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2.
Mycopathologia ; 171(6): 395-401, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21327789

ABSTRACT

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii. Zoonotic transmission to man can occur after scratches or bites of animals, mainly cats. In this study, the gamma radiation effects on yeast of S. schenckii were analyzed with a view of developing a radioattenuated vaccine for veterinary use. The cultures were irradiated at doses ranging from 1.0 to 9.0 kGy. The reproductive capacity was measured by the ability of cells to form colonies. No colonies could be recovered above 8.0 kGy, using inocula up to 10(7) cells. Nevertheless, yeast cells irradiated with 7.0 kGy already were unable to produce infection in immunosuppressed mice. Evaluation by the FungaLight™ Kit (Invitrogen) indicated that yeast cells remained viable up to 9.0 kGy. At 7.0 kGy, protein synthesis, estimated by the incorporation of [L-(35)S] methionine, continues at levels slightly lower than the controls, but a significant decrease was observed at 9.0 kGy. The DNA of 7.0 kGy irradiated cells, analyzed by electrophoresis in agarose gel, was degraded. Cytoplasmic vacuolation was the main change verified in these cells by transmission electron microscopy. The dose of 7.0 kGy was considered satisfactory for yeast attenuation since irradiated cells were unable to produce infection but retained viability, metabolic activity, and morphology.


Subject(s)
Gamma Rays , Sporothrix/radiation effects , Animals , DNA, Fungal/radiation effects , Fungal Vaccines/chemistry , Fungal Vaccines/radiation effects , Humans , Mice , Mice, Inbred BALB C , Sporothrix/growth & development , Sporothrix/metabolism , Sporothrix/pathogenicity , Sporotrichosis/microbiology , Sporotrichosis/therapy , Vaccines, Attenuated/chemistry , Vaccines, Attenuated/radiation effects
3.
Article in Russian | MEDLINE | ID: mdl-21061574

ABSTRACT

AIM: To assess increase of protective efficacy of live cold-adapted (ca) influenza vaccine after addition of adjuvant chitozan. MATERIALS AND METHODS: Used viruses: ca donor of attenuation A/Krasnodar/101/35/59 (H2N2) and epidemic strain A/Krasnodar/101/59 (H2N2); as an adjuvant--derivative of chitozan and microparticles of chitozan. Experiments were performed in outbred mice. Protective effect of immunization was measured by intranasal challenge by virulent strain of virus. Immune response was assessed by ELISA and indirect hemagglutination inhibition assay. RESULTS: During intranasal immunization of mice with intact CA donor of attenuation A/Krasnodar/101/35/59 (H2N2) addition of 1% solution of chitozan glutamate to vaccine material resulted in increased serum IgG in immunized mice and protective effect of immunization. Addition of adjuvant to ca donor strain did not influence on its ts-characteristic. It was shown that inactivated with ultraviolet radiation ca donor strain in combination with chitozan did not protect against infection caused by virulent strain A/Krasnodar/101/59, whereas the same doses of intact ca donor strain with chitozan were protective. Chitozan did not enhance replication of donor strain in upper respiratory tract of mice. CONCLUSION: Obtained data demonstrate that chitozan as a mucous-adhesive adjuvant could increase efficacy of live ca influenza vaccine.


Subject(s)
Adjuvants, Immunologic , Chitosan/immunology , Influenza A Virus, H2N2 Subtype/immunology , Influenza Vaccines/immunology , Orthomyxoviridae Infections/prevention & control , Administration, Intranasal , Animals , Antibodies, Viral/blood , Chitosan/administration & dosage , Drug Evaluation, Preclinical , Influenza Vaccines/administration & dosage , Influenza Vaccines/radiation effects , Mice , Orthomyxoviridae Infections/blood , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
4.
Parasitology ; 136(12): 1621-32, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19327194

ABSTRACT

The high level of protection which can be induced by vaccination of a range of hosts, from rodents to primates, with live radiation-attenuated schistosome larvae offers great promise for development of a human schistosome vaccine. Studies of the irradiated vaccine models benefitted from significant funding during the 1970-90s and much was learned concerning the inducers, targets and mechanisms of immunity. Less progress was made in definition of the protective antigens involved. The application of new techniques for identifying membrane and secreted antigens has recently provided new vaccine candidates and a new impetus for schistosome vaccine development. This article is intended as an overview of some of the main lessons learned from the studies of the irradiated vaccines as a backdrop to renewed interest in schistosome vaccine development.


