INFECTION AND IMMUNITY, Sept. 2004, p. 55025505 0019-9567/04/$08.000 DOI: 10.1128/IAI.72.9.55025505.2004 Copyright 2004, American Society for Microbiology.

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Vol. 72, No. 9

Protection of Black-Tailed Prairie Dogs (Cynomys ludovicianus) against Plague after Voluntary Consumption of Baits Containing Recombinant Raccoon Poxvirus Vaccine
Jordan S. Mencher,1,2 Susan R. Smith,2 Tim D. Powell,3 Dan T. Stinchcomb,3 Jorge E. Osorio,3 and Tonie E. Rocke2*
Wildlife Science Group, University of Washington, Seattle, Washington1; U.S. Geological Survey, National Wildlife Health Center, Madison, Wisconsin2; and Heska Corporation, Ft. Collins, Colorado3
Received 4 December 2003/Returned for modication 13 January 2004/Accepted 28 May 2004

Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis and signicant reservoirs of plague for humans in the western United States. A recombinant raccoon poxvirus, expressing the F1 antigen of Y. pestis, was incorporated into a palatable bait and offered to 18 black-tailed prairie dogs (Cynomys ludovicianus) for voluntary consumption; 18 negative control animals received placebo baits. Antibody titers against Y. pestis F1 antigen increased signicantly (P < 0.01) in vaccinees, and their survival was signicantly higher upon challenge with Y. pestis than that of negative controls (P < 0.01).
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Sylvatic plague, caused by the bacterium Yersinia pestis, is a disease of rodents that can afict humans, as well as other mammals, and is transmitted primarily via eas. Currently, the most signicant reservoir of plague for humans in North America is wild rodents, particularly prairie dogs and several squirrel species (11). Important trends in plague epidemiology are the increased transmission of plague from wild rodents to domestic cats as residential areas encroach on enzootic plague foci and the increased transmission of the disease from cats to their owners and veterinarians (17). Plague epizootics in prairie dog populations with mortality rates as high as 95 to 99% have been well documented for many decades (1, 5, 6, 18, 21). Not only do these outbreaks decimate local populations of prairie dogs, they also impact other species that depend on prairie dogs for food and shelter (2), such as the endangered black-footed ferret (Mustela nigripes). Although ea infestations in prairie dogs have been reduced in the past by application of insecticides to burrows (10), this method is labor intensive and generally applied after an outbreak has begun. Prophylactic control of the disease through immunization could be more effective. Recently, Osorio et al. (16) described a recombinant raccoon poxvirus (RCN) that expresses the F1 antigen of Y. pestis (herein designated RCN-F1) and protects mice from virulent plague challenge. In preliminary experiments with black-tailed prairie dogs (Cynomys ludovicianus), all animals vaccinated with RCN-F1 via intramuscular (i.m.) injection survived subcutaneous challenge with virulent Y. pestis (T. E. Rocke, unpublished data). However, efcient large-scale protection of free-ranging wildlife populations necessitates voluntary consumption of vaccine (19, 20). The purpose of this study was to investigate the ability of RCN-F1 to elicit a protective immune response

* Corresponding author. Mailing address: U.S. Geological Survey, National Wildlife Health Center, 6006 Schroeder Rd., Madison, WI 53711. Phone: (608) 270-2451. Fax: (608) 270-2415. E-mail: Tonie _Rocke@usgs.gov. Present address: Inviragen, Mt. Horeb, WI 53572. 5502

