Comparative Study of Three Different ELISA to Measure the Antibodies Against Infectious Bronchitis Virus in Vaccinated and Unvaccinated Broilers

Estudo Comparativo de Três Diferentes Modalidades de Elisa para Medir os Anticorpos Contra o Vírus da Bronquite Infecciosa em Frangos Vacinados e Não Vacinados

Estudo Comparativo de Três Diferentes Modalidades de Elisa para Medir os Anticorpos Contra o Vírus da Bronquite Infecciosa em Frangos Vacinados e Não Vacinados

Comparative Study of Three Different ELISA to Measure the Antibodies Against Infectious Bronchitis Virus in Vaccinated and Unvaccinated Broilers.

Autor(es) / Author(s)

Cardoso TC1

Oliveira C3

Da-Silva SEL1

Ferreira HL1

Pinto AA²

1-Depto. de Apoio, Produção e Saude Animal, Faculdade de Odontologia/UNESP, Laboratório Multiusuário de Imunodiagnóstico - Araçatuba

2-Depto. de Microbiologia, FCAV/ UNESP, Laboratório de Imunologia e Virologia - Jaboticabal

3-Merial Laboratório - Campinas

Correspondência / Mail Address

T. C. Cardoso

Depto. de Apoio, Produção e Saude Animal

Curso de Medicina Veterinária

Rua Clóvis Pestana, 793.

16015-050 Araçatuba - SP - Brasil.

E-mail: [e-mail]

Unitermos / Keywords

ELISA, resposta pós vacinal, vírus da bronquite infecciosa, frangos

ELISA, post vaccinal response, infectious bronchitis virus, broilers

Obsevações / Notes

The authors wish to acknowledge the excellent technical assistance of Merial Laboratories and Fundação de Amparo à Pesquisa do Estado de São Paulo for financial support.

RESUMO

No presente estudo, foram vacinados 150 frangos de corte de um dia de idade com sorotipo H120, e, após 28 dias desafiados à vacinação com o sorotipo M 41 do vírus da bronquite infecciosa das aves. Da mesma forma, foram obtidos 150 soros de aves não vacinadas para a análise. Os respectivos soros foram analisados 28, 34 e 46 dias após o desafio, examinados através das técnicas de ELISA indireto (ELISA-I), Sandwich ELISA (S-ELISA) e ELISA com bloqueio de fase líquida (ELISA-BFL) e comparados com a técnica padrão de soroneutralização (SN) para efeito de cálculo da especificidade e sensibilidade relativas, bem como os valores predictivos positivos e negativos. O cálculo do coeficiente de correlação também foi empregado para a análise de concordância. Assim, os valores da correlação encontrados foram r = 0.98 entre ELISA-BFL x SNT, r = 0.79 entre S-ELISA x SNT e r = 0.74 ELISA-I x SNT. No entanto, quando comparamos as técnicas de ELISA entre si, ELISA-BFL x S-ELISA, ELISA-BFL x ELISA-I e S-ELISA x ELISA-I os valores encontrados foram r> = 0.75, r = 0.69 e r = 0.79.

A técnica de ELISA-BFL demonstrou melhor sensibilidade relativa que as técnicas de S-ELISA e ELISA-I, mesmo 46 dias após o desafio com a estirpe heteróloga. Entretanto, apesar das técnicas de S-ELISA e ELISA-I apresentarem especificidade realtiva superiores, a melhor correlação observada foi entre as técnicas de ELISA-BFL e a SN.

ABSTRACT

Broilers were spray-vaccinated (n=150) with H₁₂₀ serotype at one-day-old, challenged after 28 days with M₄₁ IBV serotype and after bled at day 28, 34 and 46 after challenged. The respective sera were tested by the indirect ELISA (I-ELISA), sandwich ELISA (S-ELISA), liquid phase blocking ELISA (LPB-ELISA) and the standard serum neutralization test (SNT). For this purpose, a total of 300 sera samples, 150 from non vaccinated and 150 from vaccinated broilers were titrated by all the serological methods and the correlation coefficients were determined. The correlation values (r) between LPB-ELISA x SNT, S-ELISA x SNT, I-ELISA x SNT found werer = 0.98, r = 0.79, and r = 0.74, respectively. Nevertheless, the r between LPB-ELISA x S-ELISA, LPB-ELISA x I-ELISA and S-ELISA x I-ELISA were r = 0.75, r = 0.69 and r = 0.79. In fact , the I-ELISA and the S-ELISA had almost the same correlation with S-ELISA and LPB-ELISA, in contrast with the I-ELISA and LPB-ELISA. It was concluded that LPB-ELISA showed better sensitivity than I-ELISA and S-ELISA, also after 46 days challenge with the heterologous serotype. However, these two last techniques demonstrated similar specificity when the titers were compared with those obtained in SNT, even though after the heterologous serotype challenge the SNT produced better results. This study demonstrated the close relationship between LPB-ELISA and SNT assays.

