Prevalence and molecular characterization of Salmonella serovars in milk and cheese in Mansoura city , Egypt

Objective: This study was conducted to determine the prevalence of Salmonella in milk (farm bulk milk, raw market milk) and cheese (kareish, white soft cheese) samples that were collected randomly from farms, supermarkets, small vendors and shops in different districts of Mansoura city, Egypt. Materials and methods: A total of 100 farm bulk milk, raw market milk, kareish cheese and white soft cheese samples (25 of each) were screened for the prevalence of Salmonella spp. The Salmonella isolates were isolated and identified by conventional bacteriological techniques, which were further confirmed genetically by polymerase chain reaction (PCR) based on the presence of invA gene. Finally, the isolates were serotyped. Results: Salmonella could be detected in 15%(n=15/100) samples with a prevalence of 12%(n=3/25), 24%(n=6/25), 20%(n=5/25) and 4%(n=1/25) in raw market milk, raw farm bulk milk, kareish cheese and white soft cheese, respectively. The Salmonella isolates were serotyped into S. enteritidis 33.3%(n=9/27) which was the most frequent, followed by S. typhimurium 25.9%(n=7/27), S. heidelberg 14.8%(n=4/27), S. infantis 11.11%(n=3/27), S. tsevie 11.11%(n=3/27) and S. haifa 3.7%(n=1/27). Conclusion: The present study confirms the presence of Salmonella in milk and cheese samples in Mansoura, Egypt, indicating that the dairy products can act as potential sources of Salmonella infection. Thus, appropriate hygienic measures are suggestive for combating Salmonellosis in Egypt.


INTRODUCTION
Milk is considered as a high quality and nutrition-rich food for human beings (Theresa and Nicklas, 2003); this high quality is mainly referred to its composition and hygiene that are provided during the production and storage of milk.However, if the hygienic conditions are not applied properly, the milk can be contaminated by microorganisms leading to its early spoilage (Oliver et al., 2005;Nanu et al., 2007).
Salmonella are the most common pathogenic bacteria in humans and animals causing Salmonellosis.The disease is caused by numerous serovars of Salmonella, and this disease can be manifested as mild food poisoning to severe enteric fever (Jones et al., 2004).When animals are infected with Salmonellosis, high economic loss occurrs due to increased mortality and morbidity, and this diseased animals may act as the source of infections for humans through direct or indirect contacts with animals (Libby et al., 2004).Contamination of the environment and food with Salmonella mainly happens by the fecal wastes of the infected animals and humans (Ponce et al., 2008) as Salmonella colonizes mainly in the gastrointestinal tract (Abulreesh 2012).
The present study was carried out to investigate the prevalence of Salmonella in milk and cheese, and reveal out the serotypes of the Salmonella isolates.

MATERIALS AND METHODS
The technique that was used for samples collection was recommended by American Public Health Association (APHA 1992).One hundred samples (25 each of market raw milk, farm bulk tank milk, kareish cheese and white soft cheese) were obtained from different localities of Mansoura from the period between May 2014 to May 2015.The collected samples kept in an insulated ice box (4±1 0 C) to be transferred to laboratory for bacteriological examination.

Ethical statement:
The investigation was carried out according to the animal welfare code in Egypt.

Bacteriological examination:
The isolation and identification of Salmonella spp.were done according to ISO 6579 (ISO, 2002).25 mL/gm of the samples were transferred to 225 mL sterile buffered peptone water (Oxoid, CM 0509), and mixed well to obtain 10 fold dilution, then 0.1 mL of buffered peptone water cultured broth was aseptically inoculated into 10 mL of Rappaport Vassilliadis (RV) broth (Oxoid, CM 669).The culture was incubated at 41 0 C for 24 h.A loopful cultured RV broth was streaked onto Xylose-Lysine-Desoxycolate (XLD) agar (Oxoid, CM 0469) and incubated at 37 0 C for 18-24 h.The suspected colonies (pink colonies or red colonies with or without black centers) were picked up and subcultured again onto XLD agar plates (for purification) and then incubated at 37 0 C for 18-24 h.The purified colonies were subcultured onto nutrient agar slants (Oxoid, CM 0003) and incubated at 37 0 C for 24 h for further identification (biochemical, serological and molecular).All the suspected colonies were biochemically identified on the basis of Indole test, Methyl Red test, Voges proskauer test, Citrate utilization test, Triple Sugar Iron agar test and Urea Hydrolysis test.

