isolated in P/HCl became established in the host small bowel than was seen with larvae isolated in PBS; moreover, the fecundity in vitro of adult worms developing from P/HCl-isolated larvae was reduced below that for adults developing from larvae isolated from host muscle in PBS. More adult worms were recovered following infection of immune hosts with PBS-isolated larvae than were recovered from immune mice challenged with larvae isolated in P/HCl. Similar findings were observed in mice immunized by infection with Trichinella spiralis and challenged with T. pseudospiralis larvae isolated in either P/HCl or PBS. Immunization of mice with T. pseudospiralis larvae isolated by either method and challenged with larvae of T. spiralis resulted in recovery of similar percentages of the challenge inoculum.     

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.     

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%).     

Brugia pahangi in rats: increasing the number of infective larvae inoculated increases the percentage of microfilaremic rats

One hundred Brugia pahangi infective larvae (L3) caused microfilaremic (mf + ve) infection in 56% of inbred PVG rats. Adult worms were recovered consistently from infected rats but worm recovery was very low, only 1-3% of L3 inoculated survived to adulthood and the worms were dispersed in a wide range of anatomical sites. This suggested that lack of microfilaremia may be due to the low probability of male and female worms meeting in the same site and thus may be numerically and topographically based. When the number of infective larvae inoculated was increased to 500, the percentage of mf + ve infections in rats also increased to 94%, corroborating the hypothesis that lack of mf was not due to an immune response. In a further experiment all infected rats had lost both mf and adult worms by day 420. It has yet to be established whether final rejection of the parasite is due to immunity.     

Transplanted Dipetalonema viteae in the jird: effect of worm burden on parturition rates and microfilaremia

Dipetalonema viteae was studied in the jird, Meriones unguiculatus, to determine the mechanism controlling the level of peripheral microfilaremia. Jirds killed 40 days after infection served as donors of female worms of known age and reproductive status. These worms were transplanted into uninfected jirds and the resultant microfilaremias were monitored. After approximately 100 days, the recipient jirds were killed and 58% of the transplanted worms were recovered alive but depleted of sperm and microfilariae, regardless of the total number implanted in a given host. A direct linear relationship between microfilaremia and the number of recovered adult worms was found. Based on the uniform absence of sperm and microfilariae in the recovered worms it was concluded that female worms, under the conditions of the present study, do not control the peripheral microfilaremia in multi-worm infections through a reduced parturition rate.     

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)     

Studies with Brugia pahangi. 16. Precipitation antibody in infected cats detected by counterimmunoelectrophoresis

In cats infected with normal, or irradiated, infective (L3) larvae of Brugia pahangi counterimmunoelectrophoresis revealed the presence of antibody to soluble antigens derived from microfilariae, adults and infective larvae of the same parasite. Infected cats with a persistently high to moderate microfilaraemia gave positive precipitin reactions to L3, microfilarial and adult worm antigens. Cats which had become amicrofilaraemic had antibody to L3 and microfilarial antigens but not to adult worm antigen. Serum from cats inoculated with irradiated L3 larvae produced a precipitin reaction only to the L3 antigen.     

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.     

Nippostrongylus brasiliensis: local humoral responses in the lungs and intestines of rats following vaccination with irradiated larvae

Groups of rats were infected with 2000 normal larvae of Nippostrongylus brasiliensis or larvae irradiated with 10 to 120 kR. On Day 10 after infection half the animals from each group were autopsied. The remainder were challenged with 5000 unirradiated larvae on Day 15 and killed ten days later. During the experiment enteric antibody levels were estimated by coproantibody measurement. At autopsy the worm burdens were determined and worm-specific antibodies evaluated in lung extracts and serum. It was found that the levels of coproantibody detected with adult worm metabolites were positively correlated with the number of adult nematodes recovered from the intestine after primary infection. The challenge induced a similar increase of these antibodies in all immunised rats which reflected a high immunity to reinfection of vaccinated animals. Preliminary immunochemical studies suggested that the coproantibodies had SIgA properties. In lung extracts of rats immunised with larvae irradiated at 40, 80, or 120 kR and in all animals after challenge, antibodies reacting with infective larval antigens were found. Their titres were negatively correlated with serum antibody levels. The significance of bronchial and enteric antibodies in conferring protection against challenge remains to be elucidated.     

