Studies on the Isolation of Listeria monocytogenes from Food, Water, and Animal Droppings: Environmental Health Perspective

Veterinarians, Medical doctors, Environmentalists and other scholars know listeriosis by various names such as tiger river disease, silage sickness, leukocytosis, cheese sickness. Interestingly, Gustav Hulphers first discovered the bacteria and named it Bacillus hepatis. However, he did not preserve his bacterial strains, and the bacterium was later recognized as Listeria monocytogenes (Vishal. 2004). Fifteen years later, Murray et al. (1926) also identified bacteria identical to L. monocytogenes, as the cause of monocytosis in rabbit and guinea pig. The scholars preserved the isolates of the bacteria (ATCC no. 15313; ATCC no. 4428) so the credit goes to Murray et al. for isolation of L. monocytogenes for the first time. In addition to the existing knowledge, Pirie finally named the species L. monocytogenes in 1940 and thereafter it was included in the 6th edition of Bergey`s Manual of Determinative Bacteriology (1948).


Introduction
Veterinarians, Medical doctors, Environmentalists and other scholars know listeriosis by various names such as tiger river disease, silage sickness, leukocytosis, cheese sickness.Interestingly, Gustav Hülphers first discovered the bacteria and named it Bacillus hepatis.However, he did not preserve his bacterial strains, and the bacterium was later recognized as Listeria monocytogenes (Vishal. 2004).Fifteen years later, Murray et al. (1926) also identified bacteria identical to L. monocytogenes, as the cause of monocytosis in rabbit and guinea pig.The scholars preserved the isolates of the bacteria (ATCC no. 15313;ATCC no. 4428) so the credit goes to Murray et al. for isolation of L. monocytogenes for the first time.In addition to the existing knowledge, Pirie finally named the species L. monocytogenes in 1940 and thereafter it was included in the 6th edition of Bergey`s Manual of Determinative Bacteriology (1948).
In the early 1980's Listeriosis was classified under anthropozoonoses, which was changed to amphixenoses in the late 1990s.It lacks its true definition of Zoonotic disease because of involvement of an inanimate reservoir (food and Environment) as the major cause of listeriosis.Up to 1961, L. monocytogenes was regarded as the one and only species of the genus Listeria but later other species have been identified.Listeriosis is of great public health concern because of its high mortality (20 to 30%) and its common source epidemic potential.The most important aspect of this organism in food hygiene is the ability of the bacteria to survive in a wide range of temperatures and to make biofilms on various environmental surfaces, which serve as natural habitats or reservoirs (Duggan and Phillips, 1998).Direct transmission is possible, especially among veterinarians, performing gynecological interventions with aborted animals.Animals may be diseased or asymptomatic carriers of L. monocytogenes shedding the organism in their faeces.Thus, earlier it was believed that L. monocytogenes was causing disease by direct transmission from animals to humans.Today it is generally considered that ingestion is the main mode of infection with food and water being the main vehicles of infection.A listeriosis outbreak in the Maritime Provinces of Canada (1981) was indeed related to food but it was not until the outbreak of California from January to August 1985 (Linnan et al., 1988) that food was recognized as an important vehicle of Listeria transmission.According to Mead et al. (1999) food is an important vehicle of Listeria transmission in 99% of listeriosis cases.Risk assessments by the World Health Organisation (WHO) have estimated that 99% of all Listeriosis could be eliminated if the L. monocytogenes level never exceed 1000 cfu/g in food at the point of consumption.Nosocomial infection has also been described, placing physicians and other medical staff at risk.
Listeria monocytogenes is a Gram-positive rod-shaped bacterium.It is the agent of listeriosis, a serious infection caused by eating food contaminated with the bacteria.Listeriosis has been recognized as an important public health problem in the United States.The disease affects primarily pregnant women, newborns, and adults with weakened immune systems.
Listeriosis is a serious disease for humans; the overt form of the disease has mortality greater than 25 percent.The two main clinical manifestations are sepsis and meningitis.Meningitis is often complicated by encephalitis, a pathology that is unusual for bacterial infections.Microscopically, Listeria species appear as small, Gram-positive rods, which are sometimes arranged in short chains.In direct smears they may be coccoid, so they can be mistaken for streptococci.Longer cells may resemble Corynebacteria.Flagella are produced at room temperature but not at 37°C.Hemolytic activity on blood agar has been used as a marker to distinguish Listeria monocytogenes among other Listeria species, but it is not an absolutely definitive criterion.Further biochemical characterization may be necessary to distinguish between the different Listeria species ( Linnan et al., 1988).
As Gram-positive, non-spore forming, catalase-positive rods, the genus Listeria was classified in the family Corynebacteriaceae through the seventh edition of Bergey's Manual.16S rRNA cataloging studies of Stackebrandt et al. (1983) demonstrated that Listeria monocytogenes was a distinct taxon within the Lactobacillus-Bacillus branch of the bacterial phylogeny constructed by Woese (1981).In 2001, the Family Listeriaceae was created within the expanding Order Bacillales, which also includes Staphylococcaceae, Bacillaceae and others.Within this phylogeny there are six species of Listeria.The only other genus in the family is Brochothrix.

