Proposed method for agglutinating antibody titer analysis and its use as indicator of acquired immunity in pacu, Piaractus mesopotamicus.

Antibody can be assessed by agglutinating antibody titer which is a quantitative measure of circulating antibodies in serum from fish previously immunized. The antibody evaluation has been performed with different fish species, and is considered a reliable method that can be applied to confirm several hypothesis regarding acquired immunity, even in conjunction with precise methods to describe immune mechanisms. In order to provide appropriate analytical methods for future studies on the specific immune system of native fish, the present study standardized on assay to measure the serum agglutinating antibody titer produced after immunization with inactivated A. hydrophila and levamisole administration in pacu. It was possible to determine the agglutinating antibodies titer in a satisfactorily way in pacu immunized with inactive A. hydrophila, and the highest titers were observed on fish fed with levamisole.

Clonagem e expressão do gene da glicoproteína S1 do vírus da bronquite infecciosa das galinhas em Pichia pastoris / Cloning and expression in Pichia pastoris of the S1 glycoprotein gene of the chicken infectious bronchitis virus

Variações genética e antigênica são observadas com frequência elevada entre estirpes do VBIG e envolvem principalmente a glicoproteína S1. Com o objetivo de contribuir com a disponibilidade de ferramentas para o imunodiagnóstico e a imunoprofilaxia da bronquite infecciosa das galinhas foi desenvolvida uma metodologia para expressão recombinante da glicoproteína S1 na levedura Picchia pastoris. O cDNA do gene codificador dessa proteína foi obtido a partir de RNA viral de ovos embrionados infectados com a estirpe M41 do VBIG submetido à transcrição reversa (RT) e reação em cadeia da polimerase (PCR), amplificando-se a sequência codificadora de S1 acrescida de extremidades compatíveis com a clonagem no vetor usado na transformação de leveduras. A indução com metanol resultou na produção de uma proteína detectada como banda única do tamanho previsto, em western-blot, no lisado celular das leveduras transformadas. A expressão em P. pastoris mostrou ser um método eficaz para a produção recombinante da proteína S1 do VBIG, com potencial para utilização em técnicas de imunodiagnóstico da bronquite infecciosa das galinhas.

Genetic and antigenic variation are very frequently observed among IBV strains and affect mainly the S1 glycoprotein. In order to contribute to the availability of tools for immunodiagnosis and immunoprophylaxis of chicken infectious bronchitis we developed an expression system for production of recombinant S1 glycoprotein in Pichia pastoris. We obtained the cDNA from viral RNA on embryonated eggs infected with the M41 strain of IBV, by reverse transcription (RT) and polymerase chain reaction (PCR), amplifying the S1 coding sequence with extremities compatible with the vector used to transform yeast. Induction with methanol led to the production of a protein with the predicted molecular weight that was detected by Western blot in the cell lysate of transformed yeast. Expression in P. pastoris proved to be an effective method for recombinant production of S1 protein from IBV, with potential for use in immuno-diagnosis of chicken infectious bronchitis virus.

Clonagem, expressão e caracterização da nucleoproteína recombinante do vírus da bronquite infecciosa em Escherichia coli e em Pichia pastoris / Cloning, expression, and characterization of the nucleocapsid protein of infectious bronchitis virus in Escherichia coli and Pichia pastoris

O gene da proteína de nucleocapsídeo (1.230 pb) da estirpe M41 do vírus da bronquite infecciosa (VBI) foi amplificado pelas reações de transcrição reversa e em cadeia da polimerase (RT-PCR) e clonado, em seguida, em dois sistemas; pET28a - Escherichia coli e pFLD -Pichia pastoris. Os produtos recombinantes construídos para expressão (pET28a-N ou pFLD-N) foram identificados por análises de PCR e de sequenciamento de nucleotídeos. Os clones transformantes da linhagem BL21 de E. coli e da linhagem GS115 de P. pastoris foram submetidos aos protocolos apropriados de indução. A expressão da proteína N de fusão com etiqueta de poli-histidina e com massa molecular de 54 kDa foi determinada pelas técnicas de SDS-PAGE e de Western blotting, confirmando-se que ambas proteínas N recombinantes apresentaram tamanhos e antigenicidade compatíveis com a proteína N nativa do próprio VBI. O sistema E. coli expressou uma quantidade relevante da proteína N recombinante, enquanto que o sistema P. pastoris produziu uma baixa recuperação dessa proteína recombinante. A proteína N recombinante gerada pelo sistema bacteriano foi purificada em resina de níquel-sepharose. O conjunto de resultados indica que o sistema de expressão constituído por pET28a ­ E. coli é mais efetivo para produzir a proteína N recombinante do VBI destinada ao uso como antígeno para detectar anticorpos anti-virais específicos em ensaios de imunodiagnóstico para essa infecção viral.

