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.     

Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics

Okanla E. O., Stumpf J. L. &; Dusanic D. G. 1982. Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics. International Journal for Parasitology12: 251–256. BALB/c mice were immunized with either irradiated or lyophilized metacyclic, epimastigote or bloodstream forms of Trypanosoma cruzi in three weekly injections of 1 × 10⁸ trypanosomes/injection. The lyophilized trypanosomes were emulsified in equal quantities of Freund's complete adjuvant. Two weeks following the final immunization, the mice were challenged subcutaneously with metacyclics obtained from either culture or the vector Triatoma infestans. The mice challenged with metacyclics from culture included groups of mice immunized with each of the three stages, while those challenged with metacyclics from the T. infestans included mice immunized with the epimastigotes or metacyclics. Mice immunized with the irradiated epimastigotes, metacyclics and blood-stream form trypomastigote challenged with metacyclics from culture exhibited reduced parasitemias compared to mice of the control groups. Parasitemias were lowest in those mice immunized with irradiated metacyclics. The parasitemias terminated in the immunized mice before those of the control animals. No protection was detected in the mice inoculated with lyophilized trypanosomes and challenged with culture metacyclics. Groups of mice injected with either irradiated or lyophilized epimastigotes or metacyclics and challenged with metacyclics from T. infestans exhibited resistance both by reduction of the parasitemias and the duration of the parasitemias when compared to the infected control animals. This study demonstrated the comparative effectiveness in mice of irradiated and lyophilized vaccines produced from either metacyclics, epimastigotes or bloodstream forms when challenged with metacyclics obtained from culture and the vector.     

Plasmodium yoelii: induction of attenuated mutants by irradiation

When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number of blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method.     

Babesia rodhaini: the protective effect of pyruvate kinase deficiency in mice

Despite the evidence suggesting that mouse pyruvate kinase (PK) deficiency provides protection against malaria in rodents, there has been no investigation of a parallel protective effect against babesiosis caused by Babesia rodhaini. Here, we examined whether a PK-deficient co-isogenic mouse strain (CBA-Pk-1^slc) was protected against B. rodhaini infection. We demonstrated that deficiency in pyruvate kinase correlated with a significant protective effect, with survival rates of 50%, 58% and 56% in groups inoculated with 10, 10³ and 10⁵ parasitized erythrocytes, respectively. In contrast, control CBA (CBA-Pk-1⁺) mice exhibited 100% lethality, regardless of the infectious dose. In addition, CBA-Pk-1^slc mice showed decreased levels of parasitemia when compared to CBA-Pk-1⁺ mice, in groups given 10, 10³ or 10⁵ parasitized erythrocytes. These results indicate that similar to PK deficiency in rodents, PK deficiency in mice affects the in vivo growth of B. rodhaini and protects the mice from lethal babesiosis.     

Bioassay to measure effects of cooling and warming rates and protection by dimethyl sulphoxide on survival of frozen Babesia rodhaini

The percentages of Babesia rodhaini parasites that survived different rates of cooling to −79 °C were determined by titrating infectivity in CBA mice before freezing and after thawing. The cryoprotective effect of DMSO and the effect of warming rate were also assessed.When parasitized blood containing 1.5 DMSO was cooled at nominal rates of 2.5 °, 265 °, and 2785 °C/min and warmed at 4320 °C/min, the respective survival rates were 0.075, 4.9, and 0.1%, indicating the existence of an optimal cooling rate. Blood without DMSO cooled and warmed under the same conditions was over 1000 times less infective. When parasitized blood containing DMSO was cooled at 2785 °C/min and warmed at 4320 °, 24.5 °, and 1.84 °C/ min, infectivity decreased progressively with the warming rate. The degrees of haemolysis in frozen and thawed blood indicated that cooling rate was more important than an intact host cell to survival of the parasite.The growth rate of B. rodhaini in CBA mice, estimated to be one binary fission in 8.5 hr, was not affected by the addition of DMSO followed by freezing and thawing.     

