JournalofWildlifeDiseases,44(1),2008,pp.1–7 #WildlifeDiseaseAssociation2008 VACCINATION WITH F1-V FUSION PROTEIN PROTECTS BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) AGAINST PLAGUE UPON ORAL CHALLENGE WITH YERSINIA PESTIS Tonie E. Rocke,1,5SusanSmith,1 PaulMarinari,2JulieKreeger,3JeffreyT. Enama,4 and Bradford S.Powell4 1USGeologicalSurvey,NationalWildlifeHealthCenter,6006SchroederRoad,Madison,Wisconsin53711,USA 2USFishandWildlifeService,NationalBlack-FootedFerretConservationCenter,P.O.Box190,Wellington,Colorado 80549,USA 3USFishandWildlifeService,NationalBlack-FootedFerretConservationCenter,2362Highway34,Wheatland, Wyoming82201,USA 4USArmyMedicalResearchInstituteofInfectiousDiseases,BacteriologyDivision,FortDetrick,Frederick,Maryland 21702,USA 5Correspondingauthor(email:[email protected]) ABSTRACT: Previous studies have established that vaccination of black-footed ferrets (Mustela nigripes) with F1-V fusion protein by subcutaneous (SC) injection protects the animals against plague upon injection of the bacterium Yersinia pestis. This study demonstrates that the F1-V antigen can also protect ferrets against plague contracted via ingestion of a Y. pestis-infected mouse,aprobableroutefornaturalinfection.Eightblack-footedferretkitswerevaccinatedwith F1-V protein by SC injection at approximately 60 days-of-age. A booster vaccination was administered 3 mo later via SC injection. Four additional ferret kits received placebos. The animalswerechallenged6 wkaftertheboostbyfeedingeachoneaY.pestis-infectedmouse.All eight vaccinates survived challenge, while the four controls succumbed to plague within 3 days afterexposure.Todeterminethedurationofantibodypostvaccination,18additionalblack-footed ferretkitswerevaccinatedandboostedwithF1-VbySCinjectionat60and120 days-of-age.High titerstobothF1andV(meanreciprocaltitersof18,552and99,862,respectively)werefoundinall vaccinatesupto2 yrpostvaccination,whereassevencontrolanimalsremainedantibodynegative throughoutthesametimeperiod. Key words: Black-footed ferrets, F1-V protein, sylvatic plague, vaccine, Yersinia pestis. INTRODUCTION (Williams et al., 1994; Castle et al., 2001; Rocke et al., 2006). Plague, caused by the gram-negative In recent studies conducted at the US bacterium, Yersinia pestis, is a natural Geological Survey, National Wildlife threat to black-footed ferrets (Mustela Health Center (NWHC), we demonstrat- nigripes), and it has severely hindered ed that a majority of ferrets could be recent efforts to restore this endangered protected against subcutaneous (SC) Y. speciestoitshistoricrange(Barnes,1993). pestis challenge by vaccination with the The disease occurs regularly in rodents F1-V protein (Rocke et al., 2004, 2006), throughout the western USA and is a recombinant fusion protein that consists particularly devastating for prairie dogs of two known protective antigens ex- (Cynomys spp.). Ferrets, which feed pressed by Y. pestis (Powell et al., 2005). exclusively on prairie dogs, are likely Vaccinated animals that survived an initial exposed to Y. pestis by direct and indirect SC challenge with Y. pestis were com- association with their prey. They may be pletely resistant to a secondary exposure bitten by Y. pestis-infected fleas as they via consumption of a Y. pestis-infected huntandkillprairiedogsintheirburrows. mouse (Rocke et al., 2006), and a few Alternatively, and probably more impor- vaccinated animals even survived primary tantly,ferretsmaycontractplaguethrough oral challenge (Rocke, unpubl. data). direct contact or inhalation of infectious Although these results were considered aerosols as they feed on ill or dead prey promising, both of these studies were 1 Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED 01 JAN 2008 N/A - 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Vaccination with F1-V fusion protein protects black-footed ferrets 5b. GRANT NUMBER (Mustela nigripes) against plague upon oral challenge with Yersinia pestis. Journal of Wildlife Diseases 44:1-7 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Rocke, TE Smith, S Marinari, P Kreeger, J Enama, J Powell, BS 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION United States Army Medical Research Institute of Infectious Diseases, REPORT NUMBER TR-07-083 