STUDIES ON SOME MILK BORNE PATHOGENS

Assiut University web-site: www.aun.edu.eg

STUDIES ON SOME MILK BORNE PATHOGENS

SAADIA H.H. EL-SHINAWY ¹; GAMAL M. HASSAN ¹; MOHAMED A.H. El-SHATER ² and SHIMAA A. ABD El-AZIZ ²

¹ Food Hygiene Department, Faculty of Veterinary Medicine, Beni-Suef University

² Animal Health Research Institute, Egypt

Received: 26 August 2018;     Accepted: 20 September 2018

INTRODUCTION

Since ancient times, milk form a major part of human food and play a prominent role in the diet (Pal, 2014). Raw milk is known as the main transmission pathway for pathogens resulting in food borne outbreaks every year (Gillespie et al., 2003; Rey et al., 2003). Pasteurization process of milk is a heat treatment intended to reduce the number of any harmful microorganisms to a level at which they do not constitute a significant health hazard (MPI NZ, 2013).

Staphylococcus.  aureus is one of the major bacterial agents causing food borne diseases in human worldwide (EFSA, 2010). It is an opportunistic pathogen, which associated with food poisoning and food spoilage (Argudin et al., 2010). The staphylococcus food poisoning is a mild intoxication occurring after ingestion of food containing from 20ng up to 1 ug of Staphylococcal enterotoxins (SEs) which is enough to induce symptoms in human beings (Nermano et al., 2007). Clinical signs of Staphylococcal food poisoning generally disappear within 24-48 h. Deaths occur rarely and specifically in the young or elderly (Jay et al., 2005).

Corresponding author: Dr. SHIMAA A. ABD El-AZIZ

E-mail address: shimaa_ahmed1415@yahoo.com  

Present address: Animal Health Research Institute, Egypt

Listeria monocytogenes is considered as one of the most important food borne pathogen that induce serious and potentially life threatening illness known as listeriosis in humans and animals (Rahimi et al., 2012) with high mortality rate (20-30%) (Wijendra et al., 2014). It may range from non -invasive febrile gastroenteritis or influenza like symptoms especially in healthy individuals with no predisposing conditions (Aygun and Pehlivanlar, 2006) to serious invasive severe symptoms which may lead to septicemia, meningitis and abortion (Shamloo et al., 2015; Kevenk and Gulel, 2016).

Enterohemorrhagic Escherichia coli (EHEC) have emerged as an important cause of human intestinal diseases in developed countries over the past 20 years (Naylor et al., 2003). E.coli O157:H7 causes both outbreaks of diarrhea, haemorrhagic colitis and haemorragic uremic syndrome (HUS) (Baker et al., 2007). The infectious dose was very low about 100-200 organisms or even less than 10 cells in susceptible consumers which is hazardous and increased the risk of disease(CFSPH, 2009; Grant et al., 2011). In addition, its ability to produce Shiga toxins (Stxs) (formerly called Shiga-like toxins)(Beneduce et al., 2003) which is considered as best essential virulence factor for E. coli O157:H7 in human disease(Mauro and Koudelka, 2011).

Therefore, the present study aimed to study the incidence of some foodborne pathogens, as Staph. aureus,L. monocytogenes and E.coli O157:H7 in raw and pasteurized milk in El-Minia governorate with regarding to public health.

MATERIALS AND METHODS

1. Collection of Samples:

a-Raw milk: One hundred and fifty raw milk samples were purchased from 3 different sources as farms, dairy shops and collecting centers (50 each) in El-Minia governorate. All samples were collected in clean, dry, sterile containers and delivered as soon as possible to the laboratory in an insulated ice box, proved to be raw by storch's test according to the method recommended by FDA (1998) and examined at the same day.

b- Pasteurized milk: Fifty samples of pasteurized milk (bottles or cartons) were collected from different supermarkets and shops of different manufacture dates and different companies in El-Minia governorate. All bottles and cartons were thoroughly cleaned from outside and then well mixed and aseptically opened.

2- Enumeration, Isolation and identification of Staph. aureus:

By the using of Baird Parker's agar plates supplemented with egg yolk and potassium tellurite, the method recommended by APHA (2004) was applied.

