Listeria spp. in Raw Cow and Goat Meat in Port Harcourt, Nigeria

Aims: To determine the occurrence of Listeria monocytogenes and other Listeria spp. in raw cow and goat meat. Study Design: This work was based on a completely randomized design with two replication and the average values calculated for mean comparison. Place and Duration of Study: Department of Microbiology, University of Port Harcourt, Nigeria and Nigeria Institute of Medical Research, Yaba, Lagos, Nigeria. Isolation of Listeria spp. was between March 2011 and February 2012. Methodology: In this study, a total of 240 raw cow and goat meat samples were analyzed for the presence of Listeria monocytogenes and other Listeria species. The techniques recommended by the United State Department of Agriculture (USDA) revised and the Health Products and Food Methods of the Government of Canada were employed using Fraser broth and polymixin acriflavin lithium chloride ceftazidime aesculin mannitol (PALCAM) agar. Results: The results of conventional and polymerase chain reaction (PCR) characterization of the isolates revealed that 81 samples (33.75%) were positive for Listeria spp. The highest prevalence of Listeria was found in cow flesh samples (19 of 36 samples; 52.78%) followed by cow intestine samples (8 of 20; 40%) and least was goat kidney samples (5 of 28; 17.86%). Out of the 310 characterized Listeria spp., L. Original Research Article British Biotechnology Journal, 4(2): 204-214, 2014 205 monocytogenes were 4(1.29%). Other species isolated were L. innocua 20(6.45%), L. ivanovii 4(1.29%), L. seeligeri 72 (23.23%), L. welshimeri 139 (44.84%) and L. grayi 71(22.90%). No L. monocyogenes was isolated from cow samples. A higher incidence was noted during the raining season 216 (69.68%) than the dry season 94(30.32%). Conclusion: This study demonstrated the occurrence and distribution of Listeria species in retail raw cow and goat meat in Port Harcourt, Nigeria.


INTRODUCTION
Meat is the major source of protein and valuable qualities of vitamins for most people in many parts of the world, thus they are essential for the growth, repair and maintenance of body cells and necessary for our everyday activities [1]. Due to the chemical composition and biological characteristics, meats are highly perishable foods which provide excellent source for growth of many hazardous microorganisms that can cause infection in humans and spoilage of meat and economic loss [2]. Listeric infections, caused by microorganisms of the genius Listeria, occur worldwide and in a variety of animals including man [3]. Cases of listeriosis arise mainly from the ingestion of contaminated food and the disease is particularly common in ruminants fed on silage [3]. The genus Listeria comprises six species: L. monocytogenes, L. innocua, L. welshimeri, L. grayi, L. ivanovii and L. seeligeri [4]. Two of these species, L. monocytogenes and L. ivanovii, are potentially pathogenic [5]. The other four Listeria species are essentially saprophytes that have adapted for survival in soil and decaying vegetation [4]. Listeria monocytogenes, a facultative intracellular pathogen, repeatedly found in meat and meat products, raw milk, soft cheese and pasteurized dairy products, vegetables, and fish and fish products [6] is responsible for severe foodborne infections in humans of all ages but especially pregnant women, infants less than four weeks old, the elderly and immunocompromised individuals and also cause invasive disease in many ruminants including, cattle, sheep, and goats species [7][8][9]. The bacterium possesses properties that favor it as a foodborne pathogen: at variance with most other pathogens it is relatively resistant to acid and high salt concentrations; it grows at low temperature, down to freezing point, which mean it may grow in refrigerated foods, measures commonly used to control the growth of pathogens in foods [10,11]. Before the development of Listeria selective media, this property was used for selective enrichment of the bacterium from complex matrices, with Listeria outnumbering the competing flora after incubation of the enrichment culture at refrigerator temperature for weeks or months [11].
Although the occurrence of Listeria in various foods has been investigated in several countries, there is limited information regarding its prevalence in raw meat and foods in Nigeria. The present study was conducted to determine the occurrence of Listeria species in raw cow and goat meat in Port Harcourt, Nigeria.

Sample Collection
A total of 240 raw cow and goat meat samples comprising 122 cow and 118 goat meat were randomly purchased from meat vendors in three strategic markets in Port Harcourt metropolis from March, 2011 to February, 2012. The samples were kept in ice box containing ice packs and immediately transported to the Microbiology laboratory, University of Port Harcourt where they were kept in refrigerator until analyzed.

