RESUMO
A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutination (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Camelídeos Americanos/imunologia , Epitopos/imunologia , Flagelina/imunologia , Leptospira interrogans/imunologia , Leptospirose/veterinária , Lipoproteínas/imunologia , Animais , Antígenos de Bactérias/isolamento & purificação , Argentina/epidemiologia , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Camelídeos Americanos/sangue , Bovinos , Ensaio de Imunoadsorção Enzimática , Epitopos/isolamento & purificação , Flagelina/isolamento & purificação , Leptospirose/epidemiologia , Leptospirose/imunologia , Lipoproteínas/isolamento & purificação , Testes Sorológicos/veterinária , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Brucella abortus M1-luc is a mutant strain derived from S19 vaccine strain in which most of bp26 sequence has been replaced by the luciferase coding gene. Strain I2 is a double mutant derived from M1-luc in which most of omp19 has been deleted without introduction of any genetic markers. In BALB/c mice, M1-luc presented equivalent performance to S19 regarding persistence, splenomegaly and protection against challenge. Interestingly, I2 was more attenuated than S19, with no reduction of protection against challenge. In order to evaluate the potential for vaccine use of these strains in the natural host, four groups of 15 heifers, 6-month old, were either non-vaccinated or vaccinated with S19, M1-luc or I2. To at reached 17-month old, heifers were synchronized with two doses of PGF2alpha and received natural service during 60 days with two bulls. Pregnant heifers were challenged at approximately six gestation months with virulent B. abortus S2308. Blood samples post-challenge of heifers were collected for serologic test as well as specimens of aborted fetuses and premature calves for bacterial isolation and histopathological analyses. Protection levels against abortion were 78.6% for S19, 81.8% for M1-luc and 45.5% for I2, compared to the 25% that did not abort from the non-vaccinated group. These results indicate that in bovines BP26 had no influence in protective capacity of S19, correlating with the results obtained in mice. However, contrarily to what was previously observed in mice, lack of expression of Omp19 rendered in less protection capacity of S19 in the natural host.
Assuntos
Vacinas Bacterianas/imunologia , Brucella abortus/imunologia , Brucelose Bovina/prevenção & controle , Animais , Bovinos , FemininoRESUMO
cDNA clones of potato virus X (PVXcp strain), potato virus Y (PVYo strain), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTV) were used separately or combined for the detection of the corresponding RNAs in extracts of infected plants. A general method for the rapid preparation of RNA extracts without use of organic solvents (i.e. phenol) was developed for this purpose. Plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and protoplast samples, as well as vector insect extracts, were dot-blotted onto nylon or nitrocellulose membranes, subjected to sandwich nucleic acid hybridization with non-labelled specific single-stranded DNA probes followed by a biotin-labelled second step hybridization probe. Each probe was virus-specific but not strain-specific. Healthy or non-related plant extracts developed very faint or no signals. Sensitivity was tested by slot-blot hybridization. Detection levels were between 1.5 to 6 pg of viral nucleic acids and between 20 to 50 times more sensitive than standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The assay developed was tested with material that was prepared for processing in the field (combination of fresh sap with extraction solution) and tested under simple laboratory conditions for detection. It was also successfully employed for screening of germplasm for virus resistance, detection of pathogens in vector insects, plantlets grown in vitro and in more sophisticated quantitative determinations of viral replication in artificially inoculated plants and protoplasts.
Assuntos
Hibridização de Ácido Nucleico , Vírus de Plantas/isolamento & purificação , Solanum tuberosum/microbiologia , Clonagem Molecular , Sondas de DNA , Ensaio de Imunoadsorção Enzimática , Métodos , Doenças das Plantas , Vírus de Plantas/genética , RNA Viral/análise , Sensibilidade e EspecificidadeRESUMO
Mice immunized with FMDV C3 Arg 84 antigen were inoculated intraperitoneally with sarcoma 180 TG. The ascitic fluid obtained by ventral puncture contained high titers of antibodies, similar to those obtained from serum, as determined by neutralization and ELISA tests. Ascitic volumes were 10 to 20 times greater than those obtainable with.
