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USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISMANALYSIS TO DIFFERENTIATE ISOLATES OFPASTEURELLA MULTOCIDA SEROTYPE 1Authors: David S. Blehert, Keynttisha L. Jefferson, Dennis M. Heisey,Michael D. Samuel, Brenda M. Berlowski, et. al.Source: Journal of Wildlife Diseases, 44(2) : 209-225Published By: Wildlife Disease AssociationURL: https://doi.org/10.7589/0090-3558-44.2.209

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USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS

TO DIFFERENTIATE ISOLATES OF PASTEURELLA MULTOCIDA

SEROTYPE 1

David S. Blehert,1,3 Keynttisha L. Jefferson,1 Dennis M. Heisey,1 Michael D. Samuel,2

Brenda M. Berlowski,1 and Daniel J. Shadduck1

1 US Geological Survey, National Wildlife Health Center, 6006 Schroeder Road, Madison, Wisconsin 53711, USA2 US Geological Survey, Wisconsin Cooperative Wildlife Research Unit, Department of Wildlife Ecology, 204 RussellLabs, 1630 Linden Drive, Madison, Wisconsin 53706, USA3 Corresponding author (email: [email protected])

ABSTRACT: Avian cholera, an infectious disease caused by the bacterium Pasteurella multocida,kills thousands of North American wild waterfowl annually. Pasteurella multocida serotype 1isolates cultured during a laboratory challenge study of Mallards (Anas platyrhynchos) andcollected from wild birds and environmental samples during avian cholera outbreaks werecharacterized using amplified fragment length polymorphism (AFLP) analysis, a whole-genomeDNA fingerprinting technique. Comparison of the AFLP profiles of 53 isolates from the laboratorychallenge demonstrated that P. multocida underwent genetic changes during a 3-mo period.Analysis of 120 P. multocida serotype 1 isolates collected from wild birds and environmentalsamples revealed that isolates were distinguishable from one another based on regional andtemporal genetic characteristics. Thus, AFLP analysis had the ability to distinguish P. multocidaisolates of the same serotype by detecting spatiotemporal genetic changes and provides a tool toadvance the study of avian cholera epidemiology. Further application of AFLP technology to theexamination of wild bird avian cholera outbreaks may facilitate more effective management of thisdisease by providing the potential to investigate correlations between virulence and P. multocidagenotypes, to identify affiliations between bird species and bacterial genotypes, and to elucidatethe role of specific bird species in disease transmission.

Key words: Amplified fragment length polymorphism, avian cholera, DNA fingerprinting,Pasteurella multocida, waterfowl.

INTRODUCTION

Pasteurella multocida serotype 1 is theprimary causative agent of avian cholera inNorth American wild birds, and manyspecies of domestic, feral, and wild birdsare susceptible to infection (Samuel et al.,2007). Outbreaks killing thousands of wildwaterfowl occur almost annually in someareas of the United States, and the diseasehas been documented in all major flyways(Botzler, 1991; Friend, 1999).

Pasteurella multocida isolates are clas-sified based on serologic antigen presen-tation, and there are 16 somatic serotypesdesignated 1 though 16 (Rhoades andRimler, 1991). Most pathogenic P. multo-cida isolates cultured from wild waterfowlin the Pacific, Central, and Mississippiflyways of North America are serotype 1(Botzler, 1991). There have been signifi-cant annual and geographic fluctuations inthe patterns of avian cholera mortality

among wild bird species, and factorsinfluencing the initiation and the courseof an avian cholera outbreak are not wellunderstood (Rosen, 1969; Wobeser, 1992;Blanchong et al., 2006; Samuel et al.,2007).

To gain further epidemiological insightinto the dynamics of avian cholera trans-mission and disease spread among wildbirds, it is necessary to develop techniquesto differentiate P. multocida isolates of thesame serotype. The use of DNA-basedtechniques provides this ability (Owen,1989), but to date studies demonstratingthe usefulness of these techniques forunderstanding the epidemiology of aviancholera in wild birds have been limited(Wilson et al., 1995a, b; Samuel et al.,2003b; Samuel et al., 2007).

