Comparison of the protective resistance induced by 60Co-irradiated cercariae and schistosomula of the WFFS and NMRI strains of Schistosoma mansoni

Mice, CBA/HT6T6 and C57BL/10, were vaccinated with 1 X 350 or 1 X 500 Schistosoma mansoni cercariae or schistosomula attenuated with 20 or 56 krad 60Co irradiation and challenged with 200 cercariae. Protective resistance against homologous strain challenge was compared using the Winches Farm Field Station (WFFS) and Naval Medical Research Institute (NMRI) strains of S. mansoni. Maximal resistance to challenge was obtained in both strains of mice with cercariae or schistosomula of either WFFS or NMRI strain attenuated with 20 krad. Protection using organisms attenuated with 56 krad was significantly lower. Since previous studies with the two parasite strains have shown that the biological effects of irradiation are similar, the difference in the immunogenicity of the 56 krad-irradiated NMRI strain in this study from earlier studies must be due either to different local conditions for irradiation or to adaptation of the NMRI strain to a new laboratory environment. This finding may have important implications for vaccination studies and investigations of the mechanisms of immunity where radiation-attenuated parasites are used.     

Fasciola hepatica: site of resistance to reinfection in cattle

The effective site of resistance to reinfection of cattle with Fasciola hepatica was examined by recovery of challenge flukes from either the liver or body cavity. Calves infected 18 or 26 weeks previously with F. hepatica showed levels of resistance to reinfection of 56 and 94%, respectively, as assessed by recovery of flukes from the liver 15-16 weeks after challenge. Plasma glutamate dehydrogenase (EC 1.4.1.3; GLDH) enzyme activity estimations revealed only a marginal increase in these latter resistant calves compared with previously naive controls, indicating minimal liver damage as a result of migrating flukes. By comparison, when immature challenge flukes were recovered from the body cavity 4 or 14 days after infection of corresponding previously infected or naive calves, there was no significant difference in numbers. It appears, therefore, that, in cattle, resistance mechanisms are effective against challenge flukes at or soon after penetration of the liver.     

Immunization of mice with gamma-irradiated intramuscularly injected schistosomula of Schistosoma mansoni

The parameters involved in the induction of resistance against Schistosoma mansoni by injection of irradiated, artificially transformed schistosomula were studied in mice. Single intramuscular injections of 500 schistosomula exposed to radiation doses in the range 2.3 to 160 krad. resulted in significant protection (in the range 20 to 50% as assessed by reduced worm burdens) against a challenge infection administered at intervals from 3 to 24 weeks post-vaccination. However, schistosomula irradiated with 20 krad. consistently resulted in better protection than those exposed to either higher or lower radiation doses despite the persistence of stunted adults from the infections irradiated with 2.3 krad. Vaccination with 40 krad. schistosomula resulted in significant protection in terms of reduced worm and tissue egg burdens and increased survival following lethal challenge. Varying the number of irradiated schistosomula, the frequency and route of their administration, the site of challenge and the strain of host all failed to enhance the level of resistance. However, percutaneously applied, irradiated cercariae were found to be more effective in stimulating resistance (60%) than intramuscularly injected, irradiated schistosomula (40%).     

Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae.

To provide a theoretical basis for the potential development of vaccines against Onchocerca volvulus (Ov) a trial has been conducted to assess the protective efficacy of immunization of chimpanzees with X-irradiated L3 larvae. Approximately 1000 larvae were injected at 0, 1, and 7 months. The immunized animals, and unimmunized controls, were then challenged with 250 live L3. In order to provide possibly protective exposure to the immunologically distinct L4 epicuticle, a radiation dose (45 krad) was chosen which preserved about 50% of the molting ability of unirradiated larvae. Despite the presence of a strong immune response to crude adult worm extracts, and to cloned Ov antigens, at the time of challenge little or no significant protection against patent infection was observed: three of four immunized animals developed patent infection as compared to four of four controls. One immunized animal failed to become patent or to manifest the late antibody response to adult worm antigens seen in both subpatent and patent infections in this model, and may have been protected from infection. The implications of these studies for future attempts to immunize against O. volvulus are discussed.     

