Abstract
Sera collected from 442 mountain lions in 48 California counties between the years of 1987 and 2010 were tested using immunofluorescence assays and agglutination tests for the presence of antibodies reactive to Yersinia pestis, Francisella tularensis, Bartonella henselae, Borrelia burgdorferi, and Anaplasma phagocytophilum antigens. Data were analyzed for spatial and temporal trends in seropositivity. Seroprevalences for B. burgdorferi (19.9%) and B. henselae (37.1%) were relatively high, with the highest exposure in the Central Coast region for B. henselae. B. henselae DNA amplified in mountain lion samples was genetically similar to human-derived Houston-1 and domestic cat-derived U4 B. henselae strains at the gltA and ftsZ loci. The statewide seroprevalences of Y. pestis (1.4%), F. tularensis (1.4%), and A. phagocytophilum (5.9%), were comparatively low. Sera from Y. pestis- and F. tularensis-seropositive mountain lions were primarily collected in the Eastern and Western Sierra Nevada, and samples reactive to Y. pestis antigen were collected exclusively from adult females. Adult age (≥2 years) was a risk factor for B. burgdorferi exposure. Over 70% of tested animals were killed on depredation permits, and therefore were active near areas with livestock and human residential communities. Surveillance of mountain lions for these bacterial vector-borne and zoonotic agents may be informative to public health authorities, and the data are useful for detecting enzootic and peridomestic pathogen transmission patterns, particularly in combination with molecular characterization of the infecting organisms.
Introduction
Between the 1970s and 1990s, the number of human–mountain lion conflicts reported in California increased considerably, manifesting as either depredation incidents (in which mountain lions were associated with killing of domestic livestock and pets), or attacks on people (Beier 1991; Torres et al. 1996). Risk factors for mountain lion attacks on humans are likely to vary between geographic regions, but are historically hypothesized to be related to human population growth, mountain lion population growth, increasing anthropogenic activities on wild lands, and loss of mountain lion habitat through urban sprawl (Beier 1991; Torres et al. 1996; Conover 2002). Mountain lion proximity to humans and their domestic animals could pose a risk of zoonotic disease transmission, especially for pathogens that are readily transmitted across the wildlife-domestic animal-human interface.
Large temporal and spatial scales are required to study exposure to bacterial zoonoses in a wide-ranging wild animal population. We tested 442 mountain lion sera collected and archived by the California Department of Fish and Game (CDFG) during a 24-year period (1987–2010) for the presence of antibodies reactive to five bacterial vector-borne and zoonotic pathogens: Yersinia pestis, Francisella tularensis, Bartonella henselae, Borrelia burgdorferi, and Anaplasma phagocytophilum. The goals of the present study were to describe historical and contemporary temporal and spatial patterns in the distribution of mountain lion exposures to these pathogens in California. In this analysis, we consider potential enzootic and peridomestic pathogen transmission dynamics, and the implications of pathogen distribution patterns for animal and public health.
Materials and Methods
Sample collection
Mountain lion sera were collected between 1987 and 2010 by CDFG staff and collaborators. Source information was available for 390/442 samples. Sources included mountain lions killed on a depredation permit authorized by CDFG after threatening livestock or pets (n=281), mountain lions trapped and monitored for study (n=32), mountain lions killed because they posed a threat to public health and safety (n=29), dead mountain lions found by the roadside presumably hit by cars (n=23), mountain lions found dead or sick for other reasons (n=14), orphaned young mountain lions placed in captivity (n=7), mountain lions trapped and relocated for the animal's safety (n=2), and confiscated mountain lions poached or kept illegally as pets (n=2).
Whole blood was collected from live animals from the femoral vein. Dead mountain lions were typically frozen prior to thawing, necropsy, and collection of clotted blood in the heart. Following whole blood centrifugation, serum was isolated and frozen at −80°C. Animal age, sex, county of origin, and date trapped and/or killed was recorded at the time of sampling. Additional location information about the nearest city was recorded for 330 (74.7%) of 442 mountain lions. Mountain lions were classified as adult (≥2 years old) or juvenile (<2 years old) using tooth eruption and wear criteria (Ashman et al. 1983). Age and sex information was available for 431 and 438 animals, respectively.
