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Since the 1990s, oral rabies vaccination (ORV) has been used successfully to halt the westward spread of the raccoon rabies virus (RV) variant from the eastern continental USA. Elimination of raccoon RV from the eastern USA has proven challenging across targeted raccoon ( Procyon lotor ) and striped skunk ( Mephitis mephitis ) populations impacted by raccoon RV. Field trial evaluations of the Ontario Rabies Vaccine Bait (ONRAB) were initiated to expand ORV products available to meet the rabies management goal of raccoon RV elimination. This study describes the continuation of a 2011 trial in West Virginia. Our objective was to evaluate raccoon and skunk response to ORV occurring in West Virginia for an additional two years (2012-2013) at 75 baits/km 2 followed by three years (2014-2016) of evaluation at 300 baits/km 2 . We measured the change in rabies virus-neutralizing antibody (RVNA) seroprevalence in targeted wildlife populations by comparing levels pre- and post-ORV during each year of study. The increase in bait density from 75/km 2 to 300/km 2 corresponded to an increase in average post-ORV seroprevalence for raccoon and skunk populations. Raccoon population RVNA levels increased from 53% (300/565, 95% CI: 50-57%) to 82.0% (596/727, 95% CI: 79-85%) during this study, and skunk population RVNA levels increased from 11% (8/72, 95% CI: 6-20%) to 39% (51/130, 95% CI: 31-48%). The RVNA seroprevalence pre-ORV demonstrated an increasing trend across study years for both bait densities and species, indicating that multiple years of ORV may be necessary to achieve and maintain RVNA seroprevalence in target wildlife populations for the control and elimination of raccoon RV in the eastern USA.

Background: Parasites may mediate the success of biological invasions through their effect on host fitness and thus, on host population growth and stability. However, a release from the pressure of parasites is strongly related to the genetic differentiation of the host. In invasive host populations, the number of available genetic variants, allowing them to 'fight' the infection, are likely to be influenced by founder events and genetic drift. The level standing genetic variation of invasive populations may be crucial in successfully adapting to new environments and resisting diseases. We studied invasive populations of raccoon that experienced a random reduction in genetic diversity during the establishment and evaluated the relationship between host immune genetic diversity and intestinal parasites infection., Results: We distinguished two different genetic clusters that are characterized by different sets of functionally relevant MHC-DRB alleles. Both clusters were characterized by considerably different allele-parasite associations and different levels of parasite infection. The specific resistance MHC-DRB alleles explained the lower prevalence of Digenea parasites. An increased infection intensity was related to the presence of two MHC-DRB alleles. One of these alleles significantly decreased in frequency over time, causing a decrease of Digenea abundance in raccoons in consecutive years., Conclusions: Our findings suggest that intestinal parasites can exert selective pressure on an invasive host with lowered levels of immune genetic diversity and contribute to promoting local adaptation over time. The random genetic drift that created the two different genetic clusters in the invasive raccoon range imposed completely different MHC-parasite associations, strongly associated with the infection status of populations. Our findings underline the role of standing genetic variation in shaping host-parasite relationships and provide empirical support that functional genetic variation may be, at least partly, responsible for differences in the success of invasive populations.

Zoonotic pathogens such as Ebola and rabies pose a major health risk to humans. One proven approach to minimizing the impact of a pathogen relies on reducing its prevalence within animal reservoir populations using mass vaccination. However, two major challenges remain for vaccination programs that target free-ranging animal populations. First, limited or challenging access to wild hosts, and second, expenses associated with purchasing and distributing the vaccine. Together, these challenges constrain a campaign's ability to maintain adequate levels of immunity in the host population for an extended period of time. Transmissible vaccines could lessen these constraints, improving our ability to both establish and maintain herd immunity in free-ranging animal populations. Because the extent to which vaccine transmission could augment current wildlife vaccination campaigns is unknown, we develop and parameterize a mathematical model that describes long-term mass vaccination campaigns in the US that target rabies in wildlife. The model is used to investigate the ability of a weakly transmissible vaccine to (1) increase vaccine coverage in campaigns that fail to immunize at levels required for herd immunity, and (2) decrease the expense of campaigns that achieve herd immunity. When parameterized to efforts that target rabies in raccoons using vaccine baits, our model indicates that, with current vaccination efforts, a vaccine that transmits to even one additional host per vaccinated individual could sufficiently augment US efforts to preempt the spread of the rabies virus. Higher levels of transmission are needed, however, when spatial heterogeneities associated with flight-line vaccination are incorporated into the model. In addition to augmenting deficient campaigns, our results show that weak vaccine transmission can reduce the costs of vaccination campaigns that are successful in attaining herd immunity., Competing Interests: The authors have declared that no competing interests exist.

Hepatitis E virus (HEV) is a zoonotic virus which circulates in pigs and wild boars as main reservoir species. To reveal the infection rate in carnivores, we have carried out a monitoring study of raccoons, raccoon dogs, dogs and cats sampled in Brandenburg, Germany. In summary, 53.8% (43 of 80) of the raccoons, 34.3% (25 of 73) of the raccoon dogs, 56.6% (47 of 83) of dogs and 32.3% (21 of 65) of cats were tested positive for HEV-specific antibodies. No viral RNA could be detected. This first description of anti-HEV antibodies in raccoons and raccoon dogs worldwide and in dogs and cats in Germany highlights the natural host range expansion of HEV., (© 2018 Blackwell Verlag GmbH.)

