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Diagnosis of caseous lymphadenitis by ELISA in naturally infected goats from Venezuela
Small Ruminant Research 87 (2009) 92–95
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Diagnosis of caseous lymphadenitis by ELISA in naturally infected goats from Venezuela Carmen Chirino-Zárraga a,∗ , Catalina Rey-Valeirón a , Aura Scaramelli b , Lilia Carrero a a Universidad Nacional Experimental Francisco de Miranda, Departamento de Sanidad Animal, Complejo Académico Ing. José Rodolfo Bastidas, Intercomunal Coro-La Vela, Coro, Estado Falcón, Venezuela b Universidad Central de Venezuela, Facultad de Ciencias Veterinarias, Maracay, Estado Aragua, Venezuela
a r t i c l e
i n f o
Article history: Received 14 January 2009 Received in revised form 23 September 2009 Accepted 29 September 2009 Available online 29 October 2009 Keywords: Corynebacterium pseudotuberculosis Goat ELISA Caseous lymphadenitis
a b s t r a c t Due to the absence of previous reports, the goal of this work was to detect caseous lymphadenitis (CLA) in goat flocks from Venezuela using an indirect immunoenzymatic assay (ELISA). Eighteen farms were randomly selected in Falcon State, North-Western Venezuela. Blood samples were taken from 259 goats, 65 of them with abscesses. Experimental inoculations were made to healthy kids with 0.5 mL inocula containing 4.7 × 105 of Corynebacterium pseudotuberculosis to observe the kinetics of antibody response. Immunoenzymatic assays were carried out using exotoxin of C. pseudotuberculosis as antigen. Antibody response in experimentally inoculated animals was detected 2 weeks after infection. Of 259 field goat sera, 55.98% were positive by ELISA. Of 65 goats with abscesses, 67.69% had CLA demonstrated by bacteriological methods; from these, 72.73% showed antibodies by ELISA. Of the remaining goats negative to CLA, 47.62% had antibodies by ELISA. Sensitivity was calculated in 72.73% and specificity in 67.74%. The immunoenzymatic assay applied in this research could be useful to detect CLA in naturally infected goat flocks from Venezuela. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Several serodiagnostic tests have been developed worldwide to overcome the problem of clinical identification of caseous lymphadenitis (CLA) in small ruminants with variable results (Dercksen et al., 2000; Dorella et al., 2006; Seyffert et al., 2009). Immunoenzymatic methods are invaluable tools to diagnose the disease in those regions were molecular methods are not available as in Venezuela. In this work, an indirect ELISA with Corynebacterium pseudotuberculosis autochthonous strains was used to detect CLA in Venezuelan goat flocks.
2.1. Sampling Miranda County (Falcon State, North-Western Venezuela) has a goat population of 67,528 heads, distributed in 423 farms (Secretaría de Desarrollo Agrícola, 2006). From these, 18 farms with a total of 2604 animals were selected at random. Blood samples for serological tests were collected of 194 animals of both sexes and several ages or breeds with no signs of the disease, and 65 goats with clinical CLA lesions, from which pus samples were also collected and tested by bacteriological methods. Data concerning animals (age, breed, and sex) were also recorded. 2.2. Bacteriological characterization of C. pseudotuberculosis Each pus sample from field and experimental animals was processed as in Chirino-Zárraga et al. (2006).
∗ Corresponding author. Fax: +58 268 2778446. E-mail addresses: [email protected] (C. Chirino-Zárraga), [email protected] (C. Rey-Valeirón). 0921-4488/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2009.09.031
2.3. Strains of C. pseudotuberculosis Strain C32B was isolated from a Nubian male goat, 3 years old, which present two closed abscesses in superficial cervical lymph nodes; refer-
C. Chirino-Zárraga et al. / Small Ruminant Research 87 (2009) 92–95 ence strain 19410 of C. pseudotuberculosis was acquired from American Type Culture Collection (ATCC), Atlanta, USA. C32B was grown in 5% bovine blood agar (Himedia, Mumbai, India) with a modification of ter Laak et al. (1992); after 48 h in 37 ◦ C and 5% CO2 (Jones and Collins, 1986), one colony was transferred to 5 mL nutrient broth (Himedia, Bombay, India) supplemented with 0.1% Tween 80 for 24 h. From this, 1 mL was inoculated in 50 mL nutrient broth–Tween 80 for 24 h more without agitation. The bacteria were successively diluted in sterile saline solution (0.85% NaCl) to obtain colony forming units (CFU). Reference strain was cultured as directed by ATCC.
