DETECTION AND IDENTIFICATION OF SALMONELLA ISOLATED FROM CHICKENS BY POLYMERASE CHAIN REACTION (PCR)

Assiut Provincial Laboratory,

Animal Health Research Institute.

 

Detection and identification of Salmonella isolated from chickens by

polymerase chain reaction (PCR)

(With 2 Tables and 9 Figures)

 

By

Fatma M. Mohamed; M.A. Mohamed*;

Nabawia M. shata and A.M.A. Manaa

* Dept. Poultry Diseases, Fac. of Vet. Med., Assiut University, Assiut, Egypt

(Received at 4/8/2009)

 

الکشف والتعرف على معزولات السالمونيلا من الدجاج بإستخدام

إختبار البلمرة

 

فاطمة مختار محمد ، مؤمن عبد العظيم محمد ، نبوية محمود شحاتة

أحمد ممدوح مناع

 

يعتبر ميکروب السالمونيلا من الميکروبات الخطيرة لأنة أحد مسببات التسمم الغذائى للانسان والحيوان والطيور. أجريت هذه الدراسة لمعرفة نسبة تواجد السالمونيلا فى الدجاج فى محافظة أسيوط، وکذلک الأختلافات الجينية بين المعزولات المختلفة باستخدام اختبار البلمرة. وقد شملت الدراسة التعرف على التغيرات الباثولوجية التى حدثت على الانسجة المختلفة فى الدجاج المعدى صناعيا. تم فحص ۲۰۰ عينة من الأعضاء الداخلية للدجاج، کما تم عمل أختبار حساسية لمعرفة أفضل علاج للميکروب. أوضحت نتائج العزل البکتيريولوجى وجود عدد ٢٨ عترة من ميکروب السالمونيلا بنسبة ١٤٪. وللتفريق بين أنواع السالمونيلا  المعزولة أستخدمت تقنية تفاعل البلمرة المتضاعف من النوع المتعدد  m-PCR بالأستعانة ببادئات خاصة للاکتشاف النوعى للأنتيجين الجسدى والسوطى لميکروب السالمونيلا. أظهرت النتائج وجود السالمونيلا تيفيميوريوم بنسبة 57.14% والسالمونيلا أنتريديس بنسبة 42.86%. وعن طريق أختبار الحساسية تم معرفة أن الميکروب حساس للينکوسباکتين, کلورافنيکول, أريثرومايثين, سيبروفلوکساسين، ديوکسسيکلين, أسبکتينوميسين, کولستين, امبسلين, اموکسيسلين, حمض الکلاافيولنيک, والجنتاميسين. کما أظهرت الدراسة الباثولوجية وجود سمک فى جدار الأوعية الدموية وتورم فى الرئتين. هذا بالإضافة إلى وجود إرتشاح من الخلايا وحيدة النواة فى العضلات القلبية، أيضا تورم وإرتشاح خلوى بالکبد.

 

Summary

 

The occurrence and identification of Salmonella spp. using a multiplex PCR (m-PCR) in chickens, as well as histopathological changes in experimentally infected chickens and antibiotic sensitivity test were studied. 200 samples (intestine, liver) were collected from freshly dead bird for bacteriological examination. 28 Salmonella isolates were found in chicken samples with overall percentage 14%, by conventional culturing and biochemical reactions. Seven representing isolates were confirmed by a multiplex-PCR (m-PCR) using the three primers pair (ST11-F, ST15-R), (S1-F, S4-R) and Fli15-F, Fli15-R for identification of the most frequent Salmonella enterica serovars. Our results revealed that isolation rate of serovar Enteritidis was 57.14% (16/28) and 42.86% (12/28) were identified as serovarTyphimurium. Histopathologically of the experimentally infected chicken showed thickening of the alveolar wall, blood vessel wall and edema in the lung. Moreover, mononuclear cell infiltration in cardiac muscles and hepatic edema with heterophil cells infiltration in infected liver was also seen. The current study demonstrated that all of 28 Salmonella strains were susceptible to lincospectin, chloramphenicol, erythromycin, ciprofloxacin, doxycyclin, spectinomycin, colistin but were resistant to ampicillin, amoxicillin/clavulanic acid and gentamycin.

