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Research Article - (2013) Volume 4, Issue 3

Subclinical Diagnosis of Caseous Lymphadenitis Based on ELISA in Sheep from Brazil

Dayana Ribeiro1, Fernanda Alves Dorella2, Luis Gustavo Carvalho Pacheco3, Núbia Seyffert2, Thiago Luiz De Paula Castro2, Ricardo Wagner Dias Portela3, Roberto Meyer3, Anderson Miyoshi2, Maria Cecília Rui Luvizotto4 and Vasco Azevedo1,2*
1Laboratório de Genética Celular e Molecular, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
2Laboratório de Genética Celular e Molecular, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
3Laboratório de Imunologia e Biologia Molecular, Departamento de Bio-Interação, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Brazil
4Laboratório de Patologia Veterinária, Departamento de Clínica, Cirurgia e Reprodução Animal, Universidade do Estado de São Paulo, Araçatuba, Brazil
*Corresponding Author: Vasco Azevedo, Laboratório de Genética Celular e Molecular, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, Tel: + 00 5531 3409-2610, Fax: 55-31- 34092610 Email:


Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is a chronic contagious disease that affects small ruminants and still remains an important problem for many lamb-producing countries. Animals are considered clinically infected when occurs abscesses in superficial lymph nodes. Visceral or internal form can coexist which no apparent clinical signs of infection are seen. The best procedure to avoid spread of the disease is elimination of infected animals. However, as the chronic and subclinical nature of the infection of CLA alternative methods are required for detection and screening. In this study, we described the performance of indirect Enzyme-Linked Immunosorbent Assay (ELISA) for diagnosis of CLA in asymptomatics sheep. Also, test culture and biochemical identification were achieved to confirm CLA infection. The serological diagnostic was performed in sheep symptomatics (n=50) and asymptomatics (n=374) from nine flocks. Analysis reported high positivity of 71% for ELISA in 85% of asymptomatic animal for CLA with a sensitivity of 88% and specificity of 31%. Results from ELISA test in asymptomatic animals against culture for caseous lymphadenitis were more specific (97%) and permitted to exclude healthy animals without symptoms. This study concluded that C. pseudotuberculosis infection could be widely disseminated in sheep flocks in Northwestern region of the state of São Paulo, Brazil and only one screening test is not enough. The association with indirect ELISA test and culture could better indicate the real problem of CLA in sheep flocks.

Keywords: Caseous lymphadenitis; Corynebacterium pseudotuberculosis; Subclinical diagnostic; ELISA


Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is a chronic contagious disease that affects the majority of sheep-rearing areas worldwide [1-4]. The identification of infected animals, with or without clinical symptoms, is the best procedure to avoid spread and to control this disease. This identification can be realized firstly by clinical exams in which the presences of abscesses in superficial lymph nodes are observed in several animals in a group. Other bacterial pathogens are capable of producing suppurative lymphadenopathy but they tend to be sporadic in nature and are not seen as a flock problem [1].

Visceral or internal form can coexist in asymptomatic infections without any apparent clinical signs of infection, making it difficult to be diagnosed [1,4-6].

The classical or gold standard diagnosis of CLA is represented by the culture test and the identification of C. pseudotuberculosis from abscesses. This classical diagnosis may not always be advantageous or possible since chronic external lesions with little pus containing few viable organisms or visceral lesions cannot be sampled [1]. In such case, various diagnostic techniques have been developed. Enzyme-linked Immunosorbent Assay (ELISA) has already proven to be a versatile method to aid in CLA-control from herds and to identify sheep with visceral form without appealing to bacteriology resources [7-11].

Indirect ELISA test for the detection of anti-bodies against C. pseudotuberculosis secreted proteins is being performed in seroepidemiological surveys for CLA in Brazil. A high seroprevalence of this disease has been detected by using applying method either among goat (78.9%) or sheep herds (70.9%) from Minas Gerais state, despite the absence of external clinical signs of CLA in most of them (89% to 82.5%) [12,13].

Clinical examination and regular ELISA testing were able to halt the appearance of new clinical cases of CLA. Newly purchased sheep with CLA infection history should be submitted to physical examination before their introduction into naïve flocks, followed by an immediate culling of the clinically affected sheep. However, subclinical infection requests more efforts to detect and control the disease [14].

