RESUMO

Previous attempts to develop a vaccine against bovine leukemia virus (BLV) have not been successful because of inadequate or short-lived stimulation of all immunity components. In this study, we designed an approach based on an attenuated BLV provirus by deleting genes dispensable for infectivity but required for efficient replication. The ability of the vaccine to protect from natural BLV infection was investigated in the context of dairy productive conditions in an endemic region. The attenuated vaccine was tested in a farm in which the prevalence rose from 16.7% in young cattle at the beginning of the study to more than 90% in adult individuals. Sterilizing immunity was obtained in 28 out of 29 vaccinated heifers over a period of 48 months, demonstrating the effectiveness of the vaccine. As indicated by the antiviral antibody titers, the humoral response was slightly reduced compared to wild-type infection. After initial post-vaccination bursts, the proviral loads of the attenuated vaccine remained most frequently undetectable. During the first dairy cycle, proviral DNA was not detected by nested-PCR in milk samples from vaccinated cows. During the second dairy cycle, provirus was sporadically detected in milk of two vaccinated cows. Forty-two calves born from vaccinated cows were negative for proviral DNA but had antiviral antibodies in their peripheral blood. The attenuated strain was not transmitted to sentinels, further supporting the safety of the vaccine. Altogether, these data thus demonstrate that the vaccine against BLV is safe and effective in herd conditions characterized by a very high incidence. This cost-effective approach will thus decrease the prevalence of BLV without modification of production practices. After facing a series of challenges pertaining to effectiveness and biosafety, the vaccine is now available for further large-scale delivery. The different challenges and hurdles that were bypassed may be informative for the development of a vaccine against HTLV-1.

Assuntos

RESUMO

Identification of alternative attenuation targets of Mycobacterium tuberculosis (Mtb) is pivotal for designing new candidates for live attenuated anti-tuberculosis (TB) vaccines. In this context, the CtpF P-type ATPase of Mtb is an interesting target; specifically, this plasma membrane enzyme is involved in calcium transporting and response to oxidative stress. We found that a mutant of MtbH37Rv lacking ctpF expression (MtbΔctpF) displayed impaired proliferation in mouse alveolar macrophages (MH-S) during in vitro infection. Further, the levels of tumor necrosis factor and interferon-gamma in MH-S cells infected with MtbΔctpF were similar to those of cells infected with the parental strain, suggesting preservation of the immunogenic capacity. In addition, BALB/c mice infected with Mtb∆ctpF showed median survival times of 84 days, while mice infected with MtbH37Rv survived 59 days, suggesting reduced virulence of the mutant strain. Interestingly, the expression levels of ctpF in a mouse model of latent TB were significantly higher than in a mouse model of progressive TB, indicating that ctpF is involved in Mtb persistence in the dormancy state. Finally, the possibility of complementary mechanisms that counteract deficiencies in Ca2+ transport mediated by P-type ATPases is suggested. Altogether, our results demonstrate that CtpF could be a potential target for Mtb attenuation.

Assuntos

RESUMO

Cattle babesiosis is a socio-economically important tick-borne disease caused by Apicomplexa protozoa of the genus Babesia that are obligate intraerythrocytic parasites. The pathogenicity of Babesia parasites for cattle is determined by the interaction with the host immune system and the presence of the parasite's virulence genes. A Babesia bigemina strain that has been maintained under a microaerophilic stationary phase in in vitro culture conditions for several years in the laboratory lost virulence for the bovine host and the capacity for being transmitted by the tick vector. In this study, we compared the virulome of the in vitro culture attenuated Babesia bigemina strain (S) and the virulent tick transmitted parental Mexican B. bigemina strain (M). Preliminary results obtained by using the Basic Local Alignment Search Tool (BLAST) showed that out of 27 virulence genes described and analyzed in the B. bigemina virulent tick transmitted strain, only five were fully identified in the attenuated laboratory strain. In all cases, the identity and coverture of the identified genes of the wildtype strain were higher than those of the laboratory strain. This finding is putatively associated with the continuous partial loss of virulence genes in the laboratory strain after several passages of the parasite population under optimal in vitro growth conditions. The loss of virulence factors might be reflected in the absence of symptoms of the disease in cattle inoculated with the attenuated strain despite the presence of infection in the bovine host cells.