2.4. Preparation of antigen for the ELISA Three colonies from each strain, previously incubated as above, were grown in 1 L of 0.1% Tween 80–BHI broth (BBL, Le Pont de Claix, France) for 72 h and then overnight at 4 ◦ C. The culture was centrifuged at 13,800 × g for 30 min at 10◦ C. Supernatants were collected and thymerosal was added to a final concentration of 1:10,000. The protein concentration of both C32B and ATCC 19410 supernatants (12.50 and 12.64 g/L, respectively) was estimated as in Lowry et al. (1951). Supernatants containing the exotoxin were stored in aliquots of 2 mL at −20 ◦ C for further use. C. pseudotuberculosis complete bacteria to be used as antigen in ELISA were obtained as explained in Section 2.3.
2.5. Experimental inoculation Four Nubian kids of 3–5 months old, born in a flock free of C. pseudotuberculosis, clinically healthy, were maintained in isolation facilities supplied with water, hay and grass ad libitum. Two of them (1 and 2) were inoculated with 0.5 mL of C. pseudotuberculosis (strain C32B) suspension containing 4.7 × 105 CFU by subcutaneous route in the right escapular region (week 0). Fifty-six days later (week 8) a second dose of 0.5 mL containing 5.3 × 108 CFU was injected subcutaneously in the right abdominal region of both animals as in ter Laak et al. (1992). Kids 3 and 4 were inoculated subcutaneously with 0.5 mL of sterile saline solution in the right escapular region and were maintained as negative controls. Sera were taken on 41, 21 and 0 days before inoculation, and weekly until day 105 (week 15) after inoculation with C. pseudotuberculosis. Samples of the abscesses were taken from kid C1 at weeks 5 and 9. Bacteriological characterization was made as in Chirino-Zárraga et al. (2006).
2.6. ELISA To standardize the conditions of ELISA, several concentrations of antigen (exotoxin and C. pseudotuberculosis suspensions) were tested. Sera from goats with CLA were used as positive controls. Sera from 1 to 5 weeks prior to inoculation were used as negative controls. Polystyrene plates (Cliniplats EB, Labsystem, USA) were coated with increasing concentrations of C32B or ATCC 19410 exotoxin or C. pseudotuberculosis suspension (6.25, 10, 20 and 40 g/mL) diluted in bicarbonate–carbonate buffer, pH 9.6 and incubated overnight at 4 ◦ C. Wells were washed three times with 200 L phosphate saline buffer (PBS) containing 0.1% Tween 20. Nonspecific binding sites were blocked with 0.5% gelatin for 1 h at 37 ◦ C. Positive and negative sera diluted 1:100 in PBS–0.1% Tween were added in duplicate. Following incubation for 1 h at 37 ◦ C, and washed again, wells were filled with 100 L of horseradish peroxidase anti-goat immunoglobulin conjugate (Sigma® cat. A-5420, USA) diluted 1:30,000 in PBS–Tween buffer. Optimal dilution of horseradish peroxidase anti-goat immunoglobulin conjugate was previously obtained against goat IgG (Sigma® cat. I-9140, USA). After incubation of 1 h at 37 ◦ C, conjugate solution was discarded and washed as before. Each well was then filled with 100 L of ABTS (2-2 -azino-di-(3-etil-benzothiazole-sulphone-6)-dianine) (Sigma® cat.A-1888, USA) in appropriate buffer. The plates were left at room temperature in darkness for 3 h on a shaker. Absorbance values were obtained and recorded by an ELISA plate reader (Awareness, Stat Fax 2100, USA) at 405 nm. Once optimal concentration of antigen was achieved, conditions for running test with field sampled sera were the same as explained above. Evaluation of experimental sera was made with C. pseudotuberculosis suspensions as antigen. The best positive/negative sera ratio was obtained with the strain C32B exotoxin concentration of 10 g/mL so it was used for screening field samples.
