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.     

Infectivity and Immunogenicity of Irradiated Babesia rodhaini*

SYNOPSIS. Babesia rodhaini-parasitized mouse blood exposed to varied doses of γ radiation up to 30 kRad was inoculated into mice. Mice inoculated with nonirradiated B. rodhaini developed progressive infections and died 7–11 days postinoculation. Mice infected with B. rodhaini-parasitized blood exposed to doses up to and including 22 kRad developed progressive parasitemias which were delayed in comparison to mice inoculated with non-irradiated B. rodhaini. Some mice receiving parasitized blood irradiated at 26 kRad did not develop progressive parasitemias. Progressive infections were prevented by exposure to irradiation at 30 kRad. The results of 2 separate experiments revealed that one inoculation of parasitized blood exposed to 30 kRad or higher apparently stimulated a resistance to a challenge infection with nonirradiated parasitized blood. While 20 of 20 control mice died as a result of challenging infections, 9 of 28 mice previously exposed to irradiated parasitized blood survived. The injection of irradiated nonparasitized blood did not produce a discernible acquired resistance to B. rodhaini. Presumably the irradiated parasitized blood was responsible for the development of acquired resistance to B. rodhaini.     

East coast fever: 60Co-irradiation of Theileria parva in its tick vector, Rhipicephalus appendiculatus

Three experiments were carried out in which Theileria parva was irradiated in its tick vector, Rhipicephalus appendiculatus. In the first experiment, infected unfed adult ticks were irradiated at doubling doses from 4 to 32 krad. Some of the ticks were then fed for 4, 5, 6, 7 and 8 days on rabbits, and the parasites in their salivary glands examined. Five male and 5 female ticks from each irradiation dose were put onto each of a pair of susceptible cattle, whose reactions were recorded. Increasing doses of irradiation resulted in progressive destruction of the parasites. All cattle receiving ticks irradiated at doses up to and including 16 krad died of East Coast fever (ECF), and one of the cattle receiving ticks irradiated at 32 krad died.In the second experiment, recently engorged nymphs were irradiated at 1, 2 or 4 krad, and moulting nymphs at doses of 2, 4, 8, 12, 16 or 32 krad. The salivary glands of the resultant adult ticks were examined after the ticks had fed for 4, 5, 6, 7 or 8 days on rabbits. Engorged nymphs irradiated at 4 krad failed to moult, whilst moulting nymphs irradiated at 32 krad moulted but failed to attach to rabbits. Doses of irradiation survived by the ticks had no apparent morphological effect on the parasites they contained.In the third experiment, infected unfed adult ticks were irradiated at 0, 5, 10, 15, 20 25 30, 35, 40, 45, 50 or 60 krad. The ticks were fed on rabbits for 5, 6 or 7 days. Some of them were then examined morphologically, whilst others were ground in MEM/BPA and aliquots of the supernatant used to inoculate groups of 5 cattle. The reaction of these cattle, together with the morphological examination of the parasites, suggested that increasing doses of irradiation destroyed increasing numbers of parasites.     

Schistosoma mansoni: immunization of cynomolgus monkeys by injection of irradiated schistosomula.

Although the immunization of primates with irradiated schistosome cercariae has been demonstrated, no success has been reported by injection with the irradiated schistosomule stage. The present investigation was designed to test whether cynomolgus monkeys could be protectively immunized with ⁶⁰Co-irradiated Schistosoma mansoni schistosomula. Monkeys injected once with 10⁴ irradiated schistosomula (50 krad at 4 krad/min) had 52% fewer challenge worms than the control group at necropsy. Four immunizations did not induce a higher level of resistance. At 50 days post-challenge, the immunized monkeys excreted 80% fewer eggs than did the control animals. An attempt to enhance irradiated schistosomule-induced protection with tetramisole · HCl was unsuccessful.     

Immunization of mice with irradiated Trypanosoma cruzi grown in cell culture: Relation of numbers of parasites,immunizing injections,and route of immunization to resistance

Mice were given 5 or 8 weekly injectins of either 2·0 × 10⁶ or 20·0 × 10⁶ irradiated T. cruzi from cell culture (ratio of trypomastigotes to amastigotes, 1 : 1) via the intraperitoneal route or via the subcutaneous route and challenged via the subcutaneous route one week after the last injection with 5·0 × 10⁴T. cruzi in mouse blood. The irradiated parasites used were not capable of producing infections in either Vero cell cultures or C₃H mice. Mice receiving irradiated parasites were significantly protected against the challenge infection as evidenced by significantly lower mean parasitemia, lessened signs of acute disease, and reduced mortality than that observed in untreated controls. Mice receiving 5 weekly immunizing injections of irradiated parasites were more resistant to challenge than those receiving 3 in previous work. Mice receiving 8 weekly immunizing injections were not significantly more protected against challenge than those receiving 5. Mice given 5 weekly injections of 20·0 × 10⁶ irradiated parasites were significantly more resistant to challenge than those receiving 2·0 × 10⁶ irradiated parasites on the same schedule. Mice given 5 weekly intraperitoneal injections of 20·0 × 10⁶ irradiated parasites were significantly more resistant to challenge than those receiving an equivalent number of immunizing injections via the subcutaneous route.     

