Prevalence of Listeria species in camel sausages from retail markets in Aydin province in Turkey and RAPD analysis of Listeria monocytogenes isolates

Samples were taken from 100 camel sausages from the different retail markets in Aydin province in the south-west of Turkey and they were tested for the presence of Listeria spp by biochemical methods. Samples were enriched using Listeria Enrichment Broth and they were inoculated onto Listeria Selective Agar. Listeria monocytogenes was isolated from nine samples (9%), Listeria innocua from 14 samples (14%) and Listeria welshimeri from two samples(2%). A 701 bp fragment of listeriolysin O sequence for L. monocytogenes was amplified using specific primers by polymerase chain reaction (PCR) for confirmation of the identification. A random primer (OPA-11) was used in a random amplified polymorphic DNA (RAPD) assay. This detected five different band profiles amongst the L. monocytogenes isolates, indicating a relatively large amount of genetic heterogeneity amongst the nine isolates. The study has highlighted the need for improved strategies for food safety, in particular appropriate hygienic precautions to avoid contamination of sausage during the manufacturing process and appropriate preservation techniques during storage and transport, to prevent transmission of Listeria spp to consumers at home and abroad.


Introduction
A wide variety of meats and meat products, including fermented sausages, can be contaminated with Listeria spp (Johnson et al., 1990). L. monocytogenes is known to survive the commercial dry sausage manufacturing process (Incze, 1998). In Turkey, Ciftcioglu (1992) found Listeria spp in 11% of sausages, with L. monocytogenes in 2% and L. innocua in 8%. A subsequent study by Guven and Patir (1998) found Listeria spp in 16.3% of sausages, with L. monocytogenes in 11.9% and L. innocua in 11.3%. Several molecular genotyping methods have been used to type L. monocytogenes, such as DNA restriction endonuclease analysis (Wesley and Ashton, 1991), multilocus enzyme electrophoresis (Bibb et al., 1990), ribotyping (Baloga and Harlander, 1991) and pulsedfield gel electrophoresis (PFGE) (Autio et al., 1999). However, these methods are not well suited for routine use in laboratories and are time-consuming. PFGE is very discriminative, but it is labour-intensive and requires expensive apparatus (Franciosa et al., 1998). RAPD typing is suitable for differentiation of the most commonly found serotypes and for screening large panels of strains (Byun et al., 2001). The aims of this study were to determine the prevalence of Listeria spp in camel sausages at different retail markets in Aydin provience in the south-west of Turkey, to analyse genetic variability among L. monocytogenes isolates by RAPD using a random primer and to enquire whether there is a public health risk of acquiring listeriosis from consumption of camel sausage.

Material
Samples were taken from 100 camel sausages obtained from the different retail markets in Aydin province, in the south-west of Turkey.

Detection of Listeria spp
For microbiological analysis, the sausage casing was removed aseptically. A 25g sample from each sausage was added to 225ml of Listeria Enrichment Broth (LEB, Oxoid) and homogenised in a stomacher (Interscience, 78860 St Nom-France) at high speed, for one minute at room temperature and incubated at 37°C for 24 hours (primary enrichment). Then, an aliquot of 0.1ml of the culture was transferred into tubes containing 10ml LEB. The tubes were incubated for 24 hours to 48 hours at 37°C (secondary enrichment). A loopful of each enrichment culture was streaked onto Listeria Selective Agar (LSA, Oxoid) and was incubated for 24 hours, at 37°C. The suspected peer reviewed colonies with a dark brown colour or black halo were transferred onto tryptic soy agar (TSA, Difco) and incubated for 24 hours, at 37°C. The isolates were identified using conventional methods: Gram staining; the Christie, Atkins, Munch-Petersen (CAMP) test; typical umbrella motility; and fermentation of mannitol, rhamnose and xylose (Seeliger et al., 1986).