Fort Detrick, MD 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Earlier studies established that vaccination of black-footed ferrets (Mustela nigripes) with F1-V fusion protein by subcutaneous (SC) injection protected animals against plague upon injection of the bacterium Yersinia pestis. This study demonstrates that the F1-V antigen can also protect ferrets against plague contracted via ingestion of a Y. pestis-infected mouse, a probable route for natural infection. Eight black-footed ferret kits were vaccinated with F1-V protein by SC injection at approximately 60 days-of-age. A booster vaccination was administered 3 mo later via SC injection. Four additional ferret kits received placebos. The animals were challenged 6 wk after the boost by feeding each one a Y. pestis-infected mouse. All eight vaccinates survived challenge, while the four controls succumbed to plague within 3 days after exposure. To determine the duration of antibody post-vaccination, 18 additional black-footed ferret kits were vaccinated and boosted with F1-V by SC injection at 60 and 120 days-of-age. High titers to both F1 and V (mean reciprocal titers of 18,552 and 99,862 respectively) were found in all vaccinates up to 2-yr post-vaccination, whereas seven control animals remained antibody negative throughout the same time period. 15. SUBJECT TERMS Yersina pestis, plague, F1-V vaccine, black-footed ferrets, efficacy, challenge studies, wildlife biology 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF ABSTRACT OF PAGES RESPONSIBLE PERSON a. REPORT b. ABSTRACT c. THIS PAGE SAR 7 unclassified unclassified unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 2 JOURNALOFWILDLIFEDISEASES,VOL.44,NO.1,JANUARY2008 conducted in older animals that repre- NWHC, the animals were treated prophylac- tically for coccidiosis and housed individually sented less-than-ideal candidates for vac- in stainless-steel cages as described previously cination (Rocke et al., 2006) because they (Rocke et al., 2004). The animals were fed were4-to6-yr-oldspent breedersnearing a diet of raw horse meat (Toronto Small the end of their normal life span. Ferret CarnivoreDiet,MeatProducts,3347Kennedy kits that are bred in captivity for field Road #1, Scarborough, Ontario M1V 3P1, release are typically handled only twice: Canada),periodicallysupplementedwithmice (NWHC) and prairie dog meat (NBFFCC). whentheyareplaced inconditioning pens This study was reviewed and approved by at60 days-of-ageandjustbeforereleaseat NWHC’s Animal Care and Use Committee approximately 120 days-of-age. This tim- and Biosafety Committee. All personnel han- ing may align with a practical two-dose dling Y. pestis-infected animals or carcasses schedule for immunization, if vaccines were required to wear high efficiency partic- ulate air-filtered respirators with full-face administered at those age and time inter- shields, rubber aprons and boots, and double vals were shown to elicit protective surgical gloves. Research was conducted in immunity. Moreover, because ingestion compliance with the Animal Welfare Act and and/or inhalation are probably important other federal statutes and regulations relating transmission routes for Y. pestis in ferrets, to animals and experiments involving animals. an effective vaccine for field use must Experimentaldesign:Vaccineefficacytrial demonstrate protection against plague after consumption of infected prey. At approximately 60 days-of-age,eachof 12 ferretkitswasanesthetizedatNBFCC,andup Here, we establish that vaccination of to a 2 ml blood sample was drawn using ferret kits with F1-V protein confers methods described previously in Rocke et al. protection against primary oral challenge (2004). Eight of these ferrets received 0.5-ml via consumption of a Y. pestis-infected F1-V vaccine-adjuvant preparation (100 mg of mouse. Furthermore, we demonstrate the antigen)bySCinjectionbetweenthescapulae. longevity of antigen-specific antibody ti- The F1-V fusion protein and our methods of preparingthevaccine-adjuvantmixtureforSC ters in immunized ferrets, which remain injection have been described previously high up to 2 yr postvaccination (PV). (Rocke et al., 2004; Powell et al., 2005). Briefly, the antigen was diluted in Modified MATERIALSANDMETHODS