Development and standardizing edition of dot ELIZA for serological diagnosis of Maedi Visna - progressive ovine pneumonia
Abstract
Many tests are used to diagnose the Small Ruminant Lentivirus and the AGID is the standard test and the initial diagnosis is fundamental. The objective of the present study was to evaluate the dot ELIZA against the AGID test in the diagnosis of Maedi Visna - progressive ovine pneumonia. First complete viral antigen was produced by cultivating goat synovial membrane, inoculated with the K1514 strain of the virus with a10-4,2TCDI50/mL titer Standardization tests of the DE were carried out and with the results obtained, an optimal antigen concentration of 0.25 µg /well was. A total of 246 samples of sheep serum were assessed and the results compared to AGID. The sensitivity of DE was 100% and specificity 93.75%, and the agreement was 93.9%. Significant difference was observed among the results (p<0.05). The DE was more sensitive than the AGID and therefore there are perspectives for its use in screening tests, helping in the processes of transport, animal commercialization and health control. However, the need is emphasized for more analysis for the effect of registering with the official agricultural and livestock institutes.
Keywords: Maedi Visna - progressive ovine pneumonia, dot ELIZA, AGID, sensitivity, specificity
Introduction
The Small Ruminants Lentivirus (SRLV) usually determine degenerative diseases with slow progression. They include Maedi Visna virus (MVV) and the Goat Arthritis Encephalitis Virus (CAEV) that affect sheep and goats, respectively, and crossed infection has also been reported (Pisoni et al., 2005, Denner, 2007).
Among the serological tests, the most commonly used are the AGID (Agarose gel immunodiffusion) and ELISA (Enzime Linked Immunosorbent Assay) (OIE,2005). The AGID is the test most used to diagnose the SRLV and it is recommended by the OIE for use in eradication programs.
Two characteristics, sensitivity and specificity, should be observed when choosing the diagnostic test. In addition to these characteristics, currently there is a growing interest in elaborating fast, practical and low-cost tests.
The AGID is a simple, low cost test, but because it has been questioned for low sensitivity that makes it efficacious only for those animals that are already seroconverted with high anti-body indices and thus not efficacious for those that seroconvert later or that have small quantities of antibodies. The Dot-Blot is a test already developed to diagnose viral pathologies such as avian reovirus (Georgieva et al., 2002), HIV ( Herbelling et al., 1988) and Caprine Arthritic Encephalitis (Pinheiro, 2001; Pinheiro et al., 2006) but there are no reports in the literature of the use of this technique to diagnose Maedi Visna. Because of its its high sensitivity, specificity and low-cost (Pinheiro et al., 2006) it is interesting to test it in the diagnosis of infection by Maedi Visna. Therefore the objective of the present study was to standardize the Dot ELIZA for MVV diagnosis and compare the results obtained with the OIE standard test (AGID).
Material and Methods
Antigen production
The MV virus (K1514 strain) was cultivated on goat synovial membrane cells in essential minimum culture medium (MEM) with 5% bovid fetal serum (BFS) 10% gentamycine and 1% amphotericin B. The supernatants obtained were processed and analyzed according to the method used by Aragão et al.(2008). The antigen was submitted to protein doses by the Bradford method (1976) and immunoblotting and the presence of seven immunogenetic protein bands was observed (16, 27, 35, 50, 42, 63 and 123 kDa) (unpublished data). Non-inoculated supernatants were used as control and processed in the same way as the antigen.
Dot ELIZA standardization
The protein dosage showed a 2.5 µg/ µl concentration. From this value, the antigen was tittered as a means of verifying the smallest concentration capable of promoting the reaction. Nitrocellulose membranes were placed in the dot2 apparatus and sensitized with the antigen diluted in PBS (Na2HPO4 0.5 M; NaH2PO4 0.5 M; NaCl; pH 7.4) at the concentrations of 0.25; 0.5; 1.0 and 2 µg/ well and dried for 10 minutes in a hot air stream. Each concentration was made in quadruplet with two replications for each concentration. When completely dry they were then stained with Ponceau’s stain and numbered. Starting at this stage they were divided into two groups and submitted to two types of blockage. Group A., blocked with TTBS (20 mM Tris, 500 mM NaCl, 0.05 % Tween 20, pH7.5) (Georgieva, 2002) for 15 minutes and group B blocked with PBS-Tween 0.3 % for 40 minutes. After the respective times, the membranes were washed twice for two minutes with slow agitation with PBS Tween 0.05% and submitted to two different dilutions (1:25 and 1:50) of positive and negative sera (both from the commercial kit3) for 30 minutes. At the next stage, the membranes were again the washed and incubated with conjugate4 at two dilutions (1:10000 and 1:15000) for 30 minutes. After this period, the membranes were washed twice with PBS Tween 0.0 5%, twice with PBS and developed with 4 chloronaphthol[1] and diaminobenzidine (DAB)[2]. The reaction occured in one minute and was stopped with distilled water. The greatest dilution of the antigen that presented the best visual color differentiation between the positive and negative sera were considered for the effect of standardization. From this first result, a new test was carried out, which assessed the performance of the positive weak (of the American kits), and also the 1:10000, 1:15000 and 1:20000 conjugate dilutions were tested. All the stages were carried out in a chamber at 37°C under constant agitation.
AGID and samples
The method used was the micro AGID test (MAGID) standardized by Gouveia (1994) using a commercial kit7.
As a sample universe, 246 samples were used of sheep blood from herds in the state of Ceará. The blood was collected in Vacutainer tubes without anticoagulant. After collection, the blood samples were sent to the virology laboratory, where they were centrifuged at 3000 g for 10 minutes. The sera were then identified, stored in cryotubes and preserved in a freezer at -20°C until the analyses were performed. All tests were submitted to two assessors at different times.
For the statistical analysis, a comparative study was made of the results obtained in the AGID test and the Dot ELIZA. The sensitivity, relative specificity, positive predictive value and negative predictive value were assessed. The Kappa index was also calculated to assess the agreement between the tests. The Winepsicope 1.0 program was used in the analyses.
Results
Dot ELIZA standardization
All the antigen concentrations reacted specifically with the serum dilutions used, showing that any titer studied could be used to efficiently execute the tests. However, the optimum concentration considered for the effect of standardization was 0.25 µg/well viral protein. The best conjugate dilution was 1:15,000 that presented the best visual distinction among the positive and negative control sera. Both the blocks performed the same but the block with TTBS was preferred because it required only 15 minutes action. The optimal dilution value for the sera used to carry out the tests was 1:50 but the Dot ELIZA could detect the various dilutions used (1:25; 1:50) (Figs. 01 and 02).
Figure 1. Results of the standardization. The antigen dilutions are on the vertical. The serum dilutions are on the horizontal, where A= 1:25+; B= 1:25-; C= 1: 50+; D= 1:50-. Block used: PBS 0.03 %.
Figure 2. Results of the standardizations. The antigen dilutions are on the vertical. The serum dilutions are on the horizontal, where A= 1:50+; B= 1: 50-; C= 1: 25+; D= 1:25-. Block used: PBS 0.03 %. The marked area corresponds to the optimal antigen and serum dilution.
During the analysis of the 246 tests a precise distinction was verified among the positive and negative sera. Due to the use of positive and negative controls, the well considered positive was the one most visually different from the negative standard.
Dot ELIZA compared with AGID
The results of the comparison of the tests are shown in Table 01. Twenty-one (8.5%) of the 246 sera analyzed were positive in the Dot ELIZA. The test VPP was 28.5%, while the VPN was 100%, and the sensitivity and specificity were, respectively, DE 100% and 93.75%. The AGID detected the presence of antibodies for the MVV in only six of the samples analyzed (2.4%). The agreement observed among the results of the techniques was 93.9%.
