2020, Scienceline Publication

JWPR

Poultry Research

J. World Poult. Res. 10(2S): 250-262, June 14, 2020

Journal of World’^s

Research Paper, PII: S2322455X2000031-10

License: CC BY 4.0

DOI: https://dx.doi.org/10.36380/jwpr.2020.31

A Field Study on Biochemical Changes Associated with

Salmonella Infection in Ducklings

Mayada A.M. Abou Zeid^1*, Soad A. Nasef ², Gehan, I. E. Ali³and Hegazy, A.M.⁴

¹Bacteriology, Kafr El sheikh Regional Laboratory, Animal Health Research Institute, Agricultural Research Center (ARC), Egypt.

²Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agricultural Research Center (ARC), Egypt.

³Biochemistry, Kafr El sheikh Regional Laboratory, Animal Health Research Institute, Agricultural Research Center (ARC), Egypt.

⁴Poultry diseases, Kafr El sheikh Regional Laboratory, Animal Health Research Institute, Agricultural Research Center (ARC), Egypt.

^*Corresponding author’s Email: kindmemo@yahoo.com ; ORCID: 0000-0002-5733-8606

Received: 22 Feb. 2020

Accepted: 28 Mar. 2020

ABSTRACT

The present study aimed to investigate the incidence of Salmonella infection in diarrheic ducklings in Kafr El Sheikh

Governorate, Egypt. A total of 100 samples were collected from ducklings suffered from diarrhea and mortality.

Also, 50 litter samples were collected from duck farms. All specimens were collected under aseptic conditions for the

isolation of Salmonella spp. The incidence of Salmonella was 7% in pooled samples from cecum, liver, spleen and

gall bladder and 6% in litter samples. Ten strains of Salmonella spp. were serotyped, of which, S. Salamae (1 strain),

S. Miami (2 strains), S. Kentucky (4 strains), S. Paratyphi A (2 strain) and S. Magherafelt (1 strain) were

detected. Susceptibility of Salmonella isolates to 10 antimicrobial agents showed that Salmonella isolates were highly

sensitive to amikacin (100%), followed by trimethoprim/sulphamethoxazole and gentamicin (50%). While isolates

showed the highest percentage of resistance to norfloxacin (90%), followed by ciprocin (70%), flumox (70%) and

amoxicillin-clavulanic acid (70%). Virulence genes (invA, hilA, and fimA) were detected by PCR assay, all 10

Salmonella isolates showed positive results for three virulence genes, which gave specific amplicon at 284, 150, and

85 base pairs, respectively. Lethality test in five groups of three-day-old ducklings with different five isolated strains

indicated a mortality rate ranged from 20-30 % in three isolates only. The most lethal strain S. Paratyphi A was

chosen for further investigation as a pathogenicity test. IL-6 slightly decreased in the infected group in comparison to

control. The results indicated that ducks infected with Salmonella spp. significantly showed lower RBCs, Hb, PCV,

Phagocytic activity, phagocytic index, and serum albumin while, significantly had higher WBCs, neutrophil,

lymphocyte, serum globulin, uric acid, creatinine, AST and ALT concentrations compared to non-infected. It could

be concluded that Salmonella has hepatic and renal destructive effects and immunosuppressive effects.

Keywords: Biochemical changes, Ducklings, Salmonella.

INTRODUCTION

morbidity, mortality, decreased egg and meat creation in

duck. Mortality may shift from 10% to 80% or higher in

extreme episodes (Kleven and Yoder, 1998). Numerous

Salmonella serovars exist. More than 2,600 serovars are

grouped depending on the reactivity of antisera to O and H

antigens (Stevens et al., 2009 ), and the serovars from

ranches have a critical cover with those causing sicknesses

in people (Alcaine et al., 2006 ). For the control and

treatment of Salmonella, antimicrobials use is important.

However, multidrug-resistant Salmonella has emerged

and lead to treatment failure (Gong et al., 2013 ).

The Salmonella virulence is linked to a combination

of chromosomal and plasmid factors. There are different

genes such as inv, spv, fim A and stn have been identified

as major virulence genes responsible for salmonellosis.

Salmonella pathogenicity islands are huge gene tapes

inside the Salmonella chromosome that encode

Salmonella infections are a major problem in the poultry

industry. These bacteria enter the human food chain

through poultry products. Human Salmonella infections

and food-poisoning take the form of gastroenteritis, which

can result in death in highly susceptible individuals (Here

et al., 2003 ). Salmonella is a significant source of

foodborne maladies that cause morbidity and mortality

around the world. Among 94 million cases of non-typhoid

Salmonella contaminations, it was assumed that roughly

85% of the cases were initiated by nourishment root

Salmonella (Chiu et al., 2010 ).

Salmonella contaminations are too vital as both a

cause of clinical infection in duck and as a source of

nourishment borne transmission of sickness to people.

Overwhelming financial problems happen due to

To cite this paper: Abou Zeid MAM, Nasef SA , Gehan IEA and Hegazy AM (2020). A Field Study on Biochemical Changes Associated with Salmonella Infection in Ducklings. J.

World Poult. Res., 10 (2S): 250-262. DOI: https://dx.doi.org/10.36380/jwpr.2020.31

250

J. World Poult. Res., 10(2S): 250-262, 2020

determinants liable for building up particular associations

with the host. Also, it required for bacterial virulence

(Sabbagh et al., 2010 ).

Isolates were identified as Salmonella spp. based on their

colony morphology on selective media, biochemical

testing (Edwards and Ewing, 1986). Isolates that were

biochemically identified as Salmonella spp. were

confirmed serologically by using the Polyvalent

Salmonella (A-E and Vi) antisera (Benex Ltd., Shannon,

Ireland). Serological identification of Salmonella was

performed according to Grimont and Weill (2007).

Salmonella spp. enter the intestinal epithelium and

penetrates the Peyer’s patches and from the Peyer’s

patches, Salmonella spp. go toward the mesenteric lymph

nodes where it spreads to the circulatory system, leading

to transient bacteremia (Smith and Beal, 2008 ). In this

phase, there is massive chemotaxis of chemokines (IL-8,

CXC, MIP-1β) together with IL-1 and IL-6 into intestinal

mucosa Bacteria are rapidly cleared from the blood by

phagocytes in the spleen and liver, and a large fraction of

bacteria are killed by these cells (Coble et al., 2011 ).

