The adaptive immune response to Trichuris in wild versus laboratory mice: An established model system in context.

Laboratory model organisms have provided a window into how the immune system functions. An increasing body of evidence, however, suggests that the immune responses of naive laboratory animals may differ substantially to those of their wild counterparts. Past exposure, environmental challenges and physiological condition may all impact on immune responsiveness. Chronic infections of soil-transmitted helminths, which we define as establishment of adult, fecund worms, impose significant health burdens on humans, livestock and wildlife, with limited treatment success. In laboratory mice, Th1 versus Th2 immune polarisation is the major determinant of helminth infection outcome. Here we compared antigen-specific immune responses to the soil-transmitted whipworm Trichuris muris between controlled laboratory and wild free-ranging populations of house mice (Mus musculus domesticus). Wild mice harbouring chronic, low-level infections produced lower levels of cytokines in response to Trichuris antigen than laboratory-housed C57BL/6 mice. Wild mouse effector/memory CD4+ T cell phenotype reflected the antigen-specific cytokine response across the Th1/Th2 spectrum. Increasing egg shedding was associated with body condition loss. However, local Trichuris-specific Th1/Th2 balance was positively associated with worm burden only in older wild mice. Thus, although the fundamental relationships between the CD4+ T helper cell response and resistance to T. muris infection are similar in both laboratory and wild M. m. domesticus, there are quantitative differences and age-specific effects that are analogous to human immune responses. These context-dependent immune responses demonstrate the fundamental importance of understanding the differences between model and natural systems for translating mechanistic models to 'real world' immune function.

AFFINITY OF BRAZILIAN WILD MAMMAL IMMUNOGLOBULINS TO BACTERIAL PROTEINS A AND G.

Staphylococcal A and streptococcal G proteins are widely used in immunoassays when specific immunological reagents are unavailable, such as for wild animals. The affinity of bacterial proteins A and G to the immunoglobulins of seven Brazilian mammals were tested, including black-tufted marmoset (Callithrix penicillata, n = 5), golden-bellied capuchin (Sapajus xanthosternos, n = 13), woolly mouse opossum (Micoureus demerarae, n = 6), long-nosed armadillo (Dasypus novemcinctus, n = 5), collared anteater (Tamandua tetradactyla, n = 5), ocelot (Leopardus pardalis, n = 6), and vampire bat (Desmodus rotundus, n = 5). Blood samples were collected from animals that were rescued in peri-urban rainforest fragments. Sera pools of each species were tested by ELISA to determine the intensity of each bacterial protein affinity to the immunoglobulins. When comparing the affinity to both proteins, immunoglobulins from D. rotundus, S. xanthosternos, and T. tetradactyla presented a higher affinity to protein G, whereas a higher affinity to protein A was found for immunoglobulins of C. penicillata and L. pardalis. The only species that presented a very low affinity to both bacterial proteins was M. demerarae. This study can be used as a reference for further studies on the development of sensitive and specific immunodiagnostic assays to be used for the monitoring of the health of these wild mammals.

Serological Positivity against Selected Flaviviruses and Alphaviruses in Free-Ranging Bats and Birds from Costa Rica Evidence Exposure to Arboviruses Seldom Reported Locally in Humans.

Arboviruses have two ecological transmission cycles: sylvatic and urban. For some, the sylvatic cycle has not been thoroughly described in America. To study the role of wildlife in a putative sylvatic cycle, we sampled free-ranging bats and birds in two arbovirus endemic locations and analyzed them using molecular, serological, and histological methods. No current infection was detected, and no significant arbovirus-associated histological changes were observed. Neutralizing antibodies were detected against selected arboviruses. In bats, positivity in 34.95% for DENV-1, 16.26% for DENV-2, 5.69% for DENV-3, 4.87% for DENV-4, 2.43% for WNV, 4.87% for SLEV, 0.81% for YFV, 7.31% for EEEV, and 0.81% for VEEV was found. Antibodies against ZIKV were not detected. In birds, PRNT results were positive against WNV in 0.80%, SLEV in 5.64%, EEEV in 8.4%, and VEEV in 5.63%. An additional retrospective PRNT analysis was performed using bat samples from three additional DENV endemic sites resulting in a 3.27% prevalence for WNV and 1.63% for SLEV. Interestingly, one sample resulted unequivocally WNV positive confirmed by serum titration. These results suggest that free-ranging bats and birds are exposed to not currently reported hyperendemic-human infecting Flavivirus and Alphavirus; however, their role as reservoirs or hosts is still undetermined.

