Advances in diagnosis of salmonellosis and characterization of salmonella

Advances in Diagnosis of
Salmonellosis and
Characterization of Salmonella
Dr. Bhoj R singh, Principal Scientist (VM)
I/C Epidemiology; Centre for Animal Disease Research and Diagnosis
Indian Veterinary Research Institute, Izatnagar-243122, Bareilly, UP, India.
TeleFax +91-581-2302188

Why we need early diagnosis for salmonellosis ?
• Early diagnosis means nipping the problem in bud, which
is of utmost significance because:
• Any level of Salmonella leads to decreased production.
• Treatment of Sick Animals is expensive and uneconomic
in poultry industry.
• Death of animals means loss of money and product.
• Outbreak of salmonellosis results in public backlash and
decreased sales of the associated food.
• Increased work force is required to control the outbreak.
• Sampling and testing costs a lot.
• Extra record keeping is necessary for certification and
validation.
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar

Method Accuracy Detection limit
cfu/ml
Analysis time
in hours
Ease of
handling
Standard high 1-10 48-120 Complex
Modified
conventional
High 10-100 24-60 Complex
Impedimetry Good 105
-106
24-60 Easy
Immunological high 105
-106
48-60 Intermediate
DNA probes high 103
-105
22-60 Intermediate
PCR high 102
-103
22-24 Intermediate
Real time PCR high 102
-103
2-15 Intermediate
Different methods of Salmonella Detection and their sensitivity

Salmonella Antibody detection
• Whole blood agglutination test
• Rapid plate agglutination test (RPAT)
• Standard tube agglutination test (STAT)
• Passive haemagglutination test (PHAT)
• Antiglobulin test (AGT)
• Radioimmunoassay (RIA)
• Counter immuno-electrophoresis (CIE) &
• ELISA
• dot-ELISA
• AGPT

Enzyme-linked immunosorbent assays for Salmonella Enteritidis
and other Salmonella serovars
Two main basic systems are available for detection of IgG (IgY) specific for S. Enteritidis: the
indirect ELISA and the competitive ‘sandwich type’ ELISA
The indirect ELISA involves the use of a detecting antigen coated on to the wells of a
microtitre plate. After the application of a blocking reagent to reduce nonspecific binding, test
samples are applied to the wells. Specifically bound antibody in the sample is detected by an
antibody/enzyme conjugate. A variety of antigens, including LPS, flagella, SEF14 fimbriae,
Salmonella cytotoxin I, outer membrane proteins and cruder antigen preparations have been
used.
The competitive sandwich ELISA employs a specific reagent - a monoclonal antibody
(MAb) - for coating antigen to wells. This is then followed by a pure or crude antigen
preparation. Test samples are applied followed by conjugated MAb, which will not bind to the
antigen if the sample contained specific antibodies. The assay time can be shortened by
adding both test sample and conjugate together. MAbs have been prepared for LPS, flagella
and SEF14 for S. Enteritidis.
There are advantages and disadvantages to both systems. The indirect assay is simpler and
reagents are available for all Salmonella serotypes of chickens, turkeys, ducks and
mammalian hosts. The competitive ELISA can be applied to all animal species and in
general shows higher specificity. However, reagents are not available commercially for all
serotypes. There are also some affinity problems and it may be less sensitive than the
indirect assays. In the field, both systems have produced false-positive reactions.