 

Key words: Salmonella, Chickens, Multiplex-PCR, Antibiotic Resistance.

 

Introduction

 

Salmonella enterica is one of the most important leading pathogens of food-borne illness throughout the world that pose a significant health hazard to human. Infected poultry are the most frequently incriminated reservoirs of Salmonellae that can be transmitted through the food to human (Clavijo et al., 2006; Humphrey, 2006). Food-borne diseases caused by Salmonella serotypes occur at high frequency in industrialized nations and developing countries and represent an important public health problem worldwide (White et al., 2001; Lampel et al., 2000). Salmonellaenterica subsp. enterica serovar Typhimurium are the major dominating serotypes of Salmonella in poultry and poultry product (Young, et al., 2003; Gürakan et al., 2008). Salmonella spp. is one of the most important pathogens responsible for gastrointestinal infections in human, poultry and its derivatives being one of the best known sources of contamination. An increase of strains showing individual and multiple resistance against different antibiotics have been found from isolates from pigs, poultry, and cattle in recent years(Esaki et al., 2004). Standard culture methods for detecting Salmonella spp. in poultry include non-selective pre-enrichment followed by selective pre-enrichment followed by selective and differential agars (Whyte et al., 2002). These methods take approximately 4-7 days (Harvey and Price, 1979; Perales and Audicana, 1989). Since Salmonella is closely related to both public and animal health, more rapid and sensitive methods for the identification of this bacterium are required (Schrank et al., 2001). Several alternative, faster methods for the detection of Salmonella have been developed, the use of the polymerase chain reaction (PCR) being one of the most promising approaches (Candrian, 1995; Scheu et al., 1998). Rapid identification methods are based on genomic amplification techniques using distinct target DNA sequences determined by PCR. Recently, specific identification of S.enterica serovars Typhi and Paratyphi A by multiplex PCR, which detects rfbE, rfbS,viaB, and fliC genes, has been reported, and this method correctly identify S. enterica serovars Typhi and Paratyphi A and differentiate these from other Salmonella serovars that have similar antigenic structures (Hirose et al., 2002; Young et al., 2003).

The aim of this study:

1- Determination of prevalence rate of Salmonella infection among broiler chickens in Assiut.

2- Using a multiplex PCR method for Salmonella enterica serovars identification.

3- Histopathological changes in intestine and liver post infection with Salmonella.

4- Antibiotic sensitivity test to select the best treatment of Salmonella infections in chickens.

 

Materials and methods

 

Sample collections:

Two hundreds samples from internal organs (intestine and liver) were collected from diseased broiler chickens of different farms that were necropsied at Department of Poultry Diseases, Faculty of Vet. Med., Assiut University from different localities in Assiut province. The selected birds showed diarrhea, vent pasting and stunting in forty five days chickens. The post mortem examination of all the cases was performed for the all dead birds. At necropsy, gross lesions were recorded carefully, and representative tissue samples from clinically diseased birds and experimental infected birds containing lesions were fixed in 10% neutral buffered formalin for histopathological studies.

Culture procedure:

Samples were aseptically cultured into selenite F broth (Oxoid) and incubate at 41°C for 18-24 hours. Subsequently, a loop full of each broth was streaked on surface of MacConky agar, Salmonella Shigella agar (S.S. agar) and Xylose Lysine Desoxycholate agar (X.L.D. agar) for further incubation at 37°C for 24 h (FDA, 1992).

Bacteriological method for Salmonella identification:

Salmonella-typical colonies on the plates were confirmed with standard biochemical tests and procedures for Salmonella spp. (Rotger and Casadesús, 1999). Presumptive Salmonella colonies were kept at -70°C brain heart infusion broth (Difco) with the addition of 20% (v/v) glycerol. An aliquot of this storage solution was taken and incubated in 5 ml phosphate-buffered peptone water for 24 h at 37°C prior to multiplex PCR for further confirmation.         