93
2.7. Estimation of the cut off point Ten sera from health experimental animals were used as negative controls. The cut off point was established as in Sheldrake et al. (1983). Using exotoxin, the cut off point of OD405 = 0.1475. With C. pseudotuberculosis suspensions as antigen and negative control sera, cut off point of OD405 = 0.427. Five sera were used as positive controls (three experimental from weeks 10, 11 and 14 post-infection and two sera of naturally infected goats, from which C. pseudotuberculosis had been previously isolated). Specificity and sensitivity were calculated as per Tarabla (2000). 2.8. Statistical analysis Chi-square test was used for analysis of age and breed differences and for comparison between ELISA and bacteriological results (Snedecor and Cochran, 1967). The level of significance applied to data was p < 0.05. 2.9. Interpretation of results The criteria taken for considering positive or negative animals to CLA were as follows: Bacteriological isolation
ELISA
Interpretation
+ + − −
+ − + −
Positive Positive Doubtful Negative
3. Results 3.1. Pathological observations through necropsy in experimentally inoculated animals An important inflammatory response was observed through the first week post-inoculation; at second week, acute symptoms developed toward a chronic supurative state in kid 1; in kid 2, at fifth week. One abscess in scapular subcutaneous tissue was found in kid 1. Overgrown right superficial cervical and subiliac lymph nodes (4 cm × 4 cm), with seven piogranulomatous processes around were found in the kid 2. Three abscesses were also observed in inoculation site. C. pseudotuberculosis was demonstrated in cultures of the overgrown lymph nodes and the abscesses found in inoculation site in both kids. 3.2. Selection of exotoxin as antigen in ELISA Exotoxin isolated from field C. pseudotuberculosis isolates was chosen due to a best positive:negative sera ratio (≥4) compared with those of ATCC exotoxin. ELISA with C. pseudotuberculosis antigen gave a lower positive:negative sera ratio (≤2). 3.3. Development of antibodies in experimentally inoculated animals Antibody response against C. pseudotuberculosis antigen was detected at 2 weeks post-inoculation in kids 1 and 2. Average values of absorbance in both kid sera ranked between 0.250 ± 0.064 and 0.595 ± 0.07 along the experimental trial. On week 9, one week after reinoculation, antibody response elevated in both animals and maintained until the end of the trial. Uninoculated animals (kids 3 and 4) did not develop antibody response against C. pseudotuberculosis antigen (Fig. 1).
94
C. Chirino-Zárraga et al. / Small Ruminant Research 87 (2009) 92–95
Fig. 1. Antibody response against Corynebacterium pseudotuberculosis in experimentally inoculated goats, measured by an indirect ELISA (——cut off). Table 1 Comparison between isolation of Corynebacterium pseudotuberculosis and results of ELISA. Isolation of C. pseudotuberculosisa
Positive Negativeb Total
n
44 21 65
+ ELISA
− ELISA
n
%
n
%
32 10 42
72.73 47.62 64.62
12 11 23
27.27 52.38 35.38
a
As confirmed by bacteriological methods. Positive to Arcanobacterium pyogenes, Pseudomonas spp., Micrococcus spp., Staphylococcus aureus, S. caprae, Nocardia brasiliensis. b
Fig. 2. 3.4. Detection of antibodies against exotoxin in naturally infected animals Of 259 animals, 145 (55.98%) had antibodies detectable by indirect ELISA. Of 65 animals with clinical signs of the disease, 44 (67.69%) had CLA as demonstrated by bacteriological methods. From these, 32 (72.73%) showed antibodies by this assay; of 21 animals negative to CLA, 10 (47.62%) were positive by ELISA, but other microorganisms were found in the abscesses (Table 1). Using exotoxin as antigen, sensitivity was calculated in 72.73% and specificity in 67.74 %. Seroprevalences were higher in animals aged more than 24 months (14/15 = 93.33% were >54 months old); 23.53% (4/17) were kids of less than 6 months (p < 0.05).Regarding
breed, 100% (7/7), 71.62% (53/74) and 41.11% (37/90) of positive animals were cross-bred Canarian, Nubian and Creole, respectively (p < 0.05). 4. Discussion As in our results, several authors (Pépin et al., 1993; Paule et al., 2003 and others) reported enlargement of superficial lymph nodes as well as several abscesses in the inoculation sites. The antibody response increased evidently after reinoculation, which demonstrates that repeated stimuli or a chronic disease make easy the detection by immunological methods. Holstad and Teige (1988) suggested that antibodies could not avoid the establishment of bacteria in other organs and could have little impact on the recovery of the host. It is significant that 55.98% of the surveyed animals had antibodies against C. pseudotuberculosis. But, a higher prevalence (72.6%) was found in Southeast Brazil, detected by a C. pseudotuberculosis secreted proteins-based ELISA (Seyffert et al., 2009). Different levels of sensitivity and specificity using C. pseudotuberculosis cell walls or exotoxin as antigen have been obtained (ter Laak et al., 1992; Binns et al., 2007), but it seems that an ELISA against exotoxin or secreted antigens give better results (ter Laak et al., 1992; Seyffert et al., 2009). The best results were obtained in this work with exotoxin as antigen. The use of ELISA with C. pseudotuberculosis antigen to detect antibodies in field sera was discarded due
Fig. 2. Number of naturally infected goats with antibodies against Corynebacterium pseudotuberculosis exotoxin, measured by an indirect ELISA. (——cut off).
