BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, researchlibraries, and research funders in the common goal of maximizing access to critical research.

Immune Response to Recombinant Escherichia coli Iss Protein in PoultryAuthor(s): Aaron M. Lynne, Steven L. Foley, Lisa K. NolanSource: Avian Diseases, 50(2):273-276. 2006.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/7441-092105R.1URL: http://www.bioone.org/doi/full/10.1637/7441-092105R.1

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, andenvironmental sciences. BioOne provides a sustainable online platform for over 170 journals and books publishedby nonprofit societies, associations, museums, institutions, and presses.

Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance ofBioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.

Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiriesor rights and permissions requests should be directed to the individual publisher as copyright holder.

Research Note—

Immune Response to Recombinant Escherichia coli Iss Protein in Poultry

Aaron M. Lynne,A Steven L. Foley,B and Lisa K. NolanAC

ADepartment of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50010BMarshfield Clinic Research Foundation, Marshfield, WI 54449

Received 21 September 2005; Accepted 6 December 2005

SUMMARY. Colibacillosis accounts for significant losses to the poultry industry, and control efforts are hampered by limitedunderstanding of the mechanisms used by avian pathogenic Escherichia coli (APEC) to cause disease. We have found that thepresence of the increased serum survival gene (iss) is strongly associated with APEC but not with commensal E. coli, making iss, andthe protein it encodes (Iss), candidate targets of colibacillosis control procedures. To assess the potential of Iss to elicit a protectiveresponse in chickens against APEC challenge, Iss fusion proteins were produced and administered subcutaneously to four groups of2-wk-old specific-pathogen-free leghorn chickens. At 4 wk postimmunization, birds were challenged with APEC from serogroupsO2 and O78 via intramuscular injection. At 2 wk postchallenge, birds were necropsied, and lesions consistent with colibacillosiswere scored. Also, sera were collected from the birds pre- and postimmunization, and antibody titers to Iss were determined.Immunized birds produced a humoral response to Iss, and they had significantly lower lesion scores than the unimmunized controlbirds following challenge with both APEC strains. Birds that received the smallest amount of immunogen had the lowest lesionscores. Although further study will be needed to confirm the value of Iss as an immunoprotective antigen, these preliminary datasuggest that Iss may have the potential to elicit significant protection in birds against heterologous E. coli challenge.

RESUMEN. Nota de Investigacion—Repuesta inmune a la proteına recombinante Iss de Escherichia coli en aves domesticas.La colibacilosis es responsable de perdidas significativas en la industria avıcola y los esfuerzos por controlarla se ven obstaculizados

por el desconocimiento de los mecanismos utilizados por la Escherichia coli patogena aviar de causar la enfermedad. Nosotros hemosencontrado que la presencia del gen de sobrevivencia serica incrementada esta fuertemente asociado con la Escherichia coli patogenaaviar pero no con E. coli comensales, lo cual convierte a este gen y a la proteına para la cual codifica (proteına de sobrevivencia sericaincrementada) en un objetivo para los procedimientos de control de la colibacilosis. Con la finalidad de evaluar el potencial de laproteına de sobrevivencia serica incrementada de generar una respuesta protectora contra un desafıo con Escherichia coli patogenaen pollos, se generaron proteınas de fusion de la proteına de sobrevivencia serica incrementada que fueron administradassubcutaneamente a cuatro grupos de pollos leghorn libres de patogenos especıficos de dos semanas de edad. La aves fuerondesafiadas por inyeccion intramuscular a las cuatro semanas de edad con Escherichia coli patogena de los serogrupos O2 y O78. Lasaves se sacrificaron dos semanas posteriores al desafıo y se tomo registro de las lesiones consistentes con colibacilosis. Ademas, setomaron muestras de suero antes y despues de la inmunizacion y se determinaron los tıtulos de anticuerpos contra la proteına desobrevivencia serica incrementada. Luego del desafıo con ambas cepas de Escherichia coli patogenas, las aves inmunizadas generaronuna respuesta humoral contra la proteına de sobrevivencia serica incrementada y mostraron valores de lesiones menores que las avescontrol no inmunizadas. Las aves que recibieron la menor cantidad de inmunogeno tuvieron los registros de lesiones mas bajos.Aunque estudios adicionales seran necesarios para confirmar el valor de la proteına de supervivencia incrementada en suero comoun agente inmunoprotector, el presente estudio sugiere que la proteına de sobrevivencia serica incrementada puede poseer elpotencial de generar proteccion significativa en aves contra desafıos heterologos de E. coli.