INTRODUCTION

Infectious bronchitis virus contain four structural proteins: S1, S2, with the Mr of 92K and 84K, respectively, as well as the heterogeneously glycosylated membrane polypeptide p23 with an Mr of 34K, and the nucleocapsid protein with an Mr of 52K associated with RNA ( Cavanagh, 1983).

Infectious bronchitis virus (IBV) infections of chickens are diagnosed most accurately by isolating and serotyping the causal strain. However, identifying a field isolate may require a month or more of laboratory time, because the virus often needs to be adapted or undergo several blind passages to be identified as IBV before being serotyped (Darbyshire & Peters, 1984; Gelb & Killian, 1987; Brown & Bracewell, 1988).

Serological diagnosis of IBV infections has also been used as a more rapid alternative to virus isolation and identification. The antibody response of chickens after vaccination against IBV has been done by the use of the serum neutralization test (SNT), the indirect ELISA (I-ELISA) and the hemagglutination inhibition test (HIT) (Gough & Alexander, 1978; Box et al., 1988; Cardoso et al., 1996a; Cardoso et al., 1996b). Although the SNT is the only technique that can measure the neutralizing antibodies against IBV, it is rarely used because of its high cost and difficult execution. Thus, the aim of this study was to compare, for the first time for IBV serology, three different ELISAs methodologies: I-ELISA, sandwich ELISA (S-ELISA) and the liquid phase blocking ELISA (LPB-ELISA) with the standard SNT to detect antibodies against IBV from vaccinated and non vaccinated broilers serum samples.

MATERIAL AND METHODS

Virus antigen

The Mass₄₁ and H₁₂₀ of IBV were propagated by infecting 9-11-days- old embryonated specific pathogen-free (SPF) eggs, and the allantoic fluid was collected as recommended before (Cavanagh et al., 1983). The virus was further purified by previously reported methods with some modifications (Cardoso et al., 1996a). Briefly, the allantoic fluid collected and pooled from IBV-infected SPF embryonated eggs was clarified by centrifugation at 2,000g for 20min at 4º C and then submitted to 59,000g for ultracentrifugation. The viral pellet was diluted in 3mL of TNE buffer (1 mM TRIS, 0.15 M NaCl, 1mM EDTA, pH 7.0) and layered on a continuous 20-55% sucrose gradient (w/v) and centrifuged at 90,000 g for 10h at 4º C. The fractions collected from the gradient which absorbed at 254nm (viral RNA) and 280nm (total protein) were pooled and diluted in TNE buffer and the protein concentration was determined by the method of Hartrée. The virus infectivity was titrated in SPF embryonated eggs, as recommended by Gelb & Killian (1987). The same IBV strains, replicated in embryonated SPF eggs and clarified at low-speed centrifugation, was used in the serum neutralization test as homologous and heterologous serotypes.

Broilers

For this test, 300 18-day-old embrionated pathogen-free eggs were purchased from a commercial hatchery (Granja Resende, Uberlândia, MG, Brasil). They were originated from Mycoplasma gallisepticum free Arbor Acres parent flock which was vaccinated two times in rear against IBV using H₁₂₀ and killed oil vaccine (Intervet). The eggs were disinfected with formalin after arrival at Merial laboratories, (Campinas, State of São Paulo, Brasil) and hatched in a disinfected hatchery. The air of the hatchery was continuously disinfected by ultra violet light, in order to minimize the risk of airborne IBV infection.

Experimental design

Two hatches of broilers were used in two experiments. Each hatch was divided randomly into two groups of 150 birds (A and B). The group A was vaccinated with H₁₂₀ strain on day zero; Group B was the non vaccinated broilers control. Non vaccinated broilers were placed in isolators before the other group was vaccinated and placed in their isolator.

Challenge

The IBV challenge strain M41 (Witt et al., 1997) was obtained from Laboratório de Referência Animal- LARA Campinas, São Paulo, Brasil. Group A was challenged at day 28, and the respective blood samples were taken from all birds at days 28, 34 and 46. Sera were marked individually and stored at -20º C . Challenge was performed by conjuctival and intracheal routes. Each bird received one drop (0.05mL) on the conjuctiva of the right eye and 0.2mL in the trachea using a syringe and a soft rubber tube. Challenge doses were estimated at 10⁴ EID per bird.