Molecular identification:
The primarily identified Salmonella were confirmed by PCR based on the presence of invA gene.Extraction of DNA was performed by thermal cell lysis of suspected strains (Reischl et al., 2002).The DNA was used as template for the amplification of the highly conserved region of invA gene using the primers (forward: 5´-ACA GTG CTC GTT TAC GAC CTG AAT-3´; reverse: 5´-AGA CGA CTG GTA CTG ATC GAT AAT-3´) specific for all Salmonella serovars (Chiu and Ou, 1996).PCR was performed in a 25 uL reaction mixture with 2x PCR master mix solution (i-Taq TM ).Cycling conditions were optimized at initial denaturation at 95 0 C for 4 min, 35 cycles of amplification using theremocyler (Gene Amp PCR system 2700, Applied Biosystems, Foster City, CA, USA) was done with the conditions as denaturation at 95 0 C for 30 Sec, annealing at 57 0 C for 45 Sec and extension at 72 0 C for 1.5 min, followed by final extension at 72 0 C for 5 min.Vivantis Company 100-bP Plus DNA Ladder (product no.NL1407) was used.PCR products were electrophoresed in 1.5% agarose gel stained with ethidium bromide 0.5 mg/mL (Sambrook et al., 1989).The DNA band was visualized by gel documentation system.

Serological identification:
The confirmed Salmonella isolates were serotyped by slide agglutination with O polyvalent, monovalent antisera (DENKA SEIKEN Co., Japan) and tube agglutination method with H polyvalent antisera of both phase 1 and 2 for the determination of the complete antigenic formula of the isolates, as indicated by Sambrook et al. (1989).

RESULTS AND DISCUSSION
Milk and milk products are important sources of Salmonella especially for the consumers who preferred to consume raw milk.Many factors such as improper hygienic conditions in the farm, food handlers, and consumption of raw milk and milk products are the sources of Salmonella infections (Karshima et al., 2013).And, most of these cheese were sold as uncovered condition and without any container.Thus, the risk of contamination with Salmonella spp. was high.
In case of white soft cheese, the prevalence of Salmonella was 4%(n=1/25) (Table 1 PCR assay was used for detection of Salmonella before using the serological identification, as reported by Eid (2010) who found that some isolates were detected by PCR which had been detected as negative in serological examination.Also, Wang et al. (2013) reported that PCR was a rapid and accurate method for the detection of Salmonella, so it should be used firstly, as we did.

CONCLUSION
Six different Salmonella serotypes are isolated from milk and cheese samples, which may act as a source of human infection.For the prevention of Salmonella contamination in milk and milk products, we may apply proper hygienic measures during milking and handling of milk after milking.Efficient cleaning of all utensils and equipment, effective training and education the farmers to improve awareness of milk borne zoonosis and risk factors can be followed.Good Manufacturing Practice (GMP) and Hazard Analysis and Critical Control Points (HACCP) systems are suggestive to avoid Salmonella contaminaiton.

Table 1 :Figure 1 :
Prevalence of Salmonella in milk and cheese samples Agarose gel electrophoresis for PCR amplification of Salmonella invA gene product (244-bp).M: DNA marker (gene ladder 100-bp), P: positive control, N: negative control, Lane 1 to 12: positive samples.

Table 2 .
Serological typing of Salmonella isolates Salmonella in farm bulk milk.This variation might be due to the difference in hygienic and sanitation practices during milking.

Table 1 )
(ICMSF, 2006)2007)003)ilar with the report ofIbrahim et al. (2015), while ElSayed et al. (2011)reported lower prevalence of Salmonella in white soft cheese.Relatively higher prevalence of Salmonella in white soft cheese was detected byElbagory et al. (2015).Soft cheese contains high levels of moisture and pH, particularly the surface of cheese contains high moisture and pH facilitated during ripening process so that these are more exposed to microbial growth as compared to those of hard or semi-hard cheeses(Robinson, 2002).Other reasons of contamination of white soft cheese by Salmonella might be use of bad quality milk, improper heat treatment, unhygienic manufacture practice or use of contaminated brine.A PCR targeting invA gene (target size: 244-bp) was done confering rapid identification of Salmonella isolates as all Salmonella serovars have the invasion gene (invA) as a unique character (Herrera-Leon et al., 2004).Lampel et al. (2000)also reported that PCR with primers for invA gene was rapid, sensitive and specific for the detection of Salmonella in many samples.On the other hand, invA gene was widely recognized as the marker for rapid identification of Salmonella genus(Malorny et al., 2003)(Figure1).Shen et al. (2007)who reported that S. typhimurium was adapted to acid at pH 5.5 for 4 h.So, acid adaptation is an important survival mechanism enabling Salmonella spp. to persist in fermented dairy products, and possibly other acidic food products., this might be occurred manufacture, and can survive for more than 60 days in cheese(ICMSF, 2006).However, this sample proved to be the most contaminated one of the all examined samples and yielded five types of Salmonella serovars (S. typhimurium, S. enteritidis, S. tsevie, S. infantis and S. heidelberg) (Table2).Many factors determine the quality of cheese as quality of the clotted skim milk, method of the manufacture, time required to drainage the whey, quality of added salt and method of handling the finished cheese (Abou-Donia, 2008).
In this study, S. typhimurium, S. enteritidis, S. haifa and S. infantis were detected in the kareish cheese, while S. typhimurium was the most prominent one.This was agreed with