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ISOLATION, IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY TESTING OF SALMONELLA

FROM SELECTED POULTRY FARMS IN DEBRE ZEIT

Elsabeth Solomon1,Hailelule Aleme

2,Habtamu Tassew

1, Kassaw Amssalu

1, Filimon Mitiku

2, Haile Alemayehu

2

1. College of Veterinary Medicine, Mekelle University, Mekelle, Ethiopia

2. Dilla University College of Agriculture and Natural Resources, Department of Animal and range sciences,

Dilla, Ethiopia

2. Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia

ABSTRACT

A cross sectional study was conducted from December 2013 to May 2014 with the objectives to isolate, identify and

determine the antimicrobial resistance pattern of salmonella species from selected poultry farms in Debre zeit, Ethiopia.

From a total of 196 cloacal swabs collected 50 (25.5%) were found to be positive for Salmonella organisms using culture

method, and 15 (7.6%) Salmonella isolates were confirmed using biochemical tests. All culture and biochemical positive

samples were further confirmed by Polymerase Chain Reaction (PCR) through amplification of histidine transport operon

as a target gene for the presence of salmonella isolates. From culture and biochemical positive samples, gel electrophoresis

of the PCR product revealed the presence of 496bp segments in 13 (6.7%) Salmonella isolates. The statistical analysis has

revealed a significantly association between different age groups of chickens (X2 = 10.56; P = 0.005) and farms (X

2 =10.74;

P=0.013) with the percentage of Salmonella isolates. Most of the Salmonella isolates were found to be resistant against

commonly used antimicrobials such as Sulfisoxazole, Chloramphenicol and Ampicillin followed by Tetracycline,

Amoxicillin/Clavulanic acid and Cephalotin and more than half(69.3%) of the isolates were found to be multi-drug

resistant. The high prevalence of resistance to antimicrobial agents found in this study might be attributed to uncontrolled

use of antimicrobial agents as growth promoters in poultry farms. Therefore, proper treatment of chickens using

appropriate antibiotics is then quite essential.

Key words: Antimicrobials; Isolation; PCR; Poultry; Salmonella

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1. INTRODUCTION

Animal production in general and chicken production in particular plays important socioeconomic roles in developing

countries. The importance of poultry production in the national economy of developing countries and its role in improving

the nutritional status and incomes of many small scale farmers and womens has been recognized by various scholars and

rural development agencies for the last few decades (Melesse et al., 2005; Moges et al., 2010; Melesse et al.,

2011). Infectious diseases of various etiology and management problems are among the major problems in commercial

poultry production.

(Labago et al., 2003; Namata et al., 2008). Newcastle disease, Marek’s disease and infectious bursal disease are the most

devastating diseases of chickens (Lobago and Woldemeskel 2004 ; Zeleke et al., 2005). More over Bacterial disease like

salmonellosis, mycoplasmosis, and collibacilosis causes stumbling block for commercial poultry production.

Salmonellosis is a bacterial disease caused by Gram negative facultative rod shaped bacterium strains of Salmonella which

is the family Enterobacteriaceae. Chickens are the natural hosts for the highly host adapted biovar S. gallinarum and S.

pullorum, the causative agents of fowl typhoid and pullorum respectively. Fowl typhoid and pullorum disease are known as

the major obstacle of poultry industry (Medina et al., 2013; kassaye et al., 2010; Chrysostome et al., 1995; Sato et al.,

1997). Fowl typhoid is per acute, acute or chronic form of disease affecting mostly adult chickens, whereas pullorum

disease affects the very young chickens of mostly 2−3 weeks of age. Confirmatory diagnosis depends on the

bacteriological isolation and molecular characterization while serological tests can be used to detect the presence and

estimate the prevalence of infection within a flock (OIE, 2008).

Salmonellosis in poultry has been reported by various scholars from Ethiopia (Medina et al., 2013; Genet et al., 2014). As

the poultry industry develops and become more intensified, the incidence of salmonellosis including pullorum disease and

fowl typhoid become rising concern and bottleneck for the sector advancement (Genet et al., 2014).

In addition to the direct economic impact of salmonellosis in poultry production, currently resistance of Salmonella to

commonly used antimicrobials is rising concern both in the veterinary and public health sectors and has emerged as a

global problem. There are reports of high prevalence of resistance in Salmonella isolates from countries such as Taiwan

(Lauderdale et al., 2006), India (Mandal et al., 2004), The Netherlands (Duijkeren et al., 2003), France (Weill et al., 2006),

Canada (Poppe et al., 2006), and Ethiopia (Molla et al, 2003).