Key words: APEC, avian colibacillosis, immunization, Iss

Abbreviations: APEC ¼ avian pathogenic E. coli; cAMP ¼ cyclic adenosine monophosphate; CFU ¼ colony-forming unit;ELISA ¼ enzyme-linked immunosorbant assays; GST ¼ glutathione S-transferase; GST-Iss ¼ Iss protein fused to glutathioneS-transferase; IPTG ¼ isopropyl-beta-D-thiogalactopyranoside; LB ¼ Luria–Bertani; PBS ¼ phosphate-buffered Saline; PBSt ¼phosphate-buffered saline with 0.05% Tween 20; SDS-PAGE ¼ sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Colibacillosis, caused by avian pathogenic Escherichia coli(APEC), is a major problem for the poultry industry in the UnitedStates resulting in significant annual losses (2). One of the problemsin colibacillosis control is that no single bacterial trait has beenidentified that can be used as an identifier of virulent avian isolates(2) or as a target of control strategies. Previous work showed thatcomplement resistance may play an important role in APECvirulence (14,19,20,28), and the increased serum survival gene or iss(4), which is associated with E. coli complement resistance (4,6,7),was found significantly more often in APEC than it was in the E. coliisolates of apparently healthy birds (22,23). This strong association

between iss and APEC suggested that iss-centric strategies might beuseful in colibacillosis control.

For example, Iss, the protein encoded by iss (3,13), might beuseful as an immunogen capable of eliciting a protective responseagainst APEC infection in birds. If Iss could stimulate animmunoprotective response in birds, it might have wide-rangingbenefits, because iss is found in APEC of many serogroups and inAPEC isolated from various lesion types, avian host species, andforms of colibacillosis (22,23). This widespread distribution of issamong APEC suggests that an Iss-based vaccine could provide broadprotection to birds against heterologous APEC challenge. Computeranalysis of Iss’ predicted amino acid sequence has suggested thatmany portions of Iss are antigenic (13), and Iss is thought to beexposed on the bacterial surface in intact E. coli (3), meaning that itis accessible to the host’s immune system. Such observations suggestCCorresponding author.

AVIAN DISEASES 50:273–276, 2006

273

that Iss may have the ability to evoke an immunoprotective responsein birds against APEC that would have wide application.Consequently, in the present study, Iss was expressed and purified,and its potential to elicit an immunoprotective response againstheterologous APEC challenge in chickens was assessed.

MATERIALS AND METHODS

Bacterial strains and plasmids. Strains used were E. coli BL21(DE3) (Stratagene, La Jolla, CA), E. coli APEC-C-O2, and E. coliAPEC-C-O78. Escherichia coli BL21 was used for expression of GST-Issand E. coli APEC-C-O2 and APEC-C-O78 were used in experimentalchallenges. APEC-C-O2 is an APEC of the O2 serogroup, and APEC-C-O78 is an APEC of the O78 serogroup. The O2 and O78 serogroupsare among the most common ones causing disease in birds (25). Allstrains were maintained on Luria–Bertani (LB) agar (Difco, Detroit, MI)with ampicillin (100 lg/ml; Amresco, Solon, OH) where appropriate.pGEX-6P-3 (Pharmacia Biotech, Piscataway, NJ), a plasmid expressionvector designed for production of proteins fused to glutathione-S-transferase (GST), was used in these studies. pLN330 is composed ofpGEX-6P-3 with the iss gene sequence (GenBank accession numberAF0422279) without the coding region for the signal sequence (the first72 nucleotides of iss) (11). Using pLN330, Iss protein, fused toglutathione S-transferase (GST-Iss), can be expressed in E. coli BL21.Expression and solubilization of Iss fusion proteins.