Salmonella Molecular identification

Genomic DNA extraction

The liquid cultures were centrifuged at 8,700 x g for 15min, and the cell pellets were re-suspended in 1ml of sterile water. The re-suspended cells were re-centrifuged at 12,500 xg for 15min. The pelleted cells were then used for DNA extraction as followed by manufacturer instructions for QIAamp DNA miniprep kit and the concentration was determined using ultraviolet spectrophotometer at A₂₆₀.

Subtyping of Salmonella serotypes using multiplex-PCR (m-PCR):

To develop a multiplex-PCR for Salmonella serotypes identification, three pairs of primers were designed from published primer sequences as shown in (Table 1). The multiplex PCR reaction contained 5 µL of template DNA, 0.4 µM of each primer, 100 µM of each deoxynucleotide triphosphate, 1.25 mM of MgCl2 (25 mM), 2.5 U of Taq Polymerase, 5 µL of 10X PCR Buffer (Promega Corp., MI, USA), and water to bring the final reaction volume to 50 µL.

PCR was performed in a Techne Cyclogene PCR System thermocycler. The temperature program started with a PCR amplification was as follows: one initial denaturation cycle at 95 ◦C for 5 min, followed by 30 cycles of 95 ◦C for 40 s, 58 ◦C for 20 s and 72 ◦C for 20 s, and one final extension cycle of 72 ◦C for 7 min. Finally, the samples were cooled to 4°C. Fragments were separated in 1% agarose gel by unidirectional electrophoresis and visualized by staining with ethidium bromide. Fragment sizes were determined by comparison with a 1KB plus DNA ladder (Invitrogen).

 

Histopathology:

The tissues were collected from experimentally infected birds then subjected to fixation in neutral buffered formalin, trimming, washing and dehydration in ascending grades of ethanol, clearing in methylbenzoate and embedding in paraffin. 5mm thick sections were cut, stained with hematoxylin and eosin (Habib-ur-Rahman, et al, 2003).  Sections were examined in the Department of Pathology, Faculty of Veterinary Medicine, Assiut University.

 

Antibiotic sensitivity test:

Salmonella-typical colonies on the plates were culture into brain heart infusion broth over night at 37°C and culture fluently over the entire surface of nutrient agar (Difco) with sterile cotton swab. Commercial antibiotic disks containing single concentrations of each antibiotic were then placed on to the inoculated plate surface. The zone of growth inhibition around each disk after over night incubation at 37°C, were measured in millimeters. The zone diameter was interpreted using a zone size interpretation chart (Lorian, 1996).The antibiotics and their concentration were as follow. lincospectin, chloramphenicol 30µg, erythromycin 10µg, ciprofloxacin 5µg, doxycyclin 30µg, spectinomycin 10µg, colistin 25µg, enrofloxacin 5µg, ampicillin 10µg, amoxicillin/ calvulanic acid 10µg, and gentamycin 10µg (Quinn et al., 1994). 

 

Results

 

Isolation of Salmonella from the examined samples:

The isolation trials of Salmonella from collected samples yielded overall isolation rate 14% (28/200) of the examined birds. Using biochemical reactions the highest isolation rate of the 28 Salmonella isolates, 16 (57.14%) were identified as serovar Enteritidis, while 12 (42.86%) were identified as serovarTyphimurium. Of the above mentioned 28 isolates, 7 representing ones were used to confirm identification using multiplex PCR. Of these 7 isolates 4 were identified as serovar Enteritidis, while 3 were identified as serovarTyphimurium (Fig. 1).