The raccoon (Procyon lotor) is a highly adaptable carnivore that has rapidly conquered Europe over the last decades and represents a potential candidate as pathogen reservoir, bearing the risk for transmission of infectious agents, as zoonosis or spill-over, to other wild life and domestic animals and man. Comprehensive investigations of infectious diseases in raccoons require a detailed knowledge of the participating immune cell populations. To close this gap of knowledge, various antibodies were tested for cross-reactivity with leukocytes in lymphoid organs and peripheral blood of raccoons using immunohistochemistry and flow cytometry, respectively. Eight out of 16 antibodies, directed against CD3, CD79α, Pax-5, IgG, CD44, MHC class II, myeloid/histiocyte antigen (MAC387), and Iba-1 exhibited a specific immunoreaction with cells in distinct anatomical compartments in formalin-fixed paraffin-embedded lymphoid tissues. Flow cytometric analysis revealed that 7 out of 18 antibodies directed against CD11c, CD14, CD21, CD44, CD79α, MHC class I and II cross-reacted with peripheral blood-derived raccoon leukocytes. Summarized, the usefulness of several cross-reacting antibodies was determined for the characterization of raccoon immune cells in immunohistochemistry and flow cytometry, offering the opportunity to study the raccoon immune system under normal and diseased conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Blood samples are often collected from free-ranging wildlife for antibody detection. However, filter-paper (FP) strips are more cost efficient and easy to collect and store. We evaluated trapper-collected FP strips and body-cavity blood for canine distemper (CDV) and parvovirus (CPV-2) antibody detection in raccoons (Procyon lotor) and coyotes (Canis latrans). From 2008 to 2010, licensed trappers near Madison and Milwaukee, Wisconsin, US collected paired samples from harvested animals. Canine distemper antibodies were detected using virus neutralization and parvovirus antibodies were detected using hemagglutination inhibition. Titers ≥ 1:32 for CDV and ≥ 1:25 for CPV-2 were considered evidence of exposure. Using Cohen's kappa test of agreement, FP strip titers agreed with sera for CDV in coyotes (n = 28, K = 0.772) and raccoons (n = 29, K = 0.858) and for CPV-2 in coyotes (n = 40, K = 0.775) and raccoons (n = 70, K = 0.646). However, raccoons determined to be exposed to CPV-2 from sera were unexposed by FP strips in 35% of the samples. Titer results may be affected by quality and volume of blood samples, interval between collection and processing, small sample sizes, and diagnostic testing procedures. Filter-paper strips can be useful for detecting CDV and CPV-2 exposure in coyotes and raccoons with correct field sample collection and appropriate diagnostic testing procedures.

Major Histocompatibility Complex (MHC) variability plays a key role in pathogen resistance, but its relative importance compared to environmental and demographic factors that also influence resistance is unknown. We analyzed the MHC II DRB exon 2 for 165 raccoons (Procyon lotor) in Missouri (USA). For each animal we also determined the presence of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to two highly virulent pathogens, canine distemper virus (CDV) and parvovirus. We investigated the role of MHC polymorphism and other demographic and environmental factors previously associated with predicting seroconversion. In addition, using an experimental approach, we studied the relative importance of resource availability and contact rates. We found important associations between IgG antibody presence and several MHC alleles and supertypes but not between IgM antibody presence and MHC. No effect of individual MHC diversity was found. For CDV, supertype S8, one allele within S8 (Prlo-DRB(∗)222), and a second allele (Prlo-DRB(∗)204) were positively associated with being IgG+, while supertype S4 and one allele within the supertype (Prlo-DRB(∗)210) were negatively associated with being IgG+. Age, year, and increased food availability were also positively associated with being IgG+, but allele Prlo-DRB(∗)222 was a stronger predictor. For parvovirus, only one MHC allele was negatively associated with being IgG+ and age and site were stronger predictors of seroconversion. Our results show that negative-frequency dependent selection is likely acting on the raccoon MHC and that while the role of MHC in relation to other factors depends on the pathogen of interest, it may be one of the most important factors predicting successful immune response., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Local adaptation is necessary for population survival and depends on the interplay between responses to selective forces and demographic processes that introduce or retain adaptive and maladaptive attributes. Host-parasite systems are dynamic, varying in space and time, where both host and parasites must adapt to their ever-changing environment in order to survive. We investigated patterns of local adaptation in raccoon populations with varying temporal exposure to the raccoon rabies virus (RRV). RRV infects approximately 85% of the population when epizootic and has been presumed to be completely lethal once contracted; however, disease challenge experiments and varying spatial patterns of RRV spread suggest some level of immunity may exist. We first assessed patterns of local adaptation in raccoon populations along the eastern seaboard of North America by contrasting spatial patterns of neutral (microsatellite loci) and functional, major histocompatibility complex (MHC) genetic diversity and structure. We explored variation of MHC allele frequencies in the light of temporal population exposure to RRV (0-60 years) and specific RRV strains in infected raccoons. Our results revealed high levels of MHC variation (66 DRB exon 2 alleles) and pronounced genetic structure relative to neutral microsatellite loci, indicative of local adaptation. We found a positive association linking MHC genetic diversity and temporal RRV exposure, but no association with susceptibility and resistance to RRV strains. These results have implications for landscape epidemiology studies seeking to predict the spread of RRV and present an example of how population demographics influence the degree to which populations adapt to local selective pressures., (© 2014 John Wiley & Sons Ltd.)

Zoonotic wildlife diseases pose significant health risks not only to their primary vectors but also to humans and domestic animals. Rabies is a lethal encephalitis caused by rabies virus (RV). This RNA virus can infect a range of terrestrial mammals but each viral variant persists in a particular reservoir host. Active management of these host vectors is needed to minimize the negative impacts of this disease, and an understanding of the immune response to RV infection aids strategies for host vaccination. Current knowledge of immune responses to RV infection comes primarily from rodent models in which an innate immune response triggers activation of several genes and signalling pathways. It is unclear, however, how well rodent models represent the immune response of natural hosts. This study investigates the innate immune response of a primary host, the raccoon, to a peripheral challenge using the raccoon rabies virus (RRV). The extent and temporal course of this response during RRV infection was analysed using genes predicted to be upregulated during infection (IFNs; IFN regulatory factors; IL-6; Toll like receptor-3; TNF receptor). We found that RRV activated components of the innate immune system, with changes in levels of transcripts correlated with presence of viral RNA. Our results suggest that natural reservoirs of rabies may not mimic the immune response triggered in rodent models, highlighting the need for further studies of infection in primary hosts.

Beginning in 2006, point infection control operations and aerial distribution of oral rabies vaccines along the US border were performed in Quebec, Canada, to control the potential spread of raccoon rabies. A benefit-cost analysis assessed the economic efficiency of this rabies control programme into the future. In this study, a mathematical simulation model was used to determine the potential spread of raccoon rabies from the 2006 index case, and incidence rates of human post-exposure prophylaxis (PEP), animal testing and human exposure investigations were calculated. Benefits were calculated as the potential savings from reduced numbers of human PEP, animal testing and human exposure investigations owing to control, which ranged from $47 million to $53 million. Programme cost scenarios were based on projections of total expenditures, which ranged from $33 million to $49 million. Economic efficiency was indicated for approximately half of the modelled scenarios, with the greatest benefit-cost ratios resulting from reduced future programme costs., (© Published 2012. This article is a US Government work and is in the public domain in the USA.)

