Leptospirosis is a reemerging, potentially fatal zoonotic disease with a worldwide distribution caused by spirochetes in the genus Leptospira.1–4 Leptospirosis is one of the most common zoonotic diseases worldwide and is estimated to infect more than a million people annually, resulting in close to 60,000 deaths each year.5,6 Although usually recognized as a problem in developing and tropical countries, leptospirosis is increasingly reported in temperate and developed countries, attributed to globalization, urbanization, climate change, and historical underreporting.3,5,7–10
Nine pathogenic species and 300 pathogenic serovars of the Leptospira genus exist, infecting a wide diversity of mammals. Leptospires live in the renal tubules of reservoir hosts and are shed in the urine; exposure to urine-contaminated fomites transmits infection most commonly via mucous membranes or through abrasions in the skin.1,9,11–13 Clinical signs of leptospirosis range from mild to fatal, with renal and hepatic failure being the most common severe manifestations and meningitis, uveitis, pulmonary disease, and myocarditis also possible.2,10,11 In livestock, leptospirosis is an important cause of abortions and economic loss.14,15
In California, where human leptospirosis has been reportable for almost a century, 24 cases of human infection were reported between 2014 and 2018.16,17 Leptospirosis is increasingly recognized as an important infectious disease of dogs, with clusters of leptospirosis recently detected in northern California.18–20 With almost 40 million residents in California,21 humans and domestic animals in many areas live in close contact with wildlife, an interface that provides opportunity for transmission of infectious diseases, including leptospirosis. A relatively large multiple-species serosurvey22 was conducted in northern California 40 years ago, showing extensive exposure in a diversity of wildlife. Most recent studies regarding Leptospira spp in California wildlife are restricted primarily to single-species serosurveys23–25 and studies26–29 focusing on clinical leptospirosis in marine mammals.
The study reported here was a cross-sectional survey to determine prevalence of and evaluate risk factors for infection by and exposure to Leptospira spp in 12 wildlife species in close proximity to humans in California. We evaluated samples for antibodies against 6 serovars of Leptospira spp with an MAT and for pathogenic Leptospira spp DNA with a real-time PCR assay targeting the LipL32 gene.30 Potential risk factors evaluated were host species, host age, host sex, and geographic region of the state. We also assessed the genetic relatedness of Leptospira spp found in California wildlife by DNA sequencing of the rrs2 gene and MLST.
Materials and Methods
Animals and sample collection
Trapping of animals and sample collection were approved by the California Department of Fish and Wildlife (scientific collecting permit No. SC-854) and the University of California-Davis Institutional Animal Care and Use Committee. Convenience samples were obtained from 46 counties in California from April 2007 to January 2017 and included any species often seen near people as follows: squirrels (Sciurus spp and Otospermophilus beecheyi), opossums (Didelphis virginiana), striped skunks (Mephitis mephitis), raccoons (Procyon lotor), and coyotes (Canis latrans). Species were also included that humans encounter as human development expands into wildlands as follows: bobcats (Lynx rufus), mountain lions (Puma concolor), gray foxes (Urocyon cinereoargenteus), and red foxes (Vulpes vulpes).
Carcasses were donated by USDA Wildlife Services and the California Department of Fish and Wildlife and necropsied for collection of urine, blood, and kidney tissue samples. Urine samples were centrifuged at 4,000 × g for 15 minutes and the pellet resuspended in 200 μL of sterile PBS solution. All samples were frozen at −20°C until further use.
Live animals were captured in humane traps. Mesocarnivores (ie, raccoons, skunks, opossums, and foxes) were captured in live trapsa baited with canned cat food opened and placed in the trap in the evening and checked in the early morning. Squirrels were baited with oats and peanut butter; traps were opened in the early morning, checked every 3 hours throughout the day, and then closed in the evening.
All animals were anesthetized prior to sample collection. Skunks were exposed to isoflurane on a cotton ball until they became uncoordinated and then given ketamine (20 mg/kg [9.1 mg/lb], IM) and midazolam (0.5 mg/kg [0.23 mg/lb], IM). Raccoons, opossums, and foxes were anesthetized with ketamine (10 mg/kg [4.5 mg/lb], IM) and midazolam (0.5 mg/kg, IM). Squirrels were anesthetized with ketamine (up to 40 mg/kg [18.2 mg/lb], SC) and xylazine (4 mg/kg [1.8 mg/lb], SC). Animals were aged (adult vs juvenile), sexed, and uniquely ear tagged prior to release. Blood was collected from either the cephalic or lateral saphenous vein into EDTA-containing blood collection tubesb and kept cool until plasma was separated by centrifugation at 800 × g for 10 minutes. Plasma was then stored at −20°C to −80°C until use. Urine was collected aseptically by cystocentesis from animals with a palpable urinary bladder and maintained cool for transfer to the University of California-Davis, where it was processed as described for carcasses and frozen at −20°C.
