Detection of Salmonella spp. by Traditional and PCR Assays in Raw Milk, Maysan, Iraq

Abstract

Salmonella spp are characterized as rod- shaped, motile, gram- negative bacteria which has the ability to infect
animals and human. Salmonella spp occasionally causes sickness while in most cases not lead to severe
symptoms. Analyzing milk for Salmonella spp. is not routine but traditional culture methods are used to
evaluate the health condition of the dairy products. However, the antibody-based and nucleic-acid- based
methods are practical for identifying Salmonella spp. Therefore, this research was designed to evaluate the use
of traditional culture methods and PCR in detection of the presence of Salmonella spp. in raw milk samples in,
Maysan Iraq. A total number of 130 raw milk samples collected from Maysan Iraq. All the samples were
analyzed for the presence of Salmonella spp. using traditional culture method and polymerase chain reaction
(PCR). The culture method used in this experiment were done by using pre-enrichment, enrichment, selective
plating and biochemical tests. The results of this traditional technique were compared with the results obtained
from PCR method. The PCR was performed using a 284bp sequence of the invA gene. The results showed that
8 (7.07%) of samples were identified as salmonella positive using traditional culture technique but 14 (12.3%)
samples were detected as salmonella positive by PCR method. The results of the current research revealed that
the traditional culture based methods are generally time costuming and labor intensive but the development of
new rapid methods including DNA based methods such as PCR are more sensitive and have dramatically
decreased the time necessary for the detection of bacteria

Full text

Archives of Razi Institute, Vol. 77, No. 4 (2022) 1461-1465 Copyright © 2022 by
Razi Vaccine & Serum Research Institute
DOI: 10.22092/ARI.2022.359086.2369
1. Introduction
Salmonella spp. are characterized as rod- shaped,
motile, gram- negative bacteria which has the ability to
infect animals and human. Salmonella spp. occasionally
causes sickness while in most cases not lead to severe
symptoms (1, 2). It has been well documented that the
most common reported causes of foodborne sickness are
caused by Salmonella spp. (3). More than half of the
Salmonella infections occurred due to the utilization of
non-pasteurized and contaminated animal products such
Original Article
Detection of
Salmonella
spp. by Traditional and PCR Assays
in Raw Milk, Maysan, Iraq 
Dawood Saleem, H1, Fawwaz Alfarras, A2, Hameed, N. M3, Hasan Al-Zubaidi, S4, Shnain Ali,
M5, Hamood, S. A6, Hameed, S7, Hamad, D. A8, Ali Hussein, H9, Mohsin Al-Dhalemi, D10 *
1. Al-Manara College for Medical Sciences, Maysan, Iraq
2. Medical Laboratory Techniques Department, College of Medical Techology, Al-Farahidi University, Baghdad, Iraq
3. Anesthesia techniques, Al–Nisour University College, Baghdad, Iraq
4. Anesthesia Techniques Department, Al-Mustaqbal University College, Babylon, Iraq
5. Department of Dentistry, Al-Zahrawi University College, Karbala, Iraq
6. Al-Esraa University College, Baghdad, Iraq
7. Medical Device Engineering, Ashur University College, Baghdad, Iraq
8. Nursing Department, Hilla University College, Babylon, Iraq
9. Scientific Research Center, Al-Ayen University, Thi-Qar, Iraq
10. The Islamic University, Najaf, Iraq
Received 1 May 2022; Accepted 19 May 2022
Corresponding Author: dhuha14@yahoo.com
Abstract
Salmonella spp are characterized as rod- shaped, motile, gram- negative bacteria which has the ability to infect
animals and human. Salmonella spp occasionally causes sickness while in most cases not lead to severe
symptoms. Analyzing milk for Salmonella spp. is not routine but traditional culture methods are used to
evaluate the health condition of the dairy products. However, the antibody-based and nucleic-acid- based
methods are practical for identifying Salmonella spp. Therefore, this research was designed to evaluate the use
of traditional culture methods and PCR in detection of the presence of Salmonella spp. in raw milk samples in,
Maysan Iraq. A total number of 130 raw milk samples collected from Maysan Iraq. All the samples were
analyzed for the presence of Salmonella spp. using traditional culture method and polymerase chain reaction
(PCR). The culture method used in this experiment were done by using pre-enrichment, enrichment, selective
plating and biochemical tests. The results of this traditional technique were compared with the results obtained
from PCR method. The PCR was performed using a 284bp sequence of the invA gene. The results showed that
8 (7.07%) of samples were identified as salmonella positive using traditional culture technique but 14 (12.3%)
samples were detected as salmonella positive by PCR method. The results of the current research revealed that
the traditional culture based methods are generally time costuming and labor intensive but the development of
new rapid methods including DNA based methods such as PCR are more sensitive and have dramatically
decreased the time necessary for the detection of bacteria.
Keywords: PCR, biochemical tests, raw milk, Salmonella spp.

