The efficacy of the ultraviolet C pathogen inactivation system in the reduction of Babesia divergens in pooled buffy coat platelets.

BACKGROUND: Babesia spp. is an intraerythrocytic parasite that causes human babesiosis and its transmission by transfusion has been extensively demonstrated. The aim of this study was to ascertain the efficacy of an ultraviolet C (UVC)-based pathogen inactivation system in the reduction of Babesia divergens-infected platelet (PLT) concentrates and to determine the parasite's ability to survive in PLT concentrates stored under blood bank conditions. STUDY DESIGN AND METHODS: This study was conducted using in vitro cultures of B. divergens. The detection limit of the culture assay was established and, subsequently, 15 buffy coat-derived PLT concentrates (BC-PCs) were inoculated with 10(7) B. divergens-infected red blood cells. Infected BC-PCs were irradiated with 0.2 J/cm(2) UVC light using the THERAFLEX UV-Platelets method (Macopharma). Viability and parasite growth were evaluated before and after inactivation. Culture growth kinetics were monitored by DNA incorporation of [(3) H]thymidine. The ability of B. divergens to survive in PLT concentrates was also analyzed. RESULTS: The limit of detection in cultures was established at 0.1 × 10(-6) % parasites. The THERAFLEX UV-Platelets system inactivated B. divergens to below the limit of detection in 12 of 15 BC-PCs (log reduction, >6.0) and to the limit of detection (log reduction, 5.0) in three of 15. It was also demonstrated that B. divergens remains viable in BC-PCs stored up to 7 days. CONCLUSION: Since B. divergens can survive in PLT concentrates and given the performance of UVC, this system could be considered as an alternative to prevent B. divergens and other Babesia species from being transmitted through PLT transfusions.

Photosensitized inactivation of Plasmodium falciparum- and Babesia divergens-infected erythrocytes in whole blood by lipophilic pheophorbide derivatives.

BACKGROUND AND OBJECTIVES: Blood transfusions can transmit parasitic infections, such as those caused by Plasmodium (malaria), Trypanosoma cruzi (Chagas' disease), and Babesia (babesiosis). A higher degree of blood transfusion safety would be reached if methods were available for inactivating such parasites. MATERIALS AND METHODS: We evaluated the effectiveness of photosensitization using lipophilic pheophorbide and red light illumination to eradicate red blood cells infected with Plasmodium falciparum, and with Babesia divergens, in whole blood. Fluorescence microscopy and conventional fluorometry showed the specific accumulation of pheophorbide derivatives in the RBC infected with either parasite, compared with uninfected RBC. The effectiveness of different derivatives in eradicating infected RBC was first estimated in parasite cultures. RESULTS: The best photosensitizer was the N-(4-butanol) pheophorbide derivative (Ph4-OH) at 0.2 microM concentration and 5-min illumination. In whole blood, the eradication of RBC infected with B. divergens and P. falciparum was obtained with 2 microM Ph4-OH and 10 and 20 min illumination, respectively. Under these conditions of photosensitization, low levels of RBC hemolysis were noted even after 2 weeks of storage at 4 degrees C and a subsequent 48-hour incubation at 37 degrees C. No reduction of negative charges on treated RBC was noted and no increase in methemoglobin content. CONCLUSIONS: In plasma, Ph4-OH is mainly transported by high-density lipoproteins (HDL). This high affinity for HDL may explain the selective accumulation of lipophilic pheophorbide derivatives in the intracellular parasites. Photosensitization with pheophorbide derivatives may be a promising approach to inactivation of transfusion-transmissible parasites and viruses in blood bank units.

Delayed-type hypersensitivity to Babesia microti-infected erythrocytes in mice.