DNA extraction, PCR assay, and genetic analysis
Deoxyribonucleic acid was extracted from blood, urine, and kidney tissue samples with a kitc following the manufacturer's protocols incorporating negative blood controls in each extraction batch (typically 24 samples). A real-time PCR assay targeting the LipL32 gene of pathogenic Leptospira spp was performed on extracted DNA.30 Samples with a cycle threshold < 45 and a characteristic amplification plot were considered positive. One positive control (DNA extracted from cultured Leptospira interrogans serovar Pomona [serogroup Pomona]) and 3 negative controls of assay-grade water were included with each batch of reactions. Samples that were positive on a real-time PCR assay were then analyzed with a conventional PCR assay for 8 genes, including the 7 loci in Leptospira MLST scheme No. 131 and a 450-bp segment of the rrs2 gene,32 which identifies Leptospira organisms to the species level.33 Reactions were run with cited primers and conditions, and amplicons were visualized on stainedd 1% agarose gels. Bands of the expected size were excised and purified with a kite according to the manufacturer's protocol and were sequenced.f Results were examined for accuracy of base determination, and end-read errors were trimmed to yield unambiguous sequences. Sequences were compared with those in the GenBank database with a bioinformatic search tool,g and the sequences of the 7 MLST loci were compared with sequences in the Leptospira MLST database.34,h The ST was determined by means of the MLST database. A clinical sample from a northern California domestic dog (Canis lupus familiaris) with a diagnosis of leptospirosis was also included in the genetic analyses to allow for comparison with results obtained from wild carnivores.
Serologic evaluation
The MAT11 was used to evaluate samples for antibodies against 6 serovars of Leptospira spp as follows: L interrogans serovars Pomona (serogroup Pomona), Hardjo type Prajitno (serogroup Sejroe), Canicola (serogroup Canicola), Copenhageni (serogroup Icterohemorrhagiae), and Bratislava (serogroup Australis), and Leptospira kirschneri serovar Grippotyphosa (serogroup Grippotyphosa). Samples with a reciprocal titer of ≥ 100 to any serovar were considered positive for that Leptospira serovar.
Statistical analysis
Analyses were performed with open-source software,i and unless stated otherwise, values of P ≤ 0.05 were considered significant. Summary statistics and hypothesis testing were conducted for the independent variables (ie, risk factors) age class, species, sex, region, and season. Age was dichotomized as juvenile or adult. Eastern gray squirrels (Sciurus carolinensis), western gray squirrels (Sciurus griseus), and fox squirrels (Sciurus niger) were combined into the category Sciurus spp. The Fisher exact test was used to verify that no differences existed in the prevalence of Leptospira spp in these 3 squirrel species on the basis of serologic test results, PCR assay results, and combined serologic test and PCR assay results. The geographic regions were defined as follows: Bay Area (Alameda, Contra Costa, Marin, San Francisco, San Mateo, Santa Clara, and Sonoma counties), Central Coast (Monterey, Santa Cruz, and San Luis Obispo counties); Central Inland (Amador, Calaveras, El Dorado, Fresno, Inyo, Kern, Madera, Mariposa, Mono, Nevada, Placer, Sacramento, San Benito, San Joaquin, Solano, Tulare, Tuolumne, and Yolo counties), northern California (Butte, Humboldt, Lake, Lassen, Mendocino, Modoc, Napa, Plumas, Shasta, Sierra, Siskiyou, Tehama, and Yuba counties), and southern California (Ventura, Los Angeles, San Bernardino, San Diego, and Santa Barbara counties). The dry season was May through October and the wet season was November through April. The Fisher exact test was used to compare the age, sex, and seasonal distributions among species and geographic regions.
All risk factors were assessed initially for their association with the dependent variable Leptospira spp exposure (defined as positive results on serologic testing, PCR assay, or both) with univariable logistic regression. If some data for risk factors were missing for a sample, univariable models were run for those data that were available. Variables with values of P ≤ 0.2 were included in the multivariable analysis, which was run on any samples for which all risk factor data were available (ie, discarding samples from the analysis lacking data on some predictors). Two-way interactions were evaluated for significance, and confounding between variables, defined by a > 10% change in the OR, was assessed. Multicollinearity among the potential risk factors was evaluated with variance inflation factors, and overall model fit was assessed with the Hosmer-Lemeshow test.35 An optimal multivariable model was chosen to minimize the Akaike information criterion. The prevalence of each serovar was calculated as the total number of animals that had a titer ≥ 100 for that serovar per total number of animals tested.
