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1 Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte – MG, Brazil
2 Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte – MG, Brazil
3 Departamento de Bio-Interação, Universidade Federal da Bahia, Salvador – BA, Brazil
4 Universidade Estadual Vale do Acaraú, Sobral – CE, Brazil
5 Centro Nacional de Pesquisa de Caprinos, Empresa Brasileira de Pesquisa Agropecuária, Sobral – CE, Brazil
Correspondence
Vasco Azevedo
vasco{at}icb.ufmg.br
Received 11 October 2006
Accepted 23 November 2006
Abbreviations: CLA, caseous lymphadenitis; mPCR, multiplex PCR; PLD, phospholipase D.
The GenBank/EMBL/DDBJ accession number for the rpoB gene sequence of Arcanobacterium pyogenes is DQ680032.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
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Once established in a herd or flock, CLA eradication is problematic due to the inefficacy of antimicrobial therapy (Piontkowski & Shivvers, 1998; Stanford et al., 1998; Williamson, 2001). The most reliable control strategy for this disease involves vaccinating livestock and identifying and removing infected animals (Brown et al., 1986; Paton et al., 2003). However, this approach is hindered by limitations in current diagnostic techniques (Williamson, 2001; Menzies et al., 2004; Dorella et al., 2006).
Many serological tests have been proposed for CLA diagnosis, including a complement fixation test (Shigidi, 1979), synergistic haemolysis-inhibition test (Brown et al., 1987), microagglutination assay (Menzies & Muckle, 1989) and C. pseudotuberculosis phospholipase D (PLD) antigen-based ELISA (Dercksen et al., 2000). Although these tests may be of great value for the detection of subclinically infected animals, most present drawbacks, including low sensitivity, poor specificity and an inability to distinguish between previously exposed animals and those still harbouring the pathogen. Thus it is questionable whether such techniques should be employed to orient culling programmes (Williamson, 2001; Çetinkaya et al., 2002).
Accurate CLA diagnosis is based primarily upon clinical observations (external abscesses) and the identification of C. pseudotuberculosis by phenotypic and biochemical tests; this is important to differentiate this bacterium from other abscess-inducing pathogenic agents, such as Arcanobacterium pyogenes or Pasteurella multocida (Dercksen et al., 2000; Williamson, 2001; Dorella et al., 2006).
A 16S rRNA gene-based PCR assay has been developed to identify C. pseudotuberculosis isolates (Çetinkaya et al., 2002). Although this assay was useful for estimating the prevalence of CLA in the animals studied, it presented some limitations: (i) it was dependent on bacterial culture; and (ii) it was not specific enough to distinguish C. pseudotuberculosis from Corynebacterium ulcerans (Çetinkaya et al., 2002).
In order to improve C. pseudotuberculosis detection by PCR, we developed a multiplex PCR (mPCR) assay and adapted a protocol to extract bacterial genomic DNA directly from clinical samples. Amplification of multiple loci in a single reaction through mPCR is a powerful and widely used tool for the rapid and specific identification of pathogenic bacteria (Wadowsky et al., 1996; Halbert et al., 2005; O'Halloran & Cafferkey, 2005; Persson & Olsen, 2005). Our mPCR targeted three C. pseudotuberculosis genes: the 16S rRNA gene, the gene of choice for most microbial taxonomy studies (Çetinkaya et al., 2002; Khamis et al., 2005); rpoB, the RNA polymerase ß-subunit gene, currently used to study phylogenetic relationships in the genera Corynebacterium and Mycobacterium (Khamis et al., 2004, 2005; Dorella et al., 2006); and pld, which encodes the exotoxin PLD, a sphingomyelinase implicated in the virulence of C. pseudotuberculosis, C. ulcerans and Arcanobacterium haemolyticum (McNamara et al., 1995). This mPCR enabled specific identification of C. pseudotuberculosis isolates in culture and direct detection in pus samples from CLA-affected animals.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. A total of 40 caprine isolates of C. pseudotuberculosis were obtained from different sources. One type strain of C. pseudotuberculosis biovar ovis (strain CIP 102968T) and 13 strains of genetically or morphologically related bacteria were also used in the experiments.