The increasing proportion of single and multiple antimicrobial-resistant Salmonella strains isolated from human

salmonellosis cases has been associated with the widespread use of antimicrobial agents in food animal production. A

considerable number of antimicrobials commonly used in the treatment of salmonellosis and other bacterial infections of

humans are also used in veterinary practices (Wenger et al,. 1999; Gay et al,. 1994) This present a public health risk by the

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transfer of resistant Salmonella and other zoonotic bacterial pathogens or the resistant genes from food animals to humans

through consumption of contaminated food and food products.

In the current study area both small scale and large scale commercial poultry production are flourishing. As the poultry

industry further develops and become more intensified, the avian Salmonellosis can cause serous damage on the poultry

industry and human health. Hence documentation of the prevalence of the disease with confirmatory diagnosis; culture and

molecular methods will have paramount importance to design a control or prevention program and moreover the

identification of antimicrobial resistant serotypes of Salmonella will help in instituting an appropriate therapy and provide

information on the auxiliary emergence of drug resistance. Therefore, the objectives of this study were to isolate

Salmonella rganism from cloacal swab of chicken using culture, biochemical and polymerase chain reaction (PCR) and to

determine the antimicrobial susceptiblity of Salmonella organisms isolated from cloacal swab samples of chickens.

2. MATERIALS AND METHODS

2.1. Study Area

The study was carried out between November 2013 and April 2014 in Debre zeit, Ethiopia. Debre zeit is found in Oromia

regional state 47 km south east of Addis Ababa. It is located at 90

N and 400E, with human population of about 95,000.Its

altitude is about 1850 m above sea level. It experience bimodal patterns of rainfall with the main rainy season extending

from June to September. A short rainy season occurs between march and May with an average rainfall of about 80mm.The

mean annual minimum and maximum temperature are 12.300 and27.7

0 c respectively with an overall average of

18.70(CSA,2008). Highest temperatures are recorded in May and it has 61.3%mean relative humidity. The isolation of

Salmonella, identification and antimicrobial resistance determination was done at Aklilu Lema institute of pathobiology

microbiology laboratory.

2.2. Study Design and Sample Size

Cross sectional study was conducted to isolate, identify and determine antimicrobial susceptibility testing of salmonella

organisms from four selected poultry farms in Debre Zeit, Ethiopia. Farms were selected purposively based on willingness

of the farm owners. A total of 196 chickens were sampled from four purposively selected commercial poultry farms.

2.3 Study animals and Sample collection

The study animals were commercial chicken flocks that were purposively selected from four commercial poultry farms. A

total of 196 Cloacal swab samples were collected from live chickens using sterile cotton tipped swabs moistened with

buffered peptone water. The swabs were kept in properly plugged sterile test tubes and transported as soon as possible

using icebox to Aklilu lema institute of pathobiology, microbiology laboratory.

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2.4. Isolation and Identification of Salmonella

The study was conducted utilizing the conventional methods for the detection of Salmonella following the standard guide

lines given by ISO 6579: (2002). Cloacal swab samples pre-enriched in buffered peptone water were incubated for 24

hours at 370C and the pre-enrichment broth after incubation was mixed and 0.1 ml of the broth was transferred into a tube

containing 10 ml of Rappaport-Vassiliadis medium with soya (RVS broth). Another 1 ml of the pre-enrichment broth was

transferred into a tube containing 10 ml of Muller-Kauffmann tetrathionate novobiocin broth (MKTTn broth). The

inoculated RVS broth was incubated at 42o

C for 24 hours and the inoculated MKTTn broth at 37o

C for 24 hours.

After incubation for 24 hours, a loop-full of material from the RVS broth and MKTTn was transferred and streaked

separately onto the surface of Xylose lysine deoxycholate agar (XLD agar). The plates were incubated at 37o

C for 24

hours. The plates were incubated in an inverted position and after incubation; the plates were checked for growth of typical

Salmonella colonies. Characteristic Salmonella colonies, having a slightly transparent zone of reddish Color and a black

center, were sub-cultured onto the surface of XLD agar for purification. This step was repeated until the colonies in the

plate showed morphological homogeneity. All suspected colonies were streaked on the surface of nutrient agar plates, in a

manner which allowed well isolated colonies to develop. The inoculated plates were incubated at 37o

C for 24 hours. Thus

the pure culture obtained was used for biochemical and molecular confirmation.