The current study was performed to isolate and

identify Salmonella serovars isolated from ducks using

serological techniques and also to study the antibiotic

sensitivity of the isolates. PCR assay was used for

detecting Salmonella virulence genes. Also, the

pathogenicity of isolated strains and changes of

biochemical parameters and immune response during

Salmonella infection in ducks were investigated.

Antimicrobial susceptibility test of isolated

Salmonella spp.

Antimicrobial susceptibility was assessed using a

disk diffusion method according to CLSI protocols (CLSI,

2018). Sensitivity discs with variable concentrations were

used to determine the susceptibility of the isolated strains.

The following antibiotics (Bioanalyse, Epico, and

HiMedia) were used: norfloxacin, ciprocin, flumox,

amoxicillin-clavulanic acid, ampicillin, trimethoprim/

sulphamethoxazole, doxycycline, gentamicin, cefotaxime,

and amikacin. The multidrug-resistant isolates which

resistant to three or more kinds of antimicrobials (Schwarz

et al., 2010 ).

MATERIAL AND METHODS

Virulence genes of Salmonella detection by PCR

Ethical approval

The study was conducted according to the

institutional Animal Care and Use Committee (Vet.

CU20022020149)

DNA extraction

DNA extraction from examined samples was done

by using the QIAamp DNA Mini kit (Qiagen, Germany,

GmbH) with alterations from the manufacturer’s

suggestions Briefly, 10 µl of proteinase K and 200 µl of

lysis buffer was incubated with 200 µl of the tested

suspension at 56^OC for 10 min. After incubation, 100%

ethanol was put to the lysate by200 µl. The tested sample

washed and then centrifuged following the manufacturer’s

order. 100 µl of elution buffer was used to elute the

nucleic acid which provided in the kit.

Collection of samples

A total of 100 samples from 100 ducklings which

suffer from diarrhea and mortality were collected from

different farms and transported to Animal Health Research

Institute Kafr El sheikh branch for examination. Samples

from live and fresh dead birds were taken for isolation and

identification of Salmonella spp. as pooled samples from

the cecum, liver, spleen and gall bladder were collected in

sterile containers to be cultured bacteriologically. Also, 50

Litter samples were collected from duck farms from dry

areas of floor litter from the upper 2.5-5 cm of litter in

sterile plastic bags and transported to the lab for bacterial

examination.

PCR amplification

The Primerswhich used were provided by Metabion

(Germany) are listed in table 1 . Primers were used in a 25

µl of reaction mixture containing 12.5 µl of EmeraldAmp

Max PCR Master Mix (Takara, Japan), 1 µl of each primer

of 20 pmol condensations, 4.5 µl of water, and 6 µl of

DNA template. The reaction was performed in a T3

Biometra thermal cycler.

Isolation of Salmonella spp.

Samples were cultured in Rappaport Vassiliadis

Broth at 37◦C for 18 hrs. and then subcultured on

Salmonella and Shigella agar and incubated at 35° c for 24

hrs and XLD agar at 37◦C for 24-48 hrs. Salmonella was

isolated from poultry litter according to the American

Association of Avian Pathologists (AAAP) (1989).

Analysis of the PCR Products

The PCR product was isolated by electrophoresis on

1.5% agarose gel (Applichem, Germany, GmbH) in 1x

TBE buffer at room temperature using gradients of 5V/cm.

To form gel analysis, about 20 µl from products were

loaded in each gel slot. A gel pilot 100 bp DNA Ladder

251

Abou Zeid et al., 2020

(Qiagen, Germany, GmbH) and gene ruler 50 bp ladders

of the samples, the tubes were put in the adaptor sets,

where it fixed into the clamps of the Qiagen tissue Lyser.

Disruption was done in 2 minutes by high-speed (30 Hz)

shaking step. One size of 70% ethanol was put on the

cleared lysate, and the steps were done concurring to the

Decontamination of Add up to RNA from Animal Tissues

system of the QIAamp RNeasy Mini kit (Qiagen,

Germany, GmbH). N.B. On column DNase, assimilation

was done to evacuate leftover DNA.

(Fermentas, Thermo) were used to detect the segment

sizes. Gel documentation system (Alpha Innotech,

Biometra) was used for photographing the gel and the

computer software was used to analyze the data.

Lethality test

In this test, six groups of three-day-old duckling

were used (ten ducks per group for the five isolates (S.

Salamae, S. Miami, S. Kentucky, S. Paratyphi A, and S.

Magherafelt) and last group as a negative control). A day

before infection (challenge), randomly bacteriological

samples were collected from ducks and tested for

Salmonella free. Each duck was inoculated oral

inoculation (using 1 ml sterile feeding tube introduced into

the crop) with 1ml of overnight Salmonella isolates

suspension (1 × 10⁸CFU/ml). The organism was prepared

according to Osman et al. (2010). Morbidity and mortality

rates following oral inoculation were observed until 15

days (Bjerrum et al., 2003). The most lethal strain was

chosen for further investigation as pathogenicity test (its

effect on performance of duck, shedding and organ

colonization)

B. Oligonucleotide Primers: Primers which used

were provided from Metabion (Germany) are listed in

table 2.

C. Taqman RT-PCR: PCR extension was done in a

volume of 25 µl containing 3 µl of RNA format, 12.5 µl of

2x QuantiTect Probe RT-PCR Master Mix, 8.125 µl PCR

grade water, 0.5 µl from each primer of 20 pmol

condensation and 0.125 µl of each probe (30 pmol cons.)

and 0.25 µl of QuantiTect RT Mix. On the Stratagene

MX3005P real-time PCR machine The reaction was

performed.

D. Analysis of RT-PCR results: Amplification

curves and cycle threshold (CT) values were detected by

the Stratagene MX3005P software. The gene expression

difference on the RNA of the different samples was

assessed, the CT of the tested sample was compared with

the positive control group, according to the "ΔΔCT”

method mentioned by Yuan et al. (2006).

Pathogenicity study

One group of 3-day old duckling and another group

as control (20 birds for each) were separately housed in

controlled biosafety isolator. Birds were fed rations of

antibiotic-free. A day before infection (challenge) samples

were collected and tested for Salmonella free. Birds were

fasted for 12 hours to decrease crop bulk, thus expediting

the flushing of the crop. The organism was prepared

according to Osman et al. (2010). Infection dose was 1

milliliter of dilution introduced orally for all infected

ducks with 1×10⁸CFU/ml Salmonella concentration for

studying morbidity and mortality rates following oral

inoculation were observed to 45 days, The control group

was inoculated oral inoculation with1 milliliter of sterile

saline. Fecal swabs were collected for detection of fecal

shedding from all groups during the first 3 days PI, then at

weekly interval till 45 days. Moreover, at the end of each

week till 45 days, two randomly selected ducks were

sacrificed from each group for postmortem and

bacteriological examination (organ colonization).