against Y. pestis infection in black-tailed prairie dogs after voluntary ingestion of palatable vaccine-laden baits. Experimental animals. Adult black-tailed prairie dogs captured from wild colonies in South Dakota were purchased from a commercial supplier and transported to the U.S. Geological Survey National Wildlife Health Center (Madison, Wis.). Upon arrival at the National Wildlife Health Center, animals were inspected for external parasites, treated with an anthelminthic injection (Ivomec; Merck & Co., Inc, West Point, Pa.), and marked with uniquely numbered ear tags. Prairie dogs were group housed in isolation rooms with approximately 180 square ft of oor space. Beta chips covered the oor, and Rubbermaid nest boxes connected by lengths of polyvinyl chloride pipe were used to mimic a burrow system. An alfalfabased pelleted food was fed free choice (approximately 50 g per animal per day), and fresh vegetables (broccoli, carrot, green beans, and sweet potato chunks) were given once daily. Water was available ad libitum. Vaccine and bait preparation. The raccoon poxvirus-vectored recombinant plague vaccine RCN-IRES-tPA-YpF1 (designated RCN-F1 in this paper) was produced as previously described (16) and stored at 70C in 2-ml aliquots until bait production. Virus stocks were thawed and diluted to 5 107 50% tissue culture infective doses (TCID50)/ml in Hanks medium (Gibco BRL, Carlsbad, Calif.) supplemented with 5% glycerin (Sigma, St. Louis, Mo.) immediately before use. Observation of the prairie dogs food preference suggested that sweet potato was the most palatable vegetable in their laboratory diet. Finely shredded sweet potato was lightly packed in 10-g lots into wells of plastic ice cube trays, and 8 ml of liquid gelatin (9.3 g of powdered gelatin [Difco, Irvine, Calif.] in 150 ml of warmed Hanks medium) was added, followed by 1 107 TCID50 of RCN-F1 vaccine/ml in 200 l of Hanks medium with glycerin. The vaccine was gently mixed through the liquid gelatin and sweet potato. For the negative control baits, 200 l of Hanks medium with glycerin alone was inserted into the bait. The ice cube trays were then refrigerated for 30 to 90 min until the gelatin baits were solidied.

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TABLE 1. Numbers of RCN-F1 vaccine-laden baits consumed by black-tailed prairie dogs (C. ludovicianus) in relation to survival against Y. pestis challenge, days to death, and antibody titers to RCN and Y. pestis F1 and V antigens
Priming baits consumed Boosting baits consumed Anti-RCN titer prechallenge Anti-F1 titer prechallenge Survival against challenge Days to death Anti-F1 titer postchallenge Anti-V titer postchallenge

2 0 2 2 2 2 2 2 2 2 2 2 2 1 1.5 2 2 2
a

2 1 2 2 2 2 2 2 2 2 2 2 2 1.5 2 2 2 2

375 75 375 1,875 375 75 1,875 375 15 15 15 75 375 375 375 75 NDa 375

12,800 1,600 3,200 1,600 800 50 800 800 3,200 200 400 100 50 50 400 200 50 200

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No No

7 5 5 4 10 8 7 6

12,800 100 12,800 1,600 800 1,600 800 12,800 800 12,800

10,240 2,560 10,240 2,560 2,560 2,560 160 160 160 160

ND, not determined.

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To ensure that bait production did not reduce vaccine vector viability, virus was extracted from two vaccine-laden baits within 24 h after preparation by homogenization and lowspeed centrifugation. Identical processing was performed on two negative control baits containing no RCN-F1. Extracted supernatants were serially diluted (10), Vero cells were added and, after 3 days at 37C and 5% CO2, wells were stained with trypan blue and observed for disruption of the Vero cell monolayer, consistent with viral cytopathic effect (CPE). The supernatant from the vaccine-bait preparation had a titer of 1 106 TCID50/ml as compared to 2 106 TCID50/ml for the positive control sample. The difference between these two titers was probably due to incomplete extraction of virus from the bait. Even if formulation led to some reduction in viral titer, we estimate that oral consumption of one bait exposed a prairie dog to at least 2 106 TCID50 of the RCN-F1 vaccine/ml. Vaccine administration. Eighteen prairie dogs were randomly assigned to each of two isolation rooms to serve as negative control and oral vaccinate groups. Four additional animals were assigned to a third room and received the vaccine via i.m. inoculation (1 107 TCID50 of RCN-F1/ml in the right thigh on day 0 and day 23) to conrm vaccine infectivity. The groups were not matched for sex or size, although all were adults. Animals were prepared for vaccination by withholding fresh vegetables for 48 h and pelleted food for 12 to18 h. Animals were then individually identied by ear tag and placed in pet carriers with a small food dish containing a single vaccineladen or vaccine-free (placebo) bait, depending on the experimental group. After 2 to 4 h, all animals were released and bait consumption was recorded for each individual. This process was performed on days 0 and 1 (priming vaccinations) and on days 26 and 27 for negative controls and days 23 and 24 for vaccinees (booster vaccinations). Most of the animals ate both priming and boosting baits (Table 1). One animal in each of the vaccinated and negative control groups failed to consume at least one priming bait but then ate at least one boost-