Trypanosoma musculi infections in complement-deficient mice

Strains of mice (CFW, C57B1/10Sn, B10.D2/nSn, and B10.D2/oSn) were infected with Trypanosoma musculi (Trypanosoma duttoni). The complement-deficient B10.D2/oSn mice showed typical parasitemias similar to those presented by the strains possessing hemolytic complement activity. Peak parasitemias occurred 12 days postinoculation. The highest parasitemias were measured in CFW mice (657 ± 82 T. musculi/30 hi-power fields), while infections in C56BL/10Sn (528 ±44 T. musculi/30 h.p.f.), B10.D2/oSn (502 ± 20 T. musculi/30 h.p.f.) and B10.D2/nSn (512 ± 35 T. musculi/30 h.p.f.) were less severe and quantitatively comparable. The percentages of dividing forms were similar during infections in each of the strains. While parasites were detected in peripheral blood until Day 22 of infection in three of 10 C57BL/10Sn mice, none could be found at this time in blood films of CFW, B10.D2/nSn or B10.D2/oSn mice. Giemsa stained kidney imprints indicated the presence of parasites in animals of each of the strains after 33 days, when trypanosomes could no longer be detected in the peripheral blood of the mice. The minor variations in the parasitemias appeared related to the mouse strain. Complement dependent, antibody mediated immune cytolysis was not indicated as a mechanism for the elimination of T. musculi by the infected mouse.     

Babesia rodhaini: effects of the immune system in mice

Possible functions of antibody in controlling multiplication of B. rodhaini in mice have been investigated. The infectivity of parasites which have been circulating in the blood of immune hosts for 4 hr is not impaired. Clearance of parasitized red cells from the blood of immune hosts is not impaired if the parasites are prevented from leaving the red cells by the effects of radiation damage. The rate of clearance of parasitized red cells by immune hosts is very slow compared with the clearance of foreign red cells by normal hosts.     

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice. International journal for Parasitology3: 773–781. Babesia rodhaini infection was compared in irradiated, splenectomized and control mice. Although irradiation reduced the weight of the spleen by as much as 95 per cent, this reduction in size did not result in parasitaemia levels comparable to those seen in splenectomized mice, which were consistently higher. Parasitaemias were similar in irradiated and control mice, but the mean survival time in control mice was longer than that of irradiated or splenectomized mice, which were comparable. Splenectomy generally resulted in higher parasitaemias than those seen in non-splenectomized mice.Since B. rodhaini has a predeliction for invading reticulocytes, the apparent failure of irradiated mice to develop parasitaemias comparable to those of splenectomized mice, may have been due to the selective destruction of these immature red cells by irradiation.     

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.     

The proliferative response of mouse lymphoid tissues during infections with Babesia microti or Babesia rodhaini

Inchley C. J., Grieve E. M. and Preston P. M. 1987. The proliferative response of mouse lymphoid tissues during infections with Babesia microti or Babesia rodhaini. International Journal for Parasitology17: 945–950. Proliferative responses induced by the lethal protozoan parasite, Babesia rodhaini, or the non-lethal species, B. microti were measured in the lymphoid tissues of infected mice. Both stimulated equally rapid DNA synthesis in the spleen, but spleen enlargement during the first week was more pronounced after infection with B. rodhaini, suggesting an earlier influx of cirulating cells than during B. microti infections. Termination of B. rodhaini infections by chemotherapy revealed that the recruitment of cells, but not the proliferative response, was dependent on the presence of live parasites. The spleen enlargement typical of B. microti-infected mice developed during the second week, but up to half of this response could be attributed to compensatory erythropoiesis. Babesia microti, but not B. rodhaini, induced a proliferative response in peripheral lymph nodes, and transiently depressed cell division in the thymus.     

A comparison of Babesia infections in intact,surgically splenectomised,and congenitally asplenic (Dh/+) mice

Infections with Babesia rodhaini and B. microti were studied in congenitally asplenic (Dh/+) mice, surgically splenectomised mice and intact mice. Mice without spleens were more susceptible to infections than intact mice, but Dh/+ mice were less susceptible than surgically splenectomised mice, indicating that some functional splenic activity had been taken over by other tissues in Dh/+ mice. It is suggested that this functional activity may be mediated by natural killer (NK) cells, and that Dh/+ mice could prove of value in the study of babesiosis in general and NK activity in particular.Male mice were more susceptible to infection than females.     

Serum opsonins and the passive transfer of protection in Babesia rodhaini infections of rats

Serum opsonins and the passive transfer of protection in Babesia rodhaini infections of rats. International Journal for Parasitology4: 197–201. An investigation into the protective activity of serum from rats immune to B. rodhaini and the role played by opsonins in that activity was undertaken. One, three and six infections with B. rodhaini resulted in corresponding increases in the titre of specific protective antibody demonstrable by the administration of immune serum to rats. Drug control of infection resulted in a lower level of protective activity than that which developed when rats controlled infection unaided. Protective activity following recovery from a single drug controlled infection was undiminished 20 weeks after infection.Serum opsonins were detected in an in vitro culture system of normal rat peritoneal macrophages and these antibodies were specific for parasitized erythrocytes. It is suggested that opsonins were largely responsible for the protective effect demonstrated by assay in rats but that their importance, relative to other antibodies with a possible protective function, in the development of acquired immunity remains to be determined.     

