Introduction

Corynebacterium pseudotuberculosis is known worldwide to cause pseudotuberculosis or caseous lymphadenitis (CLA) in sheep and goats (Brown and Olander 1987; Dorella et al. 2006). The superficial form of the disease causes lesions, primarily on the head and neck, which are noticeable upon inspection (Gezon et al. 1991). Because of poor responses to drug therapy, long-term survival of the organism in the environment, and difficulties in detecting infected animals, it is challenging to eradicate the disease, which may continue to cause a serious problem in goats.

While multiple studies on CLA in goats have been reported in many different countries (Skalka et al. 1998; Seyffert et al. 2010), limited information is available from Korea, except in one case where C. pseudotuberculosis was isolated from a Saanen dairy goat (Shin et al. 2010). In Korea, goats are a popular livestock, and the population is approximately 250,000 (KOSIS 2010). Thus, this study was aimed at determining the serology and clinical relevance of C. pseudotuberculosis in native Korean goats (Capra hircus coreanae).

Materials and methods

Sample collection

Between November 2009 and August 2010, a total of 466 native Korean goats from 40 herds (11 to 12 samples per herd) were randomly selected for serum collection throughout the nation, and information about age, region, and season were recorded for statistical analysis. All the tested goats were reared in free stalls and had no contact with other domestic animals. None of the sampled goats had been vaccinated against CLA. The serum samples were divided into three different groups based on age: young goats (<1 year, 148 samples), adult goats (≥1 year, 284 samples), and unknown (34 samples). Sample collection regions were divided into three, i.e., the northern, central, and southern regions, according to administrative boundaries (Fig. 1). During the cold season, between October and March, 123 serum samples were obtained. During the warm season, between April and September, 343 samples were collected. The sera obtained were kept at −20 °C until testing.

Fig. 1
figure 1

To evaluate the seroprevalence of Corynebacterium pseudotuberculosis, sample collection regions were divided into three areas, i.e., northern [Gyeonggi-do (a), Gangwon-do (b), Chungcheongnam-do (c), and Chungcheongbuk-do (d)], central [Gyeongsangbuk-do (e) and Jeollabuk-do (f)], and southern [Jeollanam-do (g) and Gyeongsangnam-do (h)] regions, according to administrative boundaries

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Serology and clinical examination

All serum samples were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit (ELITEST CLA, HYPHEN BioMed, France) according to the instructions of the manufacturer.

During blood sample collection, individual goats were also examined by direct inspection and superficial lymph node palpation for CLA. Pus samples were taken from superficial abscesses of these goats using sterile syringes (Fig. 2). Part of the sampled contents was inoculated onto a 5 % sheep blood agar plate. The plate was aerobically and anaerobically incubated for 24 to 48 h at 37 °C. Isolates were identified using PCR and API Coryne kit (bioMérieux, France).

Fig. 2
figure 2

Gross appearance of native Korean goats with caseous lymphadenitis. a Superficial form of caseous lymphadenitis at the parotid lymph node. b Creamish pus taken from caseous lymphadenitis at the parotid lymph node using a sterile syringe

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DNA extraction and PCR

For PCR detection of C. pseudotuberculosis, DNA was extracted from pus samples and isolates using a DNeasy Blood and Tissue kit (Qiagen, USA) according to the manufacturer’s instructions. DNA from each pus sample and isolate was tested by a duplex PCR according to the methods described by Pacheco et al. (2007). The target genes for the duplex PCR were C. pseudotuberculosis 16S rRNA and pld, which encodes the exotoxin phospholipase D. The estimated amplicon sizes for C. pseudotuberculosis 16S rRNA and pld genes were 816 and 203 bp, respectively.

The PCR reactions were carried out in a Mastercycler® pro PCR system (Eppendorf, Germany), and the PCR amplicons were separated by agarose gel (1.5 %) electrophoresis and viewed using an UV transilluminator.

Statistical analysis

A chi-square test was performed to analyze the differences between various groups using the SPSS software (ver. 17.0; SPSS Inc., Chicago, IL). P values <0.05 were considered statistically significant. Confidence intervals (CI, 95 %) were also calculated.

Results

Among the 466 goats tested, 267 (57.3 %) were seropositive for C. pseudotuberculosis (Table 1). When prevalence was analyzed by age, 73 (49.3 %, CI 41.3–57.4) of the 148 young (<1 year) goats, 176 (62.0 %, CI 56.3–67.6) of 284 adult (≥1 year) goats, and 18 (52.9 %, CI 36.2–69.7) of 34 goats in the unknown age group were seropositive for C. pseudotuberculosis. In the analyses according to region, 51 (46.4 %, CI 37.0–55.7) of 110, 51 (65.4 %, CI 54.8–75.9) of 78, and 165 (59.4 %, CI 53.5–65.1) of 278 goat samples were seropositive in the northern, central, and southern regions, respectively. In the analyses based on season, the seroprevalence was higher in the cold season (65.0 %, 80/123; CI 56.6–73.5) than that in the warm season (54.5 %, 187/343; CI 49.3–59.8). A significant difference was observed according to age, region, and season (P < 0.05).

