Susan R. Smith

Protection of Black-Tailed Prairie Dogs (Cynomys ludovicianus) against Plague after Voluntary Consumption of Baits Containing Recombinant Raccoon Poxvirus Vaccine

ABSTRACT Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis and significant 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 significantly (P < 0.01) in vaccinees, and their survival was significantly higher upon challenge with Y. pestis than that of negative controls (P < 0.01).

Immunization of black-tailed prairie dog against plague through consumption of vaccine-laden baits

Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis and, along with other wild rodents, are significant reservoirs of plague for other wildlife and 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 three groups (n=18, 19, and 20) of black-tailed prairie dogs (Cynomys ludovicianus) for voluntary consumption, either one, two, or three times, at roughly 3-wk intervals. A control group (n=19) received baits containing raccoon poxvirus without the inserted antigen. Mean antibody titers to Y. pestis F1 antigen increased significantly in all groups ingesting the vaccine-laden baits, whereas the control group remained negative. Upon challenge with virulent Y. pestis, immunized groups had higher survival rates (38%) than the unimmunized control group (11%). The mean survival time of groups ingesting vaccine-laden baits either two or three times was significantly higher than that of animals ingesting vaccine-laden baits just one time and of animals in the control group. These results show that oral immunization of prairie dogs against plague provides some protection against challenge at dosages that simulate simultaneous delivery of the plague bacterium by numerous (3–10) flea bites.

PRELIMINARY EVALUATION OF A SIMPLE IN VITRO TEST FOR THE DIAGNOSIS OF TYPE C BOTULISM IN WILD BIRDS

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of type C botulinum toxin (Clostridium botulinum) in wild birds. This simple, antigen-capture ELISA utilizes polystyrene immunosticks as the solid substrate, chicken antitoxin (IgY) as the coating antibody, rabbit antitoxin as the primary antibody, and peroxidase-labeled goat-anti-rabbit as the secondary antibody. To evaluate the immunostick ELISA as a diagnostic test for avian botulism, known concentrations of toxin were added to heparinized blood collected from healthy birds and tested by both the ELISA and mouse bioassay. Also, blood samples from 236 bird carcasses submitted to the National Wildlife Health Center (NWHC) for cause of death determinations were tested by both procedures. Using ≤ 0.5 ml as the test volume for both procedures, the ELISA was less sensitive, detecting 0.25 ng/ml of toxin compared to 0.12 ng/ml for the mouse bioassay. Using the same volume of test sample for diagnostic submissions (≤ 0.5 ml), the ELISA was positive for 60% of the 149 clinically-diagnosed cases of botulism, whereas the mouse bioassay was positive for 79%. However, we demonstrated that with larger sample volumes (≥ 1.0 ml), the sensitivity of the ELISA may be equivalent or better than the mouse test due to the concentrating effect of the ELISA procedure. These preliminary results suggest that when adequate sample volumes are available, the immunostick ELISA can replace the mouse test for the diagnosis of botulism in wild birds.

Consumption of baits containing raccoon pox-based plague vaccines protects black-tailed prairie dogs (Cynomys ludovicianus).

Baits containing recombinant raccoon poxvirus (RCN) expressing plague antigens (fraction 1 [F1] and a truncated form of the V protein-V307) were offered for voluntary consumption several times over the course of several months to a group of 16 black-tailed prairie dogs (Cynomys ludovicianus). For comparison, another group of prairie dogs (n = 12) was injected subcutaneously (SC) (prime and boost) with 40 microg of F1-V fusion protein absorbed to alum, a vaccine-adjuvant combination demonstrated to elicit immunity to plague in mice and other mammals. Control animals received baits containing RCN without the inserted antigen (n = 8) or injected diluent (n = 7), and as there was no difference in their survival rates by Kaplan-Meier analysis, all of them were combined into one group in the final analysis. Mean antibody titers to Yersinia pestis F1 and V antigen increased (p < 0.05) in the vaccinated groups compared to controls, but titers were significantly higher (p < 0.0001) in those receiving injections of F1-V fusion protein than in those orally vaccinated with RCN-based vaccine. Interestingly, upon challenge with approximately 70,000 cfu of virulent Y. pestis, oral vaccination resulted in survival rates that were significantly higher (p = 0.025) than the group vaccinated by injection with F1-V fusion protein and substantially higher (p < 0.0001) than the control group. These results demonstrate that oral vaccination of prairie dogs using RCN-based plague vaccines provides significant protection against challenge at dosages that simulate simultaneous delivery of the plague bacterium by numerous flea bites.

Recombinant raccoon pox vaccine protects mice against lethal plague

Using a raccoon poxvirus (RCN) expression system, we have developed new recombinant vaccines that can protect mice against lethal plague infection. We tested the effects of a translation enhancer (EMCV-IRES) in combination with a secretory (tPA) signal or secretory (tPA) and membrane anchoring (CHV-gG) signals on in vitro antigen expression of F1 antigen in tissue culture and the induction of antibody responses and protection against Yersinia pestis challenge in mice. The RCN vector successfully expressed the F1 protein of Y. pestis in vitro. In addition, the level of expression was increased by the insertion of the EMCV-IRES and combinations of this and the secretory signal or secretory and anchoring signals. These recombinant viruses generated protective immune responses that resulted in survival of 80% of vaccinated mice upon challenge with Y. pestis. Of the RCN-based vaccines we tested, the RCN-IRES-tPA-YpF1 recombinant construct was the most efficacious. Mice vaccinated with this construct withstood challenge with as many as 1.5 million colony forming units of Y. pestis (7.7 x 10(4)LD(50)). Interestingly, vaccination with F1 fused to the anchoring signal (RCN-IRES-tPA-YpF1-gG) elicited significant anti-F1 antibody titers, but failed to protect mice from plague challenge. Our studies demonstrate, in vitro and in vivo, the potential importance of the EMCV-IRES and secretory signals in vaccine design. These molecular tools provide a new approach for improving the efficacy of vaccines. In addition, these novel recombinant vaccines could have human, veterinary, and wildlife applications in the prevention of plague.

