|Year : 2018 | Volume
| Issue : 3 | Page : 419-428
Evaluation of modified semisolid Rappaport Vassiliadis medium in comparison with conventional media in the isolation of Salmonella species from different samples
Soheir Abd El Rahman Abd El Samie1, Yasser Mahmoud Ismail1, Sahar Mohammed Fayed1, Shaimaa Sobhy Hamed2
1 Department of Clinical and Chemical Pathology, Benha University, Benha, Egypt
2 Department of Medicine and Surgery, Benha University, Benha, Egypt
|Date of Submission||06-Aug-2018|
|Date of Acceptance||26-Sep-2018|
|Date of Web Publication||07-Jan-2019|
Dr. Shaimaa Sobhy Hamed
Source of Support: None, Conflict of Interest: None
Objectives The goal of this study is to assess the performance of the modified semisolid Rappaport Vassiliadis media (MSRV) for detecting Salmonella spp. in comparison with conventional media especially xylose lysine desoxycholate agar (XLD agar) and Salmonella Shigella (SS) agar.
Results The analysis showed that the overall Salmonella prevalence was 10% (18/180). Of the 18 Salmonella-positive samples, 16 (89%) were isolated from patients suffering from gastroenteritis, while one Salmonella strain (5.5%) was detected in the 45 stool samples of food handlers, and one Salmonella strain (5.5%) isolated from the 45 food samples. SS culture results comparable to serotyping resulted in sensitivity, specificity, positive predictive, negative predictive, accuracy values of 83.3, 98.2, 83.3, 98.2, and 96.7%, respectively, while XLD generated 83.3% sensitivity, 97.5% specificity, 79% positive predictive value, 98.1% negative predictive value, 96.1% accuracy. Statistically, the MSRV method resulted in higher values of sensitivity (94.4%), specificity (99.4%), positive predictive value (94.4%), and negative predictive value (99.4%), and accuracy (98.9%) than those obtained by the other two culture methods.
Conclusions The current study showed that the overall diagnostic performance of MSRV for the detection of Salmonella spp., including sensitivity, specificity, and accuracy, was significantly (P<0.05) greater than that of SS and XLD culturing methods. The incidence of Salmonella in diarrheal patients was significantly (P<0.05) higher than in food handlers and food samples. The occurrence of Salmonella was not significantly (P<0.05) related to the age and the sex of diarrheal patients. Salmonella typhimurium and Salmonella lamberhurst were the most prevalent serotypes in the tested samples.
Keywords: food, modified semisolid Rappaport Vassiliadis medium, qualification, Salmonella, Salmonella Shigella medium, stool, xylose lysine desoxycholate medium
|How to cite this article:|
Abd El Samie SA, Ismail YM, Fayed SM, Hamed SS. Evaluation of modified semisolid Rappaport Vassiliadis medium in comparison with conventional media in the isolation of Salmonella species from different samples. Benha Med J 2018;35:419-28
|How to cite this URL:|
Abd El Samie SA, Ismail YM, Fayed SM, Hamed SS. Evaluation of modified semisolid Rappaport Vassiliadis medium in comparison with conventional media in the isolation of Salmonella species from different samples. Benha Med J [serial online] 2018 [cited 2019 Dec 15];35:419-28. Available from: http://www.bmfj.eg.net/text.asp?2018/35/3/419/249420
| Introduction|| |
WHO reported that Salmonella More Detailse remains one of the major global causes of foodborne diarrheal illness ,. In both developed and developing countries, Salmonellosis More Details is considered an important foodborne disease which recurrently causes a major and unacceptable threat to human public health (European Food Safety Authority, 2010). Worldwide, Salmonella spp. were estimated as the major causes of diarrheal and invasive foodborne deaths accounted for 93.8 million gastroenteritis cases, 111 000 deaths, and around 8.6 million disability-adjusted life years (Havelaar et al., 2015; Majowicz et al., 2010) ,.
The prevalence of Salmonella in food in tropical African countries was found to be the highest compared with the rest of the world .