2.4.1 Biochemical Confirmation

Biochemical identification of isolates were performed using Triple sugar iron agar (TSI), Christensen's urea agar, Simmon

Citrate Agar, Lysine iron agar (LIA) and motility test was also done For separation of motile and non-motile Salmonella

according to Cown (1985).

2.4.2 Polymerase Chain Reaction (PCR)

Few Salmonella colonies from each biochemical positive culture were taken for DNA isolation using the procedure

described by Welson. (1990) and ultimately for further confirmation with PCR. DNA extracted from Salmonella culture

was amplified by using primers specific to the gene encoding histidine transport operon. Oligonucleotide primers of 25bp

(base pair) (FF: 5’-ACTGGCGTTATCCCTTTCTCTGGTG -3’; Rev: 5’-ATGTTGTCCTGCCCCTGGTAAGAGA-3’) were

used to amplify a region of 496bp segment, histidine transport operon, of Salmonella (Rychlik and Rhoades, 1989). A PCR

assay was performed with a final volume of 25µl mixture and the reaction was allowed to run for 30 sec at 940C, 30 sec at

600C, and 45 sec at 72

0 C for denaturation, annealing and extension, respectively for about 30 cycles in a Thermocycler.

The PCR products then were electrophoresed on a 1% agarose gel containing Ethidium bromide and photographed.

Positive results were indicated by the presence of a 496-bp band seen on the gel with an ultraviolet transilluminator.

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2.5. Antimicrobial susceptibility testing

Table 1: Antimicrobial drugs and concentrations used

Antimicrobial disks Strength in microgram

Amikacin 30

Amoxicillin and clauvlonic acid 20/10(30)

Ampicillin 10

Cefoxitin 30

Ceftriaxone 30

Cephalothin 30

Chloramphenicol 30

Ciprofloxacin 5

Gentamycin 10

Kanamycin 30

Naldixic acid 30

Neomycin 30

Nitrofurantoin 100

Streptomycin 10

Sulfisoxazole 1000

Sulphamethoxazole and trimethopirim 23.75 and 1.25

Tetracycline 30

Trimethoprim 5

The antimicrobial susceptibility testing was done by the agar disk diffusion method as described by Clinical and

Laboratory Standards institute (CLSI, 2013). In brief, a 0.5 Mac-Farland standardized suspension of the bacteria was

prepared in 0.8% sterile saline and swabbed over the entire surface of Mueller Hinton agar (Oxoid) with a sterile cotton

swab. A ring of disks of each (Mast Diagnostics, UK) containing single concentrations of each antimicrobial agent was

then placed onto the inoculated surface. After overnight incubation at 37°C, clear zones of inhibition was produced by

bacterial growth and these was measured in mm using a straight line ruler. Reading of the diameter of the zone of

inhibition was done by using an interpreting chart for zone sizes and interpreted as susceptible, intermediate, and resistant

(CLSI, 2013).

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2.5. Data Management and Analysis

Different statistical models were employed to analyze the data collected using STATA version 11 software. Descriptive

statistics was used to describe the frequency and percentage of the results. Chi-sequare test was used to check the

association of potential risk factor with the occurrence of the organism. The association was taken as significant when p-

value is less than 0.05 and not significant when p-value is greater than 0.05. Antimicrobial susceptibility test result was

analyzed using WHONET version 14.0.

3. RESULTS

3.1. Isolation and Identification of Salmonella

Isolation and identification of Salmonella organisms were conducted on cloacal swab samples taken from chicken flocks

using conventional culture methods, Biochemical analysis and PCR technique.

3.2. Culture and Biochemical Characteristics

Typical Salmonella colonies with a slightly transparent zone of reddish color and a black center were observed on XLD

agar medium in an overnight culture (Fig.1). From a total of 196 cloacal swabs collected, 50 (25.5%, 50/196) were found

to be positive for Salmonella organisms by culture, and 15 (7.6%) Salmonella isolates were confirmed using biochemical

tests (Table 2 and Fig.2). All Salmonella confirmed isolates were checked for their motility and have been found to be

motile.