ResultsDot ELISA / IDGA / No. (%)
+ / + / 6 (2,4)
- / - / 225 (91,4)
+ / - / 15 (6,09)
- / + / 0
Table 1. Results obtained in the serum tests by Dot ELISA and AGID *agreement 92.9%
Regarding carrying out the tests, they were easy to perform, but the time taken to develop the results was very different. AGID required 48 to 72 hours for reading, while the Dot ELIZA required only 1.5 hours between carrying out the test and reading, that in this case was immediate. Furthermore, it was observed that neither test required expensive laboratory equipment that thus reduced costs.
The negative control made with non-inoculated cell supernatants and concentrated 500 times did not show any reaction in any of the tests.
Discussion
The DE was more sensitive than the AGID test (p<0.05). This result can be explained both because the methodological aspects by the use of nitrocellulose membranes as fixing support of the viral antigen and also because the type of antigen used to carry out the tests.
The AGID test, although it is widely used, has the disadvantage of low sensitivity. This characteristic is due, above all, to the type of antigen used. The diagnostic kits used in the present study had as antigen proteins p27 and gp135 of the CAE and OPPV virus from North America. The use of this antigen takes into consideration the fact that the proteins of the viral capsule present conserved regions for both the virus (CAE and OPPV) thus ensuring that even with the presence of another glycoprotein (gp135) variable the antibodies could be detected by cross-recognition by the existing conserved regions (Grego et al., 2002). The p27 protein stimulates strong antibody production (Brodie et al., 1998). These proteins are specific to the related virus, therefore present some distinction among the diverse strains existing. Reischak et al. (2002b) reported the need to produce viral antigens from Brazilian strains and their standard sera because, according to the authors, there was a possibility of detecting a greater quantity of positive animals and preventing the problem of false negative results.
Another aspect to consider is the fact that the infected animals suffer change in the immunogenetic reactions that vary according to the stage of the infection. The animals that had their sera analyzed did not show clinical signs of the disease and the age was not known when these animals had been infected and consequently, the infection stage. Lacerenza et al. (2006) reported that infected sheep directed their antibody production to different epitopes. Furthermore, sheep infected for longer have the capacity to produce quantities of antibodies that favor their detection (Leginagoikoa et al., 2006).
In the present study, the standardized Dot ELIZA used the complete viral antigen and the gp135 was at low levels in the final antigen after ultracentrifuging. This occurred because the concentration had been made in sucrose base, that removed much of the gp at the time of ultracentrifuging (Pinheiro et al., 2006). The use of the complete virus allowed a greater detection of the positive animals because it had a greater quantity of different proteins, overcoming the problem of the variation in the types of antibodies during infection. This statement had been reported previously by other authors (Pinheiro et al., 2006). Naeen and Goyal (1990) analyzed the use of raw antigen (without separating specific proteins) to detect bovid herpesvirus and reported that in the purification process the specificity of the test was guaranteed but the sensitivity was damaged by the lack of detection of antibodies against other proteins not present in the purified material. Varea et al. (2001) followed ELIZA and reported that infected animals presented different anti-body levels during their life and, at the time, that the increase in test sensitivity was directly linked to the titers of the specific antibodies.
In the face of the results, the DE, because it is a more sensitive and faster test, would be an alternative in MV diagnosis. These results open the way for the use of DE as a screening test due, among the factors already mentioned, to its greater sensitivity and quick results compared to the AGID.
Conclusions
Dot ELIZA is more sensitive than the AGID and there is an excellent perspective for its use in Maedi Visna - progressive ovine pneumonia - diagnosis. However, the need was shown for continuity in analyses to register it with the agricultural livestock institutions.
Acknowledgments
The authors thank Banco do Nordeste for technical and financial support in executing the experimental design and the Fundação Cearense de Apoio a Pesquisa – FUNCAP for financial support as a study grant.