Expression of GST-Iss from pLN330 was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) as previously described (11). Inducedcells were lysed with B-PER bacterial protein extraction reagent (Pierce,Rockford, IL) according to manufacturer’s instructions. Inclusion bodiescontaining GST-Iss were solubilized with inclusion body solubilizationreagent according to manufacturer protocols (Pierce). Protein wasrefolded by dialysis against decreasing concentrations of urea and finallyagainst Tris buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.5).Protein purification. Purification of GST-Iss and Iss was achieved

through use of an affinity matrix. The refolded protein was combinedwith a 50% slurry of glutathione sepharose 4B beads (Pharmacia) andincubated for 1 hr at 4 C. The mixture was added to a 10-cm3 column,and the ‘‘flow-through’’ was collected. The column was washed threetimes with phosphate-buffered saline (PBS). For GST-Iss purification,the fusion protein was eluted with glutathione elution buffer (10 mMglutathione, 50 mM Tris, pH 8.0). For Iss purification, sepharose beadswith bound fusion protein were resuspended in PreScission cleavagebuffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA,1 mM dithiothreitol) (Pharmacia). The fusion protein was cleavedusing PreScission protease (Pharmacia). The mixture was added to a 10-cm3 column, and Iss was eluted with cleavage buffer. Purification ofthe protein was assessed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) (18). Protein samples were separated on15% acrylamide Tris-HCl gels (Bio-Rad, Hercules, CA) and stainedwith Blue-Bandit protein stain (Amresco, Solon, OH).Test animals. One hundred twenty-eight 2-wk-old specific-

pathogen-free leghorn chickens were obtained from Charles RiverLaboratories (Boston, MA). All chickens were from the same hatch.

Birds were divided into eight groups of 16 as indicated in Table 1. Birdswere placed in stainless steel HEPA-filtered negative-pressure isolators1 wk before vaccination. Birds were fed and watered ad libitum andwere handled according to Institution Animal Care and Use protocols.Serology. Three days before vaccination and challenge, birds from

groups 1A, 2A, 3A, and 4A were bled from a wing vein to obtain serumsamples. Sera were used to perform antigen-specific enzyme-linkedimmunosorbant assays (ELISA) to determine the presence of antibodiesspecific for GST-Iss and purified Iss using methods previously described(10). Briefly, coating antigen (either GST-Iss or Iss) was diluted to 2–10lg/ml in coating buffer (0.02 M carbonate, 0.03 bicarbonate, pH 9.6).The wells were blocked with 3% solution of bovine serum albumin,dissolved in coating buffer. To determine antibody titers, sera wereserially diluted in phosphate-buffered saline with 0.05% Tween 20(PBSt). Following incubation with primary antibody, the antibody wasremoved and plates were washed with PBSt and incubated with goatanti-chicken alkaline-phosphatase–conjugated IgG (Sigma, St. Louis,MO). The plates were washed with PBSt and developed with P-nitrophenyl phosphate. Color development was recorded at 405 nmusing an automated ELISA plate reader (model ELx808; Biotek,Winooski, VT.).Vaccine preparation and immunization procedure. Im-

munization protocol. Each bird was given 0.5 ml of a water-in-oilemulsion containing either 50 lg, 10 lg, or 2 lg of GST-Iss per dose.At 3 wk of age, each chicken in groups 1A, 1B, 2A, 2B, 3A, and 3Breceived a 0.5-ml dose of the vaccine (Table 1). Birds in groups 4A and4B were not vaccinated (nonvaccinated controls). The injections weregiven subcutaneously in the back of the neck at the midpoint betweenthe head and body.Challenge infection. Four weeks following vaccination, each bird

