ANTIGENIC VARIATIONS BETWEEN DIFFERENT SALMONELLA SEROTYPES ISOLATED FROM CHICKENS

Dept. of Res. and Diag. of Poultry Diseases,

Animal Health Research Institute, Dokki, Giza, Egypt

Antigenic Variations Between Different Salmonella Serotypes Isolated From Chickens

(With 6 Tables and 2 Photos)

By

Jihan M. Badr and Hoda abd El Monem*

*Dept. of Biotechnology, Animal Health Research Institute,

Dokki, Giza, Egypt

(Received at 13/2/2008)

الاختلافات الأنتيجينية بين عترات السالمونيلا المختلفة المعزولة من الدجاج

جيهان مصطفى بدر ، هدى عبد المنعم

باجراء الفحص البکتريولوجى لعينات مختلفة من 750 حالة مرضية من الدجاج فى مختلف مراحل النمو أمکن عزل 42 معزولة من السالمونيلا (5,75 %). وعند التصنيف السيرولوجى لمعزولات السالمونيلا تبين أنها تنتمى الى سالمونيلا انتريتيدس (20 معزولة) , سالمونيلا تيفيميوريم (13 معزولة) , سالمونيلا انفانتيس (5 معزولات) , سالمونيلا مونتيفيديو (3 معزولات) وسالمونيلا سيرو (معزولة واحدة). وأوضحت اختبارات الحساسية المعملية لمعزولات السالمونيلا المختلفة تعدد مقاومة کل منها لتأثيرواحد أو أکثر من مضاد حيوى من المضادات الحيوية المستخدمة. کذلک أثبتت اختبارات العدوى الأصطناعية للعترات المختلفة فى الکتاکيت عمر يوم ضراوتها جميعا بدرجات متفاوتة باختلاف نوع العترة وکذلک ثبت ايجابية العترات المعزولة لاختبار الإرتباط بصبغة الکونجو الحمراء وذلک للتمييز بين العترات الممرضة والغير ممرضة. وقد أوضح التحليل الکهربائى للعترات المختلفة باستخدام طريقة س د س فصل 6-10 حلقات بروتينية مختلفة تتراوح بين 13,631 الى 200,5 کيلو دالتون وذلک حسب نوع العترة. کما أوضح اختبار الطبع المناعى لبروتينات الغشاء الخارجى لميکروبات السالمونيلا أنها تشترک فى العديد من الحلقات ذات الخصائص المناعية عند 15, 29, 35  ک.د. وکانت أعلى الحلقات البروتينية المناعية للسالمونيلا تيفيميوريم عند 35.4 ک.د., بينما کانت في السالمونيلا انتريتيدس عند 29  ک.د., وفى السالمونيلا مونتيفيديو عند 39.8 ک.د., وفى السالمونيلا انفانتيس عند 25.128ک.د , 34.091 ک.د. بينما فى السالمونيلا سيرو کانت أعلى حلقة بروتينية مناعية عند 143.08 ک.د. وفى المجمل, فقد ثبت أن استخدام کل من التحليل الکهربائى واختبار الطبع المناعى يعتبر من التقنيات الحديثة التى تتيح التشخيص السريع والدقيق للاصابة بالسالمونيلا فى الدجاج وکذلک يمکن من خلالها التعرف على الحلقات الأنتيجينية لعترات السالمونيلا المختلفة مما يمکن من أستخدامها فى أنتاج لقاح للوقاية من الاصابة بالسالمونيلا فى الدجاج.