Assuntos
Anticorpos Antivirais/biossíntese , Aphthovirus/imunologia , Líquido Ascítico/imunologia , Animais , Camundongos , Sarcoma 180 , Células Tumorais CultivadasRESUMO
Foot-and-Mouth disease virus (FMDV), contains a positive single-stranded RNA enclosed in a protein capsid. Previous studies have shown that a synthetic peptide located at the carboxyterminal end of VP1 of FMDV strain O1 Kaufbeuren (O1K) at the amino acid positions 144-159, and coupled to KLH was able to elicit high titers of neutralizing antibodies in guinea pigs and protected them against challenge with the homologous virus (8, 15). FMDV strain O1 Campos (O1 Ca) has a similar amino acid sequence to O1K, differing in amino acid 144 (leucin instead valine). We report that the O1K synthetic peptide 144-159 protects adult mice against challenge with FMDV O1Ca when the peptide is coupled to KLH with glutaraldehyde as coupling agent (Table 2) whereas protection is very low when other carrier proteins are used.
Assuntos
Anticorpos Antivirais/imunologia , Aphthovirus/imunologia , Oligopeptídeos/imunologia , Vacinas Sintéticas/imunologia , Vacinas/imunologia , Vacinas Virais/imunologia , Animais , Hemocianinas/imunologia , Testes de Neutralização , Ratos , Especificidade da Espécie , Relação Estrutura-Atividade , Proteínas Virais/imunologia , Proteínas Estruturais ViraisRESUMO
Kinetics of the humoral immune primary response and seven-day secondary response of adult CF1 mice to FMDV O1 Campos adjuvanted in aluminium hydroxide-saponin (AHS) or in oil emulsion (OE) were evaluated by means of ELISA and passive hemagglutination (PH). Analysis of the response to AHS vaccine showed that ELISA measured maximal titres of primary response at 23 days post-vaccination (dpv), and at day 17 of secondary response, while PH detected maximal titres for primary as well as secondary response around day 60 pv. Mice immunized with OE vaccine studied by ELISA presented a plateau of primary response around 40 dpv, while secondary response was maximal around 80 dpv. The same sera tested by PH showed the highest titres for primary response on day 50 pv and secondary response was maximal on day 40 pv. A criterion for the evaluation of vaccination efficiency in the murine model is proposed based on the methods employed in the determination of antibody level.
Assuntos
Anticorpos Antivirais/sangue , Aphthovirus/imunologia , Bioensaio , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Testes de Neutralização , Vacinação , Vacinas Virais/imunologia , Animais , Animais Lactentes , Anticorpos Antivirais/biossíntese , Estudos de Avaliação como Assunto , Febre Aftosa/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos/imunologiaRESUMO
Johne's disease or paratuberculosis is widespread in almost all countries and remains difficult to eradicate. Nowadays, diagnosis of Mycobacterium avium subsp. paratuberculosis (MPTB) infection is one of the main concerns. In this work, we evaluated the expression, biochemical properties and antigenicity of the Apa antigen, encoded by the gene annotated as MAP1569, in the MPTB genome. We confirmed its expression in MPTB and its glycosylation by the ConA binding assay. Although the MPTB-Apa is not an immunodominant antigen, MPTB-infected cattle showed a strong humoral response to recombinant Apa by Western blot and ELISA. Milk was also a suitable sample to be tested by ELISA. We comparatively analysed the humoral cross-reactivity to the Apa from MPTB (MPTB-Apa) and the orthologue from Mycobacterium tuberculosis (MT-Apa, identical to that from Mycobacterium bovis) in both infected and control cows. Response of M. bovis- and MPTB-infected animals against MT-Apa was similar (P=0.6985) but the response of the M. bovis-infected ones to MPTB-Apa was differential, being significantly diminished (P<0.0001). Although 6 out 45 animals from MPTB-infected herds responded to MPTB-Apa stimulation in the IFNgamma release assay, we found no significant differences when compared infected herds with non-infected ones (P=0.34). This antigen, in contrast to bovine Purified Protein Derivative (PPDb), was strongly represented in avian PPD (PPDa), as shown by the recognition of BALB/c mice hyperimmune sera against MPTB-Apa by Dot-blot immunoassay. We therefore demonstrated the antigenicity of Apa in MPTB-infected animals and a differential response to the recombinant antigen when compared to M. bovis-infected animals. These traits herein described, added to the usefulness of milk samples to detect IgG anti-Apa, could be important for routine screening in dairy cattle, considering a multiantigenic approach to overcome the lack of immunodominance.
Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias , Bovinos/imunologia , Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Reações Cruzadas , Feminino , Genes Bacterianos , Glicosilação , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Leite/imunologia , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Paratuberculose/imunologia , Paratuberculose/microbiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Especificidade da Espécie , Tuberculina/imunologiaRESUMO
This study determines whether a genetically engineered mutant of Brucella abortus, strain M-1, possesses differences in protective properties compared to the parental strain, vaccine S19. M-1 is a mutant unable to express BP26, a periplasmic protein with potential use in diagnosis. Mice vaccinated with S19 developed antibodies against BP26, while those vaccinated with M-1 did not. However, mice vaccinated with S19 or M-1 were similarly protected against challenge with pathogenic strain 2308, suggesting that the lack of BP26 does not affect the induction of the protective immune response exerted by S19. These and previous results showing that bacterial invasion and growth or replication in mouse spleens were indistinguishable between strains M-1 and S19 could indicate that the mutant is an attenuated strain which maintains the same protective properties as S19.
Assuntos
Vacina contra Brucelose/genética , Vacina contra Brucelose/imunologia , Brucella abortus/genética , Brucelose/prevenção & controle , Vacinas Sintéticas/imunologia , Animais , Brucella abortus/imunologia , Brucelose/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese InsercionalRESUMO
Brucella spp. are the causative agents of brucellosis in many different hosts, including humans. Most of the serological methods of diagnosis are based on the detection of antilipopolysaccharide antibodies, which makes the differentiation of vaccinated animals from infected animals difficult. By using molecular biology techniques, a gene that encodes a 26-kDa protein (BP26) was isolated from a Brucella abortus S19 genome lambda gt11 library. This protein is in the periplasm of B. abortus and in transformed Escherichia coli. It is exported to the periplasm via a preprotein of 29 kDa with a signal sequence of 28 amino acids. The nucleotide and amino acid sequences of this gene and protein did not show any similarity with those of previously sequenced genes. The use of this protein in Western blotting allowed the differentiation between vaccinated bovines from infected bovines and the detection of infected rams: on the other hand, sera from human patients with active brucellosis were positive, while sera from human patients with chronic brucellosis or without clinical signs were nonreactive. BP26 might be of value as an antigen for serological diagnosis of brucellosis in different mammals.
Assuntos
Proteínas de Bactérias/genética , Brucella abortus/genética , Brucelose Bovina/diagnóstico , Brucelose/diagnóstico , Genes Bacterianos , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Sequência de Bases , Brucelose/imunologia , Brucelose/veterinária , Brucelose Bovina/imunologia , Bovinos , Clonagem Molecular , DNA Bacteriano/genética , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Peso Molecular , Gravidez , Testes Sorológicos , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/imunologiaRESUMO
Se estudió un lote de 28 sueros de llama (Lama gama) de la provincia de Jujuy, Argentina, a fin de identificar antígenos inmunorreactivos contra Leptospira interrogans. Se utilizaron distintas preparaciones antigénicas de la bacteria para estudiar la inmunorreactividad mediante microaglutinación (MAT), ELISA y Western inmunoblot. Un pool de sueros bovinos positivos a la MAT fue empleado como control. Todos los sueros de llama fueron negativos mediante MAT e igual resultado se observó mediante ELISA. Dos de los 28 sueros de llama y el pool de sueros bovinos positivos, al ser evaluados por Western inmunoblot, arrojaron resultados positivos y permitieron identificar proteínas inmunorreactivas. Por MALDI-TOF se logró establecer que la proteína asociada a los dos sueros de llama inmunorreactivos era una flagelina periplásmica de Leptospira interrogans serovar Lai STR, mientras que la asociada al pool de sueros bovinos positivos a Leptospira sp. se trataba de una lipoproteína de la membrana externa de Leptospira interrogans serovar Ballum, LipL21. Estas proteínas podrían ser utilizadas en el diseño de un nuevo ELISA aplicado al diagnóstico temprano de leptospirosis, ya sea en distintos tipos de ganado como así también en reservorios silvestres.
A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutinattion (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.