was subjected to challenge with an APEC strain. Each bird in each groupwas given a 1.0-ml intramuscular injection of either 108.72 colony-forming units (CFUs) of APEC-C-O2 or 108.89 CFUs of APEC-C-O78, as indicated in Table 1. Birds were observed for 14 days followingchallenge. Birds that died were necropsied and observed for lesionsconsistent with colibacillosis; cultures of bone marrow were taken forbacterial isolation on eosin methylene blue agar. Presumptive isolateswere verified with API 20e strips (bioMerieux, Durham, NC). After theobservation period, birds that survived were euthanatized, necropsied,and observed for lesions consistent with colibacillosis. Lesions consistentwith colibacillosis, including mild or severe airsacculitis, perihepatitis,pericarditis, and/or death were scored according to a system outlinedin Table 2.Biostatistics. Efficacy of the vaccine against challenge was evaluated

by average lesion scores of vaccinates, as compared to unvaccinatedbirds. Data were analyzed by the chi-square 2-by-2 test with thesignificance at the P � 0.05 level. For a valid test, the average lesionscore of the positive control group must have been �2.0.

RESULTS

As previously described, iss was cloned into pGEX-6P-3 toproduce a GST-Iss fusion protein (11). GST-Iss expression wasinduced with IPTG; the fusion was solubilized with commercial kits,purified by affinity chromatography, and its identity confirmed by

Table 1. Vaccine level and challenge serogroup.

GroupNumberof birds

Vaccine level(lg/dose)

Challengeserogroup

1A 16 50 O21B 16 50 O782A 16 10 O22B 16 10 O783A 16 2 O23B 16 2 O784A 16 0 O24B 16 0 O78

Table 2. Scoring system.

Score Lesions

0 No lesion1 Cloudy air sacs (mild airsacculitis)2 Fibrinous airsacculitis (severe airsacculitis)3 Fibrinous airsacculitis and pericarditis4 Fibrinous airsacculitis, pericarditis, perihepatitis, or death

(must reisolate E. coli from the bone marrow of thefemur for confirmation if dead)

274 A. M. Lynne et al.

SDS-PAGE (Fig. 1). Purified Iss was obtained by cleaving Iss fromthe GST fusion partner and confirmed by SDS-PAGE (Fig. 1).

Chickens immunized with GST-Iss were able to generatea humoral immune response against GST-Iss and purified Iss ascompared to controls. Birds that received a higher dose of GST-Isshad average antibody titers of 10,000 to 100,000 against GST-Iss,whereas birds that received a lower dose had average titers of 1000(Table 3). Sera from birds also recognized purified Iss. Birds thatreceived a high dose of fusion protein had average antibody titers of10,000 against purified Iss, whereas birds that received lower doseshad lower average titers of 1000 (Table 3).

During the observation period following challenge, no birds diedthat were challenged with APEC-C-O2. For birds challenged withAPEC-C-O78, no birds died in group 1B; four birds died in group2B (three of which cultured positive for E. coli); one bird, which wasculture-positive for E. coli, died in group 3B; and two birds, bothof which were culture-positive for E. coli, ied in group 4B. Birdshad lower lesion scores when immunized with GST-Iss, regardless ofthe challenge strain used, as compared to controls. Among thevaccinated birds, those that received smaller doses of GST-Iss hadlower lesions scores when challenged with APEC of the O2 or O78serogroups (Table 4).