The nucleocapsid protein (N) gene (1,230 bp) of the M41 strain of infectious bronchitis virus (IBV) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and cloned in two systems; pET28a Escherichia coli and pFLD Pichia pastoris. The recombinant expression constructs (pET28a-N or pFLD-N) were identified by PCR and sequencing analysis. The transformant clones of BL21 strain of E. coli or GS115 of P. pastoris were submitted to appropriate inducting protocols. Expression of histidine-tagged fusion N proteins with a molecular mass of 54 kDa was determined by SDS-PAGE and Western blotting analysis, confirming that both recombinant N proteins were comparable in size and antigenicity to native IBV N protein. The E. coli system overexpressed the recombinant N protein, while the P. pastoris system produced a low yield of this recombinant protein. The bacteria expressed N protein was purified by chromatography on nickel-sepharose resin. These results indicated that the pET28a E. coli expression system is more effective to generate N recombinant protein for using as an antigen to detect anti-IBV antibodies in immuno-assays for this viral infection.

Avaliação da imunidade passiva em bezerros nascidos de vacas imunizadas com vacina contra rotavírus / Evaluation of passive immunity in calves born from cows immunized with anti-rotavirus vaccine

Com o objetivo de monitorar a imunidade passiva em bezerros alimentados com colostro de vacas imunizadas e não imunizadas com vacina contra rotavírus, foram determinados títulos de anticorpos em amostras de sangue e colostro de 26 vacas da raça Holandesa no dia do parto e de seus bezerros, à zero, às 24, 48 horas e aos sete, 14, 21, 28 dias de idade, pelo ensaio imunoenzimático. Tanto no soro sangüíneo como no colostro, os títulos dos isótipos IgG, IgG1 e IgG2 foram mais elevados no grupo dos animais vacinados, porém somente no colostro o aumento foi significativo. Os bezerros alimentados com o colostro das vacas vacinadas apresentaram títulos mais altos dos isótipos IgG, IgG1, IgG2, IgA e IgM, após a ingestão do colostro, sendo constatado aumento significativo apenas para os títulos do isótipo IgG2. Amostras positivas para rotavírus foram detectadas nos dois grupos experimentais a partir dos sete dias de idade. A vacinação materna não protegeu efetivamente os bezerros das infecções naturais por rotavírus, pois, apesar de aumentar os títulos séricos de anticorpos anti-rotavírus nos animais vacinados, não foi capaz de impedir a ocorrência da rotavirose nos bezerros alimentados com o colostro das vacas imunizadas.

Passive immune response in calves fed colostrum from immunized and nonimmunized cows by anti-rotavirus vaccine was monitored. Titers of antibodies were determined by immunoenzymatic assay in blood and colostrum sampled at parturition day from 26 Holstein cows as well as in blood from their calves collected at 0, 24, and 48 hours and seven, 14, 21, and 28 days after birth. In serum and colostrum, IgG, IgG1, and IgG2 antibody titers were higher in vaccinated animals; however, this increase was only significant in colostrum. The calves fed colostrum from vaccinated cows showed higher IgG, IgG1, IgG2, IgA, and IgM isotypes titers after the ingestion of colostrum, being evidenced significant increase only for IgG2 titers. Positive samples for rotavirus were detected in both experimental groups since seven days after birth. Results showed that maternal vaccine failed to protect effectively the calves from natural infections by rotavirus, though it increased the anti-rotavirus antibody titers in vaccinated animals, but was not capable to impair the occurrence of rotaviruses in the calves fed colostrum from immunized cows.

Extensive antigenic and genetic variation among foot-and-mouth disease type A viruses isolated from the 1994 and 1995 foci in São Paulo, Brazil.