Serum biochemical parameters

Biochemical examinations of the one ml of blood

samples were withdrawn from 3selected ducks of each

treatment via brachial vein puncture into EDTA tubes for

hematological analysis and were placed inside an icebox

and transferred to the laboratory. Hemoglobin (Hb)

according to the cyanomethemoglobin technique (Jain,

1986), red blood cell and white blood cell counts using a

Neubauer hemocytometer (Natt and Herrick, 1952) and

packed cell volume (PCV) (Britton 1963) were measured.

Differential leukocyte count was performed using blood

smears stained according to the Rosenfeld method (Lucas

andJamroz, 1961). Determination of phagocytic activity and

phagocytic index (Richardson and Smith, 1982). Also, the

blood samples were kept for 30 min at room temperature

and the serum was collected through centrifugation at

3000 RPM for 15 min and was used for Activities of

Alanine Amino Transferase (ALT) and Aspartate Amino

Transferase (AST) were determined according to Reitman

and Frankel (1957). Uric acid and creatinine were

determined according to Arliss and Entvistle, (1981), and

Michael and Malcolm (2006) respectively. Also, the serum

Detection of interleukin 6 by real-time PCR

A. RNA extraction: RNA performed from spleen

tissue samples by using a QIAamp RNeasy Mini kit

(Qiagen, Germany, GmbH) when 30 mg of the tissue

sample was added to 600 µl RLT buffer containing 10 μl

β-mercaptoethanol per 1 ml. To form the homogenization

252

J. World Poult. Res., 10(2S): 250-262, 2020

used to determine the total protein (TP) according to

Quantification of interleukin-6 mRNA expression

A linear relationship between the amount of input

RNA and the CT values for the various reactions was seen

as expected in a log10 dilution series of standard samples

for Interleukin 6 that also acted as positive controls for RT

and PCR. Regression analysis of the CT values generated

with the log10 dilution series of standards gave R2 values

for all reactions that were greater than 0.97. To account for

the variation in sampling and RNA preparations, the CT

values for cytokines and chemokines specific for each

sample were standardized using the CT value for 28S

rRNA for the same sample from a reaction completed at

the same time. Using the slopes of the Interleukin 6 and

28S rRNA log10 dilution series regression lines, the

difference in input total RNA, as represented by the 28S

rRNA, was then used to adjust cytokine- and chemokine-

specific CT values (Table 8).

(Doumas et al., 1981), albumin (Alb) according to Henry et

al., (1974), Globulins concentration (Glob) in serum was

computed by subtracting albumin concentration from total

Proteins, albumin to globulin ratio (A/G) was calculated

according to Kaneko (1989).

Statistical analysis

Statistical analysis was performed using one-way

analysis of variance using SAS software.

RESULTS

The result of serotyping of Salmonella isolates revealed

five different Salmonella serotypes (S. Salamae , S.

Miami, S. Kentucky, S. Paratyphi A, and S. Magherafelt)

with 1, 2, 4, 2 and 1 strains, respectively.

Pathogenicity study

Blood Parameters

Infection with Salmonella spp. in ducks significantly

(P˂0.05) decreased RBCs, Hb, PCV, Phagocytic activity

% and Phagocytic index while, significantly (P˂0.05)

increased WBCs, neutrophil, and lymphocyte compared

with non-infected (Table 10).

Clinical signs, Postmortem findings and mortality

rate

Clinical signs were observed in the group infected

with S. Paratyphi A 48 hours post-inoculation (PI) in the

form of extreme thirst, profuse diarrhea, huddling together

as chilled, ruffled feathers in some of them, lameness.

Staggering gait, tremors, retraction of the neck backward,

paddling movement, coma, and death. PM lesions revealed

severe congestion of all internal organs, enlargement of

the spleen, enlargement, and lobulation of the kidney,

distention of the ureters with urates. Also, the liver

appeared very pale. The mortality rate was 30%.

Kidney and liver functions related to serum

parameters

Infection with Salmonella spp. in ducks significantly

(p˂0.05) decreased serum albumin while, significantly

(p˂0.05) increased blood serum globulin, uric acid,

creatinine, AST and ALT concentrations compared with

non-infected (Table 12).

Table 1. Primer sequences, target genes, amplicon sizes, and cycling conditions.

Amplification (35 cycles)

Amplified

segment

(bp)

Target

gene

Primary

denaturation

Final

extension

Primer sequences (5'-3')

Reference

Secondary

Annealing Extension

denaturation

F:GTGAAATTATCGCCACGTTCGGGCAA

R:TCATCGCACCGTCAAAGGAACC

F:CATGGCTGGTCAGTTGGAG

94˚C

5 min.

94˚C

30 sec.

55˚C

30 sec.

72˚C

30 sec.

72˚C

10 min.

Oliveira et

al. (2003)

invA

hilA

fimA

284

150

85

94˚C

5 min.

94˚C

30 sec.

60˚C

30 sec.

72˚C

30 sec.

72˚C

7 min.

Yang et

al. (2014)

R:CGTAATTCATCGCCTAAACG

F:CCT TTC TCC ATC GTC CTG AA

R:TGG TGT TAT CTG CCT GAC CA

94˚C

5 min.

94˚C

30 sec.

50˚C

30 sec.

72˚C

30 sec.

72˚C

7 min.

Cohen et

al. (1996)

R: reverse, F: forward

253

Abou Zeid et al., 2020

Table 2. Primer sequences, target genes and cycling conditions for TaqMan RT-PCR.

Amplification (40 cycles)

Target

gene

Primers and probes sequences

(5'-3')

Reverse

transcription

Primary

denaturation

Reference

Secondary

Annealing

denaturation

and extension

F:GGCGAAGCCAGAGGAAACT

R:GACGACCGATTTGCACGTC

TaqMan probe:

(FAM)AGGACCGCTACGGACCTCCACCA (TAMRA)

F:GCTCGCCGGCTTCGA

R:GGTAGGTCTGAAAGGCGAACAG

TaqMan probe: (FAM)

28S

rRNA

Suzuki

et al.