RABORAL V-RG(®)a is a recombinant vaccine used in oral rabies vaccination (ORV) programs for wildlife in the United States. Vaccination rates for raccoons are substantially lower than vaccination rates for gray foxes and coyotes. Research suggests that the low viscosity of the oral vaccine may preclude animals from receiving an effective dose when biting into the vaccine bait delivery system. We evaluated the possibility of using two benign compounds, chitosan and N,N,N-trimethylated chitosan (TMC), to increase the viscosity of the vaccine and potentially act as adjuvants to improve the immune response in raccoons (Procyon lotor). Forty mildly sedated raccoons were orally vaccinated via needleless syringe with either RABORAL V-RG (n = 12), chitosan+RABORAL V-RG (n = 12), TMC+ RABORAL V-RG (n = 12), or no vaccine (n = 4), on day 0 and again on day 90. We collected sera every 2-4 wk for 4 mo and evaluated rabies virus-neutralizing antibodies (rVNA). Raccoons were considered responders if rVNA titers were ≥ 0.1 IU/mL. Eleven of 12 raccoons vaccinated with TMC+RABORAL V-RG responded after one dose of vaccine, as did eight of 12 vaccinated with RABORAL V-RG, and three of 12 vaccinated with chitosan+ RABORAL V-RG. Our results suggest that the inclusion of an adjuvant, such as TMC, could increase vaccine efficacy to aid in controlling rabies virus spread in wildlife reservoirs.

Multiple control methods have been used in North America to manage the spread of rabies caused by the raccoon (Procyon lotor) rabies virus variant (RRVV). Recently, oral vaccination with ONRAB(®) vaccine baits, which contain an adenovirus rabies glycoprotein recombinant, has been made available as an additional tool for rabies control. Our objectives were to estimate rabies antibody prevalence in wild-caught raccoons and striped skunks (Mephitis mephitis), and identify factors influencing the probability of being antibody positive at the individual level in these species, following oral rabies vaccination (ORV) campaigns in which ONRAB was distributed aerially in 2007-2009 in southern Québec, Canada. Following the aerial distribution of 43-155 ONRAB baits/km(2), the annual percentages of antibody-positive raccoons and skunks varied between 35% and 56% and 11% and 17%, respectively. In raccoons, the probability of being antibody positive was positively associated with age and density of ONRAB distributed, and influenced by the number of previous ORV campaigns conducted. Conversely, this probability was negatively associated with estimated abundance of raccoons in the trapping cell and proportion of residential areas near the raccoon capture location. None of the variables examined explained variation in the probability of being antibody positive in skunks. Our results indicate that the ONRAB density applied during ORV campaigns should be adjusted to account for variations in raccoon population density and presence of residential areas to increase the likelihood of creating an effective immunological barrier against RRVV. The high percentage of juvenile raccoons (annual mean =45 ± 3 [SE]%) and skunks (66 ± 2%) captured during post-ORV monitoring suggests that ORV campaigns should be conducted at least annually to account for the recruitment of naïve individuals into the populations. In Québec, the increasing use of ONRAB coincided with the elimination of rabies caused by RRVV. Nonetheless, our results indicate that improvements to this vaccine bait and/or the distribution techniques are required to increase its efficacy, especially in striped skunks.

We investigated the immune response and protection conferred in raccoons (Procyon lotor) following consumption of ONRAB(®) oral rabies vaccine baits. Forty-two wild-caught, captive raccoons were each offered an ONRAB vaccine bait; 21 controls received no vaccine baits. Blood samples collected from all raccoons before treatment, and each week posttreatment for 16 wk, were assessed for the presence of rabies virus antibody. In the bait group, an individual was considered to have responded to vaccination if serum samples from three or more consecutive weeks were antibody-positive. Using this criterion, 77% (20/26) of raccoons that consumed ONRAB baits with no observed vaccine spillage (full dose) demonstrated a humoral immune response. In the group that received a partial dose (0.05-0.90 mL vaccine recovered), 50% (8/16) of raccoons responded to vaccination. Regardless of the vaccine dose received, among the 28 raccoons that responded to vaccination 18 had antibody initially detectable at week 2 and 22 remained antibody-positive for at least 10 consecutive weeks. Kinetics of the humoral immune response suggest that the best time to conduct postbaiting surveillance for evidence of vaccination would be 6-13 wk following bait deployment, with the highest antibody prevalence expected between weeks 8-10. A sub-sample of 29 raccoons (20 ONRAB, 9 controls) was challenged with raccoon rabies virus variant 350 days posttreatment. Eight of nine controls (89%) developed rabies whereas 15/20 vaccinates (75%) survived. Survival following rabies challenge was significantly higher in raccoons presented ONRAB vaccine baits.

Two major techniques are currently used to estimate rabies virus antibody values: neutralization assays, such as the rapid fluorescent focus inhibition test (RFFIT), and enzyme-linked immunosorbent assays (ELISAs). The RFFIT is considered the gold standard assay and has been used to assess the titer of rabies virus neutralizing antibodies for more than three decades. In the late 1970s, ELISA began to be used to estimate the level of rabies virus antibody and has recently been used by some laboratories as an alternate screening test for animal sera. Although the ELISA appears simpler, safer and more efficient, the assay is less sensitive in detecting low values of rabies virus neutralizing antibodies than neutralization tests. This study was designed to evaluate a new ELISA-based method for detecting rabies virus binding antibody. This new technique uses electro-chemi-luminescence labels and carbon electrode plates to detect binding events. In this comparative study, the RFFIT and the new ELISA-based technique were used to evaluate the level of rabies virus antibodies in human and animal serum samples. By using a conservative approximation of 0.15 IU/ml as a cutoff point, the new ELISA-based technique demonstrated a sensitivity of 100% and a specificity of 95% for human samples and for experimental animal samples. The sensitivity and specificity for field animal samples was 96% and 95%, respectively. The preliminary results from this study appear promising and demonstrate a higher sensitivity than traditional ELISA methods., (Published by Elsevier Ltd.)

In North America, the raccoon rabies virus (RRV) is an endemic wildlife disease which causes acute encephalopathies and is a strong selective force on raccoons (Procyon lotor), with estimates of ∼85% of the population succumbing to the disease when epizootic. RRV is regarded as a lethal disease if untreated; therefore, no evolutionary response would be expected of raccoon populations. However, variable immune responses to RRV have been observed in raccoons indicating a potential for evolutionary adaptation. Studies of variation within the immunologically important major histocompatibility complex (MHC) have revealed relationships between MHC alleles and diseases in humans and other wildlife species. This enhances our understanding of how hosts and pathogens adapt and co-evolve. In this study, we used RRV as a model system to study host-pathogen interaction in raccoons from a challenge study and from four wild populations that differ in exposure times and viral lineages. We investigated the potential role of Prlo-DRB polymorphism in relation to susceptibility/resistance to RRV in 113 RRV positive and 143 RRV negative raccoons. Six alleles were found to be associated with RRV negative status and five alleles with RRV positive animals. We found variable patterns of MHC associations given the relative number of selective RRV sweeps in the studied regions and correlations between MHC diversity and RRV lineages. The allelic associations established provide insight into how the genetic variation of raccoons may affect the disease outcome and this can be used to examine similar associations between other rabies variants and their hosts.

