Advances in Animal and Veterinary Sciences

Download PDF Download ePUB
 Short Communication

Short Communication

Advances in Animal and Veterinary Sciences 2 (5S): 1 – 4
Special Issue–5 (2014) (Listeriosis and its public health concerns)

Prevalence of Listeria Species in Environment and Milk Samples

Sangeetha Mahadevaiah Shantha, Shubha Gopal*

    Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore – 570 006, Karnataka, India

*Corresponding author:shubhagopal_mysore@yahoo.com

ARTICLE CITATION: Shantha SM, Gopal S (2014). Prevalence of Listeria species in environment and milk samples. Adv. Anim. Vet. Sci. 2 (5S): 1 – 4.
Received: 2014–06–06, Revised: 2014–06–20, Accepted: 2014–08–20
The electronic version of this article is the complete one and can be found online at ( http://dx.doi.org/10.14737/journal.aavs/2014/2.5s.1.4 ) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

ABSTRACT

The previous notion that infection by pathogenic Listeria (listeriosis) is not an important health problem in India starts to be revised now, mainly due to changing habits of the production, distribution and storage of food, favoring the multiplication and spread of cold–tolerant bacteria like Listeria. Despite this altering scenario, data on the prevalence of Listeria on the Indian subcontinent are still rather sporadic. We investigated the occurrence of Listeria species in environmental and raw milk samples from Mysore city (moderately hot, semi–arid climate). Environmental samples included cow dung from cowshed, grazing pasture and soil samples from vegetable–cultivation land. Cold enrichment was used to recover Listeria species from raw cow milk (from individual animals) (n=130), cow dung (collected from individual animals) (n=130) and soil (n=100) samples. 10 g sample in case of cowdung and soil and 10mL in case of milk were transferred to 90 mL BHI broth and incubated at 4oC for two weeks. Aliquots from the enriched broth were streaked on Oxford and PALCAM plates, at weekly intervals. Listeria 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 the canonical panel of carbohydrates, nitrate reduction, esculin hydrolysis, methyl red and Voges Proskauer reaction. Genus– and species–specific primers were used for PCR differentiation of the isolates. 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. Strategies to reduce the incidence of Listeria in environment and milk samples should be implemented.

INTRODUCTION

Listeria is a ubiquitous organism and has been isolated from variety of sources like environmental sources, soil, sewage; surface water, animals etc., Variety of foods like raw vegetables, fruits, meat, fish, poultry, raw milk and its processing environments may also be contaminated with Listeria spp., and act as a vehicle for the dissemination of the Listeria spp. The genus Listeria has fifteen species namely Listeria monocytogenes, L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, L. rocourtiae, L. marthii, L. fleishmanii, L. weihenstephanensis, L. floridensis, L. aquatica, L. cornellensis, L. riparia, and L. grandensis) (Khelef et al., 2006; McLauchlin and Rees 2009; Bertsch et al., 2013; Halter et al., 2013; den Bakker et al., 2014) L. monocytogenes and L. ivanovii are pathogenic to humans and animals (Liu 2006).

Milk is a nutritious food which is easily contaminated during processing and acts as a good medium for the growth and multiplication of the pathogens (Agarwal et al., 2012). India is one of the largest producers of milk in the world with the highest number of cattle (Kalorey et al., 2008). Numerous outbreaks of listeriosis due to consumption of milk and milk products contaminated with L. monocytogenes are reported all over the world (Fleming et al., 1985, Dalton et al., 1997, Koch et al., 2010, CDC, 2008). Both pathogenic and nonpathogenic species of Listeria has been isolated from raw milk samples (Hayes et al., 1986; Massa et al., 1990; Kozak et al., 1996; Gaya et al., 1998; Unlu et al., 1998; Yoshida et al., 1998; Carlos et al., 2001; Aygun et al., 2006; Nero et al., 2008; Aurora et al., 2009; Vanegas et al., 2009; Sarangi et al., 2009; Jami et al., 2010; Soni et al., 2013; Rahimi et al., 2010; Jamali et al., 2013), milk products (Molla et al., 2004) and its processing environments (Chambel et al., 2007; Doijad et al., 2011).