ٍٍSUMMARY

Examination of different samples from 750 chicken cases of various growth stages, revealed the isolation of 42 Salmonella isolates (5.75%). The isolated salmonellae were serotyped as Salmonella enteritidis       (20 isolates), Salmonella typhimurium (13 isolates), Salmonella infantis (5 isolates), Salmonella montivideo (3 isolates) and Salmonella cerro    (1 isolate). Antibiogram of the isolated Salmonellae indicated multidrug- resistance to one or more than of the tested antimicrobial agents. Pathogenicity tests in one-day old chicks proved the virulence of all examined serovars with various degrees of pathogenicity and all were positive for congo red activity. SDS-PAGE protein analysis of different serovars revealed 6-10 protein bands ranged from 13.631-200.5 KD, which in relation to the isolated Salmonella serovar.  Immunoblotting of the isolated serovars revealed the presence of common protein bands at 15, 29 and 35 KD. The highest antigenicity protein band of S. typhimurium was detected at 35.4 KD, while in S. enteritidis was detected at 29KD; in S. montivideo at 39.8 KD; in S. infantis at 25.128 and 34.091 KD, while S. cerro had the highest antigenic protein band at 143.08KD. In conclusion, SDS-PAGE analysis and immunoblot provide a recent and accurate techniques for detection of salmonellosis in chickens, in addition to offer the use of immunogenicity of different detected immunogenic bands to serve as components of an effective subcellular vaccine for poultry salmonellosis.

Key words: Chickens, Salmonella, antimicrobial agents

INTRODUCTION

In the last decades, poultry and poultry products have been the main source of non-host specific Salmonella infecting humans (Shahata, 1979, Abd El-Hamid et al., 2004 and Murugkar et al., 2005). Poultry are commonly infected with a wide variety of Salmonella serovars and there has been considerable variation in the occurrence of the most common Salmonella serovars in domestic fowls in different countries and at different times.The outer membrane protein analysis has proved to be useful technique in characterization of Salmonella (Fadl et al., 2002 and Ochoa-Reparz et al., 2004). On the practical basis, detection of flock infections remains one of the most serious unsolved problems in controlling salmonellosis in poultry. Serological studies of different salmonellae revealed the presence of cross reaction between Salmonella organisms and other members of family Enterobacteriaceae such as Escherichia coli and Proteus spp., due to the presence of a common antigen (Le-Minor et al., 1982). Thus, increased interest for the control of salmonella in poultry requires the development of improved detection methods. Western immunoblotting is a convenient, sensitive specific technique for the detection of antigen and antibodies (Kim and Nagaraja, 1991). The purpose of this research work was to determine the incidence of different Salmonella serovars isolated from chickens at different stages of growth with reference to their protein analysis using SDS-PAGE as well as to study the virulence pattern of different serovars in relation to the antigenic variation between them using immunoblotting technique.

MATERIALS and METHODS

Chicken specimens:

Samples from liver, spleen, intestines (ceci and cecal tonsils), yolk sacs and bone marrows were collected under complete aseptic conditions from 750 chicken cases at different growth stages (chicks, broilers and parents) either dead (180 cases) or living ailing (550 cases) in the period from January 2006 up to March 2007. The samples were submitted to Bacteriological Unit of the Department of Diagnosis and Research of Poultry Diseases, Animal Health Research Institute, Dokki, Giza, Egypt, to be examined bacteriologically for the isolation of different salmonellae.

Isolation and identification of Salmonella from chickens

Isolation of different salmonellae from chicken samples was carried out according to Mallinson and Snoeyenbos (1994). Suspected colonies were identified morphologically, culturally and biochemically according to Holt et al., 1996 and Collier et al., 1998). Serological identification was carried out by slide agglutination test using polyvalent and monovalent [O] and [H] Salmonella antisera according to Kauffmann-White scheme described by Kauffmann (1974).

Antibiogram of the isolated salmonellae: In-vitro susceptibility testing of Salmonella isolates to various antimicrobial agents was determined using NCCLS method (NCCLS, 2003). Ten different commercial antibiotic discs (Oxoid) were used.

