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An Allele-Specific PCR Assay for the Rapid and Serotype-Specific Detection ofSalmonella PullorumAuthor(s): Atul R. Desai, Devendra H. Shah, Smriti Shringi, Mi-Jin Lee, Ying-Hua Li, Mae-Rim Cho,Jin-Ho Park, Seong-Kug Eo, John-Hwa Lee, and Joon-Seok ChaeSource: Avian Diseases, 49(4):558-561. 2005.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/7385-052205R.1URL: http://www.bioone.org/doi/full/10.1637/7385-052205R.1

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An Allele-Specific PCR Assay for the Rapid andSerotype-Specific Detection of Salmonella Pullorum

Atul R. Desai, Devendra H. Shah, Smriti Shringi, Mi-Jin Lee, Ying-Hua Li,Mae-Rim Cho, Jin-Ho Park, Seong-Kug Eo, John-Hwa Lee, and Joon-Seok ChaeA

Bio-Safety Research Institute, Department of Veterinary Internal Medicine and Pathology,College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, South Korea

Received 22 May 2005; Accepted 2 August 2005

SUMMARY. Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severeeconomic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed byserotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developeda rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in r f bS gene sequencefor the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of thisPCR assay was tested using DNA samples from Pullorum (n¼ 13), Salmonella serotypes other than Pullorum (n¼ 19), and closelyrelated non-Salmonella organisms (n¼ 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs wasobserved only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification ofPullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method ishighly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorumand differentiation from other Salmonella species.

RESUMEN. Prueba alelo especıfica de reaccion en cadena por la polimerasa para la deteccion rapida y especıfica de Salmonellaserovar pullorum.

La Salmonella serovar Pullorum, agente causal de la pullorosis en aves domesticas, es la responsable de perdidas economicasseveras para la industria avıcola en muchas partes del mundo. La deteccion definitiva de S. serovar Pullorum requiere del cultivobacteriologico seguido por la serotipificacion e identificacion bioquımica, procesos tediosos que requieren de varias semanas para serllevados a cabo. Se desarrollo una prueba alelo especıfica rapida de reaccion en cadena por la polimerasa con base en el polimorfismode la secuencia de nucleotidos del gen r f bS para la deteccion especıfica de S. serovar Pullorum y su diferenciacion de S. serovarGallinarum, con la cual se encuentra estrechamente relacionada. Se evaluo la especificidad de la prueba de reaccion en cadena por lapolimerasa empleando muestras de ADN de S. serovar Pullorum (n¼13), serotipos de Salmonella diferentes a S. Pullorum (n¼19)y organismos estrechamente relacionados diferentes a la Salmonella (n ¼ 5). La prueba de reaccion en cadena por la polimerasamostro una alta especificidad al serotipo S. Pullorum, con la presencia de un producto de 147 pares de bases unicamente en loscasos de S. serovar Pullorum, mientras que las otras muestras de ADN fueron negativas. La identificacion definitiva de cultivosbacteriologicos de S. serovar Pullorum fue posible en menos de 3 horas. Se detectaron concentraciones de ADN de S. serovarPullorum tan bajas como 100 picogramos. Esta prueba alelo especıfica de reaccion en cadena por la polimerasa mostro una altaespecificidad y sensibilidad, y puede constituirse en una herramienta molecular efectiva para la deteccion rapida y especıfica delserotipo Pullorum y su diferenciacion de otras especies de Salmonella.

Key words: allele-specific PCR, Salmonella Pullorum, Salmonella Gallinarum

Abbreviations: bp ¼ base pair; LB ¼ Luria Bertani; PCR ¼ polymerase chain reaction; PD¼ pullorum disease

Salmonella enterica serovar Pullorum is the causative agent ofpullorum disease (PD) in chickens (16). In young chicks, Pullorumcauses an acute systemic disease, while in adults, PD is manifested byloss in body weight, reduced egg production, white diarrhea, andabnormalities of the reproductive tract (16). Pullorum disease haslargely been eradicated from many countries of Europe and NorthAmerica by rigorous monitoring, employing serological testing, andculling of the serologically reactor birds. However, PD continues tooccur and cause substantial economic losses to the poultry industry inmany countries of Asia, Africa, South America, and in some parts ofEurope (11,16,17,18). Sporadic outbreaks have also occurred in com-mercial poultry flocks in countries like Germany, the United States,and the United Kingdom, which are otherwise considered to be freefrom the disease (1,7,8,13). In addition, PD is also known to bepresent in backyard and fancy breeding stocks worldwide (16). Forinstance, Pullorum was isolated from game birds in the United King-dom on 20 occasions in 1997 and 1998 (1). In Switzerland, 25 out of40 Swiss fancy-breed poultry flocks reacted positive to the routine

serologic testing for PD (21) and, in Argentina, 90% of southerngiant petrels (Macronectes giganteus) showed antibody titers indicativeof Pullorum infection (19). Therefore, continuous vigilance stillremains important in countries where PD has been controlled.