Strong delayed-type hypersensitivity (DTH) to Babesia microti was elicited when intraerythrocytic parasites (IEP) were inoculated subcutaneously into the flank of normal mice 6 to 14 days before challenge in the ipsilateral footpad with 10(8) IEP. Intraperitoneal or intravenous administration of antigen did not sensitize mice for DTH. When challenge was given 21 days after immunization, the response was approximately half of the maximum and then rose again slowly over the next 3 weeks to levels that were not significantly different from those maximal values. The response was similar in seven strains of mice, regardless of sex. The response was classified as a true DTH reaction on the basis of kinetics, histology, and the transfer of responsiveness with immune T lymphocytes of the Ly 1+ phenotype, but not with serum. The reaction was specific for IEP since control groups given two injections of red blood cells from uninfected syngeneic mice (NRBC) or one injection of NRBC or sheep red blood cells (SRBC) and one of IEP never developed significant footpad swelling. Freed parasites obtained by osmotic rupture, density gradient sedimentation, and lethally irradiated IEP were also effective for elicitation of DTH. Anti-IEP DTH was expressed in a dose-dependent fashion with 10(6), 10(7), or 10(8) parasites sufficing for immunizing inoculum as long as 10(8) parasites were used as the challenge dose. Mice immunized and challenged with 10(8) lethally irradiated IEP (60 krad, 60Co), were protected against subsequent intraperitoneal challenge with 10(8) viable IEP. If mice were infected intraperitoneally with 10(8) IEP at any time between 21 days before immunization to 2 hr after challenge, their ability to respond to immunization and challenge was profoundly depressed. These data suggest that development of a strong anti-parasite DTH response can occur in parallel with resistance to infection, but is not a rapid sequela of bloodborne infection.

The irradiation of babesia bovis. II. The immunogenicity of irradiated blood parasites for intact cattle and splenectomised calves.

Intraerythrocytic forms of B. bovis were exposed to 350 Grays (Gy) gamma irradiation and were then injected intravenously into intact two and three year old Hereford steers. One of 15 steers died on initial infection and subsequently six steers were given a virulent heterologous challenge three weeks after recovery; all six animals were highly immune. The remaining eight animals were kept under quarantine conditions for 10 months and were then challenged with a different virulent heterologous strain of B. bovis. Seven of eight were highly immune, but one animal died. Subsequently a further 12 steers were injected intravenously with 1 X 10(8) irradiated organisms. All showed only mild transient clinical signs. After 12 months quarantine in a tick-free area these animals were then challenged with a virulent heterologous strain and all 12 were shown to be highly immune. Irradiation reduced the infective dose from 1 X 10(8) to 2.5 X 10(3) parasites. These parasites multiplied at the same rate, and achieved the same maximum parasitaemia as the parent non-irradiated strain, but the disease produced by them was not severe. A dose of 2.5 X 10(3) non-irradiated parasites was lethal to all of the four animals which received it. It was concluded that irradiation had produced a predominantly avirulent parasite population.

Babesia divergens: combination of dead and live parasites in an irradiated vaccine.

Two experiments were carried out to investigate the hypothesis that the protective effect of an irradiated Babesia divergens vaccine was due to the combined inoculation of large numbers of dead parasites and small number of live ones. In the first experiment it was demonstrated that animals receiving 10(8) parasites irradiated at 25 kilorads had more severe B divergens reactions than animals in a previous experiment which had received 10(10) parasites irradiated at 24, 28 or 32 kilorads. In the second experiment it was shown that the simultaneous inoculation of 10(10) parasites killed by irradiation at 50 kilorads and 10(8) parasites irradiated at 25 kilorads resulted in the suppression of the relatively severe reactions induced by inoculation of the 25 kilorad-irradiated parasites alone. It was also shown that the simultaneous inoculation of 10(10) parasites irradiated at 50 kilorads and 10(4) live parasites similarly suppressed the anticipated reactions of the calves. These results indicated that the hypothesis was correct under the conditions of the experiment.

The irradiation of Babesia bovis. 1. The difference in pathogenicity between irradiated and non-irradiated populations.