A more recently developed genomicfingerprinting technique, fluorescent am-plified fragment length polymorphism

Journal of Wildlife Diseases, 44(2), 2008, pp. 209–225# Wildlife Disease Association 2008

209


(AFLP) analysis, provides a greater capac-ity than previous techniques to identifypolymorphic regions within a genome (Voset al., 1995) and has been applied topasteurellae epizootics in domestic birds(Amonsin et al., 2002). Amplified frag-ment length polymorphism analysis isbased on the detection of polymorphicrestriction fragments by selective poly-merase chain reaction (PCR) and providesthe capacity to examine in excess of 1,000DNA markers per isolate (Vos et al.,1995). To conduct AFLP analysis, geno-mic DNA is digested with two restrictionenzymes, followed by ligation of adaptersof defined DNA sequence to the ends ofthe restriction fragments. Ligation prod-ucts are reamplified by PCR using primersthat include a selective nucleotide at their39-end to reduce the complexity of AFLPpatterns (Geert et al., 1996). A fluorescentlabel is incorporated during selective PCRto facilitate analysis of AFLP reactionproducts using an automated DNA se-quencing instrument. Amplified fragmentlength polymorphism analysis generatesreproducible, complex patterns, referredto as genetic fingerprints, that are usefulfor distinguishing closely related organ-isms.

The objective of this study was toevaluate the utility of AFLP analysis asan epidemiological tool to distinguish P.multocida serotype 1 isolates based onregional and temporal genetic character-istics. Fifty-three serotype 1 isolates cul-tured during a laboratory challenge ofMallards (Anas platyrhynchos; Samuel etal., 2003a) and 120 serotype 1 isolatescultured from wild birds and environmen-tal samples during avian cholera outbreakswere examined. Analysis of the AFLP datarevealed that the isolates were distinguish-able.

MATERIALS AND METHODS

Bacterial isolates

Amplified fragment length polymorphismanalysis was applied to a collection of P.

multocida serotype 1 isolates from the USGeological Survey–National Wildlife HealthCenter diagnostic culture collection and fromfield studies on avian cholera epidemiology(Samuel et al., 2003b). A collection of serotype1 isolates collected during a 15-wk laboratorychallenge study of captive-reared mallardducks (Samuel et al., 2003a) was also analyzed.The challenge study was conducted as follows(for additional study details, see Samuel et al.,2003a): Six-to-eight-wk-old male Mallardswere divided into groups consisting of 30birds each, and each group was housed in aseparate isolation room (designated 4, 5, and6). Within each room birds were divided intothree groups of 10; two groups were chal-lenged with P. multocida inocula of differentenvironmental origins, and the third group wasunchallenged. Hence, six different P. multo-cida inoculae (designated 1, 2, 3, 4, 7, and 8)were used. The inocula, all originally isolatedfrom environmental samples collected incentral California (Samuel et al., 2003a), wereutilized as follows: inocula 1 and 2 (room 6),inocula 3 and 4 (room 5), inoculae 7 and 8(room 4). Pasteurella multocida was culturedfrom tissues and/or swabs collected periodi-cally from live birds during the course of thestudy, from birds that died, and followingeuthanasia of birds at the end of the study.During the study some birds inoculated withP. multocida cleared the organism, while someuninoculated birds became infected.

AFLP reaction preparation

Genomic DNA was extracted from P.multocida isolates using the Puregene DNApurification kit (Gentra Systems Inc., Minnea-polis, Minnesota, USA) according to themanufacturer’s instructions. Genomic DNAconcentration and purity were evaluated byUV spectroscopy and agarose gel electropho-resis. Amplified fragment length polymor-phism reactions were prepared as describedby Vos et al. (1995). Genomic DNA (500 ng)was digested with the restriction enzymesEcoRI and HpyCH4 IV for 3 hr and ligated,using T4 ligase (New England Biolabs, Bev-erly, Massachusetts, USA), to EcoRI (59-CTCGTA GCT GCG TAC C-39 plus 39-CAT CTGACG CAT GGT TAA-59) and HpyCH4 IV (59-GAC GAT GAG TCC TGA G-39 plus 39-TACTCA GGA CTC GC-59) double-strandedadapters. The digested and ligated DNA wasthen diluted fivefold in a 13 solution of TaqDNA polymerase reaction buffer withoutMgCl2 (Promega, Madison, Wisconsin, USA)prior to amplification by preselective PCRusing the EcoRI and HpyCH4 IV primers 59-