Figure 1: Salmonella Culture on XLD agar medium Figure 2: Biochemical test results; A) TSI B) LIA

C) Urease test D) Citrate

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Table 2: Result Summary on Biochemical Characteristics of Salmonella Isolates

Sample TSI LIA Citrate Urease

Slant/Butt H2S Gas

1 K/A + + + +

2 K/A + + + +

3 K/A + − + +

4 K/A + − + +

5 K/A + + + +

6 K/A + − + +

7 K/A + + + +

8 K/A + + + +

9 K/A + − + +

10 K/A + + + +

11 K/A + + + +

12 K/A + − + +

13 K/A + − + +

14 K/A + + + +

15 K/A + + + +

3.1.2. Polymerase Chain Reaction (PCR)

All culture and biochemical positive samples were further confirmed by PCR through amplification of histidine transport

operon as a target gene for the presence of Salmonella isolates. Gel electrophoresis of the PCR product on 1% agarose gel

revealed the presence of 496bp segments in 13 (6.6%) Salmonella isolates out of 15 culture and biochemical positive

samples (Fig.2).

Figure 3: Polymerase chain reaction for detection of Genus Salmonella

Lane M: 100bp ladder; Lane 1, 3-10, 12-15: Positive isolates (496bp); Lane 2 and 11: Negative; Lane 16:

Negative control

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3.3. Risk Factors and Existence of the Organism

In this study, the occurrence of the organism has been significantly associated with different age groups of chickens (X2 =

10.56; P = 0.005) (Table 3) and between farms (X2 =10.74; P=0.013) (Table 4). The highest salmonella level was isolated

from the 3rd

age category (Table 3) which is 40 weeks of age (12.5%) and no salmonella was isolated in first age category.

The percentage of isolation of salmonella was increased with the advancement of age in this study. Among all different

farms of cloacal swabs analyzed, Genesis and Elere farms revealed maximum isolation of Salmonella (Table 4) whereas

Salmonella was not detected in Alema farm.

Table 3: Salmonella isolates in different age groups of cloacal swab sample.

Age category

Number of tested

PCR

No of positive (%) No of Negative (%)

45 days 60 0 (0%) 60 (100%)

26 weeks 40 1 (2.5%) 39 (97.5%)

40 weeks 96 12 (12.5%) 84(87.5%)

Total 196 13(6.7%) 183(93.3%)

X2 = 10.56 P=0.005

Table 4: Description of Salmonella in cloacal swab by their farm origin

Farm name

Number of tested

PCR

No positive (%) No Negative (%)

Alema 60 0 (0%) 60 (100 %)

Efrem 40 1(2.5%) 39 (97.5%)

Elere 36 5(13.9%) 31(86.1%)

Genesis 60 7(11.7%) 53(88.3%)

Total 196 13(6.7%) 183(93.3%)

X2 =10.74 P=0.013

The present finding revealed, the association between different breeds and sex of chickens with the existence of the

organism were not statistically significant (X2 = 3.53; P= 0.06 and X

2 =2.61; P =0.106), respectively (Table 5 and 6).

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Table 5: Description of Breed associated with Salmonella isolates

Breed type

Bovans

Number of tested

PCR

No of positive (%) No of negative (%)

60 7 (11.7%) 53 (88.3%)

Cobb 500 136 6 (4.4%) 130 (95.6%)

Total 196 13(6.7%) 183(93.3%)

X2 = 3.53 P= 0.060

Table 6: Description of sex associated with Salmonella isolates.

Sex category

Number of tested

PCR

No of positive (%) No of negative (%)

Female 125 11(8.8%) 114(91.2%)

Male 71 2(2.8%) 69(97.2%)

Total 196 13(6.7%) 183(93.3%)

X2 =2.61 P =0.106

3.4. Antimicrobial Susceptibility Test

A total of 13 isolates (6.6%) were tested against 18 antimicrobial discs following CLSI guidelines. Salmonella isolates

were found to be resistant to Sulfisoxazole, Chloramphenicol and Ampicillin and followed by Tetracycline,

Amoxicillin/Clavulanic acid and Cephalotin antibiotics (Fig. 2). In the other way, all of the Salmonella isolates had been

100 % susceptible to Amikacin, Cefoxitin, Ceftriaxone, Gentamicin, Naldixic acid, Trimethoprim/Sulfamethoxazole, and

Trimethoprim and were the only antimicrobials not resistance to any of the isolates tested (Table 7). Resistance to

Kanamicin, Ciprofloxacin, Neomicin, and Nitrofurantion was not observed although the isolates had intermediate

sensitivity to these antimicrobials.

Figure 4: Percentages of antimicrobial resistance

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Table 7: Level of antimicrobial resistance of Salmonella isolates

Code Antibiotic name Breakpoint %R %I %S %R 95%C.I.