Pathogenicity tests of the isolated Salmonella serovars:

1-Pathogenicity in one-day old chicks:

Six groups (1 to 6) each of twenty, one-day old chicks which proved to be salmonella–free, were used for pathogenicity testing of various isolated Salmonella serovars. Each group was divided into two sub-groups A and B. Chicks in sub-groups A were infected orally with 3X10⁸ CFU (colony forming units) of one of the isolated serovars namely: Salmonella enteritidis, Salmonella typhimurium, Salmonella infantis, Salmonella montivideo and Salmonella cerro. Chicks in subgroups B were inoculated intrapretoneally with one of the same serovars at the same dose, while the 6^th subgroups were given 0.5 ml of sterile saline solution orally and intrapretoneally, respectively and were saved as non infected control. All chicks in different groups were kept separately and monitored for clinical signs and mortality for 14 days post-infection (Bakshi et al., 2003). Postmortem examinations accompanied by re-isolation of the infected microorganisms from internal organs of dead chicks in different groups were attempted.

2- Congo red (CR) test: All Salmonella isolates were tested for its growth status on congo red medium modified according to Berkhoff and Vinal (1986).

SDS-PAGE technique: Salmonella antigens were prepared after Ahmed et al., (1998).  Protein of various Salmonella serovars was separated by SDS-PAGE using the discontinuous buffer system described by Laemmli (1970).

Western blotting:

A- Preparation of Salmonella antisera: New Zealand white rabbits were used to produce antisera against different isolated salmonella serovars according to Kim and Nagaraja (1991).

B- Application of Western immunoblotting: To identify proteins specific for each of the isolated Salmonella serovars, Western blotting was done using the prepared salmonella antisera according to the procedure described by Talbot et al., (1984) The outer membrane proteins (OMPs) of different salmonella serovars were separated by SDS-PAGE by the method described by Laemmli (1970) and  were electrophoretically transferred to a nitrocellulose filter using transphor electrophoresis unit cell with electroblotting buffer containing 25mM Tris, 192 mM glycine, and 20% methanol, pH 8.3. The nitrocellulose filter strips were stained by Ponceau S red staining (Sigma), to check the transferred proteins and destained in distilled water. The strips were then immersed in Tris-buffered saline (TBS) containing 20 mM Tris, 500 mM NaCl, and 3% gelatin, pH 7.5, for 1hour at 37˚C. The nitrocellulose filters were rinsed briefly in Tris-Tween buffer saline (TTBS) containing 0.05% Tween-20 in TBS buffer, pH 7.5. The resulting blots were incubated for 3 hours at 37˚C with serum containing rabbit anti-Salmonella antibodies of the corresponding Salmonella serovar diluted in TTBS containing 1% gelatin. The unbound antibodies were removed by rinsing the blot in TTBS and the bound antibodies were detected by using conjugate labeled with horseradish peroxidase (HRP) and substrate 4 chlor-1-naphthol (Sigma). 

RESULTS

Table 1: Prevalence of Salmonella isolation from chickens.

Table 2: incidences, Congo red activity and antigenic structures of  Salmonella serovars isolated from chickens

Table 3: Antibiogram of salmonellae isolated from chickens.

Table 4: Pathogenicity tests of isolated Salmonella serovars in one-day old chicks.

NO.= number                              I/P=Intraperitoneal              *=days post-infection

Table 5: The molecular weights (Mol.wt.) of Salmonella serovars compared with the molecular weights of the Sigma marker

 Lane (1): protein marker    Lane (2): S.cerro   Lane (3): S.enteritidis    Lane(4): S.infantis                 Lane (5): S.montivideo     Lane (6): S.typhimurium

Table 6: The amount of protein and molecular weight of bands of western blot of Salmonella species compared with the molecular weights of western blot of bio lab-broad range prestained molecular weights marker

Photo 1: SDS-PAGE protein profile of Salmonella serovars isolated from chickens.

Lane (1): protein marker, Lane (2): S. cerro, Lane (3): S. enteritidis,  Lane (4): S. infantis, Lane (5): S. montevideo,  Lane (6): S. typhimurium

Photo 2: Western immunoblot of Salmonella serovars isolated from  chickens.