Nippostrongylus brasiliensis: mast cells and histamine levels in tissues of infected and normal rats.

The role of the spleen during Babesia microti and B. hylomysci infection was investigated by examining the course of infection in both intact and splenectomized mice. The presence of the spleen was critical during the early stages of infection to control excessive multiplication of either parasite, a role taken over by other lymphoid sites as the infection progressed. Mice splenectomized prior to or within 1 week of B. microti inoculation developed extended infections with some deaths, and others were unable to check their parasitemias. All intact mice, and those splenectomized 1 week after infection with B. microti, recovered completely with subsequent development of sterile immunity. Mice splenectomized prior to or within 1 week of B. hylomysci inoculation succumbed to hyperacute infections: Some of the intact mice, and those splenectomized 12 days after infection, recovered but continued to harbor a low-grade infection with periodical recrudescences. Erythrophagocytosis of infected and uninfected erythrocytes was detected in saline preparations and impression smears of spleen and bone marrow and rarely in blood smears of infected mice. This coincided with anemia, splenomegaly, and relatively high levels of opsonizing antibodies, especially during B. microti infection. The colloidal carbon clearance method was used to investigate the phagocytic activity of the reticuloendothelial system. Carbon clearance rates increased rapidly during both infections, but peak B. hylomysci parasitemia coincided with reticuloendothelial phagocytic depression and death of the host. Babesia microti stimulated a consistently higher reticuloendothelial phagocytic activity with higher erythrophagocytosis both in the spleen and bone marrow than did B. hylomysci.     

Babesia microti: Infectivity of parasites from ticks for hamsters and white-footed mice

To compare the infectivity of tick-transmitted Babesia microti for natural and experimental hosts, we permitted parasitized nymphal Ixodes dammini to feed on white-footed mice (Peromyscus leucopus) and hamsters. About nine-tenths of infested mice developed patent infection, but only about one-half of the hamsters become parasitemic. Tick-bitten mice become parasitemic more rapidly, but parasites became more abundant in hamsters than in mice. More than 100,000 B. microti were present in the salivary glands of nymphal ticks that remained attached to rodents for 60 hr. These parasites were collected, diluted, and inoculated into mice and hamsters. The mean infectious dose for both experimental hosts was in the range of 10,000 to 25,000 salivarian parasites. Compared to experimental hosts, natural hosts were more susceptible to tick-transmitted infection, became parasitemic more rapidly, but developed less intense parasitemias. Paradoxically, natural and experimental hosts were similarly susceptible to measured inocula of salivarian parasites.     

Precipitin responses of infected mice to exoantigens and cellular antigens of Trypanosoma musculi.

Plasma were collected from mice which had been immunosuppressed with 650 R from a cobalt-60 gamma radiation source and infected with Trypanosoma musculi. Trypanosomes were also collected from immuno-suppressed mice and from nonirradiated, infected animals. Rabbit antiserum was prepared against trypanosomes fron nonirradiated mice and employed in immunodiffusion analyses to detect trypanosome exoantigens (ExAg) in plasma of irradiated, infected mice and cellular antigens (CAg) in extracts of parasites which had been collected from immunosuppressed and nonirradiated hosts. The rabbit antiserum formed at least 3 precipitin lines with plasma from irradiated, infected mice and 8–9 precipitin lines with extracts of parasites which were obtained from immunosuppressed and untreated mice. Two of the precipitin reactions were against mouse plasma antigens (PAg). Lower levels of PAg appeared to be present in extracts of trypanosomes which were isolated from the irradiated mice than in those from nonirradiated animals.Mice synthesized antibodies against 1 ExAg which was demonstrable in immunodiffusion tests by 14 days after T. musculi infection. A single precipitin reaction was also seen after 21 days. One to 2 precipitin lines were formed with ExAg after 42 days of infection. Two to 3 precipitin lines formed between the ExAg and mouse antisera collected 98, 175 and 341 days after injection of the T. musculi.Similar immunodiffusion reactions were detected with CAg present in both the extracts of T. musculi which had been isolated from irradiated and those from nonirradiated mice and the mouse antisera. One to 2 precipitin lines were found between CAg and antisera from mice which had been infected for 14 days. Two precipitating antigen-antibody systems were seen with antisera collected after 21, 42 and 98 days and 2–3 precipitin reactions were formed between CAg and antisera collected from mice 175 and 341 days after infection.Absorption and immunodiffusion analyses conducted with rabbit and mouse antisera indicated parasite ExAg in plasma of irradiated, T. musculi infected mice were also present in preparations of CAg of the trypanosomes. The persistence of antibody and the increase in the numbers of antigen-antibody systems detected by immunodiffusion during the course of the infection may in part be related to the presence of parasites in capillaries of the kidneys long after they cannot be demonstrated in the peripheral blood of the host.     

