J Med Microbiol 55 (2006), 331-334; DOI: 10.1099/jmm.0.46373-0
© 2006 Society for General Microbiology
ISSN 0022-2615
Random amplified polymorphic DNA analysis of Campylobacter jejuni and Campylobacter coli isolated from healthy cattle and sheep
Mehmet Nuri Açik and
Burhan Çetinkaya
Department of Microbiology, Faculty of Veterinary Medicine, University of Firat, 23119, Elazig, Turkey
Correspondence
Burhan Çetinkaya
bcetinkaya{at}firat.edu.tr
Received 14 October 2005
Accepted 16 November 2005
The genetic heterogeneity among Campylobacter jejuni and Campylobacter coli isolates obtained from apparently healthy cattle and sheep was investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 348 Campylobacter isolates, consisting of C. jejuni (n=218) and C. coli (n=130), were analysed. All these isolates were successfully typed by RAPD analysis. The total numbers of band patterns defined by RAPD in cattle and sheep were 42 and 45, respectively. Of the 42 distinct types obtained from cattle, 37 types were observed in C. jejuni isolates (n=115), and the remaining 5 were in C. coli isolates (n=30). Of 45 distinct types obtained from sheep, 21 types were observed in C. jejuni isolates (n=103), and 24 were in C. coli isolates (n=100). It was concluded that a high degree of heterogeneity existed among the C. jejuni and C. coli isolates of healthy cattle and sheep.
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INTRODUCTION
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Campylobacter jejuni and Campylobacter coli are aetiological agents that cause the highest frequency of acute bacterial diarrhoea worldwide, with an estimated 2·5 million people per year affected in the USA (Mead et al., 1999). As thermophilic campylobacters are unable to grow in the environment, their reservoirs are the intestines of warm-blooded mammals and birds (Park et al., 1991). The most important route of human Campylobacter infection is considered to be the consumption of contaminated poultry and poultry products (Nadeau et al., 2002). However, the existence of Campylobacter species is well documented among many other animal species, including cattle and sheep (Stanley & Jones, 2003). The importance of cattle and sheep in campylobacteriosis is not just restricted to the contamination of milk at the farm and the carcass at slaughter, but also involves environmental and water contamination owing to the disposal of abattoir effluents and slurries to the land (Stanley & Jones, 2003).
The applicability of phenotypic methods for typing Campylobacter species is limited by the difficulty of obtaining standard antisera and phage reagents, and the lack of standardization of protocols between laboratories. In recent years, several genotypic methods have been described. One of the simplest and most cost-effective methods for the investigation of large numbers of isolates is the random amplified polymorphic DNA (RAPD) assay. This method is well recognized as a highly discriminatory tool for the molecular typing of a wide range of bacteria, including campylobacters, owing to the ability to determine polymorphisms in the entire bacterial genome. An RAPD assay that is based on the amplification of random DNA fragments using a single primer of arbitrary sequence was developed for Campylobacter spp. (Welsh & McClelland, 1990; Hernandez et al., 1995), and has been reported to be of great asset in establishing genetic diversity among C. jejuni and C. coli isolates from different sources (Hilton et al., 1997). RAPD analysis of C. jejuni and C. coli has frequently used 10-mer primers (Madden et al., 1996; Ertas et al., 2004).
While there are some reports about the genetic relationships among C. jejuni and C. coli strains of healthy cattle origin, information about the isolates of healthy sheep origin is limited. The objective of the present study was to investigate genetic heterogeneity among C. jejuni and C. coli isolates of healthy bovine and sheep origin, using an RAPD method.
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METHODS
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Bacterial isolates.
A total of 348 Campylobacter isolates, consisting of 218 C. jejuni and 130 C. coli, were used in this study. All the isolates were grown on Preston Campylobacter broth and agar, and were identified by species-specific PCR (Açik & Çetinkaya, 2005). Of the 348 isolates, 145 were obtained from various samples from healthy cattle and the remaining 203 isolates originated from healthy sheep. Of the cattle isolates, 116 (115 C. jejuni and 1 C. coli) were obtained from gall bladders, 24 C. coli were obtained from faecal samples and 5 C. coli were from intestinal contents. Of the sheep isolates, 73 (42 C. jejuni and 31 C. coli) were obtained from gall bladders, 88 (53 C. jejuni and 35 C. coli) were from intestinal contents and 42 (8 C. jejuni and 34 C. coli) were from faecal samples. While gall bladder and intestinal-content samples were collected from healthy cattle and sheep slaughtered at a local abattoir in the east of Turkey between July and September 2003, and between March and May 2004, faecal samples were collected from cattle at a local farm in the southeast of Turkey in October 2003, and from sheep of three different flocks in the east of the country in May 2004. Each individual animal was represented by only one sample; in other words, no more than one sample could be collected from the same cow or sheep. The geographical location of both cattle and sheep from which faecal samples were collected was different from those sampled for internal organs.