Salmonella cytotoxin I based ELISA
(Genus specific)
• Indirect ELISA (I-ELISA) is performed to determine Salmonella cytotoxin-I specific
antibodies to asses the infections with Salmonella irrespective of infecting serovars of
the pathogens involved. Cytotoxin I has been reported in all pathogenic strains of
Salmonella serovars (Singh and Sharma, 2000). Cytotoxin I antigens is prepared (Singh
and Sharma, 1999) using a known cytotoxigenic reference culture of S. Weltevreden (S-13
and reference anti cytotoxin (Singh and Sharma, 2000) is used.
• To determine Salmonella cytotoxin I (SCI-I) antibodies in serum samples, ELISA plates are
coated with anticytotoxin (dog) and then plates are washed thrice with PBST (Phosphate
buffer saline with 0.05% Tween-20). Remaining sites are blocked with 300µl freshly
prepared 5% (w/v) skim milk. After overnight incubation at 370
C, plates are washed with
PBST thrice. Then to the wells of ELISA plates, 100µl antigen prepared at the
concentration of 10 μg protein/well in 1M Tris is added into the wells and incubated for 2 h
at 370
C. Wells are emptied and washed with PBST and to each well , a 100 µl of diluted (1:
200 in PBS with 0.1% BSA) test serum is applied in triplicate and incubated for 2 h at 370
C
as above. Wells are emptied and washed as earlier and to each well, a 100µl of suitably
diluted (1:5000 in PBS with 0.1% BSA) anti IgG (against the test animal) HRPO conjugate is
added and incubated for 2 h at 370
C, plates are washed thrice with PBST and then
apply100µl of freshly prepared substrate (Orthophenyline diamine) to each well made in
citrate phosphate buffer (pH 4.6, 0.1M) and plates are incubated for 20 min at 370
C in dark.
The reaction is stopped with100µl of IM H2SO4 in each well. O.D. of each well is read at 492
nm with ELISA reader. Serum titre is calculated as
• Average test OD – Average Negative control OD
• ELISA titre = ————————————————————— ×100
Average Negative control O.D.

Salmonella Cytotoxin-I based dot-
ELISA (Genus specific)

Salmonella cytotoxin-I based AGPT (Genus specific)
P P
P
N
N N

Widal’s Test
• Common antigens used in Widal’s test
• Bacteria `O` Antigens `H` Antigens
• Typhi 9,12 (Vi) d
• Paratyphi A 1,2,12 a
• Paratyphi B 1,4,12 b
• Paratyphi C 6,7 (Vi) c
• `O` antigens- 1,2,4,6,7,9,12
• Capsular antigen- Vi;
• `H` antigens- a,b,c,d
• Diagnostic titres:- H- 1 : 20; O- 1:50, and Vi- 1:5.
• Incubation for tests:- H- 50o
C for 2 hr then RT for >3 hrs.
• O- 37o
C for 2-4 hrs then RT overnight.
• Vi- 37o
C or RT overnight.

Commercially available test kits
• TEK-ELISA (Organon Teknika, Cambridge UK)
• IFR-ELISA (Wyatt et al. 1995)
• Report-EIA & TECRA Salmonella Visual A (Wilson et al. 1990)
• Dulcitol-1-phosphate dehydrogenase (DPD) mab based kit (Tian et
al. 1996)
• Cytotoxin-1-antibodies based Salmonella detection protocol (Singh
et al. 2000)
• Chekit-S-enteritidis (ELISA) kit (Baumgartner et al. 1993)
• Polymyxin cloth enzyme immunoassay (Blais et al. 1997)
• 1-2 Test (Bio-control, Bothel, USA)
• Single step Salmonella (SSS) by Ampcor, Camden USA
• Iso-Grid ® of Dynal, Oslo
• Vi-TEK & Vi ELISA (Sharma et al. 1997)
• Meat Juice ELISA kit (Steinbach et al. 1999)

Factors affecting serological diagnosis
• Useful to identify infected flocks/herds rather than to identify infected individuals.
• Serologically positive reactors may no longer be infected with Salmonella
organisms, similarly, actively excreting Salmonellae may be serologically negative.
• Newborn animals are immunologically immature and do not respond serologically.
• Chickens and neonates may also acquire Salmonella antibodies passively from
their parents without having any active infection.
• Following Salmonella infections, immunoglobulin concentrations may be so high
that it may cause prozone phenomenon.
• Necessitates differentiation between vaccine response & actual infection.
• The effect of antibiotic therapy on the serological response remains unclear.
• More than 2500 different Salmonella serovars exist. It is not easy to select an
antigen and test used.
• Serological cross-reactions between different serovars could not be conclusive
about causative serovar.
• In poultry, egg yolk may be tested for immunoglobulins to Salmonella and eggs
may provide a method to screen flocks.
• Require readymade standard antigen: you need it from outside
• Require bleeding
• Sample are fragile and lost in transit
• Sera sample may be having unknown pathogens of much more hazardous
disease for which we have never thought; Ebola, Marburg, Avian influenza,
hepatitis B, HIV

Antigen identification
• Isolation
• Electrical Impedance measurement
• Antigen Capture Immunoassays
• PCR (Saiki et al. 1985 reported First PCR)
• Capillary PCR
• Multiplex PCR
• RT-PCR
Draw backs:
• Different routes of excretion of the pathogen from
host-difficult to decide the right kind of sample to be
collected.
• Irregularity in presence of antigen in host during
disease process- antigen is present or excreted only
for short time, and in different stages.