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as dairy and poultry products (4). The source of bacteria
in raw milk include, feces, teat colonization or mastitis
infection bedding (4), cross contamination from the
milking staff and auto-milker (5).
Consumption of non-pasteurized milk or dairy products
is the common amount the rural and urban populations
due to some cultural reasons or for perceived health
benefits of natural and unprocessed food (4, 6). About the
consumption of the raw and non-pasteurized dairy
products consequently caused foodborne disease, there
have been limited epidemiologic published data.
However, it has been accepted that some factors like farm
management procedure, season, number of animals on the
farm, geographical area, and hygiene significantly affect
the prevalence rate of Salmonella spp. (7, 8).
Analyzing milk for Salmonella spp. is not routine but
traditional culture methods are used to evaluate the health
condition of the dairy products. However, the antibody-
based and nucleic-acid- based methods are practical for
identifying Salmonella spp. (5). Conventional culture
methods for the identification of salmonella spp. are
generally laboratory-based, time-consuming, and labor-
intensive (9, 10). However PCR targeting specific genetic
markers represents a major advance in term of speed,
specificity and sensitivity to improve food safety (9, 10).
Therefore, this research was designed to evaluate the
use of traditional culture methods and PCR in detection
of the presence of Salmonella spp. in raw milk samples
in, Maysan, Iraq.
2. Materials and Methods
2.1. Sample Collection
In the first step the city of Maysan was divided into
10 region, then using cluster sampling method 130 raw
milk samples were collected. All the collected samples
were transported to the laboratory at 4°C.
2.2. Traditional Culture Method
The milk samples were inoculated in to selective
enrichment broths. To do this a milk sample of 25 ml was
added directly to 225 ml of Selenite Cystine broth and
Tetrathionate Broth then incubated at 43°C and 37ºC,
respectively, for 18-24 h. Selective enrichment cultures
were subcultured on to Brilliant Green Agar and Xylose
Lysine Decarboxylase Agar. Following a period of 18-24h
at 37°C, up to three identified colonies were subcultured on
Nutrient Agar at 37◦C for 18-24h. Following the incubation
completion confirmation was performed using biochemical
tests, including TSI, urease, and LI agar (11).
2.3. PCR Amplification
The DNA extraction was performed using a
commercial kit (Diatom DNA Prep 100, Galart-
Diagnosticum, Russia). The DNA samples were
obtained from the enriched culture and extracted
according to the manufactures instruction. Then the
purified DNA samples were considered as a template for
PCR tests. It has been previously mentioned that S139
and S141 primers are highly specific for the
identification of the invA gene expressed in Salmonella
spp. The primers sequence is tabulated in table 1. A 25μl
amplification mixture composed of 2.5 μl 10x PCR
buffer (500mM KCl, 200mM Tris HCl), 1.25μl dNTPs
(10mM), 1.5μl MgCl2 (50mM) was used to perform
PCR reaction using these primers. Also, the volume of
0.5 μl of each primer, combined with 0.5μl of Taq DNA
polymerase (Fermentase) and 2μl of extracted DNA
from enriched cultured samples were mixed with an
amplification mixture for performing the PCR assay. A
gradient thermocycler (Bio Rad, icycler, USA) was used
for performing the DNA template amplification. The
PCR cycling program was as follows: an initial
incubation occurred at 95 ºC for 5 min. The denaturation
step was done at 35 cycles at 94°C for 60 sec, followed
by an annealing procedure which takes place at 56º C for
30 sec, followed by elongation at 72°C for 30 sec, and
final extension happened at 72ºC for a period of 10 min.
For the final evaluation of the PCR products, the
amplicons were electrophoresed in 1.2% agarose gel. A
100bp DNA ladder was used as a size reference. A gel
documentation apparatus was used for gel
documentation, and the staining procedure with ethidium
bromide was performed prior to the gel documentation.
In this study negative and positive controls were
deionized distilled water (DDW), and S. typhymurium
(ATCC: 25923), respectively (11).