RESUMO

Hematological parameters were evaluated in broilers immunized and challenged with Eimeria tenella. Broiler chickens of Hubbard strain, females, coccidian-free, were kept in wire cages and inoculated on the third day. The experiment was designed to include five sorts of treatment with three replicates each. T1 was the negative control group, T2 received 500 attenuated sporulated oocysts by gavage, T3 was the positive control, T4 received 50 µg of sporozoite protein + Quil A vaccine, and T5 received Quil A without sporozoite protein + PBS, the last two through nasal route on days 0, 7, and 21. On the 31st day, all treatments were challenged with homologous virulent strain of E. tenella in the dose of 8.0 × 10(4) oocysts, with the exception of T1. One week later, blood sampling, lesion scores, and cecal oocyst count were carried out. The parasitological parameters showed statistical significance (p < 0.05) and there was no damage to the hematological parameters of birds (p > 0.05) by ANOVA test. The correlations suggest that the blood parameters were impaired by effects of the parasite on tissue, showing levels of hemorrhage and/or hydration.

Foram avaliados os parâmetros hematológicos em frangos de corte imunizados e desafiados com Eimeria tenella. Pintos de corte fêmeas da linhagem Hubbard, livres de coccídios, foram mantidos em baterias metálicas e inoculados no terceiro dia. O experimento foi delineado por cinco tratamentos com três repetições cada, sendo: T1 controle negativo, T2 recebeu 500 oocistos esporulados atenuados via oral, T3 controle positivo, T4 recebeu vacina contendo 50 µg de proteínas de esporozoítos + Quil A e T5 recebeu Quil A + PBS, sendo os dois últimos por via nasal nos dias 0, 7 e 21. No dia 31, todos os tratamentos foram desafiados com cepa virulenta homóloga de E. tenella na dose de 8,0 × 10(4) oocistos, exceto T1. Uma semana depois, foi realizada amostragem de sangue, escore de lesão e contagem de oocistos cecais. Os parâmetros parasitológicos apresentaram significância estatística (p < 0,05), sem que causassem prejuízos aos parâmetros hematológicos das aves (p > 0,05), pelo teste ANOVA. As correlações sugerem que os parâmetros sanguíneos foram afetados pelos efeitos do parasita no tecido, apresentando níveis de hemorragia e/ou hidratação.

Assuntos

RESUMO

Hematological parameters were evaluated in broilers immunized and challenged with Eimeria tenella. Broiler chickens of Hubbard strain, females, coccidian-free, were kept in wire cages and inoculated on the third day. The experiment was designed to include five sorts of treatment with three replicates each. T1 was the negative control group, T2 received 500 attenuated sporulated oocysts by gavage, T3 was the positive control, T4 received 50 µg of sporozoite protein + Quil A vaccine, and T5 received Quil A without sporozoite protein + PBS, the last two through nasal route on days 0, 7, and 21. On the 31st day, all treatments were challenged with homologous virulent strain of E. tenella in the dose of 8.0 × 10(4) oocysts, with the exception of T1. One week later, blood sampling, lesion scores, and cecal oocyst count were carried out. The parasitological parameters showed statistical significance (p 0.05) and there was no damage to the hematological parameters of birds (p > 0.05) by ANOVA test. The correlations suggest that the blood parameters were impaired by effects of the parasite on tissue, showing levels of hemorrhage and/or hydration.