C. Chirino-Zárraga et al. / Small Ruminant Research 87 (2009) 92–95
to a lower positive:negative sera ratio. In this work, it was found a sensitivity of 72.73% and a specificity of 67.74%. It should not be discarded cross reactions with other bacteria found in abscesses as shown in Table 1. Dercksen et al. (2000) and Kaba et al. (2001), also using C. pseudotuberculosis exotoxin in an indirect ELISA, reported lower values of sensitivity but a higher specificity if compared with these results. As Chirino-Zárraga et al. (2006) reported bacteriological methods, a higher frequency of antibodies against C. pseudotuberculosis in goats older than 24 months was observed. Few young animals were seropositive. Because CLA is a chronic disease, animals under 6 months probably had no time to be infected or develop a detectable immune response. Similarly, in Brazilian goat flocks the frequency of animals who tested positive was low in goats less than 1 year old but significantly higher in goats aged 1–2 years old (Seyffert et al., 2009). 5. Conclusion ELISA using exotoxin is a reliable method for detecting CLA in goat flocks, if it is compared with low sensitivity, costs and time-consuming of a bacteriological methods, but it has to be improved to reduce cross reactions with other pathogens. Furthermore, CLA should be precisely diagnosed in those places of Venezuela where goat breeding is the main way of living. Acknowledgments The authors wish to thank Universidad Francisco de Miranda (CI-2001-005), FONACIT-Venezuela (PEM 2001001623), Program for Graduates in Animal Health (PIPSA), and the anonymous reviewers who helped to improve this manuscript. References Binns, S.H., Green, L.E., Bailey, M., 2007. Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera. Vet. Microbiol. (127), 169–179.
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Chirino-Zárraga, C., Scaramelli, A., Rey-Valeirón, C., 2006. Bacteriological characterization of Corynebacterium pseudotuberculosis in Venezuelan goat flocks. Small Rumin. Res. (65), 170–175. Dercksen, D.P., Brinkhof, J.M., Dekker-Nooren, T., Maanen, K., Bode, C.F., Bair, G., Kamp, E.M., 2000. A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats. Vet. Microbiol. (75), 167–175. Dorella, F.A., Carvalho, L.G., Costa, S., Miyoshi, A., Azevedo, V., 2006. Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of virulence. Vet. Res. (37), 201–218. Holstad, G., Teige, J., 1988. Corynebacterium pseudotuberculosis in goats. VII. Clinical, pathological, serological and hematological changes after subcutaneous inoculation of the organism. Acta Vet. Scan. (29), 287–294. Jones, D., Collins, M.D., 1986. Irregular, nonsporing Gram positive rods. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology. Williams & Wilkins, Co., Baltimore, MD, pp. 1261–1276. Kaba, J., Kutschke, L., Gerlach, G.F., 2001. Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infections in goats. Vet. Microbiol. 78, 155–163. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin-Phenol reagent. J. Biol. Chem. (193), 265–275. Paule, B.J., Azevedo, V., Regis, L., Carminati, R., Bahia, R., Vale, V., MouraCosta, L.F., Freire, S., Nascimento, I., Schaer, R., Goes, A.M., Meyer, R., 2003. Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-␥ production, IgG avidity and antigen recognition by Western blotting. Vet. Immunol. Immunopathol. (96), 129–2139. Pépin, M., Pardon, P., Marly, J., Lantier, F., Arrigo, J.L., 1993. Acquired immunity after primary caseous lymphadenitis in sheep. Am. J. Vet. Res. (54), 873–877. Secretaría de Desarrollo Agrícola, 2006. Censo Regional de Ovinos y Caprinos. In: Resultados preliminaries (I Regional Survey on goat and sheep, Preliminary Results). Gobernación del estado Falcón, Santa Ana de Coro, Venezuela. 