Results
Samples were collected for Leptospira spp detection from 723 animals in 46 California counties between April 2007 and January 2017 (Table 1). Blood samples were collected from 657 of 723 (90.9%) animals, and kidney tissue samples, urine samples, or both were collected from 443 of 723 (61.3%) animals. Age and sex distributions did not vary significantly among species nor did the sex distribution vary significantly among geographic regions. Significant (P = 0.001) differences were found in age distributions among regions, with the Bay Area and southern California regions having an overabundance of juveniles (29.8% [36/121] and 27.5% [11/40], respectively), compared with the Central Coast (17.9% [10/56]), Central Inland (17.5% [50/286]), and northern California (9.4% [10/106]) regions. Seasonal distribution also differed between regions (P < 0.001), with samples collected from proportionately fewer animals from the Central Inland region during the wet season (47.6% [136/286]) than in other regions. The majority of samples from the Bay Area (57.0% [85/149]), Central Coast (69.5% [41/59]), northern California (67.9% [89/131]), and southern California (58.6% [17/29]) were collected during the wet season. Samples were collected from squirrels (Sciurus spp and O beecheyi) and gray foxes more often during the dry season (73.8% [79/107]), whereas samples were collected from other species more often during the wet season. Samples were collected most commonly from squirrels in the Central Inland region (87.9% [94/107]) and from red foxes in the Central Coast region (85.7% [6/7]); other host species were less geographically limited in origin. Regional and seasonal differences among species were significant (P < 0.001) for each.
Number (%) and distribution of wildlife species that had samples collected for Leptospira spp detection in California between 2007 and 2017.
| Variables | Coyotes (Canis latrans) n = 21 | Opossums (Didelphis virginiana) n = 33 | Bobcats (Lynx rufus) n = 39 | Striped skunks (Mephitis mephitis) n = 222 | Raccoons (Procyon lotor) n = 148 | Mountain lions (Puma concolor) n = 136 | Squirrels (Sciurus spp) n = 73 | California ground squirrel (Otospermophilus beecheyi) n = 34 | Gray foxes (Urocyon cinereoargenteus) n = 10 | Red foxes (Vulpes vulpes) n = 7 |
|---|---|---|---|---|---|---|---|---|---|---|
| Females* | 6 (30.0) | 12 (37.5) | 14 (37.8) | 85 (42.1) | 69 (47.3) | NA | 35 (53.8) | NA | 4 (40.0) | 3 (42.9) |
| Age class | ||||||||||
| Juvenile | 0 (0.0) | 4 (12.1) | 6 (15.4) | 42 (18.9) | 24 (16.2) | 24 (17.6) | 7 (9.6) | 9 (26.5) | 0 (0.0) | 1 (14.3) |
| Adult | 20 (95.2) | 28 (84.8) | 28 (71.8) | 157 (70.7) | 83 (56.1) | 98 (72.1) | 55 (75.3) | 24 (70.l) | 3 (30.0) | 6 (85.7) |
| Unknown | 1 (4.8) | 1 (3.0) | 5 (12.8) | 23 (10.4) | 41 (27.7) | 14 (10.3) | 11 (15.1) | l (2.9) | 7 (70.0) | 0 (0.0) |
| Season† | ||||||||||
| Dry | 6 (28.6) | 15 (45.5) | 6 (15.4) | 75 (33.8) | 50 (33.8) | 50 (36.8) | 60 (82.2) | 19 (55.9) | 8 (80.0) | 2 (28.6) |
| Wet | 15 (71.4) | 18 (54.5) | 3 (7.7) | 136 (61.3) | 97 (65.5) | 64 (47.1) | 13 (17.8) | 15 (44.l) | 2 (20.0) | 5 (71.4) |
| Unknown | 0 (0.0) | 0 (0.0) | 30 (76.9) | 11 (5.0) | 1 (0.7) | 22 (16.2) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| Region† | ||||||||||
| Bay Area | 0 (0.0) | 7 (21.2) | 3 (7.7) | 63 (28.4) | 74 (50.0) | 5 (3.7) | 1 (1.4) | 0 (0.0) | l (10.0) | 1 (14.3) |
| Central Coast | 7 (33.3) | 3 (9.1) | 2 (5.1) | 15 (6.8) | 9 (6.1) | 19 (14.0) | 0 (0.0) | 0 (0.0) | l (10.0) | 6 (85.7) |
| Central Inland | 14 (66.7) | 14 (42.4) | 10 (25.6) | 65 (29.3) | 58 (39.2) | 39 (28.7) | 61 (83.6) | 33 (97.l) | l (10.0) | 0 (0.0) |
| Northern | 0 (0.0) | 9 (27.3) | 1 (2.6) | 68 (30.6) | 7 (4.7) | 37 (27.2) | 7 (9.6) | l (2.9) | 7 (70.0) | 0 (0.0) |
| California | ||||||||||
| Southern | 0 (0.0) | 0 (0.0) | 17 (43.6) | 8 (3.6) | 0 (0.0) | 16 (11.8) | 4 (5.5) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| California | ||||||||||
| Unknown | 0 (0.0) | 0 (0.0) | 6 (15.4) | 3 (1.4) | 0 (0.0) | 20 (14.7) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
Data on sex were missing for 40 individuals.
Significant (P < 0.05) differences were found in the seasonal and regional distributions of species.
NA = Not applicable.
