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Mathematical models highlighted the importance of pathogen-mediated invasion, with the replacement of red squirrels by squirrelpox virus (SQPV) carrying grey squirrels in the UK, a well-known example. In this study, we combine new epidemiological models, with a range of infection characteristics, with recent longitudinal field and experimental studies on the SQPV dynamics in red and grey squirrel populations to better infer the mechanistic basis of the disease interaction. A key finding is that a model with either partial immunity or waning immunity and reinfection, where individuals become seropositive on the second exposure to infection, that up to now has been shown in experimental data only, can capture the key aspects of the field study observations. By fitting to SQPV epidemic observations in isolated red squirrel populations, we can infer that SQPV transmission between red squirrels is significantly (4×) higher than the transmission between grey squirrels and as a result our model shows that disease-mediated replacement of red squirrels by greys is considerably more rapid than replacement in the absence of SQPV. Our findings recover the key results of the previous model studies, which highlights the value of simple strategic models that are appropriate when there are limited data, but also emphasise the likely complexity of immune interactions in wildlife disease and how models can help infer disease processes from field data., (© 2024 The Authors. Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.)

Small mammalian hibernators use metabolic suppression to enhance survival during the winter. Torpor is punctuated by periods of euthermia used to clear metabolic by-products and damaged cell components. The current study was performed to determine if the innate immune system, specifically NLRP and AIM2 inflammasome signaling, may detect and respond to cell stress during hibernation. Nlrp3, Casp1, and Il1b genes were significantly upregulated in brown adipose tissue (BAT) during arousal with respect to the euthermic control, suggesting increased NLRP3 inflammasome priming. NLRP3, IL-18, and gasdermin D protein levels increased during torpor, indicating a lag between inflammasome priming and formation. AIM2 and gasdermin D levels increased in BAT during arousal, as did caspase-1 activity. Thus, non-shivering thermogenesis may generate pro-inflammatory triggers of inflammasome signaling. This study is the first to support a role for inflammasome signaling in sensing cellular perturbations at various points of the torpor-arousal cycle, in metabolically-active BAT, but not white adipose tissue (WAT)., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cellular immune responses were tested to determine the effect of fenbendazole on the function of lymphocytes from Bolivian squirrel monkeys (Samiri boliviensis boliviensis). Giardia-infected squirrel monkeys were treated with commercially available fenbendazole (FBZ)-medicated monkey chow. Immune responses were compared between historical controls (Giardia naïve, untreated with FBZ (control animals)) and Giardia-infected, FBZ-treated squirrel monkeys (study animals). Peripheral blood lymphocytes from study monkeys had significantly lower stimulation indices compared to control animals when cultured in vitro with concanavalin A (Con A) (p<0.0001), phytohaemagglutinin (PHA) (p<0.0001) and lipopolysaccharide (LPS) (p<0.0001). PBMCs were also analyzed for IFN-γ producing cells in response to stimulation with Con A, PHA, PWM, and LPS by the cytokine ELISPOT assay. Significantly higher responses to Con A- (p<0.0001), and PHA- (p<0.001) stimulated cultures from Giardia-infected and fenbendazole treated compared to controls. Flow cytometric analysis for expression of cell surface markers revealed a significant increase in B- and NKT-lymphocytes and significant decrease in CD14+CD16+ monocytes after FBZ treatment. Also, circulating plasma cytokines IFN-γ, TNF-α, IL-12p40, IL-1β, IL-10, IL-13, IL-1ra, IL-6 and IL-4 were significantly decreased after FBZ treatment. Comparison of hematologic parameters between controls and FBZ-treated squirrel monkeys revealed significantly lower numbers of total leukocytes, neutrophils, monocytes, and eosinophils compared to controls. However, erythrocyte indices (red cell count, hemoglobin and hematocrit were significantly higher in FBZ-treated monkeys. Our findings suggest that fenbendazole treatment may alter sensitive immune and molecular measures of inflammation. Postponing the experimental use of squirrel monkeys until at least 6 weeks after FBZ treatment should be considered., Competing Interests: The authors have declared that no competing interests exist.

Oral vaccination is an emerging management strategy to reduce the prevalence of high impact infectious diseases within wild animal populations. Plague is a flea-borne zoonosis of rodents that often decimates prairie dog (Cynomys spp.) colonies in the western USA. Recently, an oral sylvatic plague vaccine (SPV) was developed to protect prairie dogs from plague and aid recovery of the endangered black-footed ferret (Mustela nigripes). Although oral vaccination programs are targeted toward specific species, field distribution of vaccine-laden baits can result in vaccine uptake by non-target animals and unintended indirect effects. We assessed the impact of SPV on non-target rodents at paired vaccine and placebo-treated prairie dog colonies in four US states from 2013 to 2015. Bait consumption by non-target rodents was high (70.8%, n = 3113), but anti-plague antibody development on vaccine plots was low (23.7%, n = 266). In addition, no significant differences were noted in combined deer mice (Peromyscus maniculatus) and western harvest mouse (Reithrodontomys megalotis) abundance or community evenness and richness of non-target rodents between vaccine-treated and placebo plots. In our 3-year field study, we could not detect a significant positive or negative effect of SPV application on non-target rodents.

