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/lp/springer-journals/occurrence-oflisteria-monocytogenes-in-bovine-milk-in-hyderabad-nU0zd4lkN3
- Publisher
- Springer Journals
- Copyright
- Copyright © 2007 by University of Milan and Springer
- Subject
- Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
- ISSN
- 1590-4261
- eISSN
- 1869-2044
- DOI
- 10.1007/BF03175070
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Abstract
Annals of Microbiology, 57 (3) 341-344 (2007) 1 2 3 1 4 Talib Hussain CHANDIO , Aijaz Hussain SOOMRO *, Muhammad Bachal BHUTTO , Parkash DEWANI , Ghiasuddin SHAH 1 2 3 Central Veterinary Diagnostic Laboratory, Sindh, Tandojam, Pakistan; Department of Dairy Technology, Department of Parasitology, Department of Anatomy and Histology, Sindh Agriculture University, Tandojam, Pakistan Received 8 March 2007 / Accepted 13 June 2007 Abstract - In the present study 200 milk samples (50 each from cow, buffalo, market and mastitic milk) were collected from Hyderabad. Among the isolated presumptive Listeria spp., all the isolates were identified as Listeria monocytogenes and showed a prevalence of 6, 8, 10 and 12%, respectively. Through serotyping analysis all isolates were confirmed as Listeria genus, since they agglutinated with polyvalent ‘O’ serum; 12 of the 18 L. monocytogenes isolates were ascribed to serotype 1, and 6 to serotype 4. Furthermore, the pathogenicity was investigated in rabbit and mice through Anton and inoculation tests, respectively. The organism produces keratoconjunctivitis in rabbit while it causes death and presence of necrotic foci in liver, spleen and kidneys of mice. Listeria monocytogenes was found sensitive to ampicillin, chloramphenicol, ciprofloxacin, gentamycin and kanamycin. Key words: Listeria monocytogenes, bovine milk, occurrence, identification, antibiotic susceptibility. INTRODUCTION The incidence of Listeria spp. in raw bovine milk is not well documented. As in rural areas, the consumption of raw Listeria microorganisms are widely distributed in the natu- milk has traditionally been a very common practice. ral environment such as soil, sewage and water and they Therefore the objective of this study was to determine the frequently contaminate foods including a wide variety of incidence of L. monocytogenes in raw milk obtained from dairy and meat products (Pak et al., 2002; Meyer-Broseta dairy farms and retail markets of Hyderabad and its sur- et al., 2003). Among the genus Listeria, which cause the rounding areas and to check their susceptibility to different infection of listeriosis in both animals and humans, Listeria commonly used antibiotics. monocytogenes is the most pathogenic one. Listeria mono- cytogenes is a motile, Gram-positive coccobacillus, non- sporeforming, aerobic to facultative anaerobic, able to sur- MATERIALS AND METHODS vive as intracellular bacterium within leukocytes and grows over a temperature range of 0–45 °C (Rocourt, 1988; Collection of samples. The study was carried out using a Aygun and Pehlivanlar, 2006). Listeria monocytogenes can total of 200 milk samples (50 each from cow, buffalo, mar- be transmitted by the consumption of raw milk and other ket and mastitic milk) were collected randomly from milk products including homemade raw milk dairy products Hyderabad, Pakistan (Table 1). The samples were trans- and by asymptomatic personnel handling such products ported to the Microbiology Section, Central Veterinary (Potter et al., 1984). It is well known that human listerio- Diagnostic Laboratory in sterile screw capped bottles under sis is largely attributable to foodborne transmission of the refrigeration for microbiological analysis. microorganism (Meyer-Broseta et al., 2003; McLauchlin et al., 2004). Several outbreaks of listeriosis associated with Identification of Listeria spp. For the enrichment proce- the consumption of contaminated food products by L. dure, 25 ml of the sample were added to 225 ml monocytogenes have been reported (McLauchlin et al., Enrichment broth (Merck, Darmstadt, Frankfurt, Germany) 1990; Lyytikainen et al., 2000). Under unhygienic milking and incubated at 30 ºC for 24 to 48 h. One-tenth ml of the practices, indirect contamination of milk is likely to occur if enrichment broth was plated onto McBride’s Modified agar L. monocytogenes is present in feeds, faeces, udder sur- (Hitchens, 1995), and plates were incubated at 30 ºC for face, or bedding (Fedio and Jackson, 1992). However, 48 h. The suspected colonies were surface streaked on Listeria monocytogenes may also contaminate milk as a Tryptic Soy agar (Oxoid, England) with 0.6% yeast extract consequence of mastitis or abortion. Asymptomatic cows (Oxoid) and incubated at 30 ºC for 24-48 h. The presump- can shed L. monocytogenes intermittently in milk for many tive determination procedure included colony morphology, months, posing a potential threat to public health (Hird and motility, Gram staining, catalase production and haemolyt- Genigeorgis, 1990). ic activity on sheep blood agar. Isolates were confirmed by S pro- testing for nitrate reduction, urease production, H duction, MR-VP reaction, esculin hydrolysis and ability to * Corresponding author. E-mail: aijaz68@hotmail.com 342 T.H. Chandio et al. TABLE 1 - Collection of milk samples from Hyderabad city (Municipal corporation area) Sampling group Samples Origin Date of collection Place of collection collected (no.) Cow milk 50 Udder (apparently healthy) Aug.-Sept., 2005 Dairy farms located at: old cattle colony, new cattle colony, Ghumanabad and Hur camp. Buffalo milk 50 Udder(apparently healthy) Nov.-Dec., 2005 Dairy farms located at: old cattle colony, new cattle colony, Kiran shoro and Mir colony Latifabad. Market milk 50 Storage tanks (unpasteurized) Feb.-March, 2006 Milk outlets located at: Hiraabad, Tower market, Liberty market, Hussainabad, Latifabad, Phuleli, Nasim Nagar, Qasimabad, Baban Shah colony and Hashmi colony. Mastitis milk 50 Udder (clinical mastitis) May-June, 2006 Dairy farms located at: old cattle colony, new cattle colony, Ghumanabad, Wahadat colony, Hatri, Tando Yosuf, Laloo Lashari, Darya Khan Baig, Hur camp and Mir colony Latifabad. produce acid from glucose, maltose, mannitol, rhamnose blood agar colonies were dew drop like, round, low convex and xylose (Difco, Detroit, Michigan, USA). with entire margin and weak β-haemolytic. These were catalase and MR/VP positive; oxidase, H S production, Serotyping. The isolates were serotyped by slide aggluti- indol, esculin, urease and nitrate reduction negative. The nation technique using polyvalent ‘O’ antisera type 1 and 4 isolates produced acidic reaction in glucose, maltose and (Difco). rhamnose, whereas did not ferment mannitol and xylose sugars. Antibiotic susceptibility profile. Antibiotic susceptibility In the present study occurrence of L. monocytogenes in patterns of all the confirmed Listeria isolates were per- raw milk samples showed that 6, 8, 10 and 12% samples, formed by standard disc diffusion method as described by respectively from cow, buffalo, market milk and animals Bauer et al. (1966). Antibiotic discs used were ampicillin, suffering from mastitis, were positive. Overall, 9% chloramphenicol, ciprofloxacin, cotrimoxazole, erythromy- (18/200) of the raw milk samples examined were positive cin, gentamycin, kanamycin and penicillin (Oxoid). for the genus Listeria (Table 2); all the isolates were iden- tified as L. monocytogenes. No other Listeria species were Determination of pathogenicity. For pathogenicity of identified during this study. The incidence of Listeria spp. in the microorganism Anton test was performed according to raw milk samples found in this study is comparable with the Quinn et al. (1994). A loopful of L. monocytogenes culture result of Sagun et al. (2001), who reported that 6 of 250 was inoculated into the conjunctival sack of young rabbit to raw milk samples (2.4%) in Van, a city located in eastern check the virulence of the isolates. Turkey, were positive for Listeria spp., in which prevalence Furthermore, L. monocytogenes isolates were tested for of L. monocytogenes was 1.2%. In another study, the inci- mouse pathogenicity by propagating the isolates in Brain- dence of Listeria spp. was found to be 10% and L. mono- Heart Infusion (BHI, Difco), incubated at 37 ºC with shak- cytogenes 5% in 80 raw milk samples from Ankara (Erol ing at 300 rpm in a water-bath for 18-21 h. Bacterial cells and Sireli, 2002). The sources of Listeria spp. in raw milk were centrifuged at 300 x g for 30 min and washed in have been reported to be from faeces (Griffiths, 1989) and 0.85% sterile saline solution according to Murray et al. from environmental contamination during milking, storage (1985). and transport of infected cows in dairy farms and poor A total of 18 Swiss mice were inoculated with 0.1 ml silage quality (Bemrah et al., 1998). washed suspension by intraperitoneal injection. Cell viabil- ity was verified by dispersion in BHI agar plates. Mice were TABLE 2 - Incidence of Listeria monocytogenes isolated from raw observed every 24 h during the week. Infection was con- milk firmed by necropsis verifying the presence of Listeria in the spleen. Sampling Samples L. monocytogenes Serotype group collected (no.) number (%) (no. of positive) RESULTS AND DISCUSSION Cow milk 50 3 (6) 1 (2), 4 (1) Buffalo milk 50 4 (8) 1 (3), 4 (1) The phenotypic identification revealed that all the 18 iso- Market milk 50 5 (10) 1 (3), 4 (2) lates were Gram positive short rods with rounded ends; Mastitis 50 6 (12) 1 (4), 4 (2) some were curved, occur singly, in short chains and cells samples at an angle with each other in v forms. Isolates were Total 200 18 (9) 1 (12), 4 (6) determined aerobic, non-sporeforming and motile. On Ann. Microbiol., 57 (3), 341-344 (2007) 343 TABLE 3 - Antibiotic susceptibility pattern of Listeria monocytogenes Sampling Isolated Antibiotics (µg/ disc) group tested (no.) AMP (10) CIP (5) SXT (25) C (30) E (15) CN (10) K (30) P (10) SR SR SR SR SR SR SR SR (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) Cow milk 3 100 – 100 – 100 – 100 – 100 – 100 – 100 – 100 – Buffalo milk 4 100 – 100 – 75 25 100 – 100 – 100 – 100 – 100 – Market milk 5 100 – 100 – 60 40 100 – 80 20 100 – 100 – 80 20 Mastitis milk 6 100 – 100 – – 100 100 – 100 – 100 – 100 – – 100 AMP: Ampicillin, CIP: Ciprofloxacin, SXT: Sulphamethoxazole/trimethprim (Cotrimoxazole), C: Chloramphenicol, E: Erythromycin, CN: Gentamycin, K: Kanamycin, P: Penicillin. S: Susceptible, R: Resistant. Our results are in agreement with the findings of other of L. monocytogenes (McLauchlin, 1990), including vegeta- investigators (Lovett et al., 1987; Farber et al., 1988; bles, dairy products such as pasteurized milk and cheese Liewen and Plautz, 1988). However, Spanish researchers and meat products. found a really higher incidence of L. monocytogenes in the Listeria is of particular concern to the food industry and milk, being respectively 45.3% and 44.7%, (Fernandez et public health regulatory authorities because it can grow at al., 1987). refrigerator temperatures and is ubiquitous in nature On serotyping all isolates confirmed as Listeria genus (Farber and Spiers, 1987; Lamont et al., 1988). Listeria agglutinated with polyvalent ‘O’ serum; 12 of the 18 L. monocytogenes milk contamination may occur as a conse- monocytogenes isolates were ascribed to serotype-1, and 6 quence of mastitis (Gitter et al., 1980), and contaminated to serotype-4 (Table 2). Twelve Swiss mice were inoculat- raw milk may be transported to factories to be processed ed with serotype-1 strains; it was observed that all the into an array of products. However, contamination may mice had died on day 3 or 4 post inoculation. On the other come from other sources such as the dairy farm environ- side 6 mice were inoculated with serotype-4 strains and ment, water, healthy carriers and aerosols (Farber, 1992). showed no pathogenicity in the following 7 days. These The microbiological risk associated with the presence of results are in accordance with the observations of Erdeling L. monocytogenes in raw milk must be evaluated in rela- et al. (2000) and Vázquez-Salinas et al. (2001), who also tion to its final destination. When milk undergoes heat reported that serotype-1 strains are virulent for mice. treatment like pasteurization, which is then used to pro- Furthermore the development of characteristic lesions such duce dairy products, the risk is minimal. But when it is used as focal necrosis of liver, spleen and lungs, were also to make products without any heat treatment, this can be observed and L. monocytogenes cells were recovered from hazardous, especially in cheeses with short ripening peri- liver, spleen and kidneys of the dead mice. After Anton ods, where L. monocytogenes is commonly found test, the organisms produced keratoconjunctivitis within 36 (Loncaveric et al., 1995; Margolles et al., 1996). In addi- h after inoculation in rabbits; similar findings have been tion, the risk of cross-contamination during ripening and reported by Mahmood et al. (2003). post-processing steps is also an important factor. Table 3 summarises the antibiotic susceptibility profile The results of this study suggest that milk in farms from of 18 L. monocytogenes isolates from bovine milk samples. Hyderabad City is frequently contaminated with pathogen- Resistance to 3 out of 8 commonly used antibiotics was ic bacteria. Additional studies are needed to verify our observed. The majority of the isolates displayed resistance results, identify risk factors for the presence of Listeria in to erythromycin, penicillin and cotrimoxazole, whereas all raw milk, and to assess the public health impact. The the isolates were sensitive to the other antibiotics tested. prevalence of raw milk consumption from contaminated Listeria spp. is generally considered to be still susceptible farms in Hyderabad City, not only emphasizes the need to to most antibiotics (Charpentier and Courvalin, 1999). make continuous efforts to improve pasteurization proce- However, Walsh et al. (2001) reported that 10.9% of L. dures, but also necessitates the closing of unauthorized monocytogenes isolates were resistant to one or more milk collection centres and assessment of actual health antibiotics and resistance to penicillin and tetracycline was risks. more frequently observed. A similar observation has been made in our study. A higher resistance of the isolates determined from the mastitic milk to 3 out of 8 antibiotics REFERENCES used is attributed to the development of resistance due to wide and indiscriminate use of common ailments of dairy Aygun O., Pehlivanlar S. (2006). Listeria spp. in the raw milk and animals. dairy products in Antakya, Turkey. Food Contr., 17: Listeriosis is an emergent illness with a low incidence, 676–679. but with a high fatality rate, especially in immunocompro- Bauer A.W., Kirby W.M., Sherris J.C. (1966). Antibiotic suscepti- mised individuals (Norrung, 2000). Different kinds of foods bility testing by a standard single disc method. Am. J. Clin. have been reported to be associated with the transmission Pathol., 45: 493-496. 344 T.H. Chandio et al. Bemrah N., Sanaa M., Cassin M.H., Griffiths M.W., Cerf O. Lyytikainen O., Autio T., Maijala R., Ruutu P., Honkanen-Buzalski (1998). Quantitative risk assessment of human listeriosis T., Miettinen M., Hatakka M., Mikkola J., Veli-Jukka A., from consumption of soft cheese made from raw milk. Prev. Johansson T., Rantala L., Aalto T., Korkeala H., Siitonen A. (2000). An outbreak of Listeria monocytogenes serotype 3a Veter. Med., 37: 129-145. infections from butter in Finland. J. Infect. Dis., 181: 1838- Charpentier E., Courvalin P. (1999). Antibiotic resistance in Listeria spp. Antimicr. Agents Chemoth., 43: 2103.2108. Mahmood M.S., Ahmed A.N., Hussain I. (2003). Prevalence of Erdeling S., Ainsworth A.J., Austin F.W. (2000). Pathogenicity Listeria monocytogenes in poultry meat, poultry meat prod- and production of virulence factors by Listeria monocyto- ucts and other related inanimates at Faisalabad. Pak. J. genes isolates from channel catfish. J. Food Prot., 63: 613- Nutr., 2: 346-349. Margolles A., Rodrıguez A., de-los-Reyes-Gavilan C.G. (1996). Erol I., Sireli U.T. (2002). Occurrence and contamination levels Some chemical and bacteriological characteristics of regional of Listeria spp. in milk and dairy products in Ankara. FEMS cheeses from Asturias, Spain. J. Food Prot., 59: 509-515. Symposium on the Versatility of Listeria Species. 10-11 McLauchlin J. (1990). Distribution of serovars of Listeria mono- October, Izmir, Turkey. cytogenes isolated from different categories of patients with Farber J.M., Spiers J.I. (1987) Monoclonal anti-bodies directed listeriosis. Eur. J. Clin. Microbiol. Infect. Dis., 9: 210-213. against the flagellon antigens of Listeria species and their McLauchlin J., Greenwood M.H., Pini P.N. (1990). The occurrence potential in EIA-based on methods. J. Food Prot., 50: 479- of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis. Int. J. Food Microbiol., Farber J.M., Sanders G. W., Malcolm S.A. (1988). The presence 10: 255-262. of Listeria spp. in raw milk in Ontario. Can. J. Microbiol., 34: McLauchlin J., Mitchell R.T., Smerdon W.J., Jewell K. (2004). 95-100. Listeria monocytogenes and listeriosis: a review of hazard Farber J.M. (1992). Prevention and control of food-borne liste- characterization or use in microbiological risk assessment of foods. Int. J. Food Microbiol., 92: 15-33. riosis. Dairy Food Environ. Sanit., 122: 334-340. Meyer-Broseta S., Diot A., Bastian S., Riviere J., Cerf O. (2003). Fedio W.M., Jackson H. (1992). On the origin of Listeria mono- Estimation of low bacterial concentration: Listeria monocyto- cytogenes in raw bulk-tank milk. Int. Dairy J., 2: 197-208. genes in raw milk. Int. J. Food Microbiol., 80: 1-15. Fernandez Garayzabal J.F., Dominguez L., Vazquez J.A., Gomez- Murray H.W., Gerge L., Nathan C.L. (1985). Activation of mouse Lucia E., Rodriguez Ferri E.R., Suarez G. (1987). Occurrence peritoneal macrophages in vitro and in vivo by interferon of Listeria monocytogenes in raw milk. Vet. Rec., 120: 258- gamma. J. Immunol., 134: 1619-622. Norrung B. (2000). Microbiological criteria for L. monocytogenes Gitter M., Bradley R., Blampied P.H. (1980). Listeria monocyto- in foods under special consideration of risk assessment genes in bovine mastitis. Vet. Rec., 107: 390-393. approaches. Int. J. Food Microbiol., 62: 217-221. Griffiths M.W. (1989). Listeria monocytogenes: Its importance in Pak S.I., Spahr U., Jemmi T., Salman M.D. (2002). Risk factors dairy industry. J. Sci. Food Agri., 47: 133-158. for L. monocytogenes contamination of dairy products in Hird D.W., Genigeorgis C. (1990). Listeriosis in food animals: Switzerland, 1990-1999. Prev. Vet. Med., 53: 55-65. clinical signs and livestock as a potential source of direct Potter M.E., Kaufmann A.F., Feldman P.A. (1984). Un-pasteur- (nonfoodborne) infection for humans. In: Miller A.J., Smith ized milk: the hazards of a health fetish. J. Am. Med. Assoc., J.L., Somkuti G.A., Eds, Foodborne Listeriosis, Elsevier Sci. 252: 2048-2052. Publ., Amsterdam, The Netherlands, p. 31. Quinn, P.J., Carter M.E., Markey B., Carter G.R. (1994). Clinical Hitchens A.D. (1995). Listeria monocytogenes. In: FDA Veterinary Microbiology. Wolfe Publishing Co. London, pp: th Bacteriological Analytical Manual, 8 edn., AOAC 170-174. International, Arlington, VA, pp. 10.1-10.13. Rocourt J. (1988). The recognition and identification of Listeria Lamont R.J., Postlethwaite R., MacGowan A.P. (1988). Listeria species by classical methods. Infeksiyon Dergisi (Turkish J. monocytogenes and its role in human infection. J. Infect., Infection) 2: 471-485 17: 7-28. Sagun E., Sancak Y.C., Isleyici O., Ekici K. (2001). The presence Liewen M.B., Plautz M.W. (1988). Occurence of Listeria monocy- and prevalence of Listeria species in milk and herby cheese togenes in raw milk in Nebraska. J. Food Prot., 51: 840-841. in and around Van. Turk. J. Vet. Anim. Sci., 25: 15-19. Loncaveric S., Danielsson-Tham M.L., Tham W. (1995). Vázquez-Salinas C., Rodas-Suárez O., Quiñones-Ramírez E.I. Occurrence of Listeria monocytogenes in soft and semi-soft (2001). Occurrence of Listeria species in raw milk in farms on cheeses in retail outlets in Sweden. Int. J. Food Microbiol., the outskirts of Mexico City. Food Microbiol., 18: 177-181. 26: 245-250. Walsh D., Duffy G., Sheridan J.J., Blair I.S., McDowell D.A. Lovett J., Francis D.W., Hunt J.M. (1987). Listeria monocyto- (2001). Antibiotic resistance among Listeria, including genes in raw milk: detection, incidence, and pathogenicity. J. Listeria monocytogenes, in retail food. J. Appl. Microbiol., 90: Food Prot., 50: 188-192. 517-522.
Journal
Annals of Microbiology – Springer Journals
Published: Nov 20, 2009