AMC_ND20 Amoxicillin/Clavulanic

acid

14 – 17 30.8 38.5 30.8 10.4-61.1

AMK_ND30 Amikacin 15 – 16 0 0 100 0.0-28.3

AMP_ND10 Ampicillin 14 – 16 61.5 0 38.5 32.2-84.8

CEP_ND30 Cephalothin 15 – 17 23.1 38.5 38.5 6.2-54.0

CHL_ND30 Chloramphenicol 13 – 17 69.2 0 30.8 38.9-89.6

CIP_ND5 Ciprofloxacin 21 – 30 0 76.9 23.1 0.0-28.3

CRO_ND30 Ceftriaxone 20 – 22 0 0 100 0.0-28.3

FOX_ND30 Cefoxitin 15 – 17 0 0 100 0.0-28.3

GEN_ND10 Gentamicin 13 – 14 0 0 100 0.0-28.3

KAN_ND30 Kanamycin 14 – 17 0 53.8 46.2 0.0-28.3

NAL_ND30 Nalidixic acid 14 – 18 0 0 100 0.0-28.3

NEO_ND30 Neomycin 13 – 16 0 84.6 15.4 0.0-28.3

NIT_ND100 Nitrofurantoin 15 – 16 0 23.1 76.9 0.0-28.3

SOX_ND200 Sulfisoxazole 13 – 16 69.2 23.1 7.7 38.9-89.6

STR_ND10 Streptomycin 12 - 14- 0 84.6 15.4 0.0-28.3

SXT_ND1.2 Trimethoprim/Sulfametho

xazole

11 - 15- 0 0 100 0.0-28.3

TCY_ND30 Tetracycline 12 - 14- 30.8 0 69.2 10.4-61.1

TMP_ND5 Trimethoprim 11 - 15- 0 0 100 0.0-28.3

About 69.3% of Salmonella isolates were found to be multi-drug resistant as they were resistant to more than one

antimicrobial (Table 8).

Table 8: Distribution of multidrug resistance profile

Resistance profile Number of isolates Isolates (%)

AMC CEP CHL CIP 1 7.7 AMC AMP CHL CIP 1 7.7

AMC AMP CEP CHL 1 7.7

AMC AMP CCEP CHL CIP 6 46.2

Total 9 69.3

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4. DISCUSSION

Salmonella is the major cause of foodborne illness in poultry and its products, preliminary data on isolation and

identification are needed to inform public health authorities about the antimicrobial resistance and magnitude of the

problem and to monitor trends over time. Genus-specific detection of Salmonella has pronounced application to clinical

situations in which detection of Salmonella would support or confirm a clinical diagnosis and would provide important

information regarding treatment, control, and prevention. In the present study, the overall percentage of Salmonella isolates

in the total examined cloacal swabs using conventional culture and PCR method was 6.632% which is higher as compared

to the study by Kassaye et al. (2010) who reported 0.8% from cloacal swab samples in chickens and 2.6% by Mdegela et

al. (2000).

On the other way, significantly higher percentage of Salmonella isolates were reported by Yeliz et al. (2011), Alebachew

and Mekonnen (2013) and Islam et al. (2006), who reported 34% from turkey, 41.9% from Jimma and 47.7% from Tehran

respectively. This difference might be attributed to the variation of the test techniques employed (pre-enrichment steps),

the origin of sample or geographical difference and difference in management practice.

In this study, the ages of the poultries were grouped in three categories and tested for their association with the occurrence

of the organism. The highest Salmonella level was isolated from the 3rd

age category, 40 weeks of age (12.5%) and no

Salmonella was isolated in first age category, 45 days (0%). The percentage of isolation of Salmonella was increased with

the advancement of age in this study. The statistical analysis has revealed a significantly associated different age groups of

chickens (X2 = 10.56; P = 0.005). This result is in agreement with the finding of Sikder et al. (2005) who reported the

highest (30.76%) of Salmonella infection at 39 weeks of age and lowest (13.33%) at 32 weeks of age.

The occurrence of the organism has been also significantly associated with different farms (X2 =10.74; P=0.013). The

significant difference in Salmonella isolates between farms might be due to the existence of different management system

and feeding practice in different farms.