DNA extraction.
A few representative colonies from pure cultures, which were identified as C. jejuni or C. coli by species-specific multiplex PCR, following inoculation of samples onto Campylobacter selective agar containing 7 % laked horse blood (SR0048C; Oxoid) and Preston Campylobacter selective supplement (SR117E; Oxoid) (Açik & Çetinkaya, 2005), were transferred into an Eppendorf tube containing 300 µl distilled water. The bacterial suspension was treated with 300 µl TNES buffer (20 mM Tris pH 8·0, 150 mM NaCl, 10 mM EDTA, 0·2 % SDS) and proteinase K (200 µg ml–1), and was kept at 37 °C for 2 h. Following 10 min of boiling, an equal amount of phenol (saturated with Tris/HCl) was added to the suspension. The suspension was shaken vigorously by hand for 5 min and then centrifuged at 11 600 g for 10 min. The upper phase was carefully transferred into another Eppendorf tube and 3 M sodium acetate (0·1 volumes) and 95 % ethanol (2·5 volumes) were added to the suspension, which was left at –20 °C overnight to precipitate the DNA. The pellet, obtained following the centrifugation at high speed for 10 min, was washed twice with 90 and 70 % ethanol, respectively, each step was followed by 5 min centrifugation. Finally, the pellet was dried and resuspended in 50 µl distilled water.
RAPD analysis of C. jejuni and C. coli isolates.
For the RAPD analysis of C. jejuni and C. coli isolates, the reaction mixture was prepared in a total volume of 50 µl, consisting of 5 µl DNA, 10x PCR buffer (750 mM Tris/HCl, 200 mM (NH4)2SO4, 0·1 % Tween 20), 3·5 mM MgCl2, 200 µM deoxynucleoside triphosphates, 1·25 U Taq DNA polymerase (MBI Fermentas) and 1 µM OPA-11 primer (5'-CAA TCG CCG T-3') (Hernandez et al., 1995). The RAPD assay was performed in a thermal cycler with an initial denaturation step at 94 °C for 1 min, followed by 45 cycles at 94 °C for 1 min, 36 °C for 1 min and 72 °C for 2 min, then, a last step of extension at 72 °C for 5 min. PCR products were separated by electrophoresis in 1·5 % (w/v) agarose gels and visualized by ethidium bromide staining. A 100 bp DNA ladder (MBI Fermentas; SM0321) molecular mass marker was used to evaluate the size of bands.
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RESULTS AND DISCUSSION
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This study was conducted to investigate strain variations among C. jejuni and C. coli isolates of healthy bovine and ovine origin, and for this purpose an RAPD assay, which is generally considered as the most advantageous assay in terms of discrimination ability, cost and ease, was used. In total, 87 different types (42 from cattle and 45 from sheep) were defined in this analysis of 348 Campylobacter isolates of various origins. In a previous study that employed the same primer, the number of RAPD types obtained in the examination of 208 samples was reported to be even higher (n=118) (Hernandez et al., 1995). This might be expected as they were testing isolates from various sources, and in the current paper the isolates came from multiple sites in two ruminant species. Additionally, Nielsen et al. (2000) used a different primer and reported 56 RAPD types among the 80 strains examined. Subjective interpretation of the data, the choice of primers and type of samples, in addition to the variation in geographical locations, may play a role in these differences.
In the RAPD analysis of 115 C. jejuni isolates originating from the gall bladders of healthy cattle, 37 different types were obtained. Some of the RAPD types were represented by remarkably high percentages of isolates. For example, the most common types of C. jejuni were observed in 30 and 16 % of gall bladder isolates (Fig. 1
). The other types were represented by less than 6 % of the isolates (Table 1).
