The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

The Plasmodium falciparum parasite, carried by Anopheles mosquitoes, is currently a global problem due to the rising incidence of resistance of the parasite to available antimalaria drugs. Resistance and difficult treatment groups, including pregnant woman and young children, are pressing for the development of new, safe and effective prophylactic and treatment antimalarials. Because of the extensive process of developing new drugs, researchers and health care professionals have turned to combination therapy where a fast acting antimalarial is combined with slower acting drugs, such as antibiotics. The macrolide antibiotics, erytbromycin and azithromycin, have been studied to a limited extent for their potential antimalarial effect. Certain advantages, such as their safety profile (especially that of azithromycin) in pregnancy and administration to young children, motivates continual research into the advancement of the effect these drugs exude on malaria. Drug delivery systems contribute to the efficacy of medicines, conquering several difficulties of treatment with oral medication. Pheroid™ technology is a patented drug delivery system, mainly consisting of plant and essential fatty acids, and has been demonstrated to entrap, carry and deliver pharmacologically active compounds and other useful molecules. This study compared the in vitro effects of the macrolide antibiotics on the growth of a chloroquine-resistant strain (RSA 11) of Plasmodium falciparum to the effects of the macrolides entrapped in Pheroid™ vesicles on the same strain over and extended observation period of 144 hours. ELISA assays were conducted by analysing the HRP II (histidine-rich protein) levels on a pre-coated microtitre plate. The effects of the type of formulation, concentration and time were compared. The in vitro difference between erythromycin alone and entrapped in Pheroid™ vesicles were found to be statistically significant (p = 0.000000) while the effects of both formulations did not seem to be concentration dependant (p = 0.628424). Prolonged exposure was also statistically meaningful (p = 0.008268), though it seems that exposure need not exceed 96 hours. The type of formulation, in the case of azithromycin (azithromycin alone vs. azitbromycin entrapped in Pheroid™ vesicles), proved statistically significant (P = 0.002572), while neither formulation seemed concentration dependant (P = 0.427731). Prolonged exposure was found to be statistically insignificant for azithromycin (P = 0.221941). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

The Plasmodium falciparum parasite, carried by Anopheles mosquitoes, is currently a global problem due to the rising incidence of resistance of the parasite to available antimalaria drugs. Resistance and difficult treatment groups, including pregnant woman and young children, are pressing for the development of new, safe and effective prophylactic and treatment antimalarials. Because of the extensive process of developing new drugs, researchers and health care professionals have turned to combination therapy where a fast acting antimalarial is combined with slower acting drugs, such as antibiotics. The macrolide antibiotics, erytbromycin and azithromycin, have been studied to a limited extent for their potential antimalarial effect. Certain advantages, such as their safety profile (especially that of azithromycin) in pregnancy and administration to young children, motivates continual research into the advancement of the effect these drugs exude on malaria. Drug delivery systems contribute to the efficacy of medicines, conquering several difficulties of treatment with oral medication. Pheroid™ technology is a patented drug delivery system, mainly consisting of plant and essential fatty acids, and has been demonstrated to entrap, carry and deliver pharmacologically active compounds and other useful molecules. This study compared the in vitro effects of the macrolide antibiotics on the growth of a chloroquine-resistant strain (RSA 11) of Plasmodium falciparum to the effects of the macrolides entrapped in Pheroid™ vesicles on the same strain over and extended observation period of 144 hours. ELISA assays were conducted by analysing the HRP II (histidine-rich protein) levels on a pre-coated microtitre plate. The effects of the type of formulation, concentration and time were compared. The in vitro difference between erythromycin alone and entrapped in Pheroid™ vesicles were found to be statistically significant (p = 0.000000) while the effects of both formulations did not seem to be concentration dependant (p = 0.628424). Prolonged exposure was also statistically meaningful (p = 0.008268), though it seems that exposure need not exceed 96 hours. The type of formulation, in the case of azithromycin (azithromycin alone vs. azitbromycin entrapped in Pheroid™ vesicles), proved statistically significant (P = 0.002572), while neither formulation seemed concentration dependant (P = 0.427731). Prolonged exposure was found to be statistically insignificant for azithromycin (P = 0.221941). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Vaccination plays a very important part in daily life. It is essential to get vaccinated at an early age. The conventional parented method used is not always effective and not cost efficient. It requires qualified personnel and sterile conditions for administration of the vaccines. The aim of this study was to investigate the effect of chitosan, N-trimethyl chitosan chloride (TMC) and Emzaloid™ particles on the local and systemic immune response of mice after oral vaccination with Diphtheria toxoid (DT). The different formulations used were chitosan microparticles (± 10 µm), chitosan nanoparticles (± 400 nm), TMC microparticles (± 5 µm), Emzaloid microparticles (± 4 µm) and Emzaloid nanoparticles (± 500 nm). All of these formulations proved to be very good delivery systems and can entrap large amounts of the antigen. Balb/c mice were used to determine the local and systemic immune response of these formulations. The mice were vaccinated orally on three consecutive days in week 1 and 3 with 40 Lf DT per week with a total volume of 300 µl. Blood samples were taken from the mice and analysed for a systemic immune response (IgG). The same mice were used to determine the local immune response (IgA). Faeces were collected from each mouse on day 1, 3, 4, 6, 14 and 20 for analysis. An enzyme-linked immunosorbent assay (ELISA) was used to determine IgG and IgA titers. It can be concluded that chitosan nanoparticles was the only formulation with a higher response than that of the currently used vaccine. Emzaloid nanoparticles showed no significant difference in response when compared to the currently used vaccine. All the other formulations showed a much smaller response than that of the conventional method of vaccination. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Vaccination plays a very important part in daily life. It is essential to get vaccinated at an early age. The conventional parented method used is not always effective and not cost efficient. It requires qualified personnel and sterile conditions for administration of the vaccines. The aim of this study was to investigate the effect of chitosan, N-trimethyl chitosan chloride (TMC) and Emzaloid™ particles on the local and systemic immune response of mice after oral vaccination with Diphtheria toxoid (DT). The different formulations used were chitosan microparticles (± 10 µm), chitosan nanoparticles (± 400 nm), TMC microparticles (± 5 µm), Emzaloid microparticles (± 4 µm) and Emzaloid nanoparticles (± 500 nm). All of these formulations proved to be very good delivery systems and can entrap large amounts of the antigen. Balb/c mice were used to determine the local and systemic immune response of these formulations. The mice were vaccinated orally on three consecutive days in week 1 and 3 with 40 Lf DT per week with a total volume of 300 µl. Blood samples were taken from the mice and analysed for a systemic immune response (IgG). The same mice were used to determine the local immune response (IgA). Faeces were collected from each mouse on day 1, 3, 4, 6, 14 and 20 for analysis. An enzyme-linked immunosorbent assay (ELISA) was used to determine IgG and IgA titers. It can be concluded that chitosan nanoparticles was the only formulation with a higher response than that of the currently used vaccine. Emzaloid nanoparticles showed no significant difference in response when compared to the currently used vaccine. All the other formulations showed a much smaller response than that of the conventional method of vaccination. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Orientador: Iveraldo dos Santos Dutra / Banca: Samir Issa Samara / Banca: Vera Cláudia Lorenzetti Magalhães Curci / Resumo: O carbúnculo sintomático é um problema sanitário mundial, responsável por elevados coeficientes de mortalidade em bovinos e ovinos. A imunização dos animais jovens, seguida de reforço anual até 2,5 anos de idade, é a principal medida profilática. Foram realizados três experimentos distintos com intuito de avaliar as respostas sorológicas de bovinos vacinados contra o carbúnculo sintomático, pelos testes de aglutinação em placa e Elisa, empregando-se como antígenos a cepa de referência (MT) e uma cepa de campo (SP). No primeiro experimento, os bezerros foram organizados em três grupos (G1, G2 e G3) e submetidos a três protocolos distintos de vacinação empregando-se uma vacina comercial polivalente contra clostridioses. O G1 foi primovacinado aos 4 meses de idade e recebeu o reforço na desmama (8 meses). O G2 recebeu a primeira dose na desmama e reforço 30 dias após. O G3 foi vacinado somente na desmama. As coletas de soro foram realizas aos 4, 8, 9 e 10 meses de idade dos bezerros. O G1 apresentou a melhor resposta sorológica em comparação aos outros dois protocolos. Quando a avaliação dos grupos foi realizada aos 10 meses de idade, independente do protocolo empregado, a resposta sorológica foi similar. No segundo experimento, foi avaliada a imunidade natural passiva de bezerros, filhos de vacas vacinadas até 30 dias antes do parto (2ª dose), empregando-se duas vacinas comercias polivalente contra clostridioses. As coletas de soro foram realizadas aos (±)7, 45 e 90 dias de idade dos bezerros. Independente das vacinas empregadas na imunização ativa das mães, a resposta sorológica passiva dos bezerros avaliados foi similar até os 3 meses de idade. Houve uma correlação linear da resposta sorológica passiva dos bezerros com a data de vacinação das mães e o dia do parto quando empregado o teste de Elisa. No terceiro experimento, as 30 vacas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Black leg disease is one of the most important sanitary problem, responsible for high levels of mortality observed in bovines and ovines herds. The vaccination of young animals, followed by annual booter until 2,5 years-old, is the major preventive measure against outbreaks. Three distinct experiments were conducted to measure the vaccinal response from bovines. The vaccinal strains used were the reference MT and field Clostridium chauvoei isolated. Sera from vaccinated animals were tested by agglutination