Samples
• Samples should not originate from birds or eggs that
have recently been treated with antimicrobial drugs.
They can be swabs or aseptically collected samples from
affected tissues, or intestinal and cloacal contents. Other
materials to sample include eggs, eggshell surfaces,
embryos, faecal droppings and hatcher debris, especially
fluff, dust and broken eggshells.
• The nature and quantity of the sample will depend on
whether it is taken from live poultry or carcasses, and
whether lesions or faecal contamination are present.
In case of delay, samples should be stored at 0-4°C.

Steps in isolation of Salmonella
For faecal and tissue samples
• Pre-enrichment (1:10 in Buffered Peptone Water), at 35-37o
C for 18-24 hours.
• Selective enrichment broth (Tetrathionate broth, Rappaport Vassiliadis medium,
Selenite cystine medium; RV is much better, at room temp).
• Plating after 24 hour and 48 hr: on Hektoen Enteric agar with novobiocin (HEN) or
Brilliant green agar with novobiocin (BGN) or Xylose lysine citrate agar (XLT-4).
• If negative, transfer 0.1 ml to 10 ml RV after 120 hour and incubate at 37o
C for 24-48
hour and then plate as above.
• Pick up suspected colonies after 24 hour of plating on to Motility Indole Lysine (MIL)
stabs and triple sugar iron agar (TSI) slants.
• Serological confirmation.
For blood cultures: 10 ml of blood should be added to enrichment media and incubated
at 37o
C and plated daily for up to 11 days. Addition of liquoid or bile in enrichment
interferes with bactericidal action of blood and improves the Salmonella detection.
Medium of choice is 0.5 % Taurocholate or ox bile broth. Blood clots give better
results and require lesser medium. I.e. for clot from 10 ml blood 50 ml ox bile broth.
Draw backs: Take long time
Ineffective when antibiotic treatment is on
Different methods of isolation for different serovars
Intermittent excretion

Differentiation of Salmonella species and subspecies
Characters Dulcitol Lactose Sorbitol Mucate D-Tartarate Citrate Malo
nate
ONP
G
S. bongori + - + + - + - +
S. enterica ssp.
enterica
+ - + + + + - -
S. enterica ssp.
arizonae
- (-) + + - + + +
S. enterica ssp.
diarizonae
- (+) + D (-) + + +
S. enterica ssp.
houtenae
- - + - D + - -
S. enterica ssp.
indica
D (-) - + + (+) - d
S. enterica ssp.
salamae
+ + + + d + + (-)
D, delayed; ( ), variable
Other common tests are:- Indole –ve, MR +ve, VP –ve, Urease –ve, nitrate reduction +ve, Phenylalanine –ve, glucose +ve, salicin –ve, adonitol –ve,
inositol D, raffinose –ve, erythritol, esculin -ve, sucrose –ve, oxidase –ve, H2
S +(-), gelatinase –ve, lysine, ornithine +ve, arginine +ve, KCN –ve,
alginate –ve.
Common confusion occurs with Citrobacter which are lysine –ve and melibiose fermenter, a few Enterobacter may also cause some problems, they
are also usually –ve in lysine (gregoviae and aerogenes + but are + for melibiose) and for H2
S and +ve for ONPG test.

Differentiation of common Salmonella serovars
Characters H2
S Lysine Ornithine D-
tartrate
Gas in
glucose
Dulcitol Maltose Rhamnose Sorbitol
Choleraesuis D + + (+) + - + + (+)
Paratyphi A - - + - + + + + +
Typhi + + + + - - + - +
Gallinarum + + - + - + + - -
Pullorum + + + - + - - + (-)
Common Sal + + + + + + + + +

Common Non-isolation Techniques
Impedance/ conductance assays
Malthus assay:- based on antibiotic added pre-enrichment followed by
selenite based enrichment and then measuring impedance in two
cells one containing trimethylamine oxide (TMAO) and dulcitol,
another cell contain lysine. Salmonella converts TMAO to TMA
(trimethylamine). The test yields 12% false negatives and 10% false
positives
BacTrac 4100 system: based on impedance splitting method
measures impedance of medium and the electrode (M and E value
respectively). Pre-enrichment is followed by novobiocin containing
RV broth enrichment and then taking M and E value for next 22 h at
40o
C. It yields 7.4% false negatives and 4.9% false positives.
Other systems are RABIT and BACTOMETER.