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Clinical specimens. Fifty-six pus samples were collected aseptically from abscessed lymph nodes of naturally infected sheep (n=12) and goats (n=44) found in two CLA-endemic areas of Brazil. Microbiological examination, followed by biochemical identification, was used as a gold standard to confirm infection with C. pseudotuberculosis. In brief, bacteriological cultures were made of pus specimens and the resultant C. pseudotuberculosis-resembling colonies that stained Gram-positive were tested further for biochemical properties (glucose fermentation, urease and catalase) (Smibert & Krieg, 1994; Dercksen et al., 2000). Synergistic haemolysis with Rhodococcus equi ATCC 33701 and inhibition of ß-haemolysis by Staphylococcus aureus ATCC 25923 were also evaluated (Smibert & Krieg, 1994; Dercksen et al., 2000). Species identification was confirmed using the API Coryne battery (bioMérieux).
Blood samples were taken from confirmed C. pseudotuberculosis-infected animals (n=19) and from healthy animals (n=20) using the Vacutainer blood collection system (Becton Dickinson).
DNA isolation
Two different protocols were adapted for extracting DNA from pure bacterial cultures and clinical samples.
(i) Bacterial cultures. Chromosomal DNA extraction was performed in the same manner for all bacterial strains. A 20 ml 48–72 h culture was centrifuged at 4 °C and 2000 g for 20 min. Cell pellets were resuspended in 1 ml Tris/EDTA/RNase [10 mM Tris/HCl (pH 7.0), 10 mM EDTA (pH 8.0), 300 mM NaCl, 50 µg RNase A ml–1] and centrifuged again under the same conditions. Supernatants were discarded and the pellets were resuspended in 1 ml TE/lysozyme [25 mM Tris/HCl (pH 8.0), 10 mM EDTA (pH 8.0), 10 mM NaCl, 10 mg lysozyme ml–1). Samples were then incubated at 37 °C for 30 min; 30 µl 30 % (w/v) sodium N-lauroylsarcosine (sarcosyl) was added and the mixture was incubated for 20 min at 65 °C, followed by incubation for 5 min at 4 °C. DNA was purified using phenol/chloroform/isoamyl alcohol and precipitated with ethanol (Sambrook et al., 1989). DNA concentrations were determined spectrophotometrically.
(ii) Clinical samples. A DNA extraction protocol previously described for clinical samples from tuberculosis patients (Honoré-Bouakline et al., 2003) was adapted for use in this study. Briefly, 100 mg pus or the pellet of a 2 ml blood sample was resuspended in 1 ml TE/lysozyme. Samples were incubated for 1 h at 37 °C; 20 µl proteinase K (20 mg ml–1; Invitrogen) was added, followed by incubation for 2 h at 56 °C. Samples were divided into two aliquots of 500 µl, and 25 µl 30 % (w/v) sarcosyl was added to each; mixtures were incubated for 20 min at 65 °C and then for 5 min at 4 °C. DNA was purified and precipitated as described above.
Primers and mPCR conditions.
The oligonucleotide primers used in this study are listed in Table 2. Primers targeting the 16S rRNA and rpoB genes of C. pseudotuberculosis were obtained from previously published work (Çetinkaya et al., 2002; Khamis et al., 2004). Primers targeting the pld gene were designed by aligning the pld nucleotide sequences of C. pseudotuberculosis and C. ulcerans (GenBank accession nos L16587 and L16585); the reverse primer PLD-R1 was used in association with the forward primer PLD-F to amplify the pld genes of both bacteria, whilst primer PLD-R2 excluded C. ulcerans (see Fig. 1b). A primer pair targeting the mitochondrial 12S rRNA genes of Capra hircus and Ovis aries (GenBank accession nos AJ490504 and AJ490503) was designed to function as an internal amplification control for the mPCRs performed with DNA extracted directly from clinical samples of CLA. This amplification control allowed identification of samples that possessed factors inhibitory for the mPCR assay (Hoorfar et al., 2004).
