under the new naming system the salmonella enterica serovar gallinarum is divided into biovar gallinarum ( s. gallinarum ) and pullorum ( s. pullorum ) , which are identified to cause fowl typhoid and pullorum disease in poultry , respectively .
while fowl typhoid is a disease of mature birds , pullorum causes mortality of embryos and chicks .
infection with these pathogens is responsible for considerable economic losses in poultry production ( 1 , 2 ) . among diseases of poultry ,
salmonellosis is of great concern and has been responsible for serious economic losses of poultry producers ( 3 ) .
s. gallinarum and s. pullorum are non - motile , host adapted avian pathogens belonging to salmonella serogroup d ( 4 , 5 ) .
s. gallinarum and s. pullorum are very similar from the point of view of their antigenic structure ; however they are responsible for distinctly different diseases in chicken ( 2 , 6 ) .   some countries are considered free of s. gallinarum and s. pullorum ; however , infections are still sometimes reported , and are a matter of concern for the poultry industry ( 4 , 6 ) .
salmonella control efforts are complicated due to their sporadic and uneven distribution ( 7 ) .
specific characterization of salmonella isolates is therefore extremely important in order to attribute an isolate to a previously known epidemic outbreak ( 5 ) . although conventional kaufmann white scheme is still the only reliable method for serotyping of salmonella , it can not differentiate between closely related biotypes , such as s. gallinarum and s. pullorum ( 4 , 5 ) .
however , biochemical characteristics take approximately five to seven days and are very time - consuming .   recently ,
biochemical methods have been complemented by dna - based molecular techniques , because of their sensitivity and specificity .
such methods include restriction fragment length polymorphism ( rflp ) , which is sometimes associated with pcr ( pcr - rflp ) , ribotyping , pulse field gel electrophoresis ( pfge ) and variable number tandem repeat ( vntr ) ( 8 - 10 ) .
pcr- rflp is considered as a rapid test with good reproducibility for molecular typing in bacterial epidemiological studies ( 11 ) .
many researchers have focused on flagellin genes for salmonella subtyping because most of them possess the two structural genes ( flic and fljb ) that contain a hypervariable central region and a conserved flanking dna region .  
the hypervariable central region of salmonella flagellin genes makes it possible to differentiate the salmonella isolates by the pcr- rflp technique .
many studies used part i of the gene that encodes flagellin ( flic ) to differentiate serotypes .
most salmonella strains have two structural genes ( flic and flib ) that encode flagellins . non - motile strains generally exhibit these structural genes , but are unable to build up a functional flagellum ( 13 , 14 ) .
s. gallinarum and s. pullorum have been reported to possess phase 1 flagellin c gene ( flic ) ( 13 - 15 ) .
there are no data from epidemiological studies of s. gallinarum and s. pullorum isolates based on molecular typing in iran .
thus these data could be helpful in this regard and also for the rapid detection of these bacteria .
the aim of the present study was to differentiate s. gallinarum and s. pullorum isolated in iran , based on the pcr - rflp method and the hinp1i enzyme .
s. gallinarum ( n = 10 ) and s. pullorum ( n = 3 ) isolates used in this study were from clinical samples of chickens kept at the microbiology department of razi vaccine and serum research institute of karaj ( rvsri ) .
s. gallinarum ( atcc 9184 ) and s. pullorum ( atcc 9120 ) were used as positive controls and s. enteritidis ( atcc 13076 ) was used as a negative control .
all isolates were cultured on macconkey agar ( merck , germany ) for 24 hours at 37c .
briefly , for each isolate , a loopful of an overnight pure culture of bacteria was transferred into 1 ml of tris - edta ( te ) buffer 1x , boiled at 95c for 10 minutes then centrifuged at 17900  g ( 10 minutes , 4c ) , followed by addition of 1 l proteinase k ( fermentase , inc . , the netherlands ) to the supernatant .
specific primers , ctggtgatgacggtaatggt ( flicf : 866 - 885 ) and cagaaagtttcgcactctcg ( flic r : 1063 - 1044 ) , were used for the amplification of flagellin gene phase 1 ( flic ) ( 13 , 14 ) . a reaction mixture containing ,
5 l of ultra - pure water ( gibco , germany ) , 9 l of primix ( ampliqon , denmark ) , 2 l of dna and 1 l of each primer , was prepared .
the thermocycler was programmed with 1 cycle of 94 c for 5 minutes , 35 three - step cycles ; denaturation at 94c for 1 minute , annealing at 58c for 1 minute , extension at 72c for 1 minute , and a final cycle at 72c for 10 minutes .
the pcr products were electrophoresed on 1% agarose gel for 1 hour at 60 v ( 16 ) .