Other methods
Antigen capture immunoassays: Sensitivity is 103
to 106
cfu per ml and most of the commercially available
ones are serovar specific.
Commercially available systems are:
PATHSTICK, EIAFOSS, VIDAS SLM, TECRA OPUS
DIPSTICKS, DYANA Beads, Magna Beads and VICAM
beads (serovar specific).
• Genus specific Antigen Capture ELISA based on
Salmonella cytotoxin-I (Singh and Sharma, 2000)
• FAT (Singh and Sharma, 2000)
• Biken test (Singh and Sharma, 2000)
Identification of antigen with these method is less sensitive
due to requirement of large number of antigen particles
in the clinical samples.

Salmonella Cytotoxin-I based FAT
(Genus specific)

Salmonella cytotoxin-I based Biken test
(Genus specific)
N
N
P
P

Serobact Salmonella Test
A simple one step latex slide agglutination test for both
clinical and food laboratories. Serobact Salmonella is a
rapid latex slide agglutination test for the identification
of Salmonella from selective enrichment broths.
Serobact latex technology is more sensitive than direct
agglutination methods and the use of Serobact
Salmonella early presumptive identification of
Salmonella spp. saving about 24 hours than using
conventional techniques.
The test exploit Polyvalent H antisera prepared against a
comprehensive range of Salmonella flagella antigens is
coated onto latex particles.
False Positive: Specificity 97.2%. Predictive negative value 100%
False Negative: Sensitivity 100%. Predictive positive value 98.2%

REVEAL for Salmonella
The test can handle one or several samples concurrently.
Contents of the sample are wicked through the pad to a
specimen reaction zone containing colloidal gold-
labeled antibodies specific to Salmonella. Reactive
Salmonella combine with the gold-labeled antibodies
and migrate through the support until they encounter a
binding reagent zone which includes a second antibody
specific to Salmonella. When this occurs, a line appears
in the test window indicating a positive result. The rest
of the sample continues to migrate until it encounters a
second binding reagent zone. This results in the
formation of a line in the control window.

Transia Card Salmonella
This test is used directly on an enrichment
broth. It is on a sandwich - type,
immunochromatographic reaction using
highly- specific antibodies immobilised onto
a membrane and conjugated to a dye. This
allows the detection of all Salmonella
serotypes present in sample. It is based on
lipopolysaccharide (LPS) detection

Genus specific PCR- primers• 1. Product 163bp
P1- : TTATTAGGATCGCGCCAGGC
P2 : AAAGAATAACCGTTGTTCAC
• 2. inv product 284 bp (Oliveira et al., 2002)
139 : GTGAAATTATCGCCACGTTCGGGCAA
141 : TCATCGCACCGTCAAAGGAACC
• 3. Random genomic product 429 bp
ST 11: AGCCAACCATTGCTAAATTGGCGCA
ST 15 : GGTAGAAATTCCCAGCGGGTACT
• 4. Hin H2 flagellin gene (236 bp)
Hin 1750 L : CTAGTGCAAATTGTGACCGCA
Hin 1750 R : CCCCATCGCGCTACTGGTATC
• 5. H-li flagellin gene (173 bp)
H-li 1788 : AGCCTCGGCTACTGGTCTTG
H-li1789 R : CCGCAGCAAGAGTCACCTCA
• 6. GVV PQ fimbriae agf, product (261 bp)
TAF 3 : TCCGGCCCGGACTCAACG
TAF 4 : CAGCGCGGCGTTATACCG
• 7. inv A and inv gene (457 bp product)
S1: TGCTACAAGCATGAAATGG
S2: AAACTGGACCACGGTGACAA
• 8. Spv A gene based 450 bp product.
382: CAGACCACCAGTCCGGCAC
383: CAGTCAATGCTCTCTCGCTG
• 9. hisJ gene (Cohen et al. 1994) 496 bp product
Cohen 1: ACT GGC GTT ATC CCT TTC TCT GGT G
Cohen 2: ATC TTG TCC TGC CCC TGG TAA GAG A
• 10. invA product (Ferretti, et al., 2001) of 389 bp
Sal F GCTGCGCGCGAACGGCGAAG
Sal R TCCCGGCAGAGTTCCCATT
• 11. Fli-Typ 620 bp.
F CGGTGTTGCCCAGGTTGGTAAT
R ACTGGTAAAGATGGCT
• 12. A0 488 bp
AO1 GATACTGCTGAACGTAGAAGG
AO2 GCGTAAATCAGCATCTGCAGTAGC