Subject(s)
Gamma Rays , Schistosoma/immunology , Schistosoma/radiation effects , Schistosomiasis/prevention & control , Vaccination , Vaccines, Attenuated , Animals , Antibodies, Helminth/blood , Disease Models, Animal , Humans , Mice , Rats , Schistosoma/classification , Schistosoma/growth & development , Schistosomiasis/immunology , Schistosomiasis/parasitology , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
5.
Vaccine ; 27(27): 3675-80, 2009 Jun 02.
Article in English | MEDLINE | ID: mdl-19071177

ABSTRACT

We are developing a radiation attenuated Plasmodium falciparum sporozoite (PfSPZ) malaria vaccine. An important step was to determine the minimum dose of irradiation required to adequately attenuate each sporozoite. This was studied in the Plasmodium yoelii rodent model system. Exposure to 100 Gy completely attenuated P. yoelii sporozoites (PySPZ). Next we demonstrated that immunization of mice intravenously with 3 doses of 750 PySPZ that had received 200 Gy, double the radiation dose required for attenuation, resulted in 100% protection. These results support the contention that a radiation attenuated sporozoite vaccine for malaria will be safe and effective at a range of radiation doses.


Subject(s)
Malaria Vaccines/radiation effects , Plasmodium yoelii/immunology , Sporozoites/radiation effects , Animals , Dose-Response Relationship, Radiation , Female , Immunization , Malaria Vaccines/adverse effects , Malaria Vaccines/immunology , Mice , Mice, Inbred BALB C , Mice, SCID , Safety , Sporozoites/immunology , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
6.
Infect Immun ; 75(7): 3539-47, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17485453

ABSTRACT

The cytokine interplay during the development of protective immunity to the radiation-attenuated (RA) schistosome vaccine has been extensively characterized over recent years, yet the role of costimulatory molecules in the development of cell-mediated immunity is much less well understood. Here we demonstrate the importance of CD40/CD154 in vaccine-induced immunity, as CD154(-/-) mice exposed to RA schistosomes develop no protection to challenge infection. We showed that vaccinated CD154(-/-) mice have defective Th1-associated immune responses in the skin-draining lymph nodes and the lungs, with reduced or absent levels of interleukin-12p40 (IL-12p40), gamma interferon, and nitric oxide, but elevated levels of lung IL-4 and IL-5. The expression of major histocompatibility complex II (MHC-II) on antigen-presenting cells recovered from the lungs of vaccinated CD154(-/-) mice was also severely compromised. The administration of anti-CD40 monoclonal antibody (MAb) to CD154(-/-) mice did not reconstitute sustained Th1 responses in the lymph nodes or the lungs, nor did the MAb restore anti-parasite immunoglobulin G production or protective immunity. On the other hand, the administration of recombinant IL-12 (rIL-12) to CD154(-/-) mice shortly after vaccination caused elevated and sustained levels of Th1-associated cytokines, rescued MHC-II expression by lung CD11c(+) cells, and restored the appearance of inflammatory effector foci in the lungs. However, the treatment of CD154(-/-) mice with rIL-12 did not restore protection. We conclude that protective immunity to the RA schistosome vaccine is CD154 dependent but is independent of IL-12-orchestrated cellular immune mechanisms in the lungs.