the digestion solution was prepared with 10 l of the pcr product , 2 l of hinp1i buffer ( 10x ) , 1 l of hinp1l enzyme ( fermentas inc .
the netherlands ) and 17 l of ultra - pure water ( gibco , germany ) .
after incubation at 37c for 16 hours , rflps were determined by electrophoresis of the digested dna on 2% agarose gel for 2.5 hours at 60 v ( 16 ) .
sizes of the products were analyzed and compared with the 100 bp plus dna ladder ( fermentas inc .
s. gallinarum ( n = 10 ) and s. pullorum ( n = 3 ) isolates used in this study were from clinical samples of chickens kept at the microbiology department of razi vaccine and serum research institute of karaj ( rvsri ) .
s. gallinarum ( atcc 9184 ) and s. pullorum ( atcc 9120 ) were used as positive controls and s. enteritidis ( atcc 13076 ) was used as a negative control .
all isolates were cultured on macconkey agar ( merck , germany ) for 24 hours at 37c .
bacterial dna was prepared as described by paiva et al . ( 13 ) with some modifications .
briefly , for each isolate , a loopful of an overnight pure culture of bacteria was transferred into 1 ml of tris - edta ( te ) buffer 1x , boiled at 95c for 10 minutes then centrifuged at 17900  g ( 10 minutes , 4c ) , followed by addition of 1 l proteinase k ( fermentase , inc . , the netherlands ) to the supernatant .
specific primers , ctggtgatgacggtaatggt ( flicf : 866 - 885 ) and cagaaagtttcgcactctcg ( flic r : 1063 - 1044 ) , were used for the amplification of flagellin gene phase 1 ( flic ) ( 13 , 14 ) . a reaction mixture containing ,
5 l of ultra - pure water ( gibco , germany ) , 9 l of primix ( ampliqon , denmark ) , 2 l of dna and 1 l of each primer , was prepared .
the thermocycler was programmed with 1 cycle of 94 c for 5 minutes , 35 three - step cycles ; denaturation at 94c for 1 minute , annealing at 58c for 1 minute , extension at 72c for 1 minute , and a final cycle at 72c for 10 minutes .
the pcr products were electrophoresed on 1% agarose gel for 1 hour at 60 v ( 16 ) .
the digestion solution was prepared with 10 l of the pcr product , 2 l of hinp1i buffer ( 10x ) , 1 l of hinp1l enzyme ( fermentas inc .
the netherlands ) and 17 l of ultra - pure water ( gibco , germany ) .
after incubation at 37c for 16 hours , rflps were determined by electrophoresis of the digested dna on 2% agarose gel for 2.5 hours at 60 v ( 16 ) .
sizes of the products were analyzed and compared with the 100 bp plus dna ladder ( fermentas inc .
  in this study , the 197 bp fragment of the flic gene was amplified from 10 s. gallinarum and three s. pullorum and no variation in gene size was detected according to gel electrophoresis ( figure 1 ) .
sp : lanes 1 to 3 ; sg : lanes 4 to 12 , nc : negative control ( s. enteritidis ) .
digestion of s. gallinarum amplicons with hinp1i yielded two bands , of 82 and 115 bp , while no change in s. pullorum amplicons was observed , since no digestion occurred ( figure 2 ) .
m : 100 bp marker plus dna ladder ( fermentas inc . ) ; lane 1 : s. pullorum positive control ; lane 2 : s. gallinarum positive control ; lanes 3 , 4 , 5 , 7,8,10 and 12 : s. gallinarum isolates ; lanes 6 , 9 , and 11 : s. pullorum isolates .
  in this study , the 197 bp fragment of the flic gene was amplified from 10 s. gallinarum and three s. pullorum and no variation in gene size was detected according to gel electrophoresis ( figure 1 ) .
sp : lanes 1 to 3 ; sg : lanes 4 to 12 , nc : negative control ( s. enteritidis ) .
digestion of s. gallinarum amplicons with hinp1i yielded two bands , of 82 and 115 bp , while no change in s. pullorum amplicons was observed , since no digestion occurred ( figure 2 ) .
m : 100 bp marker plus dna ladder ( fermentas inc . ) ; lane 1 : s. pullorum positive control ; lane 2 : s. gallinarum positive control ; lanes 3 , 4 , 5 , 7,8,10 and 12 : s. gallinarum isolates ; lanes 6 , 9 , and 11 : s. pullorum isolates .
s. gallinarum and s. pullorum have been known as important bacterial pathogens in chicken ( 2 , 6 ) .
these serovars can not be distinguished by conventional serological methods and biochemical tests are currently complemented by molecular techniques based on flic and flib genes that encode flagellins ( 13 ) .
there has not been any research on molecular typing of s. gallinarum and s. pullorum in iran .   in the study of hong et al .