A rapid slide agglutination test based on Pullorum antigens fromstandard (O: 1, 9, 121, and 123) and variant (O: 1, 9, 121, and 122)strains has traditionally been used as an easy and cheap test for flock-based testing of Pullorum infection (16). However, this test producesvariable and erratic results (17,18), such as nonspecific reactions, anda lack of sensitivity is also suspected. Serotyping of Salmonella byconventional Kaufmann White scheme is the only reliable methodfor the definitive identification of various Salmonella serotypes.However, it can not distinguish between antigenically closely relatedbiotypes Gallinarum and Pullorum and requires further subtypingof O antigen subfactors using panels of antisera (2,5). Therefore,definitive detection of Pullorum can be made only by isolationfollowed by serotyping and biochemical testing (2,4,5,10,15,16),which tends to be a tedious and time-consuming task (taking between2 to 3 wk). It is likely, therefore, that an effective method for the rapidand serotype-specific detection of Pullorum cultures would be ofconsiderable practical value.ACorresponding author.

AVIAN DISEASES 49:558–561, 2005

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A polymerase chain reaction (PCR) can be a particularly usefultool for this purpose. In our earlier study, we reported serotype-specific nucleotide polymorphism in the r f bS gene that encodesparatose synthetase (a final step in the biosynthetic pathway ofO-side chain sugars) in Gallinarum and Pullorum (14). Of thetwo polymorphic nucleotides, the first was found at position 598(Gallinarum-specific) and the other at position 237 (Pullorum-specific) of the r f bS gene. Based on Gallinarum-specific nucleo-tide (adenine in place of guanine) at position 598, an allele-specificPCR method was developed for the serotype-specific detection ofGallinarum (14). The nucleotide polymorphism at position 237(guanine in place of adenine) seems to be a good target for thedevelopment of allele-specific PCR. However, no such PCR assayis yet available for serotype-specific detection of Pullorum. There-fore, the purpose of this study was to design and validatea new allele-specific PCR method for the serotype-specific detec-tion of Pullorum.

MATERIALS AND METHODS

Bacterial strains and DNA. Purified genomic DNAs from 10field strains of Pullorum (Table 1) were kindly provided by NationalVeterinary Research and Quarantine Services, Korea. These field strainswere isolated from outbreaks of PD that occurred in commercial poultryflocks reared in different parts of Korea, during the years 1993 and1995. Other Salmonella and non-Salmonella isolates used in this study(Table 1) were kept at �70 C in Luria Bertani (LB) broth (Difco,Sparks, MD) with the addition of 20% (v/v) glycerol. An aliquot of thissolution was taken and grown overnight in LB broth with continuousshaking (200 rpm), at 37 C. Chromosomal DNA from these isolates wasextracted using genomic DNA extraction kit (Intron, Sungnam, Korea)as per the instructions provided by the manufacturer. The culture lysateswere prepared by resuspending the bacterial colonies in 400 ll of Tris-EDTA (TE) buffer followed by boiling for 5 min. The boiled lysateswere immediately chilled on ice and, after a brief centrifugation, 5 ll ofsupernatant was used as template DNA for amplification.

Primers and PCR conditions. Based on pullorum-specificnucleotide at position 237 (guanine in place of adenine) of r f bS gene,a pair of 21-mer nucleotide primers was designed. A forward primer,r f bSP (59-GAT CGA AAA AAT AGT AGA ATT-39), complementsbases 111–131, and a reverse primer, r f bSP1.1 (59-GCA TCA AGTGAT GAG ATA ATC-39), complements bases 237–216 of the r f bSgene (the underlined base is the change included in the primer se-quence). The designing of primer r f bSP1.1 was based on the principleof allele-specific PCR described earlier (14). Amplification reactionmixtures contained 2 ll of template DNA (about 100 ng) and 23 ll ofa solution containing the following: 200 nM each deoxyribonucleoside,25 pmol of each primer (r f bSP and r f bSP1.1), 2.5 U of Super-TaqDNA polymerase (Super Bio, Suwan, Korea), 10 mM Tris hydrochlo-ride (pH 9.0), 50 mM KCl, 1.4 mM MgCl2, and 1% Triton X-100.The cycling parameters were 94 C for 5 min, followed by 30 three-stepcycles including denaturation at 94 C for 1 min, annealing at 62 C for 1min, extension at 72 C for 1 min, and a final extension cycle of 72 C for5 min. The amplification products were analyzed by electrophoresis on1.5% (w/v) agarose gel and visualized by ethidium bromide fluorescence.