Isolation of Listeria sp
The techniques recommended by the United State Department of Agriculture (USDA) revised [12] and the Health Products and Food Methods of the Government of Canada [13] were employed using Fraser broth (Oxoid, England) and polymixin acriflavin lithium chloride ceftazidime aesculin mannitol (PALCAM) agar (Oxoid, England). Twenty-five grams of each meat samples was added to a stomacher bag containing 225ml of sterile half-Fraser broth and supplements. The mixture was homogenized using a stomacher (lab-blender, Seward medical, London) at high speed for 1-2min. The test portion was incubated at 30 o C for 24h. From the pre-enrichment culture (half Fraser broth), 0.1ml was transferred into 10ml of fullstrength Fraser broth with supplements added and was incubated at 35 o C for 24-48h. From the culture obtained in Fraser broth showing evidence of darkening due to aesculin hydrolysis by Listeria sp, 0.1ml was transferred onto duplicate PALCAM plates. After spreading, plates were incubated at 37 o C for 24-48h. The plates were examined for the presence of characteristic colonies presumed to be Listeria sp-2mm grey-green colonies with a black sunken centre and a black halo on a cherry-red background, following aesculin hydrolysis and mannitol fermentation. Five typical colonies were selected randomly from a pair of PALCAM plates for confirmation and subsequent identification.

Confirmation and Identification
Colonies suspected to be Listeria were transferred onto trypticase soy agar (Becton, Dickinson and company, France) with 0.6% yeast extract (Lab M, UK ) and incubated at 37ºC for 18 to 24h, before being subjected to the following standard biochemical tests: gram staining, catalase reaction, oxidase reaction, beta haemolysis on sheep blood agar and acid production from mannitol, rhamnose and xylose. Confirmed isolates on the basis of criteria suggested by Seeliger and Jones [14] were further identified using the polymerase chain reaction (PCR).

Extraction of Listeria DNA (Deoxyribonucleic acid)
DNA was extracted by the boiling method without triton x-100 [15]. Cells were harvested by centrifugation (Eppendorf centrifuge 5418, Germany) of overnight brain heart infusion broth culture of Listeria in 2ml eppendorf tube at 10,000rpm for 2min and the supernatants discarded. The pellets were re-suspended in 1ml sterile distilled water and centrifuged after vortexing (Vortexer 59a, Denville Scientific Inc, Taiwan) at 10,000rpm for 5min. The supernatants were again discarded and the pellets resuspended in 200µl sterile water and vortexed. The suspensions were heated for 10min in a boiling bath (100 o C) (Grant GLS400, Grant Instrument, England). After cooling and vortexing, the mixtures were centrifuged at 10,000rpm for 5min. The supernatants were then transferred to a prelabelled 1.5ml eppendorf tube while the sediments were discarded. The DNA extracted was stored in deep freezer (-20 o C) until further analysis. The reactions involving MonoA, Iva1, Sel1, Wel1, Ino2, Mugra1, Siwi2, Lis1A and Lis1B were also carried out in a final volume of 25µl, containing 2.5 µl 10×pcr buffer, 1.5 µl MgCl 2 , 0.5µl DNTP (deoxynucleoside triphosphate), 0.2 µl each of appropriate primer, 0.15 µl AmphiTaq dna polymerase (all products of Solis BioDyne, Estonia), 1.5µl of appropriate DNA preparation and 18.45µl sterile distilled water. amplification following an initial denaturation at 95ºC for 3min was performed in 30 cycles, at 95ºC for 30s, 58ºC for 60s and 72ºC for 60s in a thermo cycler (Mastercycler-Eppendorf, Vapo-product,Germany). A final extension was performed for 10min at 72ºC. a 8µl aliquot of pcr product mixed with a loading dye ( 10mm, EDTA, 10% glycerol, 0.015% bromo phenol blue and 0.017% sodium dodecyl sulphate (SDS), made up to 100ml) were checked in an ethidium bromide stained 1.5% agarose (Fermentas, Life Science, Germany) and the gel read in a UV transilluminator (Genosens 1500, Clinx Science Instruments Co. Ltd, China ). Reaction mixture with the DNA of L. monocytogenes PCM 2191 serovar 01/2 (Polish Collection of Microorganisms, Poland) template serve as positive control while a reaction mixture with no DNA template was incorporated as a negative control in each reaction.