For analysis of spatial trends in disease data, and to account for some of the uneven sampling between years and counties, we grouped California counties into nine regions: Bay Area, Eastern Sierra Nevada, Western Sierra Nevada, Central Valley, North Coast, Southeast, Northcentral/Northeast, South Coast, and Central Coast, based on the distribution of known mountain lion populations and movement (Ernest et al. 2003; Fig. 1a).
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Antibody detection
Antibody testing was performed at the University of California–Davis School of Veterinary Medicine using protocols optimized for felid sera. Y. pestis antibodies were detected using passive hemagglutination and hemagglutination inhibition tests based on previously described methods (Chu 2000). Rabbit serum controls were provided by the Centers for Disease Control and Prevention (CDC). A suspect positive result was recorded if hemagglutination was observed at a serum dilution of ≥1:16 (Paul-Murphy et al. 1994; Chu 2000). Hemolyzed serum samples were diluted 1:2 in phosphate-buffered saline (PBS) prior to testing to facilitate visualization of the agglutination reaction. Reactivity to F. tularensis antigen was detected using the BD febrile antigen slide agglutination test (Becton Dickinson, San Jose, CA), according to the manufacturer's instructions, using a screening dilution of 1:80 (Magnarelli et al. 2007). Hemolyzed serum samples were screened at a 1:160 dilution to improve interpretation of reactivity. Positive control rabbit serum was provided by the manufacturer. Saline, which reacted similarly to laboratory rabbit serum, was used as a negative control. Reactions were scored 0 (no reaction) or 1+ to 4+, with ≥2+ reaction indicating a test-positive sample.
Bartonella henselae antibody detection by indirect immunofluorescence assay (IFA) was performed at a 1:64 dilution as previously described (Case et al. 2006). Serum from cats infected experimentally with B. henselae strains Houston-1 and U4, and serum from uninfected laboratory cats, were used as positive and negative controls, respectively. The slides were read using a fluorescence microscope by two to three readers, and reactions of sera from individual mountain lions were scored 0 to 4+. Scores from individual readers were averaged, and a score ≥2+ was considered seropositive.
To evaluate presumptive exposure to A. phagocytophilum and B. burgdorferi, IFAs were performed as previously described (Foley and Nieto 2011), using an FITC-labeled goat anti-cat IgG secondary antibody (KPL, Gaithersburg, MD). Sera were tested at a single dilution of 1:100 in the B. burgdorferi assay, and 1:25 in the A. phagocytophilum assay. Negative controls included water and specific-pathogen-free cat serum. Positive controls included serum from a horse experimentally infected with A. phagocytophilum strain MRK, and serum from a dog naturally infected with a California isolate of B. burgdorferi sensu stricto. Mountain lion samples were considered positive if strong fluorescence in morulae or spirochetes was detected. Tests were scored as negative, weak positive, or positive. For analysis, weak positives were deemed to be negative. Tests for these two pathogens were not performed on serum collected prior to 1991 because of possible overlap with another study.
DNA extraction, polymerase chain reaction (PCR), and sequence analysis
DNA extractions were performed with the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA), on samples with the highest seroreactivity (>3+) to B. henselae antigen, using an initial volume of 100 μL serum, and a final elution volume of 100 μL in AE buffer. Portions of the Bartonella citrate synthase-coding gene (gltA), and cell division protein-coding gene (ftsZ), were amplified as described previously (Norman et al. 1995; Zeaiter et al. 2002), with minor changes, including a 50-μL PCR reaction volume, 57°C primer annealing temperature for gltA PCR, and 72°C primer extension temperature for ftsZ PCR. Sequencing was performed by Davis Sequencing (Davis, California).