Subject(s)
CD40 Ligand/metabolism , Interleukin-12/administration & dosage , Schistosoma mansoni/immunology , Schistosomiasis mansoni/immunology , Schistosomiasis mansoni/prevention & control , Vaccines, Attenuated/administration & dosage , Animals , Antibodies, Helminth/blood , CD40 Ligand/deficiency , CD40 Ligand/genetics , Female , Interleukin-12/immunology , Lung/immunology , Lung/parasitology , Mice , Mice, Inbred C57BL , Recombinant Proteins/administration & dosage , Recombinant Proteins/immunology , Schistosoma mansoni/growth & development , Schistosoma mansoni/pathogenicity , Schistosoma mansoni/radiation effects , Schistosomiasis mansoni/parasitology , Th1 Cells/immunology , Vaccination , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
7.
J Eukaryot Microbiol ; 53(2): 151-6, 2006.
Article in English | MEDLINE | ID: mdl-16579818

ABSTRACT

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P<0.05) levels of interferon gamma, interleukin (IL)-10, and small amounts of IL-4. Antibody isotype-specific ELISA of sera from vaccinated mice exhibited both IgG1 and IgG2a isotypes of antibodies. Vaccinated mice were challenged intraperitoneally with 2 x 10(7)N. caninum tachyzoites. All vaccinated mice remained healthy and showed no obvious signs of neosporosis up to the 25th day post-challenge when the study was terminated. All unvaccinated control mice died within 1 wk of infection. Gamma-irradiated N. caninum tachyzoites can serve as an effective, attenuated vaccine for N. caninum.


Subject(s)
Coccidiosis/prevention & control , Gamma Rays , Neospora , Protozoan Vaccines , Vaccines, Attenuated , Animals , Antibodies, Protozoan/blood , Cattle , Coccidiosis/parasitology , Coccidiosis/pathology , Cytokines/metabolism , Female , Mice , Mice, Inbred C57BL , Neospora/growth & development , Neospora/immunology , Neospora/pathogenicity , Neospora/radiation effects , Protozoan Vaccines/administration & dosage , Protozoan Vaccines/radiation effects , Spleen/immunology , Vaccination , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/radiation effects
9.
Acta Biochim Biophys Sin (Shanghai) ; 37(4): 254-64, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15806292

ABSTRACT

Vaccination with ultraviolet-attenuated cercariae of Schistosoma japonicum induced protective immunity against challenge infection in experimental animal models. Our preliminary study on the transcription levels of IFN-gamma and IL-4 in splenic CD4+ T cells revealed that attenuated cercariae elicited predominantly a Th1 response in mice at the early stage, whereas normal cercariae stimulated primarily Th2-dependent responses. Further analysis on the gene profile of the skin-draining lymph nodes demonstrated that the levels of IFN-gamma were significantly higher in vaccinated mice than those in infected mice at day 4, 7 and 14 post-vaccination or post-infection. However, for IL-12 and IL-4, the potent inducers of Th1 and Th2 responses, respectively, as well as IL-10, there were no differences over the course of the experiment between the infected and vaccinated mice. To explore the underlying factors that may potentially contribute to elevated IFN-gamma in vaccinated mice, the mRNA profiles of the skin-draining lymph nodes at day 4 post-exposure were compared using oligonucleotide microarrays. Within the 847 probe sets with increased signal values, we focused on chemokines, cytokines and relevant receptors, which were validated by semi-quantitative RT-PCR. A comprehensive understanding of the immune mechanisms of attenuated cercariae-induced protection may contribute to developing efficient vaccination strategies against S. japonicum, especially during the early stage of infection.