Overall, 39.1% (257/657) of animals tested were seropositive for Leptospira spp, and 23.0% (102/443) had positive PCR assay results for Leptospira spp DNA (Figure 1; Table 2). Evidence of Leptospira spp infection or exposure was detected in all geographic regions of California and in all species. Seroprevalence for Leptospira spp was lowest in southern California (21.6% [8/37]) and highest in the Central Coast (48.2% [27/56]) region, although regional differences were not significant (P = 0.08). Prevalence of positive PCR assay results for Leptospira spp DNA was highest in northern California (28.6% [22/77]) and lowest in southern California (7.5% [3/40]); these regional differences also were not significant (P = 0.08). The prevalence of animals that tested positive for Leptospira infection or exposure on the basis of PCR assay results, serologic test results, or both was highest in the Central Coast (45.2% [28/62]) and Central Inland (44.1% [130/295]) regions. Northern California and the Bay Area also had considerable prevalences (40.9% [56/137] and 39.4% [61/155], respectively) of Leptospira spp infection or exposure, whereas the prevalence was lowest in southern California (20.0% [9/45]). In univariable analysis, all regions were associated with increased exposure to Leptospira spp, compared with southern California (Table 3).
Map of regions in California from which animals were tested by serologic and PCR assay methods for Leptospira spp. Numbers given in each region are the overall prevalence (%) of combined positive test results followed by 95% CI values.
Citation: Journal of the American Veterinary Medical Association 257, 8; 10.2460/javma.257.8.840
Numbers and percentage (95% CI) of positive serologic test, real-time PCR assay, and overall combined results for Leptospira spp infection or exposure in species of peri-urban wildlife in California between 2007 and 2017.
| Serologic test | PCR assay of kidney or urine samples | Serologic test, PCR assay, or both | ||||
|---|---|---|---|---|---|---|
| Wildlife | No. positive/No. tested | Percentage (95% CI) | No. positive/No. tested | Percentage (95% CI) | No. positive/No. tested | Percentage (95% CI) |
| Coyotes | 6/20 | 30.0 (14.5-51.9) | 2/2 | 100.0 (34.2-100.0) | 8/21 | 38.1 (20.8-59.1) |
| Opossums | 2/32 | 6.3 (1.7-20.1) | 1/6 | 16.7 (3.0-56.4) | 3/33 | 9.1 (3.1-23.6) |
| Bobcats | 10/31 | 32.3 (18.6-49.9) | 3/34 | 8.8 (3.0-23.0) | 11/39 | 28.2 (16.5-43.8) |
| Striped skunks | 78/206 | 37.9 (31.5-44.7) | 40/141 | 28.4 (21.6-36.3) | 92/222 | 41.4 (35.2-48.0) |
| Raccoons | 52/119 | 43.7 (35.1-52.7) | 23/87 | 26.4 (18.3-36.6) | 67/148 | 45.3 (37.5-53.3) |
| Mountain lions | 63/127 | 49.6 (41.1-58.2) | 28/119 | 23.5 (16.8-31.9) | 63/136 | 46.3 (38.2-54.7) |
| Eastern gray squirrels | 9/20 | 45.0 (25.8-65.8) | 0/11 | 0.0 (0.0-25.9) | 9/21 | 42.9 (24.5-63.5) |
| Western gray squirrels | 9/16 | 56.3 (33.2-76.9) | 0/4 | 0.0 (0.0-49.0) | 9/16 | 56.3 (33.2-76.9) |
| Fox squirrels | 15/36 | 41.7 (27.1-57.8) | 4/31 | 12.9 (5.1-28.9) | 18/36 | 50.0 (34.5-65.5) |
| California ground squirrels | 8/34 | 23.5 (12.4-40.0) | 0/4 | 0.0 (0.0-49.0) | 8/34 | 23.5 (12.4-40.0) |
| Gray foxes | 2/9 | 22.2 (6.3-54.7) | 1/4 | 25.0 (4.6-69.9) | 2/10 | 20.0 (5.7-51.0) |
| Red foxes | 3/7 | 42.9 (15.8-75.0) | NA | NA | 3/7 | 42.9 (15.8-75.0) |
| Overall | 257/657 | 39.1 (35.5-42.9) | 102/443 | 23.0 (19.3-27.2) | 293/723 | 40.5 (37.0-44.1) |
See Table 1 for key and for genus and species names of wildlife species.
Results of univariable logistic regression analyses of potential risk factors for Leptospira spp infection or exposure in California peri-urban wildlife that had samples collected for Leptospira spp detection between 2007 and 2017.