Statistical and spatial analyses
Confidence intervals, chi-square tests of independence, and exact tests were performed to compare prevalence by groups of interest (e.g., age, county, and animal source), using Stata 11.0 (StataCorp 2009). Chi-square tests for temporal trends were performed using Epi Info Version 6 (Centers for Disease Control and Prevention 2001). Values of p<0.05 were considered statistically significant. Maps were prepared using ArcMap version 9.1 (Environmental Systems Resource Institute 2009). Analysis for spatial, temporal (3-month time frame), and spatiotemporal clusters was performed on samples for which city of origin data were available, using the SaTScan statistic Bernoulli model (SaTScan™ version 9.0,
Results
Sera from 442 animals originating in 48 California counties were tested for the presence of antibodies reactive to Y. pestis, F. tularensis, and B. henselae antigens. Eighty-two percent (n=354) of mountain lions tested were adults, and approximately 60% of the serum samples came from males (n=267). The majority (77.6%) of animals sampled for this study were killed on depredation permits, which explains the male sex bias also observed in other populations of mountain lions depredating livestock (Suminski 1982; Torres et al. 1996; McKinney et al. 2010). An average of 18.5±14.0 samples were available for testing per year, with the majority of samples collected between 1995 and 2006 (Fig. 2). Between 1 and 78 samples were available per county, with the most animals sampled in Mendocino (n=78) and El Dorado counties (n=47) (Fig. 1a). In general, the number of samples available for testing reflected the number of CDFG depredation permits recorded (California Department of Fish and Game, 2011). Figure 1a shows the number of serum samples available for testing in each county, and the predicted distribution of mountain lion habitat. Some regions of highly suitable mountain lion habitat (e.g., Northcentral/Northeast California in Fig. 1a) were underrepresented in the sample set.
Temporal distribution of B. henselae, B. burgdorferi, A. phagocytophilum, F. tularensis, and Y. pestis exposure in California mountain lions. The number of serum samples tested for each pathogen within each 4-year period is listed above the bars. B. burgorferi and A. phagocytophilum were not tested prior to 1991.
Yersinia pestis
A total of 6 out of 428 mountain lions (1.4%, 95% CI 0.5%,3.0%) were seropositive for Y. pestis. Plague antibody titers ranged from 1:32 to 1:256. All six animals were adult females from four counties within the Western and Eastern Sierra Nevada regions: El Dorado (n=1 in 2003), Fresno (n=1 in 1989), Kern (n=2 in 1989 and 2007), and Inyo (n=2 in 1993 and 1994; Figs. 1b and 2). Five of the positive animals had been trapped and radio-collared for scientific studies. The Inyo County female sampled in 1993 was later found weak and emaciated before death. This individual was also seropositive for feline immunodeficiency virus (data not shown).
Francisella tularensis
Six of 442 mountain lions (1.4%, 95% CI 0.5%,2.9%) were seropositive for F. tularensis (Fig. 2). The animals were from five counties in the South Coast and Western and Eastern Sierra Nevada regions: El Dorado (n=1 in 1993), Inyo (n=2 in 1994), Ventura and Mariposa (n=1 each in 1998), and Fresno (n=1 in 1999; Fig. 1b). Four of these animals were male, including one juvenile from Ventura County trapped for public safety purposes. The adults were either taken on depredation permits (n=4) or found dead or sick (n=1). The animal from Mariposa County was an adult female radio-collared in 1997 who was sampled in 1998 when found dead, possibly from starvation. Several days before death, the animal appeared emaciated and lame on the roadside (L. Chow, U.S. Geological Survey).