Extraction of DNA
A few colonies from cultures were transferred into an Eppendorf tube containing 300μl of distilled water and the tubes were vortexed. Lysis was accomplished by the addition of 300μl of TNES buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 10mM EDTA, 0.2% SDS) and 200μg/ml Proteinase K. The lysis mixture was incubated at 37°C for two hours and boiled for 30 minutes. Bacterial DNA was extracted by the phenol:chloroform:isoamylalcohol procedure. All purifications and PCR reactions used L. monocytogenes serovar 1/2a: CIP 104794/ ATCC-35152 (obtained from Pasteur Institute) as the positive control and distilled water as the negative control. Primers Primers used in this study were designed by Border et al. (1990). The sequences of primer pairs were as follows: LM1 (5'-CCT AAG ACG CCA ATC GAA -3') and LM2 (5'-AAG CGC TTG CAA CTG CTC -3'). These primers amplify a 701 bp fragment on listeriolysin O sequence of L. monocytogenes. PCR PCR reactions were performed in a reaction mixture (50μl final volume) containing 5μl of 10 x PCR buffer (10mM Tris-HCl, pH 9.0, 50mM KCl, 0.1% Triton X-100), 5μl of 25mM MgCl2, 250μM of each deoxynucleoside triphosphate, 2U of Taq DNA polymerase (Fermentas, Lithuania), 50pmol of each primer and 5μl of template sample DNA. Amplification was obtained with an initial denaturation step at 94°C for five minutes, followed by 45 cycles at 94°C for 30 seconds, 52°C for one minute and 72°C for one and a half minutes. The final cycle was at 72°C for seven minutes. For all experiments, a Touchdown Thermocycler (Hybaid, Middlesex, England) was used. Ten μl of amplified products were detected by electrophoresis in 1.5% agarose, in 1 X Tris-borate-EDTA buffer. Gels were stained with ethidium bromide (0.5μg/ml). The DNA fragments were visualised by UV illumination and photographed with Polaroid film. The molecular sizes of the PCR products were compared with a 100bp DNA ladder.

RAPD analysis
The RAPD reaction mixture was prepared in a total volume of 25μl consisting of 2.5μl of 10xPCR buffer (750mM Tris-HCl, 200mM [NH 4 ] 2 SO 4 , 0.1% Tween 20, pH 8.8), 3.5mM MgCl 2 , 200μM of each deoxynucleoside triphosphates, 1.25U of Taq DNA polymerase (Fermentas, Lithuania), 1μM of OPA-11 primer (5'-CA AT CG CC GT-3'), 11μl of dsH 2 O (sterile distilled water) and 2.5μl of template DNA. Each sample was amplified through 50 cycles of denaturation (one minute at 94°C), primer annealing (one minute at 37°C) and extension (one minute at 72°C). A last cycle of extension was applied at 72°C for 10 minutes. Twenty microlitres of product was resolved by electrophoresis on a 2% agarose gel in Tris-borate EDTA buffer and visualised by staining with 0.5μg/ml ethidium bromide for 30 minutes. The agarose gels were photographed under UV light. A 1kb DNA ladder (Promega, Maddison, USA) was used as a molecular size standard. RAPD assays were performed at least three times each to check reproducibility.

Culture and PCR
Of the 100 camel sausage samples, nine (9%) contained L. monocytogenes, 14 (14%) contained L. innocua and 2 (2%) contained L. welshimeri. A 701bp fragment was amplified from all the tested L. monocytogenes strains identified by conventional procedures (Figure  1), but not from the negative control. RAPD RAPD typing using OPA-11 primer detected five different band profiles (a, b, c, d, e) on nine isolates of L. monocytogenes (Figure 2 ).

PEEr rEVIEWEd Volume 59 (6) : June, 2006
Irish Veterinary Journal discussion Studies carried out in different parts of Turkey have found Listeria spp in 11% (Ciftcioglu, 1992) to 16.3% (Guven and Patir, 1998) of sausages. In some other countries prevalence has been reported to be considerably higher (Breer and Schopfer, 1989;Farber et al., 1989).
In the present study, Listeria spp were obtained from 25% of the sampled sausages. In this and in other studies, three Listeria species have been identified: L. innocua, L. monocytogenes and L. welshimeri (Breuer and Prandl, 1988;Schmidt et al., 1988;Guven and Patir, 1998;Paziak-Domanska et al., 1999;Uyttendeale et al., 1999). In the present study, all the isolates of L. monocytogenes were successfully typed by the RAPD method using only OPA-11 primer. One random primer was used and five different profiles were observed, thus indicating a large amount of genetic heterogeneity amongst the nine isolates of L. monocytogenes.
Overall, L. innocua has been isolated from sausage more frequently than have the other two species. For instance, Paziak-Domanska et al. (1999) recovered Listeria from 15 samples of sausage, L. innocua from 10 samples, L. monocytogenes from three samples and L. welshimeri from two samples. A similar trend was evident in the present study: L. innocua from 14 samples, L. monocytogenes from nine samples and L. welshimeri from two samples. In summary, this study revealed the presence of Listeria spp in a significant proportion (25%) of samples of camel sausage on sale in different retail markets in Aydin province in the south-west of Turkey. It has indicated that a large amount of genetic heterogeneity exists among L. monocytogenes isolates from camel sausage. Thus, it has highlighted the need for improved strategies for food safety, in particular appropriate hygienic precautions to avoid contamination during the manufacturing process and appropriate preservation techniques during storage and transport to prevent transmission of Listeria spp to consumers at home and abroad.