The fate of Fasciola hepatica metacercariae following challenge infection of immune rats

Groups of rats, infected 7 weeks previously with Fasciola hepatica, together with appropriate control groups, were challenged either orally or intraperitoneally with 30 metacercariae. The mean worm recovery from the previously infected, orally challenged rats was significantly lower than from their respective controls (2.2 +/- 1.1 worms as opposed to 9.0 +/- 2.6). There was no significant difference in mean worm recovery from the previously infected, intraperitoneally challenged rats and their respective controls (5.3 +/- 3.2 worms as opposed to 6.2 +/- 1.9). Livers of the orally challenged group appeared to be largely free from secondary damage but considerable damage was evident in rats which received an intraperitioneal challenge. This evidence supports the view that the gut acts as an important barrier to metacercariae of a challenge infection. In a further experiment, young flukes were recovered from the gut, abdominal cavity and liver of immune and control rats 9, 18, 27, 36 and 45 h after oral challenge. It was found that fewer flukes successfully penetrated the guts of immune rats (3%) than those of uninfected controls (13%), again pointing to the gut as a barrier to metacercariae of a challenge infection. Protective mechanisms that may operate at the level of the gut are discussed.     

Babesia major: protection of intact calves against homologous challenge by the injection of irradiated piroplasms.

Blood from a splenectomized calf infected with Babesia major was divided into 20 ml aliquots which were γ-irradiated at doses of 0, 23.3, 27.3, 31.4, 35.4 and 39.5 krad and then inoculated into groups of three intact calves. Animals receiving non-irradiated blood had typical mild B. major reactions, but those receiving blood irradiated at 23.3, 27.3 and 31.4 krad and 2 of 3 receiving blood irradiated at 35.4 krad had minimal reactions. The remaining 4 animals had no detectable parasitaemic reactions. When the calves were challenged with a similar number (6.0 × 10⁹) of homologous parasites, they were all immune with the exception of the 4 animals which had not reacted initially. The immune status of individual cattle was reflected accurately in the results of the micro-ELISA test, which detected a significant rise in serum antibody titre of the 4 susceptible animals 7 days after challenge.     

Schistosoma mansoni: migration potential of normal and radiation attenuated parasites in naive guinea pigs

Compressed tissue autoradiography using [75Se]selenomethionine labelled parasites has been used to investigate the migration potential of normal and radiation attenuated cercariae of Schistosoma mansoni in naive guinea pigs. By Day 14 after infection. 44% of normal parasites were detected as reduced silver foci in the liver; this value corresponded well with the number of liver parasites recovered by retrograde perfusion of the hepatic portal system on Day 42 (42% of the challenge). In contrast, cercariae subjected to 50 krad of gamma irradiation failed to migrate out of the skin. The migration capacity of 20 krad irradiated parasites was less severely affected in that about half of the challenge parasites reached the lungs, but virtually none moved to the liver. These data are discussed in relation to the kinetics of immunity induced in guinea pigs by infection or vaccination with normal or radiation attenuated parasites.     

Host-parasite relationships of Fasciola hepatica in the white mouse. VIII. Successful vaccination with culture incubate antigens and antigens from sonic disruption of immature worms

Mice were successfully vaccinated using culture incubate and sonicate antigens from 16-day-old flukes. Various injection schedules using the culture incubate antigen decreased challenge worm counts by 54 to 86%. The best results were achieved when the culture incubate was injected at 12 and 24 hr of incubation. Host mortality in the natural immunity controls ranged from 33 to 42%, and in the vaccinated animals from 12.5 to 25%. Functional antigens were present by 12 hr of incubation. A single immunizing injection with the sonicate antigen decreased challenge worm counts by 86%. Two immunizing injections with this antigen decreased challenge worm counts by 82%. However, the pathologic process in the liver was more severe than in animals that received a single injection.     

