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<h1 class="Hdr1">

Short Communication


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Advances in Animal and Veterinary Sciences 2 (7): 390 – 392


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<h2 class="Hdr2">


Isolation of Salmonella from Liquid Whole Eggs Sold in Retail Outlets in Egypt, Bangladesh and not in Japan 

</h2>

<p class="p3">





<strong>Amira Ahmed</strong><span><sup>1,2*</sup></span>, 

<strong>Hajime Fukunaga</strong><span><sup>1</sup></span>, 


<strong>Takuya Mizuno</strong><span><sup>1</sup></span>, 

<strong>Takayuki Ezaki</strong><span><sup>1</sup></span>


</p>


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<div class="p4">

<ol>

<li>Department of Microbiology, Gifu University Graduate School of Medicine, 1–1 Yanagido, Gifu 501–1194, Japan</li>

<li>Department of Poultry & Rabbit Medicine, Faculty of Veterinary Medicine, Suez Canal University, Egypt </li>


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<div>        
<p><span class="Author">*Corresponding author:A.A.H.Abdelaziz@gmail.com  </span></p>

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<div id="ColorID"><p>
<span><b>ARTICLE CITATION:</b></span> 

Ahmed A; Fukunaga H; Mizuno T; Ezaki T (2014). Isolation of salmonella from liquid whole eggs sold in retail outlets in egypt, Bangladesh and not in Japan. Adv. Anim. Vet. Sci. 2 (7): 390 – 392.


<br>
<span><b>Received: </b></span>2014–07–27, <span><b>Revised: </b></span>2014–08–07, <span><b>Accepted: </b></span>2014–08–08<br>

The electronic version of this article is the complete one and can be found online at 

(<a href="http://dx.doi.org/10.14737/journal.aavs/2014/2.7.390.392 ">

http://dx.doi.org/10.14737/journal.aavs/2014/2.7.390.392 

</a>)
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited </p>
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<div>
     <h3 class="Hdr3">ABSTRACT</h3>

    <p>
    The main objective of this survey was to establish the current prevalence of Salmonella in raw egg samples from the following countries: Japan, Egypt, and Bangladesh. A total of 141 sample units of eggs were tested over a five–month period from December 2013 to April 2014. Following conventional bacteriological procedures the samples subjected for molecular identification by PCR using genus specific primers for Salmonella spp. All samples were randomly purchased and analyzed within their stated shelf life. Salmonella SPP. contamination was recorded in 17.6% (9/51) of eggs' contents of Egypt samples and recorded in 38% (8/21) of eggs' contents of Bangladesh samples while none of the samples from Japan (0/69) contaminated with Salmonella SPP in the eggs' contents. Detection of Salmonella in chicken eggs' contents needs a greater concern for effective control of Salmonellosis in poultry.
     