Dulbecco’s medium (Sigma, PO Box 14508, St. Louis, Missouri 63178, USA), mixed 1:1 Experimentalanimals with 0.2% Alhydrogel (United Vaccines, 7819 Thirty-seven black-footed ferret kits were AirportRd.,Madison,Wisconsin53562,USA), selected for this study at the US Fish and and rocked gently overnight at 4 C. Four Wildlife Service, National Black-Footed Fer- controlanimalsreceivedaplaceboof0.5 mlof ret Conservation Center (NBFFCC), Wheat- the adjuvant (without protein antigen) sus- land, Wyoming. One group of 12 kits (all pended in Dulbecco’s medium. males) was designated for the vaccine efficacy Two mo later, all 12 animals were trans- trial. The second group of 25 kits (mixed sex) portedtoNWHC.Afteranacclimationperiod was used to determine the duration of of several wk, the animals were anesthetized, antibody after vaccination. All animals were a2-mlbloodsamplewasdrawn,andabooster 60 days-of-age and were marked individually injection of F1-V was administered. About with SC embedded microchips (AVID, 78294 6 wk after the booster dose, another blood Oak Ridge Road, Folsom, Louisiana 70437, samplewasdrawn,andtheanimalswereorally USA). At NBFCC, the kits were housed with challenged by feeding each a single Y. pestis- dam and littermates until they reached infectedmouse.Six-wk-oldoutbredSwissICR 120 days-of-age. At NWHC, the animals were mice(HarlanSpragueDawley,POBox29176, housed individually as described previously Indianapolis, Indiana 46229, USA) were in- (Rocke et al., 2004). After the initial vaccina- oculatedwith.4,000cfu(in0.1 ml)wildtype tion (described later), the group of 12 animals Y.pestisstrainCO92(,20050%lethaldoses) was transported to the NWHC where they by intradermal injection. Upon death 3 days were placed in a biosafety level-three animal- after challenge, one mouse was placed in the holding facility; the other group of 25 cage of each ferret. Any carcasses or parts of remained at the NBFFCC. Upon arrival at carcassesnotingestedbyferretswithin3–4 hr ROCKEETAL.—VACCINATIONPROTECTSBLACK-FOOTEDFERRETSAGAINSTPLAGUE 3 wereremovedanddiscarded.Forthoseferrets that didnotfullyconsume theirmice, another Y.pestis-infectedmousewasgiventothemthe following day. Ferrets were monitored daily forsignsofillness,anddayofdeathwasnoted; severely debilitated animals were euthanized by CO asphyxiation. 2 Any ferrets that survived challenge were bled to determine antibody titers after 3 wk and then euthanized by intracardiac injection of Euthasol (Delmarva Laboratories, Inc., 1500 Huguenot Road, Suite 106, Midlothian, Virginia 23113, USA). In both experiments, FIGURE 1. Geometric mean titers for anti-F1 antibody and anti-V antibody in black-footed ferret dead or euthanized ferrets were immediately kits that were vaccinated with F1-V fusion protein. necropsied.Selectedtissueswerecollectedfor Animals were vaccinated via subcutaneous (SC) bacterial isolation (Rocke et al., 2004) and injection on 17 August 2004, were boosted similarly histologic examination. on 15 November 2004, and challenged with wild- type(CO92)Yersiniapestison1December2004. Experimentaldesign:Durationof antibodyresponse The second group of 25 ferrets was vacci- anti-Vlogtiterfromthelogtiterofeachofthat natedviaSCinjectionatapproximately60and same animal’s subsequent blood samples. 120 days-of-age as already described; 18 ani- Combined (anti-F1 + anti-V) log titers were mals received the F1-V vaccine-adjuvant calculatedbyaddingthelogtitersofindividual mixture, and seven animals received the animals for each antigen (Williamson et al., placebo. All of these animals remained at 1999). All statistical analyses were performed NBFFCC throughout the duration of the using SAS software (SAS Institute, Cary, study, and blood samples were drawn pre- NorthCarolina,27513USA).Statisticaldiffer- vaccination, preboost, and ,3 mo, 6 mo, and ences in change of titer between groups were 1 yr postvaccination (PV) for all 25 animals, tested separately at each blood sampling and 2 yr PV for 20 animals. period using a one-tailed Wilcoxon test. For the vaccine efficacy trial, the difference in Antibodydetermination survivorship between groups was analyzed using the Fisher two-tailed exact test. All blood samples were collected in sterile glass serum separator tubes. After