The sex specific percentage of Salmonella isolates in the present study were found to be 8.0% in females and 2.0% in

males and statistical analysis revealed there was no significant difference (X2 =2.61; P =0.106). The current finding was in

agreement with the study of Alebachew and Mekonnen (2013) in Jimma town who reported 42.2% in females and 39.1%

in males, Sikder et al. (2005) who reported 40.5% and 41% prevalence of Salmonella among chicken flocks examined in

Bangladesh. Kang et al. (2002) explained that there could not be any sexual impact on the prevalence of Salmonella

infection in male and female poultry. Even though the statistical analysis result showed no significant association between

sex groups, the slight difference in the percentage might be due to the fact that female chickens are physiologically stressed

during egg production and molting which significantly depresses the immune response of female chickens and increase the

susceptibility to Salmonella infection (Lynch et al., 2006).

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Emerging drug resistance in the foodborne bacteria isolates is of great public health concern. Although most intestinal

Salmonella infections don’t require treatment, antimicrobials may be life-saving in persons with immune suppressing

conditions or invasive illness (Lynch et al., 2006). In studies performed worldwide, there is remarkable variation in

resistance of Salmonella species from chickens to wide range of antimicrobial agents. In the present study, 6.63% of

cloacal samples were found to be infected with Salmonella and more than half of the Salmonella isolates (84.7%) tested

exhibited resistance to antimicrobials. The resistance against sulfisoxazole and chloramphenicol were found to be (69.2%)

followed by Ampicillin (61.5%), tetracyclin and Amoxicillin/Clavulanic acid (30.8%) and cephalotin (23.1%). Molla et al.

(2003) also reported (51.2%) of Salmonella strains were resistant to sulfisoxazole, (45.0%) to Amoxicillin/Clavulanic acid

and ampicillin, (41.2%) to tetracyclin and (30.0%) to chloramphenicol in their study. High resistance to penicillin (100.0%)

and (85.2%) ampicillin resistant Salmonella isolates were also reported by Yeliz et al. (2011). Resistance to tetracycline

and cephalotin were also evident in Yeliz et al. (2011) and Zhao et al. (2005).

In this study, Amikacin, Cefoxitine, Ceftriaxone, Gentamycin, Nladixic acid, trimethoprim/sulfamethoxazole, and

trimethoprim showed maximum (100.0%) antimicrobial activity against Salmonella isolates. This result is in agreement

with Molla et al. (2003) who reported all strains of Salmonella susceptible to antimicrobial effects of Naldixic acid,

Amikacin, and Gentamycin. Sensitivity of Salmonella isolates to Gentamycin has also been reported by Cardso et al.

(2006).

Multidrug resistance has been reported in many isolates of Salmonella identified from poultry farms. In the present study,

about 69.3% of Salmonella isolates were found to be multi-drug resistant as they were resistant to more than one

antimicrobial. A relatively similar finding of multidrug resistant isolates has been reported by Bayleyegn et al. (2003),

Mohammed et al. (2009) and Yeliz et al. (2011) as 63.7%, 23.5%, and 97.0 %, respectively. The presence and variation of

multidrug resistance in Salmonella isolates may be associated with time, the serovar of Salmonella, broilers versus layer,

one farm versus another and the particular antimicrobial agent (Yeliz et al,. 2011).

5. CONCLUSION

In the present study, Most of the Salmonella isolates were found to be resistant against commonly used antimicrobials such

as Sulfisoxazole, Chloramphenicol and Ampicillin followed by Tetracycline, Amoxicillin/Clavulanic acid and Cephalotin.

More than half of the Salmonella isolates were found to be multi-drug resistant as they were resistant to more than one

antimicrobial. The high prevalence of resistance to antimicrobial agents found in this study might be attributed to

uncontrolled use of antimicrobial agents as growth promoters in poultry farms which leads to resistant strains and can be

passed to human through food products. Therefore, proper treatment of chickens using appropriate antibiotics is then quite

essential.

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Competing Interests

The author(s) declare that they have no competing interests.

Author’s Contributions

Hailelule Aleme,Habtamu Tassew , Filimon Mitiku, Haile Alemayehu conceived of the study, and participated in its design

and coordination and helped to drafted the manuscript. Elsabeth Solomon carried out the Laboratory work. Kassaw

Amssalu participated in the design of the study and performed the statistical analysis. All authors read and

approved the final manuscript.

Author's Information

On behalf of the authors, I take the full responsibility for the article during submission and peer review.

Acknowledgement

The authors would like to thank the Mekelle University College of Veterinary Medicine for Financial Support

and the Aklilu Lemma Institute of Pathobiology, Addis Ababa University for unreserved assistance during data

and sample collection and Laboratory work.