210 JOURNAL OF WILDLIFE DISEASES, VOL. 44, NO. 2, APRIL 2008


GAC TGC GTA CCA ATT C-39 and 59-GATGAG TCC TGA GCG T-39, respectively, in afinal volume of 50 ml. PCR reactions contained10 ml diluted ligation mixture, 15 pmol eachEcoRI and HpyCH4 IV primers, 4 ml 40 mMdNTP mix, and 2.5 U Taq polymerase instorage buffer B (Promega). PreselectivePCR amplification conditions were an initialextension cycle at 72 C for 60 sec, followed by35 cycles of denaturation at 94 C for 50 sec,annealing at 56 C for 1 min, and extension at72 C for 2 min. Preselective PCR productswere then diluted 10-fold with double-deion-ized water, and 3 ml of the diluted preselectivemixture were used as template for selectiveamplification. Selective PCR reactions con-tained 15 pmol fluorescein phosphoramidite(FAM)–labeled EcoRI+G selective primer (59-FAM-GAC TGC GTA CCA ATT CG-39),25 pmol unlabeled HpyCH4 IV+A selectiveprimer (59-GAT GAG TCC TGA GCG TA-39),3 ml 40 mM dNTP mix, and 1.25 U Taqpolymerase in storage buffer B (Promega) ina final reaction volume of 25 ml. Selective PCRamplification conditions were 35 cycles ofdenaturation at 94 C for 50 sec, annealing at56 C for 1 min, and extension at 72 C for2 min, with a final extension at 72 C for10 min. Selective PCR reaction products werediluted fivefold with double-deionized water,and fragment analysis was conducted at theUniversity of Wisconsin–Madison Biotechnol-ogy Center using an Applied Biosystems(Foster City, California, USA) 3730 automatedcapillary DNA sequencing instrument.

AFLP data analysis

Amplified fragment length polymorphismprofiles were normalized and aligned to oneanother with respect to an internal sizingstandard using BioNumerics 3.5 (AppliedMaths Inc., Austin, Texas, USA) software’sproprietary algorithm. Continuous AFLP fin-gerprint data, in the form of densitometrydistributions, were then analyzed directlywithout attempting to identify discrete bandsbecause discretization in a nonarbitrary man-ner is not possible. Once AFLP densitometrypatterns were aligned, a Pearson productmoment correlation matrix was computed forall pairwise combinations, exported as a textfile, and imported into SAS statistical software(SAS Institute Inc., Cary, North Carolina,USA). The SAS PROC CLUSTER was usedto produce the dendrograms, using the aver-age linkage (also known as the unweighted pairgroup method with arithmetic mean [UP-GMA]; Sokal and Michener, 1958) andWARD (Ward, 1963) options for comparison.

The UPGMA and WARD dendrogram out-puts were virtually the same, and onlyUPGMA dendrograms are reported. SASPROC PRINCOMP was used to conduct aprincipal components analysis (PCA; Johnsonand Wichern, 1982) using the Pearson productmoment correlation matrix. Principal compo-nents analysis uses a different algorithm thandendrogram analysis to evaluate the data,providing a complementary assessment. Thesecond and third principal components wereused for the PCA. Although the first principalcomponent explained the majority of thevariation in the data, the first component isessentially the mean axis and typically reflectsthe average signal strength of the fingerprint.Signal strength largely reflects sample prepa-ration attributions and therefore is not usuallyan informative biologic axis on which to alignsamples. Fisher’s 2-tailed exact test was usedto determine whether specific groups ofsamples fell disproportionately on differentbranches of the dendrograms. Analysis ofvariance (ANOVA) followed by pairwise com-parisons of the means was used to test whetherthe principal components scores differedbetween groups of interest. Values of P#0.05were considered significant.

To understand the resolution AFLP analysisin distinguishing closely related bacteria, asingle genomic DNA preparation from a P.multocida serotype 1 isolate was used toconduct 10 replicate AFLP analyses. Allaspects of AFLP reaction processes wereconducted independently. A Pearson productmoment correlation matrix comparing theresulting AFLP patterns indicated an averagecorrelation of 0.92 (SD50.05) among thereplicate samples.

RESULTS

Analysis of P. multocida serotype 1 isolates from alaboratory infection trial

Amplified fragment length polymor-phism analysis was completed for the sixP. multocida inocula and for 53 isolatescultured from Mallards during the infec-tion trial. Of the 53 isolates examined, 21,12, and 20 were cultured from birdshoused in rooms 4, 5, and 6, respectively.Amplified fragment length polymorphismprofiles for each P. multocida isolate wereused to construct a dendrogram, consist-ing of four major branches, illustratinggenetic relationships among the isolates(Fig. 1). Isolates cultured from birds that

BLEHERT ET AL.—P. MULTOCIDA SEROTYPE 1 AFLP ANALYSIS 211


died (filled symbols) allocated dispropor-tionately to dendrogram branch 1, whileisolates obtained from live, apparentlyhealthy birds allocated disproportionatelyto branches 2 and 4 (open symbols). Thedistribution of P. multocida isolates cul-tured from dead birds was distinct fromthose cultured from apparently healthylive birds (P,0.001). Analysis of AFLPdata using PCA (components 2 and 3;Fig. 2) also supported the division ofbacterial isolates into the two majorgroups observed on the dendrogram, one

consisting of isolates from dead birds(filled symbols; quadrant IV), and theother consisting of isolates from live,apparently healthy birds (open symbols;quadrant I; P,0.001 for principal compo-nent 2). Further, PCA indicated that room6 bacterial isolates (circles) allocateddisproportionately to the right-hand quad-rants of the plot (Fig. 2) compared toroom 4 and room 5 isolates (diamonds andsquares, respectively; P,0.001 for princi-pal component 3).