The effect of mitomycin C treatment of infected erythrocytes on infection with Babesia microti and Babesia rodhaini in mice

In vitro treatment of Babesia microti infected erythrocytes with mitomycin C before their injection into mice prolonged the prepatent period of infection, reduced the levels of the infection in the ‘breakthrough’ parasitaemia and induced protection against reinfection. Treatment of B. microti with mitomycin C at a concentration of 25 μg ml^?1 resulted in a mean peak parasitaemia of 6.2% in the infected mice compared with 46.5% in control mice injected with untreated B. microti parasites. In addition, mice survived a normally fatal B. rodhaini infection if injected with 6.2 × 10⁷ infected erythrocytes treated with 25 μg ml^?1 mitomycin C and four of five mice survived infection with 6.2 × 10⁵ similarly treated infected erythrocytes. However, the degree of protection against B. rodhaini was dependent on the concentration of mitomycin C used to treat the parasites and treatment of 5 × 10⁷ infected erythrocytes with 50 μg ml^?1 resulted in survival of only four of the five infected mice. In addition, when 100 μg ml^?1 of mitomycin C was used to treat B. rodhaini parasites, the course of infection, although delayed, was indistinguishable from that seen in the control mice and all the mice died. The latter results and the lack of efficacy of comparable numbers of heat killed parasites suggested the necessity for sufficient, non-replicating, mitomycin C treated parasites to metabolize and produce and/or present protective antigens to the host.     

Trypanosoma musculi: Passive hemagglutination technique to measure antibody in mice

A passive hemagglutination assay was developed to measure Trypanosoma musculi-specific antibody in mice. Indicator-erythrocyte donor mice received 550 rad ⁶⁰Co 24 hr before intraperitoneal injection of 3 × 10⁴T. musculi. T. musculi antigen-coated erythrocytes were obtained from these mice on Day 9 postinfection. T. musculi antigen-coated erythrocytes obtained in this manner were used as indicator erythrocytes in a passive hemagglutination procedure. Serum from hyperimmunized mice (three consecutive infections at 21-day intervals) gave titers as high as 1:1024. Titers of 1:256 and 1:512 were obtained from singly infected mice on Days 18 and 28 postinfection, respectively. In marked contrast, nude mice infected with T. musculi did not produce a detectable agglutinating antibody response. Erythrocytes obtained from either irradiated (550 rad ⁶⁰Co) uninfected mice, nonirradiated infected mice, or normal mice did not agglutinate when combined with any of the sera tested. These data suggest the usefulness of this passive hemagglutination assay for the measurement of antibody to T. musculi in the serum of infected mice.     

Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites

Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesiabovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination.     

Bovine toxoplasmosis: experimental infections.

Three calves dosed per os with 0.75 × 10⁶, 0.75 × 10⁶ and 1.5 × 10⁶Toxoplasma oocysts developed high titres of Toxoplasma antibodies as measured by the indirect fluorescent antibody test and the dye test. Toxoplasma gondii was reisolated from 2 of these animals. Two calves given 1 × 10³ tissue cysts and 2 × 10⁶ tachyzoites intramuscularly did not develop such high fitres, but T. gondii was reisolated from the calf injected with tachyzoites.Of 4 pregnant cows given 7 × 10⁴, 7 × 10⁴ and 1.6 × 10⁵ oocysts and 1.5 × 10² tissue cysts only 2 developed significant levels of Toxoplasma antibodies. There was no evidence of Toxoplasma infection in the calves born by these cows.It was concluded that cattle do not readily acquire persistent T. gondii infections, and this may be due, in part at least, to early elimination of Toxoplasma from their tissues.     

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.     

Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst.

The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.     

Concentration and Enzyme Content of In Vitro-Cultured Babesia bigemina-Infected Erythrocytes1

ABSTRACT. Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes-lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase-were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.     

Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23 days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated.     

