www.tjprc.org editor@tjprc.org
SEROTPING AND MOLECULAR DETECTION OF DIPHASIC AND MONOPHASIC
S. TYPHIMURIUM ISOLATED FROM FRESH CHICKEN SAMPLES COLLECTED FROM
DIFFERENT LOCAL MARKETS IN NAJAF PROVINCE
DURING APRIL TO OCTOBER 2012
SANAA GHALI JABUR1 & EMAN MHAMED JAR ALLAH2
1College of Science, Kufa University, Kufa, Najaf, Iraq
2College of Science, Babylon University, Babylon, Iraq
ABSTRACT
Salmonella typhimurium is one of most importance serovars associated with human disease reported from poultry
in developing countries and during the last few years Monophasic variants of Salmonella Typhimurium-like strains,
lacking the fljB-encoded second phase H antigen, appear to be of increasing importance in infection in many Member
States such as France, Germanyand but research studies in Iraq did not or rare referred to monophasic variants.
Therefore a total of 1440 fresh chicken samples were collected from local markets in Najaf city during April to October
2012 to detect typical diphasic and monophasic S. typhimurium distribution. The results appeared the numbers and
percentages of monophasic S. Typhimurium isolates in gizzard, liver and meat were 28 isolates (5.83%), 10 isolates
(2.08%) and 3 isolates (0.6%) respectively, when it in typical diphasic S. typhimurium raised to 128 isolates (26.66 %),
71 isolates (14.79%) and 22 isolates (4.5%), respectively, hence the severity of monophasic S. typhimurium isolates in this
study may be less than it in studying typical diphasic S. typhimurium isolates.
KEYWORDS: S. typhimurium, Monophasic, Diphasic Chicken Meat
INTRODUCTION
Non-typhoidal salmonellosis (food poisoning) is a food borne disease of primary concern in developed, as well as
developing countries. Although this disease is self-limiting, can also lead to life-threatening systemic infections in
immunocompromnise patients ((Feasey et al, 2012; Graham, 2010) therefore this disease become among one of the major
public health problems that are made worldwide each year up to 1.3 billion cases of acute gastroenteritis and diarrhea
resulting in 3 million deaths annually (Razzaque et al, 2009; UL-Hassan et al, 2008).
S. enterica subspecies enterica serovar Typhimurium is considered a major Non-typhoidal Salmonella broad host
range serovar, usually associated with gastroenteritis in phylogenetically unrelated host species specially associated with
human disease worldwide, therefore these are importance to public health commonly reported from poultry in developing
countries (;CDC, 2004; Rahman et al, 2004, Sharma et al, 2005; Archambault et al, 2006).
The notation of the antigenic formulas of S. typhimurium is
O-Antigens
H-antigens of first phase: H-antigens of second phase, therefore the full antigenic formula of Salmonella
International Journal of Medicine and
Pharmaceutical Sciences (IJMPS)
ISSN(P): 2250-0049; ISSN(E): 2321-0095
Vol. 4, Issue 5, Oct 2014, 13-20
© TJPRC Pvt. Ltd.
14 Sanaa Ghali Jabur & Eman Mhamed Jar Allah
Impact Factor (JCC): 5.1064 Index Copernicus Value (ICV): 3.0
typhimurium is: 1, 4 [5], 12:i: 1, 2. Here, the somatic O-antigens are: 1, 4, [5], 12. Where i represent the H-antigens of first
phase and 1, 2 is H-antigens of second phase (EFSA, 2010; World Assembly of Delegates of the OIE, 2010;).
During the last few years Monophasic variants of Salmonella Typhimurium-like strains, lacking the fljB-encoded
second phase H antigen, with the antigenic structure 1,4,[5],12:i:- appear to be of increasing importance in many Member
States such as France, Germany, Austria, Ireland, Italy, Denmark, the Netherlands and Luxembourg. Such variants are
referred to as ‘monophasic S. Typhimurium’. Strains lacking expression of the phase one or both flagellar antigens are also
possible, but uncommon to be associated with significant disease in animals or humans. Therefore, for the purposes of this
Opinion, only the monophasic variants lacking second phase H antigens were considered. (EFSA, 2010; World Assembly
of Delegates of the OIE, 2010). Research studies in Iraq did not or rare referred to this diphasic and monophasic variants
therefore the present was aimed to differentiate the typical diphasic and monophasic S. typhimurium distribution in locally
freshly chicken samples in Najaf province.