Lane (1): S.typhimurium, Lane (2): S.cerro, Lane (3): S.enteritidis, Lane(4):S.montevideo,

Lane (5): S.infantis, Lane(6): protein marker

DISCUSSION

Bacteriological examination of different samples obtained from chicken cases at different ages revealed that out of 730 examined cases 42 Salmonella isolates were obtained with an incidence of 5.75% (Table 1).

Similar results were obtained by Hassan et al., 2003 who isolated 35 Salmonella isolates (5.51%) out of 635 examined chicken samples. On serotyping of the isolated Salmonellae (Table 2) revealed that Salmonella enteritidis was the most predominant isolated serovar (47.62%) followed by Salmonella typhimurium (30.95%). These results agreed with that obtained by Abd-Allah et al., (1995), who surveyed a large number of samples from different domestic birds and their environmental surroundings in El-Fayoum governorate and found that Salmonella enteritidis was the most prevalent isolated serovar (40%) followed by Salmonella typhimurium (24%), Salmonella montivideo (16%). However, it was noticed that during the last 10-15 years, Salmonella enteritidis has replaced Salmonella typhimurium as the commonest serovar in many countries worldwide (Poppe, 2000). Isolates of Salmonella infantis constituted 11.9% of the total number of Salmonella isolates which agreed with the results obtained by Novak and Polaharova (1993) and Hassan et al., (2003).Salmonella montivideo represented 7.14% of the isolated salmonellae, while Salmonella cerro constituted 2.38%. the same serovars were isolated from poultry samples with different incidences by many authors (Barnhart et al., 1992, Abd-Allah 1995 and Hofer et al.,1998).

Concerning Congo red binding activity, all the isolated Salmonellae were proved to be Congo red positive. It was reported  that Congo red binding activity was correlated to the invasiveness of bacteria, as fimbria promote the binding of the hydrophobic dye Congo red by the bacteria that produce such fimbria (Qadri et al., 1988). Thus, Congo red binding test may provides a simple and rapid test for screening Salmonella strains which harbor fimbria (Dorn et al., 1992).

Antibiogram of the isolated Salmonellae (Table 3) revealed multi-resistant to more than one of the tested antibiotics. However the isolates were sensitive to Ciprofloxacin, Danofloxacin and Norfloxacin (100%) followed by Cephridine and Sulfamethxazole-trimethoprim (73.8% for each).

Similar results were obtained by Lee et al., (2003) and Gorman and Adley (2004). Also, resistance to Tetracycline, Amoxycillin and Ampicillin (92.9%, 88.1% and 78.6%, respectively) were detected which agreed with Botteldoorn et al., (2004) and Johnson et al., (2005). It was reported that multi-drug resistant Salmonella serovars cause severe and septicemic salmonellosis more frequently than those which are non- resistant (Helms et al., 2002 and Gupta et al., 2003).

The high incidence of antibiotic resistance among the tested salmonellae may be due to the misuse of antibiotics in addition to under-dosing and using the antibiotics as feed additives.

The pathoginicity testing of the isolated serovars in one-day old chicks (Table 4) revealed a variation in the degree of virulence in correlation to the variation of the type of serovar and route of infection. The mortality rates ranged from 70% -100% in case of oral infection, while reached 100% mortalities in all groups of chicks infected intraperitoneally. However, many factors can influence the relative pathogenicity of Salmonella in chicks, which includes the age of the chickens at the time of infection, route of infection, presence of competing bacteria in the intestinal tract and the dose of infection (Cox et al., 1990; Cooper et al., 1994 and Bailey et al., 2001).

Mortalities appear within 24 hours after intraperitoneal infection and within 48-72 hours after oral infection. The main clinical symptoms were pyrexia, diarrheoa and inability to stand while the postmortem lesions revealed congestion of all internal organs specially in intraperitoneal infection associated with the isolation of the inoculated serovar. These results agreed with Bailey et al., (2005) who recovered Salmonella from liver, thymus, spleen, bursa and ceca within 24 hours after oral inoculation.