Inverse correlations between genetic variability and parasitism are important concerns for conservation biologists. We examined correlations between neutral genetic variability and the presence of antibodies to canine distemper virus (CDV) and feline parvovirus (FPV) in a free-ranging population of raccoons. Over 3 years there was a strong relationship between age and seroprevalence rates. Most young animals were seronegative to CDV and FPV, but the oldest age class was greater than 80 per cent seropositive to both viruses. CDV-seropositive animals had greater heterozygosity and lower measures of inbreeding compared with CDV-seronegative animals. This relationship was strongest among the youngest animals and did not occur during a 1 year CDV epidemic. In contrast, FPV-seropositive animals only had significantly lower measures of inbreeding in 1 year, perhaps because FPV-associated mortality is relatively low or primarily occurs among very young individuals that were under-represented in our sampling. These results suggest that even in large outcrossing populations, animals with lower heterozygosity and higher measures of inbreeding are less likely to successfully mount an immune response when challenged by highly pathogenic parasites.

We investigated the seroprevalence of five tick- or mite-borne zoonoses, scrub typhus (Orientia tsutsugamushi), Japanese spotted fever (Rickettsia japonica), tularemia (Francisella tularensis), Lyme disease (Borrelia afzelii and Borrelia garinii), and Q fever (Coxiella burnetii), in feral raccoons (Procyon lotor) captured in Hokkaido and Kanagawa Prefectures in Japan. Of the 559 raccoons captured in Hokkaido, 8 (1.4%), 3 (0.5%), 1 (0.2%), and 1 (0.2%) carried antibodies against O. tsutsugamushi (Gilliam type), F. tularensis, B. afzelii, and B. garinii, respectively. Of the 193 animals investigated in Kanagawa, 31 (16.1%) and 14 (7.3%) carried antibodies against O. tsutsugamushi and R. japonica, respectively, and the major serotype (27/31) of O. tsutsugamushi was Kuroki. No antibodies against C. burnetii were detected in either area examined. Therefore, feral raccoons could be an indicator of the prevalence of these four tick- or mite-borne zoonoses in the peridomestic environment in Japan.

The immunogenicity and efficacy of two rabies vaccines in wild-caught, captive raccoons (Procyon lotor) were investigated. Raccoons were fed Ontario Slim (OS) baits containing a recombinant vaccinia virus-rabies glycoprotein (VRG) oral rabies vaccine, or they were given an intramuscular (IM) injection of IMRAB(®) 3 rabies vaccine. Blood samples collected before treatment and from weeks 1 to 16 posttreatment were assessed for the presence of rabies virus antibody (RVA). There were significantly more positive responders in the group that received an IM injection of IMRAB 3 (18/27) than in the group that consumed VRG in OS baits (VRG-OS; 4/ 26). There were no significant associations among age, sex, and seroconversion. Of those animals that mounted a humoral immune response to vaccination, RVA was first detected between weeks 1 and 5, with the majority of initial seroconversions detectable at week 2. A subsample of 50 raccoons (19 VRG-OS, 18 IMRAB 3, and 13 controls) from the longitudinal serology study was challenged with live raccoon variant rabies virus 442 days after initial treatment. There were significantly more survivors in the group that received IMRAB 3 (13/18) than in the VRG-OS (5/19) or control (2/13) groups. All 15 raccoons that demonstrated a serologic response survived challenge regardless of treatment. Of the 35 raccoons with no detectable serologic response, 30 (86%) succumbed to rabies virus infection (14/15 VRG-OS, 5/7 IMRAB 3, and 11/13 controls).

Oral vaccination is an important part of wildlife rabies control programs. Currently, the vaccinia-rabies glycoprotein recombinant virus is the only oral rabies vaccine licensed in the United States, and it is not effective in skunks. In the current study, captive raccoons and skunks were used to evaluate a vaccine developed by incorporating the rabies virus glycoprotein gene into a canine adenovirus serotype 2 vector (CAV2-RVG). Seven of 7 raccoons orally vaccinated with CAV2-RVG developed virus neutralizing antibodies and survived lethal challenge. Five of 5 and 6 of 6 skunks in 2 experimental groups receiving 10-fold different dilutions of CAV2-RVG developed neutralizing antibodies and survived challenge. The results of this preliminary study suggest that CAV2-RVG stimulates protective immunity against rabies in raccoons and skunks.

A replication-competent human adenovirus vector in which the rabies virus glycoprotein gene was inserted (AdRG1.3-ONRAB) was given by direct instillation into the oral cavity to representatives of three wildlife vector species of concern in Ontario (red fox, raccoon and striped skunk) and to a variety of non-target wildlife species, domestic and laboratory species. Despite use of a relatively high dose of vaccine, no untoward clinical signs were observed. Subsequent to vaccine exposure, detection of vaccine virus in lung, spleen, intestine, liver, kidney and brain of each animal was attempted using an ONRAB-specific assay combining PCR with Southern blotting (PCR-SB). Of the 1280 tissue samples obtained from vaccinates or contact animals, 18 (1.4%) were found to be PCR-SB positive. Virus isolation attempts were performed utilizing cell culture for all PCR-SB positive tissues and a selection of PCR-SB negative tissues. Histological examination performed on all PCR-SB positive tissues failed to identify lesions attributed to the vaccine. A quantitative real-time PCR was used to determine the excretion of the vaccine in feces and in the oral cavity with 0.8% of oral swabs and 6.8% of fecal specimens found to be positive. The low rates of recovery of vaccine virus from tissues, feces and the oral cavity suggest that the likelihood of ONRAB causing a negative impact on wildlife species is unlikely.

Japanese encephalitis virus (JEV) infects numerous animal species including humans, horses and pigs. In this study, antibodies against JEV in feral raccoons (Procyon lotor), wild boars (Sus scrofa) and raccoon dogs (Nyctereutes procyonoides) in Japan were examined. The results showed that 40.7% (22 out of 54), 64.5% (40 out of 62), 69.1% (47 out of 68) and 0% (0 out of 20) of raccoons in Hyogo, Osaka, Wakayama and Hokkaido, respectively, had virus-neutralizing antibodies against JEV. In addition, 83.3% (30 out of 36) of wild boars and 63.2% (12 out of 19) of raccoon dogs in Wakayama were seropositive for JEV. There were no significant differences in seroprevalence of JEV between males and females or between adults and juveniles in these wild animals. JEV seroprevalence was compared between 37 raccoons and 30 wild boars captured in a limited period (November 2007 to February 2008), and we found that wild boars (86.7%) were significantly more seropositive for JEV antibody than raccoons (59.5%). In conclusion, JEV was prevalent in wild mammals, indicating that the possibility of JEV infection in humans may still be high in Japan. In addition, these wild animals may be good sentinels to estimate JEV infection risk in residents, as they live near humans and are not vaccinated.