Indirect ELISA (I-ELISA)

The procedure described before with some modifications introduced by Cardoso et al., (1996a) was followed. A total of 100mL/well of the different reagents used and the plates were washed five times between each step with phosphate buffered saline + 0.1% of Tween 20 pH 7.4 (PBST). The optimal concentration of each reagent (purified M₄₁ virus antigen and chicken anti- IBV serotypes reference sera) as recommended by Cardoso et al., (1996a). The anti-chicken IgG peroxidase conjugated (1:4000) was added and incubated for 1h at 37º C. Then, the plates were washed and the substrate chromogen mixture was added and the plates were read at 492nm, after blocked the reaction with HCl 2M, in a Multiskan ELISA reader. Each test serum titer was determined based on the cut off point ³ 0.2 as previously standardized (Cardoso et al., 1996a). The calculation was the average optical density (OD) expressed in relation to the positive and the negative reference serum.

Sandwich ELISA (S-ELISA)

The technique reported by Cardoso et al., (1998) was adopted, using 100mL/well of all different reagents and washings as described above. The plates were sensitized using the optimal dilution of the capture antibody (1:2000 of rabbit anti-IBV M₄₁ serotype antibody) prepared in carbonate-bicarbonate buffer 0.1M pH 9.6, for 18h at 4º C. The wells were blocked as described previously using 5% of normal rabbit serum, then an optimum dilution of the crude IBV suspension from infected allantoic fluid (M41) in PBST + 10% of skimmed milk was added to each well. The plates were incubated for 1 h at 37º C. After that, the serum samples, in a single dilution of 1:400, the anti-chicken IgG peroxidase conjugated (1:4000) and, the substrate chromogen mixture were added and incubated as described previously. The plates were read at 492nm in a Multiskan ELISA reader and, for each test serum sample, the median optical density was expressed in relation to the positive and negative reference serum as Sample/Positive values according to Cardoso et al., (1998).

Liquid phase blocking ELISA (LPB-ELISA)

The test was performed as described by McCullough et al., (1992) and Cardoso et al., (1999) with some modifications. The microplate (Nunc) wells were coated with the capture antibody diluted 1:250 or 12.5 mg/well, overnight, in 0.05 M carbonate bicarbonate buffer, pH 9.6 at 4^ºC. After five washings with phosphate-buffered saline solution containing 0.05% tween 20 (PBST), the plates were blocked with PBS containing 15% skim milk (PBSM). After incubation for 45 min at 37^ºC, the plates were ready for use. The sera to be tested, always run in duplicate single dilution of 1:8, were treated with 1% trichloroacetic acid for protein precipitation (Cardoso et al., 1999) before mixing with a fixed concentration of unpurified virus dilution (1:5), also diluted in PBS with 0.5 M NaCl. This liquid phase was executed in a separated heamagglutination plates (Nunc) and after the incubation at 37^ºC for 90 min the mixture virus-sera was transferred to the ELISA’s plates. The plates were incubated at 37^ºC for 60 min. The IBV antigen (M₄₁ strain) was stored at -70^ºC and used at a concentration of 1:5 which gave an absorbance of 1.5 at 492 nm, and the plates were washed as before. An optimal 1:4000 dilution of guinea pig detector anti-IBV serum in phosphate buffered solution containing tween and skim milk (PBSTM) was added and the plates were incubated for 60 min at 37^ºC. An optimal 1:16.000 dilution of commercial rabbit anti-guinea pig IgG conjugated to horse-radish peroxidase (Sigma) in PBSTM was added. After incubation for 60 min at 37^ºC, the plates were again washed. A mixture of 0.006% H₂O₂ and 0.4 mg o-phenylenediamine/mL of 0.1 M Na₂ HP0₄ and 0.1 M citric acid buffer, pH 5.0, was used as substrate and chromogen. After 15 min of incubation at room temperature, 2M HCl was added in order to block the enzymatic reaction, and the OD of the plate was read at 492 nm in a Titerteck multiscan reader. The respective titers were determined as recommended by Araujo et al., (1996)

Serum neutralization test

The reagents and the basic procedures used were described before (Gelb & Killian, 1987). Briefly, dilutions of the chicken sera were mixed with 100 TCID₅₀ of the Mass₄₁ and H₁₂₀ IBV serotypes in microtiter plates (Nunc, Copenhagen, Denmark) and incubated for 1h at 37^ºC. Thereafter, an equal volume of chicken embryonated kidney cells at 10⁶ cells/mL was added to each well and the plates were further incubated at 37^ºC. The reduction in virus-specific cytopathic effect was observed after 48 and 72h and the virus neutralization titer of the serum calculated by the Spearm-Karber method .