Salmonella enterica serovar Typhi (S. Typhi) and the various pathovars of S. Paratyphi are commonly referred to as typhoidal Salmonella serovars. These agents are restricted to human hosts. Salmonella serovars that fall outside of this group are typically referred to as the nontyphoidal Salmonella (NTS) serovars and are considered to have the potential to interact with human and nonhuman hosts .
For over a century, conventional methods for the isolation of salmonella are very valuable . Nevertheless, there is no ideal method for the diagnosis of Salmonella infection; the development of highly selective media continues to be of significant importance for diagnostic and treatment purposes . Plating media depend on the detection of characteristic products or byproducts and behavior of the pathogens for their isolation. For instance, agar media containing chromogenic substrates for Salmonella-specific enzymes have been developed; however, chromogenic agar media are less sensitive, but have greater specificity when compared with conventional media .
The efficacy of modified semisolid Rappaport Vassiliadis (MSRV) medium is based on the ability of Salmonella spp. to migrate through the selective medium ahead of other competing motile bacteria, thus producing opaque halos of growth surrounding the inoculating area. MSRV can be used as a plating medium for isolating Salmonella spp. (other than S. Typhi and S. Paratyphi A) from stool specimens with high sensitivity and specificity relative to chromogenic and conventional agar media ,.
Here, the basic goal of this study is to assess the performance of the MSRV media for detecting Salmonella spp. in comparison to conventional media especially xylose lysine desoxycholate (XLD) agar and Salmonella Shigella (SS) agar.
| Materials and methods|| |
The study involved 90 patients (50 men and 40 women) who developed clinical evidence of gastrointestinal illness and fever. Also, 45 rectal swabs were collected from food handlers at Benha Central Laboratory, and then 45 different food samples (raw and cooked meat) were collected. The fecal samples were submitted in a clean, container with no soap or disinfectant residue. The samples must be kept cold and transported to the laboratory within 8 h of collection. Stool, food (∼10 g of stool or food/90 ml of sterile saline solution), and food handler’s swab (10 ml of sterile saline solution) suspensions were prepared by agitation of swabs in sterile saline solution (0.85% sodium chloride) .
- MSRV agar: MSRV Agar is used for the selective enrichment of Salmonella spp. from food, animal feces, and environmental samples from the primary stage of animal production. This medium is based on the ability of salmonellae to migrate through the selective medium ahead of competing Gram-negative, motile organisms, thus producing opaque halos of growth. Novobiocin, low pH, high magnesium chloride concentration, and malachite green inhibit the growth of Gram-positive flora .
- XLD agar: XLD has been found to be a satisfactory medium for the isolation and presumptive identification of both salmonellae and shigellae. It relies on xylose fermentation, lysine decarboxylation, and production of hydrogen sulfide for the primary differentiation of Shigellae and salmonellae from nonpathogenic bacteria. Salmonellae exhaust the xylose and decarboxylate the lysine, thus altering the pH to alkaline and mimicking the Shigella reaction.
- SS agar: SS agar is a differential, selective medium for the isolation of Shigella spp. and Salmonella spp. from pathological specimens, suspected food stuffs, etc. Gram-positive and coliform organisms are inhibited by the action of the selective inhibitory components brilliant green, bile salts, thiosulfate, and citrate. Thiosulfate in combination with iron also acts as an indicator for sulfide production, which is indicated by blackening in the centers of the salmonella colonies.
| Methods|| |
- Direct plating:
- MSRV: drops from the prepared stool suspension will be inoculated in separate spots (or periphery) on the surface of MSRV medium and then incubated aerobically in an upright position at 42°C, for 20–24 h  ([Figure 1]).
|Figure 1 Motile Salmonella spp. is visible as a gray-white, turbid zone extending out from the inoculated drop.|
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- XLD: XLD agar plates were stroked with stool suspension using disposable sterile loop and then incubated at 36.0°C±1°C overnight (18–24 h) , ([Figure 2]).
|Figure 2 Salmonella colonies on xylose lysine desoxycholate agar (XLD agar) plates has a slightly transparent red halo and a black center; a pink-red zone may be seen in the media surrounding the colonies.|
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- SS agar: SS agar plates were also stroked with stool suspension using disposable sterile loop and then incubated at 36.0±1°C overnight (18–24 h) , ([Figure 3]).
|Figure 3 Salmonella colonies on Salmonella Shigella agar (SS agar) appear colorless with black centers.|
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- Indirect plating:
- Pre-enrichment: nonselective pre-enrichment allows stressed or injured Salmonellae to recover before exposure to selective enrichment media by weighting 25 g food or human feces with a sterile spatula into a flask with 25 ml of buffered peptone incubate at 36°C (±1°C) overnight (16–20 h).