DISCUSSION

As previously stated, avian colibacillosis is a costly disease to thepoultry industry, accounting for multimillion-dollar losses annually(2), making efficacious colibacillosis control measures highlydesirable. Colibacillosis control frequently focuses on managementapproaches designed to reduce predisposing conditions amongproduction birds, such as viral or mycoplasma infections (2).However, management strategies that have controlled colibacillosisin the past may not be as effective in the future. Also, use ofantimicrobial agents in animal production is being given closescrutiny at this time with limitations being placed on the use of

certain therapeutic agents in poultry production (26,27). Conse-quently, control of avian colibacillosis using vaccines in certainsituations may prove desirable.

Thus far, vaccines designed to prevent avian colibacillosis havebeen met with mixed results. Vaccines against APEC of variousserogroups have been produced (1,5,8,9,12,15,16,17,21,24). Manyof these have only been effective against homologous challenge.Kwaga et al. (17) attenuated a strain of APEC O2 serogroup bymutating the carAB operon that encodes genes for carbamoyl-phosphophatase synthetase, essential for arginine and pyrimidinebiosynthesis. This vaccine was effective in turkeys against a homol-ogous APEC challenge but not a heterologous challenge. In a studyby Peighambari and colleagues (21), APEC strains of O2 and O78serogroups were attenuated by deletion of cya and crp, which encode39,59-cyclic adenosine monophosphate (cAMP) and cAMP receptorprotein, respectively. The �cya and �crp mutant of the E. coli O2strain provided protection in chickens challenged with a homologousstrain, but again, not against heterologous challenge. This failure toprotect against heterologous challenge may prove a critical limitationof colibacillosis vaccines because APEC are quite diverse in terms ofserogroup (23).

Targeting a surface-exposed structure found in most APEC,regardless of serogroup, would seem to be a logical approach to thedevelopment of a colibacillosis vaccine. It is thought that the wide-spread distribution of iss/Iss across APEC may avoid the limitationsof some of these other vaccines. The iss gene is strongly associatedwith APEC of various serogroups but not with fecal E. coli isolatesfrom apparently healthy birds (22,23), making iss, and the protein itencodes, a potential marker for virulent avian E. coli. iss was clonedinto the plasmid pGEX-6P-3, and the resulting construct was used

Fig. 1. SDS-PAGE of the proteins used in this study. (A) SDS-PAGE of GST-Iss obtained after expression of pLN330 in E. coli BL21,followed by lysis of the cells with B-PER, solubilization of the proteinwith inclusion-body solubilization reagent, and purification withglutathione sepharose 4B. Lane S ¼ molecular size standard; lane 1 ¼E. coli BL21 cell lysate; lane 2 ¼ E. coli BL21, containing pLN330,following induction of expression with IPTG; and lane 3 ¼ purifiedGST-Iss. (B) SDS-PAGE of purified protein (Iss) obtained after cleavageof GST-Iss with PreScission protease. Lane S¼molecular size standard;lane 1 ¼ E. coli BL21 cell lysate; lane 2 ¼ E. coli BL21, containingpLN330, following induction with IPTG; and lane 3 ¼ purified Iss.

Table 3. Serum antibody titers.

Group(dose)

Anti-GST-Iss serumantibody titers

Anti-Iss serumantibody titers

Average titerpreimmu-nization

Average titerpostimmu-nization

Average titerpreimmu-nization

Average titerpostimmu-nization

1A (50 lg) 102 105 102 104

2A (50 lg) 102 104 102 104

3A (2 lg) 102 103 102 103

4A (none) 102 102 02 102

Table 4. Average lesion scores.

Vaccine level(lg/dose) Challenge

Average lesion score

O2 challengeserogroup

O78 challengeserogroup

2 Yes 0.38* 0.56A

10 Yes 0.75* 1.87B

50 Yes 1.31 1.63**None Yes 2 2.88C

None No 0 0AOne bird dead postchallenge and culture-positive for E. coli with no

macroscopic lesions.BFour birds dead postchallenge; three culture-positive for E. coli with

severe systemic lesions; and one culture-negative with no macroscopiclesions.