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Sequencing of singleplex PCR products. In order to confirm the mPCR results, ten randomly chosen isolates were tested further in singleplex PCR assays with the three C. pseudotuberculosis-specific primer pairs. PCR products were precipitated with 15 % (w/v) polyethylene glycol (Kusukawa et al., 1990) and sequenced using the DYEnamic ET Dye Terminator kit (Amersham Biosciences), according to the manufacturer's instructions. The sequences were compared with previously published 16S rRNA (GenBank accession nos X81916, X81907 and X84255), rpoB (GenBank accession no. AY492239) and pld (GenBank accession nos L16586 and L16587) C. pseudotuberculosis gene sequences, by searching for similarity using BLAST-N (Altschul et al., 1990).
Analytical specificity and sensitivity.
To evaluate the specificity of the mPCR assay, reactions were performed with genomic DNA extracted from bacterial strains taxonomically similar to C. pseudotuberculosis biovar ovis (Table 1
). Serial tenfold dilutions of DNA from C. pseudotuberculosis type strain CIP 102968T, ranging from 10 to 0.0001 ng DNA per reaction, were used to test the sensitivity of the method.
To evaluate the assay's detection limit, blood samples from goats that did not present clinical symptoms of CLA, and which were negative in ELISA tests (performed according to Paule et al., 2003), were seeded with 101–106 c.f.u. of C. pseudotuberculosis.
Statistical analysis.
An analysis to evaluate differences in the sensitivity of the mPCR assay between samples from sheep versus goats was performed with a 
2 test, using the STATISTICA software package (StatSoft).
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), only C. pseudotuberculosis biovar equi CIP 5297 yielded a similar mPCR profile, with the 816, 446 and 203 bp amplicons corresponding to the 16S rRNA, rpoB and pld genes, respectively (Fig. 1a). As in a previous study (Khamis et al., 2004), all of the corynebacteria species that we tested generated an amplicon of 
446 bp, corresponding to the rpoB gene (Fig. 1a). As expected, the pld gene product was not detected in any of the four C. ulcerans strains (Fig. 1a, lanes 4–7), illustrating the ability of the mPCR assay to differentiate this bacterium from C. pseudotuberculosis. Moreover, two other pathogenic bacteria associated with the formation of abscesses in small ruminants, A. pyogenes and P. multocida, were clearly distinguished (Fig. 1a). The 300 bp product generated by amplification of A. pyogenes genomic DNA was sequenced and assigned as the rpoB gene following a search for homology in GenBank using BLAST-N (data not shown).
To determine the sensitivity of the assay, reactions were performed with serial tenfold dilutions of purified C. pseudotuberculosis CIP 102968T DNA. The mPCR method was able to detect bacterial DNA at concentrations ranging from 10 to 0.001 ng per reaction (Fig. 2).
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Identification of bacterial isolates
The characteristic mPCR amplification profile was obtained for all 40 C. pseudotuberculosis biovar ovis field isolates tested (Table 1). Fig. 3(a) shows the mPCR profiles of ten randomly chosen C. pseudotuberculosis biovar ovis field isolates.
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Direct detection of C. pseudotuberculosis in clinical samples by mPCR
Pus samples were collected from the lymph nodes of CLA-affected sheep and goats and DNA was extracted directly from these specimens using our protocol (see Methods). A bacteriological culture was prepared from the samples as a gold standard to confirm infection by C. pseudotuberculosis. The mPCR protocol was performed as described for purified bacterial DNA; however, a non-competitive internal amplification control (12S rDNA; see Methods) was included in these reactions in order to allow differentiation between inhibited reactions, i.e. those in which there was no amplification of the internal control, and reactions in which the mPCR yielded a negative result (Hoorfar et al., 2004). Typical mPCR profiles of clinical samples are shown in Fig. 3(b).
Most of the undiluted samples were inhibitory for the mPCR assay, but more than 52 % yielded detectable results at a 1 : 2 dilution and 45 % showed amplification at a 1 : 4 dilution, whilst only 3 % showed amplification at a 1 : 8 dilution. The proportions of samples positive in the mPCR assay were not significantly different when comparing samples of sheep versus goats (P=0.60). Table 3 summarizes the results obtained for pus samples.