REFERENCES

Alebachew, k. and Mekonnen, A. (2013): A survey on Salmonella infection among chicken flocks in Jimma town,

Ethiopia. W. Appli. Sci. J. 21 (10): 1415-1420.

Cardoso, M. O., Ribeiro, A. R., Santos, L. R., Pilotto, F., Moraes, H. L. S. and Salle, C. T. P. (2006): Antibiotic

resistance in Salmonella Enteritidis isolated from broiler carcasses. Brazilian J.Microbiol. 37(3): 368−371.

Chrysostome, C. A. A.M., Bell, J.G., Demey F. and Verhulst, A. (1995): Sero prevalence to three diseases in village

chickens in Benin. Prev. Vet. Med. 22: 257-261.

CLSI, (2013): Performance standards for antimicrobial susceptibility testing; Twenty- third informational supplement.

M100-S 23.

Cown, S.T. (1985): Cown and Steel's Manual for the Identification of Medical Bacteria. 2nd

ed. Cambridge University

Press, Cambridge, UK.

CSA, (2008): Central Statistical Agency: Report on monthly average retail prices of goods and services. Stat. Bulltn. Pp.

416.

Duijkeren, E.V., Wannet, W.J.B., Houwers, D.J., and Pelt, W.V. (2003): Antimicrobial susceptibilities of Salmonella

strains isolated from humans, cattle, pigs and chickens in The Netherlands from 1984 to 2001. J. Clin. Microbiol. 41:

3574-3578.

Gay, J.M., Rice, D.H. and Steiger, J.H. (1994): Prevalence of faecal Salmonella shedding by cull dairy cattle marketed in

Washington State. J. Food Prot. 57: 95-197.

Tadele, G., Asrade, B., Bayleyegn, G, Ali, and M. S. (2014): Sero-prevalence of Fowl Typhoid and Pullorum Disease

from Apparently Healthy Chickens in Eastern Ethiopia. J. Vetrinar. Sci. Technol. 5: 156.

14

Islam, M.M., Haider, M.G., Chowdhury, E.H., Kamruzzaman, M. and Hossain, M.M. (2006): Seroprevalence and

pathological study of Salmonella infections in layer chickens and isolation and identification of causal agents. Bangl.

J. Vet. Med. 4: 79-85.

ISO, 66579-3. (2003): Microbiology of food and animal feeding stuffs-Horizontal method for the detection and

identification of Salmonella Part 3: Geneva, Switzerland.

Kang, H., Srinivasan, J. and Curtiss, R. (2002): Immune response to recombinant pneumococcal PspA antigen delivered

by live attenuated Salmonella Typhimurium serovar vaccine. J. Infect. Immun. 4: 1739-1749.

Kassaye, A., Lencho, T. and Mesele, A. (2010): Prevalence of Salmonella infection in intensive poultry farms in

Hawassa and isolation of Salmonella species from sick and dead chickens. Ethio. Vet. J. 14(2): 115-124.

Lauderdale, T.L., Aarestrup, F.M., Chen, P.C., Lai, J.F., Wang, H.Y., Shiau, Y.R., Huang, I.W. and Hung, C.L. (2006):

Multidrug resistance among different serotypes of clinical Salmonella isolates in Taiwan. Diagno. Microbiol. Infect.

Dis. 55: 149-155.

Lobago, F. and Weldemeskel, M. (2004): An outbreak of Marek’s disease in central Ethiopia. Trop. Anim. Hlth. and

Prod. 36: 397-406.

Lobago, F., Nigussie, D., Wossene, A. and Ashenafi, H. (2003): Study on the majour diseases of chickens in Debre Zeit,

Central Ethiopia. Bullet. Anim. Hlth. Prod. Afr. 51: 11-12.

Lynch, M., Painter, J., Woodruff, R., Braden, C. and Centers for disease control and prevention. (2006): Surveillance for

foodborne-disease outbreaks United States, 1998-2002. MMWR Surveill Summ. 55(10): 1-42.

Mandal, S., Mandal, M.D. and Pal, N.K. (2004): Reduced minimum inhibitory concentration of chloramphenicol for

Salmonella enterica serovar Typhi. Ind. J.Med. Sci. 58: 16-23.

Mdegela, R.H., Yongolo, M.G.S., Minga, U.M. and Olsen, J.E. (2000): Molecular epidemiology of Salmonella

gallinarum in chickens in Tanzania. Avian Pathol. 29: 457-463.

