Bayesian Spatiotemporal Pattern and Eco-climatological Drivers of Striped Skunk Rabies in the North Central Plains.

Striped skunks are one of the most important terrestrial reservoirs of rabies virus in North America, and yet the prevalence of rabies among this host is only passively monitored and the disease among this host remains largely unmanaged. Oral vaccination campaigns have not efficiently targeted striped skunks, while periodic spillovers of striped skunk variant viruses to other animals, including some domestic animals, are routinely recorded. In this study we evaluated the spatial and spatio-temporal patterns of infection status among striped skunk cases submitted for rabies testing in the North Central Plains of US in a Bayesian hierarchical framework, and also evaluated potential eco-climatological drivers of such patterns. Two Bayesian hierarchical models were fitted to point-referenced striped skunk rabies cases [n = 656 (negative), and n = 310 (positive)] received at a leading rabies diagnostic facility between the years 2007-2013. The first model included only spatial and temporal terms and a second covariate model included additional covariates representing eco-climatic conditions within a 4 km(2) home-range area for striped skunks. The better performing covariate model indicated the presence of significant spatial and temporal trends in the dataset and identified higher amounts of land covered by low-intensity developed areas [Odds ratio (OR) = 3.41; 95% Bayesian Credible Intervals (CrI) = 2.08, 3.85], higher level of patch fragmentation (OR = 1.70; 95% CrI = 1.25, 2.89), and diurnal temperature range (OR = 0.54; 95% CrI = 0.27, 0.91) to be important drivers of striped skunk rabies incidence in the study area. Model validation statistics indicated satisfactory performance for both models; however, the covariate model fared better. The findings of this study are important in the context of rabies management among striped skunks in North America, and the relevance of physical and climatological factors as risk factors for skunk to human rabies transmission and the space-time patterns of striped skunk rabies are discussed.

Comparison of anamnestic responses to rabies vaccination in dogs and cats with current and out-of-date vaccination status.

OBJECTIVE: To compare anamnestic antibody responses of dogs and cats with current versus out-of-date vaccination status. DESIGN: Cross-sectional study. ANIMALS: 74 dogs and 33 cats. PROCEDURES: Serum samples were obtained from dogs and cats that had been exposed to rabies and brought to a veterinarian for proactive serologic monitoring or that had been brought to a veterinarian for booster rabies vaccination. Blood samples were collected on the day of initial evaluation (day 0) and then again 5 to 15 days later. On day 0, a rabies vaccine was administered according to label recommendations. Paired serum samples were analyzed for antirabies antibodies by means of a rapid fluorescent focus inhibition test. RESULTS: All animals had an antirabies antibody titer ≥ 0.5 IU/mL 5 to 15 days after booster vaccination. Dogs with an out-of-date vaccination status had a higher median increase in titer, higher median fold increase in titer, and higher median titer following booster vaccination, compared with dogs with current vaccination status. Most (26/33) cats, regardless of rabies vaccination status, had a titer ≥ 12 IU/mL 5 to 15 days after booster vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that dogs with out-of-date vaccination status were not inferior in their antibody response following booster rabies vaccination, compared with dogs with current vaccination status. Findings supported immediate booster vaccination followed by observation for 45 days of dogs and cats with an out-of-date vaccination status that are exposed to rabies, as is the current practice for dogs and cats with current vaccination status.

Use of PCR to identify Leptospira in kidneys of big brown bats (Eptesicus fuscus) in Kansas and Nebraska, USA.

Bats have been implicated as potential carriers of Leptospira as a result of surveys, mostly in Australia and South America. We measured the prevalence of pathogenic leptospires in kidneys of bats from Kansas and Nebraska. From 7 August 2012 to 21 August 2012, we extracted DNA from kidneys of 98 big brown bats (Eptesicus fuscus) submitted and found negative for rabies. The DNA was processed in a two-step, seminested PCR assay with a dual-labeled Taqman probe specific for pathogenic leptospires. As a negative control, we used a saprophytic leptospire (Leptospira biflexa Patoc) and, as a pathogenic control, Leptospira interrogans Canicola. All bat kidneys were negative for pathogenic leptospires, suggesting that it is unlikely that the big brown bat, one of the most prevalent bat species in North America, is a reservoir for transmission of leptospires to dogs or humans.