Review of Oral Rabies Vaccination of Dogs and Its Application in India.

Oral rabies vaccines (ORVs) have been in use to successfully control rabies in wildlife since 1978 across Europe and the USA. This review focuses on the potential and need for the use of ORVs in free-roaming dogs to control dog-transmitted rabies in India. Iterative work to improve ORVs over the past four decades has resulted in vaccines that have high safety profiles whilst generating a consistent protective immune response to the rabies virus. The available evidence for safety and efficacy of modern ORVs in dogs and the broad and outspoken support from prominent global public health institutions for their use provides confidence to national authorities considering their use in rabies-endemic regions. India is estimated to have the largest rabies burden of any country and, whilst considerable progress has been made to increase access to human rabies prophylaxis, examples of high-output mass dog vaccination campaigns to eliminate the virus at the source remain limited. Efficiently accessing a large proportion of the dog population through parenteral methods is a considerable challenge due to the large, evasive stray dog population in many settings. Existing parenteral approaches require large skilled dog-catching teams to reach these dogs, which present financial, operational and logistical limitations to achieve 70% dog vaccination coverage in urban settings in a short duration. ORV presents the potential to accelerate the development of approaches to eliminate rabies across large areas of the South Asia region. Here we review the use of ORVs in wildlife and dogs, with specific consideration of the India setting. We also present the results of a risk analysis for a hypothetical campaign using ORV for the vaccination of dogs in an Indian state.

Mycobiome Traits Associated with Disease Tolerance Predict Many Western North American Bat Species Will Be Susceptible to White-Nose Syndrome.

White-nose syndrome (WNS), a fungal disease that has caused catastrophic population declines of bats in eastern North America, is rapidly spreading across the continent and now threatens previously unexposed bat species in western North America. The causal agent of WNS, the fungus Pseudogymnoascus destructans, can infect many species of hibernating bats, but susceptibility to WNS varies by host species. We previously reported that certain traits of the skin microbiome, particularly yeast diversity and abundance, of bat species in eastern North America are strongly associated with resistance to WNS. Using these traits, we developed models to predict WNS susceptibility of 13 species of western North American bats. Based on models derived from yeast species diversity, only one bat species, Myotis velifer, was predicted to be WNS resistant (i.e., may develop the disease, but with low mortality rates). We also screened yeasts found on western bats for P. destructans-antagonistic properties by spore germination and growth inhibition/competition assays and found the ability of yeasts to inhibit P. destructans in vitro to be strain specific. Similar to results of inhibition assays performed with yeasts isolated from bats in eastern North America, few yeasts isolated from bats in western North America inhibited P. destructans in vitro. Continued monitoring of western bat populations will serve to validate the accuracy of the mycobiome analysis in predicting WNS susceptibility, document population and susceptibility trends, and identify additional predictors to assess the vulnerability of naive bat populations to WNS. IMPORTANCE White-nose syndrome is one of the most devastating wildlife diseases ever documented. Some bat species are resistant to or tolerant of the disease, and we previously reported that certain traits of the skin mycobiome of bat species in eastern North America are strongly associated with resistance to WNS. Predicting which western bat species will be most susceptible to WNS would be of great value for establishing conservation priorities. Based on models derived from yeast species diversity, only one bat species was predicted to be WNS resistant. High susceptibility to WNS would pose a significant conservation threat to bats in western North America.

Cross-species surveillance and risk factors associated with Avian Coronavirus in North-Central and South West Regions of Nigeria.