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., Meer, R., Miyoshi, A., Azevedo, V., 2009. High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA. Res. Vet. Sci., doi:10.1016/j.rvsc.2009.07.002. Sheldrake, R.R., McGregor, G.D., Hoare, R.J.T., 1983. Somatic cell count, electrical conductivity, and serum albumin concentration for detection of bovine mastitis. J. Dairy Sci. (66), 548–555. Snedecor, G.W., Cochran, W.G., 1967. Statistical Methods, Sixth Edition. The Iowa State University Press, Iowa, p. 593. Tarabla, H.D., 2000. Epidemiología Diagnóstica (Diagnostic Epidemiology). Ctro. Pub. Univ. Nac. del Litoral, Santa Fe, Argentina, 120 pp. ter Laak, E., Bosch, J., Bijl, G., Shcreuder, B., 1992. Double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot analysis used for control of caseous lymphadenitis in goats and sheep. Am. J. Vet. Res. (53), 1125–1132.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Diagnosis of caseous lymphadenitis by ELISA in naturally infected goats from Venezuela Carmen Chirino-Zárraga a,∗ , Catalina Rey-Valeirón a , Aura Scaramelli b , Lilia Carrero a a Universidad Nacional Experimental Francisco de Miranda, Departamento de Sanidad Animal, Complejo Académico Ing. José Rodolfo Bastidas, Intercomunal Coro-La Vela, Coro, Estado Falcón, Venezuela b Universidad Central de Venezuela, Facultad de Ciencias Veterinarias, Maracay, Estado Aragua, Venezuela
a r t i c l e
i n f o
Article history: Received 14 January 2009 Received in revised form 23 September 2009 Accepted 29 September 2009 Available online 29 October 2009 Keywords: Corynebacterium pseudotuberculosis Goat ELISA Caseous lymphadenitis
a b s t r a c t Due to the absence of previous reports, the goal of this work was to detect caseous lymphadenitis (CLA) in goat flocks from Venezuela using an indirect immunoenzymatic assay (ELISA). Eighteen farms were randomly selected in Falcon State, North-Western Venezuela. Blood samples were taken from 259 goats, 65 of them with abscesses. Experimental inoculations were made to healthy kids with 0.5 mL inocula containing 4.7 × 105 of Corynebacterium pseudotuberculosis to observe the kinetics of antibody response. Immunoenzymatic assays were carried out using exotoxin of C. pseudotuberculosis as antigen. Antibody response in experimentally inoculated animals was detected 2 weeks after infection. Of 259 field goat sera, 55.98% were positive by ELISA. Of 65 goats with abscesses, 67.69% had CLA demonstrated by bacteriological methods; from these, 72.73% showed antibodies by ELISA. Of the remaining goats negative to CLA, 47.62% had antibodies by ELISA. Sensitivity was calculated in 72.73% and specificity in 67.74%. The immunoenzymatic assay applied in this research could be useful to detect CLA in naturally infected goat flocks from Venezuela. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Several serodiagnostic tests have been developed worldwide to overcome the problem of clinical identification of caseous lymphadenitis (CLA) in small ruminants with variable results (Dercksen et al., 2000; Dorella et al., 2006; Seyffert et al., 2009). Immunoenzymatic methods are invaluable tools to diagnose the disease in those regions were molecular methods are not available as in Venezuela. In this work, an indirect ELISA with Corynebacterium pseudotuberculosis autochthonous strains was used to detect CLA in Venezuelan goat flocks.
