Prevalence of Listeria Species in Environment and Milk Samples

Received: Revised: Accepted: 2014–06–06 2014–06–2

ISSN: 2307-8316 (Online); ISSN: 2309-3331 (Print) Raw milk (n=130), soil (n=100) and cow dung (n=130) samples from Mysore city were studied for the presence of Listeria spp. Raw cow milk and cow dung samples from the individual cow and soil samples from vegetable-cultivation land and Pasture were collected. All the samples were collected in UV sterilized polythene covers and brought to the laboratory. The samples were processed on the same day of collection.
Isolation of Listeria spp. was done by following cold enrichment method as per Dhanashree et al., 2003 with slight modifications for the isolation of Listeria spp. 10 ml in case of cowdung and soil and 10mL in case of milk were transferred to 90 mL of Brain Heart Infusion Broth (BHI, Hi-Media Laboratories, Mumbai). The sample was incubated at 4 o C for 48 h to six week. The enriched broth was streaked on Oxford and PALCAM agar plates and incubated at 30 °C for 24 h.
Grey green colonies with black sunken centres from PALCAM agar plates and black colonies with black sunken centre from Oxford agar, suspected to be Listeria spp. were picked up and cultured on Brain Heart Infusion Agar (BHI, Hi-Media Laboratories, Mumbai). All the suspected isolates were subjected to phenotypic and genotypic characterization. Phenotypic characterization included standard biochemical tests such as catalase test, motility at 25 °C and 37 °C, acid production from glucose, mannitol, rhamnose, xylose and α methyl D mannoside, nitrate reduction, hydrolysis of esculin, methyl red test and Voges Proskauer test.
The confirmation of the Listeria isolates was done by Polymerase Chain Reaction (PCR). Lis1A and Lis1B primer pairs were used for the identification of genus Listeria (Bubert et al., 1992). Then the positive isolates were subjected to species identification by using primer pairs Mono A and Mono B for L. monocytogenes, Ino 2 and Lis1B for L. innocua, Wel 1 and Lis 1B for L. welshimeri, Sel 1 and Lis1B for L. seeligeri, Iva1 and Lis 1B for L. ivanovii (Bubert et al., 1999).
The isolates were grown on BHI agar plates for 24 h at 30 °C. A single colony was transferred to 100 μl of sterile distilled water and heated at 100 °C for 10 minutes in a dry bath (Bangalore Genei Pvt. Ltd., Bangalore) followed by cooling at 4 °C. This served as crude DNA lysate.
The DNA amplification reaction was performed in a Master Cycler gradient thermocycler (Eppendorf, Hamburg, Germany) with a pre-heated lid in PCR tubes (0.5 ml). The cycling conditions for PCR with the primer pair Lis1A and Lis1B included an initial denaturation of DNA at 94 °C for 5 min followed by 30 cycles each of 45 s denaturation at 94 °C, 60 s annealing at 50 °C and 3 min extension at 72 °C, followed by a final extension of 10 min at 72 °C.
The PCR conditions for identification of species started with an initial denaturation temperature of 94 °C for 5 min and were completed with the final elongation step at 72 °C for 8 min. Amplification conditions varied in the denaturation, annealing and elongation step with the different primer pairs. The details of the primers used in the study are as given in the  The PCR products were separated in a 1.2% agarose gel along with a DNA ladder (Lambda 1Kb fermentas) and analyzed using a gel documentation system.
In the present study Listeria spp. was isolated from 1-2% of the samples tested (Table 2). Listeria spp. has been isolated from raw milk, soil and cow dung samples from different parts all over the world. In India few reports are available on Table 1: Primers used in the study and the species identified. (Bubert et al., 1999;Bubert et al., 1992) the incidence of Listeria. In our study, ten isolates were suspected to be Listeria on PALCAM plates and among them three samples were found to be positive for Listeria spp. The isolates were confirmed as L. innocua, L. ivanovii and L. seeligeri. (Figure 1) Figure 1: Identification of the isolates using the genus specific and species specific primer pairs; Lane M -1 Kb Marker; Lane 1-Control L. monocytogenes EGD-e; Lane 2 -4 -Isolates tested with the primer pair Lis1A and Lis1B; Lane 5-7 Listeria species confirmed with species specific primers L. ivanovii was isolated from 1% of the soil samples, L. seeligeri from 0.76% of the cowdung samples and L. innocua from 0.76% of the raw milk samples tested. The presence of L. innocua in milk correlates with the results reported by Dhanashree et al. 2003. Singh et al., (2008 found that out 51 isolates from milk 13 isolates were confirmed as L. monocytogenes. In 2003 Gianfranceschi et al., reports that 17.4% of dairy products were found positive for L. monocytogenes. 60.6% of milk samples from Tiruchirapalli were found positive for L. monocytogenes (Shrinithivihanshini et al., 2011). 16.7% of L. monocytogenes was isolated from raw milk samples commercialized in Portugal (Mena et al., 2004) Raw milk in Malaysia was assessed for the presence of Listeria spp. by Chye et al. (2004). They reported that 4.4% of raw milk samples were positive for Listeria spp. Among them 1.9% were L. monocytogenes, 2.1% were L. innocua and 0.6% were L. welshimeri. Latorre et al. (2009) reported the incidence of L. monocytogenes in milk and fecal samples of cows. 7.1% of fecal samples and 7.3% of milk samples were positive for L. monocytogenes. 16% of the fecal samples of mammals and bird were found positive for L. monocytogenes (Kalorey et al., 2006). Same kind of results was reported by Zaytseva et al., (2007). Soil samples from agriculture fields and animal inhabited areas were examined for the presence of Listeria by Moshtaghi et al. (2003) and found 17.7% of Listeria spp. among them 5.4% were L. monocytogenes, 1.5% were L. ivanovii, 7.7% were L. innocua and 3.1% were L. welshimeri.
The results of our study showed the incidence of Listeria spp. in raw milk, soil and cow dung samples. Very strict measures should be taken to ensure that milk samples are not contaminated by external sources.