The immunocompetence handicap hypothesis (ICHH) proposes that testosterone has both beneficial effects on male reproductive potential and negative effects by suppressing the immune system. However, support for the ICHH has been variable and an alternative hypothesis suggests that testosterone may be acting indirectly via cortisol to suppress immunity (the stress-linked ICHH). A third hypothesis is that increased energetic investment in immunity results in the suppression of testosterone. We tested these hypotheses in male Cape ground squirrels ( Xerus inauris ) through two separate manipulations: first, by triggering a strong immune response using a lipopolysaccharide (LPS) injection and, secondly, by increasing circulating testosterone using silastic testosterone implants. Responding to an immune challenge significantly reduced testosterone, supporting the immune suppression hypothesis, while increasing circulating testosterone had no effect on immunocompetence, body mass, ectoparasite abundances or cortisol levels, failing to support either the ICHH or stress-linked ICHH. Our results add to the increasing body of literature that challenges the ICHH, and we conclude that the trade-off between testosterone and immunity is mediated through immune activation and not through testosterone in male Cape ground squirrels. Being able to test the ICHH, stress-linked ICHH and immune suppression hypotheses in a free-ranging mammal gives us a unique opportunity to examine the mechanisms mediating this trade-off., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities., (© 2017 Wiley Periodicals, Inc.)

Sylvatic plague, caused by Yersinia pestis, frequently afflicts prairie dogs (Cynomys spp.), causing population declines and local extirpations. We tested the effectiveness of bait-delivered sylvatic plague vaccine (SPV) in prairie dog colonies on 29 paired placebo and treatment plots (1-59 ha in size; average 16.9 ha) in 7 western states from 2013 to 2015. We compared relative abundance (using catch per unit effort (CPUE) as an index) and apparent survival of prairie dogs on 26 of the 29 paired plots, 12 with confirmed or suspected plague (Y. pestis positive carcasses or fleas). Even though plague mortality occurred in prairie dogs on vaccine plots, SPV treatment had an overall positive effect on CPUE in all three years, regardless of plague status. Odds of capturing a unique animal were 1.10 (95% confidence interval [C.I.] 1.02-1.19) times higher per trap day on vaccine-treated plots than placebo plots in 2013, 1.47 (95% C.I. 1.41-1.52) times higher in 2014 and 1.19 (95% C.I. 1.13-1.25) times higher in 2015. On pairs where plague occurred, odds of apparent survival were 1.76 (95% Bayesian credible interval [B.C.I.] 1.28-2.43) times higher on vaccine plots than placebo plots for adults and 2.41 (95% B.C.I. 1.72-3.38) times higher for juveniles. Our results provide evidence that consumption of vaccine-laden baits can protect prairie dogs against plague; however, further evaluation and refinement are needed to optimize SPV use as a management tool.

Plague impacts prairie dogs (Cynomys spp.), the endangered black-footed ferret (Mustela nigripes) and other sensitive wildlife species. We compared efficacy of prophylactic treatments (burrow dusting with deltamethrin or oral vaccination with recombinant "sylvatic plague vaccine" [RCN-F1/V307]) to placebo treatment in black-tailed prairie dog (C. ludovicianus) colonies. Between 2013 and 2015, we measured prairie dog apparent survival, burrow activity and flea abundance on triplicate plots ("blocks") receiving dust, vaccine or placebo treatment. Epizootic plague affected all three blocks but emerged asynchronously. Dust plots had fewer fleas per burrow (P < 0.0001), and prairie dogs captured on dust plots had fewer fleas (P < 0.0001) than those on vaccine or placebo plots. Burrow activity and prairie dog density declined sharply in placebo plots when epizootic plague emerged. Patterns in corresponding dust and vaccine plots were less consistent and appeared strongly influenced by timing of treatment applications relative to plague emergence. Deltamethrin or oral vaccination enhanced apparent survival within two blocks. Applying insecticide or vaccine prior to epizootic emergence blunted effects of plague on prairie dog survival and abundance, thereby preventing colony collapse. Successful plague mitigation will likely entail strategic combined uses of burrow dusting and oral vaccination within large colonies or colony complexes.

The endangered black-footed ferret (Mustela nigripes) is affected by plague, caused by Yersinia pestis, both directly, as a cause of mortality, and indirectly, because of the impacts of plague on its prairie dog (Cynomys spp.) prey base. Recent developments in vaccines and vaccine delivery have raised the possibility of plague control in prairie dog populations, thereby protecting ferret populations. A large-scale experimental investigation across the western US shows that sylvatic plague vaccine delivered in oral baits can increase prairie dog survival. In northern Colorado, an examination of the efficacy of insecticides to control fleas and plague vaccine shows that timing and method of plague control is important, with different implications for long-term and large-scale management of Y. pestis delivery. In both cases, the studies show that ambitious field-work and cross-sectoral collaboration can provide potential solutions to difficult issues of wildlife management, conservation and disease ecology.

Monkeypox (MPX) is a zoonotic disease endemic in Central and West Africa and is caused by Monkeypox virus (MPXV), the most virulent Orthopoxvirus affecting humans since the eradication of Variola virus (VARV). Many aspects of the MPXV transmission cycle, including the natural host of the virus, remain unknown. African rope squirrels (Funisciurus spp.) are considered potential reservoirs of MPXV, as serosurveillance data in Central Africa has confirmed the circulation of the virus in these rodent species [1,2]. In order to understand the tissue tropism and clinical signs associated with infection with MPXV in these species, wild-caught rope squirrels were experimentally infected via intranasal and intradermal exposure with a recombinant MPXV strain from Central Africa engineered to express the luciferase gene. After infection, we monitored viral replication and shedding via in vivo bioluminescent imaging, viral culture and real time PCR. MPXV infection in African rope squirrels caused mortality and moderate to severe morbidity, with clinical signs including pox lesions in the skin, eyes, mouth and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, fast systemic spread, and long periods of viral shedding. Shedding and luminescence peaked at day 6 post infection and was still detectable after 15 days. Interestingly, one sentinel animal, housed in the same room but in a separate cage, also developed severe MPX disease and was euthanized. This study indicates that MPXV causes significant pathology in African rope squirrels and infected rope squirrels shed large quantities of virus, supporting their role as a potential source of MPXV transmission to humans and other animals in endemic MPX regions.