Distribution of oral rabies vaccine (ORV) is an effective but costly strategy to control raccoon rabies. Because of high costs, ORV for raccoon rabies in the U.S. has been limited primarily to epizootic areas, leaving extensive raccoon rabies regions without any ORV intervention. Several cost scenarios for ORV application in raccoon rabies enzootic and epizootic regions were modelled in New York State to obtain estimated costs of ORV baits per scenario and potential savings compared with a uniform ORV baiting strategy. These cost scenarios modelled at the census tract, level the application of ORV baits at different densities according to levels of risk defined by the observed number of raccoon rabies cases per km2 and the expected number of cases per km2 estimated with a Poisson regression model. Bait purchase costs were lower using the modelled cost scenarios than a uniform baiting strategy, for both the NYS enzootic region and the Long Island epizootic zone. The proportion of savings for the NYS enzootic region was 29.57%, and the proportion of savings for the Long Island epizootic zone was 38.9%. Use of these cost scenarios to determine bait distribution by rabies risk level should be considered to maximize efficacy and reduce costs of ORV interventions.

Background: The USDA, Wildlife Services cooperative oral rabies vaccination (ORV) program uses a live vaccinia virus-vectored (genus Orthopoxvirus) vaccine, Raboral V-RG (V-RG), to vaccinate specific wildlife species against rabies virus in several regions of the U.S. Several naturally occurring orthopoxviruses have been found in North America, including one isolated from asymptomatic raccoons (Procyon lotor). The effect of naturally occurring antibodies to orthopoxviruses on successful V-RG vaccination in raccoons is the focus of this study., Results: Overall, raccoons pre-immunized (n = 10) with a recombinant raccoonpox virus vaccine (RCN-F1) responded to vaccination with V-RG with lower rabies virus neutralizing antibody (VNA) titers than those which were not pre-immunized (n = 10) and some failed to seroconvert for rabies VNA to detectable levels., Conclusion: These results suggest that the success of some ORV campaigns may be hindered where raccoonpox virus or possibly other orthopoxvirus antibodies are common in wildlife species targeted for ORV. If these areas are identified, different vaccination strategies may be warranted.

Ohio's oral rabies vaccination (ORV) program was established to prevent the westward spread of the raccoon (Procyon lotor) rabies virus (Lyssavirus, Rhabdoviridae) in Ohio, USA. The program, which targets raccoons, distributes vaccine-bait units (VBU) at a target density of 75 units/km2. Few studies have examined the relationship of VBU density and target population density to the prevalence of rabies virus-neutralizing antibodies (RVNA). We conducted experimental VBU distributions in August 2003 and August 2004, 150 km west of the ORV zone where there was no history of raccoon rabies. We measured change in RVNA titers in blood collected from live-trapped raccoons before and after VBU distributions. A closed population mark-recapture estimate of the size of the target population was 91 raccoons/km2, compared to the realized VBU distribution density of 70 units/km2. Surprisingly, 41% of 37 serum samples were RVNA-positive (>or=0.05 IU/ml) before VBU distribution in 2003, but all titers were <0.25 IU/ml. Although viable VBUs were distributed in August 2003, only 21% of 315 samples were RVNA-positive before VBU distribution in 2004, but 9% had titers>or=0.25 IU/ml. Tetracycline (biomarker in bait) prevalence in teeth indicated that 57% of raccoons ingested VBUs after distribution in 2003, and 54% ingested VBUs after distribution in 2004. However, only 8% and 11% of sera were positive for RVNA (>or=0.05 IU/ml) after VBU distribution in 2003 and 2004, respectively. Only 4-5% of sera collected after bait distribution had titers>or=0.25 IU/ml each year. The standard distribution density of 75 VBUs/km2 was insufficient to produce a population-wide immunoprotective response against rabies infection in our high-density target population. Presence of RVNA in a presumed naïve population before baiting demonstrates that estimating prevalence of RVNA after oral rabies vaccination can be problematic without knowledge of background titers and seasonal changes in prevalence of RVNA before and after baiting.

Oral vaccination is an important tool currently in use to control the spread of rabies in wildlife populations in various programs around the world. Oral rabies vaccination (ORV) of raccoons represents the largest targeted program to control wildlife rabies in the United States. Currently, the vaccinia-rabies glycoprotein recombinant virus vaccine (V-RG) is the only licensed oral rabies vaccine in the US. In the current study, captive raccoons were used to evaluate two previously described constructs of a rabies virus vaccine developed by reverse genetics (SPBNGAS and SPBNGAS-GAS) for immunogenicity and efficacy compared to the V-RG vaccine. Four of five control animals succumbed to rabies virus after severe challenge, while three of five animals vaccinated orally with SPBNGAS succumbed. No mortality was observed for animals administered SPBNGAS-GAS or the V-RG vaccine. The results of this preliminary study suggest that SPBNGAS-GAS provides comparable efficacy to V-RG. Additional studies will be needed to determine the duration of immunity and optimal dosage of SPBNGAS-GAS and to examine its efficacy in other reservoir species.

An epidemic model for rabies in raccoons is formulated with discrete time and spatial features. The goal is to analyze the strategies for optimal distribution of vaccine baits to minimize the spread of the disease and the cost of implementing the control. Discrete optimal control techniques are used to derive the optimality system, which is then solved numerically to illustrate various scenarios.

Oral rabies vaccination (ORV) baiting programs for control of raccoon (Procyon lotor) rabies in the USA have been conducted or are in progress in eight states east of the Mississippi River. However, data specific to the relationship between raccoon population density and the minimum density of baits necessary to significantly elevate rabies immunity are few. We used the 22-km2 US National Aeronautics and Space Administration Plum Brook Station (PBS) in Erie County, Ohio, USA, to evaluate the period of exposure for placebo vaccine baits placed at a density of 75 baits/km2 relative to raccoon population density. Our objectives were to 1) estimate raccoon population density within the fragmented forest, old-field, and industrial landscape at PBS: and 2) quantify the time that placebo, Merial RABORAL V-RG vaccine baits were available to raccoons. From August through November 2002 we surveyed raccoon use of PBS along 19.3 km of paved-road transects by using a forward-looking infrared camera mounted inside a vehicle. We used Distance 3.5 software to calculate a probability of detection function by which we estimated raccoon population density from transect data. Estimated population density on PBS decreased from August (33.4 raccoons/km2) through November (13.6 raccoons/km2), yielding a monthly mean of 24.5 raccoons/km2. We also quantified exposure time for ORV baits placed by hand on five 1-km2 grids on PBS from September through October. An average 82.7% (SD = 4.6) of baits were removed within 1 wk of placement. Given raccoon population density, estimates of bait removal and sachet condition, and assuming 22.9% nontarget take, the baiting density of 75/ km2 yielded an average of 3.3 baits consumed per raccoon and the sachet perforated.