Recovery, distribution, and development of Acanthocheilonema viteae third- and early fourth-stage larvae in adult jirds

Living third- and fourth-stage larvae (L3 and L4) of Acanthocheilonema viteae were recovered quantitatively from adult Meriones unguiculatus within the first 10 days after subcutaneous inoculation of 60 arthropod-derived larvae (mL3). The average recovery of the inoculated larvae was about one third (28.5%), and the majority (87.7%) were found in muscular tissues. Seventy-two hours after inoculation, larvae could be isolated from all body locations, although the majority still was found near the site of inoculation. Morphological and biometrical data indicated that, at least until molting, the development of the larval population was not synchronous, with molting occurring over a period of 48 hr on days 7 and 8 postinoculation. The stomatal rings of postinvasive L3's and L4's were distinguishable structurally and could be used as stage-specific determinants. Immediately after infection, L3's showed a linear growth in diameter; rapid longitudinal growth started after the molt, leading to a doubling in the length of L4's within 4 days. The time course of shedding was reconstructed in detail using isolated L3/L4 intermediates.     

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.     

Brugia pahangi: production of a monoclonal antibody reactive with the surface of infective larvae.

Monoclonal antibodies against infective third-stage larvae (L3) of Brugia pahangi were generated from mice immunized with L3 antigens. The monoclonal antibodies were L3 stage-specific or stage-nonspecific. A BpG1 monoclonal antibody (IgG1 subclass) showing L3 stage-specificity was examined in detail. BpG1 recognized the surface of B. pahangi L3 and also reacted with the surface of Brugia malayi L3 but not with the surface of filarial worms of other genera, such as Acanthocheilonema viteae and Litomosoides carinii. BpG1 promoted cellular adhesion to the surface of B. pahangi L3. BpG1 bound on living L3 was shed but the shedding rate was relatively slow. The surface antigen recognized by BpG1 had a molecular weight of 58 kDa. It was stable to heat and periodate treatments but sensitive to trypsin digestion and was released from living L3 by SDS but not by Triton X-100 or CTAB. Preincubation of L3 with BpG1 significantly reduced the recovery rate of worms compared with the preincubation with a monoclonal antibody (IgG1 subclass) against the inner tissues of B. pahangi L3 or control supernatant of P3U1 myeloma cells. This result suggests that the antigen containing the BpG1 epitope may be one of the targets of a protective immune response against Brugia infection.     

Some observations on a possible role of lung and fecal IgA antibodies in immunity of rats to Nippostrongylus brasiliensis

Factors influencing lung IgA antibody responses to Nippostrongylus brasiliensis infections and the role of lung and fecal IgA antibodies in immunity to this nematode were studied in rats. Hooded Lister rats were vaccinated subcutaneously with infective larvae radio-attenuated at 80-180 kr or with a single dose of infective larvae somatic proteins administered intravenously or intragastrically, and then challenged 14 days later with normal larvae. It was found that optimal lung IgA antibody responses depended more on the duration of the antigenic stimulation than on the quantity of antigenic material present, although a threshold amount was required. However, comparisons of lung anti-larval IgA antibody levels in rats resistant or susceptible to challenge indicated that these antibodies were not directly involved in specific host protective immunity. Levels of haemagglutinating fecal antibodies reacting with adult nematode metabolites were correlated with the numbers of adult worms recovered from the intestines following vaccination and also with the degree of resistance to reinfection. However, preincubation of adult (day 5) nematodes in media containing the IgA fraction of fecal globulins from primary infected rats did not reduce the ability of these worms to establish and survive in naive rats.     

Concomitant immunity in a rodent model of filariasis: the infection of Meriones unguiculatus with Acanthocheilonema viteae

In an attempt to study the occurrence of concomitant immunity in filarial infections, jirds (Meriones unguiculatus) were experimentally infected with Acanthocheilonema viteae, and patent animals were superinfected with a defined dose of A. viteae stage 3 larvae (L3). Infected animals harbored significantly less worms deriving from the superinfection than the control group (P < 0.05, 56.2%, and 63.4% protection), as shown by analysis of female worms 6 wk after superinfection on the basis of their developmental status and their length. This protection was not due to contact with L3 antigens because a significant reduction of worm burdens deriving of a superinfection was also observed after subcutaneous implantation of a single female worm (P < 0.05, 40.2% and 64.9% protection). The induced protective responses target L3 and restrict their migration because an established infection resulted in a reduction of L3 recovery (95.6% and 94.3%, P < 0.001) from tissues of jirds at day 5 after superinfection. Other data show that L3 from a superinfection are trapped within eosinophil-rich granulomas, which is likely to create unfavorable conditions for the worms and to lead to later death. Taken together, established A. viteae-infections partially protect hosts against homologous superinfection by an immune-mediated mechanism and, thus, regulate the population density of the parasites within the host by concomitant immunity.