During hibernation, significant changes occur in the systemic and intestinal immune populations. We found that the lungs of hibernating 13-lined ground squirrels (Ictidomys tridecemlineatus) also undergo shifts in immune phenotype. Within the population of mononuclear cells, the percentage of T cells increases and the percentage of CD11b/c(+) cells decreases in hibernators. E-selectin, which promotes endothelial attachment, increases during arousal from torpor. Levels of the anti-inflammatory cytokine interleukin (IL)-10 in the lung are lower during hibernation while levels of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α remain constant. Expression of suppressor of cytokine signaling (SOCS) proteins is also decreased in torpid hibernators. Our data point to a unique immune phenotype in the lung of hibernating ground squirrels in which certain immunosuppressive proteins are downregulated while some potentially inflammatory proteins are maintained or amplified. This indicates that the lung houses an immune population that can potentially respond to antigenic challenge during hibernation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The hypothalamic-pituitary-adrenal (HPA) axis releases glucocorticoids (GCs), or stress hormones, during the vertebrate stress response. GCs can both enhance and suppress the immune system depending on whether the experienced stressor is acute or chronic and what aspect of immune function is measured. More research is needed to fully understand how the immune system reacts to stressors. In this study, we examined the effects of chronically raised GCs on innate immune function in Belding's ground squirrels (Urocitellus beldingi). We measured immune function with a bacteria killing ability (BKA) assay, an integrative and functional assessment of an animal's ability to clear a bacterial infection. All studies to date have examined how acute stressors or repeated social stressors impact BKA. This study is the first to our knowledge to investigate how chronically raised GCs impact BKA both before and after an immune challenge. We noninvasively raised GCs in treatment squirrels for six days and then gave them, and a group of untreated (control) squirrels, an injection of lipopolysaccharide (LPS) to stimulate their innate immune system. Treatment squirrels exhibited lower BKA after, but not before, being challenged with LPS. These results suggest that experiencing chronic stress may not be detrimental to immune functioning until an individual is challenged with an infection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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

ONRAB(®) is a recombinant human adenovirus type 5 (HAd5) with the rabies glycoprotein gene incorporated into its genome. ONRAB(®) has been used in Canada as an oral rabies vaccine in target wildlife species such as: red fox (Vulpes vulpes), raccoon (Procyon lotor), and striped skunk (Mepthis mephitis). We evaluated the safety of ONRAB(®) in non-target wildlife species likely to contact the vaccine baits during oral rabies vaccine campaigns in the United States. We investigated the effects of oral inoculation of high titer ONRAB(®), approximately ten times the dose given to target species, in wood rats (Neotoma spp.), eastern cottontail rabbits (Sylvilagus floridanus), Virginia opossums (Didelphis virginiana), eastern wild turkeys (Meleagris gallopavo silvestri), and fox squirrels (Sciurus niger). We performed real-time polymerase chain reaction (PCR) on fecal swabs, oral swabs, and tissues, including lung, liver, kidney, small intestine, large intestine, and when appropriate nasal turbinates, to detect ONRAB(®) DNA from inoculated animals. By seven days post-inoculation, turkeys, opossums, and cottontails had all stopped shedding ONRAB(®) DNA. One wood rat and one fox squirrel still had detectable levels of ONRAB(®) DNA in fecal swabs 14 days post-inoculation. Real-time PCR analysis of the tissues revealed some ONRAB(®) DNA persisting in certain tissues; however, there were no significant gross or histologic lesions associated with ONRAB(®) in any of the species studied. Our results suggest that many non-target species are not likely to be impacted by the distribution of ONRAB(®) as part of oral rabies vaccination programs in the United States., (Published by Elsevier Ltd.)

Mammalian hibernation consists of periods of low metabolism and body temperature (torpor), interspersed by euthermic arousal periods. The function of both the innate and adaptive immune system is suppressed during hibernation. In this study, we analyzed the humoral adaptive immune response to a T-cell independent (TI-2) and a T-cell dependent (TD) antigen. Thirteen-lined ground squirrels were immunized in summer or during hibernation with either a TI-2 or TD antigen on day 0 and day 14. Blood was drawn on day 0, 7, 14, 21 and 28. Both types of antigens induced a significant rise in antibody titer in summer animals. Much to our surprise, however, only immunization with the TD antigen, and not with the TI-2 antigen induced a humoral response in hibernators. Flow cytometric analysis of CD4 (helper T-lymphocytes), CD8 (cytotoxic T-lymphocytes) and CD45RA (B-lymphocytes) in blood, spleen and lymph nodes ruled out massive apoptosis as explanation of the absent TI humoral response during hibernation. Rather, reduced TI-2 stimulation of B-lymphocytes, possibly due to lowered serum complement during torpor, may explain the reduced antibody production in response to a TI-2 antigen. These results demonstrate that hibernation diminishes the capacity to induce a TI-2 humoral immune response, while the capacity to induce a humoral response to a TD antigen is maintained., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Immunoenhancing attributes of melatonin (Mel) on the immunocompromised state induced by glucocorticoid is well known, but the involvement of their receptors in the modulation of immunity has never been studied in any rodent. The present study explores the role of Mel and its receptors (MT1 and MT2) in amelioration of immunocompromised state induced by a synthetic glucocorticoid, dexamethasone (Dex) in a tropical rodent Funambulus pennanti. Immune parameters viz. DTH response, Lymphocyte proliferation, cytokine (IL-2) and antibody production were assessed following pretreatment of Mel and Dex alone or in combination. Mel enhanced the IL-2 production, thymic and splenic lymphocyte proliferation thereby increasing T helper cell associated immune responses and anti-KLH-IgG production. MT1 and MT2 receptor expression was downregulated following Dex treatment while glucocorticoid receptors (GR) expression was downregulated in Mel treated groups suggesting that the immunomodulatory effects of glucocorticoids and Mel are mediated via their receptors. To gain further insights on the role of Mel receptors, we used nonselective melatonin receptor antagonist luzindole which resulted in reversal of most of the immunomodulatory actions of Mel. Therefore, it may be suggested that a physiological cross talk exist between Mel and GR which is of high adaptive significance in wild animals for balancing the immunity during ecologically stressful conditions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Differences in how males and females respond to foreign antigens are common across taxa. Such sexual differences in the immune system are predicted to be greater in species with high promiscuity and sociality as these factors increase the likelihood of disease transmission. Intense sperm competition is thought to further this sexual dichotomy as increased investment in spermatogenesis likely incurs additional immunological costs. Xerus inauris, a ground squirrel found throughout southern Africa, is extremely social and promiscuous with one of the highest male reproductive investments among rodents. These life-history attributes suggest males and females should demonstrate a large dichotomy in immunity. Contrary to our prediction, we found no difference in spleen mass between the sexes. However, we did find significant biases in leukocyte types and red blood cell counts, possibly reflecting responses to parasite types. Among males, we predicted greater investments in spermatogenesis would result in reduced immunological investments. We found a negative association between testes and spleen size and a positive relationship between testes and number of lice suggesting trade-offs in reproductive investment possibly due to the costs associated with spermatogenesis and immunity. We suggest when measuring sexual differences in immunity it is important to consider the effects of reproductive pressures, parasite types, and life history costs.