    </p>
 
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<div>
  
  <h3 class="Hdr3"></h3>
    <p>
    The consumption of raw or lightly cooked eggs, egg containing foods in particular mayonnaise, desserts and sauces has been associated with high percentage of many human Salmonellosis outbreaks. 
</p><p>The intestinal tract is the primary reservoir of Salmonella in poultry birds leading to contamination of chicken eggs in cloacal region through horizontal route. Transovarian contamination is another important route of Salmonella egg contamination. Several studies have isolated Salmonella spp. from ovaries and oviducts of naturally and experimentally infected hens. The presence of Salmonella in the reproductive tract was consistent with the production of Salmonella contaminated eggs in the albumen, the yolk or the both (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Keller1995" target="_self">Keller et al., 1995</a>; <a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Gast1990" target="_self">Gast and Beard, 1990</a>; <a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Gast1993" target="_self">Gast, 1993</a>; <a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Timoney1989" target="_self">Timoney et al., 1989</a>). Food borne Salmonella out–breaks still represents a great hazard in Egypt (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Ibrahim2013" target="_self">Ibrahim et al., 2013</a>), Bangladesh (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Barua2013" target="_self">Barua et al., 2013</a>) and in a lower extent in Japan (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Suzuki2009" target="_self">Suzuki H, and Yamamoto S, 2009</a>).
</p><p>The goal of this study was to determine the incidence of Salmonella in raw egg samples sold in the following countries: Japan, Egypt, and Bangladesh, using both conventional method, and a specific PCR detection technique.
</p><br><p>
Eggs were purchased from a retail outlets in a residential area of (Gifu Prefecture, Japan; Kafr Elsheik, Dkhalia, and Ismailia cities, Egypt; Dhaka, Bangladesh) over a period of 5–months (December 2013–April 2014). A total of 141 eggs' content were analyzed for the presence of Salmonella. In order to collect the eggs' contents, eggs' surfaces were sterilized by swabbing with 70% alcohol, drying with the air and then cracked with a sterile knife. Yolks and whites were mixed thoroughly, so that their contents pooled; each pool consisted between 6 and 10 eggs.
</p><br><p>
According to The World Organization for Animal Health 2008, the conventional culture methods for detection of Salmonella in food include non–selective pre–enrichment followed by selective enrichment, plating on selective and differential agars, and biochemical tests.
</p><br><p>
A 25 gram portion of the pooled sample was added to 225 ml BPW and mixed well before incubation at 35° C for 24 hours.
</p><br><p>
1ml samples were taken and mixed with 10 ml of Rappaport Vassialidis broth (RV) and incubated at 35° C for 24 hours.
</p><br><p>
A loopful of each enriched sample was streaked on selective agar medium deoxycholate hydrogen sulphide lactose agar (DHL) plate (EIKEN, Tokyo, Japan), using such medium enables testing of the bacterium for lactose fermentation and H2S production. Suspect colonies are confirmed by molecular identification.
</p><br><p>
The DNA was released by heating at 100° C for 5 min. The cellular debris was pelleted by centrifugation at 8000 rpm for 5 min, and the clear supernatant fluid containing nucleic acids was conserved until it was used in subsequent PCR assays.
</p><br><p>
The DNA samples (5 µL) were amplified in an optimized 20 µL reaction mixture consisting of 2.5 µL of each primer 0.4 µM /reaction mixture, (10 µL) 2× SYBER Premix Extaq buffer (TAKARA, Japan). The reaction mixture was incubated in a programmable DNA thermal cycler (Thermogen, model QB–0224B). Salmonella Genus specific primers 139 bp (AMR No DCR227, Japan), which were based on the invA gene of Salmonella, were used in the PCR assay. 
</p><p>A reagent blank containing all the components of the reaction mixture with the exception of template DNA (which was replaced by sterile distilled water) was included with every PCR assay. Furthermore, positive DNA control was included, which was prepared from Salmonella sp., respectively. The cycling parameters used were initially denaturated at 95° C for 1 min followed by 40 cycles of amplification of 10 s at 95° C, and 10 s at 60° C. Then, the PCR tubes were held at 4° C.
</p><br><p>
The PCR products were analyzed by gel electrophoresis. Five microliters of each sample was loaded onto 2.0% of agarose gel in 1×TAE buffer at 100 V/cm for
</p><p>30 min. The gel was stained with ethidium bromide and electrophoresed products were visualized with a UV transilluminator (FAS–III, Toyobo, Osaka, Japan). A 100–bp ladder (Toyobo, Japan) was used as a molecular weight marker.
</p><p>Growth of Salmonella was detected on DHL agar plates as shown in Figure 1 whitish colonies (non–lactose fermenter) with H2S production indicated by a blackening of the colonies due to a formation of iron sulfide.
</p>

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<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig1_496.PNG" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig1_496.PNG" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig1_496.PNG" target="new">Figure 1: </a></b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig1_496.PNG"><span>Shows the growth of Salmonella on DHL plates with its whitish (non– lactose fermenter) colonies, and blackish colouration because of H2S production</a></p>
</div>
<div>