In a controlled experiment, 16 wild-trapped raccoons were exposed to 1 of 2 genetically modified live pseudorabies virus (PRV) vaccines used in swine. One vaccine had genes deleted for thymidine kinase (TK(-)) and glycoprotein G (gG(-)); the other had an additional deletion for glycoprotein E (gE(-)). These vaccines were administered orally and intranasally at four dose levels: 10(3), 10(4), 10(5), and 10(6) TCID(50). The 21 days survival rate was 37.5% for the gG(-)TK(-) vaccine; all of the survivors developed antibodies to PRV. All animals receiving the gG(-)gE(-)TK(-) vaccine survived; 75% (all except the lowest dose) developed anti-PRV antibodies. Survivors were challenged intranasally with a 3.2x10(3) TCID(50) dose of the virulent wildtype PRV Shope strain. Two of the remaining three gG(-)TK(-) vaccinated raccoons survived the challenge; for the gG(-)gE(-)TK(-) vaccine, the survival rate was 50% (4/8). The raccoons with higher vaccine-induced antibody titers were more likely to survive the challenge with the virulent PRV; there was a 100% mortality rate for raccoons lacking detectable anti-PRV antibodies. This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent virus.

During 1993-96 the Ontario Ministry of Natural Resources (Canada) implemented a research project to evaluate the efficacy of three candidate baits to deliver oral rabies vaccine to wild raccoons (Procyon lotor). Extensive field testing revealed that raccoon acceptance of Sugar-Vanilla baits (SV) at densities of 200/km2 and 400/km2, hand-placed in urban habitats of Scarborough (Ontario) during 1993, was 74% and 82%, respectively. Raccoon density in those areas averaged 11/km2. Aerial placement of SV baits in rural habitats in Barrie (Ontario) during 1993 and 1994, yielded raccoon acceptance levels of 58% with a density of 100 baits/km2, 59% at 75 baits/km2, and 47% at 50 baits/km2. Raccoon acceptance of SV baits was significantly lower in areas baited at the density of 50/km2. Acceptance of Cheese baits (CH) at a density of 75 baits/km2 was 52%. During 1996 trials in Barrie, modified SV baits with blister packs protruding through the matrix yielded raccoon acceptance values of 51% at a bait density of 54/km2, whereas acceptance of regular SV baits was 39% at a density of 51 baits/km2. Pooling of bait acceptance data for all years revealed that bait acceptance was highest for adult male raccoons. Raccoon density in rural habitats (Barrie, Ontario) where the studies took place, averaged 11-13/km2. Puncture and impact testing of blister packs in baits suggested that they would adequately serve as a vehicle to contain oral rabies vaccine for delivery to raccoons via baits.

Oral rabies vaccine-laden baits, with a tetracycline biomarker, were distributed in Pinellas County (Florida, USA) by helicopter drop and from cars from January to April 1997. A total of 130,320 baits was distributed throughout the county, yielding an average bait density of 185 baits per km2. Bait ingestion was estimated by microscopic detection of tetracycline in tooth and bone samples from 244 raccoons (Procyon lotor), 33 opossums (Didelphis virginianus), 31 feral cats, and two gray foxes (Urocyon cinereoargenteus) that were trapped during February-April 1997. Active surveillance consisted of 17 trapping sites that were further categorized by six community descriptors. Passive surveillance consisted of animals that were collected as nuisance animals by Pinellas County Animal Services. The proportion of tetracycline positive raccoons was compared between collection techniques, among trapping sites, vegetation communities, and age and sex categories. Since there was no statistically significant difference in the frequency of tetracycline positive raccoons trapped during active surveillance (59%, 110/187) and passive surveillance (53%, 30/57), the data were pooled, resulting in a tetracycline positive frequency of 57% (140/244). The range in the positive tetracycline frequency established for raccoons from the 17 active surveillance sites was 9% (1/11) to 100% (3/3). The tetracycline positive frequency for raccoons ranged from 25% (3/12) at the dumpster sites to 78% (14/18) at the landfills. Juvenile male raccoons (71%, 34/48) were the most commonly marked age and sex class and adult females (42%, 21/50) were the least commonly marked age and sex class. Eighty-five percent (28/33) of the opossums, 3% (1/31) of the feral cats, and 50% (1/2) of the gray foxes were tetracycline positive.

Eight 8-wk-old raccoon pups (Procyon lotor) with maternal canine distemper virus (CDV) neutralizing antibodies (NAb) and 24 8-wk-old seronegative pups were administered a commercial modified-live CDV vaccine (Galaxy, D, Solvay Animal Health, Inc., Kitchener, Ontario, Canada). All 24 seronegative raccoons had detectable serum CDV NAb titers 14 days after the initial dose. Titers rose to maximum levels 4 wk post-vaccination. Mean titers for groups of vaccinated seronegative pups were maintained between 1:256 and 1:2,048 for the remainder of the 3 mo observation period. Geometric means of the serum CDV NAb titer of eight seronegative pups given a single vaccine dose at 8 wk of age did not differ significantly from those of eight pups that were given serial doses at 8, 12, and 16 wk of age, or from those of eight pups vaccinated once at 16 wk of age. Seven unvaccinated 8-wk-old raccoon pups used as controls remained seronegative throughout the trial. Seven out of eight 8-wk-old pups with maternal antibodies, vaccinated at 8, 12, and 16 wk of age, failed to develop a rise in their CDV NAb titers until at least 18 wk of age, 2 wk after the third vaccination. Titers in eight unvaccinated raccoons with maternal antibodies declined steadily to undetectable levels at 20 wk of age. A half-life of 10.55 days was calculated for maternally-derived CDV NAb in raccoon pups. Sixteen vaccinated raccoons were protected from clinical disease following experimental oronasal challenge with a virulent raccoon strain of CDV, 13 to 23 wk after vaccination. Serum CDV NAb titers at the time of challenge ranged from 1:12 to 1:384 and increased during the period of observation. Three of four unvaccinated seronegative raccoons used as controls failed to mount any detectable CDV NAb and were euthanatized after developing clinical signs of canine distemper 26, 29, and 30 days post-challenge (PC). Necropsies confirmed the diagnosis. The fourth control raccoon exhibited transient equivocal clinical signs, mounted a sluggish humoral response, but was clinically normal when euthanatized 42 days PC. In this raccoon, there was focal non-suppurative encephalitis with intranuclear inclusion bodies typical of CDV infection.