Infectious bronchitis viral (IBV) (Avian coronavirus) diseases is among the major reproductive diseases affecting the avian production in Africa. There is scanty information on its current status and vaccination compliance among captive wild birds (CWB) and indigenous chickens (LC) in Nigeria. This study aimed to assess the exposure and the risk factors associated with IBV in CWB and LC from North-central and South west regions of Nigeria. Sera samples from 218 LC and 43 CWB were examined for IBV IgG using enzyme linked immunosorbent assay. Also, owners of LC and managers of CWB were interviewed using a pre-tested structured checklist. An overall IBV prevalence of 42.9% (112/261) was obtained. Captive wild birds and indigenous chickens had 11.6% (5/43) and 49.1% (107/218) prevalence respectively with a significant difference (p< 0.0001, OR= 7.3, 95% CI= 2.8-19.3). Also, geo-location indicated significant difference in IBV exposure among birds (p<=0.034). Furthermore, the study showed that there had never been laboratory screening on all acquired wild birds for exposure to infectious agents in the study location while none of these birds (LB/CWB) had history of vaccination. Since IBV is endemic in Nigeria, the use of vaccine for prophylactic measure should be advocated among LC and CWB owners in order to avoid unnecessary losses. Also, the essence of screening for infectious agents in newly acquired wild birds should be considered crucial for health sustenance and public safety.

Serological Responses of Raccoons and Striped Skunks to Ontario Rabies Vaccine Bait in West Virginia during 2012-2016.

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/km2 followed by three years (2014-2016) of evaluation at 300 baits/km2. 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/km2 to 300/km2 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.

Serosurvey of West Nile virus (WNV) in free-ranging raptors from Brazil.

West Nile virus (WNV) is a mosquito-borne Flavivirus that can affect birds, horses, and humans, and is the only zoonotic Flavivirus that has been identified in six continents. In Brazil, until 2010, there was no evidence of WNV circulation. Recently, the virus was isolated from a horse with encephalitis, and the first human cases were registered in Brazil. Despite that, there is still no information on the enzootic cycle of this virus in birds or wildlife. This study aimed to investigate whether there is evidence of WNV circulation among wild birds from Southern Brazil. For this, we used free-living wild raptors (live-trapped or rescued) as potential sentinels to investigate the presence of WNV antibodies using ELISA and plaque reduction neutralization test (PRNT) assay. In addition, the presence of nucleic acids from Flavivirus family members was investigated. None of the birds sampled presented clinical findings compatible with WNV. Of the 200 serum samples from birds of prey belonging to 21 species, ten (5%) were positive for the presence of WNV antibodies on ELISA testing. The PRNT test did not confirm the ELISA results, but indicated that three birds had possibly been exposed to Saint Louis encephalitis virus (SLEV). All samples were negative for Flavivirus RNA. The results presented here evince the need for permanent surveillance for emerging flaviviruses in Brazil, as well as for a contingency policy in the case of human/animal outbreaks, particularly in high-risk areas.

Differences in the growth rate and immune strategies of farmed and wild mallard populations.

Individuals reared in captivity are exposed to distinct selection pressures and evolutionary processes causing genetic and phenotypic divergence from wild populations. Consequently, restocking with farmed individuals may represent a considerable risk for the fitness of free-living populations. Supportive breeding on a massive scale has been established in many European countries to increase hunting opportunities for the most common duck species, the mallard (Anas platyrhynchos). It has previously been shown that mallards from breeding facilities differ genetically from wild populations and there is some indication of morphological differences. Using a common-garden experiment, we tested for differences in growth parameters between free-living populations and individuals from breeding facilities during the first 20 days of post-hatching development, a critical phase for survival in free-living populations. In addition, we compared their immune function by assessing two haematological parameters, H/L ratio and immature erythrocyte frequency, and plasma complement activity. Our data show that farmed ducklings exhibit larger morphological parameters, a higher growth rates, and higher complement activity. In haematological parameters, we observed high dynamic changes in duckling ontogeny in relation to their morphological parameters. In conclusion, our data demonstrate pronounced phenotype divergence between farmed and wild mallard populations that can be genetically determined. We argue that this divergence can directly or indirectly affect fitness of farmed individuals introduced to the breeding population as well as fitness of farmed x wild hybrids.

Faecal parasites increase with age but not reproductive effort in wild female chimpanzees.