Amplified fragment length polymor-

FIGURE 1. Dendrogram analysis of Pasteurella multocida serotype 1 isolates cultured from Mallardsduring a laboratory infection trial. Isolates cultured from birds that died are represented by filled symbols, andisolates cultured from live, apparently healthy birds are represented by open symbols. Dendrogram branches1–4 are labeled, and inocula 1, 2, 3, 4, 7, and 8 are identified numerically to the left of symbols (gray)indicating in which room they were used: room 4 (diamonds), room 5 (squares), and room 6 (circles).



phism profiles were also used to infer thegenetic correlation of parental inocula tothe descendant isolates cultured duringeach infection trial. For this analysisrelatedness was determined by comparingthe correlation (determined from thePearson product moment correlation ma-trix) of the AFLP profile generated from asingle P. multocida isolate from each birdto the AFLP profiles of both parentalinocula used within the infection trialisolation room. The inoculum with themost closely correlated AFLP profile to adescendant isolate was identified as theparent. For room 4, five isolates werelikely descendants of inoculum 7, and 15

isolates were likely descendants of inocu-lum 8; for room 5, four isolates were likelydescendants of inoculum 3, and sevenisolates were likely descendants of inocu-lum 4; for room 6, no likely descendants ofinoculum 1 were isolated, but 19 isolateswere likely descendants of inoculum 2.For each infection trial one isolate wasequally correlated to both inocula used,and these isolates were excluded from thisanalysis. A plot (not shown) of thecorrelation of each bacterial isolate to themost closely related inoculum within eachinfection trial yielded a line described bythe following equation (correlation toparent520.0053week of isolation+0.89).

FIGURE 2. Principal components analysis of Pasteurella multocida serotype 1 isolates cultured fromMallards during a laboratory infection trial. Isolates from birds that died are represented by filled symbols,and isolates from live, apparently healthy birds are represented by open symbols. Quadrants I–IV are labeled,and inocula 1, 2, 3, 4, 7, and 8 are identified numerically and with symbols (gray) indicating in which roomthey were used: room 4 (diamonds), room 5 (squares), and room 6 (circles).



This analysis indicated that over time thecorrelation of parental inocula to descen-dant isolates decreased (slope520.005;P50.046). Serial P. multocida isolationsfrom individual birds were insufficient toevaluate genetic changes in the bacteria atgreater resolution than the populationlevel.

Analysis of P. multocida serotype 1 isolates fromwild birds and environmental samples

One-hundred-twenty serotype 1 isolatesfrom a variety of wild bird species (72total; Table 1) and environmental samples(48 total; Table 2) were examined usingthe AFLP technique. Isolate collectionlocations included California (89 isolates),Hawaii (one isolate), Iowa (two isolates),Missouri (one isolate), Nebraska (14isolates), Utah (12 isolates), and Wisconsin(one isolate). The predominant bird spe-cies from which P. multocida was culturedincluded Eared Grebes (Podiceps nigri-collis; 20 isolates), Snow Geese (Chencaerulescens; 13 isolates), and RuddyDucks (Oxyura jamaicensis; 12 isolates).To facilitate the comparison of bacterialisolates by collection date, the followingcategories were established: January 1981to March 1997 (20 isolates), November1997 to April 1998 (62 isolates), October1998 to March 1999 (20 isolates), andJanuary 2000 to October 2004 (18 iso-lates). These categories were determinedbased on the availability of isolates sur-rounding significant avian cholera mortal-ity events from 1997 to 1999. The largestgroup of bird and environmental sampleswas collected in central California (61isolates) during the winter of 1997/1998(54 of the 61 isolates).