Serogroup specific PCR : Important
diagnostic tool particularly in identification of
rough strains (Hoorfar et al. 1999).
• Serogroup Primers (5’-3’) Amplicon size bps
• B B1 : AGAATATGTAATTGTCAG 882
• B2 : TAACCGTTTCAGTAGTTC
• C-1 C1 : GGTTCCATAAGTATATCT 471
C2 : CTGGATACGAACCCGTAT
• C2-C3 C21 : ATGCTTGATGTGAATAAG 820
• C31 : CTAATCGAGTCAAGAAAG
• A&D D1 : TCACGACTTACATCCTAC 720
• D2 : CTGCTATATCAGCACAAC

Multiplex PCR for Salmonella in faeces
Amplifies 491 bp product for inv (chromosomal) gene segment,
795 bp product for spvA gene segment on virulence plasmid

Various probes for Salmonella genus
1. Random chromosomal fragemnt
• TS11: GTCACGGAAGAAGAGAAATCCGTACG Tsen et al. 1991
• TS21: TACATCGTAAAGCACCATCGCAATA
• TS31: AGACCACTGACCCAGCCTAATCAA
2. Random chromosomal fragment
• ST15 rev: GAGTACCCGCTGGGAATTTCTAC Olsen et al. 1995
• InvA gene S3: CTGGTTGATTTCCTGATCGC Stone et al. 1994
3. IS200 is not present in S. Agona, S. Arizonae, S. Dar-es-Salam, S.
Panama, S. Infantis, S. Virchow and S. I.9, 12 : Z. Detection limit for S.
Typhi is 103-4 cfu/ml. In IS 200 a tandem repeat of 0.3 kb I used for
cross hybridization (Gilbert et al. 1990). Colorimetric single phase
hybridization assay (CdorDNAH) can detect Salmonella by use of 16S
and 23 S rRNA based probes but it can not detect S. subspp. V. and
gave 7% false positive due to cross reaction with Citrobacter freundii
(Curiale et al. 1990) and detection limit is 108-109cfu/ml. It is produced
by Genetrack (earlier used radiolabelled probe but now enzyme
labelled probes are used) AOAC approved (Flowors et al., 1987).
Specificity and sensitivity of non-radioactive rRNA based
oligonucleotide probs are comparable with culture method and results
are given usually in 48 hr.

Salmonella BAX System (Automated PCR)
Time to Perform: 1-hour-to-1-day
An automated system to quickly and accurately.
The BAX system cycler/detector is used to load the
prepared samples. In less than four hours, computer-
generated results are clearly displayed on the screen. A
single tablet of integrated PCR reagents combines
sample and control primers, plus reagents that overcome
inhibition in chocolate and other challenging food types.
There is no need to run a separate control. Process up to
96 samples in one batch. BAX for screening Salmonella
can work with even the most difficult samples.
Specificity 98%; Sensitivity 98%

Commercial probe based tests
• Gene-Trak Salmonella Microwell and tube
assays: Test is based on microtiter colorimetric
absorbance reading. The DNA hybridization test
employs Salmonella-specific DNA probes which
are directly labeled with horseradish peroxidase.
A colorimetric endpoint is then used for the
detection of Salmonella in food samples
following broth culture enrichment.
• Sensitivity: 1-5 CFU/25g. Testing time: 1.5 hours
(after 40 to 48 hour enrichment).
• Tests per kit: Up to 98