Medina, E., Fanos T., Mesula G., Teferi D. and Tariku J. (2013): Sero and media culture prevalence of Salmonellosis in

local and exotic chicken, Debre Zeit, Ethiopia. African J. Microbiol. Res. 7(12): 1041-1044.

Melesse, A., Maak, S. and vonLengerken, G. (2005): The performance of naked neck and their F1 crosses with Lohmann

White and New Hampshire chicken breeds under long-term heat stress conditions. Ethio. J. Anim. Prod. 5 (1): 91-106.

Melesse, A., Tiruneh, W. and Negesse, T. (2011): Effects of feeding Moringa stenopetala leaf meal on nutrient intake

and growth performance of Rhode Island Red chicks under tropical climate. Trop. Subtrop. Agroecosyst. 14: 485-492.

Moges, F., Melesse, A. and Dessie, T. (2010): Assessment of village chicken production system and evaluation of the

productive and reproductive performance of local chicken ecotype in Bure district, North West Ethiopia. Afr. J. Agric.

Res. 5(13): 1739-1748. Mohammad, M.S.D., Mahnaz, T., Latif G., Shabnam M., Mariam, S., Rounak B., Mohammad K.S.Y. and Mohammad

R.Z. (2009): Characterization of antibiotic resistant patterns of salmonella serotypes isolated from beef and chicken

sample in Tehran. Jundi. J. Microbiol. 2(4): 124-131.

Molla, B., Mesfin. A. and Alemayehu, D. (2003): Multiple antimocrobial-resistant Salmonella serotypes isolated from

chicken carcass and giblets in Debre Zeit and Adis Ababa, Ethiopia. Ethio. J. Hlth Develop. 17 (2): 131-149.

Nwanta, J. A., Egege , S .C., Alli-Balogun, J. K. and. Ezema, W. S. (2008): Evaluation of prevalence and seasonality of

Newcastle disease in chicken in Kaduna, Nigeria. World. Pltr. Sci. J. 64: 416- 425.

OIE, (2008): Fowl typhoid and pullorum disease. In: Terrestrial Manual. Office International des Epizooties (OIE), Paris,

France. Pp. 538-548.

Poppe, C., Martin, L., Muckle, A., Archambault, M., McEwen, S. and Weir, E. (2006): Characterization of antimicrobial

resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada.

Canad. J. Vet. Res. 70: 105-114.

Rychlik, W. and Rhoades, R.E. (1989): A computer program for choosing optimal oligonucleotides for filter

hybridization, sequencing, and in vitro amplification of DNA. Nucleic Acids Res. 17: 8543. 8551.

Sato, Y., Sato, G., Tuchili, L., Pandey, G.S., Nakajima, A., Chimana, H. and Sinsungwe, H. (1997): Status of Salmonella

Gallinarum-Pullorum infections in poultry in Zambia. Avian Dis. 41: 490-495.

Sikder A, Islam M, Rahman M. and Rahman M. (2005): Seroprevalence of Salmonella and Mycoplasma gallisepticum

infection in the six model breeder chicken flocks farms at Patuakhali District in Bangladesh. Int. J. Chicken flocks Sci.

4(11): 905-910.

15

Wegner H.C. and Aarestrup, F.M., Gerner-Smidt P. and Bager, F. (1999): Transfer of antibiotic resistant bacteria from

animals to man. Acta. Vet. Scand. Suppl. 92: 51-57.

Weill, F.X., Guesnier, F., Guibert, V., Timinouni, M., Demartin, M., Polomack, L. and Grimont, P.A.D. (2006):

Multidrug resistance in Salmonella enterica serotype Typhimurium from human in France (1993-2003). J.

Clin.Microbiol. 44: 700-708.

Yeliz, Y., Zafer, G., Sebnem, P. and Nurhan, E. (2011): Incidence and antibiotic resistance of Salmonella spp. on raw

chicken carcasses. Food. Res. Inter. 44: 725–728..

Zeleke, A., Sorri, T., Gelaye, E. and Ayelet, G. (2005): Newcastle disease in village chickens in the southern and rift

valley district in Ethiopia. Inter. J. pltr. Sci. 4(7): 507-510.

Zhao, S., Fedorka-Cray, P. J., Friedman, S., Mcdermott, P. F., Walker, R. D., Qaiyumi, S., et al. (2005): Characterization

of Salmonella Typhimurium of animal origin obtained from the National Antimicrobial Resistance Monitoring

System. Foodborne Pathog. Dis. 2: 169−181.