Natural habitats of Listeria and incidence of disease
Until about 1960, Listeria monocytogenes was thought to be associated almost exclusively with infections in animals, and less frequently in humans.However, in subsequent years, listeriae, including the pathogenic species L. monocytogenes and L. ivanovii, began to be isolated from a variety of sources, and they are now recognized to be widely distributed in Nature.In addition to humans, at least 42 species of wild and domestic mammals and 17 avian species, including domestic and game fowl, can harbor listeriae.Listeria monocytogenes is reportedly carried in the intestinal tract of 5-10% of the human population without any apparent symptoms of disease.Listeriae have also been isolated from crustaceans, fish, oysters, ticks, and flies (Doyle and Schoeni, 1986).
The term listeriosis encompasses a wide variety of disease symptoms that are similar in animals and humans.Listeria monocytogenes causes listeriosis in animals and humans; L. ivanovii causes the disease in animals only, mainly sheep.Encephalitis is the most common form of the disease in ruminant animals.In young animals, visceral or septicemic infections often occur.Intra-uterine infection of the fetus via the placenta frequently results in abortion in sheep and cattle (Doyle and Schoeni, 1986).
The true incidence of listeriosis in humans is not known, because in the average healthy adult, infections are usually asymptomatic, or at most produce a mild influenza-like disease.Clinical features range from mild influenza-like symptoms to meningitis and/or meningoencephalitis.Illness is most likely to occur in pregnant women, neonates, the elderly and immunocompromised individuals, but apparently healthy individuals may also be affected.In the serious (overt) form of the disease, meningitis, frequently accompanied by septicemia, is the most commonly encountered disease manifestation.In pregnant women, however, even though the most usual symptom is a mild influenza-like illness without meningitis, infection of the fetus is extremely common and can lead to abortion, stillbirth, or delivery of an acutely ill infant (David et al., 1994).
In humans, overt listeriosis following infection with L. monocytogenes is usually sporadic, but outbreaks of epidemic proportions have occurred.In 1981, there was an outbreak that involved over 100 people in Canada.Thirty-four of the infections occurred in pregnant women, among whom there were nine stillbirths, 23 infants born infected, and two live healthy births.Among 77 non pregnant adults who developed overt disease, there was nearly 30% mortality.The source of the outbreak was coleslaw produced by a local manufacturer (David et al., 1994.)In 1985, in California, 142 people developed overt listeriosis.Of these, 93 cases were perinatal, and among the 49 cases that were in non pregnant individuals, 48 were immunocompromised.Thirty fetuses or newborn infants died and 18 adults died.The source of the bacteria was a certain brand of "pasteurized" soft cheese that apparently got contaminated with non pasteurized (raw) milk during the manufacturing process (Kenneth, 2004)..In 2002, a multistate outbreak of Listeria monocytogenes infections with 46 culture-confirmed cases, seven deaths, and three stillbirths or miscarriages in eight states was linked to eating sliced turkey deli meat.One intact food product and 25 environmental samples from a poultry processing plant yielded L. monocytogenes.Two environmental isolates from floor drains were indistinguishable from that of outbreak patient isolates, suggesting that the plant might be the source of the outbreak (Kenneth, 2004).
Listeria monocytogenes, commonly referred to as Listeria, is a pathogen that causes listeriosis, a serious human illness.It is unlike most other foodborne pathogens because it can grow at proper refrigeration temperatures.In addition, Listeria is widely distributed in nature, and the organism has been recovered from farm fields, vegetables, animals and other environments such as food processing facilities, retail stores and home kitchens and readyto-eat foods (Kenneth, 2004).