2.1. Sampling Miranda County (Falcon State, North-Western Venezuela) has a goat population of 67,528 heads, distributed in 423 farms (Secretaría de Desarrollo Agrícola, 2006). From these, 18 farms with a total of 2604 animals were selected at random. Blood samples for serological tests were collected of 194 animals of both sexes and several ages or breeds with no signs of the disease, and 65 goats with clinical CLA lesions, from which pus samples were also collected and tested by bacteriological methods. Data concerning animals (age, breed, and sex) were also recorded. 2.2. Bacteriological characterization of C. pseudotuberculosis Each pus sample from field and experimental animals was processed as in Chirino-Zárraga et al. (2006).
∗ Corresponding author. Fax: +58 268 2778446. E-mail addresses: [email protected] (C. Chirino-Zárraga), [email protected] (C. Rey-Valeirón). 0921-4488/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2009.09.031
2.3. Strains of C. pseudotuberculosis Strain C32B was isolated from a Nubian male goat, 3 years old, which present two closed abscesses in superficial cervical lymph nodes; refer-
C. Chirino-Zárraga et al. / Small Ruminant Research 87 (2009) 92–95 ence strain 19410 of C. pseudotuberculosis was acquired from American Type Culture Collection (ATCC), Atlanta, USA. C32B was grown in 5% bovine blood agar (Himedia, Mumbai, India) with a modification of ter Laak et al. (1992); after 48 h in 37 ◦ C and 5% CO2 (Jones and Collins, 1986), one colony was transferred to 5 mL nutrient broth (Himedia, Bombay, India) supplemented with 0.1% Tween 80 for 24 h. From this, 1 mL was inoculated in 50 mL nutrient broth–Tween 80 for 24 h more without agitation. The bacteria were successively diluted in sterile saline solution (0.85% NaCl) to obtain colony forming units (CFU). Reference strain was cultured as directed by ATCC.
2.4. Preparation of antigen for the ELISA Three colonies from each strain, previously incubated as above, were grown in 1 L of 0.1% Tween 80–BHI broth (BBL, Le Pont de Claix, France) for 72 h and then overnight at 4 ◦ C. The culture was centrifuged at 13,800 × g for 30 min at 10◦ C. Supernatants were collected and thymerosal was added to a final concentration of 1:10,000. The protein concentration of both C32B and ATCC 19410 supernatants (12.50 and 12.64 g/L, respectively) was estimated as in Lowry et al. (1951). Supernatants containing the exotoxin were stored in aliquots of 2 mL at −20 ◦ C for further use. C. pseudotuberculosis complete bacteria to be used as antigen in ELISA were obtained as explained in Section 2.3.
2.5. Experimental inoculation Four Nubian kids of 3–5 months old, born in a flock free of C. pseudotuberculosis, clinically healthy, were maintained in isolation facilities supplied with water, hay and grass ad libitum. Two of them (1 and 2) were inoculated with 0.5 mL of C. pseudotuberculosis (strain C32B) suspension containing 4.7 × 105 CFU by subcutaneous route in the right escapular region (week 0). Fifty-six days later (week 8) a second dose of 0.5 mL containing 5.3 × 108 CFU was injected subcutaneously in the right abdominal region of both animals as in ter Laak et al. (1992). Kids 3 and 4 were inoculated subcutaneously with 0.5 mL of sterile saline solution in the right escapular region and were maintained as negative controls. Sera were taken on 41, 21 and 0 days before inoculation, and weekly until day 105 (week 15) after inoculation with C. pseudotuberculosis. Samples of the abscesses were taken from kid C1 at weeks 5 and 9. Bacteriological characterization was made as in Chirino-Zárraga et al. (2006).