MATERIALS AND METHODS
Collection of Chicken Sample (Study Area and Period)
The research was focused on Najaf province. The study population included only the fresh chickens (that use as
human food) from 15 different locally markets regions that distribute in Najaf during the period extended from April to
October 2012, a total of 480 chicken were taken from four locally markets randomly in each region as 8 chicken
(weighed 1-1.5 Kg) from each market, putted into separate plastic bags, cooled in an ice box and immediately transported
to laboratory. The different samples of each chicken (liver, gizzard and meat) collected individually, homogenized and
diluted.
Isolation and Identification of S. typhimurium
Twenty five grams from each Meat, Liver and gizzard of each chicken were individually homogenized and diluted
in 225ml of Tetrathonat broth medium then from each one of sample, serial dilution were performed to10- . One milliliter
from each dilution was cultured on selective enrichment XLD medium in three replicates, incubation at 43°C for 24 h.
for isolation of enteric pathogens Salmonella, and incubation was extended to 48h to increase visibility of H2S Production.
(Wallace et al, 2011).
Calculate the Percentage of Typical Diphasic and Monophasic S. typhimurium Isolates
The presumptively positive Salmonella isolates were confirmed by the biochemical method (Wallace et al, 2011)
and S. typhimurium with slide agglutination test and Multipluex PCR assay and then the percentage of typical diphasic and
monophasic of S. typhimurium isolates Calculated by equation below (Ali, 2007)
Serological Test
All isolates identified biochemically as Slamonella were serologically examined by a slide agglutination test that
performed with Polyvalent "O" Antisera, specific O antisera (SIFIN), Polyvalent H antisera And specific H antisera
(SIFIN), The somatic (O) and flagellar (H) antigens were characterized by slide agglutination with commercially available
anti-sera (SIFIN, Germany) and the serotype was assigned according to the Kauffmann-White scheme.
Serotyping and Molecular Detection of Diphasic and Monophasic S. typhimurium Isolated from Fresh 15
Chicken Samples Collected from Different Local Markets in Najaf Province during April to October 2012
www.tjprc.org editor@tjprc.org
(Wallace et al, 2011; Saraj and Stefan. 2009).
Molecular Differentiation of a Typical Diphasic S. Tphimurium and Monophasic 4, [5], 12: I
In order to differentiate Salmonella strains belonging to the somatic antigen group “B” and sharing the same first
flagellar antigen ‘i’, a multiplex PCR used to support the serological identification of the antigens. (Tennant et al, 2010).
DNA Extraction
DNA Extraction was performed depending on method mentioned by Tennant et al, (2010).
· A strain of Salmonella spp. was spreaded on a non selective agar tube
· The tube incubated at 37°C ± 1°C for 18-24 hours
· four hundred ?ls of nuclease free water were added in a 1,5ml or 2ml dnase free tube and suspend the bacteria
· The bacterial cells mixed (e.g. using a vortex)
· Absorbance was reared at 600nm. Correct absorbance to be comprised between 1 and 2.
· The tube Incubated at 100°C for 15min
· Centrifuge at 20°C to 25°C for 5 min. at 10,000g
· The supernatant collected and stored at -20°C
Multipluex PCR Assay Primers
The list of primers used to genes for identification and differentiation of diphasic S. typhimurium and monophasic
4,[5],12:I:-using multipluex PCR assay were (BioNeer). These primers are given in Table 1.
Table 1: Primer Sequence of the Genes for Differentiation of Diphasic
S. typhimurium and Monophasic 4, [5], 12: I:-Using Multipluex PCR Assay
Preparing the Primers
The Bioneer primers were prepared depending on manufacturer instructions by dissolving the lyophilized product
with TE buffer molecular grad after spinning down briefly. Working primer tube was prepared by diluted with TE buffer
molecular grad. The final picomoles depended on the procedure of each primer.
A Multiplex PCR Cycling Profiles
A multiplex Polymerase chain reaction assays were carried out in a 20 ?l reaction volume, and the a multiplex
PCR amplification conditions performed with a thermal cycler were specific to each single primer set. (Tennant et al,
2010). (Table 2) represents the amplification conditions depending on their reference procedure.