Nine foot-and-mouth disease virus (FMDV) type A isolates recovered from the field FMD foci in São Paulo State, Brazil, during 1994 and 1995 (a period preceding the last reported focus of FMD in 1996 in this state) were compared among themselves and with the reference vaccine strain A(24)Cruzeiro. The techniques used were sandwich ELISA, virus neutralization (VN), polyacrylamide gel electrophoresis (PAGE) of the structural polypeptides and direct sequencing of the VP1-coding region (1D gene). Results of VN were recorded as serological relationships "R" and those from ELISA were expressed as percentage of the homologous reaction "r". ELISA and VN gave comparable results (correlation coefficient, 0.936) allowing assignment of these field viruses to four groups which were distinct from the A(24)Cruzeiro strain. PAGE and 1D nucleotide sequencing were also able to distinguish between these viruses. The high level of genetic and antigenic variation found when comparing the A(24)Cruzeiro vaccine strain and type A strains recovered from the last identified foci of FMD came from a formerly endemic area where vaccination with polyvalent vaccines (O(1)Campos, A(24)Cruzeiro and C(3)Indaial) had been extensively applied. The similarity between the results of the serological and genetic analyses suggest that the antigenic differences found are mainly located in the 1D protein.

Detection of infectious bronchitis virus in experimentally infected chickens by an antigen-competitive ELISA.

An antigen-competitive enzyme-linked immunosorbent assay (Ag-C-ELISA) was developed for the detection of infectious bronchitis virus (IBV) antigens, M41 strain, in tissues from experimentally infected chickens, or in allantoic fluid harvested from inoculated embryonated eggs. The detection limit of IBV in the Ag-C-ELISA was 10(4.1) median embryo infective doses (EID(50))/well. Tracheal and lung samples from chickens vaccinated with 10(2.5) EID(50) of live attenuated infectious bronchitis (H120) vaccine were negative in the direct detection Ag-C-ELISA. The results indicate that the Ag-C-ELISA has the potential to detect IBV, either directly in tissue samples or when combined with the passage of material in embryonated eggs, thereby constituting an alternative method for the diagnosis of IBV.

Detection and quantification of antibodies to Newcastle disease virus in ostrich and rhea sera using a liquid phase blocking enzyme-linked immunosorbent assay.

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method. The cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement (kappa = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Antibody response to Newcastle disease vaccination in a flock of young partridges (Rhynchotus rufescens).

Ten young partridges (Rhynchotus rufescens) were vaccinated with the lentogenic strain of Newcastle disease virus. Another eight unvaccinated birds were kept in close contact with the treated flock. Antibodies levels were measured over the course of 3 mo in all birds using the hemagglutination inhibition (HI) test and the liquid-phase blocking enzyme-linked immunosorbent assay (LPB-ELISA). The LPB-ELISA was standardized, and the results were compared with those obtained with the HI test. Antibodies increased after 23 days postvaccination in 16 birds with no side effects as determined by both the HI test and the LPB-ELISA.

A double antibody sandwich ELISA for rapid diagnosis of virus infection and to measure the humoral response against infectious bursal disease on clinical material.

A double antibody sandwich ELISA (DAS-ELISA) was developed and employed for simultaneous direct detection of infectious bursal disease virus (IBDV) from bursal samples and to measure the humoral response, using the same basic immunoreagents. The purified and non-purified antigen, capture antibody and chicken hyperimmune sera were prepared, and standardized for this purpose. The DAS-ELISA was applied to both 80 bursal suspensions and 224 corresponding serum samples from vaccinated and non-vaccinated commercial flocks. Bursae samples were collected at 2 weeks of age, and submitted to histological examination, virus isolation in specific pathogen-free chickens embryos, and the DAS-ELISA technique. Serum titres obtained in indirect ELISA and serum neutralization test were compared with those in DAS-ELISA. The agreement was 80% between DAS-ELISA, and the conventional techniques, with high sensitivity (87%) and specificity (90%).

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Babesia bovis in cattle.

A method for the isolation of Babesia bovis merozoites from infected erythrocytes (Machado et al., 1994) and an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-B. bovis antibodies were developed. This ELISA utilizes a soluble, alkali-digested B. bovis antigen. Sera from calves experimentally infected with B. bovis were screened by this technique from day 9 to day 233 postinfection (PI). Maximum titers were reached between days 29 and 149 PI. Sera from calves (n = 62), heifers (n = 38) and cows (n = 49), raised in tick-infested areas of São Paulo State, showed higher antibody levels in heifers and cows. A higher percentage of negative sera (19.4%) was found among calves. Sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and immunoblotting have identified proteins of similar molecular mass in the two species. Sera from calves experimentally infected with B. bovis reacted with homologous antigens at the level of 95, 66 and 23 kDa. The same serum reacted with the 23 kDa band of heterologous antigen. Sera from calves experimentally infected with B. bigemina recognized 82, 66, 58, 36 and the 23 kDa polypeptides of homologous and heterologous antigens. The experimental ELISA described may prove to be a practical serological test for bovine Babesia infection with the choice of specific test antigen for B. bovis and B. bigemina.