Bartonella henselae
We detected a statewide B. henselae seroprevalence in mountain lions of 37.1% (164/442, 95% CI 32.6%,41.8%; Table 1 and Fig. 1b). Seroprevalences varied significantly across regions (p<0.01). Among counties in which there were 10 or more samples tested, seroprevalences ranged from 9.1% (95% CI 0.23%,41.3%) in San Diego County, to 76.9% (95% CI 46.2%,95.0%) in Santa Cruz County (Table 1). Seroprevalences of B. henselae in the intensively-sampled El Dorado and Mendocino counties were 34.0% and 34.6%, respectively. When data were analyzed by region, B. henselae seroprevalence in Central Coast mountain lions (62.7%, 95% CI 48.1%,75.9%), was significantly higher than that in Eastern Sierra Nevada (11.1%, 95% CI 1.4%,34.7%), the North Coast (33.3%, 95% CI 25.1%,42.4%), and Western Sierra Nevada (32.5%, 95% CI 25.5%,40.2%; Table 1 and Fig. 1b). An apparent linear increase in the seroprevalence of B. henselae was observed over the 24-year study period, with animals sampled from 1994–2000 being 2.8 times more likely to be seropositive, and animals sampled from 2001–2008 being 5.2 times more likely to be seropositive, than animals sampled from 1987–1993 (Fig. 2; p<0.01). Animal source category, age, and sex were not associated with an increased risk of B. henselae exposure.
NT, not tested.
Seven serum samples with exceptionally strong immunofluorescence reactions to B. henselae were chosen for DNA extraction and PCR using Bartonella spp. gltA and ftsZ primers. Three samples (FM96002, FM96036, and FM00023) produced PCR products indicative of the presence of Bartonella using both primer sets. Samples FM96036 and FM00023 were 100% identical to B. henselae Houston-1 (ATCC 49882) at the gltA (252 bp) and ftsZ (764 bp) loci (GenBank accession numbers L38987 and AF061746), with the exception of two base pairs of the FM00023 ftsZ sequence, which could not be resolved despite sequencing in both directions. Sample FM96002 was also 100% identical to Houston-1 at the gltA locus, but was 100% similar to B. henselae strain UCD-U4 (U4) (Chung et al. 2004) at the ftsZ locus (762 bp).
Borrelia burgdorferi and Anaplasma phagocytophilum
Sera collected between 1991 and 2010 were tested for exposure to B. burgdorferi (n=408) and A. phagocytophilum (n=421; Table 2). The overall seroprevalences of B. burgdorferi and A. phagocytophilum were 19.9% (81/408, 95% CI 16.1%,24.1%) and 5.9% (25/421, 95% C.I. 3.9%,8.6%), respectively. Mountain lion exposure to B. burgdorferi was higher in adults than juveniles (p=0.02). No significant difference in seroprevalence was detected between source category, year group, or region for either pathogen (Figs. 1c, d, and 2, and Table 2). SaTScan analysis of A. phagocytophilum results revealed a temporal cluster of seropositive mountain lions sampled between December 2005 and March 2006 (p=0.07).
The number of serum samples tested for A. phagocytophilum antibodies is in parentheses.
NT, not tested; CI, confidence interval.
Discussion
California free-ranging mountain lions had high levels of exposure to B. henselae and B. burgdorferi, low levels of exposure to A. phagocytophilum, and rare exposure to Y. pestis and F. tularensis. Analysis of archived mountain lion serum revealed temporal and spatial trends in vector-borne and zoonotic pathogen distribution, as well as areas of potentially elevated risk to wildlife, domestic animals, and public health.
Mountain lions were likely infected with B. henselae, the agent of cat-scratch disease (CSD), by cat fleas or ticks while in close proximity to free-roaming domestic cats and their ectoparasites (Yamamoto et al. 1998; Chang et al. 2001, 2002). The statewide B. henselae seroprevalence of 37.1% detected here is similar to the 28.5% and 35% seroprevalences reported in mountain lions in previous studies (Yamamoto et al. 1998; Chomel et al. 2004). Regional variations in B. henselae seroprevalence (Fig. 1b) are similar to patterns of bobcat and pet cat exposures in other California studies (Jameson et al. 1995; Chomel et al. 2004). As hypothesized for pet cats, wild felid exposure is likely associated with flea vector abundance driven by environmental variables such as annual precipitation and average daily temperature (Jameson et al. 1995). B. henselae DNA isolated in a subset of mountain lion sera was most similar to organisms isolated in domestic cats (U4) and humans (Houston-1) (Regnery et al. 1992; Chung et al. 2004), supporting the hypotheses that mountain lions can serve as enzootic reservoirs of CSD (Yamamoto et al. 1998; Chomel et al. 2004).
