Incidence of Listeria monocytogenes in milk from producers in the Maseru area

Thesis (M. Tech. (Biomedical Technology)) -- Central University of technology, Free State, 2013The objective of this study was to determine the prevalence of Listeria monocytogenes and also to assess the general hygiene of fresh milk in the Maseru area, Lesotho. A total of 200 milk samples (40 pasteurised and 160 raw milk samples) were used for the research. Raw milk samples were collected from the local farmers at the Dairy reception as they bring it for selling. Pasteurised milk samples were bought from different milk selling points in the Maseru area. The total aerobic plate count, total coliform count and total E. coli count for 160 raw milk samples and 40 pasteurised samples were performed to determine the quality of milk. Milk was enriched in selective broths to increase detection sensitivity and was directly plated on selective agars for direct bacterial enumeration. About 54.4% of the of the raw milk samples had total aerobic plate counts greater that 200 000 cfu/ml while 55.6% (89/160) of the raw samples had high counts of greater than 20 cfu/ml for total coliforms, and 21.9% (35/160) of the samples had higher than expected total E. coli counts. High total coliform count was detected in 17.5% (7/40) of the pasteurised milk samples and about 67.5% (27/40) of these samples exceeded the limit for total aerobic plate counts. The counts exceeded the milk standards for pasteurised milk. Phosphatase activity was detected in seven pasteurised milk samples, whereas 33 tested negative for phosphatase activity. Some pasteurised milk samples tested positive for coliform counts which exceeded the maximum limits according to national standards for pasteurised milk. However, most of the pasteurised samples (82.5%) had acceptable counts of less than 20 cfu/ml. API and PCR were used for confirmation and amplification of the isolated Listeria strains. The prevalence of Listeria was found to be (3.75%). Listeria species were found in 6 out of 200 samples tested (160 raw milk samples and 40 pasteurised milk), and were only detected in the raw milk samples. Five species belonged to Listeria monocytogenes and one was Listeria innocua. None of the Listeria was detected in the pasteurised milk samples. Serotyping was done through multiplex PCR with D1, D2, FlaA and GLT primers to determine the serovar groups of L. monocytogenes. All six isolates revealed 214 bp gene which identifies the serotypes in Lineages I or III. The genetic fingerprinting of the isolated Listeria was also determined. Enterobacterial Repetitive Intergenic Consensus (ERIC) sequence-based PCR was used to generate DNA fingerprints with ERIC specific primers. On the basis of ERIC-PCR fingerprints, three different DNA patterns could be discriminated among the analysed isolates. Three L. monocytogenes isolates showed similar DNA banding patterns, while two isolates both had different profiles. A questionnaire was used to determine consumption of raw (unpasteurised) milk or pasteurised milk and its products and it was completed by 300 households from the community. Although there was no indicated prevalence of raw (unpasteurised) milk consumption from the community, participants indicated symptoms alleged to consumption of pasteurised milk and/or milk products. According to community perception some of the dairy products consumed were allegedly implicated in food poisoning illnesses experienced. Participants indicated more symptoms with both fresh and sour milk consumption than in cheese and yogurt consumption