<p>Molecular characterization using PCR produced positive amplification of 139 bp fragments of invA genes (100%) specific for all members of Salmonella SPP.
</p><p>From the previous results, it could be concluded that Salmonella–specific PCR test (Figure 2) in conjunction with traditional isolation methods could be effective in providing a more accurate profile of the prevalence of Salmonella in poultry products.
</p>

</div>
<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_496.PNG" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_496.PNG" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_496.PNG" target="new">Figure 2: </a></b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_496.PNG"><span></a></p>
</div>
<div>



<p>
The results of Salmonella incidence in eggs from pooled samples are shown in Table 1. Salmonella contamination was recorded 17.6% (9/51) of eggs contents of Egypt samples and recorded in 38% (8/21) of eggs contents of Bangladesh samples while none of the samples from japan (0/69) contains Salmonella contamination in the egg’s contents.
</p>


</div>
<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table1_496.PNG" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/imagesfiles/Table1_496.PNG" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table1_496.PNG" target="new">Table 1: </a></b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table1_496.PNG"><span>The prevalence of Salmonella isolated from eggs' content</a></p>
</div>
<div>


<p>Since Salmonella is an important zoonotic bacterium with poultry as largest single reservoir of Salmonella (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Gupta1999" target="_self">Gupta et al., 1999</a>), the assessment of the contamination level and site of contamination are the utmost importance in deciding the control strategies against Salmonella in chickens.
</p><p>Moreover, the use of a three step protocol (pre–enrichment, selective enrichment and selective plating) as specified in the FDA's Bacteriological Analytical Manual (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#US Food Drug2002" target="_self">US Food Drug Administration, 2002</a>) was found satisfactory for the recovery of Salmonella spp.
</p><p>The present study demonstrated widespread contamination by Salmonella in eggs retailed in Egypt, similar average of contamination of poultry and or poultry products showed previously by (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Ibrahim2013" target="_self">Ibrahim et al., 2013</a>, <a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Ibrahim2014" target="_self">Ibrahim et al., 2014</a>) 
</p><p>Our results reinforce previous investigations, suggesting that Salmonella as one of the predominant pathogens circulating in poultry in Bangladesh (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Barua2012" target="_self">Barua et al., 2012</a>, <a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Barua2013" target="_self">2013</a>) and plays a role as a source of human infections (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Barua2014" target="_self">Barua et al., 2014</a>), and consistent with the previous studies indicating the very low internal eggs' contamination in Japan (<a href="http://nexusacademicpublishers.com/table_contents_detail/4/350/html/#Suzuki2009" target="_self">Suzuki H, and Yamamoto S, 2009</a>).
</p><p>This study emphasized the need to implement proactive measures of hygienic practices, surveillance programs for laying hen flocks should be optimized, and the application of Hazard Analysis and Critical Control Point (HACCP) in the preparation and processing of foods in order to reduce Salmonella contamination of eggs in retail and to reduce the risk of human infection.


</p>
  
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<div>
<h3 class="Hdr3">REFERENCES</h3>
 

<p class="p2"> <a name="Barua2012"></a>
Barua H, Biswas PK, Olsen KEP, Christensen JP (2012). Prevalence and characterization of motile Salmonella in commercial layer poultry farms in Bangladesh. PLoS ONE 7, e35914, http://dx.doi.org/10.1371/journal.pone.0035914.<br>
<a href="http://dx.doi.org/10.1371/journal.pone.0035914" target="_blank">http://dx.doi.org/10.1371/journal.pone.0035914</a>
</p>

<p class="p2"> <a name="Barua2013"></a>
Barua H, Biswas PK, Olsen KEP, Shil SK, Christensen JP (2013).Molecular characterization of motile serovars of Salmonella entericA from breeder and commercial broiler poultry farms in Bangladesh. PLoS ONE 8, e57811, http://dx.doi.org/10.1371/journal.pone.0057811. <br>
<a href="http://dx.doi.org/10.1371/journal.pone.0057811" target="_blank">http://dx.doi.org/10.1371/journal.pone.0057811</a>
</p>