| Variables | No. of animals | Coefficient | OR | P value |
|---|---|---|---|---|
| Host species | ||||
| Coyotes | 21 | 1.817 | 6.15 | 0.016 |
| Opossums | 33 | Referent | NA | NA |
| Bobcats | 39 | l.368 | 3.93 | 0.051 |
| Striped skunks | 222 | l.957 | 7.08 | 0.002 |
| Raccoons | 148 | 2.113 | 8.27 | < 0.001 |
| Mountain lions | 136 | 2.155 | 8.63 | 0.001 |
| Squirrels (Sciurus spp) | 73 | 2.275 | 9.73 | 0.001 |
| California ground squirrels | 34 | 1.124 | 3.08 | 0.123 |
| Gray foxes | 10 | 0.916 | 2.50 | 0.358 |
| Red foxes | 7 | 2.015 | 7.50 | 0.039 |
| Host age | ||||
| Juvenile | 117 | Referent | NA | NA |
| Adult | 502 | l.432 | 4.19 | < 0.001 |
| Host sex | ||||
| Female | 287 | Referent | NA | NA |
| Male | 396 | 0.291 | l.34 | 0.066 |
| Region | ||||
| Bay Area | 155 | 0.954 | 2.60 | 0.019 |
| Central Coast | 62 | 1.192 | 3.29 | 0.008 |
| Central Inland | 295 | 1.148 | 3.15 | 0.003 |
| Northern California | 137 | 1.017 | 2.70 | 0.013 |
| Southern California | 45 | Referent | NA | NA |
| Season | ||||
| Dry season (May through October) | 291 | Referent | NA | NA |
| Wet season (November through April) | 368 | 0.167 | 1.18 | 0.297 |
Animals were considered to be positive for Leptospira spp exposure in the regression analyses if they had positive results for either real-time PCR assay, serologic testing, or both.
See Tables l and 2 for remainder of key.
Among species evaluated, seroprevalence varied significantly (P < 0.001) from a low value in opossums (6.3% [2/32]) to a high value in western gray squirrels (56.3% [9/16]). Leptospira spp DNA was not found in eastern or western gray squirrels nor in California ground squirrels (O beechyi), but otherwise was detected in all species evaluated by PCR assay, with the highest prevalence of positive PCR assay results for Leptospira spp in coyotes (100% [2/2]) followed by striped skunks (28.4% [40/141]), raccoons (26.4% [23/87]), gray foxes (25.0% [1/4]), and mountain lions (23.5% [28/119]). Differences in prevalence of positive PCR assay results for Leptospira spp DNA among species were significant (P = 0.001). Exposure overall was most common in western gray squirrels (56.3% [9/16]) and least common in opossums (9.1% [3/33]). Univariable logistic regression with opossums as the referent indicated elevated exposure rates in coyotes, striped skunks, raccoons, mountain lions, Sciurus spp, and red foxes (Table 3).
On univariable analysis, adult animals had significantly (P < 0.001) greater odds than juvenile animals to have had Leptospira spp exposure (OR, 4.2; 95% CI, 2.6 to 7.1). Host sex (P = 0.07) and season (P = 0.30) were not significantly associated with exposure.
Geographic region, host species, host age class, and host sex were included in the multivariable model. Because of missing data for ≥ 1 potential risk factor, 119 animals were excluded, leaving data from 604 animals included in the multivariable analysis. The model demonstrated good overall fit (Hosmer-Lemeshow test, P = 0.75). All risk factors were significant (Table 4). After adjusting for age, sex, and host species, animals from southern California continued to have significantly lower odds than those from other regions of California to show evidence of Leptospira spp infection or exposure. Coyotes, bobcats, striped skunks, raccoons, mountain lions, and tree squirrels had significantly greater odds than opossums to be positive for Leptospira spp exposure in the multivariable model. Compared with juveniles, adult animals had almost 5 times the odds (OR, 4.9; 95% CI, 2.93 to 8.59) of Leptospira spp exposure, and males (compared with females) had 1.5 times the odds (OR, 1.5; 95% CI, 1.04 to 2.12) of exposure to Leptospira spp after adjusting for other risk factors.
Results of multivariable logistic regression analysis of potential risk factors for Leptospira spp infection or exposure in California peri-urban wildlife that had samples collected for Leptospira spp detection between 2007 and 2017.
| Variables | No. of Animals | Coefficient | OR | P value |
|---|---|---|---|---|
| Host species | ||||
| Coyotes | 20 | 1.602 | 4.96 | 0.038 |
| Opossums | 32 | Referent | NA | NA |
| Bobcats | 29 | 2.325 | 10.23 | 0.003 |
| Striped skunks | 197 | 2.191 | 8.95 | < 0.001 |
| Raccoons | 107 | 2.442 | 11.49 | < 0.001 |
| Mountain lions | 116 | 2.487 | 12.03 | < 0.001 |
| Squirrels (Sciurus spp) | 60 | 2.585 | 13.26 | < 0.001 |
| California ground squirrels | 33 | 1.127 | 3.09 | 0.138 |
| Gray foxes | 3 | 1.339 | 3.81 | 0.332 |
| Red foxes | 7 | 1.870 | 6.49 | 0.067 |
| Host age | ||||
| Juvenile | 116 | Referent | NA | NA |
| Adult | 488 | 1.592 | 4.91 | < 0.001 |
| Host sex | ||||
| Female | 251 | Referent | NA | NA |
| Male | 353 | 0.393 | 1.48 | 0.032 |
| Region | ||||
| Bay Area | 119 | 1.320 | 3.74 | 0.009 |
| Central Coast | 56 | 1.584 | 4.88 | 0.003 |
| Central Inland | 283 | 1.371 | 3.94 | 0.003 |
| Northern California | 106 | 1.162 | 3.20 | 0.017 |
| Southern California | 40 | Referent | NA | NA |
Animals were considered to be positive for Leptospira spp exposure in the regression analyses if they had positive results for either real-time PCR assay, serologic testing, or both.