Some effects of time,temperature and feeding on infection rates with Babesia bovis and Babesia bigemina in Boophilus microplus larvae

Dalgliesh R. J. and Stewart N. P. 1982. Some effects of time, temperature and feeding on infection rates with Babesia bovis and Babesia bigemina in Boophilus microplus larvae. International Journal for Parasitology12: 323–326. Percentages of larval ticks in which Babesia bovis and B. bigemina parasites could be detected (infection rates) were determined after the larvae had been exposed to temperatures between 9°C and 27°C for periods of 1–35 days and then either fed on calves or heated at 37°C to stimulate babesial development. Infection rates with both species increased during 2–4 weeks after the larvae hatched, regardless of the temperature of exposure. Infection rates with B. bovis were higher after exposure of larvae to 14°C than to 27°C. This effect was less pronounced with B. bigemina. Infection rates were higher in fed larvae than in unfed, ‘heat stimulated’ larvae. The findings indicate that infected larval ticks become more efficient vectors of Babesia during the first 2–4 weeks after hatching and that repeated sampling of a tick population is necessary to determine valid infection rates.     

Plasmodium berghei: biological variation in immune serum-treated mice.

Antiserum was obtained from mice which had been immunized with irradiated Plasmodium berghei parasitized erythrocytes and which survived subsequent challenge. This antiserum suppressed P. berghei infections in mice; parasitemia and mortality were delayed 7–8 days as compared to those of control animals. Parasites surviving in antiserum-treated animals were isolated by inoculation of blood into normal recipients. When antiserum was tested against this derived parasite population, there was no observable effect on parasitemia or mortality. The derived parasites also exhibited a decreased virulence for mice. This work confirms the previous observation that antiserum treatment can result in a biologically variant population of P. berghei.     

Trypanosoma evansi: Therapeutic efficacy of diminazine aceturate in crossbred calves, Bos taurus and B. indicus

The therapeutic efficacy of diminazine aceturate (Berenil) against Trypanosoma evansi infection (surra) in calves was determined. Seven crossbred calves, 8 to 12 months old, were inoculated subcutaneously with 5 × 10⁷ parasites of a virulent strain of T. evansi. All of the calves developed clinical infection and exhibited symptoms associated with chronic surra. Forty-one to forty-four days after inoculation, five of the calves were treated with a single dose of diminazine aceturate at 10 mg/kg body weight intramuscularly. The two remaining calves were left as infected but untreated controls. The treated calves were found free of the infection on repeated blood smear examinations and diagnostic challenge tests in mice 12 hr to 16 days later. One of the five calves, which was treated at an advanced stage of the disease, died on the fifth day after treatment. Splenectomy of the treated calves, 16 to 19 days after treatment, did not result in relapse of parasitemia, indicating that diminazine aceturate at 10 mg/kg had a rapid trypanocidal effect in surra of calves. Both of the controls died of the disease on the 45th and 47th day.     

The effects of gamma-irradiation on migration and survival of Schistosoma mansoni schistosomula in mice

Infections with irradiated Schistosoma mansoni were established by intramuscular (i.m.) injection of mechanically transformed schistosomula. A dose of 2.3 krad. allowed persistence of a small proportion of worms to adulthood, and of these survivors the majority of the female worms were sexually sterile. However, a small proportion of 2.3 krad.-irradiated females and a larger proportion of similarly irradiated males were capable of pairing successfully with non-irradiated partners. Radiation in the range 2.3 to 10 krad. resulted in slightly reduced peak recoveries from the lungs while 20 krad. resulted in a much reduced and 40 krad. a virtual absence of survival to the lung stage. Increasing doses of radiation in the range 2.3 to 10 krad. resulted in successively fewer parasites reaching the liver. Thus, the major sites of the radiation-induced mortality appeared to be as follows: 2.3 krad., mainly in the liver; 4 krad., in the lungs and liver; 10 krad., mainly in the lungs; 20 krad., at the injection site and in the lungs and 40 krad., mainly at the injection site. The infections studied here showed reduced survival following exposure to high doses of radiation compared with the infections, established as percutaneously applied cercariae, which have been reported by other workers. Possible reasons for the disparity are discussed.     

Schistosoma mansoni: Interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice

Mechanically transformed schistosomula of Schistosoma mansoni were irradiated with levels of ⁶⁰Co irradiation between 2.5 and 54 krad, cryopreserved by the two-step addition of ethanediol and rapid cooling technique, and were injected intramuscularly into groups of mice which were perfused 40 days later. The schistosomula were either irradiated and then cryopreserved (IC) or cryopreserved and then irradiated in the frozen state (CI). Development into adult worms was prevented with 4 krad for IC schistosomula, but for CI schistosomula a small number of worms (1.6%) was recovered using 8.8 krad. A dose of 4 krad was sufficient to prevent development of unfrozen controls (I), but for schistosomula irradiated while exposed to ethanediol (EI), a dose of 7 krad was required. Using the different protocols, the peak levels of protection against a challenge infection were achieved with 9 (IC) and 16 krad (CI), compared to 20 krad for unfrozen schistosomula (I) reported previously. The highest level of protection (65%) was achieved with CI schistosomula. Possible interactions between the radioprotective and damaging effects of cryopreservation are discussed.     

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.