L. monocytogenes has been isolated from organically fertilized soils (Szymczak et al., 2013) and Botzler et al., (1974) reported that L. monocytogenes can survive in soil and water. Listeria spp. has been isolated from the fecal samples of the mammals and birds (Yadav et al., 2009; Yadav et al., 2011).

The presence of Listeria spp. in variety of foods, milk and environmental samples has been reported from all over the world and few reports are available on the incidence of Listeria in India. The present study was undertaken to study the incidence of Listeria spp. in raw cow milk, cow dung and soil from vegetable–cultivation land and grazing Pasteur.


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 oC for 48 h to six week. The enriched broth was streaked on Oxford and PALCAM agar plates and incubated at 30 oC 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 oC 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 oC for 10 minutes in a dry bath (Bangalore Genei Pvt. Ltd., Bangalore) followed by cooling at 4 °C. This served as crude DNA lysate.

PCR amplification was performed in 50 μl reaction mixture containing 5 μl of 10X PCR buffer; 1 μl of 10 mM dNTP mix; 0.5 μl of 10 μM of each primer; 0.25 μl of 5U/μl of Taq polymerase; 4 μl of 25 mM MgCl2; 2 μl DNA template; 36.75 μl of distilled water. All the reagents used in PCR were purchased from Fermentas.

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 oC for 5 min followed by 30 cycles each of 45 s denaturation at 94 oC , 60 s annealing at 50 oC 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 oC for 5 min and were completed with the final elongation step at 72 oC 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 Table 1.

For Mono A and Mono B primer pair denaturation temperature is 94 oC for 45 s, annealing temperature of 55 oC for 60 s and elongation step at 72 oC for 60 s. For Ino2 and Lis1B pair denaturation temperature is 94 oC for 45 s, annealing temperature of 62 oC for 60 s and elongation step at 72 oC for 45 s. For Sel1, Lis1B pair, Wel1, Lis1B pair and Iva1, Lis 1B pair denaturation temperature is 95 oC for 30 s, annealing temperature of 62 oC for 30 s and elongation step at 72 oC for 90 s.

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 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).

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.

ACKNOWLEDGEMENTS

Dr. Shubha Gopal thank University Grants Commission, New Delhi Govt. of India for their financial support. Grant No. F.No.39–201/2010 (SR).

Sangeetha M S thanks University Grants Commission, New Delhi, Govt. of India, for providing the Rajiv Gandhi National Fellowship. F.14-2(SC)/2010(S-III)

CONFLICT OF INTEREST

The author confirms that this article has no conflict of interest.

REFERENCES

Agarwal A, Awasthi V, Dua A, Ganguly S, Garg V, Marwaha SS (2012). Microbiological profile of Milk: Impact on Household Practices. Indian JPublic Health, 56(1): 88 – 94
http://dx.doi.org/10.4103/0019-557X.96984
PMid:22684182

Aurora R, Prakash A, Prakash S (2009). Genotypic characterization of Listeria monocytogenes isolated from milk and read to eat indigenous milk products. Food Control, 20: 835 – 839
http://dx.doi.org/10.1016/j.foodcont.2008.10.017

Aygun O, Pehlivanlur S (2006). Listeria spp. in the raw milk and dairy products in Antakya, Turkey. Food Control, 17: 676 – 679
http://dx.doi.org/10.1016/j.foodcont.2005.09.014

Bertsch D, Rau J, Eugster MR, Haug MC, Lawson PA, Christophe Lacroix C, Meile L (2013). Listeria fleischmannii sp. nov., isolated from cheese. Int. J. Syst. Evol. Microbiol., 63: 526 – 532
http://dx.doi.org/10.1099/ijs.0.036947-0
PMid:22523164

Botzler RG, Cowan AB, Wetzler TF (1974). Survival of Listeria monocytogenes in soil and water. J. Wildl. Dis., 10: 204 – 212
http://dx.doi.org/10.7589/0090-3558-10.3.204
PMid:4210764

Bubert A, Hein I, Rauch M, Lehner A, Yoon B, Goebel W, Wagner M (1999). Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl. Environ. Microbiol., 65: 4688 – 4692
PMid:10508109 PMCid:PMC91627