CTwo birds dead postchallenge and culture-positive for E. coli withsevere systemic lesions.*Significantly different from nonvaccinated controls (P � 0.05).**Significantly different from nonvaccinated controls (P � 0.05).

(The Mann-Whitney U-test was used to determine the statistic.)

Immune response to Iss 275

to produce a GST-Iss fusion protein. Because Iss alone is quite small,there was concern that it might not elicit a strong immune response.Therefore, GST was selected as a fusion partner in an effort to elicita stronger immune response. There was also some concern that themajority of antibodies elicited by GST-Iss would be directed againstGST and not Iss, because GST is approximately three times the sizeof Iss. However, birds immunized with GST-Iss were able to produceantibody titers against GST-Iss and Iss that were significantly dif-ferent from unimmunized controls. Also, Iss did stimulate animmunoprotective response against heterologous challenge. Para-doxically, lower doses seemed to offer better protection than didhigher doses, a result we cannot account for at this time. Much moreresearch will be needed to determine if use of iss/Iss based vaccinesand other control strategies are practical. Based on the results of thisstudy, we feel that an iss/Iss based control strategy is worthy of futureconsideration and study.

REFERENCES

1. Arp, L. H. Effect of passive immunization on phagocytosis of blood-borne Escherichia coli in spleen and liver of turkeys. Am. J. Vet. Res. 43:1034–1040. 1982.

2. Barnes, H. J., J. P. Vaillancourt, and W. B. Gross. Colibacillosis. In:Diseases of Poultry, 11th ed. Y. M. Saif, ed. Iowa State University Press,Ames, IA. pp. 631–652. 2003.

3. Barondess, J. J., and J. Beckwith. bor Gene of phage lambda, involvedin serum resistance, encodes a widely conserved outer membrane lipoprotein.J. Bacteriol. 177:1247–1253. 1995.

4. Binns, M. M., D. L. Davies, and K. G. Hardy. Cloned fragments ofthe plasmid ColV, I-K94 specifying virulence and serum resistance. Nature279:778–781. 1979.

5. Bolin, C. A., and A. E. Jensen. Passive immunization with antibodiesagainst iron-regulated outer membrane proteins protects turkeys fromEscherichia coli septicemia. Infect. Immun. 55:1239–1242. 1987.

6. Chuba, P. J., M. A. Leon, A. Banerjee, and S. Palchaudhuri. Cloningand DNA sequence of plasmid determinant iss, coding for increased serumsurvival and surface exclusion, which has homology with lambda DNA.Mol. Gen. Genet. 216:287–292. 1989.

7. Chuba, P. J., S. Palchaudhuri, and M. A. Leon. Contributions of traTand iss genes to the serum resistance phenotype of plasmid ColV2-K94.FEMS Microbiol. Lett. 37:135–140. 1986.

8. Deb, J. R., and E. G. Harry. Laboratory trials with inactivatedvaccines against Escherichia coli (O78 K80) infection in fowls. Res. Vet. Sci.20:131–138. 1976.

9. Deb, J. R., and E. G. Harry. Laboratory trials with inactivatedvaccines against Escherichia coli (O2:K1) infection in fowls. Res. Vet. Sci. 24:308–313. 1978.10. Foley, S. L., S. M. Horne, C. W. Giddings, T. R. Gustad, E. D.

Handegard, M. Robinson, and L. K. Nolan. Monoclonal antibodies to avianEscherichia coli Iss. Avian Dis. 47:79–86. 2003.11. Foley, S. L., S. M. Horne, C. W. Giddings, M. Robinson, and L. K.

Nolan. Iss from a virulent avian Escherichia coli. AvianDis. 44:185–191. 2000.

12. Gyimah, J. E., B. Panigrahy, and J. D. Williams. Immunogenicity ofan Escherichia coli multivalent pilus vaccine in chickens. Avian Dis. 30:687–689. 1986.13. Horne, S. M., S. J. Pfaff-McDonough, C. W. Giddings, and L. K.