2.6. ELISA To standardize the conditions of ELISA, several concentrations of antigen (exotoxin and C. pseudotuberculosis suspensions) were tested. Sera from goats with CLA were used as positive controls. Sera from 1 to 5 weeks prior to inoculation were used as negative controls. Polystyrene plates (Cliniplats EB, Labsystem, USA) were coated with increasing concentrations of C32B or ATCC 19410 exotoxin or C. pseudotuberculosis suspension (6.25, 10, 20 and 40 g/mL) diluted in bicarbonate–carbonate buffer, pH 9.6 and incubated overnight at 4 ◦ C. Wells were washed three times with 200 L phosphate saline buffer (PBS) containing 0.1% Tween 20. Nonspecific binding sites were blocked with 0.5% gelatin for 1 h at 37 ◦ C. Positive and negative sera diluted 1:100 in PBS–0.1% Tween were added in duplicate. Following incubation for 1 h at 37 ◦ C, and washed again, wells were filled with 100 L of horseradish peroxidase anti-goat immunoglobulin conjugate (Sigma® cat. A-5420, USA) diluted 1:30,000 in PBS–Tween buffer. Optimal dilution of horseradish peroxidase anti-goat immunoglobulin conjugate was previously obtained against goat IgG (Sigma® cat. I-9140, USA). After incubation of 1 h at 37 ◦ C, conjugate solution was discarded and washed as before. Each well was then filled with 100 L of ABTS (2-2 -azino-di-(3-etil-benzothiazole-sulphone-6)-dianine) (Sigma® cat.A-1888, USA) in appropriate buffer. The plates were left at room temperature in darkness for 3 h on a shaker. Absorbance values were obtained and recorded by an ELISA plate reader (Awareness, Stat Fax 2100, USA) at 405 nm. Once optimal concentration of antigen was achieved, conditions for running test with field sampled sera were the same as explained above. Evaluation of experimental sera was made with C. pseudotuberculosis suspensions as antigen. The best positive/negative sera ratio was obtained with the strain C32B exotoxin concentration of 10 g/mL so it was used for screening field samples.
93
2.7. Estimation of the cut off point Ten sera from health experimental animals were used as negative controls. The cut off point was established as in Sheldrake et al. (1983). Using exotoxin, the cut off point of OD405 = 0.1475. With C. pseudotuberculosis suspensions as antigen and negative control sera, cut off point of OD405 = 0.427. Five sera were used as positive controls (three experimental from weeks 10, 11 and 14 post-infection and two sera of naturally infected goats, from which C. pseudotuberculosis had been previously isolated). Specificity and sensitivity were calculated as per Tarabla (2000). 2.8. Statistical analysis Chi-square test was used for analysis of age and breed differences and for comparison between ELISA and bacteriological results (Snedecor and Cochran, 1967). The level of significance applied to data was p < 0.05. 2.9. Interpretation of results The criteria taken for considering positive or negative animals to CLA were as follows: Bacteriological isolation
ELISA
Interpretation
+ + − −
+ − + −
Positive Positive Doubtful Negative
3. Results 3.1. Pathological observations through necropsy in experimentally inoculated animals An important inflammatory response was observed through the first week post-inoculation; at second week, acute symptoms developed toward a chronic supurative state in kid 1; in kid 2, at fifth week. One abscess in scapular subcutaneous tissue was found in kid 1. Overgrown right superficial cervical and subiliac lymph nodes (4 cm × 4 cm), with seven piogranulomatous processes around were found in the kid 2. Three abscesses were also observed in inoculation site. C. pseudotuberculosis was demonstrated in cultures of the overgrown lymph nodes and the abscesses found in inoculation site in both kids. 3.2. Selection of exotoxin as antigen in ELISA Exotoxin isolated from field C. pseudotuberculosis isolates was chosen due to a best positive:negative sera ratio (≥4) compared with those of ATCC exotoxin. ELISA with C. pseudotuberculosis antigen gave a lower positive:negative sera ratio (≤2). 3.3. Development of antibodies in experimentally inoculated animals Antibody response against C. pseudotuberculosis antigen was detected at 2 weeks post-inoculation in kids 1 and 2. Average values of absorbance in both kid sera ranked between 0.250 ± 0.064 and 0.595 ± 0.07 along the experimental trial. On week 9, one week after reinoculation, antibody response elevated in both animals and maintained until the end of the trial. Uninoculated animals (kids 3 and 4) did not develop antibody response against C. pseudotuberculosis antigen (Fig. 1).