Schistosoma mansoni: vaccination of mice with 10-krad-irradiated, cryopreserved schistosomules

Protection against a Schistosoma mansoni cercarial challenge was evaluated in mice immunized with a vaccine composed of 10-krad-irradiated, cryopreserved schistosomules. The level of resistance induced in C57B1/6 or NMRI (CV) mice increased with the number of schistosomules injected. Up to 83% reduction in challenge worm burden was achieved when 5000 schistosomules were injected per mouse. Intramuscular injection of the vaccine was superior to subcutaneous. Multiple immunizations, up to 3 at 4-week intervals, did not increase the resistance induced by a single immunization. A high level of protection developed in as little as 2 weeks and was maintained through at least 12 weeks postimmunization. The vaccine irradiated with 10 krad from either a 60-cobalt or 137-cesium source induced equivalent levels of resistance, and no differences were found in the immunogenicity of vaccines comprised of organisms irradiated as cercariae or as 1- to 3-hr-old schistosomules. These findings are basic to the development of a cryopreserved, live vaccine against schistosomiasis of humans or domestic animals.     

Revaccination of Cattle with Bacille Calmette-Guérin Two Years after First Vaccination when Immunity Has Waned,Boosted Protection against Challenge with Mycobacterium bovis

In both humans and animals, controversy exists concerning the duration of protection induced by BCG vaccine against tuberculosis (TB) and whether revaccination enhances protection. A long-term study was undertaken to determine whether BCG-vaccinated calves would be protected against challenge with Mycobacterium bovis 2½ years after vaccination and to determine the effect of revaccination after 2 years. Seventy–nine calves were divided into five groups (n = 15–17 calves/group) with four of the groups vaccinated subcutaneously with 10⁵ CFU of BCG Danish at 2–4 weeks of age and the fifth group serving as non-vaccinated controls. Three of the four BCG-vaccinated groups were revaccinated 2 years after the initial vaccination. One BCG-vaccinated group was revaccinated with BCG. A second group was vaccinated subcutaneously with a TB protein vaccine consisting of biopolyester particles (Biobeads) displaying two mycobacterial proteins, ESAT-6 and Antigen 85A, mixed with an adjuvant. A third group was vaccinated with TB proteins from M. bovis culture filtrate, mixed with an adjuvant. Twenty-three weeks after the BCG revaccination, all animals were challenged endotracheally with virulent M. bovis and a further 13 weeks later, animals were killed and necropsied to determine protection against TB. The BCG-vaccinated animals produced positive tuberculin caudal fold intradermal (15 of 62 animals) and IFN-γ TB test responses (six of 62 animals) at 6 months after vaccination, but not at subsequent time-points compared to the non-vaccinated animals. Calves receiving a single vaccination with BCG vaccine 2½ years prior to challenge were not protected against TB, while those revaccinated with BCG 2 years after the initial vaccination displayed significant reductions in lung and pulmonary lymph node lesion scores compared to the non-vaccinated animals. In contrast, no reduction in lesion scores was observed in the animals revaccinated with the TB protein vaccines with their immune responses biased towards induction of antibody.     

The response of lambs to vaccination and challenge with Trichostrongylus colubriformis: Effect of plane of nutrition on,and the inter-relationship between,immunological responsiveness and resistance

Wagland B. M., Steel J. W., Windon R. G. and Dineen J. K. 1984. The response of lambs to vaccination and challenge with Trichostrongylus colubriformis: effect of plane of nutrition on, and the inter-relationship between, immunological responsiveness and resistance. International Journal for Parasitology14: 39–44. Merino lambs weaned at 8 weeks of age were fed either ground and pelleted (high plane, HP) or chopped (low plane, LP) lucerne hay ad libitum to achieve an approximate 2-fold difference in liveweight gain. When aged 17 and 21 weeks, 15 of the 20 lambs in each diet group were vaccinated with 80,000 irradiated T. colubriformis larvae. At 25 weeks, vaccinated and unvaccinated lambs were treated with anthelmintic and one week later challenged with 30,000 normal larvae. Four weeks after challenge the animals were killed for worm counts. After vaccination HP lambs had higher titres of antibodies to the parasite and after challenge had lower worm egg outputs, and lower worm burdens than LP lambs. Immunological responsiveness (serum titre of complement-fixing antibodies against worm antigen) and manifestations of resistance (eggs produced per female worm per day and percent protection calculated from worm counts) were significantly correlated within dietary groups. Percent protection and egg production per female worm were highly correlated (r = ?0.81) in individual animals pooled over dietary groups, suggesting that both manifestations of resistance respond to essentially the same immunological mechanism. Failure to obtain significant correlation between weight gain pre-vaccination and immunological and resistance parameters indicated that animal production and resistance to infection are not genetically linked. Negative correlation of weight gain during the vaccination period with serum antibody titre at challenge suggests that the developing immune response competes with weight gain for limited physiological resources of the animal.     