As indicated on the dendrogram(Fig. 3a), the isolates segregated into fourmajor clusters (labeled 1–4) consisting of12 branches (labeled A–L). Cluster 1(branches A and B) contained six isolates(three bird and three environmental) fromCalifornia (three isolates) and Nebraska(three isolates; Fig. 3a). Cluster 2 (branchesC–F) contained 47 isolates (38 bird and

nine environmental) from California (27isolates), Iowa (one isolate), Missouri (oneisolate), Nebraska (nine isolates), Utah(eight isolates), and Wisconsin (one iso-late; Figs. 3a and 3b). Cluster 3 (branchesG–K) contained 66 serotype 1 isolates (31bird and 35 environmental) from Califor-nia (59 isolates), Iowa (one isolate),Nebraska (two isolates), and Utah (fourisolates; Figs. 3a and 3c). A serotype 3type strain was located on divergentbranch K within cluster 3. A singleserotype 1 isolate cultured from a Hawai-ian Coot (Fulica americana alai) was theonly member of cluster 4 (branch L).

Amplified fragment length polymor-phism results were analyzed to determinewhether dendrogram branching patternswere related to collection locations orcollection dates (Fig. 3a). Collection loca-tion categories analyzed included centralCalifornia, the Salton Sea in southernCalifornia, the Midwest (Iowa, Missouri,Nebraska, and Wisconsin, collectively),and the Great Salt Lake in Utah. CentralCalifornia isolates (51/61) allocated dis-proportionately to cluster 3 (Figs. 3a and3c; P,0.001). Salton Sea (19/25) andMidwestern isolates (12/18) allocated dis-proportionately to cluster 2 (Figs. 3a and3b; P,0.001). The majority of Great SaltLake isolates (8/12) allocated to cluster 2,but this result was not determined to besignificant (P50.22). Analysis of isolatesbased on collection dates indicated thatisolates collected between January 1981and March 1997 (14/20) allocated dispro-portionately to cluster 2 (Figs. 3a and 3b;P,0.001). Isolates collected between No-vember 1997 and April 1998 (50/62)allocated disproportionately to cluster 3(Figs. 3a and 3c; P,0.001). Although themajority of isolates collected betweenOctober 1998 and March 1999 (12/20)and between January 2000 and October2004 (11/18) allocated within cluster 2,these groupings were not determined tobe significant (P50.16 and 0.21, respec-tively).

Principal components analysis of the



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wild bird and environmental isolate AFLPdata using components 2 and 3 supportedthe allocation of isolates into the samebranch-based clusters observed on thedendrogram (data not shown). Whenexamined based on collection location,PCA supported the existence of a visuallydistinct cluster of isolates from centralCalifornia (Fig. 4, open circles; P,0.001for component 2). This same cluster,consisting primarily of isolates culturedfrom samples collected between Novem-ber 1997 and April 1998, was alsoapparent by PCA when samples wereidentified based on collection date(P,0.001 for component 2; data notshown). Statistical comparison of thecontributions of regional (P,0.001 forcomponent 2) and temporal (P50.026 forcomponent 2) genetic characteristics ofthe isolates to the observed clustersindicated that both exerted significantcontributions.

A more detailed analysis of the P.multocida isolates collected in centralCalifornia established that the majority ofthese isolates (48/61) allocated to branchesH (30 isolates) and I (18 isolates) of thedendrogram (Figs. 3a and 3c). Examina-tion of the central California isolates basedon specific collection locations (16 sitesdistributed among the Sacramento andSan Joaquin Valley Refuge Complexes)indicated that although branch H isolateswere cultured from samples collected atboth the Sacramento and San JoaquinValley Refuge Complex sites, branch Iisolates were only cultured from samplescollected at San Joaquin Valley RefugeComplex sites (Fig. 5). Furthermore,branch I isolates were exclusively culturedduring the 1997/1998 season (Tables 1and 2). In contrast, consistent with theirbroader local distribution within centralCalifornia, branch H isolates were alsoidentified at collection sites outside ofcentral California, including the SaltonSea, the Great Salt Lake, and the Mid-western United States (Tables 1 and 2 andT

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TABLE 2. Summary of P. multocida serotype 1 isolates cultured from environmental samples.