Subject(s)
Cytokines/blood , Interferon-gamma/blood , Schistosoma japonicum/immunology , Skin/immunology , Transcription Factors/blood , Vaccination/methods , Vaccines, Attenuated/administration & dosage , Animals , Female , Gene Expression Profiling/methods , Larva/immunology , Larva/radiation effects , Mice , Mice, Inbred C57BL , Schistosoma japonicum/radiation effects , Ultraviolet Rays , Vaccines, Attenuated/radiation effects
10.
J Exp Biol ; 206(Pt 21): 3803-8, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14506215

ABSTRACT

Annually, malaria causes >300 million clinical cases and 1 million deaths, is responsible for the loss of >1% of gross domestic product (GDP) in Africa and is a serious concern for travelers. An effective vaccine could have a dramatic impact on the disease. For 20 years, scientists have tried to develop modern, recombinant 'subunit' malaria vaccines. This has been difficult. In fact, there is only one recombinant protein vaccine on the market for any disease, and no vaccines based on synthetic peptides, recombinant viruses, recombinant bacteria or DNA plasmids. Most vaccines are based on attenuated or inactivated whole pathogens or material derived directly from the infectious agent. It is in that context that our recent report summarizing the protection of humans with attenuated Plasmodium falciparum (Pf) sporozoites produced at four different sites over 25 years is important. In studies utilizing live mosquitoes as the vaccine delivery mechanism, there was complete protection against malaria in 93% of volunteers (13/14) and 94% of challenges (33/35). Sanaria's goal is to develop and commercialize a non-replicating, metabolically active Pf sporozoite vaccine. Three practical questions must be addressed before manufacturing for clinical trials: (1) can one administer the vaccine by a route that is clinically practical; (2) can one produce adequate quantities of sporozoites; and (3) can sporozoites be produced with the physical characteristics that meet the regulatory, potency and safety requirements of regulatory authorities? Once these questions have been answered, Sanaria will demonstrate that the vaccine protects >90% of human recipients against experimental challenge with Pf sporozoites, can be produced with an efficiency that makes it economically feasible, and protects >90% of African infants and children from infection, and has severe morbidity and mortality. By producing a vaccine for travelers, Sanaria will provide the infrastructure, regulatory foundation and funds necessary to speed licensure, manufacturing and deployment of the vaccine for the infants and children who need it most.


Subject(s)
Malaria Vaccines/immunology , Malaria, Falciparum/prevention & control , Plasmodium falciparum/immunology , Sporozoites/immunology , Vaccination/methods , Animals , Plasmodium falciparum/radiation effects , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
11.
Can Vet J ; 42(5): 368-74, 2001 May.
Article in English | MEDLINE | ID: mdl-11360859

ABSTRACT

The viability of an attenuated live virus rabies vaccine in a bait targeted to red foxes was examined under various operational conditions in a series of experiments in Ontario. The virus was relatively stable over a 28-day period in the field, losing a mean 0.5, s = 0.2 log10 of virus titer. The micro-environment into which the bait was placed (open cultivated field, grassy meadow, wooded grove, sun or shade) did not make an appreciable difference in the viability of the virus. There was no significant difference (P < or = 0.05) between mean ambient temperatures and the temperature of fluids in blister packs of baits placed in sun or shade. Sixty-three percent of foxes fed baits exposed to sun and shade conditions for 21 days (titer 10(6.2) tissue culture infective doses per 1 mL) developed rabies virus-neutralizing antibodies. Storage of vaccine baits at -30 degrees C prior to bait distribution was important in maintaining virus viability.


Subject(s)
Foxes , Rabies Vaccines/administration & dosage , Rabies/veterinary , Vaccination/veterinary , Animals , Drug Stability , Rabies/prevention & control , Rabies Vaccines/radiation effects , Rabies Vaccines/standards , Rabies virus/immunology , Rabies virus/physiology , Rabies virus/radiation effects , Sunlight/adverse effects , Temperature , Time Factors , Vaccination/methods , Vaccination/standards , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/radiation effects , Vaccines, Attenuated/standards
12.
J Helminthol ; 59(1): 57-60, 1985 Mar.
Article in English | MEDLINE | ID: mdl-3989264