Nolan. Cloning and sequencing of the iss gene from a virulent avianEscherichia coli. Avian Dis. 44:179–184. 2000.14. Ike, K., K. Kawahara, H. Danbara, and K. Kume. Serum resistance

and aerobactin iron uptake in avian Escherichia coli mediated by conjugative100-megadalton plasmid. J. Vet. Med. Sci. 54:1091–1098. 1992.15. Kariyawasam, S., B. N. Wilkie, and C. L. Gyles. Construction,

characterization, and evaluation of the vaccine potential of three geneticallydefined mutants of avian pathogenic Escherichia coli. Avian Dis. 48:287–299. 2004.16. Kariyawasam, S., B. N. Wilkie, D. B. Hunter, and C. L. Gyles.

Systemic and mucosal antibody responses to selected cell surface antigensof avian pathogenic Escherichia coli in experimentally infected chickens.Avian Dis. 46:668–678. 2002.17. Kwaga, J. K., B. J. Allan, J. V. van der Hurk., H. Seida., and A. A.

Potter. A carAB mutant of avian pathogenic Escherichia coli serogroup O2 isattenuated and effective as a live oral vaccine against colibacillosis in turkeys.Infect. Immun. 62:3766–3772. 1994.18. Laemmli, U. K. Cleavage of structural proteins during the assembly

of the head of bacteriophage T4. Nature 227:680–685. 1970.19. Nolan, L. K., S. M. Horne, C. W. Giddings, S. L. Foley, T. J.

Johnson, A. M. Lynne, and J. Skyberg. Resistance to serum complement, iss,and virulence of avian Escherichia coli. Vet. Res. Commun. 27:101–110.2003.20. Nolan, L. K., R. E. Wooley, and R. K. Cooper. Transposon

mutagenesis used to study the role of complement resistance in the virulenceof an avian Escherichia coli isolate. Avian Dis. 36:398–402. 1992.21. Peighambari, S. M., D. B. Hunter, P. E. Shewen, and C. L. Gyles.

Safety, immunogenicity, and efficacy of two Escherichia coli cya crp mutantsas vaccines for broilers. Avian Dis. 46:287–297. 2002.22. Pfaff-McDonough, S. J., S. M. Horne, C. W. Giddings, J. O. Ebert,

C. Doetkott, M. H. Smith, and L. K. Nolan. Complement resistance-relatedtraits among Escherichia coli isolates from apparently healthy birds andbirds with colibacillosis. Avian Dis. 44:23–33. 2000.23. Rodriguez-Siek, K. E., C. W. Giddings, C. Doetkott, T. J. Johnson,

and L. K. Nolan. Characterizing the APEC pathotype. Vet. Res. 36:241–256. 2005.24. Roland, K., K. Karaca, and D. Sizemore. Expression of Escherichia

coli antigens in Salmonella typhimurium as a vaccine to prevent airsacculitisin chickens. Avian Dis. 48:595–605. 2004.25. Sojka, W. J., and R. B. A. Carnaghan. Escherichia coli infection in

poultry. Res. Vet. Sci. 2:340–352. 1961.26. Sundolf, S. F. Enrofloxacin for poultry; opportunity for hearing. U.S.

Food and Drug Administration. Available from: http://www.fda.gov/OHRMS/DOCKETS/98fr/103100a.htm. 2000.27. Sundolf, S. F. Abbott Laboratories’ sarafloxacin for poultry;

withdrawal of approval of NADAs. Available from: http://www.fda.gov/OHRMS/DOCKETS/98fr/043001h.htm. 2001.28. Vidotto, M. C., E. E. Muller, J. C. de Freitas, A. A. Alfieri, I. G.

Guimaraes, and D. S. Santos. Virulence factors of avian Escherichia coli.Avian Dis. 34:531–538. 1990.

276 A. M. Lynne et al.