CollectionDendrogram

ID no.cBrancha Date State County Locationb Latitude Longitude

A 17 Mar 1996 NE Clay RWB WMD, Sandpiper 40u389N 97u589W —A 8 Jan 1998 CA Colusa Colusa NWR, T15A 39u099N 122u039W —A 3 Apr 1998 NE Phelps RWB WMD, Johnson 40u349N 99u209W —C 7 Jan 1998 CA Merced Los Banos WMA, Gadwall Unit 2 37u039N 120u479W 12C 7 Jan 1998 CA Merced Los Banos WMA, Gadwall Unit 2 37u039N 120u479W 14C 8 Jan 1998 CA Colusa Colusa NWR, T15A 39u099N 122u039W 18C 14 Jan 1998 CA Imperial Salton Sea NWR 33u119N 115u379W 41C 15 Jan 1998 CA Colusa Delevan NWR, T6 39u209N 122u079W 16C 15 Jan 1998 CA Colusa Delevan NWR, T6 39u209N 122u079W 15C 2 Feb 1998 CA Merced Merced NWR, Mud Slough Unit 5 37u049N 120u469W 13C 2 Apr 1998 NE Phelps RWB WMD, Funk 40u319N 99u139W 87E 3 Apr 1998 NE Phelps RWB WMD, Johnson 40u349N 99u209W 40G 23 Jan 1998 CA Colusa Delevan NWR, T16 39u199N 122u079W 92H 17 Mar 1996 NE Fillmore RWB WMD, Mallard Haven 40u279N 97u459W 99H 20 Mar 1997 NE Clay RWB WMD, Sandpiper 40u389N 97u589W 88H 25 Nov 1997 CA Colusa Colusa NWR, T15A 39u099N 122u039W 67H 7 Jan 1998 CA Merced Los Banos WMA, Gadwall Unit 2 37u039N 120u479W 70H 7 Jan 1998 CA Merced Los Banos WMA, Gadwall Unit 2 37u039N 120u479W 71H 7 Jan 1998 CA Merced Los Banos WMA, Gadwall Unit 3 37u029N 120u469W 82H 8 Jan 1998 CA Colusa Colusa NWR, T13A 39u099N 122u039W 60H 15 Jan 1998 CA Stanislaus San Joaquin River NWR, East

Page Lake37u409N 121u129W 62

H 15 Jan 1998 CA Stanislaus San Joaquin River NWR, EastPage Lake

37u409N 121u129W 63


37u409N 121u129W 64


37u409N 121u129W 68


37u409N 121u129W 69


37u409N 121u129W 72

H 15 Jan 1998 CA Colusa Delevan NWR, T6 39u209N 122u079W 81H 15 Jan 1998 CA Colusa Delevan NWR, T6 39u209N 122u079W 83H 15 Jan 1998 CA Stanislaus San Joaquin River NWR, East


H 15 Jan 1998 CA Stanislaus San Joaquin River NWR, Page Lake 37u409N 121u129W 104H 23 Jan 1998 CA Colusa Delevan NWR, T16 39u199N 122u079W 98H 30 Jan 1998 CA Colusa Delevan NWR, T15A 39u199N 122u059W 61H 3 Feb 1998 CA Merced Los Banos WMA, Gadwall Unit 1 37u039N 120u479W 80H 3 Feb 1998 CA Merced Los Banos WMA, Gadwall Unit 1 37u039N 120u479W 106H 4 Feb 1998 CA Merced Merced NWR, Arena Plains Unit 37u179N 120u449W 105I 7 Jan 1998 CA Merced Los Banos WMA, Gadwall Unit 2 37u039N 120u479W 112I 7 Jan 1998 CA Merced Los Banos WMA, Gadwall Unit 2 37u039N 120u479W 115I 7 Jan 1998 CA Merced Los Banos WMA, Gadwall Unit 2 37u039N 120u479W 116I 15 Jan 1998 CA Stanislaus San Joaquin River NWR, Page Lake 37u409N 121u129W 102I 15 Jan 1998 CA Stanislaus San Joaquin River NWR, Page Lake 37u409N 121u129W 111I 15 Jan 1998 CA Stanislaus San Joaquin River NWR, East


I 2 Feb 1998 CA Merced Merced NWR, Mud Slough Unit 5 37u049N 120u469W 101I 2 Feb 1998 CA Merced Merced NWR, Mud Slough Unit 5 37u049N 120u469W 120I 3 Feb 1998 CA Merced Los Banos WMA, Gadwall Unit 1 37u039N 120u479W 118I 3 Feb 1998 CA Merced Los Banos WMA, Gadwall Unit 1 37u039N 120u479W 121I 4 Feb 1998 CA Merced Merced NWR, Arena Plains Unit 37u179N 120u449W 103



Fig. 3c). Collection dates for all branch Hisolates ranged from 1996 to 2004.