Genetic characterization and phylogenetic analysis of skunk-associated rabies viruses in North America with special emphasis on the central plains.

Across North America the skunk acts as a reservoir for several rabies virus variants. Some of these variants are geographically restricted in range as is the case for the California skunk variant and two distinct variants present in Mexico. In contrast the North Central and South Central skunk rabies viruses are dispersed in overlapping ranges over large areas of the Midwestern region of the United States with the former extending into southern parts of the Canadian prairies. Despite this extensive range, there has been only very limited molecular characterization of these two viral variants. This study has examined the genetic diversity of the rabies viruses associated with North American skunks, with particular emphasis on the South Central skunk variant which was found to comprise three distinct geographically restricted groups of viruses that could in some cases be further sub-divided. The phylogenetic relationships of these groups and sub-groups allowed us to infer the likely direction of spread of these variants in some instances. Patterns of amino acid replacement of North American skunk-associated rabies viruses for both the nucleoprotein and glycoprotein products are also examined. These patterns reflect the virus phylogeny but no amino acid residues associated specifically with the skunk host were identified.

Contrasting landscape epidemiology of two sympatric rabies virus strains.

Viral strain evolution and disease emergence are influenced by anthropogenic change to the environment. We investigated viral characteristics, host ecology, and landscape features in the rabies-striped skunk disease system of the central Great Plains to determine how these factors interact to influence disease emergence. We amplified portions of the N and G genes of rabies viral RNA from 269 samples extracted from striped skunk brains throughout the distribution of two different rabies strains for which striped skunks were the reservoir. Because the distribution of these two strains overlapped on the landscape and were present in the same host population, we could evaluate how viral properties influenced epidemiological patterns in the area of sympatry. We found that South Central Skunk rabies (SCSK) exhibited intense purifying selection and high infectivity, which are both characteristics of an epizootic virus. Conversely, North Central Skunk rabies (NCSK) exhibited relaxed purifying selection and comparatively lower infectivity, suggesting the presence of an enzootic virus. The host population in the area of sympatry was highly admixed, and skunks among allopatric and sympatric areas had similar effective population sizes. Spatial analysis indicated that landscape features had minimal influence on NCSK movement across the landscape, but those same features were partial barriers to the spread of SCSK. We conclude that NCSK and SCSK have different epidemiological properties that interact differently with both host and landscape features to influence rabies spread in the central Great Plains. We suggest a holistic approach for future studies of emerging infectious diseases that includes studies of viral properties, host characteristics, and spatial features.

Effects of anesthesia and surgery on serologic responses to vaccination in kittens.

OBJECTIVE: To determine the effects of anesthesia and surgery on serologic responses to vaccination in kittens. DESIGN: Prospective controlled trial. ANIMALS: 32 specific-pathogen-free kittens. PROCEDURES: Kittens were assigned to 1 of 4 treatment groups: neutering at 7, 8, or 9 weeks of age or no neutering. All kittens were inoculated with modified-live virus vaccines against feline panleukopenia virus (FPV), feline herpesvirus (FHV), and feline calicivirus (FCV) at 8, 11, and 14 weeks of age and inactivated rabies virus (RV) at 14 weeks of age. Serum antibody titers against FPV, FHV, and FCV were determined at 8, 9, 11, 14, and 17 weeks of age; RV titers were determined at 14 and 17 weeks of age. RESULTS: Serologic responses of kittens neutered at the time of first vaccination (8 weeks) were not different from those of kittens neutered 1 week before (7 weeks) or 1 week after (9 weeks) first vaccination or from those of kittens that were not neutered. In total, 31%, 0%, 69%, and 9% of kittens failed to develop adequate titers against FPV, FCV, FHV, and RV, respectively, by 17 weeks of age. CONCLUSIONS AND CLINICAL RELEVANCE: Neutering at or near the time of first vaccination with a modified-live virus vaccine did not impair antibody responses in kittens. Many kittens that were last vaccinated at 14 weeks of age had inadequate antibody titers at 17 weeks of age. Kittens may be vaccinated in the perioperative period when necessary, and the primary vaccination series should be extended through at least 16 weeks of age.