ABSTRACT

Twenty-one mice were injected intramuscularly with 2000 Schistosoma mansoni schistosomula irradiated at 20 krad and cryopreserved; three mice were killed on each of days 0, 2, 5, 9, 19, 28 and 44 days after infection and muscle from the site of injection in the left hind leg, the lungs and livers removed for histological examination. Schistosomula were seen in sections from the leg muscle from days 0 to 19 inclusive, in the lungs from day 2 to day 28 inclusive and in the livers from days 9 to 28 inclusive. Most schistosomula were seen in sections of the leg muscle with considerably fewer parasites occurring in the lungs and especially the livers. Granulomatous reactions comprising eosinophils, polymorphs, plasma cells and macrophages were first seen in the leg muscle on day 2, in the lungs on day 5 and in the liver on day 19. The peak inflammatory reactions appeared to occur between days 5 and 9, 9 and 19 and 28 and 44 respectively in the three tissues. The pathology is discussed in relation to the dose of irradiation required to attenuate the schistosomula for optimal immunogenicity.


Subject(s)
Schistosoma mansoni/immunology , Schistosomiasis/pathology , Vaccines/radiation effects , Animals , Freezing , Larva/immunology , Larva/radiation effects , Male , Mice , Mice, Inbred CBA , Preservation, Biological , Schistosoma mansoni/radiation effects , Schistosomiasis/parasitology , Schistosomiasis/prevention & control , Time Factors , Vaccines/immunology , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
13.
J Helminthol ; 59(1): 51-5, 1985 Mar.
Article in English | MEDLINE | ID: mdl-3989263

ABSTRACT

Three sheep were vaccinated with two doses of 3 krad-irradiated cryopreserved Schistosoma bovis schistosomula containing 20,000 and 17,000 organisms respectively, injected intramuscularly 23 days apart after storage in liquid nitrogen for between 9 and 46 days. A challenge of 5360 S. bovis cercariae was administered percutaneously approximately four weeks after the last vaccine dose to these animals and to three controls. Post-challenge the vaccinated animals gained significantly more weight (27% v. 9%), produced fewer eggs in their faeces, showed a smaller reduction in PCV values (-18% v. -27%) and were over-all in better condition than control animals. At perfusion 49.1% fewer adult worms were found in the vaccinated sheep than in controls. The tissue egg burdens were similar in both groups. Histopathologically both groups were similar except that fewer and smaller egg lesions were observed in the livers of vaccinated animals.


Subject(s)
Schistosoma/radiation effects , Schistosomiasis/veterinary , Sheep Diseases/prevention & control , Vaccines/radiation effects , Animals , Cobalt Radioisotopes , Dose-Response Relationship, Immunologic , Feces/parasitology , Female , Freezing , Larva/immunology , Larva/radiation effects , Male , Parasite Egg Count/veterinary , Preservation, Biological , Schistosoma/immunology , Schistosomiasis/prevention & control , Sheep , Vaccines/immunology , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
14.
Parasite Immunol ; 5(6): 567-75, 1983 Nov.
Article in English | MEDLINE | ID: mdl-6657295

ABSTRACT

Eleven strains of inbred mice were examined for their ability to develop resistance to challenge Schistosoma mansoni infection as a result of previous exposure to homologous cercariae that had been attenuated by high-dose irradiation. Two strains, C57B1/6J and BALB/c, demonstrated consistently high levels of vaccine-induced immunity (means of 64% and 58% resistance, respectively, when compared to control groups of the same strain) and were designated as 'high responder' strains to vaccination. Six other strains fell into an intermediate category, demonstrating moderate, yet statistically significant, levels of immunity resulting from vaccination (means of 30-50% resistance). Only one of the strains examined consistently failed to respond to vaccination by the development of significant levels of immunity to challenge infection. Animals of the P/N strain demonstrated a mean of only 15% resistance to challenge in five experiments and have been classified as 'low responders' to vaccination. P/N mice have previously been characterized as deficient in their ability to mount delayed hypersensitivity reactions, produce lymphokine and display macrophage activation for cytolysis of extracellular and intracellular targets in other experimental systems, suggesting that these immune responses may be critical to the establishment of vaccine-induced resistance to S. mansoni infection. The availability of high and low responder mouse strains should facilitate a genetic approach to characterization of the immune effector mechanism(s) of vaccine-induced resistance to S. mansoni infection.