DISCUSSION

This work demonstrated that P. multo-cida serotype 1 isolates are geneticallyheterogeneous and that AFLP analysis is auseful technique for distinguishing them.Amplified fragment length polymorphismanalysis of serotype 1 isolates culturedduring a 3-mo laboratory infection trial ofMallards (Samuel et al., 2003a) indicatedthat the bacteria underwent detectablegenetic changes during the trial. Temporalgenetic change among the isolates wasdemonstrated by graphic analysis (data notshown), which indicated a decrease in thecorrelation of each isolate cultured duringthe infection trial to its most closelyrelated parent. The y-intercept (0.89)indicated that the isolates at the beginningof the trial were similar to the parentalinocula within the resolution of thetechnique, 0.92 (SD50.05). By the endof the trial, however, descendent isolateswere 0.83 correlated to the parentalinocula. The decrease in the correlationof descendant to parental AFLP patternsover the course of the infection trial isconsistent with the finding that as a resultof single nucleotide changes and geneticrecombination, bacterial genomes are in acontinuous state of flux (Levin and Berg-strom, 2000). Analyses to determinewhether the observed genetic changesamong the P. multocida isolates examinedaffected functional properties (e.g., viru-lence) of the bacteria were beyond the

scope of this project. However, genomeplasticity may enhance a bacterium’sability to adapt to diverse natural environ-ments (Earl et al., 2007), and geneticchange leading to fluctuations in P.multocida virulence levels provides apossible mechanism to explain observedtemporal variations in North American P.multocida restriction enzyme fingerprints(Wilson et al., 1995b; Samuel et al., 2003b)and avian cholera mortality patterns (Lehret al., 2005; Blanchong et al., 2006).Unlike previously employed techniquesfor differentiating P. multocida serotype1 isolates, including restriction enzyme(Wilson et al., 1995a, b; Samuel et al.,2003b) and serotyping (Rhoades andRimler, 1991) analyses, AFLP analysisdoes not currently allow the ready assign-ment of isolates to predefined groupings.It does, however, allow comparison of therelationships among collections of P.multocida isolates, and provides a contextfor genetic changes that accumulate overthe short term (during a disease outbreak)in contrast to geographic and temporalgenetic differences among P. multocidafrom one outbreak season to the next.

A pattern revealed by both dendrogram(Fig. 1) and principal components (Fig. 2)analyses of the infection trial AFLP datawas the presence of a cluster of P.multocida isolates cultured from live birdsdistinct from those cultured from deadbirds. We hypothesize that this patternresulted from genetic drift leading todivergence of the P. multocida isolatescultured from live birds later in the trialcompared to isolates cultured from dead

CollectionDendrogram

ID no.cBrancha Date State County Locationb Latitude Longitude

I 4 Feb 1998 CA Merced Merced NWR, Arena Plains Unit 37u179N 120u449W 119K 24 Nov 1997 CA Siskiyou Tule Lake NWR 41u559N 121u399W 36

a Designations refer to dendrogram branches defined in Figure 3a.b NWR 5 national wildlife refuge; RWB WMD 5 Rain Water Basin wetlands management district; WMA 5 wildlife

management area.c Numbers refer to isolate identifiers in Figures 3b and 3c.

TABLE 2. Continued.



birds during the first 2 wk. Additionally,segregation of all trial 6 isolates along withinoculum 2 from the other inocula andisolates was consistent with the determi-nation that 19 of 20 trial 6 isolates likelydescended from inoculum 2. Thus, theability to distinguish identified descen-dants of inoculum 2 throughout theinfection trial demonstrated the utility ofAFLP analysis to track bacteria withdistinct genetic characteristics.

The capacity of AFLP analysis todistinguish P. multocida serotype 1 isolatesduring epidemiologic investigations ofavian cholera outbreaks was demonstratedby using this technique to characterize 120isolates cultured from wild birds (Table 1)and environmental samples (Table 2). The

most distinct epidemiological cluster of P.multocida isolates examined originatedfrom samples collected in central Califor-nia (Figs. 4 and 5), and the majority ofisolates from this region, regardless ofwhether they were collected from birds orfrom environmental samples, allocated tobranches H and I of the dendrogram(Figs. 3a and 3c). The inability to distin-guish P. multocida isolates originatingfrom birds or the environment by AFLPanalysis is consistent with the transmissionof bacterial isolates between birds andtheir environment within an outbreak area(Samuel et al., 2004). Within centralCalifornia, branch H isolates exhibited awider distribution pattern than branch Iisolates (Fig. 5). The wider spatial and

FIGURE 3. Dendrogram analyses of Pasteurella multocida serotype 1 isolates cultured from wild birds andenvironmental samples. (a) Dendrogram in which each branch was truncated at its root representative of allisolates included in this study. Four major clusters (1–4) and 12 branches (A–L) are labeled. Locationcategories (C.CA 5 central California, GSL 5 Great Salt Lake, HI 5 Hawaii, Mid 5 Midwestern states,N.CA 5 northern California, and SS 5 Salton Sea) for each group of isolates are labeled. The number ofisolates from each location category precedes the location designation. (b) and (c) Details of all isolates withinclusters 2 and 3, respectively. Symbols indicate the source of each isolate, and dendrogram identificationnumbers are defined in Tables 1 and 2.