Energy investment in reproduction is predicted to trade off against other necessary physiological functions like immunity, but it is unclear to what extent this impacts fitness in long-lived species. Among mammals, female primates, and especially apes, exhibit extensive periods of investment in each offspring. During this time, energy diverted to gestation and lactation is hypothesized to incur short and long-term deficits in maternal immunity and lead to accelerated ageing. We examined the relationship between reproduction and immunity, as measured by faecal parasite counts, in wild female chimpanzees (Pan troglodytes schweinfurthii) of Kibale National Park, Uganda. While we observed higher parasite shedding (counts of eggs, cysts and larvae) in pregnant chimpanzees relative to cycling females, parasites rapidly decreased during early lactation, the most energetically taxing phase of the reproductive cycle. Additionally, while our results indicate that parasite shedding increases with age, females with higher fertility for their age had lower faecal parasite counts. Such findings support the hypothesis that the relatively conservative rate of female reproduction in chimpanzees may be protective against the negative effects of reproductive effort on health. This article is part of the theme issue 'Evolution of the primate ageing process'.

Plasma carotenoids and immunity in a despotic avian scavenger.

Carotenoids are pigmented compounds acquired through diet that have important functions as antioxidants and immune modulators. We studied the association between immunity and circulating carotenoids in Andean condors (Vultur gryphus). We evaluated the relationship between α-, ß-, and γ-globulin blood concentrations and different circulating carotenoids in two groups of Andean condors that differ in their mean health status, rehabilitating (suffering different pathologies), and wild individuals (trapped when displaying their physiological behavior). In rehabilitating individuals, α-, ß-, and γ-globulin concentrations were higher than in wild individuals. This shows that rehabilitating individuals were developing an immune response associated with the pathologies that they were suffering at the time of sampling. In addition, circulating carotenoids were lower in rehabilitating than in wild individuals. We found negative correlations between α-, ß-, and γ-globulins and different circulating carotenoids in rehabilitating individuals, but not in wild condors. Xanthophylls were strongly related to α-, ß-, and γ-globulin blood concentrations in rehabilitating, but not in wild condors. Our results suggest that there is a potential relationship between circulating carotenoids and immunity in the Andean condor. Given that this species may display a carotenoid-based pigmentation, our results could suggest that a trade-off between the immune system and external coloration could operate in this species, which may have implications in their access to food resources and mate selection and, thus, in their conservation.

Genetic and antigenic characterization of H5 and H7 avian influenza viruses isolated from migratory waterfowl in Mongolia from 2017 to 2019.

The circulation of highly pathogenic avian influenza viruses (HPAIVs) of various subtypes (e.g., H5N1, H5N6, H5N8, and H7N9) in poultry remains a global concern for animal and public health. Migratory waterfowls play important roles in the transmission of these viruses across countries. To monitor virus spread by wild birds, active surveillance for avian influenza in migratory waterfowl was conducted in Mongolia from 2015 to 2019. In total, 5000 fecal samples were collected from lakesides in central Mongolia, and 167 influenza A viruses were isolated. Two H5N3, four H7N3, and two H7N7 viruses were characterized in this study. The amino acid sequence at hemagglutinin (HA) cleavage site of those isolates suggested low pathogenicity in chickens. Phylogenetic analysis revealed that all H5 and H7 viruses were closely related to recent H5 and H7 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds in Asia and Europe. Antigenicity of H7Nx was similar to those of typical non-pathogenic avian influenza viruses (AIVs). While HPAIVs or A/Anhui/1/2013 (H7N9)-related LPAIVs were not detected in migratory waterfowl in Mongolia, sporadic introductions of AIVs including H5 and H7 viruses into Mongolia through the wild bird migration were identified. Thus, continued monitoring of H5 and H7 AIVs in both domestic and wild birds is needed for the early detection of HPAIVs spread into the country.

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.

Noninvasive measurement of mucosal immunity in a free-ranging baboon population.