Over the past 40 yr, epizootics of plague (Yersinia pestis) in northern Arizona have reduced populations of the Gunnison's prairie dog (Cynomys gunnisoni), with the exception of a large population found in the Aubrey Valley (AV). To examine potential mechanisms accounting for their survival, we collected prairie dog serum samples in 2005-2006 from AV and a neighboring population near Seligman (SE), Arizona. We quantified gene expression at 58 diverse immune proteins using a multiplexed enzyme-linked immunosorbent assay panel. We found a subset of proteins important in coagulation and inflammation (tissue factor [TF], calbindin [Cal], and thrombopoietin [TPO]) and T-cell responses (CD40L and CD40) that were present in AV at levels two to eight times greater than SE. These results suggest that AV and SE animals might differ in their ability to mount an immune response.

The redwood chipmunk contributes to the maintenance of tick-borne diseases in northern California. The range of redwood chipmunks overlaps that of western black-legged ticks and tick-borne disease, including granulocytic anaplasmosis and Lyme borreliosis. Chipmunks have high Anaplasma phagocytophilum PCR- and seroprevalence, are infested with a diversity of Ixodes spp. ticks, and are reservoir competent for Borrelia burgdorferi. We hypothesized that chipmunks could maintain tick-borne disease on the forest floor while also potentially bridging infection to arboreal sciurids as well. We used radio-telemetry to evaluate chipmunk movement and use of trees, characterized burrows, described prevalence of tick-borne disease, and identified ticks on these chipmunks. A total of 192 chipmunks from Hendy Woods, Mendocino County, California, USA, was evaluated between November 2005 and April 2009. The mean density was 2.26-5.8 chipmunks/ha. The longest detected life span was 3 years. Female weights ranged from 80-120 g and males from 80-180 g. The A. phagocytophilum and Borrelia spp. seroprevalence was 21.4% and 24.7%, respectively, and PCR prevalence for these pathogens was 10.6% and 0%, respectively. Ixodes spp. ticks included I. angustus, I. ochotonae, I. pacificus, and I. spinipalpis. The mean infestation level was 0.92 ticks/chipmunk. Based on telemetry of 11 chipmunks, the greatest distance traveled ranged from 0.14-0.63 km for females and 0.1-1.26 km for males. Areas occupied by chipmunks ranged from 0.005-0.24 km(2) for females and 0.006-0.73 km(2) for males. On 3 occasions, chipmunks were found in trees. Burrows were identified under a moss-covered redwood log, deep under a live redwood tree, under a Douglas fir log, in a clump of huckleberry, in a root collection from an overturned Douglas fir tree, and in a cluster of exposed huckleberry roots. The biology of the redwood chipmunk has multiple features that allow it to be an important reservoir host for tick-borne disease in northwestern California., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

An inverse relation exists between melatonin and androgen in most of the seasonally breeding rodents, but the regulation of their receptors in modulation of immune function has never been reported. The present study accessed the expression pattern of melatonin receptor types (mt1R & mt2R), immune parameters (lymphoid organs weight, leucocyte count, delayed type hypersensitivity and lymphocyte proliferation) in spleen and thymus whereas androgen receptor (AR) expression in thymus of Funambulus pennanti during reproductively active phase. In-vivo melatonin treatment (Mel) and castration (Cx) significantly increased mt1R expression, immune parameters in spleen and thymus but decreased AR expression in thymus only when compared with sham control (Con) squirrels as AR expression was not observed in spleen. Mel alone or in combination with testosterone (T) to Cx squirrels significantly increased mt1R expression, immune parameters in spleen and thymus but decreased AR expression in thymus. T alone in Cx squirrels significantly decreased mt1R expression, immune parameters in spleen and thymus but increased thymic AR expression significantly. In-vitro thymocyte culture supported our in-vivo findings. Mel significantly increased mt1R expression, lymphocyte proliferation, IL-2 secretion but decreased AR expression. T alone significantly decreased aforementioned three parameters but increased AR expression. Combined treatment of Mel and T bring back all parameters to control level. Though we found high mt2R expression, but no significant change has been observed. Thus, present study suggests a clear-cut trade-off relation between mt1R and AR expression that might be acting as an important mediator in seasonal adjustment of immune function in tropical rodents.