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The only previously reported molecular method employed for specific identification of C. pseudotuberculosis isolated from pus samples of CLA-affected animals, based on the amplification of a 16S rRNA gene fragment (Çetinkaya et al., 2002), has two major drawbacks: (i) dependence on bacterial culture; and (ii) an inability to distinguish the bacterium C. ulcerans.
Our mPCR assay was specific enough to differentiate C. pseudotuberculosis from C. ulcerans (Fig. 1a). These two bacteria have been reported to possess 99.7 % similarity between their 16S rRNA genes and 93.6 % between their rpoB genes (Riegel et al., 1995; Khamis et al., 2004); most of their biochemical properties are similar and both may eventually produce diphtheric toxin (Riegel et al., 1995; DeWinter et al., 2005). The high genomic similarity may explain the inability of a previously published study to differentiate these two bacteria by PCR (Çetinkaya et al., 2002). As C. ulcerans is also a PLD producer, the reverse primer used to amplify the pld gene in our study was designed so that it possessed a mismatch in its 3' end (two Gs instead of two Cs) (Fig. 1b), allowing differentiation from C. pseudotuberculosis due to the absence of the 203 bp amplicon.
Although C. ulcerans is primarily recognized as a veterinary pathogen, there has been a marked increase in the number of human infections (DeWinter et al., 2005; De Zoysa et al., 2005) and it appears that domestic cats and dogs can be potential reservoirs (De Zoysa et al., 2005). In this context, our mPCR would be able to identify bacteria collected from the nasal discharge of domestic animals and could differentiate bacteria cultured from samples from diphtheria patients by using the primer pair PLD-F/PLD-R1, which allows amplification of the pld gene of C. ulcerans (see Fig. 1b).
Çetinkaya et al. (2002) reported a weaker amplification of the 816 bp 16S rRNA gene amplicon in biovar equi of C. pseudotuberculosis when compared with that of biovar ovis. This result was attributed to a mismatch (an A instead of a G) five bases from the 5' end of the forward primer used (16S-F; Table 2). However, this result was not reproducible under the conditions used in our study. We propose that amplification of the 16S rRNA gene using primers 16S-F/16S-R is not sufficient to differentiate the two biovars of C. pseudotuberculosis. It is interesting that infection of small ruminants with biovar equi is not known to occur (Spier et al., 2004).
The mPCR assay was able to confirm the identification of 40 C. pseudotuberculosis field isolates and reliably detected this bacterium in a reaction mixture containing as little as 0.001 ng of genomic DNA, illustrating its reproducibility and high sensitivity.
A DNA extraction protocol was adapted to recover bacterial genomic DNA directly from clinical samples of CLA-affected animals. This strategy offers advantages over bacteriological culture, including reduced time of analysis and the ability to detect unviable bacteria. The detection limit of the goat blood spiking experiment was 103 c.f.u. C. pseudotuberculosis (ml sample)–1. However, this value may underestimate the number of viable organisms, as intracellular bacteria such as C. pseudotuberculosis and R. equi have a tendency to clump when cultured (Harrington et al., 2005). A detection limit above 103 c.f.u. (ml sample)–1 would help explain the inability of the mPCR assay to detect C. pseudotuberculosis in blood samples from infected sheep and goats.
On the other hand, the mPCR efficiently detected this bacterium in a high proportion (94.6 %) of confirmed CLA-infected pus samples. In contrast to serological CLA testing (Menzies & Muckle, 1989; Dercksen et al., 2000), the mPCR sensitivity was the same for both sheep and goats (Table 3).
As our mPCR assay provides an efficient, accurate, rapid and reproducible method for the identification of cultured C. pseudotuberculosis and its direct detection in pus samples, we propose that it may be used instead of bacteriological culture as a confirmatory CLA test.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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to a phospholipase D enzyme-linked immunosorbent assay for diagnosis of Corynebacterium pseudotuberculosis infection in experimentally infected goats. Vet Microbiol 100, 129–137.[CrossRef][Medline] production, IgG avidity and antigen recognition by Western blotting. Vet Immunol Immunopathol 96, 129–139.[CrossRef][Medline]
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