- Selective enrichment: with a micropipette or sterile loop a measure of 0.1 ml (100 µl or one drop by loop) of the pre-enrichment broth will be transferred to 10 ml Rappaport-Vassiliadisbroth. Incubate Rappaport-Vassiliadis at 41.5±0.5°C overnight (18–24 h) .
- Spread on agar plates: after selective enrichment the three agar plates, MSRV, XLD agar, and SS agar were inoculated by the same approach previously described with direct plating. The test sample is considered presumptively positive for motile Salmonella spp. on MSRV, when the growth of migrated cells is visible as a gray-white, turbid zone extending out from the inoculated drop, but considered negative, when the medium remains blue-green around the inoculation drops. On the XLD plates, a typical Salmonella colony has a slightly transparent red halo and a black center; a pink-red zone may be seen in the media surrounding the colonies ([Figure 2]). The resulting Salmonella colonies on SS agar will appear colorless with black centers ([Figure 3]).
- Biochemical Identification: according to the WHO  isolates suspected of being salmonella on the previous media were inoculated on the following biochemical test: motility indole, ornithine, citrate (Simmons), urea hydrolysis, hydrogen sulfide, or triple sugar iron test, and lysine decarboxylase.
- Serotyping: serological identification was carried out, based on the slide agglutination test, according to the Kauffman–White scheme which classifies the genus Salmonella into serotypes according to combinations of somatic O (using polyvalent and monovalent O antisera) and flagellar H antigens (using H grouping and factors antisera).
- Statistical analysis: performances of the comparable three medium were analyzed using SPSS version 20 (IBM Corp, released 2011, IBM SPSS Statistics for Windows, Version 20.0; IBM, Armonk, New York, USA), with differences considered significant at P value less than 0.05. Analyses of diagnostic sensitivity, diagnostic specificity, and positive and negative predictive values were examined using Microsoft Excel 2016 (Microsoft Excel version 3, 2013; Microsoft Corp., Redmond, Washington, USA).
| Results|| |
A total of 180 samples were examined for the detection of Salmonella; 90 stool samples from patients suffering from gastroenteritis, 45 stool samples from food handlers, and 45 food samples of raw and/or cooked meat, chicken, and fish) were collected. The analysis showed that the overall Salmonella prevalence was 10% (18/180). Of the 18 Salmonella-positive samples, 16 (89%) were isolated from patients suffering from gastroenteritis, while one (5.5%) Salmonella strain was detected in the 45 stool samples of food handlers, and one Salmonella strain (5.5%) was isolated from the 45 food samples ([Table 1]).
|Table 1 The results of MSRV agar, SS agar, and XLD agar for the isolation of Salmonella from 180 samples (90 stool samples of patients suffering of gastroenteritis, 45 stool samples of food handlers, and 45 food samples) in comparison to serotyping|
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In detail, SS and XLD culture methods, comparable to serotyping, carried out for the detection of Salmonella spp. in the 90 stool samples of diarrheal patients resulted in 14 true positives, 71 true negative, three false positives, and two false negative samples, thus generating an area under the curve (AUC), sensitivity, specificity, and accuracy values of 0.92, 87.5, 95.9, and 94.4%, respectively; while, on the same samples the MSRV method produced 15 true positives, 73 true negatives, one false positive, and one false negative sample, which resulted in 0.96 AUC, 93.8% sensitivity, 98.6% specificity, and 96.1% accuracy ([Table 3]).
|Table 2 Comparison of age and sex findings on Salmonella-positive samples|
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|Table 3 the performance of SS agar, XLD agar and MSRV agar in comparison to serotyping for the prediction of salmonella-positive stool samples among diarrheal patients|
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In the 45 stool samples of food handlers, SS, XLD, and MSRV culture methods were 100% in concordance with the serotyping results ([Table 4]).