centrifuga- tion of blood samples, the serum was trans- RESULTS ferred to 2-ml polypropylene tubes and frozen Vaccineefficacy at220 Cforfutureanalyses.Antibodiesagainst F1 and V antigens were measured using an All eight vaccinated ferrets developed enzyme-linked immunosorbent assay (ELISA) significantantibodytiterstobothF1andV as previously described (Rocke et al., 2004), after their first vaccine dose as compared withslightmodificationsasfollows.AllELISA’s were conducted at NWHC. Coating of ELISA to both their prevaccination titers plates with V antigen was performed with the (P,0.0001)andthetitersofcontrol-group same concentration ofprotein(1 mg/ml) but in animals (P,0.005). After the second a 100-ml volume instead of 50 ml. Serum vaccine dose, all eight vaccinates had samples were serially diluted fourfold starting significantly increased (Fig. 1) anti-F1 at1:640. The highest dilution that was positive (definedasexceedingthemeanoffournegative and anti-V titers (at least fourfold and as control samples by three standard deviations) much as 64-fold; P,0.0001). In contrast, was considered to be the end point, and its the titers of control animals remained reciprocalvaluewasrecordedasthetiter. negative throughout the experiment. All eight vaccinates survived oral chal- Statisticalanalysis lenge with a Y. pestis-infected mouse with Antibody titers were transformed by calcu- no signs of illness, whereas the four latingthelog ofthetiter.Changeinlogtiter 10 was then calculated by subtracting an in- control animals that died succumbed to dividual animal’s pre-inoculation anti-F1 or plague within 3 days of ingesting a Y. 4 JOURNALOFWILDLIFEDISEASES,VOL.44,NO.1,JANUARY2008 FIGURE 3. Geometricmeantitersofanti-F1and FIGURE 2. Survival of black-footed ferrets vacci- anti-Vantibodiesinblack-footedferretkitsvaccinat- nated with F1-V protein and control animals that ed with F1-V protein at 0, 3mo, 6mo, and 1yr received a placebo after challenge via consumption (n518) and 2yr (n514) postvaccination. Animals ofaY.pestis-infectedmouse. received the first vaccine dose in August 2004 and receivedasimilarboostinOctober2004. pestis-infected mouse (Fig. 2). Gross and negative throughout the 2-yr duration of histologic lesions consistent with plague the study. were observed in all dead control animals, including marked congestion and hemor- DISCUSSION rhage in the liver, spleen, lymph nodes, and intestines. Yersinia pestis was isolated In this study, we found that black- from nasal swabs and liver in every case. footed ferret kits vaccinated and boosted Antigen-specific titers of all survivors SC with F1-V protein were protected rose significantly after challenge (Fig. 1). against plague contracted via ingestion of Meananti-F1andanti-Vantibodytitersof a Y. pestis-infected mouse. All vaccinated surviving vaccinates were significantly animals developed high antibody titers higher (P50.0187 and 0.0368, respective- againstF1andVantigens,survivedoralY. ly) than their mean prechallenge titers. pestis challenge, and showed a significant Upon euthanasia and necropsy of surviv- increase in antigen-specific titers after ing vaccinates at the end of the experi- challenge. Together with our earlier stud- ment, no obvious lesions characteristic of ies(Rockeetal.,2004,2006),theseresults plague were observed at the organ or demonstrated that the F1-V protein is an tissue level, and no Y. pestis was isolated effective vaccine against plague for black- from tested tissue samples. footed ferrets, eliciting protective immu- nity against both an oral challenge (con- Durationofantibody sumption of infected prey) and an SC All18vaccinatesdevelopedasignificant injection of the bacteria (simulated flea- increase in antibody titers to F1 and V bite exposure). Our results are consistent antigens (Fig. 3) after the initial vaccine with numerous other studies that have dose (P,0.0001) at 60 days-of-age and an demonstrated the protective efficacy of even higher increase in titer after the F1-V fusion protein against Y. pestis booster injection (P,0.0001) at 120 days- infection in mice (Anderson et al., 1998; of-age. Antibody titers remained high for Williamson etal.,1999;Glynnetal.,2005; at least 2 yr PV (Fig. 2), and there was no Powelletal.,2005)andreaffirmtheutility difference in combined log titers (anti-F1 of F1-V protein as a plague vaccine. and anti-V) between males and females The antibodies elicited against Y. pestis (P50.3584). Controls remained antibody antigens after captive ferret kits were ROCKEETAL.