(2009)

50˚C

30 min.

94˚C

15 min.

94˚C

15 sec.

60˚C

1 min.

IL-6

AGGAGAAATGCCTGACGAAGCTCTCCA (TAMRA)

R: reverse, F: forward

Table 3. Incidence of Salmonella spp. isolated from

ducklings and duck farms, Egypt

Table 6. Mortality rates in ducks infected with different

Salmonella isolates through oral inoculation.

No. of

positive

samples

No. of

infected duck

No. of

deaths

Percentage

No. of

samples

Percentage

%

Strain

Types of samples

%

0

20

0

30

20

S. Salamae

S. Miami

S. Kentucky

S. Paratyphi A

S. Magherafelt

10

0

2

0

3

2

Pooled samples from cecum,

liver, spleen and gall bladder

100

7

Litter samples

50

3

6

Table 4. The results of antimicrobial susceptibility test of

Salmonella spp. (n=10) isolated from ducklings, Egypt

Table 7. Fecal shedding and mortality of experimentally-

infected ducks with Salmonella Paratyphi A

Susceptible

Intermediate Resistant

Number of positive

ducks for Salmonella

Antimicrobial

agent

Mortality

(number)

Time

No.

%

No.

%

No.

%

shedding (%)

Norfloxacin

Ciprocin

Flumox

1

-

10

-

3

-

30

9

7

90

70

1st day

-

2

1

2

-

12 (60)

13 (72.2)

15 (88.2)

14 (100)

7 (70)

-

2nd day

Amoxicillin–

Clavulanic acid

Ampicillin

Trimethoprim /

Sulphamethoxazole

Doxycycline

Gentamicin

Cefotaxime

Amikacin

3rd day

3

5

30

50

-

7

5

70

50

End of 1^stweek

End of 2^ndweek

End of 3^rdweek

End of 4th week

End of 5th week

After 45 days

3 (37.5)

3 (50)

3

5

30

50

2

4

8

-

20

40

80

-

5

1

-

50

10

-

1 (25)

2

20

-

0

10

100

-

Table 5. Detection of invA, hilA, fimA virulence genes by

PCR in Salmonella serotypes isolated from ducklings and

litter duck farms, Egypt.

Table 8. Reisolation of Salmonella from internal organs

of experimentally infected ducks with Salmonella

Paratyphi A (n=18)

Gene

S. Paratyphi A

invA

hilA

fimA

Serotype

S. Salamae

S. Miami

Organ

No

10

12

%

1+ve strain

2+ve strain

1+ve strain

2+ve strain

1+ve strain

2+ve strain

Liver

55.5

66.6

Spleen

S. Kentucky

S. Paratyphi A

4+ve strains

2+ve strain

4+ve strains

2+ve strain

4+ve strains

2+ve strain

Gall bladder

Cecum

7

11

38.8

61.1

S. Magherafelt

1+ve strain

+ve: positive

254

J. World Poult. Res., 10(2S): 250-262, 2020

Table 9. Detection of interleukin-6 mRNA and 28S rRNA in spleen of control and Salmonella infected ducklings by real-

time PCR

28S rRNA

Individual

Interleukin-6

Groups

Sample No.

Fold change

Mean

CT

Individual

CT

Mean

CT

Individual

Collective

1

20.53

23.68

-

Control

20.42

23.59

20.81

2

1

2

3

20.30

20.38

20.64

20.77

23.49

20.50

20.89

21.04

-

8.2821

7.5685

7.4643

Infected

20.60

7.7633

CT: cycle threshold

Table 10. Effect of Salmonella challenge on some blood

Table 11. Effect of Salmonella challenge on some serum

parameters of ducklings

biochemical parameters of ducklings

Non-infected

Items

Infected ducks

Items

Non-infected ducks

Infected ducks

ducks

RBCs x106 /mm3

WBCs x103 /mm3

2.12± 0.06^a

1.12 ± 0.12^b

51 ± 0.32^a

Total protein (g/dl)

Albumin (g/dl)

Globulin (g/dl)

5.94 ±0. 08^a

4.8 ±0. 03^a

0.80 ±0. 08^b

5.86 ±0. 05^a

2.69±0.09^b

26.5 ± 0.87^b

Hb (g/dl)

PCV%

Lymphocyte%

Neutrophil%

8.08 ± 0.12^a

34.98 ± 0.33^a

29.93±1.81^b

54.3±1.61^b

5.6± 0.32^b

18.48 ± 1.76^b

36.38±1.11^a

63.4±1.75^a

1.1366 ±0. 11^a

Uric acid (mg/dl)

Creatinine (mg/dl)

AST (u/ml)

7.74 ±0. 05^b

0.67 ±0. 08^b

42 ±0. 46^b

49±0.27^b

11.04 ±0. 02^a

1.46 ±0. 03^a

67.3 ±0. 38^a

82.3 ±1. 2^a

Monocyte%

6.12±0.20^a

5.40±0.22^a

Esinophil%

Basophil%

Phagocytic activity%

Phagocytic index

2.8±0.09^a

5.6±0.53^a

2.01±0.05^a

4.08±0.23^a

25.93±1.22^b

1.180.32^b

35.23±1.61^a

1.96± 0.12^a

ALT (u/ml)

Values are expressed as mean ± standard error. Different superscript

letters within the same row indicate a significant difference (p ≤0.05).

letters within the same row indicate a significant difference (p ≤0. 05).

Figure 1. Detection of invA virulence gene in Salmonella isolates. Agarose gel showing polymerase chain reaction

amplification of products of invA virulence gene of Salmonella. Lane L: 100-600 bp molecular size marker. Lane Pos:

Control positive Salmonella invA virulence gene at 284 bp. Lane 1,2,3,4,5,6,7,8,9 and 10: samples positive for invA gene.

255

Abou Zeid et al., 2020

Figure 2. Detection of hilA virulence gene in Salmonella isolates. Agarose gel showing polymerase chain reaction

amplification products of hilA virulence gene of Salmonella Lane L: 100-600 bp molecular size marker. Lane Pos: Control

positive Salmonella hilA virulence gene at 150 bp. Lane 1, 2, 3,4,5,6,7,8,9 and 10: samples positive for hilA gene.

Figure 3. Detection of fimA virulence gene in Salmonella isolates. Agarose gel showing polymerase chain reaction

amplification products of fimA virulence gene of Salmonella. Lane L: 50-1000 bp molecular size marker. Lane Pos: Control

positive Salmonella fimA virulence gene at 85 bp. Lane 1, 2, 3,4,5,6,7,8,9 and 10: samples positive for fimA gene.