Assuntos
Animais , Bovinos , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Camelídeos Americanos/imunologia , Epitopos/imunologia , Flagelina/imunologia , Leptospira interrogans/imunologia , Leptospirose/veterinária , Lipoproteínas/imunologia , Antígenos de Bactérias/isolamento & purificação , Argentina/epidemiologia , Western Blotting , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Camelídeos Americanos/sangue , Ensaio de Imunoadsorção Enzimática , Epitopos/isolamento & purificação , Flagelina/isolamento & purificação , Leptospirose/epidemiologia , Leptospirose/imunologia , Lipoproteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Testes Sorológicos/veterináriaRESUMO
cDNA libraries, representative of potato viruses X (PVXc strain) and Y (PVY degrees strain) genomes were obtained. A PVX cDNA cloned fragment was sequenced and biotinylated to be used as hybridization probe for the detection of purified virus or nucleic acid extracts of infected plants. Dot hybridization assay was sensitive to detect 4 ng of viral particles, corresponding to about 200 pg of viral RNA. The level of detection in infected plant extracts was as effective as that obtained with the ELISA. The presence of biotinylated PVY cDNA in the hybridization mixture did not affect sensitivity of the PVX detection assay, suggesting that a single diagnostic assay for several potato viruses and virus-related pathogens could be developed.
Assuntos
Sondas de DNA , Hibridização de Ácido Nucleico , Vírus de Plantas/isolamento & purificação , RNA Viral , Sequência de Aminoácidos , Sequência de Bases , Biotina , DNA , Dados de Sequência Molecular , Vírus de Plantas/genética , RNA Viral/genéticaAssuntos
Derivados de Benzeno/metabolismo , Monitoramento Ambiental/métodos , Animais , Derivados de Benzeno/química , Cromatografia Líquida de Alta Pressão , Daphnia/metabolismo , Eucariotos/metabolismo , Peixes/metabolismo , Modelos Teóricos , Valor Preditivo dos Testes , Análise de Regressão , Solubilidade , Espanha , Especificidade da EspécieRESUMO
Un hexadecapéptido correspondiente a la secuencia 144-159 de la proteína estructural VP1 del virus de la fiebre aftosa (CFA), cepa O1K, fue sintetizado por métodos químicos. El mismo, acoplado covalentemente a distintas proteínas portadoras, demostró su capacidad de inducir anticuerpos neutralizantes del VFA cepa O1 Campos (O1Ca) en ratones adultos. La protección fue del 100% cuando se lo acopló a la hemocianina de un molusco (KLH) y fue inoculado en cantidad de 60 microng, dividida en dos dosis y adyuvada con mezcla oleosa. Las proteínas utilizadas como portadoras: albúmina bovina, purificado proteico de Mycobacterium (PPD), gliadina y células de Brocelas produjeron una respuesta inmune menor que la KLH
Assuntos
Camundongos , Animais , Aphthovirus/imunologia , Peptídeos/biossíntese , Ensaio de Imunoadsorção EnzimáticaRESUMO
Kinetics of the humoral immune primary response and seven-day secondary response of adult CF1 mice to FMDV O1 Campos adjuvanted in aluminium hydroxide-saponin (AHS) or in oil emulsion (OE) were evaluated by means of ELISA and passive hemagglutination (PH). Analysis of the response to AHS vaccine showed that ELISA measured maximal titres of primary response at 23 days post-vaccination (dpv), and at day 17 of secondary response, while PH detected maximal titres for primary as well as secondary response around day 60 pv. Mice immunized with OE vaccine studied by ELISA presented a plateau of primary response around 40 dpv, while secondary response was maximal around 80 dpv. The same sera tested by PH showed the highest titres for primary response on day 50 pv and secondary response was maximal on day 40 pv. A criterion for the evaluation of vaccination efficiency in the murine model is proposed based on the methods employed in the determination of antibody level.
RESUMO
Kinetics of the humoral immune primary response and seven-day secondary response of adult CF1 mice to FMDV O1 Campos adjuvanted in aluminium hydroxide-saponin (AHS) or in oil emulsion (OE) were evaluated by means of ELISA and passive hemagglutination (PH). Analysis of the response to AHS vaccine showed that ELISA measured maximal titres of primary response at 23 days post-vaccination (dpv), and at day 17 of secondary response, while PH detected maximal titres for primary as well as secondary response around day 60 pv. Mice immunized with OE vaccine studied by ELISA presented a plateau of primary response around 40 dpv, while secondary response was maximal around 80 dpv. The same sera tested by PH showed the highest titres for primary response on day 50 pv and secondary response was maximal on day 40 pv. A criterion for the evaluation of vaccination efficiency in the murine model is proposed based on the methods employed in the determination of antibody level.