Babesia bigemina: immune response of cattle inoculated with irradiated parasites

Effects of various radiation dosages on the infectivity and immunogenicity of Babesia bigemina were studied. Calves infected with 1 × 10¹⁰, B. bigemina parasitized erythrocytes exposed to 24 krad developed progressive parasitemias. Some calves receiving 1 × 10¹⁰ parasitized erythrocytes irradiated at 36 krad did not develop progressive infections. Progressive infections were prevented by exposure to irradiation at 48 and 60 krad. A degree of acquired resistance to infection with B. bigemina developed in calves after inoculation with parasites irradiated at 48 and 60 krad. The resistance developed was sufficient to suppress multiplication of the Babesia and to permit calves to survive otherwise severe clinical infections due to challenge with nonirradiated parasites. Irradiated parasites were frozen without apparent loss of immunizing properties.     

Efficacy of UV-irradiated larval vaccine of Ancylostoma ceylanicum (Looss, 1911) in golden hamsters (Mesocricetus auratus)

A vaccination trial in golden hamsters with UV-irradiated infective larvae of Ancylostoma ceylanicum was attempted. One oral vaccination of hamsters with 100 infective larvae irradiated by means of UV-tube (390 nm) at different time intervals induced the development of resistance. As the time exposure of irradiation was increased, there was a corresponding decrease in the subsequent worm establishment. A high level of protection afforded by larvae irradiated for 15 min UV-exposure was recorded giving 99.0% and 95.0% worm reduction against the challenge doses of 100 and 1000 normal larvae respectively. There was no marked difference in worm establishment in hamsters vaccinated either orally or subcutaneously, followed by oral challenge. In the vaccinated hamsters, the manifestations of resistance at 15 min UV-exposure were shown by marked reduction in worm establishment and highly reduced epg in pellets with significantly higher blood haemoglobin levels compared with those given normal larvae as vaccine and challenge controls.     

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.     

Oesophagostomum radiatum: successful vaccination of calves with high molecular weight antigens

Extracts of adult Oesophagostomum radiatum were resolved into four fractions by gel filtration chromatography on a Superose 12 column. ELISA assays on these four fractions showed that the antibodies produced by naturally infected calves predominantly reacted with the void volume fraction (VV). Three trials were conducted in which calves were vaccinated with the VV fraction of adult extract. When compared to untreated controls, vaccination with the VV of adult extract significantly (P less than 0.05) reduced worm establishment and faecal egg output after challenge infection relative to untreated controls.     

Fasciola hepatica in rats: transfer of immunity by serum and cells from infected to F. hepatica naive animals

Immune, hyperimmune, and nonimmune serum samples were collected from inbred rats following 10 to 15 weeks of one [5 metacercariae (mc)/rat], two (5 mc followed by 30 mc/rat) or no (uninfected) exposure to Fasciola hepatica. Lymphoid cells also were collected from these donors. Inbred, naive rats in groups receiving immune serum, hyperimmune serum, nonimmune serum (serum control), immune cells, hyperimmune cells, and nonimmune cells (cell control) received intraperitoneally either a total of 20 ml of serum or a total of 3 x 10(8) viable lymphoid cells. A challenge infection of 30 mc/rat was administered orally at about the time of serum or cell transfer. The transfer of immunity was evaluated by examining recipient rats for parasites 4 and 8 weeks after challenge. Some hematological parameters and the precipitating antibody response of the recipients were monitored also. Hyperimmune serum, unlike immune serum, consistently provided a significant degree of protection in recipient rats. The precipitating antibody titre of this serum was higher than that obtained from the immune donor group. The importance of a second sensitization to obtain sufficiently potent serum was demonstrated. Lymphoid cells from infected donors did not consistently confer protection on recipients. Thus, the expression of protective immunity against F. hepatica seemed to be more dependent on the presence of antibodies than on cells. The hematological parameters of the recipients, in general, supported this observation. The precipitating-antibody response of protected rats was lower than that of unprotected animals following challenge, presumably because the development of fewer worms in the former provided less antigenic stimulation.     