VACCINATION WITH F1-V FUSION PROTEIN PROTECTS BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) AGAINST PLAGUE UPON ORAL CHALLENGE WITH YERSINIA PESTIS

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, 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 postvaccination, 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 postvaccination, whereas seven control animals remained antibody negative throughout the same time period.

RECOMBINANT F1-V FUSION PROTEIN PROTECTS BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) AGAINST VIRULENT YERSINIA PESTIS INFECTION

Abstract Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague, caused by the bacterium Yersinia pestis, and this disease has severely hampered efforts to restore ferrets to their historic range. A study was conducted to assess the efficacy of vaccination of black-footed ferrets against plague using a recombinant protein vaccine, designated F1-V, developed by personnel at the U.S. Army Medical Research Institute of Infectious Diseases. Seven postreproductive black-footed ferrets were immunized with the vaccine, followed by two booster immunizations on days 23 and 154; three control black-footed ferrets received a placebo. After the second immunization, antibody titers to both F1 and V antigen were found to be significantly higher in vaccinates than controls. On challenge with 7,800 colony-forming units of virulent plague by s.c. injection, the three control animals died within 3 days, but six of seven vaccinates survived with no ill effects. The seventh vaccinate died on day 8. These results indicate that black-footed ferrets can be immunized against plague induced by the s.c. route, similar to fleabite injection.

Toxicoinfectious Botulism in Commercial Caponized Chickens

Abstract During the summer of 2003, two flocks of commercial broiler chickens experienced unusually high death losses following caponizing at 3 wk of age and again between 8 and 14 wk of age. In September, fifteen 11-wk-old live capons were submitted to the Iowa State University Veterinary Diagnostic Laboratory for assistance. In both flocks, the second episode of elevated mortality was associated with incoordination, flaccid paralysis of leg, wing, and neck muscles, a recumbent body posture characterized by neck extension, and diarrhea. No macroscopic or microscopic lesions were detected in affected chickens. Hearts containing clotted blood and ceca were submitted to the National Wildlife Health Center in Madison, WI. Type C botulinum toxin was identified in heart blood and ceca by mouse bioassay tests. Enzyme-linked immunosorbent assay tests on heart blood samples were also positive for type C botulinum toxin. Clostridium botulinum was isolated from the ceca and genes encoding type C botulinum toxin were detected in cecal contents by a polymerase chain reaction test. Chickens are less susceptible to botulism as they age, and this disease has not previously been documented in broilers as old as 14 wk of age. Wound contamination by spores of C. botulinum may have contributed to the unusually high death losses following caponizing.

Further development of raccoon poxvirus-vectored vaccines against plague (Yersinia pestis).

In previous studies, we demonstrated protection against plague in mice and prairie dogs using a raccoon pox (RCN) virus-vectored vaccine that expressed the F1 capsular antigen of Yersinia pestis. In order to improve vaccine efficacy, we have now constructed additional RCN-plague vaccines containing two different forms of the lcrV (V) gene, including full-length (Vfull) and a truncated form (V307). Mouse challenge studies with Y. pestis strain CO92 showed that vaccination with a combination of RCN-F1 and the truncated V construct (RCN-V307) provided the greatest improvement (P=0.01) in protection against plague over vaccination with RCN-F1 alone. This effect was mediated primarily by anti-F1 and anti-V antibodies and both contributed independently to increased survival of vaccinated mice.

THE INNATE IMMUNE RESPONSE MAY BE IMPORTANT FOR SURVIVING PLAGUE IN WILD GUNNISON'S PRAIRIE DOGS

Abstract Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis, with ≥99% mortality reported from multiple studies of plague epizootics. A colony of Gunnisons prairie dogs (Cynomys gunnisoni) in the Aubrey Valley (AV) of northern Arizona appears to have survived several regional epizootics of plague, whereas nearby colonies have been severely affected by Y. pestis. To examine potential mechanisms accounting for survival in the AV colony, we conducted a laboratory Y. pestis challenge experiment on 60 wild-caught prairie dogs from AV and from a nearby, large colony with frequent past outbreaks of plague, Espee (n = 30 per colony). Test animals were challenged subcutaneously with the fully virulent Y. pestis strain CO92 at three doses: 50, 5,000, and 50,000 colony-forming units (cfu); this range is lethal in black-tailed prairie dogs (Cynomys ludovicianus). Contrary to our expectations, only 40% of the animals died. Although mortality trended higher in the Espee colony (50%) compared with AV (30%), the differences among infectious doses were not statistically significant. Only 39% of the survivors developed moderate to high antibody levels to Y. pestis, indicating that mechanisms other than humoral immunity are important in resistance to plague. The ratio of neutrophils to lymphocytes was not correlated with plague survival in this study. However, several immune proteins with roles in innate immunity (VCAM-1, CXCL-1, and vWF) were upregulated during plague infection and warrant further inquiry into their role for protection against this disease. These results suggest plague resistance exists in wild populations of the Gunnisons prairie dog and provide important directions for future studies.