The predicted mountain lion habitat in California overlaps extensively with the known range of the western black-legged tick Ixodes pacificus (Furman and Loomis 1984; Torres et al. 1996; Foley et al. 1999), the far-western vector of both B. burgdorferi, the agent of Lyme disease (LD), and A. phagocytophilum, the agent of human granulocytic anaplasmosis (HGA) (Clover and Lane 1995; Richter et al. 1996). Exposure to B. burgdorferi was prevalent in Mendocino, Sonoma, and Nevada counties (23.7%, 30.0%, and 21.4%, respectively), where some of the highest LD incidence rates in the state occur (California Department of Public Health, 2012). Antibodies reactive to A. phagocytophilum were detected in 12% of mountain lions previously tested in the North Coast range and Sierra Nevada foothills (Foley et al. 1999). In our study, mountain lions from the North Coast and Western Sierra Nevada had a seroprevalence of 9.5% and 5.5%, respectively. Because of the diversity of Borrelia and Anaplasma species with varying zoonotic potential in California (Postic et al. 2007; Foley et al. 2009; Girard et al. 2009, 2011), molecular characterization of tick-borne organisms infecting mountain lions will be essential to better understand their sentinel and reservoir potential for LD and HGA.
Francisella tularensis, the agent of tularemia, is found in a wide range of wildlife species, and is transmitted via arthropod bite, direct contact, ingestion, and inhalation. There is substantial overlap between the list of species that are susceptible to infection with, or are amplifying hosts of, F. tularensis in nature and known prey items for mountain lions (Emmons et al. 1976; Feldman 2003; Villepique et al. 2011). Yersinia pestis, the agent of plague, is a highly virulent pathogen in humans as well as wild and domestic felids (Rust et al. 1971; Tabor and Thomas 1986; Eidson et al. 1991; Gasper et al. 1993; Wild et al. 2006), and cases of plague transmission to humans via domestic cats and wild carnivores are numerous (Gage et al. 1994, 2000; Wong et al. 2009). Mountain lions are most likely exposed to Y. pestis when bitten by infected fleas of a rodent prey item or via consumption of infected prey (Gage et al. 1994). Interestingly, all six plague-seropositive mountain lions in this study were females. Female mountain lions in this study may have had a greater potential for exposure to plague and other diseases infecting small mammals due to their tendency to take smaller prey compared to males (Torres et al. 1996).
The low exposure rates of mountain lions to F. tularensis and Y. pestis found in this study may be related to the low sensitivity of agglutination tests using hemolyzed serum, or poor temporal and spatial overlap of mountain lion sampling with enzootic reservoirs and vectors, for example in northeastern California where plague activity is well documented (Smith et al. 2010). Considering the 40% plague seroprevalence detected in an earlier California mountain lion study (Paul-Murphy et al. 1994), and 8.8% plague seroprevalence detected in 295 mountain lions sampled in 34 California counties between 1984 and 2008 (California Department of Public Health, Vector-Borne Disease Section, unpublished data), humans are at risk for exposure to plague if they come into contact with California mountain lion carcasses (Wong et al. 2009).
Findings from this study may be used to evaluate the human risk of exposure to Y. pestis, F. tularensis, B. henselae, B. burgdorferi, and A. phagocytophilum, in rural areas of the North Coast, Western Sierra Nevada, and Central Coast, where livestock depredation by mountain lions is most frequent. The next step for advancing our understanding of the transmission dynamics and epidemiological significance of mountain lion exposures to these pathogens is to characterize the genetic diversity of infecting organisms in residents, domestic pets, and wild felids.
Footnotes
Acknowledgments
We greatly appreciate the contributions of various wildlife researchers from academic institutions, CDFG field biologists, and Wildlife Investigations Laboratory (Rancho Cordova, CA) staff members, who over the years collected the mountain lion serum used in this study. We thank Misty Cain, Sarah Krycia, and Joey Tse, who assisted in the laboratory and with data collection.
Author Disclosure Statement
No competing financial interests exist.