Babesia bovis parasites attenuated by 35 krads gamma irradiation and parasites not exposed to irradiation, were injected into intact 2-year-old Hereford steers. All five animals receiving non-irradiated blood died but the five animals which received irradiated blood were only mildly affected. Highly significant differences were observed in changes to plasma fibrinogen, serum fibrinogen-like proteins, packed cell volume, partial thromboplastin time, prothrombin time, blood kinins, and plasma kininogen levels in the control animals but non-significant changes in these parameters occurred in the group receiving iradiated blood. Significant changes in the antiplasmin alpha 2M, and the antithrombin levels occurred in control cattle but not in the group receiving irradiated blood. Parasite multiplications rates and maximum parasitaemias were similar in both groups. Irradiation reduced the dose of living parasites from 1 x 10(8) to 2.5 x 10(3), but this was not the reason for the mild reactions. It was concluded that irradiation had selected an avirulent parasite population.

The effects of irradiation on Babesia maintained in vitro.

Blood infected with Babesia radhaini, B major or B divergens was irradiated to different absorbed doses between 0 and 120 krad, and then maintained in vitro in the presence of 3H hypoxanthine for 24 h. The effects of irradiation were measured by the ability of the parasites to incorporate 3h hypoxanthine and, in the case of B rodhaini, by the ability of the parasites to infect mice. B major and B divergens were slightly more radio-resistant than B rodhaini, but all showed a progressive fall in ability to incorporate 3H hypoxanthine with increasing doses of irradiation, except at low doses of irradiation when there was increased uptake of 3H hypoxanthine. In the case of B rodhaini there was close correlation between the ability of the parasites to incorporate 3H hypoxanthine and their infectivity for mice. Both types of activity were abolished at doses of 40 krad and above.

Babesia divergens: the immunisation of splenectomised calves during irradiated piroplasms.

Groups of three splenectomised calves were inoculated with 1 . 2 x 10(10) Babesia divergens-infected erythrocytes irradiated at 24, 28, 32, 36 and 40 kilorads. Control calves were inoculated with 1 . 2 x 10(7) or 1 . 2 x 10(4) non-irradiated parasites. While control animals all experienced severe reactions, animals receiving blood irradiated at 24, 28 and 32 kilorads had mild reactions and were solidly immune to an homologous challenge of 1 . 1 x 10(4) Babesia-infected erythrocytes. Animals receiving parasites irradiated at 36 and 40 kilorads had limited ability to resist the challenge.

Protection of cattle against Babesia major by the inoculation of irradiated piroplasms.

Two experiments were carried out on Babesia major in splenectomised calves. In the first one, the parasite was titrated and a linear relationship was observed between infective dose (10(2)--10(7) parasites) and onset of disease. In the second experiment aliquots of 1.2 X 10(10) parasites were irradiated at doses of 24, 28, 32, 36 and 40 krads and inoculated into groups of calves, while control calves received 1.2 X 10(7) or 1.2 X 10(4) parasites. Animals which received parasites irradiated at 24 or 28 krads had mild reactions and were immune to homologous challenge. Animals receiving parasites irradiated at the higher dose levels had limited ability to resist the challenge.

[Effect of ionizing radiation on the virulent and immunogenic properties of Babesia ovis]. / Vliianie na ionizirashtata radiatsiia vurkhu virulentnite i imunogennite svoistva na Babesia ovis.

The effect was followed up of ionizing radiation at rates of 20, 25, 30, 32, 35, 40 and 50 krad on the virulence and the immunogenic properties of B. ovis. The experiments were carried with 22 sheep (20 test and 2 donor) according to the rate of treatment into 7 test groups with a total of 17 animals, and 1 control with 3 sheep. One month after infection with irradiated blood reinfection was carried out of 8 animals (of different groups) that had recovered, using virulent untreated blood. The titer of the antibodies in the remaining sheep was followed up by means of the complement-fixation test. It was found that irradiation at 20-25 krad lowered the virulence of the Babesia organisms, but these could provoke the disease in an acute form and cause death. The animals that survived after being infected with the indicated doses developed a comparatively good immunity. It is considered that the most appropriate dose of irradiation is 30 krad. 32 and 35 krad do not kill Babesiae; the parasites thus irradiated, however, cannot protect the animals at reinfection. It has been found that Babesiae irradiated at the rates of 40 and 450 krad do not cause an infection process, and at reinfection the animals respond almost in the same way as the controls.