Photoperiodic regulation of melatonin receptor types on target tissues, such as lymphatic organs, has never been explored for any seasonal breeder. In the present study, we accessed the high affinity membrane melatonin receptors MT1 and MT2 expression dynamics in lymphoid organs (i.e., spleen and thymus) of a seasonally breeding rodent Funambulus pennanti during two major reproductive phases (i.e., active and inactive), when the internal hormonal (melatonin and gonadal steroid) as well as the ecological conditions were entirely different. Photoperiod regulates circulatory melatonin level; hence, we noted the effect of different photoperiodic regimes (long; 16L:8D and short; 10L:14D photoperiod) equivalent to summer and winter daylength on membrane melatonin receptor MT1 and MT2 expression in spleen and thymus. We have correlated the melatonin receptor expression with two major hormones varying seasonally (i.e., melatonin and testosterone) also being responsible for modulation of immunity of a seasonal breeder. Differential immunoreactivity of MT1 and MT2 receptor in spleen and thymus of F. pennanti suggests an involvement of both the receptor types in signal transduction of photoperiod for seasonal immunomodulation, because in the tropical zone, a slight difference (1:45-2 h) in daylength may change reproductive physiology and immunity of animals for adaptation. Our above suggestion receives strong support from the experiment of photoperiodic exposure on MT1 and MT2 expression at the translational level, where long daylength decreased the circulatory melatonin level and melatonin receptor expression in both lymphatic tissues. On the other hand, under short daylength, expression of MT1 and MT2 receptor increased in both spleen and thymus along with concomitant increase in circulatory melatonin level. Differential hormonal level of melatonin and gonadal hormones during reproductively active and inactive phase and its direct relation with melatonin receptor expression dynamics in lymphoid organs could be responsible for seasonal adjustment of immunity and reproduction.

A comparative analysis of low linear energy transfer (LET) gamma-radiation-induced damage in the lymphatic tissue of a tropical seasonal breeder, Indian palm squirrel (Funambulus pennanti), during its reproductively active phase (RAP) and inactive phase (RIP) was performed with simultaneous investigation of the effects of long-term melatonin pre-treatment (100 microg/100 g body weight). A total of 120 squirrels (60 during RAP and 60 during RIP) were divided into 12 groups and sacrificed at 4, 24, 48, 72 and 168 h following 5 Gy gamma-radiation exposure; control groups were excluded from exposure. Total leukocyte count and absolute lymphocyte count (ALC) and melatonin only of peripheral blood, stimulation index, thiobarbituric-acid-reactive substances (TBARS) level, superoxide dismutase (SOD) activity, and the apoptotic index of spleen as analysed by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) noted at observed time-points were significantly reduced in melatonin pre-treated groups during RAP and RIP. Long-term melatonin pre-treatment mitigated radiation-induced alterations more prominently during RIP, as assessed by ALC, TBARS, SOD, TUNEL and caspase-3 activity, at some time-points. Our results demonstrate an inhibitory role of melatonin on caspase-3 activity in splenocytes during RAP and RIP following gamma-radiation-induced caspase-mediated apoptosis. Hence, we propose that melatonin might preserve the viability of immune cells of a seasonal breeder against background radiation, which is constantly present in the environment.

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

There is growing evidence that populations of the northern flying squirrel (Glaucomys sabrinus) are declining in the eastern United States, perhaps due to competition with the southern flying squirrel (Glaucomys volans). Potential causes include parasite-mediated or apparent competition from the shared intestinal nematode, Strongyloides robustus, which has been shown to detrimentally affect the northern flying squirrel but not the southern flying squirrel. To investigate this hypothesis, we conducted a preliminary study on the parasite community of both flying squirrel species from sites in Pennsylvania where the two species occur sympatrically and where G. sabrinus is now considered endangered at the state level. We compared these parasite communities with those from northern flying squirrels from northern New York where the southern flying squirrel is absent. We found eight species of gastrointestinal parasites (Pterygodermatites peromysci, Lemuricola sciuri, Syphacia thompsoni, Syphacia spp., Capillaria spp., Citellinema bifurcatum, Strogyloides robustus, and an unidentifiable cestode species) in both species of flying squirrels examined for our study. The parasite-mediated competition hypothesis was partially supported. For example, in Pennsylvania, S. robustus was overdispersed in southern flying squirrels, such that a small proportion of the hosts carried a large proportion of the worm population. In addition, we found S. robustus to be present in northern flying squirrels when the species were sympatric, but not where southern flying squirrels were absent in New York. However, there was no association between S. robustus and the body condition of flying squirrels. We detected a potential parasite community interaction, as S. robustus abundance was positively associated with P. peromysci.

Hibernation is associated with a prolonged fast (5-8 mo) which has the potential to affect intestinal immunity. We examined several aspects of the intestinal immune system in summer (non-hibernating) and hibernating ground squirrels. Peyer's patches were largely unaffected by hibernation, but numbers of intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) were greater in hibernators compared with summer. Hibernator IEL were less mature as demonstrated by low numbers of cells expressing activation-associated markers and co-receptors. Compared with summer, the percentage of B cells was higher and percentage of T cells was lower in the hibernator LPL. Hibernation was associated with greater mucosal levels of IFN-gamma, TNF-alpha, IL-10 and IL-4, but IL-6 and TGF-beta were unchanged. Mucosal IgA levels were greater in entrance and torpid hibernators compared with summer. The results suggest that modifications of the intestinal immune system during hibernation may help preserve gut integrity throughout the winter fast.

This study evaluates binding of bacterial lipopolysaccharide (LPS) by splenic macrophages from golden-mantled ground squirrels (Spermophilus lateralis, GMGS), a hibernating mammal, at a variety of in vitro incubation temperatures to determine whether this aspect of immune function is effective at low body temperatures. LPS-binding by ground squirrel macrophages was compared to that of rat splenic macrophages. Macrophages were collected from squirrels at discreet stages in their annual cycle and incubated with fluorescein-labeled LPS (LPS-FITC). The percentage of GMGS that bound LPS-FITC did not change as a function of hibernation season or as a function of incubation temperature. The total amount of LPS-FITC bound per cell was similarly unaffected by season or temperature, however, macrophages from torpid squirrels bound more LPS-FITC than cells from normothermic squirrels. Macrophages of golden-mantled ground squirrels bind LPS at a wide range of temperatures throughout their annual cycle; an ability shared between hibernators and non-hibernators alike.