3- Isolation and identification of L. monocytogenes:

By the using of PALCAM agar, the plates were incubated at 35°C for 48 hours according to Hitchins (2001).

4- Isolation and identification of E. coli O157 H7:

By the using of cefixime tellurite-sorbitol MacConkey medium, the incubation was at 37ᵒC for 24 hrs ±2 hrs according to De Boor and Heuvelink (2000).

5- Serological confirmation of the E. coli isolates: All the suspected isolates were serologically identified by slide agglutination according toKok et al. (1996) by using rapid diagnostic E.coli antisera sets (DENKA SEIKEN Co., Japan), non sorbitol fermenting (NSF) colonies were subjected to agglutination test by using monvalent O157and H7 antisera (Oxoid).

6- Molecular characterization of L. monocytogenes and Staph. aureus by PCR:

6.1. Extraction of DNA for PCR:

DNA was extracted from colonies by using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) Catalogue no.51304 according to the manufacturer's instructions.

6.2. DNA amplification:

Conventional PCR was carried out for identification of the DNA extracts to confirm the presence of genetic material of genus Staph.aureus and L.monocytogenes. Amplification of target genes (clfA and 16S rRNA) respectively was carried out in a thermal cycler (Biometra, Germany) using 25μl reaction volume containing (12.5 Emerald Amp GT PCR mastermix (2x premix), 5.5 μl PCR grade water, 1 μl Forward primer (20 pmol), 1 μl Reverse primer (20 pmol) and 5 μl Template DNA.

Oligonucleotide primers sequences.

Cycling conditions of the different primers during cPCR

6.3. Detection and identification of PCR product.

The amplified DNA fragments were resolved by agarose gel electrophoresis, stained with ethidium bromide (0.5 μg/ml) and the gels can be screened and pictured under UV light.

RESULTS

Table 1: Incidence of  Staph .aureus in the examined raw milk samples.

Table 2 : Statistical analytical results of Staph. aureus count/ml in the examined milk samples.

Table3: Frequency distribution of the positive samples based on their Staph. aureus count/ml.

Table 4: Acceptable and unacceptable samples of the examined milk samples based on their Staph. aureus count/ml. according to the Egyptain standards (2005).

Table 5: Incidence of  L. monocytogenes in the examined raw milk samples.

Table 6: Some E.coli serotypesisolatedfrom the examined raw  milk samples.

Table 7: Incidence of Staph .aureus, L. monocytogenes and E. coli O157:H7 in the examined pasteurized milk samples.

Photo1: PCR results for Staph. aureus clfA gene and L. monocytogenes 16S rRNA gene showing positive amplification 638 bp of Staph. aureus clfA gene and 1200 bp of L. monocytogenes 16S rRNA gene, L showed [Gelpilot100 bp plus ladder (Qiagen, 100-1500 bp)].

DISCUSSION

The results illustrated inTable 1 revealed that, 96 (64%) of the examined raw milk samples were contaminated with Staph. aureus. Nearly similar findings 52% and 56.66% was reported by Pourhassan and Taravat (2011); El-Jakee et al. (2013), respectively. However, higher results of Staph.aureus isolated from cowʼs milk reported by Al-Tarazi et al. (2003); Ekici et al. (2004) in percentages of 80 and 75 % respectively. While, lower results were estimated by Jahan et al. (2015); Ayele et al. (2017) in percentages 25.3 and 23.4% respectively.

Staph. aureus are wide spread in nature, up to 50% of humans may carry this organism in their nasal passages, throats and on their hair and skin. So it is good indicator of the personal hygiene of the workers with respiratory infections (Kamat et al., 1991; Harvey and Gilmour, 1996).

The results in Table 4 provedthat 10%, 60%, 50% and 96% of the milk samples from farms, collecting centers, dairy shops and pasteurized milk respectively were acceptable for the Staph. aureus cfu/ml according to the Egyptian standards (2005).

The obtained results reported in Table 5 reveals that raw milk was contaminated with L. monocytogenes with an overall incidence of 8%. Nearly similar results were recorded by El-Malt and Abdel–Hameed (2009); El-Marnissi et al. (2013); Kevenk and Gulel (2016). Meanwhile, these results disagreed with those recorded by Sammarco et al (2005); Aygun and Pehlivanlar (2006); Atil et al. (2011) who failed to isolate L. monocytogenes.