The outer membrane proteins (OMPs) of salmonella and its compositions have been a subject of growing interest during the last few decades (Roushdy, 1998). In this work, (OMPs) of the isolated Salmonella serovars have been analyzed and electrophoresis' profiles have been determined by sodium dodocyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and resolved into protein bands as shown in Photo (1) and Table (5)

SDS-PAGE results revealed the determination of 6-10 protein bands ranging from 13.631 -200.5 kilo Dalton (KD). Nearly similar results were obtained by El-Reedy et al. (2007) who identified about 12 different protein bands ranging from 22-289 KD by SDS-PAGE analysis of 12 different salmonella serotypes isolated from poultry. Also there were common protein bands at 15, 26 and 35 KD identified in all isolated Salmonella serovars which agreed with the results obtained by Ames (1973), Sarasombath et al. (1988) and Joradat and Zawistowski (1998) who detected the presence of 35 KD protein band in all examined salmonellae. On the other hand, S. cerro was characterized by 3 deeply stained protein bands at 120, 60.15 and 26 KD.; S. enteritidis was characterized by 4 deeply stained protein bands at 150.5, 100.66, 75.1 and 620.5 KD; S. infantis has 3 deeply stained protein bands at 180.2, 75 and 62.112 KD; S. montivideo characterized by 2 deeply stained protein bands at 150.9 KD and 100 KD, while S. typhimurium obtained 5 deeply stained protein bands at150, 100.25, 75.4, 62.71 and 36.532 KD. It is clear that the protein composition in relation to Salmonella serovars which agreed with the results obtained by Roushdy (1998). 

SDS-PAGE immuonoblot procedure provided a rapid method for providing serological evidence of infection with Salmonella (Chart et al., 1997). In this study, SDS-PAGE immuonoblotting detected 10-13 antigenic protein bands ranged from 11.8-147.3 KD as shown in Photo (2) and Table (6) with the detection of more protein bands which could not be detected in gel electrophoresis. This may be due to that immunoblot is more specific technique than gel electrophoresis so any epitopes can be captured by antibodies. It was noticed that the highest antigenicity protein band of S. typhimurium was detected at 35.4 KD,  while  in S. enteritidis was detected at 29 KD; in S. montivideo at 39.8 KD; in S. infantis at 25.128 and 34.091 KD while S. cerro had the highest antigenic protein band at 143.08. Similar results obtained by Nese et al. (2003) who found that Salmonella typhimurium isolates contain OMPs have the highest antigenicity common fractions at 36-43 KD. Moreover, SDS-IMMUNOBLOT of the five Salmonella serovars showed the presence of common protein bands at 15, 29 and 35 KD which may constitute the common genus antigen.  These results agreed with that obtained by Nasef (1995) who studied the immunogenicity of Salmonella common protein detected at 29 KD and revealed the immunologic specificity of this band against antisera of different studied salmonella serotypes. Also Fathi (2004) recognized the 35 KD protein band in all examined salmonellae.

On the other hand, it was noticed the presence of shared immunogenic protein bands between different Salmonella serovars which may be the reason of cross reaction between Salmonella serovars. Similar results were obtained by Timoney et al. (1990) and Van Zijderveld et al. (1992) who observed the presence of cross reaction between different Salmonella serotypes specially group B and D1 in ELISA based on whole flagella antigen. Also, Cooper and Thorn (1996) reported that rabbit sera raised against Salmonella montivideo reacted strongly with Salmonella enteritidis flagellins. The cross reactions may be attributed to common epitopes present on different flagellins.

In conclusion, this study has been able to identify the differences in the organization of the proteins of the isolated Salmonella serovars which demonstrated the potential use of SDS-PAGE analysis and immuonoblotting as a recent and accurate techniques for detection of salmonellosis in chickens and the use of immunogenicity of different detected immunogenic bands to serve as components of an effective subcellular vaccine for poultry salmonellosis.

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