Subject(s)
Disease Models, Animal , Mice, Inbred Strains/immunology , Schistosomiasis/immunology , Vaccines/immunology , Animals , Female , Male , Mice , Mice, Inbred A/immunology , Mice, Inbred BALB C/immunology , Mice, Inbred C3H/immunology , Mice, Inbred C57BL/immunology , Mice, Inbred CBA/immunology , Mice, Inbred Strains/genetics , Schistosoma mansoni/immunology , Schistosoma mansoni/radiation effects , Vaccination/methods , Vaccines/radiation effects , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
15.
Zh Mikrobiol Epidemiol Immunobiol ; (4): 98-101, 1982 Apr.
Article in Russian | MEDLINE | ID: mdl-7080770

ABSTRACT

The results obtained in the study of the specific potency of rabies vaccine prepared from sheep brain tissue and inactivated by UV irradiation indicate that, even in the presence of the lowest immunogenicity index (0.5), 5-6 injections of the vaccine, made not daily, but at interval of 3 and 7 days, induced the production of antibodies in the titers not lower than those resulting from 14-20 daily injections of the same vaccine or Fermi vaccine. The preparation inactivated by UV irradiation should be introduced for therapy according to the shortened immunization schedule with intervals, taking into account the immunogenicity index.


Subject(s)
Brain/immunology , Rabies Vaccines/immunology , Ultraviolet Rays , Adolescent , Adult , Aged , Animals , Antibodies, Viral/analysis , Humans , Immunization Schedule , Middle Aged , Rabbits , Rabies Vaccines/administration & dosage , Rabies Vaccines/radiation effects , Rabies virus/immunology , Sheep/immunology , Time Factors , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vaccines, Attenuated/radiation effects
17.
Zh Mikrobiol Epidemiol Immunobiol ; (11): 28-30, 1980 Nov.
Article in Russian | MEDLINE | ID: mdl-6160700

ABSTRACT

The study of the effect of gamma globulin introduced in different doses (0.5 and 0.25 ml/mg) in combination with Fermi rabies vaccine (observations on humans were made) and with cerebral rabies vaccine inactivated by UV irradiation (in animal experiments) demonstrated that the injection of the higher doses of gamma globulin resulted in lower geometrical mean of antibody titers. Therefore, in combined administration of rabies vaccine and gamma globulin for postexposure rabies prevention it is advisable to reduce the dose of gamma globulin by one-half.


Subject(s)
Antibody Specificity , Immunity, Active , Rabies Vaccines/administration & dosage , Animals , Antibodies, Viral/analysis , Bites and Stings/immunology , Dose-Response Relationship, Immunologic , Humans , Rabies Vaccines/radiation effects , Rats , Ultraviolet Rays , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/radiation effects , gamma-Globulins/administration & dosage
18.
Zh Mikrobiol Epidemiol Immunobiol ; (6): 39-41, 1979 Jun.
Article in Russian | MEDLINE | ID: mdl-380240

ABSTRACT

Tests in volunteers showed that the reactogenicity of rabies vaccine prepared from sheep brain and inactivated with ultraviolet rays was not greater than the reactogenicity of Fermi vaccine. At the same time it was found to have a higher activity when injected both in the form of 5% suspension (in full and decreased doses) and with brain tissue content as low as 2.5%.


Subject(s)
Antigens, Viral/immunology , Brain/immunology , Rabies Vaccines/radiation effects , Ultraviolet Rays , Vaccines, Attenuated/radiation effects , Adolescent , Adult , Animals , Antibodies, Viral/analysis , Bites and Stings/drug therapy , Clinical Trials as Topic , Drug Evaluation , Humans , Immunization , Middle Aged , Rabies/prevention & control , Rabies Vaccines/immunology , Rabies virus/immunology , Sheep/immunology , Time Factors , Vaccines, Attenuated/immunology
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