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3. Results and Discussion
Previously published research revealed that several
illnesses are transmissible via the consumption of raw
dairy products. It has been well established that these
products are considered major vehicles for the
transmission of pathogens (12, 13). Due to the high
prevalence of Salmonellosis and pervasiveness of
Salmonella spp. isolation and characterization of these
food-transmitted pathogens has of great importance for
the prevention and control of the disease prevalence
(14). There have been introduced several different
technology for detection of Salmonella spp. including
traditional and novel culture technique (14),
immunomagnetic separation, EIA and ELISA-based
assay incorporating fluorescent and molecular
techniques such as DNA hybridization and PCR-based
assay. Almost all rapid test protocol need a selective
enrichment stage (15). In this study we compared
traditional culture method with PCR- based assay, to
compare number of 130 raw milk samples from 10
different regions of the city of maysan were analyzed.
The results of the current research showed that out of
130 raw collected milk samples from 10 dairy markets
from different region of maysan by using traditional
culture technique 9 (7.06%) samples were
characterized as Salmonella positive, while with using
PCR method 16 (12.4%) samples were identified as
Salmonella – positive (Figure 1).
For Enterobacteriaceae, specially for Salmonella
growth there have been at least 8 plate agar media
commercially produced. Most of selective agar media
for isolation of Salmonella spp. rely on one or both of
two identifiers including lactose fermentation and
hydrogen sulfide production, in combination with
various concentrations of inhibitors (14). Bennett,
Greenwood (11) reported that 5% of serotypes of
Salmonella isolated from clinical samples did not
produce hydrogen sulfide. The hydrogen sulfide
production measurements combined with acid
production tests from lactose are inadequate for the
identification of Salmonella spp. from commensal
bacteria. Also, the majority of the media do not easily
yield colorless Salmonella colonies (non-lactose
fermenting colonies). In a previously published study it
is revealed that milk samples may carry several
microorganisms that may compete with Salmonella in
the enrichment medium. Therefore, the coexistence of
Salmonella spp. with other residence bacterial in milk
that some of them have very similar biochemical
properties to those of Salmonella spp. may increase the
false-positive tested samples or keeping the total
number of Salmonella lower than the detection limit on
culture plate (5).
Timing plays a pivotal role in quality control in the
processing of a food product, therefore any attempts to
carry out routine monitoring must take into
consideration. The development of new rapid
techniques including DNA-based techniques in
Table 1. Sequence of oligonuclotid used as primers in the PCR assay
Primer
Sequence (5
-3
)
Target gene
Amplicon fragment (bp)
Reference: No
S139-F
GTG AAA TTA TCG CCA CGT TCG GGC AA
Inv A
284
(15)
S141-R
TCA TCG CAC CGT CAA AGG AAC C
Figure 1. Agaros gel electrophoresis of PCR product of InvA
gene of Salmonella spp.:Lane M ,100 bp Marker; Lane N:
Negative control; Lane P:Positive control (S.Typhimurium:
ATCC: 25923); Lane 1to4 Salmonella spp. isolated from raw
milk

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identifying bacterial species has dramatically reduced
the necessary time for the characterization of bacteria
(5). Previously in a study for the validation and
standardization of PCR for the detection of five major
foodborne pathogens including Salmonella conducted
by Chen the results showed that the most selective
primer set was found to be 139-147, which targets the
invA gene (15). These set of primers could identify all
types of Salmonellae and showed no cross reactions
with other organisms (1, 2).
The results of the previous published studies have
shown a wide range of Salmonella contamination in
raw milk (1, 3, 6). The results of a study conducted by
Steele, McNAB (16) showed only 0.17% Salmonella
positive samples from of 350 bulk tank. Kessel (5)
found Salmonella spp. in 12% in Maryland, USA from
200 bulk tank milk samples. The results of a study
conducted by Ekici, Bozkurt (17) revealed that from 36
dairy farms in Van, Turkey Salmonella spp. did not
isolated in any of the samples. The findings of a study
conducted by Jayarao and Henning (8) showed that out
of 131 bulk tank milk sample 6.1% of the samples were
Salmonella positive. In all the above mentioned studies
enrichment culture techniques was used followed by
isolation on selective agars.
Therefore we can conclude that raw milk
contamination with Salmonella should not be
considered a rare case. The results obtained from
bacteriological quality control of raw milk in the
current research showed relatively high contamination
rate of Salmonella. The growth potential for Salmonella
in improperly stored raw milk and in other dairy
products, represents a serious public health risk,
particularly for susceptible populations.
Authors' Contribution
Study concept and design: A. F. A. and M. S. A.
Acquisition of data: N. M. H.
Analysis and interpretation of data: H. D. S.
Drafting of the manuscript: D. A. H.
Critical revision of the manuscript for important
intellectual content: S. H. A., S. H. and D. M. A.
Statistical analysis: H. A. H.
Administrative, technical, and material support: S. A.
H. and D. M. A.
Ethics
All the procedures were approved by the ethics
committee of the Al-Manara College for Medical
Sciences, Maysan, Iraq.
Conflict of Interest
The authors declare that they have no conflict of
interest.
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