—VACCINATIONPROTECTSBLACK-FOOTEDFERRETSAGAINSTPLAGUE 5 vaccinated with F1-V protein appear to lastatleast2 yr(thelasttimepointtested) and may possibly last longer, since they showed little drop in titer. Although we did not challenge these animals, we believe their titers continued to be pro- tective against plague. Anderson et al. (1998)demonstratedthatF1-V-vaccinated mice retained antibodies protective against pneumonic plague for as long as 1 yr, the last time point tested in their study. Furthermore, other investigators have shown that the combined anti-F1 and anti-V log titer correlates better with protection against plague in mice than do FIGURE 4. Combined anti-F1 and anti-V anti- either of the individual log-transformed body titers of black-footed ferrets immunized with anti-F1 or anti-V titers (Williamson et al., F1-Vfusionproteinthateitherdiedorsurvivedupon 1999; J. Adamovicz, pers. comm.). By Y. pestis challenge. Data are combined from this study and previous studies (Rocke, et al., 2006, analyzing titer data (Fig. 4) generated unpubl. data) and include animals that received from vaccinated black-footed ferrets in either1or2subcutaneous(SC)injectionsofvaccine this study, a previous study (Rocke et al., at a dose of 100mg F1-V protein per animal and 2006), and unpublished data (Rocke), we animalschallengedeitherbySCinjectionofYersinia determined that the mean combined anti- pestis or via consumption of a Y. pestis-infected mouse. The horizontal lines indicate the combined F1 and anti-V log titer of vaccinates that anti-F1andanti-Vlogtitersatwhich50%(7.97)and survived Y. pestis challenge in our studies 90% (9.05) of the animals would survive Y. pestis (9.1461.05, n521) was significantly challengeasdeterminedbyprobitanalysis(with95% higher (P,0.0009) than the combined confidence intervals of 6.91 to 8.57 and 8.50 to log titer of vaccinated ferrets that died 13.26,respectively). upon challenge (7.6960.68, n511). We used probit analysis (SAS) to examine this risk, prairie dogs used for ferret feeding relationship further and found that an are quarantined for at least 21 days. animal with a combined log titer of 9.05 Vaccination of captive ferrets with F1-V (confidence interval of 8.50 to 13.26) had protein should reduce the risk of Y. pestis a 90% chance of surviving Y. pestis exposure even further. This vaccine could exposure. The combined log titers of also be used to immunize ferret kits that vaccinated ferrets 1 yr PV ranged from are released as part of reintroduction 8.62 to 10.43, with a mean of 9.5660.64. programs and whenever wild ferrets are These values suggest that the majority of trappedformonitoringprograms.In2004, these animals were probably protected a study was begun to measure survival against plague 1 yr PV. At 2 yr PV, rates of both field-born and field-released combined log titers ranged from 8.02 to ferret kits vaccinated with F1-V in com- 11.03, with a mean of 9.22. Probit analysis parison to animals not vaccinated. Al- suggests that all the vaccinated animals though survival data are not available yet, had a 50% chance of surviving Y. pestis six field-released vaccinates trapped ap- exposure (Fig. 4) and most (8/14) had proximately 1 yr later had significant a greater than 90% chance of survival. antibody titers to F1 and V (combined At NBFFCC, zoos, and other facilities log titer range of 7.42 to 9.83). where prairie dog meat is fed to captive Finally, although trapping and vaccina- ferrets, there is a minimal but continual tion is a labor-intensive prospect, the risk of Y. pestis exposure. To mitigate this vaccine might also be useful when plague 6 JOURNALOFWILDLIFEDISEASES,VOL.44,NO.1,JANUARY2008 threatens important ferret populations and long-term efficacy of single-dose subunit that have been reestablished in their vaccines against Yersinia pestis in mice. Amer- ican Journal of Tropical Medicine and Hygiene native range. In 2005, plague killed 58:793–799. numerous prairie dogs on the Pine Ridge BARNES, A. M. 