Ecoimmunological patterns and processes remain understudied in wild primates, in part because of the lack of noninvasive methods to measure immunity. Secretory immunoglobulin A (sIgA) is the most abundant antibody present at mammalian mucosal surfaces and provides an important first line of defense against pathogens. Recent studies show that sIgA can be measured noninvasively in feces and is a good marker of mucosal immunity. Here we validated a commercial ELISA kit to measure fecal IgA in baboons, tested the robustness of its results to variation in collection and storage conditions, and developed a cost-effective in-house ELISA for baboon fecal IgA. Using data from the custom ELISA, we assessed the relationship between fecal IgA concentrations and gastrointestinal parasite burden, and tested how sex, age, and reproductive effort predict fecal IgA in wild baboons. We find that IgA concentrations can be measured in baboon feces using an in-house ELISA and are highly correlated to the values obtained with a commercial kit. Fecal IgA concentrations are stable when extracts are stored for up to 22 months at -20°C. Fecal IgA concentrations were negatively correlated with parasite egg counts (Trichuris trichiura), but not parasite richness. Fecal IgA did not vary between the sexes, but for males, concentrations were higher in adults versus adolescents. Lactating females had significantly lower fecal IgA than pregnant females, but neither pregnant nor lactating female concentrations differed significantly from cycling females. Males who engaged in more mate-guarding exhibited similar IgA concentrations to those who engaged in little mate-guarding. These patterns may reflect the low energetic costs of mucosal immunity, or the complex dependence of IgA excretion on individual condition. Adding a noninvasive measure of mucosal immunity will promote a better understanding of how ecology modulates possible tradeoffs between the immune system and other energetically costly processes in the wild.

Neutralizing antibodies against Rift Valley fever virus in wild antelope in far northern KwaZulu-Natal, South Africa, indicate recent virus circulation.

Rift Valley fever (RVF) is a zoonotic viral disease of domestic ruminants in Africa and the Arabian Peninsula caused by a mosquito-borne Phlebovirus. Outbreaks in livestock and humans occur after heavy rains favour breeding of vectors, and the virus is thought to survive dry seasons in the eggs of floodwater-breeding aedine mosquitoes. We recently found high seroconversion rates to RVF virus (RVFV) in cattle and goats, in the absence of outbreaks, in far northern KwaZulu-Natal (KZN), South Africa. Here, we report the prevalence of, and factors associated with, neutralizing antibodies to RVFV in 326 sera collected opportunistically from nyala (Tragelaphus angasii) and impala (Aepyceros melampus) culled during 2016-2018 in two nature reserves in the same area. The overall seroprevalence of RVFV, determined using the serum neutralization test, was 35.0% (114/326; 95%CI: 29.8%-40.4%) and tended to be higher in Ndumo Game Reserve (11/20; 55.0%; 95%CI: 31.5%-76.9%) than in Tembe Elephant Park (103/306; 33.6%; 95%CI: 28.4%-39.3%) (p = .087). The presence of antibodies in juveniles (6/21; 28.6%; 95%CI: 11.3%-52.2%) and sub-adults (13/65; 20.0%; 95%CI: 11.1%-37.8%) confirmed that infections had occurred at least until 2016, well after the 2008-2011 RVF outbreaks in South Africa. Odds of seropositivity was higher in adults than in sub-adults (OR = 3.98; 95%CI: 1.83-8.67; p = .001), in males than in females (OR = 2.66; 95%CI: 1.51-4.68; p = .001) and in animals collected ≤2 km from a swamp or floodplain compared with those collected further away (OR = 3.30; 95%CI: 1.70-6.38; p < .001). Under similar ecological conditions, domestic and wild ruminants may play a similar role in maintenance of RVFV circulation and either or both may serve as the mammalian host in a vector-host reservoir system. The study confirms the recent circulation of RVFV in the tropical coastal plain of northern KZN, providing the basis for investigation of factors affecting virus circulation and the role of wildlife in RVF epidemiology.

Diet May Drive Influenza A Virus Exposure in African Mammals.