ing bait. One animal in the negative control group failed to eat any baits and was eliminated from further analyses. Y. pestis challenge. Six weeks post-priming vaccination, all animals were challenged with the CO92 wild-type isolate of Y. pestis (provided by the U.S. Army Medical Research Institute of Infectious Diseases). Stock aliquots of the bacteria, prepared and quantied as previously described (16), were diluted 1,000-fold in sterile saline. A volume of 0.2 ml of this solution was administered to each prairie dog by s.c. injection in the right hip region. Plate counts of the challenge inoculum indicated a dose of 132,000 CFU (6,600 mouse 50% lethal doses), and concurrent mouse tests conrmed its virulence. Because as many as 11,000 to 24,000 bacteria per bite may be regurgitated by ea vectors (3), our challenge dose represents approximately that delivered by 6 to 12 infectious ea bites. Several attempts were made prior to this experiment to determine a 50% lethal dose for our Y. pestis challenge inoculum in blacktailed prairie dogs (Rocke, unpublished). However, unlike inbred mice, reproducible results could not be achieved with prairie dogs. Prairie dogs were monitored for 21 days for signs of illness or death, after which all survivors were humanely euthanized. All carcasses were frozen for future necropsy. Survival rates of orally vaccinated prairie dogs differed signicantly (P 0.01) from those of negative controls via the Fisher exact test (22). Ten of the 18 (55.6%) vaccinees survived challenge (Table 1) compared to only 2 of 17 (11.8%) controls that ingested one placebo bait. In contrast, time to death for those animals that did not survive challenge was not signicantly different (P 0.2) between the vaccinated and control groups via the Mann-Whitney ranks test (22). Although not included in our analyses, two of four animals vaccinated via the i.m. route also survived challenge. Plague-induced mortality in challenged animals was veried by isolation of Y. pestis-specic DNA sequences from tissue culture by PCR. Selected frozen carcasses were thawed and necropsied, and tissue samples from lung, liver, and spleen were cultured in brain heart infusion broth (Difco) and on

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blood agar plates (Becton-Dickinson, Franklin Lakes, N.J.) at 28C for up to 72 h. The DNA was subsequently extracted from the culture broth and stored at 20C. For PCR, primers specic for the Y. pestis F1 gene (12) were used to amplify DNA fragments that were fractionated and directly visualized using standard techniques. Y. pestis DNA fragments were recovered by PCR from the lungs of 10 of 12 necropsied animals that had succumbed to plague challenge and from the liver or spleen of 4 of the 12. Y. pestis DNA was not recovered from any sampled tissue of the three necropsied animals that survived challenge. Anti-RCN antibody. Blood samples (300 l) were collected from the medial saphenous vein of each prairie dog before the priming and booster vaccinations and before challenge; blood samples were also obtained from survivors postchallenge. Serum was collected and stored at 20C until analyses. A modication of a low antibody titer microneutralization assay was used to determine serum anti-RCN antibody titers. Prairie dog serum samples were serially diluted and tested for the ability to neutralize 1 103 PFU/ml in microtiter plates. After incubation for 2 h at 37C, incubated samples were added to duplicate wells of previously seeded 96-well plates containing Vero cells. After additional incubation, xation, and staining, CPE was scored in relation to negative control wells and the highest dilution of serum with reduced infection (25 to 50% reduction in CPE) was recorded. Of 17 vaccinees tested, all developed anti-RCN antibody titers of 1:15 or higher (maximum, 1:1,875) postboost (Table 1); the serum from one individual was not tested because it was hemolytic. The geometric mean titer (GMT) of the oral vaccinees was 1:176, while all the negative controls had anti-RCN antibody titers of 1:15. All four animals immunized with RCN-F1 by i.m. injection developed anti-RCN antibodies, conrming infectivity of the virus; their GMT was 1:839. Anti-F1 antibody titers. Antibody titers to Y. pestis F1 antigen were determined by using a modied enzyme-linked immunosorbent assay ELISA protocol (4) with F1 antigen supplied by the Centers for Disease Control and Prevention. Briey, serum samples were serially diluted fourfold from 1:50 to 1:12,800; test samples were run in duplicate. Each plate also contained four replicates of a negative control serum sample and two replicates of a positive control serum sample. A horseradish peroxidase-labeled anti-prairie dog immunoglobulin G custom prepared by Bethyl Laboratories (Montgomery, Tex.) was diluted 1:100 and used as the secondary antibody. Titers 1:50 were treated as equal to 1:50, and those 1:12,800 were treated as equal to 1:12,800. Baseline antibody titers against F1 antigen were all 1:50. Fourteen of the 18 vaccinees developed anti-F1 antibody titers of 1:100 after vaccination (Table 1). The postprime GMT of oral vaccinees was 1:177, and their postboost GMT was 1:416. Reciprocal titers were log10 transformed, and the difference in each animals transformed titer from baseline to postprime and from postprime to postboost was calculated. Anti-F1 antibody titers increased signicantly in vaccinated animals after both the initial dose (P 0.01, Wilcoxon signed rank test) and the second booster dose (P 0.01; Fig. 1). These data demonstrate that voluntary ingestion of RCN-F1-laden baits by prairie dogs elicited a humoral immune response to F1 protein. Anti-V antibody. Antibody titers to another Y. pestis protein, V antigen, was measured similarly by enzyme-linked immu-