REFERENCES
Aragão, M. A. C. et al. 2008. Maedi-Visna Vírus: produção de antígeno, análise protéica e antigênica. Arq. Inst. Biol. 75, 423-429.
Bradford, M., 1976. A rapid and sensitive met.hod for microgram quantities of protein utilizing the principle of protein - dye binding. Anal Biochem. 72, 245 -248.
Brodie, S.J. et al., 1998. Current concepts in the epizootiology, diagnosis, and economic importante of ovine progressive pneumonia in North America: A review. Small Ruminant Res. 27, 1-17.
Denner, J. , 2007.Transspecies transmissions of retroviruses: New cases. Virology. 369, 229-233.
Georgieva, M. et al., 2002 A Dot-immunobinding assay to dtect antibodies against avian reoviruses. J. Exp. Pathol.518, .35-38.
Gouveia, A. M. 1994. Padronização de microtécnica de imunodifusão em gel de agarose para diagnóstico de lentivírus Pneumonia Progressiva Ovina (OPP) - Maedi-Visna (MVV) - Artrite Encefalite Caprina (CAEV).EMBRAPA Caprinos, 4 p.p.
Grego et al. , 2002. Genetic heterogeneity of small ruminant lentiviruses involves immunodominant epitope of capsid antigen and affects sensitivity of single-based immunoassay. Clin. Diag. Labor. Immunol. 9, 828-832.
Herbelling, R.L. et al., 1988. Dot-immunobinding assay compared with enzyme-linked immunosorbent assay for rapid and specific dtecttion of retroviruses antibody induced by human or simian acquired immunodeficiency syndrome.J. Clin. Microbiol. 26, 765-767.
Lacerenza, D. et al., 2006. Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes. Vet. Immunol. Immunopathol. 112, 264-271.
Leginagoikoa, I. et al., 2006. Horizontal Maedi-Visna virus (MVV) infection in adult dairy-sheep raised Ander varyng MVV-infection pressures investigated by ELISA an PCR. Res. Vet. Sci.,80, 235-241.
Naeen, K. e Goyal, M. S. , 1990. A dot immunobinding assay on nitrocellulose for the detection of bovid herpesvirus-antibodies. J. Vet. Diag. Invest., 2, p.107-110.
Office International des Epizooties. Código sanitário para los animales terrestres (2005). Disponível em: <http// Acesso em: 15.05. 2006.
Pinheiro, R.R. Vírus da Artrite Encefalite Caprina: Desenvolvimento e padronização de ensaios imunienzimáticos (ELISA e Dot-Blot) e estudo epidemiológico no Estado do Ceará. 2001. 115f. Tese (doutorado em medicina veterinária preventiva e epidemiologia) - Universidade Federal de Minas Gerais, Belo Horizonte, 2001.
Pinheiro et al., 2006. Desenvolvimento de dot-blot para detecção de anticorpos para o vírusda Artrite Encefalite Caprina em caprinos.Rev. Port. Ciên. Vet.,101, 51-56.
Pisoni, G. et al., 2005. Phylogenetic analysis of small-ruminant lentivirus subtype B1 in mixed flocks: Evidence for natural transmission from goats to sheep. Virology, 339, 147-152.
Reis, J.K.P. e Leite, R.C., 1994. An enzime-linked immunosorbent assay (ELISA) test for the diagnosis of equine infectious anemia in Brazil. Prev. Vet.. Méd, 20, 261-267.
Reischak, D et al., 2002b. Importância da escolha dos reagentes para o diagnóstico de infecção por lentivirus de pequenos ruminantes em caprinos. Vet. Not.. 8, 51-56.
Varea, R. et al., 2001. Early detection of Maedi Visna (ovine progressive pneumonia) virus seroconversion in field sheep samples. J. Vet. Diag. Invest. 13, 301-307.
[1]3,3´-Diaminobenzidine (DAB) – Sigma. Cat. Nº D 5637
[2] 4-Cloro-1-Naphto – Sigma. Cat. Nº C 8890