<p class="p2"> <a name="Barua2014"></a>
Barua H, Biswas PK, Talukder KA, Olsen KE, Christensen JP (2014). Poultry as a possible source of non–typhoidal Salmonella enterica serovars in humans in Bangladesh. Vet. Microbiol. 168: 372 – 380. <br>
<a href="http://dx.doi.org/10.1016/j.vetmic.2013.11.020" target="_blank">http://dx.doi.org/10.1016/j.vetmic.2013.11.020</a><br>
PMid:24355536 
</p>

<p class="p2"> <a name="Gast1990"></a>
Gast RK, Beard CW (1990). Production of Salmonella Enteritidis–contaminated eggs by experimentally infected hens. Avian Dis. 34:438–46. <br>
<a href="http://dx.doi.org/10.2307/1591433" target="_blank">http://dx.doi.org/10.2307/1591433</a><br>
<a href="http://dx.doi.org/10.2307/1591394" target="_blank">http://dx.doi.org/10.2307/1591394</a><br>
PMid:2196046 
</p>

<p class="p2"> <a name="Gast1993"></a>
Gast RK (1993). Detection of Salmonella Enteritidis in experimentally infected laying hens by culturing pools of egg contents. Poult. Sci. 72: 267 – 74. <br>
<a href="http://dx.doi.org/10.3382/ps.0720267" target="_blank">http://dx.doi.org/10.3382/ps.0720267</a><br>
PMid:8441730 
</p>

<p class="p2"> <a name="Gupta1999"></a>
Gupta V, Ray P, Sharma M (1999). Antimicrobial resistance pattern of Shigella and non–typhi Salmonella isolated from patients with diarrhoea. Indian J. Med. Res. 109: 43−45.
</p>

<p class="p2"> <a name="Ibrahim2013"></a>
Ibrahim MA, Emeash HH, Ghoneim NH, Abdel–Halim MA (2013). Seroepidemiological studies on poultry Salmonellosis and its public health importance. J. World's Poult. Res. 3(1): 18 – 23.
</p>

<p class="p2"> <a name="Ibrahim2014"></a>
Ibrahim WA, Abd El–Ghany WA, Nasef SA, Hatem ME (2014). A comparative study on the use of real time polymerase chain reaction (RT–PCR) and standard isolation techniques for the detection of Salmonellae in broiler chicks. Int. J. of Vet. Sci. Med. 2 (1): 67 – 71. <br>
<a href="http://dx.doi.org/10.1016/j.ijvsm.2013.11.001" target="_blank">http://dx.doi.org/10.1016/j.ijvsm.2013.11.001</a>
</p>

<p class="p2"> <a name="Keller1995"></a>
Keller LH, Benson CE, Krotec K, Eckroade RJ (1995). Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens. Infect. Immun. 63: 2443 – 9. <br>
PMid:7790055 PMCid:PMC173326
</p>

<p class="p2"> <a name="Suzuki2009"></a>
Suzuki Hodaka, Yamamoto Shigeki (2009). Literature survey of Salmonella contamination in eggs and egg products in the world Bulletin of National Institute of Health Sciences. 127: 74 – 83.
</p>

<p class="p2"> <a name="Timoney1989"></a>
Timoney JF, Shivaprasad HL, Baker RC, Rowe B (1989). Egg transmission after infection of hens with Salmonella enteritidis phage type 4. Vet. Rec. 125: 600 – 1. <br>
PMid:2692277 
</p>

<p class="p2"> <a name="US Food Drug2002"></a>
US Food Drug Administration (2002). Bacteriological analytical manual. Chap. 5. Salmonella AOAC International, Gaithessburg Md Available at http: 11 www.cfsan.felagovebam/bam–5.html.
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