Response of feral cats to vaccination at the time of neutering.

OBJECTIVE: To determine whether administration of inactivated virus or modified-live virus (MLV) vaccines to feral cats at the time of neutering induces protective serum antiviral antibody titers. DESIGN: Prospective study. ANIMALS: 61 feral cats included in a trap-neuter-return program in Florida. PROCEDURES: Each cat received vaccines against feline panleukopenia virus (FPV), feline herpes virus (FHV), feline calicivirus (FCV), FeLV, and rabies virus (RV). Immediately on completion of surgery, vaccines that contained inactivated RV and FeLV antigens and either MLV or inactivated FPV, FHV, and FCV antigens were administered. Titers of antiviral antibodies (except those against FeLV) were assessed in serum samples obtained immediately prior to surgery and approximately 10 weeks later. RESULTS: Prior to vaccination, some of the cats had protective serum antibody titers against FPV (33%), FHV (21%), FCV (64%), and RV (3%). Following vaccination, the overall proportion of cats with protective serum antiviral antibody titers increased (FPV [90%], FHV [56%], FCV [93%], and RV [98%]). With the exception of the FHV vaccine, there were no differences in the proportions of cats protected with inactivated virus versus MLV vaccines. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that exposure to FPV, FHV, and FCV is common among feral cats and that a high proportion of cats are susceptible to RV infection. Feral cats appeared to have an excellent immune response following vaccination at the time of neutering. Incorporation of vaccination into trap-neuter-return programs is likely to protect the health of individual cats and possibly reduce the disease burden in the community.

Results of vaccination of Asian elephants (Elephas maximus) with monovalent inactivated rabies vaccine.

OBJECTIVE: To evaluate the humoral immune response of Asian elephants to a primary IM vaccination with either 1 or 2 doses of a commercially available inactivated rabies virus vaccine and evaluate the anamnestic response to a 1-dose booster vaccination. ANIMALS: 16 captive Asian elephants. PROCEDURES: Elephants with no known prior rabies vaccinations were assigned into 2 treatment groups of 8 elephants; 1 group received 1 dose of vaccine, and the other group received 2 doses of vaccine 9 days apart. All elephants received one or two 4-mL IM injections of a monovalent inactivated rabies virus vaccine. Blood was collected prior to vaccination (day 0) and on days 9, 35, 112, and 344. All elephants received 1 booster dose of vaccine on day 344, and a final blood sample was taken 40 days later (day 384). Serum was tested for rabies virus-neutralizing antibodies by use of the rapid fluorescent focus inhibition test. RESULTS: All elephants were seronegative prior to vaccination. There were significant differences in the rabies geometric mean titers between the 2 elephant groups at days 35, 112, and 202. Both groups had a strong anamnestic response 40 days after the booster given at day 344. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the ability of Asian elephants to develop a humoral immune response after vaccination with a commercially available monovalent inactivated rabies virus vaccine and the feasibility of instituting a rabies virus vaccination program for elephants that are in frequent contact with humans. A 2-dose series of rabies virus vaccine should provide an adequate antibody response in elephants, and annual boosters should maintain the antibody response in this species.

Detection of cellular immunity to rabies antigens in human vaccinees.