The sera of 16 species of wild animals representing 5 classes of vertebrates were assayed for amebicidal activity against species of Naegleria. The greatest activity was observed for sera of bullfrogs, muskrats, and raccoons, all of which are animals associated with water. In contrast, the sera from animals such as toads, box turtles, sparrows, and squirrels exhibited minimal or no amebicidal activity. In general, pathogenic Naegleria tended to be less susceptible than nonpathogenic Naegleria to the lytic effect of raccoon serum. Heat-inactivated serum was not amebicidal, suggesting that perhaps complement may be involved in the serum-mediated lysis of amebas.

Any cost-benefit analysis of the use of an oral vaccine to control raccoon rabies should include calculating both costs and benefits in terms of $/unit area. Further, cost savings must be adjusted to match the stages of an epizootic: pre-epizootic, epizootic, and post-epizootic. A generic model, which can be adapted to different sites, illustrates the use of threshold analysis to link distribution costs, cost savings, bait density, and vaccine price. Initial results indicate the need to lower the cost of the vaccine, continue research to determine optimal bait densities, and examine distribution plans that do not require continued protection of areas in which raccoon rabies was eliminated through previous vaccination programs.

Captive-bred raccoons (Procyon lotor) developed immune resistance to infestation by the larval stage of the ixodid tick, Ixodes scapularis, the vector of Borrelia burgdorferi, following repeated applications of both nymphs and larvae. Resistance was expressed as a significant decrease in the proportion of engorged larvae recovered from each cohort. Resistance to nymphs was not noted, but, only two such cohorts were applied. Utilizing an enzyme-linked immunosor-bent assay (ELISA) developed to detect raccoon serum antibodies to tick salivary gland antigens, raccoons evidenced a two to ten-fold increase in anti-tick salivary extract antibody titer following the application of two cohorts of nymphs and eights cohorts of larvae. The tick saliva antigens recognized by both pre- and post-exposure raccoon sera were evaluated by Western blotting. The production of antibodies correlated with the development of resistance to infestation, suggesting that the resistance was immune-mediated and could be measured by anti-tick salivary extract antibody titers. Resistance in exposed raccoons prevents nearly 90% of larvae from prolonged feeding. Prolonged feeding is required for engorgement and the transmission of various infectious agents, such as B. burgdorferi.

We report the case of a 39-year-old asthmatic patient who developed acute rhinoconjunctivitis, Quincke's edema, and asthma after repeated exposure to a raccoon (Procyon lotor) which a friend kept at home. Skin prick and scratch tests with native raccoon hair and epithelium, as well as RAST, were clearly positive. Elimination of contact with the raccoon and avoiding the home of his friend led to a cessation of the patient's acute exacerbations.

Successful field oral vaccination and protection against viral diseases have so far been achieved only with live-attenuated or live-recombinant virus vaccines. In this communication, we present data that demonstrate that a glycoprotein derived from recombinant baculovirus-infected insect cells is efficacious as an oral vaccine. The glycoprotein (G) of rabies virus (Evelyn Rokitnicki Abelseth strain) was abundantly expressed in a baculovirus expression system and oral vaccination of racoons with the baculovirus-expressed G protein resulted in the production of rabies virus-neutralizing antibodies and protection against a lethal challenge with a street rabies virus. The potential for using the baculovirus-expressed G protein for oral immunization of wildlife is discussed.

Canine adenovirus type 2 (CAV2) has been proposed for recombinant vaccines to control rabies in wild animals. To evaluate the suitability of CAV2 as a safe vector for the genetically engineered vaccines, seven wild-caught raccoons (three males and four females) were administered CAV2 per os. Two of the animals were euthanatized on each of post-infection days 3, 6, and 14, and one was euthanatized on day 21. Two other control raccoons (a male and a female) were also euthanatized on day 21. Microscopic pulmonary lesions of multifocal necrotizing bronchiolitis with basophilic intranuclear inclusions were seen in 3/4 raccoons euthanatized on post-infection days 3 and 6. Ultrastructural examination of lungs with pulmonary lesions revealed hexagonal viral particles characteristic of adenoviruses. CAV2 is potentially pathogenic for raccoons, and this susceptibility should be of concern to developers of recombinant vaccines who intend to use CAV2 as a vaccine vector.

More than 2,500 sera from approximately 30 wild and domestic species in southern Texas were tested for neutralizing antibodies to Venezuelan equine encephalitis (VEE). Virus isolations were also attempted from blood and tissue samples of many of the wild specimens. VEE neutralizing substances were present in a variety of species collected prior to the 1971 epizootic, suggesting that VEE was present and perhaps enzootic in this area before the recent epizootic. Serologic results of this study suggest that deer (Odocoileus virginianus) and feral swine (Sus scrofa) may serve as good indicators or sentinels of VEE transmission. The reservoir of VEE was not established, but results of the study suggest that a number of species or a combination of animal host populations including deer, feral swine, and peccaries (Pecari angulatus) may be involved in the eizootiologyof VEE in southern Texas.

The prevalence of antibodies to Toxoplasma gondii was investigated among five species of wild carnivores in Norther Ccalifornia. The highest prevalence was among bobcats (Lynx rufus), with 15 of 21 tested being serologically positive. Other results included serological evidence of toxoplasmosis in two of seven raccoons (Procyon lotor), one of three badgers (taxidea taxus) and two of three coyotes (Canis latrans). Two gray foxes (Urocyon cinereoargenteus) were serologically negative. Oone badger with an indirect hemagglutination antibody titer of 1:8192 was found to harbor T. gondii in its brain tissues.

A series of coupled differential equations was used to model the temporal dynamics of rabies in raccoons in the mid-Atlantic region of the United States. The model takes explicit account of the development of natural immunity to rabies and was used to evaluate culling and vaccination elimination strategies. For habitats typical of the mid-Atlantic states, and given the assumptions of the model, it was estimated that elimination of rabies in raccoons by culling may involve the annual removal of over 32% of the raccoon population or the yearly vaccination of up to 99% of the susceptible fraction. Assuming a constant marginal cost for both culling and vaccination, the model suggests that, whatever the actual cost of each method, the cheapest strategy will always involve either culling or vaccination alone. A combined strategy of culling and vaccination will be cheaper than culling alone only when the per capita cost of vaccination is around one-fifth or less the per capita cost of culling.