256

J. World Poult. Res., 10(2S): 250-262, 2020

Figure 4. Clinical signs and postmortem lesions in experimentally Salmonella infected ducks. (A): staggering gait. (B):

whitish diarrhea. (C): yellowish liver. (D): congested liver and intestine 1^stweek post-infection. (E): congested kidney after

45 days post-infection.

257

Abou Zeid et al., 2020

Figure 5. Expression of IL-6 and 28S rRNA in spleen of ducks following infection with Salmonella. The data are fold

changes in mRNA determined by quantitative RT-PCR.

Dakahlia and Damietta Governorates by 9.6% Batikh

(2018), who isolated Salmonella from broiler chicken farm

litter by 8%, but these results differ from Shamoon et al.,

(1998) who isolated Salmonella from ducks in open

houses which was 16.6%, Abd-El-Rahman et al. (2000),

who reported that the percentage of isolation was 20%

DISCUSSION

In the present work, Salmonella was isolated from

ducklings and litter with a rate of 7% and 6%, respectively

(Table 3 ), this rate appears to be similar to Abd El- Tawab

et al. (2015) who isolated Salmonella from ducks in

258

J. World Poult. Res., 10(2S): 250-262, 2020

from 10 duck flocks in North Sinai. Hoszowski and Wasyl

genes were detected by 100% and 82.61% respectively in

retail beef meat samples.Transmission of infection is

generally considered to occur orally. Enormous bacterial

increase happens inside the intestine and tissue attack

happens quickly. Under test conditions, mortality created

by a harmful strain may change from 25% to 100%

between distinctive inbred lines (Barrow et al., 1987).

This study examined the lethality of Salmonella

strains using 3-day-old ducklings (Table 6 ). The

observations were done during 15 days and showed a low

rate of morbidity rate (weakness, lethargy, and low growth

rate) while, the mortality rate reached 30%. Similar result

was reported by Batikh (2018 ), who detected that the

mortality was 28.6% in ducklings after inoculated orally

with S.Bargny, S.Enteritidis and S.Kentucky strains.

The results indicated the pathogenesis of

experimentally infected duckling with Salmonella

Paratyphi A (Tables 7 and 8; Figure 4). Pathogenesis

studies associated with virulent strains suggested that

organisms multiply in the liver and spleen after the

invasion and then disseminate to other organs, producing a

systemic infection (Barrow et al., 1987). Ducks are very

resistant to infection produced by Salmonella, they are

possibly reservoirs of it and may shed it in the feces and

pollutethe environment (Barrow et al., 1999). The present

study revealed that colonization of the cecum and

shedding of S. Paratyphi A in the feces was detected in the

feces since 24 h post-infection, a similar result was

reported by Ribeiro et al. (2005), who detected S. Kottbus

in the feces of broiler chicks since 24 h until 42 days post-

infection.

(2005), who detected Salmonella in duck broilers with a

percentage of 14.3%, Adzitey and Huda (2012), who

detected Salmonella in duck floor swab and transport crate

swab with percentage of 13.3% for each.

The result of serotyping of 10 Salmonella isolates

using "O", 'H" and "Vi" antisera are illustrated, which

clarified that the serotype of Salmonella spp obtained from

positive Salmonella samples were S. Salamae (1 Strain), S.

Miami (2 strain), S. Kentucky (4 Strain), S. Paratyphi A (2

Strain), and S. Magherafelt (1 strain). The most prevalent

serovar was S. Kentuky (4 strains), these results agreed

with Elgohary et al. (2017), who reported that S. Kentuky

is the most prevalent serovar in diarrheic young duckling

and slaughtered ducks (2 serovars) for each, but these

results disagree with Guran et al. (2017), who found that

S. Kentucky has been rarely reported in ducks, however, it

has been reported in other animals, such as chicken

According to the results concerning antimicrobial

susceptibility tests presented in table 4, 10 isolates showed

the highest percentage of resistance (90%) to norfloxacin,

followed by ciprocin, flumox, and amoxicillin-clavulanic

acid by 70% for each. these results were higher than those

reported by Abd El-Tawab et al. (2015), who detected that

the resistance to amoxicillin and ampicillin /sulbactam

was 50 % and 60% respectively in ducks isolates, but

results of the present study were lower than Mohamed et

al., (2015), who detected that the amoxicillin was 80%

sensitive to Salmonella isolated from broilers, while in this

study Salmonella isolates were amikacin sensitive by

100%, followed by trimethoprim/sulphamethoxazole,

gentamicin by 50% for each. Similar result was reported

by Abd El-Tawab et al. (2015), who reported that

Salmonella was sensitivity to amikacin by 100% and

sulfa+trimethoprim by 60% in ducks isolates. All isolates

were screened by PCR analysis for the presence or

absence of three selected virulence genes (invA, hilA and

fimA) (Table 5; Figures 1, 2 and 3). The most common

virulence gene which presents in Salmonella, invA gene,

was used as a PCR target gene for the detection of

Salmonella (Dong et al., 2014). Also, PCR screening

analysis detected the presence of invA, hilA, and fimA in

all Salmonella isolates. These result agreement with Abd

El-Tawab et al. (2017), who detected that the percentage

of Salmonella Typhimurium virulence genes invA and hilA

were 100 % for each which isolated from clinically

mastitic milk samples of cattle cows, Malorny et al.

(2003), who revealed that in the studied strain that invA

gene was detected in the rate of 100% and Thung et al.

(2018), who detected Salmonella invA and hilA virulence

Effect of Salmonella infection on interleukin 6

In the bacteremia phase, there is massive chemotaxis

of chemokines (IL-8, CXC, MIP-1β) together with IL-1

and IL-6 into the intestinal mucosa. Bacteria are rapidly

cleared from the blood by phagocytes in spleen and liver,

and a large fraction of bacteria are killed by these cells

(Coble et al., 2011 ). IL-6 decreased in the infected group

in compared to control one (Table 9, Figure 5 ), these

differed from (Kaiser et al., 2000 ), who reported that IL-6

is usually indicative of the initiation of an acute-phase

response and is produced following infection with S.

Typhimurium in vitro model of avian cell culture.