FIG. 1. Geometric mean anti-F1 antibody titers in black-tailed prairie dogs (C. ludovicianus) that consumed RCN-F1 vaccine-laden baits (vaccinees) or placebo baits (negative controls). Columns labeled with different letters indicate a signicant difference from 0 in median change in titer between bleeds (Wilcoxon signed ranks test; P 0.05). Downloaded from iai.asm.org by on January 22, 2008

nosorbent assay in samples collected prechallenge and from postchallenge survivors, using V protein provided by the U.S. Army Medical Research Institute for Infectious Diseases; the samples were serially diluted fourfold from 1:160 to 1:10,480 and tested in duplicate as described above. Signicant anti-V antibody titers were not detectable prior to challenge but increased signicantly postchallenge in 8 of 12 animals that survived infection (Table 1). Survival in relationship to antibody titers. Logistic regression (14) plotting the probability of surviving challenge against transformed postbooster anti-F1 antibody titer resulted in an odds ratio of 23 (95% condence interval, 1.39 to 377.5), indicating a weak but signicant relationship (P 0.05) between anti-F1 antibody titer and survival. All eight vaccinees with prechallenge anti-F1 titers 1:800 survived challenge with Y. pestis, whereas most animals (vaccinees and controls) with titers 400 (23 of 27) died upon challenge. There was no signicant relationship between anti-RCN antibody titer and survival of prairie dogs (P 0.05). Antibody titers to F1 and V antigens, but not necessarily both, increased postchallenge in all but one survivor of Y. pestis challenge (Table 1). We were surprised that two unvaccinated animals survived challenge with 130,000 CFU and believe that this suggests that innate resistance to Y. pestis may occur in some prairie dogs. The nature of this resistance is unknown. Interestingly, the two control animals that survived challenge developed high antibody titers to V antigen (2,560 and 10,240) but not to F1 (50 and 100). Conclusions. This is the rst report of protection against plague in prairie dogs elicited through voluntary consumption of a vaccine-laden bait. Prairie dogs that ingested at least one RCN-F1 vaccine-laden bait were more likely to survive challenge (56%) with Y. pestis than control animals that consumed placebo baits (12%). In our laboratory trial, we used sweet potato gelatin baits because they were easy to produce and suited our requirements. More stable, weather-resistant baits will need to be developed for future eld trials. Like vaccinia virus and other orthopoxviruses, RCN infects mucosal tissue and, thus, it is ideally suited for use as an oral viral vector. Also,

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RCN is highly attenuated and shown to be safe in numerous animal species, including black-footed ferrets (Rocke, unpublished), raccoons, skunks, bobcats, cats, dogs, and sheep (7, 8, 9, 13). Following immunization of mice and cats with RCNvectored vaccines in other studies, RCN infection was limited to the site of exposure and no viremia or viral shedding was detected (15). Oral vaccination through consumption of vaccine-laden baits may have application in managing plague epizootics in free-ranging prairie dog populations. Immunization of natural prairie dog colonies via oral, vaccine-laden baits could directly reduce plague mortality in exposed individuals and could also reduce the source of bacteria for ea transmission.
We thank Mansour Samadpour, University of Washington, for the generous use of his laboratory facilities and equipment; May Chu, Centers for Disease Control and Prevention, for supplying F1 antigen; Arthur Friedlander, U.S. Army Medical Research Institute for Infectious Diseases, for supplying V antigen and the Y. pestis strain used for challenge; and the animal care staff of the USGS-National Wildlife Health Center for their assistance in completing this study. Editorial comments of P. Nol and A. Bersch were much appreciated. Funding for this project was provided by the USGS-Biological Resources Division, U.S. Fish and Wildlife Service, the National Fish and Wildlife Foundation, the Turner Foundation, and the National Wildlife Federation.
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