BACKGROUND: Influenza A viruses (IAVs) represent repeatedly emerging pathogens with near worldwide distribution and an unclear nonavian-host spectrum. While the natural hosts for IAV are among waterfowl species, certain mammals can be productively infected. Southern Africa is home to diverse avian and mammalian fauna for which almost no information exists on IAV dynamics. METHODS: We evaluated 111 serum samples from 14 mammalian species from Namibia for the presence of IAV-specific antibodies and tested whether host phylogeny, sociality, or diet influence viral prevalence and diversity. RESULTS: Free-ranging African mammals are exposed to diverse IAV subtypes. Herbivores developed antibodies against 3 different hemagglutinin (HA) subtypes, at low prevalence, while carnivores showed a higher prevalence and diversity of HA-specific antibody responses against 11 different subtypes. Host phylogeny and sociality were not significantly associated with HA antibody prevalence or subtype diversity. Both seroprevalence and HA diversity were significantly increased in carnivores regularly feeding on birds. CONCLUSIONS: The risk of infection and transmission may be driven by diet and ecological factors that increase contact with migratory and resident waterfowl. Consequently, wild mammals, particularly those that specialize on hunting and scavenging birds, could play an important but overlooked role in influenza epizootics.

The genetic architecture of helminth-specific immune responses in a wild population of Soay sheep (Ovis aries).

Much of our knowledge of the drivers of immune variation, and how these responses vary over time, comes from humans, domesticated livestock or laboratory organisms. While the genetic basis of variation in immune responses have been investigated in these systems, there is a poor understanding of how genetic variation influences immunity in natural, untreated populations living in complex environments. Here, we examine the genetic architecture of variation in immune traits in the Soay sheep of St Kilda, an unmanaged population of sheep infected with strongyle gastrointestinal nematodes. We assayed IgA, IgE and IgG antibodies against the prevalent nematode Teladorsagia circumcincta in the blood plasma of > 3,000 sheep collected over 26 years. Antibody levels were significantly heritable (h2 = 0.21 to 0.57) and highly stable over an individual's lifespan. IgA levels were strongly associated with a region on chromosome 24 explaining 21.1% and 24.5% of heritable variation in lambs and adults, respectively. This region was adjacent to two candidate loci, Class II Major Histocompatibility Complex Transactivator (CIITA) and C-Type Lectin Domain Containing 16A (CLEC16A). Lamb IgA levels were also associated with the immunoglobulin heavy constant loci (IGH) complex, and adult IgE levels and lamb IgA and IgG levels were associated with the major histocompatibility complex (MHC). This study provides evidence of high heritability of a complex immunological trait under natural conditions and provides the first evidence from a genome-wide study that large effect genes located outside the MHC region exist for immune traits in the wild.

Detection of antibodies against flavivirus over time in wild non-human primates from the lowlands of Costa Rica.

Two-hundred-nine free ranging non-human primates from 31 locations throughout Costa Rica were captured and released between 1993 and 2012, and blood samples, sera or plasma were collected, to detect antigens and antibodies, and so assess the distribution of active and passive flavivirus infections over time. A competitive enzyme-linked immunoassay for the detection of antibodies was used to determine the distribution of past flavivirus infections over time, while Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used to detect active West Nile Virus (WNV) and Dengue virus (DENV) infections. The first serological evidence of flavivirus in these animals was determined in 1993, at the same time when DENV re-emerged in humans from Costa Rica. An increase in the number of seropositive wild monkeys to flavivirus was determined over time in the country (11.3% seropositivity in 1993-1996, 20.7% in 2001-2008, and finally 52.9% in 2010-2012). Furthermore, the presence of DENV2 was detected in samples from four howler monkeys collected in 2001-2002, whereas DENV2, DENV3, and DENV4 were found in samples from four white-faced monkeys, and WNV in three howler monkeys living in the Pacific coast of Costa Rica during 2010-2012. The habitat where the positive PCR individuals lived were characterized as fragmented forests, having temperatures ranging from 26°C to 28°C, altitudes below 250 meters above sea level, high precipitation during 7 to 9 months (1500-4000 mm), and a marked dry season of 3 to 5 months. All these animals were living near mangroves; however, they did not show clinical signs of illness at the time of sampling. Results obtained show that the number of seropositive wild non-human primates to flavivirus were increasing during time in the country, longitudinal studies are needed to investigate their role as sentinels of these viruses and to determine if flavivirus infections can affect these species.

ONRAB® oral rabies vaccine is shed from, but does not persist in, captive mammals.

ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41 days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6 h to 3 days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies.