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Standardization of an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies anti-Corynebacterium pseudotuberculosis in sheep
Small Ruminant Research 116 (2014) 229–232
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Standardization of an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies anti-Corynebacterium pseudotuberculosis in sheep Alessandra Figueiredo Castro Nassar a,∗ , Simone Miyashiro a , Fábio Gregori b , Rosa Maria Piatti a , Gabriela Terezinha Daniel a , Lilian Gregory b a b
Instituto Biológico, Av. Conselheiro Rodrigues Alves, 1252, Vila Mariana, São Paulo, SP CEP: 04014-002, Brazil Faculdade de Medicina Veterinária e Zootecnia-USP, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 1 August 2013 Received in revised form 21 October 2013 Accepted 23 October 2013 Available online 1 November 2013 Keywords: C. pseudotuberculosis Sheep Microbiological culture PCR ELISA
a b s t r a c t Caseous lymphadenitis (CLA) is a contagious disease caused by Corynebacterium pseudotuberculosis. It occurs worldwide and is responsible for economic losses in sheep raising. The aim of this study was to standardize an indirect ELISA to detect antibodies anti-C. pseudotuberculosis in sheep. Serum samples from animals previously tested serially using microbiological culture and PCR from the puncture of lymph nodes and lungs, made up an initial panel with the aim of standardizing the test. When it was applied to field samples, the ELISA reaction showed sensitivity of 100% and specificity of 100% based on microbiological culture and PCR results. Although microbiological culture is the gold standard in C. pseudotuberculosis diagnosis, the ELISA standardized in this study seems to be advantageous because it is faster to process the samples, and may be used as a screening test, or even for animals trade, once the disease is often unapparent, making clinical and microbiological diagnosis difficult to be performed. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Caseous lymphadenitis (CLA) is a chronic suppurative disease that affects small ruminants. The disease is characterized by abscesses in superficial and mesenteric lymph nodes, as well as internal organs, and is caused by the bacterium Corynebacterium pseudotuberculosis (Baird and Fontaine, 2007; Guimarães et al., 2009). CLA is found all over the world and has economic impact in sheep raising, leading to weight loss, carcass condemnation, and decreased reproductive efficiency and wool production (Lloyd et al., 1990). CLA diagnosis is based on clinical diagnosis, in which abscesses are seen in superficial lymph nodes. Laboratory examination, isolation and bacterial identification,
considered to be the gold standard, should be carried out in caseous material drained from the abscesses and organs (Alves and Pinheiro, 2002; Baird and Fontaine, 2007). Serological tests, such as gel immunodiffusion (Burrell, 1988), western-blotting and recombinant ELISA with PLD gene (Menzies et al., 1994) have some advantages in situations in which sheep do not show evident signs of infection, with internal abscesses in lungs and mediastinal lymph nodes (Binns et al., 2007). The aim of this study was to develop a sensitive and specific indirect ELISA to detect antibodies anti-C. pseudotuberculosis in sheep in order to contribute to the control and prevention of the disease in this animal species. 2. Materials and methods 2.1. Animals status classification
∗ Corresponding author. Tel.: +55 11 50871721; fax: +55 11 50871791. E-mail addresses: [email protected], [email protected] (A.F.C. Nassar). 0921-4488/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2013.10.016
In order to select the positive animals for CLA, we collected 103 serum samples paired with punctures of enlarged superficial lymph nodes in sheep from São Paulo and Minas Gerais States herds. To confirm the
230
A.F.C. Nassar et al. / Small Ruminant Research 116 (2014) 229–232
positive status, microbiological culture and PCR procedures were performed for puncture contents. For negative animals selection, serum samples from 43 animals were collected in an abattoir from animals with absence of enlarged superficial lymph nodes and no internal organs suggestive lesions. To confirm the negative status, fragments of lung and mediastinal lymph nodes from all animals were collected for microbiological culture and PCR procedures.
0.05% hydrogen peroxide 30%, and incubated at 37 ◦ C for 10 min, followed by the addition of 50 L of the blocking solution (sulfuric acid 4 N). Reading was carried out in a microplate reader Robonik® model Readwell Plate – ELISA Plate Analyzer with a 492-nm filter. Absorbance values obtained were expressed as arithmetic means of the duplicates. All washing steps were carried out in the automatic Washwell Plate ELISA Washer, Robonik® , with 250 L of washing solution per well.
2.2. Microbiological culture
2.5. Statistical analysis
Material aspirated from lymph nodes and organs collected in the slaughterhouse were suspended 1:5 (w/v) in sterile saline solution 0.85%, and 10 L of the suspension were seeded on 5% sheep blood agar and incubated for 48 h at 37 ◦ C. After the incubation period, when bacterial multiplication occurred in the medium, bacterioscopy by Gram stain was performed. Bacterial species were identified according to Quinn et al. (2011) and Koneman et al. (2008).
The cutoff point of the ELISA was determined by means of the receiver operating characteristics (ROC) curve based on the performance evaluation of 42 positive and 43 negative samples on the standardized indirect ELISA. Relative sensitivity and specificity were defined using the ELISA results for the serum samples, having the isolation and identification of C. pseudotuberculosis as the gold standard. For repeatability of the ELISA reaction, 8 sera were selected, four positive and also four negative for CLA in microbiological culture and PCR. Each sample was tested in quadruplicate in four different plates, in a way to determine the variance among the repetitions.
2.3. PCR DNA extraction was performed using DNAzol (Invitrogen® ) from the supernatant of the lymph node and organ suspension (lungs and mediastinal lymph nodes) diluted 1:5 (w/v) in saline sterile solution 0.85%. For the PCR technique, primers described by Pacheco et al. (2007) were used. DNA amplification was carried out using 10 L of DNA added of 40 L of the PCR mix containing 1.25 U Taq DNA polymerase (Invitrogen® ), 200 M of each deoxynucleotide, buffer (10 mM Tris–HCl, pH 8.0; 50 mM KCl); 2 mM MgCl2 , and 0.5 M of each primer. The amplification protocol was: denaturation at 95 ◦ C for 3 min; 30 cycles of 95 ◦ C for 1 min; 58 ◦ C for 40 s, and 72 ◦ C for 1 min, final extension at 72 ◦ C for 7 min. Amplified products were resolved by electrophoresis in 1.0% agarose gel added of Sybr Safe (Invitrogen® ) 0.5 g/mL. 2.4. ELISA 2.4.1. Antigen preparation One strain of C. pseudotuberculosis isolated and confirmed by biochemical identification and PCR was seeded on sheep blood agar 5% and incubated at 37 ◦ C for 48 h. Then, it was transferred to brain heart infusion (BHI) broth incubated for 48 h at 37 ◦ C for growth until it got cloudy. After this period, the bacterial suspension was centrifuged at 960 × g for 10 min, and the sediment was resuspended in 10 mL of PBS, pH 7.4. After being washed twice with PBS, it was resuspended in 2 mL of the same buffer. Bacterial cells were broken by ultrasound (T7-thornitron) for 12 min at 60 Hz. The mass obtained was centrifuged at 960 × g for 10 min and protein concentration was assessed using the colorimetric method Micro BCA Protein Assay Kit (Pierce® ). The antigen was placed in microtubes (1.5 mL per tube) and stored at −20 ◦ C (Binns et al., 2007). 2.4.2. Standardization of indirect ELISA For the indirect ELISA reaction, 96-well polystyrene flat-bottom plates were used (Costar). Antigen and serum dilutions were carried out by chessboard titration of the immunoreagents in order to standardize their concentrations. In the initial stage of the standardization a total of 10 plates were used with 6 positive and 6 negative serum samples, according to the results obtained in the microbiological culture and PCR. Microplates were sensitized with 100 L of C. pseudotuberculosis antigen solution in the following concentrations: 2.5 g/mL, 4 g/mL, 7.5 g/mL, and 10 g/mL in bicarbonate–carbonate buffer 0.1 M, pH 9.6, incubated for 4 ◦ C for 18 h. After this incubation, plates were washed three times with PBS-Tween solution (PBS pH 7.4/0.1% Tween 20). Blocking of the reaction with 1% non-fat powdered milk in PBS, pH 7.4 was performed for 1 h at 37 ◦ C. After that, plates were washed three times with PBSTween solution. One hundred microliters of the diluted test sera (1:20, 1:50, 1:100 and 1:200), in duplicate, were added to the dilution buffer of the sample (PBS pH 7.4/0.01% Tween 20/1% non-fat powdered milk) and incubated for 1 h at 37 ◦ C. After that, plates were washed three times with PBS-Tween solution. One hundred microliters of the peroxidasemarked anti-sheep IgG conjugate diluted 1:400 in PBS-Tween solution were added, and the plates were incubated for 1 h at 37 ◦ C. After that, the plates were washed three times with PBS-Tween solution. The reaction was developed with the use of 2 mg of ortho-phenylenediamine (OPD) chromatogenous solution in citrate-phosphate solution pH 5.0 added of
3. Results 3.1. Animals status From 103 puncture samples, 42 (42/103) both positive resulted in microbiological culture and PCR for C. pseudotuberculosis. On negative animals selection, samples from 43 animals without suggestive lesions, negative resulted (43/43) both in microbiological culture and PCR, for C. pseudotuberculosis. This way, we have got to work with 42 positive serum samples and 43 negative serum samples for indirect ELISA performance evaluation. 3.2. ELISA 3.2.1. Standardization of indirect ELISA Protein concentration of the antigen for the indirect ELISA was 75 g/mL. Considering the criteria for the absorbance inside the acceptable ranges of mean optical density (positive ≤2.00) and the greatest antigen dilution, antigen concentration adopted per plate was 4 g/mL, and serum dilution, 1:200. These concentrations have presented absorbance inside the acceptable ranges of mean optical density and differences between positive and negative values. For the positive samples, mean optical density was 1.88 ± 0.43, and for the negative samples, 0.71 ± 0.18. 3.3. Statistical analysis The cutoff point of the ELISA was determined by means of ROC curve, using the SigmaPlot software version 12.1, based on a panel of 85 sera (42 positive and 43 negative). Positive samples were those that presented OD > 1.1 (95% CI 0.9159–1.0), and the negative ones were those that presented OD < 1.1 (95% CI 0.9178–1.0). Fig. 1 shows the result of the dot plot demonstrating that there was no superposition in optical density of the populations that has been positive and negative in the gold standard when the previously defined cutoff value was adopted. Regarding to the test repeatability, it presented standard deviation ranging from 0.047 to 0.085 for positive sera from 0.010 to 0.043
A.F.C. Nassar et al. / Small Ruminant Research 116 (2014) 229–232
Fig. 1. Results of the indirect ELISA for relative sensitivity of the sera using the ROC curve.
for negative sera. Similarly, the variation coefficient ranged from 2.516 to 5.657 for positive from 1.256 to 8.6885 to negative sera. 4. Discussion Fontaine and Baird (2008) described that animals experimentally infected with C. pseudotuberculosis by subcutaneous route developed primary foci of infection in mediastinal lymph nodes and lungs. Menzies et al. (1994) stated that it is impossible to be sure that a population of negative animals is actually not infected and animals have never got in contact with the disease. On the recently work, besides the clinical and macroscopic findings suggestive of C. pseudotuberculosis infection, the samples were serially tested by microbiological culture and PCR, in order to overcome the limitations of classification in positive or negative populations. Nowadays, there are many ELISAs that use several antigens of the bacterium, such as bacterial cell wall and exotoxin (phospholipase D) our recombinant exotoxin (Binns et al., 2007; Chirino-Zárraga et al., 2009; Menzies et al., 1994). The indirect ELISA standardized in this study was performed with the use of the lysed cells supernatant, despite being a partially purified antigen, it may be detected C. pseudotuberculosis antibodies, presenting on 100% of sensitivity and specificity when it is compared to PCR and microbiology culture. In a study carried out by Dercksen et al. (2000), four ELISA were analyzed for the detection of antibodies in sheep, and the test that presented the best sensitivity (79 ± 5%) and specificity (99 ±1%) was the one that used the exotoxin (phospholipase D) as the antigen, which was described by Menzies et al. (1994). On the other hand, Kaba et al. (2001) standardized an ELISA with raw bacterial wall extract with sensitivity and specificity of 85% and 96%, respectively. Seyffert et al. (2010) carried out a study on the prevalence of the bacterium in Minas Gerais
231
region using the indirect ELISA with C. pseudotuberculosis raw antigen, and sensitivity and specificity were 93.5% and 100%, respectively. The results for sensitivity and specificity presented in the field under the curve for the two populations of the test, and also the repeatability enabled the complete differentiation of the populations of animals that were positive and negative in the standardized ELISA. The variation coefficients found inside the acceptable range were ≤15% (Crowther, 2009). The distinct sensitivity and specificity values described by different authors can be due different methodologies for selection of animals, either naturally of experimentally infected, latency period, or cross reaction with other similar microorganisms (Seyffert et al., 2010). The indirect ELISA reported here has advantages in the quick diagnosis of CLA in sheep when it is compared due to microbiological culture and PCR, in terms of sensitivity, specificity and time, it has given the simplicity of the test and antigen preparation, it’s automation, also the small amount of sample used. Moreover this methodology may be used in the diagnosis, in the trade of animals, and furthermore epidemiology studies in Brazil. References Alves, F.S.F., Pinheiro, R.R., 2002. Linfadenite Caseosa – Recomendac¸ões e Medidas Profiláticas. Agrop. Catarinense 13, 12–14. Baird, G.J., Fontaine, M.C., 2007. Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis. J. Comp. Pathol. 137, 179–210. Binns, S.H., Green, L.E., Bailey, M., 2007. Development and validation of ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera. Vet. Microbiol. 123, 169–179. Burrell, D.H., 1988. A simplified double immunodiffusion technique for detection of Corynebacterium ovis antitoxin. Res. Vet. Sci. 28, 234–237. Chirino-Zárraga, C., Rey-Valerion, C., Scaramelli, A., Carrero, L., 2009. Diagnosis of caseous lymphadenitis by ELISA in naturally infected goats from Venezuela. Small Rumin. Res. 87, 92–95. Crowther, J.R., 2009. Methods in molecular biology, the ELISA guidebook. In: Validation of Diagnostic Test for Infectious Diseases. Humana Press, Austria, pp. 291–334. Dercksen, D.P., Brinkhof, J.M.A., Dekker-Nooren, T., Van Maanem, K., Bode, C.F., Baird, G., Kamp, E.M., 2000. A comparison of four serological test for the diagnosis of caseous lymphadenitis in sheep and goats. Vet. Microbiol. 75, 167–175. Fontaine, M.C., Baird, G.J., 2008. Caseous lymphadenitis. Small Rumin. Res. 76, 42–48. Guimarães, A.S., Seyffert, N., Bastos, B.L., Portela, R.W.D., Meyer, R., Carmo, F.B., Cruz, J.C.M., Mcculloch, J.A., Lage, A.P., Heinemann, M.B., Miyoshi, A., Azevedo, V., Gouveia, A.M.G., 2009. Caseous lymphadenitis in sheep flocks of the State of Minas Gerais, Brazil: prevalence and management surveys. Small Rumin. Res. 87, 86–91. Kaba, J., Kutschke, L., Gerlach, G.F., 2001. Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infection in goats. Vet. Microbiol. 78, 155–163. Koneman, E.W., William, M.J., Schreckenberger, P.C., Winn, W.C., Allen, S.D., Woods, G.L., 2008. Diagnóstico microbiológico: texto e atlas colorido. In: Bacilos Gram-positivos Aeróbicos e Facultativos. Guanabara Koogan, Rio de Janeiro, pp. 760–851. Lloyd, S., Lindsay, H.J., Slater, J.D., Jackson, P.G.G., 1990. Corynebacterium pseudotuberculosis infection (caseous lymphadenitis) in goats. Goat Vet. Soc. 11, 55–65. Menzies, P.I., Muckle, C.A., Hwang, Y.T., Songer, G.J., 1994. Evaluation of an enzyme-linked immunosorbent assay using an Escherichia coli recombinant phospholipase D antigen for the diagnosis of Corynebacterium pseudotuberculosis infection. Small Rumin. Res. 13, 193–198. Pacheco, L.G.C., Pena, R.R., Castro, T.L.P., Dorella, F.A., Bahia, R.C., Carminati, R., Frota, M.N.L., Oliveira, S.C., Meyer, R., Alves, F.S.F., Miyoshi, A., Azevedo, V., 2007. Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid
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detection of this pathogen in clinical samples. J. Med. Microbiol. 59, 480–486. Quinn, P.J., Markey, B.K., Leonard, F.C., FitzPatrick, E.S., Fanning, S., Hartigan, P.J., 2011. Chapter 17, Corynebacterium species. In: Veterinary Microbiology and Microbial Disease, 2nd ed. Wiley-Blackwell, Oxford, UK, pp. 207–210.