Effect of Salmonella infection on hematological

parameters

Infection with Salmonella spp. in ducks significantly

(P˂0.05) decreased RBCs, Hb, PCV, Phagocytic activity

% and Phagocytic index while, significantly (P˂0.05)

259

Abou Zeid et al., 2020

increased WBCs, neutrophil, and lymphocyte compared

Salmonella species and therefore is used as a golden

marker in the genetic diagnosis of Salmonella species. It is

concluded that Salmonella had immunosuppressive effects

and destructive effects on the liver and kidney.

with noninfected (Table 10). Infection with Salmonella

spp. in ducks significantly (P˂0.05) decreased serum

albumin while, significantly (P˂0.05) increased blood

serum globulin, uric acid, creatinine, AST and ALT

concentrations compared with non-infected (Table 10).

Changes that happened in the blood picture and

biochemical values are a mirror of the changes that

occurred in the tissues and organs as a result of bacterial

infection. These findings agreed with those reported by,

(Assoku et al., 1970 ) and (Kokosharov, 2006), who

discussed that there were decreased in RBCs, Hb and PCV

in poultry infected with S. Gallinarium. Increased values

of WBCs, neutrophil, and lymphocyte agreed with

(Morgulis, 2002 ), who recorded that Leukocytosis is

usually due to heterophilia, and common causes are

general infections due to septicemias caused by infectious

agents, such as Salmonella and disagreed with Allan and

Duffus (1971), found no changes in lymphocyte counts

during fowl typhoid and Assoku et al. (1970), worked at S.

Gallinarium in birds, and noticed that the count of

lymphocyte was lower than the normal values. There were

no important changes in the percentage of eosinophil,

monocyte, and basophil, these results were coordinated

with previous results (Cardoso et al., 2003 and Freitas

Neto et al., 2007 ). Decreased level in Phagocytic activity

% and Phagocytic index agreed with (Belih et al., 2016 ).

There was a decrease in albumin, AST, ALT and

increased globulin (Table 11 ), which agreed with (Freitas

Neto et al., 2007), who reported that serum albumin was

lower while ALT and AST were higher in S. Gallinarium

infection. This may be due to the inability of protein

synthesized by the liver which reflects lesion intensity,

visibly proven by hepatomegaly and loss of protein by the

affected kidney. Therefore, the damage in the glomerular

filtration barrier, inflammation of the renal parenchyma or

epithelial damage of the tubules leads to the presence of

plasma proteins in the urine (Relford and Lees, 1996 ).

In the present work, Salmonella infection

significantly increased serum creatinine and uric acid

levels in Salmonella infected group, that agreed with

Hegazy et al. (2014).

REFERENCES

Abd El-Tawab AA, Nabih AM, Agag MA, Ali A and Marwah H (2017).

Molecular studies of virulence genes of Salmonella Typhimurium

causing clinical mastitis in dairy cattle. Benha Veterinary Medical

Journal, 33(2): 27-37. Available at: http://www.bvmj.bu.edu.eg

Abd El-Tawab A, El-Hofy FI, Ammar AM, Nasef SA and Nabil NM

(2015). Molecular studies on antimicrobial resistance genes in

Salmonella isolated from poultry flocks in Egypt. Benha

Veterinary Medical Journal, 28(2): 176-187. Available at:

http://www.bvmj.bu.edu.eg

Abd-El-Rahman, Mahmoud

A

and Moussa HMM (2000).

Bacteriological and histopathological studies on Salmonella

isolates from ducks in North Sinai. Egyptian Journal of

Agricultural

Research,

78(1):15-24.

Available

at:

https://www.cabdirect.org/cabdirect/abstract/20013148120

Adzitey F, Rusul G and Huda N (2012). Prevalence and antibiotic

resistance of Salmonella serovars in ducks, duck rearing and

processing environments in Penang, Malaysia. Food Research

International,45(2):947-952.

Available

at:

https://www.sciencedirect.com/science/article/pii/S0963996911001

530

Alcaine SD, Soyer Y, Warnick LD, Su WL, Sukhnan S , Richards J,

Fortes ED, McDonough P, Root T P, Dumas NB, Gro¨hn Y and

Wiedmann M (2006). Multilocus sequence typing supports the

hypothesis that cow- and human-associated Salmonella isolates

represent distinct and overlapping populations. Applied and

Environmental Microbiology, 72: 7575–7585. Available at:

https://aem.asm.org/content/aem/72/12/7575.full.pdf

Allan D and Duffus WPH (1971). The immunopathology in fowls

(Gallus domesticus) of acute and subacute Salmonella Gallinarum

infection. Research in Veterinary Science, 12(2): 140-151. DOI:

https://doi.org/10.1016/S0034-5288(18)34207-3

American Association of Avian Pathologists (AAAP) (1989).

A

Laboratory manual for the isolation and identification of avian

pathogens. Third Edition. Kendall/Hunt. USA.

Arliss JO and Entvistle WM (1981). Enzymatic determination of uric

acid. Clinical Chemistry Acta, 118:301-309

Assoku RKG, Penhale WJ and Buxton A (1970). Haematological

changes in acute experimental Salmonella Gallinarum infection in

chickens. Journal of Comparative Pathology, 80(3): 473-485. DOI:

https://doi.org/10.1016/0021-9975(70)90080-0

Barrow PA, Huggins MB, Lovell MA and Simpson JM (1987).

Observations on the pathogenesis of experimental Salmonella

Typhimurium infection in chickens. Veterinary Science, 42:194-

199. DOI: https://doi.org/10.1016/S0034-5288(18)30685-4

Barrow PA, Lovell MA, Murphy CK and Page K (1999). Salmonella

infection in a commercial line of ducks; experimental studies on

virulence, intestinal colonization and immune protection.

CONCLUSION

Eidemiology

and

Infection,

123(1):121-32.

DOI:

https://doi.org/10.1017/S0950268899002605

Results of antibiotic sensitivity demonstrated that

amikacin could be used for ducks against Salmonella

infection. Molecular analysis showed that virulence genes

of invA, hilA and fimA were found in all Salmonella strains

isolated from ducklings. The invA gene is present only in

Batikh M. (2018). Epidemiological studies on transmission of avian

pathogens from fish farms to water fowls in Kafr El-Sheikh

governorate. Doctor of Philosophy Thesis, Poultry disease

department, Faculty of Veterinary Medicine, Cairo University,

Egypt.

260

J. World Poult. Res., 10(2S): 250-262, 2020

Belih S, EL-Hadad S, Amen G and Basiony MR (2016). Influence of

Guran HS, Mann D, Alali WQ (2017).