See Tables 1 and 2 for remainder of key.
Antibodies against L interrogans serovar Pomona (serogroup Pomona) were most commonly detected overall, with 77.8% (200/257) of samples that were serologically positive having a reciprocal titer ≥ 100 to that serovar (Table 5). Because animals often had titers ≥ 100 for multiple serovars, the sum of all positive samples for all serovars exceeded the total number of animals tested. Serovar Pomona antibodies predominated in raccoons, striped skunks, coyotes, red foxes, bobcats, and mountain lions. The second most commonly detected serovar was L interrogans serovar Copenhageni (serogroup Icterohemorrhagiae), with a reciprocal titer of ≥ 100 found in 47.5% (122/257) of seropositive samples; this serovar predominated in gray foxes, opossums, and all squirrels. The serovar L interrogans Bratislava (serogroup Australis) was found in 22.3% (55/247) of seropositive samples primarily in mountain lions, bobcats, and striped skunks. Less common serovars were L interrogans Hardjo type Prajitno (serogroup Sejroe) in raccoons, mountain lions, and striped skunks; L interrogans serovar Canicola (serogroup Canicola) in raccoons, striped skunks, and mountain lions; and L kirschneri serovar Grippotyphosa (serogroup Grippotyphosa) in coyotes, raccoons, striped skunks, and mountain lions. The GenBank accession numbers for DNA sequences were as follows: MN477295-MN477307, MN485806-MN485870, and MN491809-MN491846.
Percentage (95% CI) of Leptospira spp-seropositive California peri-urban wildlife on the basis of MAT results against 6 serovars between 2007 and 2017.
| Wildlife | No. of animals | Pomona | Hardjo | Grippotyphosa | Canicola | Copenhageni | Bratislava | All serovars |
|---|---|---|---|---|---|---|---|---|
| Coyotes | 20 | 20.0 (8.1-41.6) | 0.0 (0-16.1) | 5.0 (0.9-23.6) | 0.0 (0-16.1) | 30.0 (14.5-51.9) | 5.0 (0.9-23.6) | 30.0 (14.5-51.9) |
| Opossums | 32 | 3.1 (0.6-15.7) | 0.0 (0-10.7) | 0.0 (0-10.7) | 0.0 (0-10.7) | 6.3 (1.7-20.1) | 3.1 (0.6-15.7) | 6.3 (1.7-20.1) |
| Bobcats | 31 | 29.0 (16.1-46.6) | 0.0 (0-11.0) | 0.0 (0-11.0) | 0.0 (0-11.0) | 19.4 (9.2-36.3) | 6.5 (1.8-20.7) | 32.3 (18.6-49.9) |
| Striped skunks | 206 | 36.4 (30.1-43.2) | 1.5 (0.5-4.2) | 1.0 (0.3-3.5) | 1.9 (0.8-4.9) | 7.3 (4.5-11.7) | 4.9 (2.7-8.7) | 37.9 (31.5-44.7) |
| Raccoons | 119 | 37.8 (29.6-46.8) | 6.7 (3.4-2.7) | 1.7 (0.5-5.9) | 6.7 (3.4-12.7) | 19.3 (13.2-27.3) | 17.6 (11.8-25.5) | 43.7 (35.1-52.7) |
| Mountain lions | 127 | 44.9 (36.5-53.6) | 2.4 (0.8-6.7) | 0.8 (0.1-4.3) | 0.8 (0.1-4.3) | 25.2 (18.5-33.4) | 12.6 (7.9-19.5) | 49.6 (41.1-58.2) |
| Eastern gray squirrels | 20 | 0.0 (0-16.1) | 0.0 (0-16.1) | 0.0 (0-16.1) | 0.0 (0-16.1) | 40.0 (21.9-61.3) | 5.0 (0.9-23.6) | 45.0 (25.8-65.8) |
| Western gray squirrels | 16 | 6.3 (1.1-28.3) | 0.0 (0-19.4) | 0.0 (0-19.4) | 0.0 (0-19.4) | 56.3 (33.2-76.9) | 0.0 (0-19.4) | 56.3 (33.2-76.9) |
| Fox squirrels | 36 | 13.9 (6.1-28.7) | 0.0 (0-9.6) | 0.0 (0-9.6) | 0.0 (0-9.6) | 30.1 (18.0-46.9) | 2.8 (0.5-14.2) | 41.7 (27.1-57.8) |
| California ground squirrels | 34 | 0.0 (0-10.1) | 0.0 (0-10.2) | 0.0 (0-10.2) | 0.0 (0-10.2) | 20.6 (10.3-36.8) | 5.9 (1.6-19.1) | 23.5 (12.4-40.0) |
| Gray foxes | 9 | 0.0 (0-29.9) | 0.0 (0-29.9) | 0.0 (0-29.9) | 0.0 (0-29.9) | 22.2 (6.3-54.7) | 0.0 (0-29.9) | 22.2 (6.3-54.7) |
| Red foxes | 7 | 42.9 (15.8-75.0) | 0.0 (0-35.4) | 0.0 (0-35.4) | 0.0 (0-35.4) | 14.3 (2.6-51.3) | 0.0 (0-35.4) | 42.9 (15.8-75.0) |
Bratislava = Leptospira interrogans serovar Bratislava (serogroup Australis). Canicola = Leptospira interrogans serovar Canicola (serogroup Canicola). Copenhageni = Leptospira interrogans serovar Copenhageni (serogroup Icterohemorrhagiae). Grippotyphosa = Leptospira kirschneri serovar Grippotyphosa (serogroup Grippotyphosa). Hardjo = Leptospira interrogans serovar Hardjo type Prajitno (serogroup Sejroe). Pomona = Leptospira interrogans serovar Pomona (serogroup Pomona).