The challenge of diagnosing CLA in subclinical infections highlights the necessity for rapid, practical and available diagnostic test for CLA infection. The purpose of this study was to describe the performance of the indirect ELISA to detect CLA in asymptomatic sheep from Northwestern region of the state of São Paulo, Brazil. Additionally, bacterial culture and biochemical identification were realized in samples from asymptomatic animals and positive at indirect ELISA test to confirm CLA infection.

Materials and Methods


Samples were collected from 424 sheep during the months of June and July 2007, from nine farms located in the Northwestern region of the state of São Paulo, Brazil.

The inclusion of sheep flocks in this study required the existence of apparently healthy animals with heterogeneous distribution of age, male and female, breeds and husbandry methods.

Each animal was clinically examined and classified as symptomatic or asymptomatic for CLA. Animals were considered asymptomatic when apparently healthy, without any abscess, and which could be living or not with sheep presenting clinical signs of CLA.

Bacteriological and serum samples

Bacteriological samples were collected by Fine Needle Aspiration Biopsy (FNAB). Following sterilization of the skin, FNAB was performed preferentially in the right prescapular lymph node of the animals, using a 23 G or 25 G needle and a 10 ml syringe coupled to a cytoaspirator. A single aspirate was performed, using more than 5 ml suction. Needle and syringe were transported as a unit and kept on ice until their storage at-20°C.

Blood samples were taken using serum collection tubes and 21 G needle (BD Vacutainer Blood Collection Serum Tubes and Eclipse™ Blood Collection Needle 21 G, Becton-Dickinson, Franklin Lakes, New Jersey). Serum samples were separated by centrifugation (800g/ 10 min) and stored at-20°C.

Enzyme linked immunosorbent assay (ELISA)

An indirect ELISA was performed to identify total immunoglobulins directed towards C. pseudotuberculosis secreted antigens in sheep sera, as previously described [8,13]. Briefly, 96-well microplates (Nunc MaxiSorp, eBiosciense, San Diego, California) were coated with 100 μl per well of the supernatant of a 48-hour C. pseudotuberculosis culture in Brain Heart Infusion (BHI) broth, and then 1:100 diluted sheep serum samples were assayed in duplicate. Appropriate positive and negative control samples were included in all plates. Reads were taken in a microplate reader (Bio-Rad 550 Microplate Reader, Bio-Rad Laboratories, Hercules, California) at 490 nm.

The cut-off point to define seropositivity was previously described [13]. Therefore, it was calculated as the mean absorbance at OD490 nm observed for all truly negative sera (n=150), plus three standard deviations (OD490nm=0.32) [13,15]. Mean values for positive, negative, and blank control samples were 1.38, 0.15 and 0.05, respectively.

Bacterial culture and biochemical identification

The material in the needle and syringe of each collected sample was washed with 400 μl of sterile water and stored at -80°C with cryopreservation for microbiological examination. Twenty μl of each sample were spread on BHI agar plates and incubated at 37°C for 48 h. C. pseudotuberculosis-like colonies (yellowish, dry, with irregular borders) that stained Gram-positive were tested further for biochemical properties.

The ability of C. pseudotuberculosis to ferment carbohydrates was evaluated in basic Cystine Tryptic Agar medium containing 1% dehydrated carbohydrates, including glucose, sucrose, maltose, mannose, mannitol, xylose, and ribose. The isolates were also evaluated for catalase and urease production, nitrate reduction and alkaline phosphatase activity, along with motility and H2S production [15].

Synergistic haemolysis with Rhodococcus equi (ATCC 33701) and inhibition of β-haemolysis by Staphylococcus aureus (ATCC 25923) were also evaluated [15].

Statistical analysis

Statistical analysis was done with nonparametric statistics by entering frequencies into 2×2 tables. Thus, Chi-square Test determined the association between indirect ELISA test and clinical exam information (symptomatic and asymptomatic). Sensitivity, specificity, predictive values and accuracy were calculated with positive and negative controls. The degree of concordance among the diagnostic exams was measured by the Kappa coefficient. The alpha level for significance was 5%. The statistical analyses were made with the program OpenEpi version 2.3.


This study evaluated the diagnostic performance of indirect ELISA method to detect CLA infection by secreted antigens in sheep sera from nine flocks, which were realized in association with clinical inspection. For this purpose, serum samples (n=424) were used in the test.

The distribution of the analyzed samples had mainly female (n=388), ages ranged from 1 to 12 months and originating from 3 kinds of breeds, as shown in Table 1.