Table 1 Seroprevalence of Corynebacterium pseudotuberculosis in 466 native Korean goats according to age, region, and season
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Among the 466 goats tested, 34 (7.3 %) were presumptively diagnosed with CLA by superficial abscesses. In most cases, parotid, mandibular, and cervical lymph nodes were affected, and the affected lymph nodes were characterized by enlargement and hairless skin around them (Fig. 2). All cases yielded a creamish pus (Fig. 2b). Of these 34 goats, C. pseudotuberculosis was isolated from 24 (70.6 % of goats with CLA lesions; Table 2). All isolates were identified as C. pseudotuberculosis with biochemical tests and PCR. In total, 95.8 % (23/24) of the goats in which C. pseudotuberculosis was isolated were adults (data not shown). Of the 10 goats with superficial abscesses from which C. pseudotuberculosis was not isolated, two goats were positive for Staphylococcus aureus (Table 2).

Table 2 Comparison between Corynebacterium pseudotuberculosis isolation and ELISA results in 34 native Korean goats with superficial abscesses
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When the pus samples from the 24 goats in which C. pseudotuberculosis was isolated were assessed by duplex PCR for the detection of C. pseudotuberculosis 16S rRNA and pld genes, all 24 samples showed estimated amplicon sizes of 816 and 203 bp for C. pseudotuberculosis 16S rRNA and pld genes, respectively (Fig. 3).

Fig. 3
figure 3

Duplex PCR detection of Corynebacterium pseudotuberculosis 16S rRNA and pld genes. Lane M, 100-bp DNA ladder; Lanes 1–3, field isolates of C. pseudotuberculosis; Lane 4, a positive control (ATCC43926) of C. pseudotuberculosis biovar ovis; and Lane 5, a negative control. The DNA fragments produced were analyzed on agarose gel (1.5 %) and visualized by ethidium bromide staining and UV transillumination. Estimated sizes of amplicons are indicated on the right for C. pseudotuberculosis 16S rRNA and pld genes at 816 and 203 bp, respectively

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Discussion

In the present study, CLA prevalence was higher in adult goats (≥1 year, 62.0 %) than in young goats (<1 year, 49.3 %), which was statistically significant (P < 0.05). Consistent with the data obtained from another study, seroprevalence tended to increase with age (Seyffert et al. 2010). One possible explanation for the higher seroprevalence in adult goats is that the frequency of abscess discharge increases with age.

When analyzed by region, a statistically significant difference was observed (P < 0.05). The authors cautiously suspect that the difference was attributed to the degree of sanitation at each farm. However, because of a lack of information, this study has the limitation of being unable to deduce the exact reason for the significant difference observed according to region.

According to season, the seroprevalence was significantly higher in the cold season than that in the warm season (P < 0.05). Physical transfer of purulent discharges from superficial CLA lesions has been suggested as an important mode of infection (Ashfaq and Campbell 1980). In the cold season, native Korean goats are typically raised in dense populations within barns for warmth. The authors suggest that this type of handling makes transmission easier due to close contact with infected goats. Furthermore, low temperatures could extend the survival period of C. pseudotuberculosis in the environment (Augustine and Renshaw 1986).

Among 34 goats that were presumptively diagnosed with CLA by direct inspection and superficial lymph node palpation, C. pseudotuberculosis was isolated from 24 (70.6 %) goats. Of the 10 goats in which C. pseudotuberculosis was not isolated, S. aureus was identified in two. These results are partially consistent with those from another study showing that Arcanobacterium pyogenes and S. aureus infections were characterized by superficial abscesses in goats (Gezon et al. 1991). The failure to isolate C. pseudotuberculosis from pus samples may be due to specimens being collected from exudates from the center of the abscess, or from inspissated pus, which may not yield bacterial growth.

Because C. pseudotuberculosis was isolated from goats that showed positive ELISA results, the authors suggest that the ELISA kit used was sensitive in detecting C. pseudotuberculosis infections. These findings are consistent with a previous study in which ELISA plates coated with recombinant phospholipase D were highly specific and sensitive for detecting C. pseudotuberculosis infection (Menzies et al. 1994). Moreover, Ellis et al. (1990) suggested that the recombinant antigen could be useful for overcoming problems with nonspecific cross-reaction in serological assays. Despite reports of cross-reaction with Mycobacterium paratuberculosis (Pepin et al. 1987) and of no significant relationship between the extent of CLA lesions and antibody titer (Ellis et al. 1990), ELISA has been shown effective in assessing the eradication of CLA in sheep and goats (Dercksen et al. 1996; Baird and Malone 2010). While only 6.3 % (29/461) of the tested goats presented CLA lesions, a high seroprevalence of 57.3 % (264/461) for C. pseudotuberculosis was observed. This could be explained by a high frequency of internal CLA lesions, which could not be evaluated in this study, because all the animals were alive at the time of testing.

Typical goat production in Korea involves an intensive handling system with little management practice. Furthermore, there is no licensed vaccine against CLA currently available. To the best of our knowledge, this study describes for the first time the prevalence of CLA in native Korean goats. Considering the high seroprevalence and bacterial isolation from most of the superficial CLA lesions, it is suspected that many internal CLA lesions also exist. This study indicates that C. pseudotuberculosis infection is widespread among goats in Korea and that bacterial infection was associated with superficial abscess. Thus, appropriate control programs need to be established to prevent transmission of C. pseudotuberculosis.