 

Specificity of the multiplex polymerase chain reaction:

For multiplex PCR, 3 primer pairs were used, (ST11-F, ST15-R), (S1-F, S4-R) and (Fli15-F, Fli15-R). The results revealed that no amplification could be observed among non-Salmonella strains as a negative control, but a specific amplification (429bp) could be detected in all Salmonella strains for the genus Salmonella, whereas             (Fli15-F, Fli15-R) primer pair could amplify a 650bp fragment among the tested strains of S. Typhimurium only. However, (S1-F, S4-R) primer pair could amplify a 250bp fragment, examined strains of S. Enteritidis (Table2).

 

Pathology:

Gross examination of the infected birds showed marked lesions in many organs. The lung appeared severely congested (Fig.2). The intestinal and cecal walls were congested (Fig.3). Liver was enlarged with dark red discoloration and friable consistency (Fig.4). Hemorrhages were also demonstrated at the junction between proventriculus and gizzard (Fig.5).

            The aim of histopathological examination is detect the pathological action of the Salmonalla microorganisms on different tissues and organs. Our results revealed severes acute systemic lesions in almost all body parts. H&E stained sections of intestine exhibited mucosal epithelial cell necrosis, the submucosal blood vessels appeared dilated, engorged with blood with edema in their walls. Extravascular red blood cells (RBCS) could be seen mixed with infiltration of a granular mononuclear cells and heterophils in the submucosa. Hepatic lesions were characterized by multifocal necrosis of hepatocytes and vascular congestion (Figs 6 & 7). The lung revealed thickening of the alveolar wall due to massive infiltration with heterophils and lymphocytes. Degenerated alveolar wall were fused forming emphysemas (Fig.8). Cardiac muscles demonstrated heterophilic and mononuclear cell infiltration (Fig.9).

 

Antibiotic sensitivity test:

All 28 Salmonella isolates were susceptible to the antimicrobial effect of lincospectin, chloraphenicol, erythromycin, ciprofloxacin, doxycyclin, spectinomycin, colistin, but they were resistant to ampicillin, amoxicillin/clavulanic acid and gentamycin.

 

Table 1: Primers used in this study for multiplex-PCR identification of the most frequent Salmonella.

 

(1)     Salmonella sp., (2) S. entertidis, (3) S. typhimuriym

 

Table 2: Specificity of the multiplex PCR

 

Strains	Number of strains	PCR-positive results by m-PCR with amplified products of
		429 pb	620 pb	250 pb
S. Enteritidis	3	3	0	3
S. Typhimurium	4	4	4	0

 

 

 

 

 

 

 

 

 

Fig. 1: Mutltiplex PCR amplification from various suspected Salmonella strains, S. Typhimurium lanes (1,2,3,6) and S. Enteritidislanes    (4 and 5), M, 1KB plus DNA ladder (Invitrogen).

 

 

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LEGENDS

 

Fig.2: Lungs of infected chickens showing different stages of pneumonia 

Fig.3: Intestine of infected chickens showing focal necrotic intestinal lesions.

Fig.4: Liver and heart of infected chicken showing perihepatitis and pericarditis.

Fig.5: Hemorrhages in the junction between proventriculus and gizzard (arrow). 

Fig.6: Liver of infected chicken showing congestion and oedema with heterophil cell infiltration.

Fig.7: Lung of infected chicken showing effusion and thickening of the degenerated alveolar wall due to massive heterophilic and mononuclear cell infiltration.

Fig.8: Lung of infected chicken showing septal and prevascular edema. 

Fig.9: Cardiac muscles showing heterophilic and mononuclear cell infiltration.

 

Discussion

 

Salmonella enterica are recognized as major food-borne pathogens in the world (Gatto et al., 2006). The predominant serotypes change over time and differ from one geographical area to another. All over the world, as well as in our country, the most often isolated serotype is S. Enteritidis (Gatto et al., 2006). In this study, the Salmonella detection rate was the highest (14%) in chicken carcasses. These results are in general agreement with previous results that Salmonella in poultry products were generally low and ranged from 8% in Albania (Beli et al., 2001), 12% in turkey (Ozbey and Ertas, 2006), 23-34% in Belgium (Uyttendaele et al., 1998, 1999), 25% in United Kingdom (Plummer et al., 1995), 26% in Ireland (Duffy et al., 1999), 36% Malaysia (Rusul et al., 1996) to 43% in USA (Bokanyi et al., 1990). However, Antunes et al. (2003) mentioned that poultry samples are frequently contaminated with Salmonella (60%) which belong to 10 different serotypes. The controversy in the results could be attributed to several factors such as differences in origin, time period and age of the diseased birds, sampling procedure, and differences in methodology applied to detect the pathogen (Bryan and Doyle, 1995; Uyttendaele       et al., 1999). 