See Table 1 for genus and species names of wildlife species.
For the 102 samples that were positive by real-time PCR assay, the rrs2 gene was successfully amplified and sequenced from 7 raccoons, 11 striped skunks, 1 bobcat, and 4 mountain lions. All 7 loci from MLST scheme No. 1 were successfully amplified in 5 raccoons, 6 striped skunks, and 2 mountain lions. The rrs2 gene and the pfkB gene (1/7 MLST loci) were successfully amplified and sequenced from the domestic dog clinical case sample. There was 100% sequence homology among all samples for each of the genes evaluated. The rrs2 sequence shared 100% sequence identity to L interrogans. The MLST was ST 140, which corresponded to 18 isolates in the Leptospira MLST database. Of these 18 isolates, all were L interrogans, and of the 7 isolates for which the serovar or serogroup was reported, 5 were identified as L interrogans serovar Pomona, 1 as L interrogans serovar Guaratuba (serogroup Pyrogenes), and 1 as L interrogans serogroup Grippotyphosa. The GenBank accession numbers for DNA sequences are MN477295-MN477307, MN485806-MN485870, and MN491809-MN491846.
Discussion
Results of the present study indicated that pathogenic Leptospira spp are common and widespread throughout peri-urban wildlife species in California. We found evidence of infection in all regions of the state and in all host species evaluated, with prevalence exceeding 40% in raccoons, striped skunks, mountain lions, bobcats, tree squirrels, California ground squirrels, and red foxes. Unlike many previous studies in North American wildlife that used only serologic methods to investigate the epidemiology of Leptospira spp,36–44 our study also used PCR assay to determine whether a host was actively infected. Nearly a quarter of animals were PCR assay-positive for Leptospira spp DNA, which could even be an underestimate given limits of sensitivity of the PCR assay. These data provide crucial information regarding the risk of environmental contamination and thus risk to other animals, including humans and domestic animals.
Forty years ago, an antibody-based survey of wildlife in northern California found widespread exposure to Leptospira spp in a diversity of wildlife, although differences in methods make direct comparisons difficult.22 In that study, a cutoff value was used that considered animals seropositive at much lower titers than in the present study, which would have overestimated the prevalence considerably. Authors also used a different panel of serovars, some of which have now been elevated to new species taxonomically, and it is unclear which reference strains were used in their MAT. Nevertheless, overall their results agree with ours, including that there are multiple circulating Leptospira spp cycles in California among wildlife, that infection is common, and that carnivores appear generally more commonly infected than rodents. Most other studies of peri-urban wildlife in North America have been limited to a more restricted set of host species, but had results similar to ours for particular species. A large serosurvey from 2011 to 2017 found seroprevalences of 60% in striped skunks, 41% in raccoons, 29% in coyotes, and 34% to 35% in foxes for Leptospira spp serovar in the Midwest and eastern United States.42 Seroprevalence was 57% in California ground squirrels in Oregon43; 33% in raccoons in Ontario, Canada44; and 45% in fox squirrels in Colorado.45 In Connecticut, 36% of raccoons, 13% of skunks, and 5% of eastern gray squirrels were seropositive.39 In California, Leptospira spp exposure has been reported in those species we tested here as well as wild boars (Sus scrofa), black bears (Ursus americanus), mule deer (Odocoileus hemionus), California sea lions (Zalophus californianus), Pacific harbor seals (Phoca vitulina richardsii), and northern elephant seals (Mirounga angustirostris).23,25–28,46
Opossums, which are common peri-urban wildlife that frequently enter yards and even homes, had the least evidence of Leptospira spp infection of any animal we tested. Similarly, of 28 opossums tested in Connecticut, none was positive on MAT.39 Low prevalence could occur if opossums are truly less susceptible to infection or less likely to be exposed, or if some sample bias in our study underestimated their true exposure rate. However, during experimental infection, opossums failed to mount strong antibody responses.47 In our study, the single opossum with positive PCR assay results for Leptospira spp was serologically negative, which supports the possibility that this wildlife species remains a source of environmental contamination with Leptospira spp despite its own low seroprevalence.