94
C. Chirino-Zárraga et al. / Small Ruminant Research 87 (2009) 92–95
Fig. 1. Antibody response against Corynebacterium pseudotuberculosis in experimentally inoculated goats, measured by an indirect ELISA (——cut off). Table 1 Comparison between isolation of Corynebacterium pseudotuberculosis and results of ELISA. Isolation of C. pseudotuberculosisa
Positive Negativeb Total
n
44 21 65
+ ELISA
− ELISA
n
%
n
%
32 10 42
72.73 47.62 64.62
12 11 23
27.27 52.38 35.38
a
As confirmed by bacteriological methods. Positive to Arcanobacterium pyogenes, Pseudomonas spp., Micrococcus spp., Staphylococcus aureus, S. caprae, Nocardia brasiliensis. b
Fig. 2. 3.4. Detection of antibodies against exotoxin in naturally infected animals Of 259 animals, 145 (55.98%) had antibodies detectable by indirect ELISA. Of 65 animals with clinical signs of the disease, 44 (67.69%) had CLA as demonstrated by bacteriological methods. From these, 32 (72.73%) showed antibodies by this assay; of 21 animals negative to CLA, 10 (47.62%) were positive by ELISA, but other microorganisms were found in the abscesses (Table 1). Using exotoxin as antigen, sensitivity was calculated in 72.73% and specificity in 67.74 %. Seroprevalences were higher in animals aged more than 24 months (14/15 = 93.33% were >54 months old); 23.53% (4/17) were kids of less than 6 months (p < 0.05).Regarding
breed, 100% (7/7), 71.62% (53/74) and 41.11% (37/90) of positive animals were cross-bred Canarian, Nubian and Creole, respectively (p < 0.05). 4. Discussion As in our results, several authors (Pépin et al., 1993; Paule et al., 2003 and others) reported enlargement of superficial lymph nodes as well as several abscesses in the inoculation sites. The antibody response increased evidently after reinoculation, which demonstrates that repeated stimuli or a chronic disease make easy the detection by immunological methods. Holstad and Teige (1988) suggested that antibodies could not avoid the establishment of bacteria in other organs and could have little impact on the recovery of the host. It is significant that 55.98% of the surveyed animals had antibodies against C. pseudotuberculosis. But, a higher prevalence (72.6%) was found in Southeast Brazil, detected by a C. pseudotuberculosis secreted proteins-based ELISA (Seyffert et al., 2009). Different levels of sensitivity and specificity using C. pseudotuberculosis cell walls or exotoxin as antigen have been obtained (ter Laak et al., 1992; Binns et al., 2007), but it seems that an ELISA against exotoxin or secreted antigens give better results (ter Laak et al., 1992; Seyffert et al., 2009). The best results were obtained in this work with exotoxin as antigen. The use of ELISA with C. pseudotuberculosis antigen to detect antibodies in field sera was discarded due
Fig. 2. Number of naturally infected goats with antibodies against Corynebacterium pseudotuberculosis exotoxin, measured by an indirect ELISA. (——cut off).
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to a lower positive:negative sera ratio. In this work, it was found a sensitivity of 72.73% and a specificity of 67.74%. It should not be discarded cross reactions with other bacteria found in abscesses as shown in Table 1. Dercksen et al. (2000) and Kaba et al. (2001), also using C. pseudotuberculosis exotoxin in an indirect ELISA, reported lower values of sensitivity but a higher specificity if compared with these results. As Chirino-Zárraga et al. (2006) reported bacteriological methods, a higher frequency of antibodies against C. pseudotuberculosis in goats older than 24 months was observed. Few young animals were seropositive. Because CLA is a chronic disease, animals under 6 months probably had no time to be infected or develop a detectable immune response. Similarly, in Brazilian goat flocks the frequency of animals who tested positive was low in goats less than 1 year old but significantly higher in goats aged 1–2 years old (Seyffert et al., 2009). 5. Conclusion ELISA using exotoxin is a reliable method for detecting CLA in goat flocks, if it is compared with low sensitivity, costs and time-consuming of a bacteriological methods, but it has to be improved to reduce cross reactions with other pathogens. Furthermore, CLA should be precisely diagnosed in those places of Venezuela where goat breeding is the main way of living. Acknowledgments The authors wish to thank Universidad Francisco de Miranda (CI-2001-005), FONACIT-Venezuela (PEM 2001001623), Program for Graduates in Animal Health (PIPSA), and the anonymous reviewers who helped to improve this manuscript. References Binns, S.H., Green, L.E., Bailey, M., 2007. Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera. Vet. Microbiol. (127), 169–179.
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