Statistical analysis

The sensitivities of the ELISAs were calculated as the percentages of positives between 7 and 18 days post-challenge. The specificity was calculated as the percentage of negatives in the non challenged broilers, using the same cut off values: I-ELISA ³ 0.2; S-ELISA ³ 0.3 and LPB ³ 0.6, as determined before (Cardoso et al., 1996a; Cardoso et al., 1998; Cardoso et al., 1999). The sensitivity of each of the serotype-specific SN test was calculated as the percentage of positives 18 days after homologous IBV infection. The specificity of each of SNT was calculated as the percentage of negative in the heterologously challenged group A, also 18 days post challenge. In order to calculate the correlation coefficient the usual measurement of precision was used of the relation of variables X (SNT) and Y ( the three different ELISAs) and the comparison between the ELISAs for antibodies titers obtained 18 days after heterologous IBV infection. The respective predictive positive and negative values were calculated as recommended by Witt et al., (1997).

Reproducibility of the serological assays

The reproducibility of the ELISAs for antibody detection was determined using the OD values for serum dilutions of negative and positive controls, tested on twenty different days. These values were subjected to statistical analysis by the Students t test. The ELISAs did not detect Newcastle disease, Reovirus or Influenza A antibodies (data not shown).

RESULTS

Analysis of post-vaccinated challenged broilers sera tested by the ELISAs.

ELISAs were also compared with respect to the reactions obtained with sera from 150 post-vaccinated challenged brolilers. Using the calculations described in the Materials and Methods, the results shown on Table 1 demonstrate that LPB-ELISA was the most sensitive ELISA (100%) until 46 day after challenge with the heterologous serotype. However I-ELISA and S-ELISA were equally sensitive, when the pre- challenge antibody response was analyzed.

Analysis of non-vaccinated broilers sera by the ELISAs.

The three different ELISA were compared regarding the reactions obtained with sera from 150 non-vaccinated broilers (Table 2). The lowest specificity rate was seen with I-ELISA and the LPB-ELISA. In contrast, both S-ELISA and the standard SNT presented the same specificity (99.3%). Therefore, the sensitivity of all ELISA tested was satisfactory.

Comparison between the three different ELISAs

Most of the ELISA comparison gave a significant correlation, especially when the titers obtained by LPB-ELISA were compared to S-ELISA. (Figure 2A and 2C). Those results demonstrated that LPB-ELISA and I-ELISA comparison resulted in the lowest correlation coefficient r = 0.69 (Figure 2B). In contrast, data showed in figure 2A and 2C are showed almost the same coefficient, r = 0.75 and r = 0.79 respectively.

Comparison of ELISA with SNT results

A relationship could be seen between the SNT titers and those obtained from LPB-ELISA (Figure 1A) r = 0.98. Moreover, S-ELISA and I-ELISA demonstrated almost the same coefficient, r = 0.79 and r = 0.74 respectively ( Figure 1B and 1C).

DISCUSSION

Sequential infections of animals or humans with viruses that exist as multiple antigen often induce complex antibody response patterns. So, sequential IBV inoculations with heterologous virus types produced similar serum antibody responses in chickens. (Gough & Alexander, 1978; Darbyshire & Peters, 1984; Gelb & Killian, 1987; Brown & Bracewell, 1988; Ignajatovic & Galli, 1995). In fact, antibody production to the original infecting virus predominates, even after subsequent infections with the heterologous virus types. However, the results presented on table 1 demonstrated that the SN titres were higher before, and after the challenge with the M₄₁ strain. Another interesting finding was the similarity sensitive between LPB ELISA and SN test even 46 days after the challenge with the M₄₁ strain, according to the results also found by Witt et al., (1997). It was confirmed when the correlation coefficient determined showed high agreement between the two techniques r = 0.98, results also found by Cardoso et al., (1998), Esterhuysen et al., (1995), Mc Cullough et al., (1992) and Araujo et al., (1996). The I-ELISA and LPB-ELISA (Figure 2B) comparison resulted in a lowest correlation coefficient r = 0.69, but significant at p<0.0005. However, I-ELISA and S-ELISA presented the same antibody response before and also 46 days after the challenge, and with the heterologous strain. The specificity of all ELISA was satisfactory, with a few false negative serum detection (Hamblin et al., 1986). It is important to emphasize, in poultry, high specificity of serological test is more relevant than high sensitivity, since low sensitivity can be compensated by using a large number of serum samples, as demonstrated by Witt et al., (1997). Therefore, in this study the ELISA technique may have a number of advantages over the serum neutralization test, but the exact interpretation of the serological results in IBV infections serotyping may be necessary. Virus isolation and serotyping are the correct laboratory routine for IBV, based on means of establishing the virus serotype that caused an IBV infection. Thus, the LPB-ELISA is considered a useful methodology for IBV antibody detection, since under heterologous challenge with wide serotypes. The demonstration of a rise in antibody titre between acute and convalescent disease-phase serum would be valuable information, also before virus isolation attempts.

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Publication Dates

• Publication in this collection
  30 Oct 2002
• Date of issue
  Jan 2001