Symptoms and disease process
L. monocytogenes causes listeriosis, a serious infection with high hospitalization rates for those who become ill.People at highest risk for a severe case include the elderly, the fetuses of pregnant women, and the immunosuppressed.It is unique among foodborne pathogens since its incubation time (time from ingestion of cells to illness) is at least seven days.Listeriosis is a rare disease with a high mortality rate, causing about 43 percent of the food poisoning deaths in the United States.
L. monocytogenes can also cause mild, flu-like symptoms in healthy individuals when consumed at very high levels.A person with listeriosis has fever, muscle aches and occasional gastrointestinal symptoms such as nausea or diarrhea.If infection spreads to the nervous system, symptoms such as headache, stiff neck, confusion, loss of balance, or convulsions can occur.Infected pregnant women may experience only a mild, flu-like illness; however, infections during pregnancy can lead to miscarriage or stillbirth, premature delivery or infection of the newborn (Vishal, 2004).

Primary routes of transmission
Foods can become contaminated with L. monocytogenes along the continuum from farm to fork, in the produce growing environment, during processing, or during handling and preparation in retail establishments and consumers' kitchens (ILSI, 2005).
The primary route of transmission is through the ingestion of contaminated food.The International Life Sciences Institute in 2005 described high-risk foods for causing listeriosis as those with the following properties: 1. have the potential for contamination with L. monocytogenes; 2. support the growth of L. monocytogenes to high numbers; 3. are ready-to-eat; 4. require refrigeration; and 5. are stored for an extended period of time (ILSI, 2005).
Because Listeria is abundant in nature and can be found almost anywhere, there can be a constant re-introduction of the organism into the food plant, retail setting, foodservice establishment and home.It is difficult to totally eliminate this contaminant from the foodhandling environment, but the goal is to control it as effectively as possible, especially where it can contaminate ready-to-eat, refrigerated foods.
Although L. monocytogenes is the only member of the Listeria family that causes human illness, the presence of any member of the Listeria family in a food processing environment may indicate that conditions are favorable for L.monocytogenes proliferation(ILSI,2005)..

Control
Effective control of L. monocytogenes requires prevention of contamination (to the extent possible) and prevention of growth through time/temperature or formulation control.Knowledge of potential harborage sites is important, as contamination is more likely to occur when the organism has become established in a niche.Food processing plant surveys have found Listeria in the following locations (listed approximately in the order of prevalence): cleaning aids such as brushes, sponges, etc.  product and/or equipment wash areas  food contact surfaces  condensate  walls and ceilings

Compressed air (ILSI, 2005)
Control of Listeria relies on detecting and managing harborage sites with thorough and frequent cleaning.This includes daily cleaning of floors and drains, and adequate attention to less frequently cleaned areas such as HVAC systems, walls, coolers and freezers.Also, damaged equipment, cracks, crevices and hollow areas must be part of sanitation and inspection schedules.It is essential to avoid creation of aerosols during cleaning, especially of floors and drains, to avoid spread of contaminants.
The organism is killed by normal food pasteurization and cooking processes, and is typically sensitive to most sanitizers at recommended rates.Contamination may occur after the cooking process in the processing environment, at retail locations and in the home.For example, post pasteurization contamination of food products can occur when the organism is dispersed via an aerosol.Prevention of growth is essential to avoid the potential for illness, because L. monocytogenes can grow at refrigerated temperatures, defeating one of the traditional food safety measures.
L. monocytogenes can survive on cold surfaces and can also multiply slowly at 34° F. It has also been shown to grow to a water activity as low as 0.92 and over a pH range of 4.4-9.4(ICMSF, 2004).Because the organism can grow under refrigeration, effective labeling to ensure product rotation in retail settings is an important control measure for ready-to-eat products.