16 Sanaa Ghali Jabur & Eman Mhamed Jar Allah
Impact Factor (JCC): 5.1064 Index Copernicus Value (ICV): 3.0
Table 2: The Multiplex PCR Amplification Conditions of Identification and Differentiation
of Typical Diphasic S. Tphimurium and Monophasic 4,[5],12:I:
Preparation of Agarose Gel
Agarose gel was prepared by adding 1 gm of agarose powder to 100 ml of TBE buffer previously prepared
(90 ml D.W were added to 10 ml TBE buffer l0X) in conical flask, the final concentration was 1 X and pH 8. The conical
flask was placed in boiling water bath until it become clear and then allowed to cool to 50°C, and 1.5 ?l ethidium bromide
at concentration of 0.5 mg/ml was added. The agarose poured kindly in equilibrated gel tray earlier set with two combs
fixed in the end and in the middle, and the two ends of gel tray were sealed. The agarose allowed solidifying at room
temperature for 30 minutes. The comb was removed gently from the tray and the seal was removed from the ends of the
tray. The comb made wells used for loading DNA samples.
Multiplex PCR Product Analysis
Agarose Gel Electrophoresis
The amplified PCR products were detected by agarose gel electrophoresis and visualized by staining with
ethidium bromide. PCR products were loaded to the agarose gel wells: 5?l from single product to single well in known
sequence, followed by 100 bp ladder to one of the wells in each row. The gel tray was fixed in electrophoresis chamber. IX
TBE buffer was added to the chamber until covering the surface of the gel. The electric current was performed at 60 volt
for 1.5 hour.
Electrophoresis Results
The electrophoresis result was detected by using gel documentation. The base pair of DNA bands were measured
according to the ladder. The positive results were distinguished when there was DNA band equal to the target product size.
Finally, the gel was photographed using gel documentation saving picture
Statistical Analysis
The data were analyzed statistically, using the least significance differences test (LSD), in experiment of one
factor and analysis of variance (ANOVA) at the P value level of 0.05 (Daniel, 1999).
RESULTS AND DISCUSSIONS
Serological Characteristics of S. Typhimurium
In order to confirm the identification of Salmonella isolates and for obtain the serovar Typhimurium, serological
examination was done for isolates which represented biochemical test of Salmonella, there are only 369/662 (55.7%)
isolates were appeared positive agglutination results with Anti-salmo. Gr B serra and Anti-salmo. poly-H 1&2 sera where
262/662 (39.2%) of isolates gave positive results with Anti-Salmonella Hi sera which represent phase 1 and were 221/662
(33.3%) appear agglutination with Anti-salmonella H2 sera which represent phase 2, Table 3.
Serotyping and Molecular Detection of Diphasic and Monophasic S. typhimurium Isolated from Fresh 17
Chicken Samples Collected from Different Local Markets in Najaf Province during April to October 2012
www.tjprc.org editor@tjprc.org
Table 3: Frequency of S. typhimurium Serotyping in Liver, Meat and Gizzard of Chicken Samples
The final results in Anti-Salmonella Hi (phase1) and Anti- Salmonella H2(phase2) experiments indicated that
total S. typhimurium isolates were either 262 isolates or 221 isolates because the final test to indicate serovars of S.
typhimurium depend on results of this two tests( EFSA, 2010) but the difference between number of S. typhimurium
isolates in this two serotyping tests may be related to lacking of fljB-encoded second phase H antigen, (Hopkins et al,
2010, Alcaine et al, 2006 ) or to different mutations and deletions have been associated with the lack of phase 2 flagella
expression genes (Garaizar et al, 2002) .
Molecular Study for Identification and Differentiation of Typical Diphasic and Monophasic S. Typhimurium
However serological test indicated S. typhimurium isolates were 262 isolates which appeared first flagellar phase
but 221 isolates from it do not appeared flagellar phase 2. So in order to differentiate S. typhimurium strains belonging to
the somatic antigen group “B” (group that contain S. typhimurium serovar) and sharing the same first flagellar antigen
‘i’, a multiplex PCR used to support the serological identification of the flagellar antigens. This test appeared 262 isolates
(100%) have first flagellar antigen ‘i’ gene but second flagellar antigen gene (fljB allele) that encoded phase 2 ‘1,2’ was
positive only in 221 isolates 221/262(84.35%),( figure 1, table 4).
Table 4: Result of Multiplex PCR for Salmonella typhimurium Isolates
Gene Serotyping Phase
Number of Isolate %
Positive Negative
fliB-fliA
intergenic region
of the flagellin
gene cluster
Phase 1 262(100) 0
flj B Phase 2 221(84.35%) 41(15.64%)
Depending on data mentioned above the Monophasic variants of Salmonella Typhimurium-like strains, lacking
the fljB-encoded second phase H antigen appeared only in 41isolates 41/262(15.64%)( 5).