In vivo effect of dexamethasone and melatonin on immunomodulation has been investigated by studying the lymphocyte proliferation to the mitogen Con A from various lymphoid tissues including bone marrow cells of a seasonally breeding rodent adult male F. pennanti during reproductively inactive phase (October to December). During this phase, animal faces the maximum challenges of the nature (hypothermic stress, scarcity of food and shelter). Dexamethasone treatment (60 microg/day/squirrel) for 60 consecutive days significantly decreased the thymus and spleen activity. The lymphoid tissues mass, total leukocyte, lymphocyte count of peripheral blood, bone marrow and T-cell mediated immune function was also significantly suppressed following the dexamethasone treatment but treatment of melatonin (25 microg/squirrel/day) along with dexamethasone significantly restored the suppressed immune status in squirrels. Further, histological study of the thymus showed profound changes in the cellularity with a depletion of thymocytes in the cortex region of thymic lobules and increased in connective tissues and spindle cells. Melatonin treatment alone increased thymocytes density in thymic cortex, clearly suggesting that melatonin counteracted the experimentally induced immune stress by dexamethasone. Therefore, in nature during reproductively inactive phase of the squirrel a high level of melatonin was noted, that is required to combat nature's stress, which might have increased the internal level of corticoids., (Copyright 2004 Elsevier Inc.)

We investigated the relative importance of environmental factors versus host phenotype in determining parasite prevalence in Eurasian red squirrels (Sciurus vulgaris). One hundred and forty-three fecal samples of 116 different squirrels collected in 2000 and 2001 from five study areas in the Italian Alps, were examined for intestinal protozoans. Two species of Eimeria were present with a medium to high prevalence in both years and in all areas, while two other species were rare, occurring only in some areas and not in all years. Cryptosporidium parvum had a high prevalence in the two study areas of the Western Alps, while in the three areas of the Central Alps it was recorded only once. The prevalence of Eimeria sciurorum and C. parvum fluctuated in parallel with squirrel density, suggesting a possible correlation between the presence of these protozoans and host density. A gender effect on E. sciurorum prevalence at low density could be explained by different space use patterns and social organization of males and females. C. parvum occurred more frequently in young squirrels, suggesting an acquired immunity in adults, but age-related susceptibility was not found for eimerian species. The coccidian community was more similar within than between regions, and study area and year were key parameters in predicting coccidia infection. There was no evidence of competition between coccidian species, but one positive interaction between E. sciurorum and E. andrewsi was observed. Our results suggest that the effects of geographic region, area features, and year effects probably related to fluctuations in host population density, were more important than individual phenotypic host characteristics in structuring the coccidian assemblage and determining levels of parasite prevalence in red squirrel populations.

Numbers of leucocytes in squirrels with gametocytes of Hepatozoon in their blood (infected) were compared with animals without gametocytes (uninfected). Typical values for leucocytes/mm3 blood in uninfected squirrels were: leucocytes 5.7 x 10(3), granulocytes 3.4 x 10(3), lymphocytes 2.0 x 10(3) and monocytes 0.3 x 10(3) cells. Infection caused an increase in monocytes, lymphocytes and granulocytes, and there was a significant positive association between parasitaemia level and numbers of both total leucocytes and monocytes. Infected animals had more uninfected monocytes/mm3 blood than did uninfected animals. The proportions of monocytes were more variable over time in infected animals, but no shift between infected and uninfected status was detected. Transfer of serum from infected squirrels to mice resulted in elevated counts of total blood leucocytes, monocytes and granulocytes, but not of lymphocytes, as compared with controls. Serum from squirrels with high parasitaemias had a more marked effect than serum from squirrels with low parasitaemias. Results indicate an infection-related monocytosis, possibly controlled by cytokines, that increases the number of cells available for invasion by gametocytes, thus enhancing the chances of parasite transmission.

Little is known about the changes in the immune system that coincide with the annual cycle of hibernating mammals. This study investigates classical pathway complement activity in the serum of the golden-mantled ground squirrel, a mammalian hibernator. Complement activity varied significantly among discreet stages of the annual cycle and is lowest during torpor and greatest during stages of arousal. C3 mRNA levels follow a pattern similar to that of complement-mediated hemolysis throughout the year but do not vary significantly among hibernation states. The classical pathway of the serum complement system is able to function in vitro at 5 degrees C, although at a slower rate than at 34 degrees C. The classical pathway of the serum complement system is active throughout all phases of the annual cycle of the golden-mantled ground squirrel.

Melatonin (MEL) regulation of seasonal variation in immunity has been studied extensively in temperate mammals. This report is the first on a tropical mammal, the Indian palm squirrel, F. pennanti. In response to the annual environmental cycle, we studied the rhythms of plasma MEL and the immune parameters of total blood leucocytes, absolute blood lymphocytes and blastogenic responses of blood, thymus and spleen lymphocytes. We found that in parallel with MEL all the immune parameters increased during the month of April onward, when natural day length, temperature, humidity and rainfall were increasing. Maximum values occurred during November (reproductively inactive phase) when the values of all the physical factors were comparatively low. Lowest values occurred during January-March (reproductively active phase) when the values of the physical factors were lowest. In order to establish a clear interrelationship between the pineal MEL and the immune system function, we manipulated these squirrels with exogenous MEL (25mg/100 g B wt/day) at 1730 h during their pineal inactive phase (March) while another group was pinealectomized (Px) during November when their pineal was active. The MEL injection significantly increased all the immune parameters, while Px decreased them significantly. Hence, we suggest that MEL is immuno-enhancing for this tropical squirrel, and plays an important role in the maintenance of its immunity in accordance with the seasonal changes in environmental factors and gonadal status.