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Environmental Health -Emerging Issues and Practice 184 Since this organism continues to elicit concern among consumers, regulators, processors and retailers, studies need to be carefully designed to ensure validity (ILSI, 2005).

Background on challenges to the zero tolerance initiative
The FDA/Food Safety and Inspection Service risk assessment reinforces epidemiological conclusions that food borne Listeriosis is a moderately rare, although severe, disease.A study by the Food Products Association showed it is likely that low levels of L. monocytogenes are consumed routinely with limited effect.It is believed that 5 percent of the general population may be asymptomatic carriers of Listeria, but the percentage may be higher in particular groups, such as slaughterhouse workers.
Extensive risk assessments and analyses have been conducted by the Food Safety and Inspection Service (FSIS, 2005), U.S. Food and Drug Administration/FSIS , World Health Organization/Food and Agriculture Organization and International Life Sciences Institute to identify factors that contribute to risk of illness (Gombas et al., 2003).This research is important because the prevalence in the food supply does not match the rate of illness in the population, and because the outcome of illness in susceptible individuals is very severe.These assessments have generally concluded that the ability of a food to support growth of Listeria enhances risk.Because of this, US FDA issued draft Compliance Policy Guidelines in February, 2008 based on the ability of a product to support growth of L. monocytogenes (ILSI, 2005).The USDA retains a zero tolerance policy for RTE foods with respect to L. monocytogenes, while other countries allow up to 100 Cfu/g in certain foods (ILSI, 2005).