Epidemiological Survey on the Infection of Intestinal Flukes in Residents of Muan-gun, Jeollanam-do, the Republic of Korea

Infection status of intestinal flukes was investigated in residents of Muan-gun, Jeollanam-do, the Republic of Korea. Total 1,257 fecal samples of residents were examined by formalin-ether sedimentation technique and Kato-Katz thick smear method. Helminth eggs were detected from 95 (7.6%) residents, and eggs of heterophyid flukes and Clonorchis sinensis were found from 62 (4.9%) and 40 (3.2%) cases, respectively. The larger heterophyid eggs, somewhat dark-brown in color and 37.7 × 21.5 µm in average size, and found in 32 (2.6%) out of 62 egg positive cases of heterophyid flukes. To confirm the adult flukes, we performed worm recovery from 12 cases after praziquantel treatment and purgation with MgSO₄. A total of 1,281 adult flukes, assigned to 7 species, were recovered from 9 cooperative cases. Heterophyes nocens (total 981 specimens) was collected from 9 cases, Stictodora fuscata (80) from 7, Gymnophalloides seoi (75) from 5, Pygidiopsis summa (140) from 3, Stellantchasmus falcatus (3) from 2, and Stictodora lari and Acanthotrema felis (each 1 worm) from 1 case each. The intrauterine eggs of S. fuscata collected from the recovered worm were identical with the larger heterophyid eggs detected in the stool examination. By the present study, it was confirmed that A. felis is a new intestinal fluke infecting humans, and residents in Muan-gun, Jeollanam-do are infected with variable species of intestinal trematodes.     

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.     

Brucella abortus strain RB51 vaccination in elk. II. Failure of high dosage to prevent abortion

Brucella abortus strain RB51 is used as a vaccine because it induces antibodies that do not react on standard serologic tests for brucellosis allowing differentiation between vaccination and infection. Strain RB51 was evaluated in captive elk (Cervus elaphus) to determine if vaccination protected against abortion following experimental challenge. Thirty elk were vaccinated intramuscularly with 1.0 x 10(10) colony-forming units (CFU) of strain RB51 in March 1998. Fourteen of these were given a booster dose of 1.13 x 10(10) CFU exactly 1 yr later. All vaccinated elk seroconverted via a modified dot blot assay to strain RB51 with the booster group having higher titers (P < or = 0.001). Seventeen other elk served as unvaccinated controls. All elk were bred and determined pregnant using pregnancy-specific protein B analysis. Elk were challenged in March 2000 with 1.1 x 10(7) CFU of B. abortus strain 2308 administered intraconjunctivally and all elk seroconverted to strain 2308. Fifteen of 17 control elk aborted; 16 of 16 elk given a single vaccination aborted (P = 0.44); and 13 of 14 elk given a booster aborted (P = 0.86). There were two viable calves in the control group and one in the booster group. Strain 2308 was recovered from fetuses and nonviable calves in all groups. Based on the results of this and other studies, the use of strain RB51 to prevent abortion in elk cannot be recommended.     

Trichinella spiralis: antifecundity and antinewborn larvae immunity in swine

The development of antifecundity and antinewborn larvae immunity in swine infected with Trichinella spiralis was investigated. In primary infections, adult female worm fecundity dropped sharply after 3 weeks, although adults could be recovered from the small intestine for at least 7 weeks after infection. In challenge infections of pigs infected previously, adult female worm fecundity was depressed up to 51% and the adults were expelled within 3 weeks. Since immune pigs are almost completely resistant to the secondary establishment of muscle larvae, this suggested the existence of immune effector mechanisms also acting on the newborn larvae. This was supported by observations, using an indirect fluorescent antibody assay, that pig antibody bound to the surface of the newborn larvae. Passive transfer of immune pig serum resulted in a large reduction in muscle larvae burden in both infected pig and rat recipients. Adult female worm fecundity in such immune serum recipients was reduced only by 20% and worm survival in the intestine was unaffected. These results indicate that immunity to the newborn larvae, in addition to antifecundity effects, are responsible for the high levels of acquired resistance to T. spiralis in swine.