Specificity and sensitivity of allele-specific PCR. Thespecificity of the designed primers was tested by applying the PCRassay on the genomic DNAs from the Salmonella and non-Salmonellastrains listed in Table 1. Separately, the specificity of PCR primers wastested by applying this PCR assay on the template DNA prepared frommixed culture lysates containing Pullorum SP-11, Typhimurium,Escherichia coli, Pseudomonas putida, and Proteus mirabilis. In order todetermine the sensitivity, Pullorum (SP-11) DNA was serially dilutedand subjected to the PCR amplifications as described above. Theamplified products were analyzed on a 1.5% (w/v) agarose gel stainedwith ethidium bromide and the sensitivity was determined on the basisof visualization of the products in the agarose gel.

r f bS PCR, cloning, and sequencing analysis. A 720-base pair(bp) region of r f bS gene from Pullorum strains (ATCC 13036 andATCC 10398) was amplified using a PCR method essentially as describedearlier (12,14). The PCR products were purified with GFX PCR DNApurification kit (Amersham Biosciences, Chalfont St. Giles, Bucking-hamshire, UK) and cloned into pGEMT-Easy vector (Promega,Madison, WI). The cloned DNAs were sequenced by cycler sequencerusing ABI prism377 DNA sequencer (Genotech, Daejeon, Korea).

RESULTS

Initially, this allele-specific PCR was applied on the purified DNAfrom Pullorum (SP-11) and Gallinarum (ATCC 9184) strains. PCRconditions that could generate an expected PCR product weredetermined after several rounds of amplifications using differentannealing temperatures and MgCl2 concentrations (data not shown).Using primer pair r f bSP and r f bSP1.1, a unique amplificationproduct of 147 bp was observed only in the case of Pullorum (Fig.1a). Subsequently, the purified genomic DNAs from 10 field strainsof Pullorum as well as all other bacterial strains were tested by thisallele-specific PCR assay. Other Salmonella serotypes (includingGallinarum) and non-Salmonella organisms failed to produce anyamplicons (Fig. 1a). These results were consistent when the templateDNA in the form of culture lysates prepared from mixed cultureswere tested (Fig. 1b), indicating that the primers employed in thisstudy are highly specific for Pullorum. These results also indicate thatnone of the non-Pullorum strains contains a cited nucleotide change(guanine in the place of adenine) at position 237. The PCR assay wasable to detect Pullorum DNA at a concentration as low as 100 pg(Fig. 2), indicating that the allele-specific PCR assay is highlysensitive. A 720-bp region of r f bS gene amplified from Pullorumstrains ATCC 13036 and ATCC 10398 was sequenced and thesequences were registered in the GenBank under accession numbersDQ074436 and DQ074437. The comparative sequence analysesperformed at NCBI GenBank blast server showed that the r f bS

Table 1. Salmonella and non-Salmonella isolates used in this study.A

Salmonella entericaserotype or other species Serogroup Strains and sources

Pullorum D S-11 (NVRQS)Pullorum D ATCC 13036Pullorum D ATCC 10398Pullorum D Field isolatesB (10)Gallinarum D ATCC 9184Gallinarum D Field isolatesC (14)Dublin D S-37 (NVRQS)Enteritidis D ATCC 13076Typhi D ATCC 19430Typhimurium B ATCC 13311Escherichia coli — EC 031107Klebsiella pneumoniae — ATCC 25306Proteus mirablis — ATCC 25933Pseudomonas putida — DBS-CBNUShigella flexernii — ATCC 12022

ASalmonella strains were obtained from NVRQS, National VeterinaryResearch and Quarantine Services; DBS-CBNU, Department ofBiological Sciences, Chonbuk National University, Korea; ATCC,American Type Culture Collection, Rockville, MD.

BPurified genomic DNAs were obtained from NVRQS, Korea.CField strains of Gallinarum isolated from outbreaks of fowl typhoid

from commercial poultry in Chonbuk and Chungnam provinces ofKorea. Figures in parentheses show numbers of strains.

Specific detection of Salmonella Pullorum by PCR 559

sequences determined in this study were 100% similar or identicalto Pullorum (SP-11) r f bS gene sequence (accession AF442475)registered earlier in the GenBank database.