Salmonella prevalence

sodium butyrate on Salmonella infection in broiler chicks. Benha

Veterinary Medical Journal, 31(2): 21-32. Available at:

http://www.bvmj.bu.edu.eg

associated with chicken parts with and without skin from retail

establishments in Atlanta metropolitan area, Georgia. Food

Control,

73:462–7.

DOI:

https://doi.org/10.1016/j.foodcont.2016.08.038

Bjerrum CL, Engberg RM and Pedersen K (2003). Infection models for

Salmonella Typhimurium DT110 in day-old and 14-day-old broiler

chickens kept in isolators. Avian Diseases, 47(4): 1474-1480. DOI:

https://doi.org/10.1637/7051

Hegazy AMa , Barakat Mb , Sabreen E Fadlb and El-Keredy MS Abeer

(2014). Effect of Pedicoccus acidilactici on immunity, production

and lipid profile in broilers. Alexandria Journal of Veterinary

Sciences, 41: 35- 46. DOI: https://doi.org/10.5455/ajvs.158314

Britton CJ (1963). Disorders of the Blood, 9th ed. I. A. Churchill, Ld.

London. The United Kingdom.

Henry RJ, Canmon DC and Winkelman JW (1974). Determination of

calcium by atomic absorption spectrophotometry. In: Henry RJ,

Cannon DC, Winkelman JW (eds) Clinical chemistry: principles

and techniques, 2^ndEd. Harper and Row, Maryland, p 657.

Cardoso A LSP, Tessari EN and Castro AG (2003). Estudo

hematológico em aves inoculadas com Salmonella Gallinarum.

Arquivos do Instituto Biológico, 70(1): 35-42. Available at:

http://www.biologico.sp.gov.br/uploads/docs/arq/V70_1/cardoso.p

df

Here L, Wagenaar JA; Vanknapen F and Urlings BAP (2003). Passage

of Salmonella through the crop and gizzard of broiler chickens fed

with fermented liquid feed. Avian Pathology, 32: 173-181. DOI:

https://doi.org/10.1080/0307945021000071597

Chiu LH, Chiu CH, Horn YM, Chiou CS, Lee CY, Yeh CM, Yu CY,

Wu CP, Chang CC and Chu C (2010). Characterization of 13

multi-drug resistant Salmonella serovars from different broiler

chickens associated with those of human isolates. BMC

Hoszowski A and Wasyl D (2005). Salmonella prevalence and resistance

to antibiotics in Poland. Medycyna Weterynaryjna, 61(6):660-663.

Microbiology,10:86.

https://link.springer.com/content/pdf/10.1186/1471-2180-10-86.pdf

Available

at:

Jain NC (1986). Schaulm Veterinary Haematology 4^thEdition, Lea and

Febiger. Philadelphia.

CLSI

(2018). Performance standards for antimicrobial disk

Kaiser P, Rothwell L , Galyov EE ,Barrow PA, Burnside J and Wigley

P (2000). Differential cytokine expression in avian cells in

response to invasion by Salmonella Typhimurium, Salmonella

Enteritidis and Salmonella Gallinarum. Microbiology, 12:3217–

3226. DOI: https://doi.org/10.1099/00221287-146-12-3217

Kaneko JJ (1989). Clinical biochemistry of domestic animals.4^th

Edition. Academic Press lnc. New York .146-159:612-647.

susceptibility tests; approved standard 12^thEdition.

Coble DJ, Redmond SB, Hale B and Lamont SJ (2011). Distinct lines of

chickens express different splenic cytokine profiles in response to

Salmonella Enteritidis challenge. Poultry Science, 90(8): 1659-

1663.

Cohen HJ, Mechanda SM and Lin W (1996). PCR amplification of the

fimA gene sequence of

Salmonella Typhimurium, a specific

Kleven SH and Yoder HW (1998). Mycoplasmosis. In: HG Purchase,

LH Arp, CH Domermuth and JE Pearson (Eds). A Laboratory

manual for the isolation and identification of avian pathogens.

method for detection of Salmonella spp. Applied and

Environmental Microbiology, 62(12):4303-8. Available at:

https://aem.asm.org/content/62/12/4303.short

Kokosharov T (2006).Changes in the protein profile in birds with

experimental acute fowl typhoid. Bulgarian Journal of Veterinary

Dong P, Zhu L, Mao Y, Liang R, Niu L, Zhang Y, Li K and Luo X

(2014). Prevalence and proﬁle of Salmonella from samples along

the production line in Chinese beef processing plants. Food

Medicine,9(3):

189-192.

Available

at:

https://pdfs.semanticscholar.org/f4bf/8e577513bb09afc12adb63c8e

82ae443fe0f.pdf

Control,

38:54–60.

DOI:

https://doi.org/10.1016/j.foodcont.2013.09.066

Lucas AM and Jamroz C (1961). Atlas of avian hematology. US

Department of Agriculture. Washington, DC, Agriculture

Monograph 25.4th Edition, American Association of Avian

Pathologists, Kenett Square, pp. 74-88.

Doumas BT, Bayso DD, Carter RJ, Peters T and Schaffer R (1981).

Determination of total serum protein. Clinical Chemistry, 27:

1642-1643.

Edwards PR, Ewing WH ( 1986). Edwards and Ewing’s identification of

Enterobacteriaceae (4^thEdition.), New York, U.S.A: Elseviers

Science Publishing Co., Inc.

Malorny B, Hoorfar J, Bunge C and Helmuth R ( 2003). Multicenter

validation of the analytic accuracy of Salmonella PCR: toward an

international standard. Applied Environmental Microbiology, 69:

Elgohary AH, Abu Elnagai AAM, Ibrahim HS, Hedia RH, Dorgham SM

and Elgabry EA (2017). Detection of virulence genes of

Salmonella in diarrheic ducks by using polymerase chain reaction

(PCR). Egyptian Journal of Veterinary Science, 48(1): 11-21.

290-296.