|Table 4 the performance of SS, XLD, and MSRV agar in comparison to serotyping for the prediction of salmonella-positive stool samples among food handlers|
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With the 45 food samples, the SS culture method yielded all tested sample Salmonella negative, which in comparison to serotyping resulted in one false negative sample ([Table 5]), while the XLD isolation method resulted in one false positive and one false negative sample that generated AUC, sensitivity, specificity, and accuracy values of 0.49, 0.0, 97.7, and 95.6%, respectively. The isolation results of Salmonella from food samples using the MSRV method were totally in agreement with the serotyping results. According to primary colonial appearance on SS Agar, XLD and MSRV medium, the results showed that 24 of 180 samples from the stool of diarrheal patients and food handlers, and food samples were morphologically identified as Salmonella-positive colonies ([Table 6]). Out of the 24 samples, 14 were Salmonella-positive by the three culture methods and serotyping, 2 samples gave growth only on SS agar (both were -ve by serotyping), 2 samples were grown only on XLD agar (both were serotyping negative), 3 samples gave colony only on MSRV agar (all were serotyping positive), 2 samples were positive on SS and XLD mediums (one was serotyping positive), and one sample gave salmonellae colony on MSRV and XLD mediums (serotyping-negative). Further, serotyping showed that 18 of 24 suspected Salmonella samples were confirmed as being Salmonella-positive samples ([Table 1] and [Table 6]).
|Table 5 the performance of SS, XLD, and MSRV culturing methods in comparison to serotyping for the prediction of salmonella-positive food samples|
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|Table 6 The results of serotyping conducted on 24 samples morphologically showed salmonella colonies on SS and/or XLD, and/or MSRV plates|
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Serologically, the serotypes identified were 33.3% (6/18) Salmonella typhimurium, 27.8% (5/18) Salmonella lamberhurst, and 16.7% (3/18) Salmonella tsevie. Further, only one (5.5%) isolate was identified for Salmonella anatum, Salmonella butantan, Salmonella paratyphi C, and Salmonella tumodi.
| Discussion|| |
The cornerstone for the diagnosis of diarrheal illness is the microbiologic analysis of the stool, so stool culture testing has still been routinely used for most patients with diarrheal illness ,.
In this study, 180 (90 diarrheal patient, 45 stool of food handlers, and 45 different food sample) samples were examined to estimate the performance of MSRV agar in comparison to SS agar, and XLD agar medium for primary isolation of Salmonella strains. The final analysis showed that the Salmonella prevalence was 10% (18/180). This percentage was closely related with the rate of isolation by Ruiz Gomez et al. , 11.3% (197/1717). However, it was higher than the rate recorded by Maddocks et al.  which was 1.8% of the 500 clinical stool samples. The Centers for Disease Control and Prevention (CDC) reported that 1.4 million cases, 15 000 hospitalizations, and 400 deaths are caused by Salmonella infections in the USA every year . On the other hand, the incidence of Salmonella at the level of stool sampled from patients suffering from gastroenteritis was 17.8% (16/90) ([Table 1] and [Table 2]), which was lower than the rate isolated with Stuart and Pivnick , 29.3% (a total of 72 salmonellae from the 246 specimens) and nearly close to the rate of Salmonella in the stool sample detected by Aguirre et al. , was 19.30% (n=83/430). A more recent study on a total of 13 327 diarrheal patients, salmonella was reported in 1.1% (147/13 327) of patients stool sample . The discrepancy between our results and Lee et al.  study may be in the choice of sample, as the stool culture for salmonella is not requested except if there is strong evidence of presence of bacterial dysentery , and difference in environment and epidemiological background.