DISCUSSION

In order to increase the sensitivity, specificity, and speed for thedetection of Salmonella, several DNA-based methods have beendeveloped (3,6). However, the method for differentiating Pullorumfrom other group D Salmonella strains, such as Gallinarum andEnteritidis, that are primarily isolated from poultry, is not yetavailable. The r f bS gene encoding paratose synthatase is found to beuniquely present in the serotype belonging to Salmonella serogroupD (9,20). DNA sequencing has shown that the r f bS gene fromPullorum contains a nucleotide polymorphism at position 237 withrespect to the Gallinarum and other serogroup D Salmonellaserotypes (12). Based on the above nucleotide polymorphism,a PCR-RFLP method was developed to differentiate betweengenetically closely related Pullorum and Gallinarum strains (12).However, this method required two steps in which the 720-bpfragment of r f bS gene was amplified, and agarose gel electrophoresis

was performed to verify the amplification of the desired fragment.This was followed by TfiI and PleI digestion of the PCR amplicons;and to identify the polymorphism in the r f bS gene, the result wasvalidated by a new electrophoresis. The allele-specific PCR assaydeveloped in this study is based on the similar principle but has theadvantage that it can differentiate Pullorum from Gallinarum as wellas other Salmonella strains belonging to serogroup D using a singletube reaction without the need of restriction enzyme analysis of thePCR products. We also determined whether Pullorum strainsisolated from clinical outbreaks of PD in different geographicregions would also have the same characteristic nucleotide poly-morphism and could thus be rapidly distinguished from Gallinarum.We analyzed the purified genomic DNA of 10 field isolates ofPullorum that were isolated during the years 1993–95 fromoutbreaks of PD in various commercial poultry flocks located indifferent geographic regions of Korea. The designed primers, basedon characteristic point mutation at position 237 of r f bS gene,selectively and invariably amplified the 147-bp amplicon from allthese isolates. These results also confirm the earlier reports ofa polymorphic site located at position 237 of the r f bS gene, whichuniformly distinguishes Pullorum (guanine) from non-Pullorumisolates (12,14), indicating that the PCR assay was reliable andaccurate. The specificity did not alter when this PCR assay wasapplied on the culture lysates of Pullorum mixed with otherunrelated bacteria. Moreover, this PCR assay could be directlyapplied on boiled culture lysates, which further reduced the time forconfirmation without the need for extraction of a bacterial DNA. Inconclusion, using this PCR assay, the definitive detection ofPullorum cultures could be achieved in less than 3 hr. This allele-specific PCR assay is rapid and specific, as well as sensitive, and maywork out as a novel tool for the rapid and serotype-specific detectionof Pullorum in veterinary microbiology or a Salmonella referencelaboratory that routinely processes Pullorum cultures.

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Fig. 1. Electrophoresis on agarose gel showing the 147-bp PCR product after amplification with allele-specific primers. M, DNA size marker (100-bp ladder). (a) Amplifications were performed with chromosomal DNA from Pullorum ATCC 13036 (1), Pullorum ATCC 10398 (2), PullorumS-11 (3), Pullorum field strains (4–6), Gallinarum ATCC 9184 (7), Gallinarum field strains (8–10), Enteritidis ATCC 13076 (11), Dublin S-37(12), Typhi ATCC 19430 (13), Typhimurium ATCC 13311 (14), E. coli (15), water control (16). (b) Amplifications were performed with templateDNA prepared from mixed culture lysates of Pullorum SP-11 with E. coli (1), Pseudomonas putida (2), Typhimurium (3), Proteus mirabilis (4). Lanes5–9 show the results of PCR performed with pure culture lysates of E. coli, P. putida, P. mirabilis, Pullorum, and Typhimurium, respectively.

Fig. 2. Agarose gel electrophoresis of serial 10-fold dilution ofPullorum (SP-11) DNA amplified by an allele-specific PCR assay.Lanes: M, DNA size marker (100-bp ladder); 1–5 show the results using10,000, 1000, 100, 10, and 1 pg DNA, respectively.

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ACKNOWLEDGMENTS

We thank Dr. Hee-Soo Lee for providing purified genomic DNAsfrom field strains of Pullorum. This work was supported in part by theresearch grant of Brain Korea 21 project E001 and the Bio-SafetyResearch Institute, Chonbuk National University. Dr. D. H. Shahreceived the grant of the Post-Doc program (2004–2005) of ChonbukNational University, Jeonju, Korea.

Specific detection of Salmonella Pullorum by PCR 561