TNF production has been studied in peritoneal macrophages and splenic T cells of Arctic Yakutian ground squirrel (Citellus Undulatus Pallas) in hibernating and awake animals in winter and in prehibernating autumn as well as in active euthermic spring-summer animals. A high level of TNF production in macrophages of ground squirrel is observed over the active period and during arousals in winter. There are no significant season variations in TNF production in splenic T lymphocytes of ground squirrels. This suggests the major role of activated macrophages in the arousals of hibernating animals. T lymphocyte proliferation in ground squirrels in the active period is higher than in winter, and the most significant seasonal variations are found in T cell mitogenic response, which increases in spring-summer period. Evidence is presented that functional activity of macrophages of squirrel in autumn has much in common with that in winter rather than in spring-summer period.

The serological survey of white hares (n = 8), squirrels (n = 118), and Asian chipmunks (n = 486) in the dark coniferous forests of Middle Siberia revealed tick-borne encephalitis virus antihemagglutinins only in the former two species (37.5 +/- 17.1 and 7.6 +/- 2.4%, respectively) and in the squirrel, there is a close seasonal relation between the parameters of immune interbred and those virophoricity of taiga tick nymphs.

Major histocompatibility complex (MHC) class I genes encode cell surface molecules that bind and present immunogenic peptides to cytolytic T lymphocytes. We have developed a model using the MHC (Scab) of tassel-eared squirrels, Sciurus aberti, to investigate the evolution of MHC genes in subspecies that have been effectively isolated in comparable, if not identical, environments. Class I cDNAs from single S. aberti aberti and S. aberti kaibabensis squirrels were cloned and sequenced. These two cDNA transcripts differed by nucleotide substitutions that were predominantly located in codons impacting the peptide binding site, and nonsynonymous substitutions exceeded synonymous substitutions at these sites. These sequences also differed by the insertion of two amino acids in a beta-strand adjacent to position 45 in pocket B of the peptide binding site that may result in a deeper pocket with altered peptide specificity. This indel is present in additional Scab class I sequences, and class I sequences in five subspecies carry identically sized insertions. Phylogenetic analyses of exons 2, 3, and 4 with neighbor-joining and maximum parsimony methods depict that Scab class I sequences diverged at a point intermediate between murid class I sequences and class I sequences of primates, carnivores, and artiodactyls. The relative relatedness of Scab class I sequences to those in the latter group appears to be founded in relative similarities in exons 2 and 3, which encode the peptide binding site. These results bring into question the use of a single model for rodent class I sequences. Moreover, they demonstrate that the inclusion of exons 2 and 3 in phylogenetic analyses of class I may obscure true phylogenetic relationships, perhaps due to convergence through strong selective pressure.

Blood samples of 34 Eastern chipmunks trapped in the vicinity of an Aedes albopictus population near New Alsace, IN, were tested for neutralizing antibodies to La Crosse (LAC) virus and other California group viruses. Two samples were positive for LAC antibodies. Analysis of mosquito bloodmeals demonstrated that both Ae. albopictus and Ae. triseriatus from the site fed on chipmunks. This note documents the first record of LAC antibodies in sylvan rodents from Indiana, the presence of LAC virus in the vicinity of Ae. albopictus and that wild Ae. albopictus feed on Eastern chipmunks.

To study the persistence of Y. pestis capsular antigen, or fraction 1 (F1), in the body of less important plague carriers in the Mountain Altai and Transbaikal natural foci, as well as in experimentally infected ticks, the liquid-phase competitive radioimmunoassay (RIA) was used for the first time. In this study RIA showed, due to its sensitivity, doubtless advantages over traditional methods, such as the passive hemagglutination (PHA) test and the antibody neutralization (AN) test, and made it possible to detect F1 in picogram amounts. RIA revealed that F1 persisted in Siberian long-tailed gophers for up 14 months after the infection of the animals in diffusion chambers and for 7 months after their infection by subcutaneous injection. Experiments on Daurian pikas confirmed that, in comparison with the PHA and AN tests, RIA ensured fourfold effectiveness in the detection of antigen F1. The study of infected mites revealed that antigen F1 could be retained in them for more than a year and detected by RIA techniques in 10% of cases. The data obtained in this investigation indicate that the persistence of microorganisms should be studied mainly with the use of new-generation tests, and RIA, being one of the most sensitive techniques, deserves wide approval and introduction into the practical work of institutions intended for plague control.

Spotted souslik (Spermophilus suslicus) interferon was induced in different cell cultures with two strains: Random (NDV-R) and Hertfordshire (NDV-H) of Newcastle disease virus. Both strains were good interferon inducers in cultures of lung cells (SL), kidney cells (SK) and embryo cells (SE). There were no significant differences in the amount of interferon produced in cells at different passage levels but the yield of interferon depended on the donor of cells. The highest interferon titers were produced 24h after infection of SK cells with NDV-R at multiplicity of infection 100 or .10 TCID50 per cell. UV-irradiation decreased the interferonogenic properties of NDV-R. Priming with 100 U/ml of 10 U/ml of homologous interferon significantly enhanced interferon production in souslik cells. However, the interferon concentrations higher than 100 U/ml caused a distinct inhibition in the subsequent interferon production ("blocking"). The pretreatment of cells with L-ascorbic acid slightly increases the yield of interferon produced in souslik cells after induction with NDV-H. Calcium chloride at nontoxic concentrations added together with the virus-inducer practically exhibited no effect on the interferon production in SL cells.