Foram avaliados os parâmetros hematológicos em frangos de corte imunizados e desafiados com Eimeria tenella. Pintos de corte fêmeas da linhagem Hubbard, livres de coccídios, foram mantidos em baterias metálicas e inoculados no terceiro dia. O experimento foi delineado por cinco tratamentos com três repetições cada, sendo: T1 controle negativo, T2 recebeu 500 oocistos esporulados atenuados via oral, T3 controle positivo, T4 recebeu vacina contendo 50 µg de proteínas de esporozoítos + Quil A e T5 recebeu Quil A + PBS, sendo os dois últimos por via nasal nos dias 0, 7 e 21. No dia 31, todos os tratamentos foram desafiados com cepa virulenta homóloga de E. tenella na dose de 8,0 × 10(4) oocistos, exceto T1. Uma semana depois, foi realizada amostragem de sangue, escore de lesão e contagem de oocistos cecais. Os parâmetros parasitológicos apresentaram significância estatística (p 0,05), sem que causassem prejuízos aos parâmetros hematológicos das aves (p > 0,05), pelo teste ANOVA. As correlações sugerem que os parâmetros sanguíneos foram afetados pelos efeitos do parasita no tecido, apresentando níveis de hemorragia e/ou hidratação.

RESUMO

Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80 percent. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain.

Salmonella enterica serovar Gallinarum (SG) é o agente do tifo aviário, doença severa que provoca mortalidade em até 80 por cento do plantel de aves. SG encontra-se entre os poucos sorotipos de Salmonella que são agentes etiológicos de enfermidade específica, à semelhança de Salmonella Typhi em seres humanos podendo, portanto, servir de modelo experimental para outras salmoneloses hospedeiro especíifcas. Além dos mecanismos de virulência, a bactéria utiliza mecanismos de sobrevivência para permanecer no hospedeiro. A ativação desses mecanismos pode ou não estar associada à ativação dos mecanismos de virulência. Entre os mecanismos fisiológicos, está a produção de vitamina B12 que Salmonella spp. realiza em ambientes anaeróbicos, como quando encontra-se intracelularmente no organismo hospedeiro. Neste estudo, analisou-se a infecção de aves por cepas de SG, que tiveram genes alterados que participam da biossíntese de vitamina B12. Foram produzidos mutantes de SG contendo os genes cbiA e cobS alterados e um terceiro, contendo ambos os genes alterados. A sobrevivência e a ação patogênica de SG não foi modificada pela alteração simples de um dos genes, mas tornou a cepa de SG completamente atenuada quando os dois foram modificados. A mortalidade provocada pela cepa selvagem de SG foi de 64,52 por cento, enquanto que não observou-se mortalidade alguma no grupo de aves infectadas com SGNal r"cobs"cbiA. Estudos futuros deverão ser realizados para elucidar este processo fisiológico bacteriano e para avaliar a utilização desta cepa de SG como cepa vacinal.

RESUMO

Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain.

RESUMO

Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain.

Salmonella enterica serovar Gallinarum (SG) é o agente do tifo aviário, doença severa que provoca mortalidade em até 80% do plantel de aves. SG encontra-se entre os poucos sorotipos de Salmonella que são agentes etiológicos de enfermidade específica, à semelhança de Salmonella Typhi em seres humanos podendo, portanto, servir de modelo experimental para outras salmoneloses hospedeiro especíifcas. Além dos mecanismos de virulência, a bactéria utiliza mecanismos de sobrevivência para permanecer no hospedeiro. A ativação desses mecanismos pode ou não estar associada à ativação dos mecanismos de virulência. Entre os mecanismos fisiológicos, está a produção de vitamina B12 que Salmonella spp. realiza em ambientes anaeróbicos, como quando encontra-se intracelularmente no organismo hospedeiro. Neste estudo, analisou-se a infecção de aves por cepas de SG, que tiveram genes alterados que participam da biossíntese de vitamina B12. Foram produzidos mutantes de SG contendo os genes cbiA e cobS alterados e um terceiro, contendo ambos os genes alterados. A sobrevivência e a ação patogênica de SG não foi modificada pela alteração simples de um dos genes, mas tornou a cepa de SG completamente atenuada quando os dois foram modificados. A mortalidade provocada pela cepa selvagem de SG foi de 64,52%, enquanto que não observou-se mortalidade alguma no grupo de aves infectadas com SGNal r"cobs"cbiA. Estudos futuros deverão ser realizados para elucidar este processo fisiológico bacteriano e para avaliar a utilização desta cepa de SG como cepa vacinal.