A nonradioactive multi-parameter flow cytometry assay was developed to identify antigen-specific lymphocytes in human subjects previously vaccinated against rabies virus and was subsequently compared to the standard tritiated thymidine method. A cell tracking dye, carboxyfluorescein succinimidyl ester, was used in combination with surface label for CD4 and CD8 cells in order to determine the response of lymphocytes to killed rabies virus in an antigen recall assay. The rabies virus-specific lymphocyte response was compared to the humoral immune response in each of ten vaccinated and five non-vaccinated subjects. Lymphocyte responses to rabies virus were observed in all ten vaccinated subjects; some noted as early as 3 days after stimulation while others were not until 7 days after stimulation. There was good agreement between the proliferation index of the CFSE assay and the simulation index of the [3H]thymidine assay (kappa statistic=0.73). An inverse relationship was detected between the level of rabies virus neutralizing antibody (RVNA) and the lymphocyte response to inactivated rabies virus in the vaccinated subjects. The association between cytokines production and level of humoral and cellular response was investigated in four representative subjects. Two vaccinated subjects with high proliferation indices and low RVNA titers produced Th1 type cytokines to rabies virus stimulation, whereas two vaccinated individuals with low proliferation indices and high RVNA titers responses did not produce these cytokines.

Circumstances of bat encounters and knowledge of rabies among Minnesota residents submitting bats for rabies testing.

Minnesota residents who submitted a bat to the Minnesota Department of Health for rabies testing in 2003 were surveyed by telephone regarding the circumstances of the bat encounter and their knowledge of bats and rabies. Of 442 bats submitted for testing, 12 (3%) tested positive for rabies, and 410 (93%) tested negative; 17 (4%) bats were unsuitable for testing, and three (1%) had equivocal results. A case-control study found that rabid bats were more likely than non-rabid bats to be found in September, found outside, found in a wooded area, unable to fly, acting ill, or acting aggressively. Rabid bats were not more likely than non-rabid bats to be found during the day or to have bitten someone. While most persons submitting bats for rabies testing were aware that bats can carry rabies, few knew they should submit the bat for testing until they sought the advice of an animal control officer, veterinarian, or healthcare provider.

The influence of homologous vs. heterologous challenge virus strains on the serological test results of rabies virus neutralizing assays.

The effect that the relatedness of the viral seed strain used to produce rabies vaccines has to the strain of challenge virus used to measure rabies virus neutralizing antibodies after vaccination was evaluated. Serum samples from 173 subjects vaccinated with either purified Vero cell rabies vaccine (PVRV), produced from the Pittman Moore (PM) seed strain of rabies virus, or purified chick embryo cell rabies vaccine (PCECV), produced from the Flury low egg passage (Flury-LEP) seed strain of rabies virus, were tested in parallel assays by RFFIT using a homologous and a heterologous testing system. In the homologous system, CVS-11 was used as the challenge virus in the assay to evaluate the humoral immune response in subjects vaccinated with PVRV and Flury-LEP was used for subjects vaccinated with PCECV. In the heterologous system, CVS-11 was used as the challenge virus in the assay to evaluate subjects vaccinated with PCECV and Flury-LEP was used for subjects vaccinated with PVRV. Although the difference in G protein homology between the CVS-11 and Flury-LEP rabies virus strains has been reported to be only 5.8%, the use of a homologous testing system resulted in approximately 30% higher titers for nearly two-thirds of the samples from both vaccine groups compared to a heterologous testing system. The evaluation of equivalence of the immune response after vaccination with the two different vaccines was dependent upon the type of testing system, homologous or heterologous, used to evaluate the level of rabies virus neutralizing antibodies. Equivalence between the vaccines was achieved when a homologous testing system was used but not when a heterologous testing system was used. The results of this study indicate that the strain of virus used in the biological assays to measure the level of rabies virus neutralizing antibodies after vaccination could profoundly influence the evaluation of rabies vaccines.

Reducing the cost of post-exposure rabies prophylaxis: efficacy of 0.1 ml PCEC rabies vaccine administered intradermally using the Thai Red Cross post-exposure regimen in patients severely exposed to laboratory-confirmed rabid animals.