Liquid-phase blocking sandwich enzyme-linked immunosorbent assay for detection of antibodies against foot-and-mouth disease virus in water buffalo sera.

OBJECTIVE: To develop and apply the liquid-phase blocking sandwich ELISA (BLOCKING-ELISA) for the quantification of antibodies against foot-and-mouth disease virus (FMDV) strains O1 Campos, A24 Cruzeiro, and C3 Indaial. DESIGN--Antibody quantification. SAMPLE POPULATION: 158 water buffalo from various premises of Sao Paulo State-Brazil. The sera were collected either from systemically vaccinated or nonvaccinated animals. PROCEDURE: The basic reagents of BLOCKING-ELISA (capture and detector antibodies, virus antigens, and conjugate) were prepared and the reaction was optimized and standardized to quantify water buffalo antibodies against FMDV. An alternative procedure based on mathematical interpolation was adopted to estimate more precisely the antibody 50% competition titers in the BLOCKING-ELISA. These titers were compared with the virus-neutralization test (VNT) titers to determine the correlation between these techniques. The percentages of agreement, cutoff points, and reproducibility also were determined. RESULTS: The antibody titers obtained in the BLOCKING-ELISA had high positive correlation coefficients with VNT, reaching values of 0.90 for O1 Campos and C3 Indaial, and 0.82 for the A24 Cruzeiro (P < 0.0005). The cutoff points obtained by use of the copositivity and conegativity curves allowed determination of high levels of agreement between BLOCKING-ELISA and VNT antibody titers against the 3 FMDV strains analyzed. CONCLUSIONS: The results characterized by high correlation coefficients, levels of agreement, and reproducibility indicate that the BLOCKING-ELISA may replace the conventional VNT for detection and quantification of antibodies from water buffalo sera to FMDV.

Rapid coagglutination test for the detection and typing of foot and mouth disease virus.

Protein A containing Staphylococcus aureus was used to develop a coagglutination (COA) test for the detection and typing of foot and mouth disease virus (FMDV) O, A and C serotypes in infected cells and tissues. Different batches and amounts of guinea pig anti-FMDV sera were assessed to optimize the preparation of COA conjugates. The sensitivity and specificity of the COA Test for the detection of FMDV O, A and C serotypes and heterologous viruses was also characterized. Comparison between the COA Test and complement fixation test for the detection and typing of FMDV obtained from extracts of tongue epithelial tissues from infected cattle revealed high agreement in the results and indicated a potential application of the COA Test for the direct diagnosis of viruses.

A pro-inflammatory effect of foot and mouth disease virus on immune and non immune guinea pigs

A estirpe O 1campos do vírus da febre aftosa (VFA) usada como agente indutor do teste de pleurisia foi capaz de desencadear um efeito pró-inflamatório em cobaias normais e imunes. A atividade pró-inflamatória do VFA, detectada em dois intervalos de pleurisia (24 e 48 horas) foi demonstrada, somente por quimiotaxia de leucócitos mononucleares (MN), no primeiro intervalo e por efeito edematogênico, migraçäo de MN e polimorfonucleares (PMN), no último intervalo de reaçäo. Os perfis de reaçäo inflamatória induzida pelo VFA em cobaias imunes (imunizadas com vacinas oleosas anti-VFAO 1 campos), aos 7 e aos 30 dias pós-vacinaçäo (PV) apresentaram intensidades mais baixas do que as observadas em cobaias normais, embora nas cobaias com 7 dias de vacinaçäo a quimiotaxia de leucócitos totais e de PMN tenha sido similar àquela encontrada nos animais normais, no intervalo de 48 horas de reaçäo. Ademais, nas cobaias com 30 dias PV, o VFA induziu um aumento significante no volume de exsudato e na infiltraçäo de MN, no intervalo de 24 horas, sendo que os valores de todos os parâmetros do exsudato inflamatório caíram a níveis normais, no segundo intervalo de reaçäo. Nas cobaias imunes foi observada uma associaçäo negativa entre o aumento no título de anticorpos soro-neutralizantes, de 7 para 30 dias PV e as intensidades dos parâmetros inflamatórios pleurais. O teste de pleurisia revelou-se um procedimento adequado para avaliar a atividade pró-inflamatória do VFA