Seyffert, N., Guimarães, A.S., Pacheco, L.G.C., Portela, R.W., Bastos, B.L., Dorella, F.A., Heinemann, M.B., Lage, A.P., Gouveia, A.M.G., Meyer, R., Miyoshi, A., Azevedo, V., 2010. High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA. Res. Vet. Sci. 88, 50–55.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Standardization of an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies anti-Corynebacterium pseudotuberculosis in sheep Alessandra Figueiredo Castro Nassar a,∗ , Simone Miyashiro a , Fábio Gregori b , Rosa Maria Piatti a , Gabriela Terezinha Daniel a , Lilian Gregory b a b
Instituto Biológico, Av. Conselheiro Rodrigues Alves, 1252, Vila Mariana, São Paulo, SP CEP: 04014-002, Brazil Faculdade de Medicina Veterinária e Zootecnia-USP, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 1 August 2013 Received in revised form 21 October 2013 Accepted 23 October 2013 Available online 1 November 2013 Keywords: C. pseudotuberculosis Sheep Microbiological culture PCR ELISA
a b s t r a c t Caseous lymphadenitis (CLA) is a contagious disease caused by Corynebacterium pseudotuberculosis. It occurs worldwide and is responsible for economic losses in sheep raising. The aim of this study was to standardize an indirect ELISA to detect antibodies anti-C. pseudotuberculosis in sheep. Serum samples from animals previously tested serially using microbiological culture and PCR from the puncture of lymph nodes and lungs, made up an initial panel with the aim of standardizing the test. When it was applied to field samples, the ELISA reaction showed sensitivity of 100% and specificity of 100% based on microbiological culture and PCR results. Although microbiological culture is the gold standard in C. pseudotuberculosis diagnosis, the ELISA standardized in this study seems to be advantageous because it is faster to process the samples, and may be used as a screening test, or even for animals trade, once the disease is often unapparent, making clinical and microbiological diagnosis difficult to be performed. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Caseous lymphadenitis (CLA) is a chronic suppurative disease that affects small ruminants. The disease is characterized by abscesses in superficial and mesenteric lymph nodes, as well as internal organs, and is caused by the bacterium Corynebacterium pseudotuberculosis (Baird and Fontaine, 2007; Guimarães et al., 2009). CLA is found all over the world and has economic impact in sheep raising, leading to weight loss, carcass condemnation, and decreased reproductive efficiency and wool production (Lloyd et al., 1990). CLA diagnosis is based on clinical diagnosis, in which abscesses are seen in superficial lymph nodes. Laboratory examination, isolation and bacterial identification,
considered to be the gold standard, should be carried out in caseous material drained from the abscesses and organs (Alves and Pinheiro, 2002; Baird and Fontaine, 2007). Serological tests, such as gel immunodiffusion (Burrell, 1988), western-blotting and recombinant ELISA with PLD gene (Menzies et al., 1994) have some advantages in situations in which sheep do not show evident signs of infection, with internal abscesses in lungs and mediastinal lymph nodes (Binns et al., 2007). The aim of this study was to develop a sensitive and specific indirect ELISA to detect antibodies anti-C. pseudotuberculosis in sheep in order to contribute to the control and prevention of the disease in this animal species. 2. Materials and methods 2.1. Animals status classification
∗ Corresponding author. Tel.: +55 11 50871721; fax: +55 11 50871791. E-mail addresses: [email protected], [email protected] (A.F.C. Nassar). 0921-4488/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2013.10.016
In order to select the positive animals for CLA, we collected 103 serum samples paired with punctures of enlarged superficial lymph nodes in sheep from São Paulo and Minas Gerais States herds. To confirm the
230
A.F.C. Nassar et al. / Small Ruminant Research 116 (2014) 229–232
positive status, microbiological culture and PCR procedures were performed for puncture contents. For negative animals selection, serum samples from 43 animals were collected in an abattoir from animals with absence of enlarged superficial lymph nodes and no internal organs suggestive lesions. To confirm the negative status, fragments of lung and mediastinal lymph nodes from all animals were collected for microbiological culture and PCR procedures.
0.05% hydrogen peroxide 30%, and incubated at 37 ◦ C for 10 min, followed by the addition of 50 L of the blocking solution (sulfuric acid 4 N). Reading was carried out in a microplate reader Robonik® model Readwell Plate – ELISA Plate Analyzer with a 492-nm filter. Absorbance values obtained were expressed as arithmetic means of the duplicates. All washing steps were carried out in the automatic Washwell Plate ELISA Washer, Robonik® , with 250 L of washing solution per well.