Variable Group Positive(n) Positive (%) Total Tested (n)
Sex Females 288 96% 388
Males 13 4% 36
Breed Santa Inês 172 57% 232
Suffolk 15 5% 18
Mixed 114 38% 174
Age (months) 1–12 41 14% 89
13–24 209 69% 282
25–36 51 17% 53
Clinical Symptom Asymptomatic 257 85% 374
  Symptomatic 44 15% 50

Table 1: Distribution of results based on ELISA for C. pseudotuberculosis in Northwestern region of the state of São Paulo, Brazil.

CLA lesions in symptomatic sheep were most commonly observed in the prescapular lymph nodes right and left, followed by right parotid lymph node, right popliteal lymph node and right crural lymph node. The frequency of positive and negative animals from each farm, according to indirect ELISA Test, was represented in Figure 1.


Figure 1: Distribution of positive and negative animals by ELISA from sheep farms in Northwestern region of the state of São Paulo, Brazil.

A 2×2 table was used to evaluate the association between clinical inspection (symptomatic and asymptomatic) and indirect ELISA test results (Table 2), from a total of 424 sheep clinically examined, where 50 were symptomatic and 374 asymptomatic (Table 1). According to results shown in Table 2, indirect ELISA test showed 88% (95% CI: 76- 94%) of sensitivity and 31% (95% CI: 27-36%) of specificity.

    Symptomatic Asymptomatic Total
ELISA + 44 257 301
- 6 117 123
  Total 50 374 424
Chi square Test (p=0.002)

Table 2: Association of results obtained from indirect ELISA based on clinical examination.

In order to evaluate the veracity of the results obtained from indirect ELISA test and clinical exams, regarding asymptomatic and false positive animals (n=257), bacterial culture and biochemical identification were realized in samples collected by FNAB procedure (n=113). The performance of the screening procedure (indirect ELISA test) against a gold standard (Culture test) was analyzed in a 2×2 table (Table 3).

According to the obtained results (Table 3), by combining two diagnostic methods (Culture vs. indirect ELISA test), it was confirmed that 2 animals were truly positive and 31 were truly negative resulting in a 97% of specificity (95% CI: 84-99%).

Parameter Estimate Lower - Upper 95% CIs
Sensitivity 2.469% (0.6797, 8.5631)
Specificity 96.88% (84.26, 99.451)
Positive Predictive Value 66.67% (20.77, 93.851)
Negative Predictive Value 28.18% (20.62, 37.221)
Diagnostic Accuracy 29.2% (21.61, 38.161)
Cohen’s kappa -0.0037 (-0.04164 - 0.03409)

Table 3: Performance of results from ELISA test in asymptomatic animals against culture for caseous lymphadenitis.


Corynebacterium pseudotuberculosis infection is widely disseminated in small ruminants’ worldwide [2]. The present study identified the high widespread of CLA and its possible presence in all sheep herd tested in Northwestern region of the state of São Paulo, Brazil. The higher seropositivity was predominant in adults (13 to 24 months old), females and asymptomatic sheep. CLA was found ranging from 35% to 98% of seropositivity.

Curiously, even though great sanitary and zootechnical procedures were observed in two of nine farms (number 4 and 6, Figure 1), they showed the higher level of positivity. These herds were semi-intensive to intensive husbandry system, composed mainly by national type of race (Santa Inês) and the owners had enough knowledge about clinical occurrence of CLA. However, recently-acquired animals were not isolated from the others neither tested to CLA. Thus, the early identification is crucial of control and eradication programs, as described in previous epidemiological studies [9,10,13].

Some researchers have been reporting strategies to control and reduce the incidence of CLA in affected flocks. The most significant effects were seen when ELISA was regular used in association with clinical inspections in affected flocks from the United Kingdom [16]. These serological tests have been used as a research tool in others countries, as Brazil. However, such programs may be hindered by the difficulties associated with detection of subclinically infected animals [4,13,17]. Therefore, in the present study, we employed the indirect ELISA for detecting humoral responses in asymptomatics (n=374) and symptomatics (n=50) sheep. The higher sensitivity of 88% and low specificity of 31% presented 257 samples as false positive with 71% of seropositivity.