Bubert A, Kohler S, Goebel W (1992). The homologous and heterologous regions within the iap gene allow genus– and species specific identification of Listeria spp. by polymerase chain reaction. Appl. Environ. Microbiol., 58: 2625 – 2632
PMid:1514809 PMCid:PMC195830

Carlos VS, Oscar R–S, Irma Q–R (2001). Occurrence of Listeria species in raw milk in farms on the outskirts of Mexico City. Food Microbiol., 18: 177 – 181
http://dx.doi.org/10.1006/fmic.2000.0389

Chambel L, Sol M, Fernandes I, Barbosa M, Zilhão I, Barata B, Jordan S, Perni S, Sharma G, Adriao A, Faleiro L, Requena T, Pelaez C, Andrew PW, Tenreiro R (2007). Occurrence and persistence of Listeria spp. in the environment of ewe and cow's milk cheese dairies in Portugal unveiled by an integrated analysis of identification, typing and spatial–temporal mapping along production cycle. Int. J. Food Microbiol., 116: 52 – 63
http://dx.doi.org/10.1016/j.ijfoodmicro.2006.12.035
PMid:17337311

Chyle FY, Abdullah A, Ayob MK (2004). Bacteriological quality and safety of raw milk in Malaysia. Food Microbiol., 21: 535 – 541
http://dx.doi.org/10.1016/j.fm.2003.11.007

Dalton CB, Austin CC, Sobel J, Hayes PS, Bibb WF, Graves LM, Swaminathan B, Proctor ME, Griffin PM (1997). An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk. New Engl. J Med., 336: 100 – 105
http://dx.doi.org/10.1056/NEJM199701093360204
PMid:8988887

den Bakker HC, Warchocki S, Wright EM, Allred AF, Ahlstrom C, Manuel CS, Stasiewicz MJ, Burrell A, Roof S, Strawn L, Fortes ED, Nightingale KK, Kephart D, Wiedmann M (2014). Five new species of Listeria (L. floridensis sp. nov, L. aquatica sp. nov., L. cornellensis sp. nov. L. riparia sp. nov., and L. grandensis sp. nov.) from agricultural and natural environments in the United States. Int. J. Syst. Evol. Microbiol.: ijs.0.052720–0v1–ijs.0.052720–0.

Dhanashree B, Otta SK, Karunasagar I, Goebel W, Karunasagar I (2003) Incidence of Listeria species in clinical and food samples in Mangalore, India. Food Microbiol., 20: 447 – 453
http://dx.doi.org/10.1016/S0740-0020(02)00140-5

Doijad S, Barbuddhe SB, Garg S, Kalekar S, Rodrigues J, D'costa D, Bhosle S, Chakraborty T (2011). Incidence and genetic variability of Listeria species from three milk processing plants. Food Control, 22: 1900 – 1904
http://dx.doi.org/10.1016/j.foodcont.2011.05.001

Fleming DW, Cochi SL, MacDonald KL, Brondum J, Hayes PS, Plikaytis BD, Holmes MB, Audurier A, Broome CV, Reingold AL (1985). Pasteurized milk as a vehicle of infection in an outbreak Listeriosis. The New Engl. J. Med., 312(7): 404 – 407
http://dx.doi.org/10.1056/NEJM198502143120704
PMid:3918263

Gaya P, Sanchez J, Medina M, Nunez M (1998). Incidence of Listeria monocytogenes and other Listeria species in raw milk produced in Spain. Food Microbiol., 15: 551 – 555
http://dx.doi.org/10.1006/fmic.1997.0175

Gianfranceschi M, Galtuso A, Tartaro S, Aureli P (2003). Incidence of Listeria monocytogenes in food and environmental samples in Italy between 1990 and 1999: Serotype distribution in food, environmental and clinical samples. Eur. J. Epidemiol., 18: 1001 – 1006
http://dx.doi.org/10.1023/A:1025849532417 PMid:14598931