2.2. Microbiological culture
2.5. Statistical analysis
Material aspirated from lymph nodes and organs collected in the slaughterhouse were suspended 1:5 (w/v) in sterile saline solution 0.85%, and 10 L of the suspension were seeded on 5% sheep blood agar and incubated for 48 h at 37 ◦ C. After the incubation period, when bacterial multiplication occurred in the medium, bacterioscopy by Gram stain was performed. Bacterial species were identified according to Quinn et al. (2011) and Koneman et al. (2008).
The cutoff point of the ELISA was determined by means of the receiver operating characteristics (ROC) curve based on the performance evaluation of 42 positive and 43 negative samples on the standardized indirect ELISA. Relative sensitivity and specificity were defined using the ELISA results for the serum samples, having the isolation and identification of C. pseudotuberculosis as the gold standard. For repeatability of the ELISA reaction, 8 sera were selected, four positive and also four negative for CLA in microbiological culture and PCR. Each sample was tested in quadruplicate in four different plates, in a way to determine the variance among the repetitions.
2.3. PCR DNA extraction was performed using DNAzol (Invitrogen® ) from the supernatant of the lymph node and organ suspension (lungs and mediastinal lymph nodes) diluted 1:5 (w/v) in saline sterile solution 0.85%. For the PCR technique, primers described by Pacheco et al. (2007) were used. DNA amplification was carried out using 10 L of DNA added of 40 L of the PCR mix containing 1.25 U Taq DNA polymerase (Invitrogen® ), 200 M of each deoxynucleotide, buffer (10 mM Tris–HCl, pH 8.0; 50 mM KCl); 2 mM MgCl2 , and 0.5 M of each primer. The amplification protocol was: denaturation at 95 ◦ C for 3 min; 30 cycles of 95 ◦ C for 1 min; 58 ◦ C for 40 s, and 72 ◦ C for 1 min, final extension at 72 ◦ C for 7 min. Amplified products were resolved by electrophoresis in 1.0% agarose gel added of Sybr Safe (Invitrogen® ) 0.5 g/mL. 2.4. ELISA 2.4.1. Antigen preparation One strain of C. pseudotuberculosis isolated and confirmed by biochemical identification and PCR was seeded on sheep blood agar 5% and incubated at 37 ◦ C for 48 h. Then, it was transferred to brain heart infusion (BHI) broth incubated for 48 h at 37 ◦ C for growth until it got cloudy. After this period, the bacterial suspension was centrifuged at 960 × g for 10 min, and the sediment was resuspended in 10 mL of PBS, pH 7.4. After being washed twice with PBS, it was resuspended in 2 mL of the same buffer. Bacterial cells were broken by ultrasound (T7-thornitron) for 12 min at 60 Hz. The mass obtained was centrifuged at 960 × g for 10 min and protein concentration was assessed using the colorimetric method Micro BCA Protein Assay Kit (Pierce® ). The antigen was placed in microtubes (1.5 mL per tube) and stored at −20 ◦ C (Binns et al., 2007). 2.4.2. Standardization of indirect ELISA For the indirect ELISA reaction, 96-well polystyrene flat-bottom plates were used (Costar). Antigen and serum dilutions were carried out by chessboard titration of the immunoreagents in order to standardize their concentrations. In the initial stage of the standardization a total of 10 plates were used with 6 positive and 6 negative serum samples, according to the results obtained in the microbiological culture and PCR. Microplates were sensitized with 100 L of C. pseudotuberculosis antigen solution in the following concentrations: 2.5 g/mL, 4 g/mL, 7.5 g/mL, and 10 g/mL in bicarbonate–carbonate buffer 0.1 M, pH 9.6, incubated for 4 ◦ C for 18 h. After this incubation, plates were washed three times with PBS-Tween solution (PBS pH 7.4/0.1% Tween 20). Blocking of the reaction with 1% non-fat powdered milk in PBS, pH 7.4 was performed for 1 h at 37 ◦ C. After that, plates were washed three times with PBSTween solution. One hundred microliters of the diluted test sera (1:20, 1:50, 1:100 and 1:200), in duplicate, were added to the dilution buffer of the sample (PBS pH 7.4/0.01% Tween 20/1% non-fat powdered milk) and incubated for 1 h at 37 ◦ C. After that, plates were washed three times with PBS-Tween solution. One hundred microliters of the peroxidasemarked anti-sheep IgG conjugate diluted 1:400 in PBS-Tween solution were added, and the plates were incubated for 1 h at 37 ◦ C. After that, the plates were washed three times with PBS-Tween solution. The reaction was developed with the use of 2 mg of ortho-phenylenediamine (OPD) chromatogenous solution in citrate-phosphate solution pH 5.0 added of
