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

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

Immunity to Litomosoides carinii in Mastomys natalensis. 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.     

Fecundity of Litomosoides carinii (Nematoda, Filarioidea) in vivo and in vitro

Several parameters concerning the reproduction of Litomosoides carinii were assessed using quantitatively infected cotton rats (Sigmodon hispidus). The course of embryogenesis from the fertilization of eggs to the delivery of the first microfilariae was observed by daily autopsies during prepatency. The duration of embryogenesis in vivo could thus be determined as 18 +/- 2 days. The contents of embryos in the uteri of female worms had been examined at various intervals. At the onset of patency 7-8 weeks p.i. the females were 71 +/- 6 mm long and on average contained 308 X 10(3) embryos/female, of which 19% were pathologically altered. In the middle of patency 16-20 weeks p.i. the females had grown up to 100 +/- 11 mm in length and now contained 509 X 10(3) embryos/female, 25% of them were pathologically altered, the others were normally developed. A positive correlation between the body length of a female worm and its number of embryos in utero was evident. Additionally the percentage of pathologically altered embryos was increased with respect to the age of the worms. The calculated fecundity of a female L. carinii in vivo of around 20 X 10(3) microfilariae/female per day had been confirmed with worms maintained in vitro. Three combinations of media and serum supplements were used and their influence on embryogenesis evaluated.     

Recovery of adult stages and microfilaraemia after low dose inoculation of third stage larvae of Litomosoides carinii in Sigmodon hispidus

Because of the negative binomial distribution of filarial third stage larvae (L3) in their vectors, under natural conditions only a few are usually transferred per bite. After an inoculation of 5 L3 per animal into eight host animals at least one developed a long lasting patency based on one reproductive female only. After an inoculation of 15 L3, three of eight animals developed long lasting patency, harbouring between two and five fertile females. The rates of adult stages recovered were 0.43 and 0.30 respectively. The parasitaemias of the six patent animals in both experimental groups increased with the number of reproductive females present (r = 0.89, p = less than 0.005). All non-patent animals which were mf-negative in the pleural fluid and lung blood as well had a single sex or no worm load. In only one animal was there an apparently normal but non-reproductive pair of worms without any pathological alterations of the host animal. Encapsulated adult worms were found rarely, but independent of the final worm load or inoculation dose and always beside normal adults. In three of the 16 animals inoculated with 5 or 15 L3 patency passed after 10-12 weeks p.i., in two others it seemed to pass soon. After inoculation of 30, 40 or 60 L3 per animal patency passed early in about one half of 105 animals, when they were observed up to 24-36 weeks p.i. In conclusion all types of host defensive reactions are already visible after inoculation with such small doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experiments in vitro with Litomosoides carinii (Nematoda: Filarioidea). I. Maintenance of adult females and microfilariae as well as release of microfilariae in different culture media (author's transl)

Embryos of L. carinii continue intrauterine development to microfilariae and are totally released into the medium within 5--6 days when the latter (Tc 199) is changed daily and air is used as the gas phase. Oogenesis or further fertilization of eggs, however, does not occur in vitro in any of the media examined by us. One female releases 140 X 10(3) microfilaria/day on an average in vitro within 5--6 days. Mean initial numbers of 300 X 10(3) Mf/female/day are observed. Addition of equine serum inhibits microfilarial release in vitro; normal cotton rat serum prolongs survival of females while total numbers of released microfilariae or retained embryonic stages are not increased. The serum of post-patent animals does not influence the numbers of released microfilariae or their viability or survival of females. Microfilariae released in vitro in Tc 199 + 33% normal cotton rat serum survive for more than 8 days, when air is used as the gas phase and the medium is changed daily. Microfilariae isolated from the blood of patent animals survive for at most 6 days, at a 48-hourly change of medium survival does not even exceed 4 days.     

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.     

Suppression of nodulation in soybeans by superoptimal inoculation with Bradyrhizobium japonicum

Symbiotic nodulation of the primary roots of soybeans ( Glycine max L. Merrill cv. Pride 216) is regulated by the plant, and is suppressed in response to a high inoculum dose of Bradyrhizobium japonicum USDA strain I–110 (ARS)⁺ applied at one time to the root. If an optimal dose is followed 10 h later by a superoptimal dose, nodules from the first inoculum near the base of the primary root are suppressed in a dose-dependent way similar to that observed after single inoculations. The nodules which appear are probably derived from infections initiated by the bacteria in both inocula.     

Litomosoides carinii: mode of action in vitro of benzothiazole and amoscanate derivatives with antifilarial activity

It is suggested that the recently developed benzothiazole and amoscanate derivatives with antifilarial activity exert their action in vitro by an inhibition of mitochondrial-derived respiration. It was confirmed that the drugs CGP 20376, 21835, 20308, 21306, and 6140 cause a rapid immobilization in vitro of the adult filarial worm, Litomosoides carinii, the time required being similar to rotenone at the same concentration. The other drugs investigated, CGPs 20309, 21833, 24589, 23518, and 13231, were also effective; however, they required much longer incubation times. Submitochondrial particles (SMP) were prepared from Ascaris muscle and rat liver. The concentration of drug causing 50% inhibition of respiration (IC50) was calculated. It was found that the drugs most rapidly inhibiting respiration have IC50s for NADH oxidase of less than 25 microM in both Ascaris and rat liver SMP. This effect on SMP respiration could be overcome by using succinate as a substrate, indicating the site of inhibition to be within complex I of the mitochondrial respiratory chain. Further experiments showed that whereas the respiratory chain's NADH:ferricyanide reductase was unaffected by these drugs, there were pronounced effects on both Ascaris and rat liver NADH:quinone reductase activity. This suggests that the inhibition within complex I occurs after the flavoprotein dehydrogenase, but before the site of the quinone reduction. The other compounds examined, which had a slower effect on motility, also showed inhibition of the NADH oxidase, but not to as great an extent as the aforementioned compounds. The compounds most active against motility were also most effective at inhibiting respiration in intact adult L. carinii. Analysis of the aerobic end products produced by L. carinii showed that acetate production was greatly reduced even in the presence of low concentrations of the drugs. There was also a slight decrease in lactate production. However, a direct effect on the glycolytic pathway was ruled out by two observations. One, that the production of lactate from cell-free extracts of L. carinii is unaffected by the presence of the drugs, and secondly, that a protozoan, Giardia lamblia, reliant on glycolysis for energy production, can survive for long periods of time in the presence of high concentrations of the drugs. A correlation can be observed between the time for immobilization of the filarial worm and the strength of inhibition of mitochondrial respiration. Therefore, it is suggested that, at least in vitro, the mechanism of toxicity of these antifilarials in L. carinii is due to the blocking of the respiratory chain at a site similar to that of rotenone.     

The genetic regulation of host response to inoculation with Mycobacterium bovis (BCG) and Mycobacterium tuberculosis H37RV

Vaccination with M. bovis (BCG) essentially prolonged survival time (ST) of several strain mice, with the exception, of CBA/N, infected with M. tuberculosis H37Rv. ST of CBA/N, differing from CBA by xid mutation, was not prolonged by vaccination. Mouse strains with alternative alleles of BCG gene (s and r) and fzy gene as a genetic marker for Bcg5 were used for segregation analysis. It was shown that ST, the level of DTH reaction of mice infected with M. tuberculosis H37Rv, and protective effect of BCG vaccination did not depend on Bcg gene. However, Bcg gene, apparently, regulate the DTH response to PPD in mice only vaccinated with M. bovis (BCG).