The lacking of fljB-encoded second phase H antigen which characterized Monophasic S. Typhimurium- Like
strains may be related to mutations and deletions in fljB-encoded second phase H antigen. There are some investigators
reported such mutations and deletions leading to this resultes, Garaizar et al.( 2002) found at least some of the S. 4,5,12,
i:– isolates from Spain appear to be characterized by deletion of a large fragment, including fljB and hin, encoding a DNA
invertase essential for fljB expression. Where some researches referred to lacking fljB-encoded second phase H antigen,
(Hopkins et al, 2010; Alcaine et al, 2006).
18 Sanaa Ghali Jabur & Eman Mhamed Jar Allah
Impact Factor (JCC): 5.1064 Index Copernicus Value (ICV): 3.0
Figure 1: Ethidium Bromide- Stained Agarose Gel of Multiplex PCR Amplified Products from DNA of Some
S. Typhimurium Isolates Extracted that Amplified With Primers FFLIB(F) and RFLIA(R) (Encoded Flib-Flia
Intergenic Region of the Flagellin Gene Cluster) and Sense-59(F) and Antisense-83(R) (Encoded Fljb Allele).
Lane (Lad) Show DNA Marker (100 Bp). Lane (1-3), (5-10) and Lane(12-13) Show Positive Results with both
Flib-Flia Intergenic Region And Fljb Allele While Lane(4) And (11) Show Positive Results Only with Flib-Flia
Intergenic Region and Nagative Results with Fljb Allele
Numbers and Percentage of Typical Diphasic S. Typhimurium and Monophasic Variants of S. Typhimurium-
Like Strains in Fresh Chicken Food Samples
The numbers and percentages of monophasic S. Typhimurium isolates in gizzard, liver and meat were 28 isolates
(5.83%), 10 isolates (2.08%) and 3 isolates (0.6%) respectively, when it in typical diphasic S. typhimurium raised 128
isolates (26.66 %), 71 isolates (14.79%) and 22 isolates (4.5%), respectively ( table 5).
Table 5: Number and Percentage of Typical Diphasic S. typhimurium and Monophasic Variants of Salmonella
Typhimurium-Like Strains in Liver, Meat and Gizzard of Fresh Chicken Food Source
The data reported to ECDC do not suggest infections with the monophasic variants to be more severe. This
conclusion is based on the percentage of strains isolated from blood compared to faeces, as this ratio was in the same range
or even lower, than for other S. Typhimurium (which had 1.8% isolated from blood and 95% from faeces). ( EFSA, 2010);
therefore, this part of our study investigated of the number and percentage of monophasic S. Typhimurium isolates
compared with that’s of typical S. Typhimurium.
As regard the data in Table 5 the severity of monophasic S. Typhimurium isolates in this study may be less than it
in studying typical diphasic S. typhimurium isolates depend on (EFSA, 2010).
Serotyping and Molecular Detection of Diphasic and Monophasic S. typhimurium Isolated from Fresh 19
Chicken Samples Collected from Different Local Markets in Najaf Province during April to October 2012
www.tjprc.org editor@tjprc.org
REFERENCES
1. Alcaine S. D, Soyer Y, Warnick L. D, Su W. L, Sukhnanand S, Richards J, Fortes E.D, McDonough P, Root
T. P, Dumas N.B, Grohn Y. and Wiedmann M. (2006). Multilocus sequence typing supports the hypothesis that
cow- and human-associated Salmonella isolates represent distinct and overlapping populations. Appl Environ
Microbiol, 72, 7575-7585.
2. Ali, Z. M. (2007).A Bacteriological and Genetic study of Enteropathogenic E. coli in AL-Najaf AL-Ashraf
Governorate. A Thesis of the (Ph.D)- College of Education for Girls/University of Kufa.
3. Archambault M, Petrov P, Hendriksen R S, Asseva G, Bangtrakulnonth A, Hasman H, Aarestrup F M. (2006).
Molecular characterization and occurrence of extended-spectrum _-lactamase resistance genes among Salmonella
enterica serovar Corvallis from Thailand, Bulgaria, and Denmark. Microb. Drug Resist, 12: 192-198.
4. CDC (Centers for Disease Control and Prevention). (2004). Salmonella Surveillance: Annual Summary.
Atlanta, Georgia: US Department of Health and Human Services.
5. Daniel, W.W. (1999). Biostatistics, a foundation for analysis in the Health Sciences, 7th edition. Wiley company.
6. European Commission. (2000). Salmonella. In: Opinion of the scientific committee on veterinary measures
relating to public health on food-borne zoonoses. Health and Consumer Protection Directorate General,
Directorate B - Scientific Health Opinions, Unit B3-Management of Scientific Committees II, 12th April 2000.
pp. 24-27.