Halter EL, Neuhaus K, Scherer S (2013). Listeria weihenstephanensis sp. nov., isolated from the water plant Lemna trisulca taken from a freshwater pond. Int. J. Syst. Evol. Microbiol., 63: 641 – 647
http://dx.doi.org/10.1099/ijs.0.036830-0
PMid:22544790

Hayes PS, Feeley JC, Graves LM, Ajello GW, Fleming DW (1986). Isolation of Listeria monocytogenes from Raw Milk. Appl. Environ. Microbiol., 51(2): 438 – 440
PMid:3082282 PMCid:PMC238890

Jamali H, Radmelia B, Thong KL (2013). Prevalence, characterization and antimicrobial resistance of Listeria species and Listeria monocytogenes isolates from raw milk in farm bulk tanks. Food Control, 34: 121 – 125
http://dx.doi.org/10.1016/j.foodcont.2013.04.023

Jami S, Jamshidi A, Khanzadi S (2010). The presence of Listeria spp. in raw milk samples in Mashhad, Iran. World Appl. Sci. J., 10(2): 249 – 253

Kalorey DR, Kurkure NV, Warke SR, Rawool DB, Malik SVS, Barbuddhe SB (2006). Isolation of pathogenic Listeria monocytogenes in faeces of wild animals in captivity. Comp Immunol. Microbiol., 29:295 – 300
http://dx.doi.org/10.1016/j.cimid.2006.07.002
PMid:17034860

Kalorey DR, Warke SR, Kurkure NV, Rawool DB, Barbuddhe SB (2008). Listeria species in bovine raw milk: A large survey of central India. Food Control, 19: 109 – 112
http://dx.doi.org/10.1016/j.foodcont.2007.02.006

Khelef N, Lecuit M, Buchrieser C, Cabanes D, Dussurget O, Cossart P (2006). Bacteria: firmicutes, Cyanobacteria. In M. Dworkin (Ed.), Prokaryotes A handbook on the biology of bacteria, Springer, New York, Volume 4: pp. 404 – 476

Koch J, Dworak R, Prager R, Becker B, Brockmann S, Wicks A, Wichmann–Schauer H, Hof H, Werber D, Stark K (2010). Large listeriosis outbreak linked to cheese made from Pasteurized milk, Germany, 2006–2007. Foodborne pathog. Dis., 7(12): 1581 – 1584
http://dx.doi.org/10.1089/fpd.2010.0631
PMid:20807110

Kozak J, Balmer T, Byrne R, Fisher K (1996). Prevalence of Listeria monocytogenes in foods: Incidence in dairy products. Food Control, 7(4/5): 215 – 221
http://dx.doi.org/10.1016/S0956-7135(96)00042-4

Latorre AA, Kessel JASV, Karns JS, Zurokowski MJ, Pradhan AK, Zadoks RN, Boor KJ, Schukken YH (2009). Molecular Ecology of Listeria monocytogenes: Evidence for a Reservoir in milking equipment on a dairy farm. Appl. Environ. Microbiol., 75(5): 1315 – 1323
http://dx.doi.org/10.1128/AEM.01826-08
PMid:19114514 PMCid:PMC2648159

Liu D (2006). Identification, subtyping and virulence determination of Listeria monocytogenes, an important foodborne pathogen. J. Med. Microbiol., 55: 645 – 659
http://dx.doi.org/10.1099/jmm.0.46495-0
PMid:16687581

Massa S, Cesaroni D, Poda G, Trovatelli LD (1990). The incidence of Listeria spp. in soft cheeses, butter and raw milk in the provenance of Bologia. J. Appl. Bacteriol., 68: 153 – 156
http://dx.doi.org/10.1111/j.1365-2672.1990.tb02560.x
PMid:2318744

McLauchlin J, Rees CED (2009). Genus Listeria Pirie 1940a, 383AL. In Bergey's Manual of Systematic Bacteriology. The Firmicutes (2nd Ed.), Springer, New York, ISBN 978–0–387–95041–9
PMCid:PMC2756898

Mena C, Almeida G, Carneiro L, Teixeira P, Hogg T, Gibbs PA (2004). Incidence of Listeria monocytogenes in different food products commercialized in Portugal. Food Microbiol., 21: 213 – 216
http://dx.doi.org/10.1016/S0740-0020(03)00057-1