Although these results suggested that ELISA remains a good alternative to CLA-screening programs, it is questionable whether only one diagnostic test should be employed to drive CLA eradication programs [4,18]. Several factors can interfere with the interpretation of ELISAs, specifically: (i) the nature of the circulating antibodies against C. pseudotuberculosis, which may decline during periods of pathogen dormancy, resulting in high numbers of false-negatives; and most importantly, (ii) the inability to distinguish between previously exposed animals and those still harboring the pathogen [1,9,10].

In order to confirm ELISA results and to better understand the real state of CLA infection in asymptomatic sheep, we included bacterial culture and biochemical identification in our analysis. Then, we had a new specificity of 97% with only 2% of prevalence. Based on our findings, culture and indirect ELISA test completed each other and would be a great alternative to confirm or exclude CLA infection. We believe that this association could better translate the real prevalence of properties affected and the necessity to control CLA spread in Brazil.


We are grateful to the farmers who helped us to acquire the samples for this study. This work was financial supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil) and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brasil).


  1. Baird GJ, Fontaine MC (2007) Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis. J Comp Pathol 137: 179-210.
  2. Dorella FA, Pacheco LG, Oliveira SC, Miyoshi A, Azevedo V (2006) Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of virulence. Vet Res 37: 201-218.
  3. Paton MW, Walker SB, Rose IR, Watt GF (2003) Prevalence of caseous lymphadenitis and usage of caseous lymphadenitis vaccines in sheep flocks. Aust Vet J 81: 91-95.
  4. Williamson LH (2001) Caseous lymphadenitis in small ruminants. Vet Clin North Am Food Anim Pract 17: 359-371.
  5. Jesse FFA, Sang SL, Saharee AA, Shahirudin S (2011) Pathological changes in the organs of mice model inoculated with Corynebacterium pseudotuberculosis organism. Pertanika Journal of Tropical Agricultural Science 34: 145-149.
  6. Kaba J, Kutschke L, Gerlach GF (2001) Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infections in goats. Vet Microbiol 78: 155-163.
  7. Binns SH, Green LE, Bailey M (2007) Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera. Vet Microbiol 123: 169-179.
  8. Carminati R, Bahia R, Moura Costa LF, Paule BJA, Vale VL, et al. (2003) Determinação da sensibilidade e da especificidade de um teste de ELISA indireto para o diagnóstico de linfadenite caseosa em caprinos. R Ci Méd Biol 2: 88-93.
  9. Dercksen DP, Brinkhof JM, Dekker-Nooren T, Maanen K, Bode CF, et al. (2000) A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats. VetMicrobiol 75: 167-175.
  10. Schreuder BE, ter Laak EA, Dercksen DP (1994) Eradication of caseous lymphadenitis in sheep with the help of a newly developed ELISA technique. Vet Rec 135: 174-176.
  11. ter Laak EA, Bosch J, Bijl GC, Schreuder BE (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.
  12. Guimarães AS, Seyffert N, Bastos BL, Portela RWD, Meyer R, et al. (2009) Caseous lymphadenitis in sheep flocks of the state of Minas Gerais, Brazil: Prevalence and management surveys. Small Rumin Res 87: 86-91.
  13. Seyffert N, Guimarães AS, Pacheco LG, Portela RW, Bastos BL, et al. (2010) High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacteriumpseudotuberculosis secreted proteins-based ELISA. Res Vet Sci 88: 50-55.
  14. Baird GJ, Malone FE (2010) Control of caseous lymphadenitis in six sheep flocks using clinical examination and regular ELISA testing. Vet Rec 166: 358-362.
  15. Frey A, Di Canzio J, Zurakowski DA (1998) A statistically defined endpoint titer determination method for immunoassays. J Immunol Methods 221: 35-41.
  16. Gerhardt P, Murray RGE, Wood WA, Krieg NR (1994) Methods for general and molecular bacteriology. (3rd edn) American Society for Microbiology, Washington, USA.
  17. Windsor PA (2011) Control of caseous lymphadenitis. Vet Clin North Am Food Anim Pract 27: 193-202.
  18. Cetinkaya B, Karahan M, Atil E, Kalin R, De Baere T, et al. (2002) Identification of Corynebacterium pseudotuberculosis isolates from sheep and goats by PCR. Vet Microbiol 88: 75-83.
Citation: Ribeiro D, Dorella FA, Pacheco LGC, Seyffert N, de Paula Castro TL, et al. (2013) Subclinical Diagnosis of Caseous Lymphadenitis Based on ELISA in Sheep from Brazil. J Bacteriol Parasitol 4:170.

Copyright: © 2013 Ribeiro D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.