and Enzyme Linked Immunosorbent Assay (Elisa), both standardized for the present study. First experiment, calves were divided into three groups (G1, G2 and G3); and submitted to three vaccination schedule with a polyvalent vaccine. The G1 received first vaccine at 4 months of age and a subsequent booster after calving (8 month-old). The G2 received first vaccine dose after calving and booster at 30 days after. The G3 received only one vaccine dose at 8 months. The sera were colleted at 4, 8, 9 and 10 months for all groups studied. The G1 group showed the best serological response at 10 months of age in comparison to G2 e G3 and control. Moreover, at 10 months of age all groups presented similar levels of serological response. The second experiment, the natural immunity of calves, separated from their mothers vaccinated 30 days before calving with two polyvalent vaccines. The respective serum was colleted at (±) 7, 45 and 90 days of age. All calves presented similar serological response at 3 months of age, independent of vaccinal strain used. The third experiment, 30 heifers, Nelore race, aged above 4 years-old, without vaccination against black leg, were vaccinated with two Clostridium strains. When the SP strain was used the serological response was considered good in G3 (first experiment), second and third experiment for agglutination assay. To compare both techniques, agglutination... (Complete abstract click electronic access below) / Mestre

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Made available in DSpace on 2014-06-11T19:27:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-19Bitstream added on 2014-06-13T18:48:02Z : No. of bitstreams: 1 araujo_rf_me_jabo.pdf: 248554 bytes, checksum: e7d4bd27c73efa391fb0fac3082dd622 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O carbúnculo sintomático é um problema sanitário mundial, responsável por elevados coeficientes de mortalidade em bovinos e ovinos. A imunização dos animais jovens, seguida de reforço anual até 2,5 anos de idade, é a principal medida profilática. Foram realizados três experimentos distintos com intuito de avaliar as respostas sorológicas de bovinos vacinados contra o carbúnculo sintomático, pelos testes de aglutinação em placa e Elisa, empregando-se como antígenos a cepa de referência (MT) e uma cepa de campo (SP). No primeiro experimento, os bezerros foram organizados em três grupos (G1, G2 e G3) e submetidos a três protocolos distintos de vacinação empregando-se uma vacina comercial polivalente contra clostridioses. O G1 foi primovacinado aos 4 meses de idade e recebeu o reforço na desmama (8 meses). O G2 recebeu a primeira dose na desmama e reforço 30 dias após. O G3 foi vacinado somente na desmama. As coletas de soro foram realizas aos 4, 8, 9 e 10 meses de idade dos bezerros. O G1 apresentou a melhor resposta sorológica em comparação aos outros dois protocolos. Quando a avaliação dos grupos foi realizada aos 10 meses de idade, independente do protocolo empregado, a resposta sorológica foi similar. No segundo experimento, foi avaliada a imunidade natural passiva de bezerros, filhos de vacas vacinadas até 30 dias antes do parto (2ª dose), empregando-se duas vacinas comercias polivalente contra clostridioses. As coletas de soro foram realizadas aos (±)7, 45 e 90 dias de idade dos bezerros. Independente das vacinas empregadas na imunização ativa das mães, a resposta sorológica passiva dos bezerros avaliados foi similar até os 3 meses de idade. Houve uma correlação linear da resposta sorológica passiva dos bezerros com a data de vacinação das mães e o dia do parto quando empregado o teste de Elisa. No terceiro experimento, as 30 vacas... / Black leg disease is one of the most important sanitary problem, responsible for high levels of mortality observed in bovines and ovines herds. The vaccination of young animals, followed by annual booter until 2,5 years-old, is the major preventive measure against outbreaks. Three distinct experiments were conducted to measure the vaccinal response from bovines. The vaccinal strains used were the reference MT and field Clostridium chauvoei isolated. Sera from vaccinated animals were tested by agglutination and Enzyme Linked Immunosorbent Assay (Elisa), both standardized for the present study. First experiment, calves were divided into three groups (G1, G2 and G3); and submitted to three vaccination schedule with a polyvalent vaccine. The G1 received first vaccine at 4 months of age and a subsequent booster after calving (8 month-old). The G2 received first vaccine dose after calving and booster at 30 days after. The G3 received only one vaccine dose at 8 months. The sera were colleted at 4, 8, 9 and 10 months for all groups studied. The G1 group showed the best serological response at 10 months of age in comparison to G2 e G3 and control. Moreover, at 10 months of age all groups presented similar levels of serological response. The second experiment, the natural immunity of calves, separated from their mothers vaccinated 30 days before calving with two polyvalent vaccines. The respective serum was colleted at (±) 7, 45 and 90 days of age. All calves presented similar serological response at 3 months of age, independent of vaccinal strain used. The third experiment, 30 heifers, Nelore race, aged above 4 years-old, without vaccination against black leg, were vaccinated with two Clostridium strains. When the SP strain was used the serological response was considered good in G3 (first experiment), second and third experiment for agglutination assay. To compare both techniques, agglutination... (Complete abstract click electronic access below)