Purified chick embryo cell rabies vaccine (PCECV) administered as 0.1 ml intradermally according to the Thai Red Cross (TRC) regimen could reduce the cost of PEP by up to 84% when compared to the traditional five-dose Essen regimen. To confirm the efficacy of 0.1 ml of PCECV using the TRC regimen, a clinical trial was conducted in 113 patients presenting with category III exposures from confirmed rabid animals at two bite referral centres in the Philippines. Patients were monitored monthly for 1 year after exposure. PCECV was well tolerated, no vaccine-related serious adverse events occurred and all patients were alive 1 year after their initial exposure.

Vaccination of Egyptian fruit bats (Rousettus aegyptiacus) with monovalent inactivated rabies vaccine.

Twenty-six captive, adult Egyptian fruit bats (Rousettus aegyptiacus) were tested for the presence of rabies virus neutralizing antibodies (RVNA) using a rapid fluorescent focus inhibition test before and after vaccination. The bats were randomly assigned into three treatment groups: group A (n = 10) bats each received one 0.1-ml dose of monovalent inactivated rabies vaccine, group B (n = 10) bats each received two 0.1-ml doses of vaccine given 30 days apart, and group C (n = 6) bats remained unvaccinated. Plasma was collected from all bats before vaccination and on days 14, 30, 60, and 360. All bats were seronegative before vaccination, and all unvaccinated animals remained negative throughout the study. Rabies virus neutralization titers remained above 0.5 IU/ml from day 30 through day 360 for both vaccinated groups. Group B had significantly higher titers on day 60. This study demonstrated a measurable humoral immune response after vaccination with an inactivated rabies vaccine, with two doses producing a higher level of RVNA. This study confirms the feasibility of a rabies vaccination program for Egyptian fruit bats.

Serologic evidence of nonfatal rabies exposure in a free-ranging oncilla (Leopardus tigrinus) in Cotapata National Park, Bolivia.

A clinically healthy free-ranging oncilla (Leopardus tigrinus) was live-trapped in Boliva in 2000. Based on serology, we concluded that this animal was exposed to feline panleukopenia virus, Toxoplasma gondii, and rabies virus. The rabies virus-neutralizing antibody titer (>70 IU/ml) in this oncilla was unusual for an asymptomatic animal exposed to street virus and at a level expected in animals exposed to a large amount of virus, clinically affected, or vaccinated. Based on a subsequent 18 mo of radiotracking, we concluded that the oncilla had a nonfatal exposure to rabies virus.

Induction of protective immunity by topic application of a recombinant adenovirus expressing rabies virus glycoprotein.

The objective of this study was to determine if a replication defective recombinant adenovirus expressing rabies virus glycoprotein (Adrab.gp) given through a non-invasive vaccination route (by topical application) onto the skin (NIVS) could elicit an immune response and/or protection against rabies. Groups of mice were immunized by NIVS with various doses of Adrab.gp. For comparison, groups of mice were immunized intramuscularly, subcutaneously, or intradermally with Adrab.gp. Mice received two booster immunizations at 1 and 2 months after the first immunization. Virus neutralizing antibody (VNA) titers were measured at day 21 after the first and second immunizations and at day 14 after the third immunization. Fifty percent of the mice immunized by NIVS with 2 x 10(7) and 2 x 10(8)pfu Adrab.gp vaccine developed VNA, whereas none of the control mice or the mice immunized by NIVS with the lowest dose (2 x 10(6)pfu) of Adrab.gp virus developed VNA. However, this low dose induced high titers of VNA in mice immunized by parenteral routes. Two weeks after the last immunization, all the mice were challenged with a lethal dose of rabies virus. More than 70% of the animals immunized by NIVS with > or = 2 x 10(7)pfu Adrab.gp virus survived the challenge, whereas all the mice in the negative control group and the group immunized by NIVS with the lowest dose of Adrab.gp succumbed to rabies. Taken together, the results suggest that NIVS with Adrab.gp can induce VNA production and protection against lethal challenge with rabies virus in mice.