3. Results 3.1. Animals status From 103 puncture samples, 42 (42/103) both positive resulted in microbiological culture and PCR for C. pseudotuberculosis. On negative animals selection, samples from 43 animals without suggestive lesions, negative resulted (43/43) both in microbiological culture and PCR, for C. pseudotuberculosis. This way, we have got to work with 42 positive serum samples and 43 negative serum samples for indirect ELISA performance evaluation. 3.2. ELISA 3.2.1. Standardization of indirect ELISA Protein concentration of the antigen for the indirect ELISA was 75 g/mL. Considering the criteria for the absorbance inside the acceptable ranges of mean optical density (positive ≤2.00) and the greatest antigen dilution, antigen concentration adopted per plate was 4 g/mL, and serum dilution, 1:200. These concentrations have presented absorbance inside the acceptable ranges of mean optical density and differences between positive and negative values. For the positive samples, mean optical density was 1.88 ± 0.43, and for the negative samples, 0.71 ± 0.18. 3.3. Statistical analysis The cutoff point of the ELISA was determined by means of ROC curve, using the SigmaPlot software version 12.1, based on a panel of 85 sera (42 positive and 43 negative). Positive samples were those that presented OD > 1.1 (95% CI 0.9159–1.0), and the negative ones were those that presented OD < 1.1 (95% CI 0.9178–1.0). Fig. 1 shows the result of the dot plot demonstrating that there was no superposition in optical density of the populations that has been positive and negative in the gold standard when the previously defined cutoff value was adopted. Regarding to the test repeatability, it presented standard deviation ranging from 0.047 to 0.085 for positive sera from 0.010 to 0.043
A.F.C. Nassar et al. / Small Ruminant Research 116 (2014) 229–232
Fig. 1. Results of the indirect ELISA for relative sensitivity of the sera using the ROC curve.
for negative sera. Similarly, the variation coefficient ranged from 2.516 to 5.657 for positive from 1.256 to 8.6885 to negative sera. 4. Discussion Fontaine and Baird (2008) described that animals experimentally infected with C. pseudotuberculosis by subcutaneous route developed primary foci of infection in mediastinal lymph nodes and lungs. Menzies et al. (1994) stated that it is impossible to be sure that a population of negative animals is actually not infected and animals have never got in contact with the disease. On the recently work, besides the clinical and macroscopic findings suggestive of C. pseudotuberculosis infection, the samples were serially tested by microbiological culture and PCR, in order to overcome the limitations of classification in positive or negative populations. Nowadays, there are many ELISAs that use several antigens of the bacterium, such as bacterial cell wall and exotoxin (phospholipase D) our recombinant exotoxin (Binns et al., 2007; Chirino-Zárraga et al., 2009; Menzies et al., 1994). The indirect ELISA standardized in this study was performed with the use of the lysed cells supernatant, despite being a partially purified antigen, it may be detected C. pseudotuberculosis antibodies, presenting on 100% of sensitivity and specificity when it is compared to PCR and microbiology culture. In a study carried out by Dercksen et al. (2000), four ELISA were analyzed for the detection of antibodies in sheep, and the test that presented the best sensitivity (79 ± 5%) and specificity (99 ±1%) was the one that used the exotoxin (phospholipase D) as the antigen, which was described by Menzies et al. (1994). On the other hand, Kaba et al. (2001) standardized an ELISA with raw bacterial wall extract with sensitivity and specificity of 85% and 96%, respectively. Seyffert et al. (2010) carried out a study on the prevalence of the bacterium in Minas Gerais
231