Real time PCR
Quantitative real-time polymerase chain reaction (PCR) provides an accurate
method for determination of levels of specific DNA and RNA sequences in
tissue and clinical samples. It is based on detection of a fluorescent signal
produced proportionally during amplification of a PCR product. Data
acquisition and analysis by real-time PCR is short due to automation. DNA
and RNA can be quantified using this detection system without laborious
post-PCR processing.
The key to the detection is estimation of fluorescence emitted either from
fluorescent probes, primers or dyes binding to dsDNA. A probe (ie, TaqMan)
is designed to anneal to the target sequence between the traditional forward
and reverse primers. The probe is labeled at the 5' end with a reporter
fluorochrome (usually 6-carboxyfluorescein [6-FAM]) and a quencher
fluorochrome (6-carboxy-tetramethyl-rhodamine [TAMRA]) added at any T
position or at the 3' end.3 The probe is designed to have a higher Tm than
the primers, and during the extension phase, the probe must be 100%
hybridized for success of the assay. As long as both fluorochromes are on
the probe, the quencher molecule stops all fluorescence by the reporter.
However, as Taq polymerase extends the primer, the intrinsic 5' to 3'
nuclease activity of Taq degrades the probe, releasing the reporter
fluorochrome. The amount of fluorescence released during the amplification
cycle is proportional to the amount of product generated in each cycle.
Although, primers originally designed for end-point PCR, works well for real
time PCR, needs standardization to have adequate specificity or sensitivity.

Sources:
Chen S, Yee A, Griffiths M, et al. Int J Food Microbiol 1997;35:239-250.
Eyigor AA, and Tayfun K, Carli B. Avian Diseases: Vol. 47, No. 2, pp. 380–386
Some primers and probes used in RT-PCR of Salmonella: Although same probes
and primers can be used for diagnosis with RT-PCR as used otherwise in PCR, some
people have tried other probes and primers too

Characterization of Salmonella isolates
Virulence markers
• In vitro tests-
– Conventional &
– Molecular
• In vivo tests-
– Conventional &
– New biomodels

Conventional in vitro tests
Congo red dye binding assay
P
N

DNase test
P
P
P
P
P
P
P
N
N
N
N
N

Rings around mercuric chloride disk
Golden hallo around acriflavine
disks,
Ring around crystal violet disk
(All three are believed to be plasmid
mediated characterts)

Effect of plasmid curing on golden hallo reaction around acrifalavine disk
Original A14 strain
A14 strain after curing

Detection of Haemolysins
Haemolysis of washed goat RBCs by S. Paratyphi E436 (A), E44b (B) and S. Typhi, E206(C), (D)
E345 strains. After incubation at 37O
C for 24 hours.

Haemolysis of washed goat RBCs by S. Gallinarum S54 (A), and B haemolytic strains
of Streptococcus aureus (B, C, D). After incubation at 37O
C for 24 hours.

Molecular in vitro tests
• Virulence plasmid detection by isolation of plasmid
– Detection by PCR
– Detection by Probes
• Detection of stn gene for enterotoxin, hly/ sly gene for haemolysins
– cytolysin gene (sly A)
Sal L 1 AGG AGA TGA AAT TGG AAT CGC CA
Sal L 2 TGC CCC TGC ACC TCA ATC GTG AG
stm-O1 CGC AGG TTC TGA ATG CGG AA
STM-O2 TAA TAC CTG CTG TAG CAA GG
– Detection of stn (Product size 617 bp) gene for enterotoxin, hly/ sly gene for haemolysins etc.
Stn-1 5’ TTG TGT CGC TAT, CAC TGG CAA CC 3’
STN-2 5’ ATT CGT AAC CCG CTC TCG TCC 3’

Plasmid profiling
(Many different kinds of plasmids are there in Salmonella strains)

Conventional in vivo models
Mouse model
Retention
of urine in
chronic
salmonell
osis
Loss of
hair,
necrosis of
tail
Control

• 12 day old chick embryo inoculation
Healthy Intra-allantoic S. Gallinarum
inoculated on 12th
day

Rabbit ligated ileal loop assay
Inoculated with cytotoxic enterotoxin
Inoculated with cytotonic
enterotoxin

Vasopermeability factor test assay
Dermonecrosis associated with Salmonella cytotoxin Red zone associated with Salmonella enterotoxin
Negative

New in vivo models
Germinating maize seed model
Inoculated with non-pathogenic rough S. Typhimurium
Inoculated with pathogenic S. Typhimurium

Advances in diagnosis of salmonellosis and characterization of salmonella

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