Listeria monocytogenes in the human and animal environment
Listeriosis is essentially a food borne disease caused by Listeria monocytogenes and to some extent L. ivanovii.The disease conditions vary from severe invasive forms that affect immunocompromised patients to febrile gastroenteritis and perinatal infections associated with fetal loss or abortion in humans and animals (Siegman-Igra et al., 2002).Although rare, the disease is reported (Lyautey et al., 2007) to have a very high mortality rate (20-50%), thus making it of serious public health concern.Despite the general consensus that food is the primary route of transmission of this disease, wastewater has long been reported to be a potential reservoir for Listeria species and possible source of transmission (Paillard et al., 2005;Arslan and Ozdemir, 2008).Watkins and Sleath (1981) reported the prevalence of Listeria species in sewage at numbers far higher than those of Salmonella species.And recent studies suggest that Listeria species readily survive conventional wastewater treatment processes even after tertiary treatment (Paillard et al., 2005).
With reports of inadequate removal of Listeria pathogens from wastewater coming from the developed world (Paillard et al., 2005), one can safely presume that wastewater treatment plants in developing countries such as South Africa are inefficient at removing these pathogens from wastewater influents prior to discharge of the final effluents into the receiving waters for obvious reasons.Most studies (Mackintosh and Colvin, 2003;Obi et al., 2007;Obi et al., 2008) in the area of water quality in South Africa had focused almost exclusively on drinking or potable water supply with scanty reports in the literature on treated wastewater effluent as a source of pathogens for receiving waters.This may have serious public health implications as about 80 % of South Africans are reported to depend on surface water bodies for drinking, domestic and agricultural purposes (Mackintosh and Colvin, 2003).It is little surprise therefore that about 43, 000 deaths (mostly children) are reported annually in South Africa due to diarrhea diseases (Mara, 2001).The situation is amongst the worst in the Eastern Cape Province due to high level of poverty, low level of sanitation, and lack of appropriate infrastructure (Mackintosh and Colvin, 2003).While reports in the media suggests that cholera may be responsible for the majority of these infections, actual diagnosis suggests that these diseases could have been caused by any other waterborne pathogen apart from Vibrio species.A case in point was seen in the report of the Daily Dispatch of Thursday, 30th of January 2003, where out of 446 cases of water related diseases reported to the Eastern Cape health authorities, only 25 (5.6 %) were confirmed to be cholera and yet the disease was termed a "cholera outbreak" without ascertaining the true identities of the pathogens responsible for over 84% of reported cases.
There is a general belief that the larger population of bacteria species grow as adherent to surfaces in all nutrient-sufficient aquatic ecosystems and that these sessile bacterial cells differ profoundly from their planktonic (free-living) counterpart (Costerton et al., 1978).It has also been reported that the existence of pathogens as free-living or plankton-associated cells, is critical to their survival in the environment as well as their transmission from one host to another (Donlan and Costerton, 2002).Several studies have revealed the preponderance of Listeria species to exist as biofilms attached to surfaces such as stainless steel, glass and propylene (Mafu et al., 1990), and food and food processing environments (Lunden et al., 2000).There is however little or no report in the literature on Listerio-plankton association in the natural environment.Understanding the distribution of Listeria cells as free-living or plankton-associated niches may provide clues on how best to reduce the survival potentials of these pathogens in the environment and during wastewater treatment, and consequently reduce their ability to interact with human and animal populations.
Nwachukwu et al., 2010 reported the isolation of Listeria monocytogenes from two anthropogenic lakes from Lokpa-Ukwu, Abia State (Nigeria).The identification of isolates was based on cultural and morphological appearances.The identity of isolates was confirmed by biochemical tests.The results of this study revealed that the pathogen was present in 22 out of 24 samples of water from Lake A, giving a prevalence rate of 91.67% while in lake B, the prevalence rate observed was 79.17%.This high prevalence rate is not surprising as the organism is quite ubiquitous in soil, water, and animal dung samples.Listeria monocytogenes is water and food borne bacterial pathogen that is ubiquitous in nature and shows ability to persist in its environment for prolong time (Yutaka et al., 2004).: Quin et al., 1999;Slustsker and Schuchat, 1999 ;Afnor, 2000 ;EUAHS,2007 )., 1991).Control of L. monocytogenes in the food processing environment has been the subject of a number of scientific publications (Fenlon et al., 1996).L. monocytogenes can multiply slowly at refrigeration temperatures, thereby challenging an important defense against food-borne pathogens.(Doyle et al, 2001).

Listeria monocytogenes in food and food industries
Most cases of human listeriosis occur sporadically -that is, in an isolated manner without any apparent pattern.However, much of what is known about the epidemiology of the disease has been derived from outbreak-associated cases, in which there is an abrupt increase in reports of the disease.With rare exceptions, foods that have been reported to be associated with outbreaks or sporadic cases of listeriosis have been foods that can support the growth of L. monocytogenes and that are ready-to-eat (including coleslaw, fresh soft cheese made with unpasteurized milk, frankfurters, deli meats, and butter) USFDA, 2003.
Outbreaks of listeriosis are often associated with a processing or production failure (Slutsker and Schuchat 1999); this association has been less evident among sporadic cases (Slutsker and Schuchat 1999).
In addition to this information obtained from reported cases of listeriosis, contamination data (largely obtained from samples of foods collected at retail or during storage before sale) are available in published scientific literature, government documents and industry documents, or were made available to us from unpublished government and industry documents (Doyle et al, 2001).These contamination data show that L. monocytogenes has been detected to varying degrees in unpasteurized and pasteurized milk, high fat dairy products, soft unripened cheese (cottage cheese, cream cheese, ricotta), cooked ready-to-eat crustaceans, smoked seafood, fresh soft cheese (queso fresco), semi-soft cheese (blue, brick, monterey), soft-ripened cheese (brie, camembert, feta), deli-type salads, sandwiches, freshcut fruits and vegetables, and raw molluscan shellfish (Doyle et al, 2001).However, these data also show that most RTE foods do not contain detectable numbers of L. monocytogenes.For many RTE foods, contamination of foods with L. monocytogenes can be avoidede.g., through the application of current good manufacturing practices that establish controls on ingredients, listericidal and listeristatic processes, segregation of foods that have been cooked from those that have not, and sanitation.Sanitation controls include effective environmental monitoring programs designed to identify and eliminate L. monocytogenes in and on surfaces and areas in the plant (Doyle et al, 2001).The critical control points for food manufacturing industries will include one or more of the following:  A packaging line is moved or modified significantly.