Remarkably, the sensitivity (88.9%), specificity (99.4%), and accuracy (98.3%) values produced by direct culturing on Modified semi-solid Rappaport Vassiliadis Agar were close to that of indirect isolation in current study ([Table 1] and [Table 7]). This result was higher than the recovery rates of direct plating reported by Srijan et al. (2015) (85.3%), and Dusch and Altwegg (1995), (63.4%), while was lower than Ruiz Gomez et al. (1998), (89.2%). The reasons, of high recovery rate recorded here by direct plating, were that the number of samples used in previous studies was significantly higher than those used in our study.
|Table 7 sensitivity, specificity, positive predictive value and negative predictive value, and accuracy values generated by MSRV media, SS media, and XLD media comparable to serotyping for detection of Salmonella isolates in 180 samples|
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Given the food handlers, 2.22% (1/45) of the rectal swabs were salmonella positive ([Table 1] and [Table 3]), which is close to Aklilu et al. , who isolated Salmonella from 3.5% of food handlers stool. But, in Nigeria, a higher rate was obtained, where 13.2% (7/53) of apparently healthy food handlers were positive for Salmonella spp. .
In Africa, diarrheal disease agents, particularly nontyphoidal Salmonella, were responsible for 70% of foodborne diseases. Moreover in Africa, nontyphoidal Salmonella enterica was responsible for more than half (32 000) of the global deaths (59 000) . Here, salmonella was isolated from 2.22% (1/45) of food samples ([Table 1] and [Table 4]), which was significantly lower than Gelinski et al. , who detected it in 17% (17/100) of Brazilian sausages.
The data showed that there was no significant (P>0.05) relation between the age and/or the sex of diarrheal patients, with the occurrence of salmonella in the tested stool samples ([Table 2]).
Statistically, the results showed that the MSRV culturing method has significantly higher (P<0.05) agreement with the serotyping result or performance than the other two methods at the overall level ([Table 1]) and at the level of diarrheal patients and food samples ([Table 3] and [Table 5]), but at the level of food handlers group no difference in the performance of the three culturing methods was noticed for the isolation of salmonella ([Table 4]).
In comparison to serotyping, SS culturing method resulted in 15 true positives, 159 true negatives, three false positives, and three false negative samples. Thus, SS generated sensitivity, specificity, positive predictive, negative predictive, and accuracy values of 83.3, 98.2, 83.3, 98.2, and 96.7%, respectively, while, the XLD generated 15 true positives, 158 true negatives, four false positives, and three false negative samples, which resulted in 83.3% sensitivity, 97.5% specificity, 79% positive predictive value, 98.1% negative predictive value, and 96.1% accuracy. Further, the MSRV method generated 17 true positives, 161 true negatives, one false positive, and one false negative sample, which statistically resulted in higher values of sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, as follows, 94.4, 99.4, 94.4, 99.4, and 98.9%, respectively, than those obtained by the other two culture methods ([Table 6]). The sensitivity values, 100%, and 96.4%, of MSRV agar after enrichment generated within the studies of Dusch and Altwegg (1995), and Ruiz Gomez et al. (1998) was higher than the values recorded here before 88.9% and after enrichment and 94.4%, respectively.
Whenever MSRV was evaluated in comparison to other media it was always with the highest value in terms of performance. Ruiz et al.  found that sensitivities and specificities of MSRV and SS agar were 95.1 and 98.1, and 69.1 and 99.3, respectively. Also, MSRV was the most sensitive (100%) medium tested and was a very specific medium for the isolation of NTS from stool specimens, comparable to Hektoen enteric agar (HE), Rambach agar (Ra), SM-ID medium (SM), xylose lysine Tergitol 4 agar (XLT4), novobiocin-brilliant green-glycerol-lactose agar (NBGL) . Similarly, Srijan and Wongstitwilairoong  suggested that the use of MSRV over MacConkey (MAC) and Hektoen enteric (HE), and XLD agars for the isolation of nontyphi Salmonella species from human stools was more efficacious, while the study of Ruiz Gomez et al.  reported that the yield from MSRV was significantly greater than that from SS (P<0.0001), generating sensitivity values of 89.2% (173/176 Salmonella strains were isolated) on MSRV and 64.4% (125/176) on SS by direct plating, and after enrichment in selenite broth, the yield from MSRV was 96.4% (n=187) and from SS it was 88.1% (n=171).
Notably, the specificity values of MSRV obtained in the current study was superior to the values reported by previous studies. To clarify, Ruiz Gomez et al.  found that SS, after enrichment, had higher specificity (100%) than the MSRV (97.3% specificity), which mean that the SS culturing method yielded a lower number of false positive samples than MSRV. Also, Ruiz and Nunez  reported similar conclusions.