Development of resistance to coccidiosis in the absence of merogonic development using X-irradiated Eimeria acervulina oocysts

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic development in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.     

Trypanosoma musculi and Trichinella spirails: Concomitant infections and selection for resistance genotypes in mice

Trypanosoma musculi infections were given to mice of different strains before, at the same time, and after an infection with 400 Trichinella spiralis. Examined parameters of the host response to T. spiralis were worm rejection, antifecundity responses, development of immunological memory, and muscle larvae burden. After dual infection, each mouse strain showed characteristic effects on resistance to T. spiralis. This was due to a dynamic interaction between the genes controlling rejection of T. spiralis and those influencing T. musculi growth. C3H mice develop high trypanosome parasitemias. This impairs worm expulsion and the development of memory to T. spiralis when Trypanosoma infections take place on the same day or 7 days before. The C57B1/6 mouse develops low parasitemias and T. musculi infections on the same day, or 7 days before T. spiralis, delaying worm rejection only slightly despite the overall weak capacity of B6 mice to expel worms. NFR-strain mice are strong responders to T. spiralis and also develop low parasitemias. Trypanosome infections on the same day, or after T. spiralis, produce a delay in worm rejection; the former is comparable to C3H mice. However, NFR mice alone showed enhanced rejection of worm when T. musculi infections preceded T. spiralis by 7 days. An unusual feature of C3H mice was that T. musculi infections 7 days before T. spiralis increased antifecundity responses at the same time that worm expulsion was inhibited. Trypanosome infections can therefore modulate distinct antihelminth immune responses in different directions simultaneously. The different outcomes of dual infections compared with single infections provides another selective mechanism by which genetic polymorphisms can be established and maintained in the vertebrate host.     

Plasmodium chabaudi chabaudi: effect of low parasitemias on immunity in CB6F1 mice.

We examined the effect that low parasitemias have on the immune response of CB6F1 mice infected with Plasmodium chabaudi chabaudi AS. Ascending parasitemias were stopped by chloroquine treatment when they were between 1.6 and 9.4%. Mice that suffered low parasitemias developed good immunity to homologous reinfection but, contrary to what happened in mice that suffered full parasitemias, they did not develop immunity to heterologous reinfection with Plasmodium yoelii 17XL. Total IgG antiparasite antibody responses were similar in mice that suffered low or full parasitemia, both in primary infection and after reinfection. At the level of isotypes, IgM, IgG1, IgG2b, and IgG3 responses were similar in mice that suffered low or full parasitemias, but after reinfection, mice that suffered low parasitemias responded with higher levels of IgG2a than mice that suffered full parasitemias. Mice that suffered low parasitemias did not have splenomegaly but their immunity to homologous reinfection was diminished after splenectomy in a manner similar to that of splenectomized mice that suffered full parasitemia. CB6F1 mice can develop homologous immunity even if exposed to low parasitemias but cannot develop heterologous immunity unless exposed to high parasite loads.     

Studies on cell fusion between Babesia rodhaini-infected mouse erythrocytes and baby hamster kidney cells.

Heterokaryons were formed by fusion of B. rodhaini-infected mouse erythrocytes and baby hamster kidney (BHK) cells, using Sendai virus. The erythrocyte membrane rapidly lysed inside the BHK cell cytoplasm releasing free parasites. There was no evidence that parasite multiplication occurred inside the BHK cells, nor that parasitized BHK cells were infective for mice.Transient erythrocyte homokaryons were observed in some preparations.The approach indicates a possible method for the in vitro cultivation of Babesia.     

The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice

Irvin A. D., Young E. R. and Purnell R. E. 1978. The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. International Journal for Parasitology8: 19–24. Blood and mice infected with Babesia microti and B. rodhaini, and from cattle infected with B. divergens and B. major, was incubated in Eagles medium for 24 h in the presence of tritiated purines and pyrimidines. Uptake of these compounds was assessed by liquid scintillation counting and by autoradiography. Hypoxanthine, adenosine and adenine were readily incorporated by all four species of parasites. Thymine, thymidine and uridine were generally not incorporated. Uptake of [³H]hypoxanthine by B. microti occurred within minutes of exposure to the precursor. The amount of [³H]hypoxanthine incorporated by B. rodhaini-infected erythrocytes was proportional to the percentage of parasitized cellsThe results suggest that structural analogues of hypoxanthine and other purines may be incorporated and act against intra-erythrocytic Babesia.