Statistical Analysis
The distribution of the Listeria species in the various meat types and parts were subjected to analysis of variance (ANOVA) and Duncan [18] to determine means that differed.

RESULTS AND DISCUSSION
Out of a total of 240 meat samples analyzed, 81(33.75%) were positive for Listeria. Listeria species were isolated from all the beef and goat meat parts examined ( Table 1). The level of contamination of meat samples by Listeria monocytogenes and other Listeria species varied and was highest in cow flesh (19 of 36 samples, 52.78%), followed by cow intestine (8 of 20 samples, 40%) and least was goat kidney (5 of 28 samples, 17.86%).  All 310 Listeria isolates identified as L. monocytogenes 4(1.29%), L. innocua 20(6.45%), L. seeligeri 72(23.23%), L. ivanovii 4 (1.29%), L. welshimeri (139%) and L. grayi 71(22.90%) by conventional method were also positive using the PCR assay (Fig. 1). There were no significant difference (p=.05) in the occurrence of Listeria among the different meat parts examined. The highest occurrence of Listeria was in July (15.81%); it was also high in August (12.58%), both being the peak of the raining season in Port Harcourt (Fig. 2). Overall, a higher incidence was noted during the raining season 216 (69.68%) than the dry season 94(30.32%).

Fig. 2. Monthly Distribution of Isolated Listeria Species
The importance of raw meat and meat products as a vehicle for the transmission of various diseases, especially in countries where hygienic standards are not strictly enforced has been well documented [19] It is not surprising that L. monocytogenes and other Listeria spp. were found in meat considering its widespread occurrence in nature, especially in plants, and their associations in nature with other bacteria that are well established in meats make it reasonable to find it in meats [20].
Overall, 33.75% (81 of 240) of all meat samples in this study were contaminated with Listeria species. This incidence of Listeria in the meat examined coincides with the report of a 0 to 68% prevalence of Listeria in fresh meat [21]. This can be attributed to the consumption of contaminated silage or other feeds, fecal contamination during evisceration and from handlers and slaughterhouses [3,[22][23][24].
In this study, L. welshimeri was the predominant Listeria species isolated from the beef and goat meat samples; other Listeria species were less common ( Table 2). Our result differed from those already reported [20,25,26] that stated that L. monocytogenes and L. innocua are most often reported with most investigators reporting one or the other to be most predominant and L. welshimeri coming third or next after L. innocua, followed by L. seeligeri and lastly L. ivanovii in meat among this five related species. L. monocytogenes accounted for 4(1.29%) of the 310 Listeria isolated. None was from the beef samples. Previous studies in Nigeria, India, Serbia and Bangkok, reported the inability to isolate L. monocytogenes in raw-goat, -beef and -meat [22,[27][28][29]. However, a number of authors reported a 4.65% and 6.4% (Bulgeria), 5.1% (Ethiopia), 6.66% (India), 17.7% (Portugal), 20% (Greek), 31% (Denmark) and 35% (Spain) prevalence of L. monocytogenes from raw-beef, -goat andmeat [10,[30][31][32][33][34][35], making obvious that the occurrence of L. monocytogenes varies from one place and one author to the other.
The distribution of the Listeria species (Fig. 2) shows that the highest occurrence was in July (15.81%). The occurrence was also high for August (12.58%), both being the peak of the raining season in Port Harcourt, Nigeria. This is comparable with a high incidence of Listeria in beef and goat meat during fall in Iran [36].
The inability of the Lis1A and Lis1B and MonoA and Lis1B primer combinations to produce the expected PCR product for all Listeria species and L. monocytogenes respectively, occasioned the use of primer combination; LI1 and U1 (Fig. 1) and LM1 and LM2 targeting the more conserve genomes of all Listeria and L. monocytogenes respectively. However, the others produced the expected PCR product, amongst them Siwi2 and Lis1B (Fig. 3) for L. seeligeri, L. ivanovii and L. welshimeri, the most predominant of the Listeria species isolated.

CONCLUSION
This study has demonstrated the presence and distribution of L. monocytogenes and other Listeria species in raw meat examined in Port Harcourt. The high percentage of the isolation of Listeria suggests the need for improved food safety through proper hygienic measures during processing of food and meat products.