In the present study the statistical outputs of enrichment plating on MSRV agar were relatively higher than direct plating but was not significant. Most studies conducted for isolation of Salmonella spp. on MSRV medium concluded that enrichment followed with plating on the media resulted in better performance. Also, Aspinall et al. (1992) found that MSRV medium when combined with direct culture and selenite enrichment, 98.9% of 183 strains of Salmonella spp., from faeces, were recovered. Also, Ruiz et al. (1996) reported that after enrichment in selenite broth, the yield from MSRV (96.4%) was also significantly greater than that from SS (88.1%). The results here indicated that screening of samples for the presence of salmonellae on SS and XLD media is labor-intensive because of the high number of colonies (e.g. Proteus spp. and Citrobacter spp.) that resemble Salmonella spp. This results in a low specificity and, consequently, in additional costs for subsequent identification . While MSRV medium resulted in lowest false positives and negatives, and the direct plating produced sensitivity and specificity values close to the indirect plating, and higher than the values of SS and XLD mediums. Hence, direct plating on MSRV could shorten the detection time of Salmonella spp.
Of the serotyping conducted in our study resulted in seven Salmonella enterica serotypes S. typhimurium was the highest identified serotype with (6/18) 33.3%; five were isolated from diarrheal patients and one from rectal swabs of food handlers. These results agree with previous studies of Kelly et al. , who found that Salmonella enterica is an important human pathogen, especially S. typhimurium was the most common serotypes in Europe and North. Similarly, in Africa S. typhimurium was among the major serovar implicated and together with Salmonella enteritidis were responsible for 91% of the cases of invasive nontyphoidal Salmonella (iNTS) disease .
Then, S. lamberhurst come here in the next rank after S. typhimurium was isolated at a rate of 27.8% (n=5/18). In the United States, only one case of S. lamberhurst was reported to CDC between 1999 and 2009 .
Also, in our study three S. tsevie (16.7%) were isolated from the stool samples of diarrheal illness (2/3), and food samples (1/3). In Egypt, S. tsevie were isolated in four broiler chicken flocks in Qalyubia Governorate at a rate of 3.92% (n=2) , which may suggest the link between the source of contamination with S. tsevie and the illness. In the USA, there were five cases of laboratory-confirmed S. tsevie from human sources reported to the CDC between 1999 and 2013 ,.
Furthermore, S. paratyphi C has been isolated from one (5.5%) of the diarrheal patients ([Table 7]). This organism was classified as a member of serogroup C1 , and can cause typhoid in humans but, unlike S. typhi, also occasionally can infect animals . This pathogen was reported as a minor cause of enteric fevers in South America, Asia, and the USA . However, in British Guiana, a part of Africa, occasional outbreaks of enteric fever caused by S. paratyphi C have been reported . Also it was incriminated in an outbreak in South Africa . In the USA, there were nine cases of Salmonella paratyphi C from human sources reported to the CDC between 1999 and 2013 ,.
In the current study, S. anatum was detected in only one stool sample of diarrheal patients. It is considered one of the top 20 Salmonella serotypes in the USA . Recently, S. anatum was responsible for five hospitalized ill people and one death in a multistate outbreak in the USA .
Finally, S. butantan was also detected in a stool sample of diarrheal patients. This organism and Butantan var. (15+) are rarely reported, only five laboratory-confirmed isolates from human sources were reported to CDC between 1999 and 2013 ,.
| Conclusion|| |
The current study has shown that the overall diagnostic performance of MSRV for the detection of Salmonella, including sensitivity, specificity, and accuracy was significantly (P<0.05) greater than that of SS and XLD culturing methods. The incidence of salmonella in diarrheal patients (17.8%) was significantly (P<0.05) higher than in food handlers (2.2%) and food samples (2.2%). The occurrence of salmonella in male diarrheal patients was higher than females, however statistically was not significant (P<0.05). S. Typhimurium was the most prevalent serotype in the tested samples.
This study was technically supported and provided with the necessary space, material and equipment of processing and completion by the Department of clinical and Chemical Pathology, Benha University.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]