, the pcr- rflp flagella typing scheme was successfully applied for serotype identification of 112 salmonella isolates obtained from poultry and poultry environments ( 17 ) . also , our methods were successful in differentiating these two biotypes by pcr- rflp .
they showed that pcr - rflp with hinp1i was successful in differentiating the two biotypes .
these results suggested that the variable regions of flic could be used as a genetic marker and allow differentiation of these biotypes from each other and pcr - rflp with hinp1i for these biotypes is a valuable tool for identification of non - motile serotypes of salmonella ( 14 ) .
our study was done on ten s. gallinarum and three s. pullorum and the same result was obtained using hinp1i enzyme . in another study , practical application of restriction patterns of flic gene using a mixture of endonucleases ( taqi and scai ) to differentiate s. gallinarum and s. pullorum was performed . according to their results this method with the used enzymes was not useful to differentiate s. gallinarum from s. pullorum ( 15 ) but in our study a different enzyme , hinp1i , could differentiate these two biotypes . in the kisiela et al .
the results obtained from our research and theirs showed that fimh and flic gene with restriction enzyme scai and hinp 1i could differentiate s. gallinarum from s. pullorum . in another investigation done by paiva et al .
pcr amplicons ( 197 bp ) of 14 s. pullorum and 22 s. gallinarum that had various results on biochemical tests , were digested with the hinp1i enzyme and the same results were obtained . in our research
the bacterial isolates had different results for biochemical tests but the same pattern for pcr - rflp ( 13 ) . a study done by menghistu et al .
( 8) on s. gallinarum showed three patterns in 12 s. gallinarum isolates by pcr - rflp using the restriction enzyme alui .
our results showed that the hinp1i enzyme could be the same as alui in such investigation to identify s. gallinarum from s. pullorum . in the present study
, we were able to demonstrate that the use of flic gene restriction patterns is a useful method for allowing the differentiation between s. gallinarum and s. pullorum isolated in iran ; including those with atypical biochemical behavior .
therefore , our results support that this method may be adopted to differentiate s. gallinarum from s. pullorum .  
several sequences of the gene encoding phase 1 flagellin ( flic ) are available ( 19 , 20 ) .
the distal parts of the flic alleles are conserved regions , making this gene in any serotype suitable for easy amplification , whereas the central region of the flic gene is hyper variable , making it a target for differentiation among salmonella serotypes ( 14 , 15 ) .
s. gallinarum and s. pullorum 
 flic gene represent allelic variants and differ only in two codons , including 316 and 339 , which shows that the hinp1i enzyme recognizes one cleavage site in s. gallinarum ( codon 316 ) , but not in s. pullorum ( 14 , 21 ) . in the present study by using the applied technique
since s. gallinarum and s. pullorum are important in industry , thus the accurate identification of them with molecular technique can be effective in this area .
our literature review in iran showed that there is n't any publication in this field .
also our results demonstrated that pcr - rflp with flic gene and hinp1i endonuclease could be effective in detecting s. gallinarum and s. pullorum .
the result indicated that there was no limitation in this technique for differentiation of these biotypes .
 background : salmonella spp . is the major bacterial pathogen in poultry and is responsible for significant economic losses of the poultry industry in many parts of the world . among salmonella spp . 
 , salmonella 
 gallinarum and salmonella . 
 
 pullorum are the most common causative agents of chicken salmonellosis resulting in high mortality and morbidity.objectives:the aim of this study was to identify s. gallinarum and s. pullorum by using the polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) method.materials and methods : in this study , 13 samples of salmonella , isolated from local poultry , were obtained from razi type culture collection ( rtcc ) . for the pcr - rflp method based on the flic gene , 
 extracted dna was used as a template for amplifying of the flic gene ( 197bp ) using specific primers . 
 pcr products were subjected to digestion using hinp1i restriction endonuclease.results:for the pcr , 197 bp flic fragment was amplified from all 13 isolates . 
 ten out of 13 were s. 
 gallinarum and the other three were s. pullorum . 
 as part of the pcr - rflp , two fragments were obtained ( 82 bp and 115 bp ) for all s. 
 gallinarum , whereas no digestion was observed in s. pullorum , and 197 bp fragment was seen.conclusions:pcr-rflp with flic gene and hinp1i endonuclease were successfully applied to differentiate the two biotypes . 
 the results suggested that this technique could be effective in detecting s. gallinarum and s. pullorum . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Bacterial Strains
3.2. DNA Extraction
3.3. Amplification of the fliC Gene by PCR
3.4. PCR-RFLP
4. Results
4.1. Amplification of the fliC Gene
4.2. PCR-RFLP Analysis
5. Discussion