Molla B, Yilma R, Alemayehu D (2004). Listeria monocytogenes and other Listeria species in retail meat and milk products in Addis Ababa, Ethiopia. Ethiop. J. Health Dev., 18(3): 208 – 212

Moshtaghi H, Garg SR, Mandokhot UV (2003). Prevalence of Listeria in soil. Indian J. Exp Biol., 41: 1466 – 1468
PMid:15320505

Nero LA, de Maltos MR, Barros de AF, Ortolani MBT, Beloti V, de Melo Franco BDG (2008). Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil: Occurrence and interference of indigenous micro biota in their isolation and development, Zoonoses Public Health, 55: 299 –305
http://dx.doi.org/10.1111/j.1863-2378.2008.01130.x
PMid:18489543

Rahimi E, Ameri M, Momtaz H (2010). Prevalence and antimicrobial resistance of Listeria species isolated from milk and dairy products in Iran. Food Control, 21: 1448 – 1452
http://dx.doi.org/10.1016/j.foodcont.2010.03.014

Sarangi LN, Panda HK, Priyadarshini A, Sahoo S, Palani TK, Ranbhijuli S, Senapati S, Mohanty DN (2009). Prevalence of Listeria species in milk samples of cattle of Odisha. Indian J. Comp. Microbiol., Immunol. Infect. Dis., 30(2): 135 – 136

Singh P, Prakash A (2008). Isolation of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes from milk samples sold under market conditions at Agra region, Acta Agric. Slov., 92(1): 83 – 88

Soni DK, Singh RK, Singh DV, Dubey SK (2013). Characterization of Listeria monocytogenes isolated from Ganges water, human clinical and milk samples of Varanasi, India. Infect., Genet. Evol., 14: 83 – 91
http://dx.doi.org/10.1016/j.meegid.2012.09.019
PMid:23201044

Srinithivihahshini ND, Sheela MM, Mahamuni D, Chithra DR (2011). Occurrence of Listeria monocytogenes in Food and ready to Eat food products available in Thiruchirapalli, Tamil Nadu, India. World J. Life Sci. Med. Res., 1(4): 70 – 75

Szymczak B, Szymczak M, Sawicki W, Dąbrowski W (2013). Anthropogenic impact on the presence of L. monocytogenes in soil, fruits and vegetables. Folia Microbiol., 59: 23 – 29
http://dx.doi.org/10.1007/s12223-013-0260-8
PMid:23775320 PMCid:PMC3889501

Unlu GV, Unlu M, Bakici MZ (1998). Incidence of Listeria spp. from raw milk in Sivas. Tr. J. Medical sciences, 28: 389 – 392

Venegas MC, Vasquez E, Martinez AJ, Rueda AM (2009). Detection of Listeria monocytogenes in raw whole milk for human consumption in Columbia by real time PCR. Food Control, 20: 430 – 432
http://dx.doi.org/10.1016/j.foodcont.2008.07.007

Yadav MM, Roy A (2009). Prevalence of Listeria spp. including Listeria monocytogenes from apparently healthy sheep of Gujarat State, India. Zoonoses Public Health, 56: 515 – 524
http://dx.doi.org/10.1111/j.1863-2378.2008.01201.x
PMid:19243567

Yadav MM, Roy A, Bhandari B, Jain RG (2011). Prevalence of Listeria species including L. monocytogenes from apparently healthy animals at Baroda Zoo, Gujarat State, India. J. Threat. taxa, 3(7): 1929 – 1935

Yoshida T, Kato Y, Sato M, Hirai K (1998). Sources and routes of contamination of raw milk with Listeria monocytogenes and its control. J. Vet. Med. Sci., 60(10): 1165 – 1168
http://dx.doi.org/10.1292/jvms.60.1165
PMid:9819776

Zaytseva E, Ermolaeva S, Somov GP (2007). Low genetic diversity and epidemiological significance of Listeria monocytogenes isolated from wild animals in the far east of Russia. Infect. Genet. Evol., 7: 736 – 742.
http://dx.doi.org/10.1016/j.meegid.2007.07.006
PMid:17716956