The sources of Listeria spp. in raw milk have been reported to be faecal (Griffiths, 1989) and environmental contamination during milking, storage and transport, infected cows in dairy farms and poor silage quality (Bemrah et al., 1998). High contamination of milk with L. monocytogenes in autumn and winter when silages are fed. Broseta et al.  (2003) also reported that contamination of raw milk with L. monocytogenes is usually more common in the winter, most likely because silage feeding in many parts of the world is more common in that season. In addition, a large number of studies have indicated that clinical listeriosis in ruminants is often associated with feeding poor quality silage (Boerlin et al., 2002).

Silage is not widely used as animal feed in El-Minia governorate, Egypt.; the animals were fed with dry feed and some green grass. Therefore, the contamination source of L. monocytogenes in raw milk in this study may be of fecal and environmental contamination during milking, storage and transport, infected animals in dairy farms. Furthermore, most cattle and sheep farms in Egypt do not have adequate hygiene precautions and animals live in a natural environment together with people.

E.coli O157: H7 could not be detected in all of the examined samples using conventional culture, biochemical and serological methods. Serological identification of the isolated E. coli are listed in Table 6 where serotypes O114, O142, O86a and O 148 were detected.

The obtained results was in line with the results obtained by  Coia et al. (2001); Dontorou et al. (2003); Meshref (2007); Issa et al. (2010); Zeinhom (2011) who couldn’t isolate E. coli O157 H7 from raw milk examined in  Netherlands, Greece, Turkey and Egypt respectively. On the contrary the current results disagreed with others like Abdel Khalek et al. (2001); Amer and Soliman (2004); El-Gedawy et al. (2016) who reported 3, 1 and 1 % of raw milk examined in Egypt were contaminated with E. coli O157:H7.

The failure in detection of E. coli O157:H7 in milk is mainly returned to its presence sporadically at very low levels among very high levels of competitor organisms (Siriken et al., 2006). It can be thought that there is very low risk, but on the other hand, this bacterium can survive for more than 50 days in municipal reservoir and lake water (Wang and Doyle, 1998) and dairy cattle are asymptomatic carriers of this bacterium (Zhao et al., 1995; Wang et al., 1997; Heuvelink et al., 1998), thus increasing the risk for transmission through cattle to cattle, milk and milk products and to other foods.

As recorded in Table 7 it was apparent that out of the 50 examined samples of pasteurized milk, Staph. aureus was isolated from 2 samples (4%). This result was similar to that obtained by Vahedi et al. (2013). While higher percentages were recorded by Gu¨ndog˘an et al. (2006); Rall et al. (2008); De Oliveira et al. (2011).  Unlike, Leite et al. (2002); Ayele et al. (2017) did not detect Staph. aureus in the pasteurized milk in Salvador and Ethiopia respectively.

The presence of Staph. aureus in pasteurized samples can be explained either by post-pasteurization contamination or by the presence of heat resistant strain. When heat treatment process is ineffective; faulty pasteurization will not destroy all foodborne pathogens (Kadariya et al., 2014). Insufficiently cleaned milk equipment was the most frequently incriminated source of pasteurized milk contamination with Staph. aureus.

It was clear from the data showed in Table 7 that L. monocytogenes could not be detected in all of the examined samples. These results are in agreement with those obtained by Baek et al. (2000); Padilha et al. (2001); Abd El Aal and Atta (2009). On the contrary, the current results disagreed with others like, Ahmed and Hussein (2005); Seyoum et al. (2015) as they reported 4 and 1 % of pasteurized milk examined were contaminated with L. monocytogenes.

Similarly E. coli O157:H7 couldn't be detected in pasteurized milk, unlike Upton and Coia (1994) who could isolated it after occurring an outbreak of E. coli O157 infection associated with pasteurized milk supply. This outbreak resulted in 9 children developing haemolytic uraemic syndrome (HUS) and one elderly woman developing thrombotic thrombocytopenia purpura (TTP).

It was concluded that, some milk borne pathogens like Staph. aureus and L. monocytogenes could be isolated from rawmilk, in which it is unsafe for human consumption.

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