Available

at:

https://aem.asm.org/content/aem/69/1/290.full.pdf

Michael P and Malcolm W ( 2006). Measurement of serum creatinine-

current status and future goals. The Clinical Biochemist

Reviews,27(4): 173-184. Available at:

Available

at:

https://ejvs.journals.ekb.eg/article_3994_d62ce1329fe53bd4c4daee

776968a688.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1784008/pdf/cbr2

7_4p173.pdf

Freitas Neto OC, Arroyave W, Alessi AC, Fagliari JJ and Berchieri A

(2007). Infection of commercial laying hens with Salmonella

Gallinarum: clinical, anatomopathological and haematological

studies. Brazilian Journal of Poultry Science, 9(2): 133-141. DOI:

http://dx.doi.org/10.1590/S1516-635X2007000200010

Mohamed T shaaban, Hegazy AM and Sami I Menisy (2015).

Microbiological and molecular studies on Salmonella spp. isolated

from broilers in Kafr El.sheikh governorate, Egypt. Journal of

Bioscience and Applied Research, 1(2):59-67.

Morgulis M (2002). Imunologia aplicada. In: Macari M, Furlan RL,

Gonzalez E editors. Fisiologia aviária aplicada a frangos de corte.

Jaboticabal: FUNEP/ UNESP, pp.375-429.

Gong J, Xu M , Zhu C, Miao J, Liu X, Xu B, Zhang J, Yu Y and Jia X

(2013). Antimicrobial resistance, presence of integrons and biofilm

formation of Salmonella Pullorum isolates from eastern China

(1962-2010).

https://doi.org/10.1080/03079457.2013.788129

Avian

Pathology,

42:

290–294.

DOI:

Natt MP and Herrick CA (1952). A new blood diluent for counting the

erythrocytes and leucocytes of the chicken. Poultry Science,

31:735-738. DOI: https://doi.org/10.3382/ps.0310735

Grimont PA and Weill FX (2007). Antigenic formulae of the

Salmonella Serovars, WHO Collaborating Center for Reference

and Research on Salmonella, Paris. (9^thEdition). Available at:

http://www.pasteur.fr/sante/clre/cadrecnr/salmoms-index.html

Oliveira SD, Rodenbusch CR, Ce MC, Rocha SLS and Canal CW

(2003). Evaluation of selective and non- selective enrichment PCR

procedures for Salmonella detection. Lett. Applied Microbiology,

261

Abou Zeid et al., 2020

36:

217-221.

DOI:

https://doi.org/10.1046/j.1472-

Shamoon GN, Ali TS and Al-Atar MY (1998). Isolation of Salmonella

765X.2003.01294.x

from local ducks. Iraqi Journal of Veterinary Sciences, 11(2):75-

68.

Osman KM, Moussa IMI, Yousef AMM, Aly MM, Radwan MI and

Alwathnani HA (2010). Pathogenicity of some avian Salmonella

serovars in two different animal models: SPF chickens and

BALB/c mice. Environment and We An International Journal of

Science and Technology,5: 65-78.

Smith AL and Beal R ( 2008). The avian enteric immune system in

health and disease. In: Davison F, Kaspers B, Schat KA, eds.

Avian immunology, London: Academic Press: 243–71.

Stevens MP, Humphrey TJ, and Maskell DJ (2009). Molecular insights

into farm animal and zoonotic Salmonella infections. Philosophical

Transaction of the Royal Society B. London,364: 2709–2723. DOI:

https://doi.org/10.1098/rstb.2009.0094

Reitman S and Frankel S (1957). A colorimetric method for the

determination of serum glutamic oxaloaceitic and glutamic pyruvic

transaminases. American Journal of Clinical Pathology, 28:56-63.

Relford RL and Lees GE (1996). Nephrotic syndrome in dogs: diagnosis

and treatment. Journal of Compendium on Continuing Education

Practicing Veterinarian, 18:279-292.

Suzuki K, Okada H, Itoh T, Tada T, Mase M, Nakamura K, Kubo M and

Tsukamoto K (2009). Association of increased pathogenicity of

Asian H5N1 highly pathogenic avian influenza viruses in chickens

with highly efficient viral replication accompanied by early

destruction of innate immune responses. Journal of Virology, 83

Ribeiro SAM, Berchieri Jr A, Orsi MA, Mendonça AO and Ferrati AR

(2005). Experimental infection by Salmonella enterica subsp

enterica serovar Kottbus in day-old broiler chickens. Brazilian

(15):

7475–7486.

Available

at:

https://jvi.asm.org/content/jvi/83/15/7475.full.pdf

Journal

of

Poultry

Science,

7(2):

107-112.

DOI:

http://dx.doi.org/10.1590/S1516-635X2005000200007

Thung TY, Radu S, Mahyudin NA, Rukayadi Y, Zakaria Z, Mazlan N,

Tan BH, Lee E, Yeoh SL, Chin YZ, Tan CW, Kuan CH, Basri DF

and Wan Mohamed Radzi Che WJ (2018).Prevalence, virulence

genes and antimicrobial resistance profiles of Salmonella serovars

from retail beef in Selangor, Malaysia. Frontiers in Microbiology,

8: 2697. DOI: https://doi.org/10.3389/fmicb.2017.02697

Richardson MD and Smith H (1982). Differentiation of extracellular

from ingested Candida albicans blastospores in phagocytosis tests

by staining with fluorescein-labelled concanavalin A. Journal of

Immunological

Methods,

52(2):241–244.

DOI:

https://doi.org/10.1016/0022-1759(82)90050-3

Yang X, Brisbin J,Yu H,Wang Q,Yin F,Zhang Y, Sabour P, Sharif S

Sabbagh SC, Forest CG, Lepage C, Leclerc JM and Daigle F (2010).

Uncovering distinctive features in the genomes of Salmonella

enterica serovars Typhimurium and Typhi. FEMS. Microbiological

and Gong

J

(2014).Selected lactic acid-producing bacterial

isolates with the capacity to reduce Salmonella translocation and

virulence gene expression in chickens.PLOS ONE, 9(4): e93022.

Letters,305(1):1–13.

DOI:

https://doi.org/10.1111/j.1574-

.Available

at:

6968.2010.01904.x

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3984083/

Schwarz S, Silley P, Simjee S, Woodford N, van Duijkeren E, Johnson

AP and Gaastra (2010). Assessing the antimicrobial

susceptibility of bacteria obtained from animals. Journal of

Antimicrobial Chemotherapy, 65(4): 601-604. DOI:

https://doi.org/10.1093/jac/dkq037

Yuan JS, Reed A, Chen F and Stewart CN (2006). Statistical analysis of

real-time PCR data. BMC Bioinformatics, 7(1):85. Available at:

https://link.springer.com/content/pdf/10.1186/1471-2105-7-85.pdf

W

262