temporal distribution of branch H isolatesin central California was also consistentwith a more diverse national distributionpattern for branch H isolates. Samuel etal. (2005) demonstrated that healthy wildwaterfowl have the potential to transmit P.multocida to other birds and locations.Accordingly, the wider spatial and tempo-ral distribution of branch H isolates, bothlocally and nationally, may have resulted

from the movements of carrier birds.Identification of a group of bacterialisolates primarily associated with a distinctgeographic region and a single seasonindicates that AFLP analysis provides apotentially useful tool to further elucidatethe transmission of P. multocida betweenbird species, across the landscape, andover time.

Both dendrogram (Figs. 3a and 3b) andprincipal components (Fig. 4) analysesrevealed that Great Salt Lake and SaltonSea P. multocida isolates clustered togeth-er. The majority of Great Salt Lakeisolates analyzed for this study werecultured from Eared Grebes, while SaltonSea isolates were primarily cultured fromEared Grebes and Ruddy Ducks (Ta-ble 1). Large numbers of Eared Grebesmove between the Great Salt Lake and theSalton Sea each year (Jehl, 1993; Jehl etal., 1999). Thus, interactions betweenGreat Salt Lake and Salton Sea EaredGrebe populations may facilitate thetransmission of disease agents betweenthese two sites resulting in genetic ho-mogenization, as measured by AFLPanalysis, of the P. multocida isolatescultured from waterfowl of this geographicregion. Application of AFLP analysis to P.multocida isolates from additional sitesalong waterfowl migratory routes mayprovide information on the transmissionof avian cholera useful to wildlife resourcemanagers for predicting where futuredisease outbreaks might occur.

The previously perceived homogeneityof P. multocida serotype 1 has been alimiting factor in understanding the epi-demiology of avian cholera infections inwild bird populations (Samuel et al.,2007). Using AFLP analysis, we havedemonstrated that the P. multocida ge-nome is subject to genetic drift and thatthere is sufficient diversity among P.multocida serotype 1 isolates to distinguishregional and temporal epidemiologic pat-terns. The utility of this technique fortracing avian cholera outbreaks amongwild birds was most clearly exemplified

FIGURE 3. Continued.



by the group of genetically distinct P.multocida isolates originating from centralCalifornia bird and environmental sam-ples. Further application of AFLP tech-nology to the study of avian choleraepidemiology has the potential ability tolink bacterial isolates from the environ-ment to those from infected hosts, todetermine spatiotemporal patterns of iso-

late types, to identify specific affiliationsbetween P. multocida genotypes and birdspecies, to correlate virulence with bacte-rial genotypes, and to understand the roleof specific bird species in transmitting orspreading this disease. Accomplishment ofthese goals will provide wildlife resourcemanagers with the means to predict localbird population impacts during ongoing

FIGURE 4. Principal components analysis of Pasteurella multocida serotype 1 isolates cultured from wildbirds and environmental samples. Each isolate is designated according to its collection location; the clusterencompassing 49 of the 61 central California P. multocida isolates is circled.



FIGURE 5. Distribution of Pasteurella multocida serotype 1 isolates cultured from wild birds andenvironmental samples collected in central California. Isolates are represented on the map according to theircollection location using dendrogram branch designations from Figure 3a. Collection sites are indicatedwith triangles.



avian cholera die-offs, to identify birdspecies that may be at greatest risk forinfection during a mortality event, and tocorrelate bird movement patterns with thepotential for disease spread. Amplifiedfragment length polymorphism analysispromises to be a valuable tool to bothenhance understanding of the epidemiol-ogy of avian cholera infection and moreeffectively manage this disease among wildbirds.

ACKNOWLEDGMENTS

This work was supported by the Salton SeaAuthority through Wisconsin CooperativeWildlife Research Unit Research Work Order83 and by the USGS Status and TrendsProgram through the Office of the EasternRegional Executive for Biology. We thank C.Franson (USGS–National Wildlife HealthCenter), H. Ip (USGS–National WildlifeHealth Center), and C. Soos (EnvironmentCanada) for providing helpful suggestionsduring the preparation of this manuscript.

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Received for publication 9 April 2007.



Documents

USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS … · GAC TGC GTA CCA ATT C-3 and 5 -GAT GAG TCC TGA GCG T-3 , respectively, in a final volume of 50 m l. PCR reactions contained