Sera and livers from 40 Richardson ground squirrels were examined for evidence of ground squirrel hepatitis virus (GSHV) infection and hepatocellular carcinoma. Twenty-five sera were obtained from fully grown adult ground squirrels and 15 from young ground squirrels estimated to be between 6-8 weeks of age. All animals had been caught in the wild and had spent less than 1 month in captivity. Sixteen sera (40%) had at least one serologic marker of GSHV (2 with GSHV surface antigen, 3 GSHV core antigen, 5 GSHV antibody to core antigen and 11 GSHV antibody to surface antigen). Two cases of hepatocellular carcinoma were detected, both in adult ground squirrels. Tumour tissue and adjacent normal liver tissue were negative for GSHV surface and core antigen by direct immunofluorescence in both livers and negative for GSHV-DNA by molecular hybridization in the one tumour examined. These results indicate that: A) GSHV is geographically more widespread than previously considered; B) viral transmission occurs early in life, probably via the vertical route; and C) hepatocellular carcinoma is a relatively common finding in these animals while still in their wild state. Any causal relationship between hepatocellular carcinoma and GSHV infection in these animals, however, has yet to be demonstrated.

A new cell line derived from a woodchuck hepatocellular carcinoma serially transplanted in athymic nude mice has been established and named WH257GE10. The original tumor in the nude mouse system produces woodchuck hepatitis surface antigen and albumin. In addition, woodchuck hepatitis virus DNA is integrated into cellular DNA. Adaptation of the cells to the in vitro culture condition was completed after 15 months with the doubling time of 40 hr. The morphologic features of the cell by light microscopy are of an epithelial type. The modal chromosome number is 36 and the karyotype is mainly metacentric, similar to that observed in normal woodchuck liver cells. Ornithine and tyrosine aminotransferase activities were detected. Production of albumin was demonstrated in the cytoplasm by indirect immunofluorescence. Integration of woodchuck hepatitis virus DNA was shown by Southern blot analysis, although the secretion of woodchuck hepatitis surface antigen was not detected. This cell line provides an excellent in vitro model to study human hepatocellular carcinoma related to hepatitis B virus.

Two squirrels aged 16 weeks and three muskrats aged 24 weeks were subcutaneously infected with a dose of 400 SMicLD50 of the extraneurally passaged "236" strain of Tahyna virus. Viremia was detected in one squirrel (48 and 96 hours post infection) and in two muskrats (24 and 48 hours p.i.). Seroconversion was demonstrated by plaque-reduction neutralization test (PRNT) 21 days p.i. in all animals.

California encephalitis (CE) virus (snowshoe hare subtype) was isolated from 1 of 38 pools comprising 970 unengorged female Aedes canadensis mosquitoes and from 3 of 152 pools containing 5,676 A. communis mosquitoes which were collected in the Yukon Territory, Canada between latitudes 61 and 66 degrees N during June and July 1974. During four summers 1971 through 1974, this virus was recovered from 26 of 648 pools derived from 30,686 mosquitoes of 4 species. Isolation of CE virus from 1 of 109 pools of Aedes sp. larvae collected during May 1974 suggests maintenance of this virus over winter by transovarial transfer. Infectivity has been maintained in Culiseta inornata mosquitoes which were held continuously at 32 degrees F for 138 days. Neutralizing antibodies to CE virus were detected in 705 of 4,913 (14%) mammals collected during summers 1971 through 1974, including 430 of 1,076 (40%) snowshoe hares (Lepus americanus), 266 of 3,610 (7%) ground squirrels (Citellus undulatus) and 9 of 227 (4%) red squirrels (Tamiasciurus hudsonicus).

Transmission rates of La Crosse (LAC) virus observed in Aedes triseriatus females that had engorged on chipmunks with antibody to LAC and had been mated by infected males 5-11 days later (24%, 69/288) were 40% lower than in those fed on chipmunks without antibody (38%, 112/293). Similar results were obtained in three separate trials using males infected 1) by inoculation with prototype LAC virus, 2) transovarially with a field strain, or 3) transovarially with the field strain following passage through a viremic chipmunk. Similar rates were also observed in trials with F2 and F3 progeny of several strains of Ae. triseriatus collected from LAC-endemic and non-endemic areas. Reduction of oral transmission by venereally infected females mated by transovarially infected males following engorgement of antibody in chipmunks or other vertebrates could be important in the natural control of LAC virus, since most adult chipmunks sampled in endemic areas have antibodies neutralizing LAC. Ten-fold higher rates of venereal infection found in females mated by infected males 5 or more days after engorgement on LAC antibody-negative chipmunks than in those mated without prior engorgement extend previous findings of higher rates of transmission after engorgement on laboratory mice to include the natural vertebrate host.

Mosquitoes (eggs, larvae, and adults), small woodland animals, and residents of an area where California encephalitis is endemic were studied to elucidate the host-vector cycle of La Crosse virus. Elementary schoolchildren from surrounding communities and gray squirrels from another area were tested to compare the prevelence of serum antibodies to La Crosse virus in areas where the disease is endemic with the prevalence of these antibodies in areas where the disease is not endemic. From 1971 to 1974, eight isolations of La Crosse virus were made. Three of the isolates were from adult mosquitoes, one from Aedes triseriatus eggs, and four from A. triseriatus larvae. The isolation of virus from field-collected eggs and larvae confirms previous studies from Wisconsin that suggest that La Crosse virus overwinters in eggs of the mosquite A. triseriatus. In an area where California encephalitis is endemic, 10 of 19 small woodland animals (53%), which are the natural hosts of A. triseriatus, had hemagglutination-inhibiting and neutralizing antibodies to La Crosse virus. In contrast, none of 10 squirrels from an area where the disease is not endemic had such antibodies. Fourteen of 79 residents of this area (17.7%) had both types of antibody. Eleven of the 14 seropositive residents lived in one small sector of the community studied, an indication that foci of La Crosse virus activity may be very localized. Elementary schoolchildren from surrounding communitites had a significantly lower prevalence of hemagglutination-inhibiting antibody to La Crosse virus than did the residents of the area where California encephalitis was endemic.