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Used equipment is brought from storage or another plant and installed into the process flow.

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An equipment breakdown occurs.

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Construction or major modifications are made to an area where RTE foods are processed or exposed (e.g., replacing refrigeration units or floors, replacing or building walls, modifications to sewer lines).

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A new employee, unfamiliar with the operation and L. monocytogenes controls, has been hired to work in, or to clean equipment in, the area where RTE foods are processed or exposed.

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Personnel who handle RTE foods touch surfaces or equipment likely to be contaminated (e.g., floor, trash cans) and do not change gloves or follow other required procedures before handling the food.

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Periods of heavy production make it difficult to clean the floors of holding coolers as scheduled.

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A drain backs up.

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Product is caught or hung-up on equipment.(Stagnant product in a system can be a major site of microbial growth during production.) Raw or under-processed foods are placed in an area designated for cooked foods.

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Frequent product changes on a packaging line cause you to change packaging film, labels, forming pockets or molds, line speeds, etc.  Personnel are used interchangeably for packaging raw and cooked foods.

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Increased production causes you to perform wet cleaning of lines that have been taken down from production in the same room as lines that are running product.

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Heat exchangers have become compromised (e.g., with pinholes). Equipment parts, tubs, screens, etc. are cleaned on the floor.

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Waste bins in the RTE area are not properly maintained, cleaned and sanitized.

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Personnel handling RTE foods may come into contact with these items and then contaminate the foods and/or food contact surfaces.

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Re-circulating pumps and lines are not cleaned and sanitized. Indiscriminate use of high-pressure hoses in cleaning.

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Inappropriate use of footbaths in dry processing areas.

Traditional method
Current methods for identification of L. monocytogenes rely on physiological and biochemical methods.These include Gram stain morphology, catalase, motility, beta haemolysis on blood agar and oblique illumination of colonies on blood free agar.

a. Isolation methods
Conventional methods for the isolation of L. monocytogenes from food, water, and soil immediately that have gained acceptance for international regulatory purposes include the United States Food and Drug Administration (FDA) method (Hitchins, 1998), the Association of Official Analytical Chemists (AOAC) official method , the ISO 11290 Standards , the United States Department of Agriculture (USDA)-Food Safety and Inspection Service (FSIS) method (FSIS, 2002).)and the French Standards (AFNOR, 2000;AFNOR, 2004).Depending on the nature of the sample, a particular method might be more suitable than others.Listeria monocytogenes is positive with the S. aureus streak and negative with R. equi, whereas the test with L. ivanovii gives the reverse reactions (Quinn, 1999).

Polymerase Chain Reaction (PCR) Enrichment and DNA extraction
A total of 25 g or 25 ml of food sample, soil, water, animal droppings, is incubated in 225 ml of Listeria enrichment broth (Oxoid, England) at 30 ± 1 °C for 24 and 48 h.For DNA isolation, 1 ml of suspension after 24 h and after 48 h is necessary.
Homogenate is centrifuged at 1800 × g for 5 min and the supernatant is discarded.The pellet is re-suspended in 100 μl of 0.5 % TRITON X-100 (Sigma, Germany) and the whole process is repeated.Homogenate is incubated at 95 °C for 5-10 min.2 μl of Proteinase K [20 mg/ml] (Promega, USA) are added to the homogenate after cooling and it is incubated at 55 °C for 2 h.Proteins are removed with a phenol-chloroform-isoamylalcohol [25:24:1] solution (Sigma, Germany).DNA is precipitated with ice-cold absolute ethanol at -70 °C for 2h, Centrifuged at 3500 × g for 10 min.Pellet is dried and DNA is re-suspended in 30 μl of sterile distilled water.
Hot start is at 94 °C for 2 min.The reaction mixtures are subjected to 35 cycles consisting of heat denaturation at 94 °C for 30 s, primer annealing at 60 °C for 30 s and DNA extension at 74 °C for 1 min.Finally, the samples are maintained at 74 °C for 5 min for the final extension of DNA.These incubation conditions are the same for second round-nested PCR, except those LIP1 and LIP2, since these primers require 45 cycles.
The second round employed primers LIP1 and LIP2 (Simon et al. 1996) directed against nucleotides 634-654 and 886-907 of first product amplified by PRFA1 and PRFA2. 2 μl of completed first round reaction mixture are added to each reaction as target DNA.
Remaining components are the same as in the first round.

Aim and objectives of the study
The aims of this work are to: i. Investigate the occurrence of Listeria monocytogenes in poultry droppings from farms in the study area.ii.Study the pattern of antibiotic susceptibility of isolates of L. monocytogenes from the environment.

Study area
Poultry droppings were collected from three different farms.These farms include: David's Poultry Farm, Harrison's Farm, and Paul's Poultry Farm, all in the Okigwe Local Government Area of Imo State, Nigeria.

Sample collection
The poultry droppings were collected using sterile universal bottles, the screw caps were carefully removed using sterile hand gloves and with the help of an applicator stick, a large gentamycin, lincocin respectively.L. monocytogenes strains from Farm A showed 75% resistance to floxapen and Ampicillin.L. monocytogenes showed 83% susceptibility to tarivid (Quinolone).
The L. monocytogenes strains from Farm A showed a susceptibility of 83.3%, 83.3% in Lincocin and Tarivid respectively.This observation is in accordance with the report of Walsh et.al, (2001) where L. monocytogenes isolates were more susceptible to Drovid than floxapen.
Walsh et.al, 2004 also reported high resistance of L. monocytogenes to Penicillin.This drug rate of resistance to β-lactains and few quinolones could be as a result of drug abuse, and
Studies on the Isolation of Listeria monocytogenes from Food, Water, and Animal Droppings: Environmental Health Perspective 185 www.intechopen.com

Table 1 .
Potential Sources of L. monocytogenesScenarios That Could Lead to Contamination with L. monocytogenes agricultural commodities, and food processing environments (particularly cool damp areas) (NACMCF The selective Media for the isolation of Listeria species include Oxford Agar, Palcam Agar, Listeria Selective Agar (Oxoid, Basingstoke, England), and Nutrient Agar Supplemented with esculine bile salt.Listeria spp.isolates.It is used in the ISO and AOAC protocols and it is considered to be optional in the FDA and USDA-FSIS methods.The test is simple to perform and easy to read.It consists of streaking a ß-haemolytic Staphylococcus aureus (ATCC strain 49444 or 25923, NCTC strain 7428 or 1803) and Rhodococcus equi (ATCC strain 6939, NCTC strain 1621) in single straight lines in parallel, on a sheep blood agar plate or a double-layered agar plate with a very thin blood agar overlay.The streaks should have enough separation to allow test and control Listeria strains to be streaked perpendicularly, in between the two indicator organisms, without quite touching them (separated by 1-2 mm).After incubation for 24-48 hours at 35-37°C (12-18 hours if using the thin blood agar overlay),a positive reaction consists of an enhanced zone of ß-haemolysis, at the intersection of the test/control and indicator strains.

Table 2 .
List of primers for detection of Listeria monocytogenes using polymerase chain Reaction 7.

Table 4 .
).The highest susceptibility rate of 77.8% was observed on Tarivid and L. monocytogenes isolates from Farm 2 Number that remained susceptible to the antibiotic, NR; Number that was resistant to the given antibiotics Antibiotic Susceptibility patterns of L. monocytogenes isolated from different poultry droppings. Ns;