The isolation trials adopted in the current study yielded that the overall isolation rate was 14% (28/200) of the examined birds. Using biochemical reactions, the serovar Enteritidis was identified from 16/28 with a percentage 57.14%, while 42.86% (12/28) were identified as serovarTyphimurium. These results simulates recent reports from England and Spain that S. Enteritidis and S. Typhimurium are the most frequent serotype with incidences of 60% and 86%, respectively (Gatto et al., 2006).

A Multiplex PCR-based assay (m-PCR) with three sets of primers was developed for the detection of the most common serotypes of S. enterica as S. Enteritidis and S. Typhimurium. This is a very rapid and simple molecular method for serotyping common Salmonella, the specific sequence could be detected in all Salmonella enterica serotypes. The time for serotyping is dramatically reduced to only 5 hrs. The method is basic and does not need specialized staff and a large collection of antisera. The assay may be applied in any clinical facility which has PCR and electrophoresis equipment.

In accordance with (Hirose, et al, 2002) a multplix PCR gave a positive result for all Salmonella strains yielding a specific fragment of 429 bp at the genus level and an additional distinct 250 bp amplified product of sefA, which allowed identification of S. Enteritidis, whereas an additional 620-bp amplified band was observed only in samples of S. Typhimurium serotype. Random amplified polymorphic DNA analysis with primer 3 is of potential use as a serotype-specific marker for S. Typhimurium, (Gürakan et al., 2008).

Our result reveals that all the 28 Salmonella isolates were susceptible to the antimicrobial effect of lincospectin, chloramphenicol, erythromycin, ciprofloxacin, doxycyclin, spectinomycin, colistin, but they were resistant to ampicillin, amoxicillin/clavulanic acid and gentamycin. This is partially consistent with results of (Zahraei Salehi et al., 2005) that 30 Salmonella strains were susceptible to the antimicrobial effect of cefotaxime, tylosin, colistin, ciprofloxacin, enrofloxacin, gentamycin, chloramphenicol, cephalotin and cefotaxime and resistent to trimethoprim, nalidixic acid, flumequine, tetracycline, neomycin, streptomycin, kanamycin and amikacin. However our results disagrees with the findings of Kristiansen et al. (2003) that Salmonella Typhimurium DT104, is resistant to streptomycin, chloramphenicol, amoxicillin, sulfonamides, and tetracyclines.

Both gross and histopathological lesions of Salmonella infections in chicken are a great diagnostic tool besides bacteriological studies (Dhillon et al., 2001). The gross lesions demonstrated in the current study are in a general agreement with those of Talha et al. (2001), Habib-ur-Rahman et al. (2003) that include hepatic enlargement,  hepatic and intestinal congestion besides dark red discoloration and fleshy consistency of the lung. Histopathologically, the present observations support previous studies (Talha et al., 2001; Habib-ur-Rahman et al., 2003; Haider et al., 2004)and are expressed by congestion and hemorrhages with infiltration of mononuclear cells in the intestinal mucosa and submucosa, myocarditis, extravasated blood aggregates and edema. These pathological lesions could be attributed to the direct action of Salmonella which is a facultative intracellular microorganism (Buxton and Jean, 1963; Ruby et al., 2003).   

 

Acknowledgements

 

 The authors would like to acknowledge the head of the Department of Poultry Diseases for the facilities offered to complete this work. We also would like to thank all technical staff in the Laboratory of Poultry Diseases in the Department of Poultry Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt.

 

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