region using the indirect ELISA with C. pseudotuberculosis raw antigen, and sensitivity and specificity were 93.5% and 100%, respectively. The results for sensitivity and specificity presented in the field under the curve for the two populations of the test, and also the repeatability enabled the complete differentiation of the populations of animals that were positive and negative in the standardized ELISA. The variation coefficients found inside the acceptable range were ≤15% (Crowther, 2009). The distinct sensitivity and specificity values described by different authors can be due different methodologies for selection of animals, either naturally of experimentally infected, latency period, or cross reaction with other similar microorganisms (Seyffert et al., 2010). The indirect ELISA reported here has advantages in the quick diagnosis of CLA in sheep when it is compared due to microbiological culture and PCR, in terms of sensitivity, specificity and time, it has given the simplicity of the test and antigen preparation, it’s automation, also the small amount of sample used. Moreover this methodology may be used in the diagnosis, in the trade of animals, and furthermore epidemiology studies in Brazil. References Alves, F.S.F., Pinheiro, R.R., 2002. Linfadenite Caseosa – Recomendac¸ões e Medidas Profiláticas. Agrop. Catarinense 13, 12–14. Baird, G.J., Fontaine, M.C., 2007. Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis. J. Comp. Pathol. 137, 179–210. Binns, S.H., Green, L.E., Bailey, M., 2007. Development and validation of ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera. Vet. Microbiol. 123, 169–179. Burrell, D.H., 1988. A simplified double immunodiffusion technique for detection of Corynebacterium ovis antitoxin. Res. Vet. Sci. 28, 234–237. Chirino-Zárraga, C., Rey-Valerion, C., Scaramelli, A., Carrero, L., 2009. Diagnosis of caseous lymphadenitis by ELISA in naturally infected goats from Venezuela. Small Rumin. Res. 87, 92–95. Crowther, J.R., 2009. Methods in molecular biology, the ELISA guidebook. In: Validation of Diagnostic Test for Infectious Diseases. Humana Press, Austria, pp. 291–334. Dercksen, D.P., Brinkhof, J.M.A., Dekker-Nooren, T., Van Maanem, K., Bode, C.F., Baird, G., Kamp, E.M., 2000. A comparison of four serological test for the diagnosis of caseous lymphadenitis in sheep and goats. Vet. Microbiol. 75, 167–175. Fontaine, M.C., Baird, G.J., 2008. Caseous lymphadenitis. Small Rumin. Res. 76, 42–48. Guimarães, A.S., Seyffert, N., Bastos, B.L., Portela, R.W.D., Meyer, R., Carmo, F.B., Cruz, J.C.M., Mcculloch, J.A., Lage, A.P., Heinemann, M.B., Miyoshi, A., Azevedo, V., Gouveia, A.M.G., 2009. Caseous lymphadenitis in sheep flocks of the State of Minas Gerais, Brazil: prevalence and management surveys. Small Rumin. Res. 87, 86–91. Kaba, J., Kutschke, L., Gerlach, G.F., 2001. Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infection in goats. Vet. Microbiol. 78, 155–163. Koneman, E.W., William, M.J., Schreckenberger, P.C., Winn, W.C., Allen, S.D., Woods, G.L., 2008. Diagnóstico microbiológico: texto e atlas colorido. In: Bacilos Gram-positivos Aeróbicos e Facultativos. Guanabara Koogan, Rio de Janeiro, pp. 760–851. Lloyd, S., Lindsay, H.J., Slater, J.D., Jackson, P.G.G., 1990. Corynebacterium pseudotuberculosis infection (caseous lymphadenitis) in goats. Goat Vet. Soc. 11, 55–65. Menzies, P.I., Muckle, C.A., Hwang, Y.T., Songer, G.J., 1994. Evaluation of an enzyme-linked immunosorbent assay using an Escherichia coli recombinant phospholipase D antigen for the diagnosis of Corynebacterium pseudotuberculosis infection. Small Rumin. Res. 13, 193–198. Pacheco, L.G.C., Pena, R.R., Castro, T.L.P., Dorella, F.A., Bahia, R.C., Carminati, R., Frota, M.N.L., Oliveira, S.C., Meyer, R., Alves, F.S.F., Miyoshi, A., Azevedo, V., 2007. Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid
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detection of this pathogen in clinical samples. J. Med. Microbiol. 59, 480–486. Quinn, P.J., Markey, B.K., Leonard, F.C., FitzPatrick, E.S., Fanning, S., Hartigan, P.J., 2011. Chapter 17, Corynebacterium species. In: Veterinary Microbiology and Microbial Disease, 2nd ed. Wiley-Blackwell, Oxford, UK, pp. 207–210.
Seyffert, N., Guimarães, A.S., Pacheco, L.G.C., Portela, R.W., Bastos, B.L., Dorella, F.A., Heinemann, M.B., Lage, A.P., Gouveia, A.M.G., Meyer, R., Miyoshi, A., Azevedo, V., 2010. High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA. Res. Vet. Sci. 88, 50–55.