Two infectious raccoon poxvirus (RCN) recombinants for expressing rabies virus surface spike glycoprotein (G) were produced by homologous recombination between raccoon poxvirus DNA and chimeric plasmids previously used for production of vaccinia virus recombinants. Expression of G protein was controlled by vaccinia virus promoter P7.5 (early/late class) or by P11 (late class). Immunoprecipitation of infected cell extracts indicated that both of the RCN recombinants directed faithful expression of G protein. Raccoons that were fed polyurethane baits loaded with either recombinant quickly developed high levels of rabies virus neutralizing antibodies and were protected when challenged with lethal raccoon rabies street virus.

Wildlife species from 38 of Florida's 67 counties were surveyed over a 10 year period for the presence of antibody to the five major arboviruses circulating in the state. The routine screening of 7891 sera from wild birds and mammals via the hemagglutination-inhibition (H1) test with selected reactors subjected to serum neutralization testing has 1) provided information regarding geographic distribution and seasonality of circulation of these viruses 2) identified enzootic foci of infection and those species of wildlife most commonly infected and 3) documented the potential value of certain wild mammals as indicators of St. Louis Encephalitis and Venezuelan equine encephalomyelitis virus activity prior to the detection of human cases. Limited studies of Tamiami and Tensaw virus on sera from mammals collected for other purposed provided additional baseline information on the activity of these viruses in Florida mammals. Isolations of eastern equine encephalomyelitis virus were made from the heart of a loggerhead shrike (Lanius excubitor), Tensaw virus from the brain of a gray fox (Urocyon cinereoargenteus), and Keystone virus from the heart of a bluejay (Cyanocitta cristata).

An attenuated strain of canine adenovirus type-2 (CAV-2) was administered orally to 2 foxes (Vulpes fulva), 6 raccoons (Procyon lotor), a skunk (Mephitis mephitis), and a mongoose (Herpestus auropunctatus). Blood was collected weekly from the animals to monitor CAV-2 virus-neutralizing antibody titers. All animals had increases in titers. Sera from 8 foxes, 30 mongooses, 52 raccoons, and 22 skunks trapped in the field had naturally occurring antibody to CAV-2.

Ninety percent of foxes fed commercial ERA vaccine in a specially designed bait developed rabies serum neutralizing antibodies. The vaccine bait did not cause clinical signs of rabies when consumed by foxes, raccoons, skunks, dogs, cats, cattle and monkeys. When presented, in the laboratory, to wild rodents of the species Microtus, Mus musculus and Peromyscus, the vaccine baits caused vaccine-induced rabies only in Mus musculus. Laboratory mice of the CD-1 and CLL strain were susceptible to vaccine-induced rabies; however, studies showed that transmission of virus to other animals did not occur. These studies suggest that the vaccine bait described could be useful in a rabies control program in areas where foxes and wild dogs are the principal vectors.

Eighty-three wild mammals of ten species were inoculated with a first mouse passage level Venezuelan encephalitis (VE) virus isolated from south Texas in 1971 during the VE outbreak. Rodents were highly susceptible to VE infection and circulated VE virus at levels ranging from 10(7.7) to 10(11.5) suckling mouse intracranial lethal doses per ml for 2 to 4 days. These levels of virus could easily infect vector mosquitoes. Mortality was high in all species of rodents except Sigmodon hispidus, adult Neotoma micropus, and adult Peromyscus leucopus. Lagomorphs were susceptible to VE infection but circulated VE virus at or near mosquito threshold levels for 1 day only. Raccoons and opossums were relatively resistant to VE infection or circulated low levels of virus in the blood. Juvenile animals of four species were more susceptible and experienced higher viremias than did adults. In two of the four, mortality was higher in the juveniles than in adults. A consideration of results obtained during this study and those of other investigators led to the conclusion that equines were of major importance, that canines, lagomorphs and rodents were of minor importance, and that other species tested were of no importance as virus amplifiers in VE epizootics.

Mink virus enteritis, feline panleukopenia and canine parvovirus-2 were inoculated separately into groups of raccoon, mink, red fox and striped skunk. Raccoons were highly susceptible to mink virus enteritis and feline panleukopenia, with animals developing clinical illness, and several dying within six to ten days of inoculation with lesions typical of parvovirus infection. Both viruses were shed in high titre in the feces of infected raccoons, and high antibody titres were stimulated. Raccoons inoculated with canine parvovirus-2 showed no signs; shedding of virus was sporadic though moderate titres of antibody developed. Mink inoculated with mink virus enteritis and feline panleukopenia developed signs and lesions of early parvovirus infection. No signs or significant lesions followed canine parvovirus-2 inoculation. Shedding of virus was heavy (mink virus enteritis) or sporadic (feline panleukopenia and canine parvovirus-2), though good serological responses were elicited to all three viruses. Red fox showed no signs of infection, shed all three viruses only sporadically, and the serological response was strong only to feline panleukopenia. Skunks developed low antibody titres, but no signs, and did not shed virus. Antibody to parvovirus was found in 79.2% of 144 wild red foxes; 22.3% of 112 wild raccoons; 1.3% of 157 wild skunks and 6/7 coyotes in southern Ontario. The likely significance of these viruses to wild and captive individuals and populations of these carnivores is discussed.

Wild and domestic animals from 3 geographic-climatologic areas in northern California were tested for antibodies against Toxoplasma gondii. A total of 2,796 serum samples representing 37 species of wild mammals, 35 species of wild birds, and 5 species of domestic animals were tested by the indirect hemagglutination test. Of 1,174 wild mammal serums tested, 10.8% were positive, which compared with 14.7% of the 1,221 domestic mammal serums. Of 229 wild carnivores tested, 45% were seropositive, including 69% of 86 bobcats, 28% of 58 coyotes, 48% of 25 raccoons, 27% of 26 gray foxes, 22% of 32 striped skunks, a civet cat, and a mink. Serologic evidence of infection was found in 38% of 47 rural domestic cats, but none of the 7 dogs tested was seropositive. Of 160 murid rodents (rats and house mice) in rural habitats, 4% were seropositive, which compared with 2% of 399 cricetine rodents (mostly deer mice) collected from wilderness habitats. Seven percent of 56 wild Artiodactyla (deer and feral pigs) were seropositive, which compared with 15% of 1,048 domestic sheep tested. Of 401 birds tested, 3.5% had antibodies against T gondii. The highest prevalence of antibodies among birds was in crows (14%). Toxoplasma was isolated from 1 raven, by mouse inoculation. In general, the highest prevalence of seropositive carnivores, rodents, and sheep was in the coastal region below 100 ft elevation, where the weather is cool and damp for much of the year. In the central valley the highest prevalence among sheep was in areas under irrigation. The prevalence of antibodies was lowest in the mountain areas, where climatologic extremes prevail at various seasons of the year.