7. (EFSA) European Food Safety Authority (2010). Scientific Opinion on monitoring and assessment of the
public health risk of “Salmonella Typhimurium-like” strains1. EFSA Journal; 8(10):1826.
8. European Commission. (2000). Salmonella. In: Opinion of the scientific committee on veterinary measures
relating to public health on food-borne zoonoses. Health and Consumer Protection Directorate General,
Directorate B - Scientific Health Opinions, Unit B3-Management of Scientific Committees II, 12th April 2000.
pp. 24-27.
9. Feasey N. A, Dougan G, Kingsley R. A, Heyderman R. S, Gordon M. A. (2012). Invasive non-typhoidal
salmonella disease: an emerging and neglected tropical disease in Africa. Lancet 379: 2489–2499.
10. Garaizar J, Porwollik S, Echeita A, Rementeria A, Herrera S, Wong R. M. Y, Frye J, Usera M. A, McClelland
M. (2002). DNA microarray-based typing of an atypical monophasic Salmonella enterica serovar. J Clin
Microbiol, 40, 2074-2078.
11. Graham S. M. (2010). Nontyphoidal salmonellosis in Africa. Curr Opin Infect Dis 23: 409–414.
12. Hopkins K. L, Nair S, Kirchner M, Guerra B, Granier S, Lucarelli C, Porrero C, Jakubczak A, Threlfall E. J. and
Mevius D.(2010). Genetic variation in emerging multidrug-resistant Salmonella enterica 4, [5], 12:i:- from seven
European countries. 110th General Meeting of the American Society for Microbiology, San Diego, CA,
May 23-27.
13. MacFaddin, J. F. (2000). Biochemical Tests for the Identification of Medical Bacteria, 3rded. Philadelphia:
Lippincott Williams and Wilkins,
20 Sanaa Ghali Jabur & Eman Mhamed Jar Allah
Impact Factor (JCC): 5.1064 Index Copernicus Value (ICV): 3.0
14. Narapati D. (2007). prevalence and antimicrobial resistance of Salmonella in import wed chickens Carcasses in
Bhutan. Master of Veterinary Public health. chiang MAI University and freie univerittat berlin.109 page.
15. Rahman, H, Kumar, A, Murugkar, H.V, Bhattacharyya, D.K, Shome, B.R, Shome, R. (2004):
Some epidemiological features of Salmonella Typhimurium isolates from north east India. Indian Journal of
Animal Sciences 74 (6), 609-611.
16. Razzaque M. A, Bedair M, Abbas S, Al-Mutawa T.(2009). Economic impact of calf mortality on dairy farms in
Kuwait. Pakistan Vet J, 29(3): 97-101.
17. Saraj U. S, Stefan S. (2009). Plasmid fingerprinting and virulence gene detection among indigenous stains of
Salmonella enterica serovar Enteritidis. J Ayub Med Coll Abbottabad.21(2):83-86
18. Sharma, J. K, Bedi, J. S, Gill, J. P. S, Sharma, C. S, Aulakh, R. S, Bedi, S.K. (2005): Prevalence of
enterotoxigenic Escherichia coli and Salmonella Typhimurium in meat and meat products. Journal of Food
Science and Technology 42 (1), 56-58.
19. Tennant S. M, Diallo S, Levy H, Livio S, Sow S. O, Tapia M, Fields P. I, Mikoleit M, Tamboura B, Kotloff
K. L, Nataro J. P, Galen J. E, Levine M. M. (2010). Identification by PCR of non-typhoidal Salmonella enterica
serovars associated with invasive infections among febrile patients in Mali. PLoS Neglected Tropical Diseases, 4,
e621.
20. UL-Hassan M, Hussain M. I, Shahzadi B, Shaheen M, Mahmood M. S, Rafique A,
Mahmood-ul-H. M.(2008). Occurrence of some zoonotic microorganisms in faecal matter of house rat
(Rattus rattus) and house mouse (Mus musculus) trapped from various structures. Pakistan Vet J, 28(4): 171-174.
21. Wallace H. A, Andrew J, Thomas H. (2011). Bacteriological Analytical Manual. Chapter 5. USDA/FSIS
Microbiological Laboratory Guidebook, 3rd Edition. U. S. Food and Drug Administration.
22. World Assembly of Delegates of the OIE. (2010). OIE Terrestrial Manual 2010. Chapter 2.9.9.
— Salmonellosis. NB: Version adopted by the World Assembly of Delegates of the OIE in May 2010.