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Fig. 1. RAPD types of C. jejuni isolates obtained from the gall bladder samples from cattle, using the OPA-11 primer. M, 100 bp molecular mass marker (MBI Fermentas; SM0321); lanes 1–11, different types (lane 6 represents the most frequenttype).
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In the RAPD analysis of 30 C. coli isolates originating from various samples from cattle, 5 different types were obtained. The most common type was represented by 40 % of intestinal-content samples and 33 % of faecal samples. All five different types were observed in faecal isolates. Four distinct types were obtained from intestinal isolates. The type observed from the gall bladder isolate was similar to one of the types obtained from faecal and intestinal-content isolates (Table 1
). In the analysis, identical types were obtained from samples from different locations. The overall results suggest that the degree of heterogeneity among Campylobacter isolates from healthy cattle is relatively high, although some of the types of Campylobacter isolates were represented by a high percentage, and identical types of C. coli isolates were obtained from different locations. The fact that identical types were obtained from field and abattoir isolates suggests that there was no influence of location on the diversity in cattle.
Similar findings were also made for C. jejuni and C. coli isolates originating from intestinal contents, gall bladders and faecal samples of healthy sheep. In the RAPD analysis of 103 C. jejuni isolates originating from various samples from sheep, 21 different types were obtained (Table 2). Of these, nine types were obtained from gall bladders, ten from intestinal contents and two from faecal samples. The most common types were represented by 63 % of faecal samples, 26 % of gall bladders and 19 % of intestinal contents. Other types were detected at much lower percentages. In the analysis of 100 C. coli isolates originated from sheep, 24 different types were obtained (Table 2). Of these, seven types were obtained from gall bladder samples, eight from intestinal contents and nine from faecal samples. The most common types were represented by 48 % of gall bladder samples, 35 % of faecal samples and 34 % of intestinal-content samples (Fig. 2). Other types were detected at much lower percentages. Two of the types obtained from faecal isolates were observed in all three flocks, but the remaining seven distinct types were obtained in only one flock. The absence of identical types from field and abattoir isolates indicates that the geographical location might play a role in strain variation in sheep.
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Fig. 2. RAPD types of C. coli isolates obtained from the intestinal contents and gall bladders of healthy sheep, using the OPA-11 primer. M, 100 bp molecular mass marker (MBI Fermentas; SM0321); lanes 1–8, types obtained from intestinal-content samples (lane 2 represents the most frequent type); lanes 9–15, types obtained from gall bladder samples (lane10 represents the most frequent type).
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Information about the genetic relationships among C. jejuni and C. coli strains of healthy sheep origin is scarce. Scates et al. (2003) reported 8 distinct band types in the examination of 30 liver samples in sheep. To our knowledge, no studies investigating genetic heterogeneity among Campylobacter strains isolated from other types of sample from sheep have been conducted hitherto. The results of the present study showed that the number of RAPD profiles among C. coli isolates from sheep was higher than from cattle. On the contrary, the number of band types of C. jejuni isolates was higher in cattle.
The RAPD assay has been proved to have excellent discrimination ability due to the fact that the entire genome is the target in genotyping. A common opinion exists among researchers that the discrimination of an RAPD assay is higher than that of PFGE (Nielsen et al., 2000). However, the major disadvantage of the RAPD assay appears to be its poor reproducibility and repeatability. Hernandez et al. (1996) noted that 17 % of their Campylobacter isolates could not be typed, due to DNase activity, using the arbitrarily primed-PCR fingerprint method . In this study, RAPD analysis was successfully applied in typing 100 % of C. jejuni and C. coli isolates. Moreover, the assay was repeated at least twice and identical results were obtained. The findings reported by Hilton et al. (1997) also supported the idea that RAPD may successfully be used to type Campylobacter species originating from various samples.
In conclusion, it was observed that a high degree of heterogeneity existed among C. jejuni and C. coli isolates of healthy cattle and sheep origin. RAPD analysis appears to be a valuable tool in epidemiological surveillance, and for investigating the distribution of types in animals, the environment and humans.
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ACKNOWLEDGEMENTS
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The technical support and help in sample collection of Dr H. Ongor, Dr M. Karahan and Mr M. Bazna is gratefully acknowledged. This work was supported by the Scientific Research Council of Firat University (FUBAP 800) and was part of a PhD thesis produced by M. N. Açik.
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REFERENCES
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INTRODUCTION
METHODS