1993. A review of plague and its Indian Reservation in South Dakota. This relevance to prairie dog populations and the outbreak occurred just 48 km south of the black-footed ferret. In Proceedings of the Symposium on the management of prairie Conata Basin, where the largest number dog complexes for the reintroduction of the of black-footed ferrets resides (about 250 black-footed ferret, J. L. Oldemeyer, D. E. animals), representing half of the free- Biggins, B. J. Miller and R. Crete (eds.). US ranging population. In an effort to protect FishandWildlifeServiceBiologicalReport,93: at least some of these animals should the 28–37. disease move northward, 100 ferrets were CASTLE,K.T.,D.BIGGINS,L.G.CARTER,M.CHU,K. INNES, AND J. WIMSATT. 2001. Susceptibility of captured and vaccinated with F1-V fusion the Siberian polecat to subcutaneous and oral proteinantigen.Thateffortisstillongoing, Yersinia pestis exposure. Journal of Wildlife and the findings will be reported sepa- Diseases37:746–754. rately.Currently,theF1-Vvaccineusedin GLYNN,A.,G.J.ROY,B.S.POWELL,J.A.ADAMOVICZ, this study is strictly available for animal L. C. FREYTAG, AND J. D. CLEMENTS. 2005. Protection against aerosolized Yersinia pestis testing on a limited basis. However, challenge following homologous and heterolo- commercial production of this or a similar gous prime-boost with recombinant plague vaccine is planned upon further definition antigens. Infection and Immunity 73: 5256– of its efficacy in preventing plague in 5261. animals. MENCHER, J., S. R. SMITH, J. E. OSORIO, D. STINCHCOMB,ANDT.E.ROCKE.2004.Protection Even though we demonstrated that ofblack-tailedprairiedogs(Cynomysludovicia- administration of F1-V elicited a robust nus)againstplagueaftervoluntaryconsumption protectiveresponse,weviewvaccinationof of baits containing recombinant raccoon poxvi- rus vaccine. Infection and Immunity 72: 5502– ferrets as anemergency, stop-gapmeasure 5505. to prevent plague in free-ranging popula- POWELL,B.,G.ANDREWS,J.ENAMA,S.JENDREK,C.R. tions of ferrets. A more effective strategy BOLT, P. WORSHAM, J. PULLEN, W. RIBOT, H. B. may be to devise a practical method to HINES, L. SMITH, D. HEATH, AND J. ADAMOVICZ. directly control the disease in prairie dogs, 2005.Designandtestingforanon-taggedF1-V fusionproteinasvaccineantigenagainstbubonic the primary source of infection for ferrets. and pneumonic plague. Biotechnology Progress This wildlife disease management goal 21:1490–1510. appears to be approachable through tar- ROCKE, T. E., J. MENCHER, S. R. SMITH, A. M. geted immunization of prairie dogs using FRIEDLANDER, G. P. ANDREWS, AND L. A. oral baits laden with a different plague BAETEN. 2004. Recombinant F1-V fusion pro- tein protects black-footed ferrets (Mustela ni- vaccine (Mencher et al., 2004). gripes)againstvirulentYersiniapestisinfection. Journal of Zoo and Wildlife Medicine 35: 142– ACKNOWLEDGMENTS 146. We thank T. Hoffman, S. Hwa, C. Pauly, ———, P. NOL, P. MARINARI, J. KREEGER, S. SMITH, and P. Vertz for technical assistance, D. G. P. ANDREWS, AND A. M. FRIEDLANDER. 2006. Heiseyforstatisticaladvice,andJ.Adamovicz, Vaccination as a potential means to prevent S. Dawe, and E. Hofmeister for editorial plague in black-footed ferrets. In Recovery of review. This study was funded bytheUS Fish theblack-footedferret:progressandcontinuing and Wildlife Service and the US Geological challenges, J. E. Roelle, B. J. Miller, J. L. Survey. Opinions, conclusions, and recom- GodbeyandD.E.Biggins(eds.).USGeological mendations are the author’s and not neces- Survey Scientific Investigations Report, 2005- sarily endorsed by the US Army. 5293:243–247. WILLIAMS, E. S., K. MILLS, D. R. KWIATKOWSKI, LITERATURECITED E. T. THORNE, AND A. BOERGER-FIELDS. 1994. Plague in a black-footed ferret (Mustela ni- ANDERSON,G.W.,JR.,D.G.HEATH,C.R.BOLT,S.I. gripes). Journal of Wildlife Diseases 30: 581– WELKOS, AND A. M. FRIEDLANDER. 1998. Short 585. ROCKEETAL.—VACCINATIONPROTECTSBLACK-FOOTEDFERRETSAGAINSTPLAGUE 7 WILLIAMSON,E.D.,P.M.VESEY,K.J.GILLHESPY,S. model. Clinical and Experimental Immunology M. ELEY, M. GREEN, AND R. W. TITBALL. 1999. 116:107–114. AnIgG1titretotheF1andVantigenscorrelates with protection against plague in the mouse Receivedforpublication21November2006.