In our study, southern California appeared to have the lowest rates of Leptospira spp as has been reported for California mule deer and sea lions.23,26 Southern California has a drier climate than other regions we assessed, and leptospires require moisture to live for any duration of time outside of the host.2 Leptospires can survive for months in fresh water or wet soil,48,49 which accounts for the higher overall (on the basis of serologic testing and PCR assay) prevalence found in the wetter northern areas of California. The fact that there was no difference across wet and dry seasons is likely because animals remain chronically infected or at least persistently seropositive.
Interestingly, however, leptospirosis is well documented in marine mammals along the California coast including southern California where California sea lions experience repeated epidemics attributable to L interrogans serovar Pomona.24,26,29 This is the same serovar that was most commonly detected in mule deer in a state-wide survey,23 and in our study, L interrogans serovar Pomona was detected in many host species in all regions of California. We also found serum antibodies against L interrogans serovar Copenhageni in gray foxes, opossums, and all 4 species of squirrels. Although the serovar with the highest titer may not always be the infecting serovar, because of the paradoxical reactions that can occur with MAT,15 the predominance of these 2 serovars, each in a different set of host species, suggests the presence of at least 2 major Leptospira spp serovars in California peri-urban wildlife.
In addition to serologic classification methods, Leptospira spp strains can also be classified to ST with the use of MLST.50 Use of the 7 housekeeping genes in MLST allows for a finer-scale genetic classification, compared with species determination by means of a single gene.51,52 Unfortunately, we were able to amplify and type strains only from raccoons, striped skunks, mountain lions, and bobcats (likely because amplicons from the other species were too weak; the Taqman screening PCR assay is more sensitive than the PCR assay associated with the MLST genes), all of which had serologic evidence of L interrogans serovar Pomona. However, our finding of 100% sequence identity in all samples and for all genes analyzed strongly supports the hypothesis that Leptospira spp circulating in California wildlife are shared among multiple host species and likely spill over into domestic animals. The ST identified in our study was ST 140, which was identified in the Leptospira MLST database as L interrogans serovar Pomona from a human in Australia and 4 samples without geographic location provided. This ST has also been reported from a human in Sri Lanka identified as L interrogans serovar Grippotyphosa and from an opossum (scientific name not provided) in Brazil identified as L interrogans serovar Guaratuba. In a study51 in Tahiti, ST 140 was also 1 of 3 circulating STs; in that study, ST 140 was found only in domestic pigs whereas 2 other STs were found in rats (Rattus spp) and humans in locations where all 3 host species were present, providing evidence that sequence typing is a useful tool in epidemiological studies of leptospirosis.
Although MLST provides finer-scale genetic discrimination than the use of a single gene, it may, in some cases, be less discriminatory than serologic classification schemes of Leptospira spp53 as occurred in our study where strains from some seropositive host species could not be typed. Concurrent use of genetic and serologic classification schemes is common but can be difficult to resolve. Within a single Leptospira spp, multiple serogroups (and serovars) can be found, and conversely, a single serogroup may include isolates from different Leptospira spp.2 Sequence typing with MLST does not always correspond to serologic classifications; for example, at the time the MLST scheme used in our study was developed, of the 190 STs identified, 18 (including ST 140, identified in our samples) included isolates from > 1 serovar.31 Another limitation of MLST is that the available database is limited and would benefit from additional strains and more detailed metadata. With its increasing availability and decreased costs, whole genome sequencing will increase the ability to discriminate between circulating Leptospira spp strains in the future and shed more light on the epidemiological and ecologic nature of this pathogen.52
In many parts of California, humans and domestic animals are in daily direct or indirect contact with peri-urban wildlife. Our finding of widespread common Leptospira spp in numerous species of wildlife highlights the potential for exposure to infectious disease at the urban-wildland interface. Each geographic region will be distinct in its representation of host species and Leptospira strains, but our data indicate that multiple-host studies with the use of both serologic testing and PCR assay can yield valuable insights. In California, the multiple Leptospira spp strains detected by serologic testing, combined with apparent host species partitioning of different serogroups, may indicate that more than 1 epidemiological cycle of Leptospira spp is present in California wildlife.
Acknowledgments
This study was partially funded by Zoetis.
The authors declare that there were no conflicts of interest
The auhors thank Deana Clifford, Jaime Rudd, Shannon Chandler, Rebecca Mihalco, Jane Riner, Molly Church, Elle Glueckert, Ashley Hill, and Jane Sykes for their invaluable assistance.
ABBREVIATIONS
| MAT | Microscopic agglutination test |
| MLST | Multiple locus sequence type |
| ST | Sequence type |
Footnotes
Tomahawk Live Trap, Hazlehurst, Wis.
Becton, Dickinson and Company, Franklin Lakes, NJ.
DNAeasy Blood and Tissue Kit, Qiagen, Valencia, Calif.
GelStar, Lonza, Rockland, Me.
QIAquick Gel Extraction Kit, Qiagen, Valencia, Calif.
ABI Prism 3730 DNA sequencer, University of California, Davis Sequencing Facility, Davis, Calif.
BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed Apr 20, 2020.
PubMLST. Leptospira spp.pubmlst.org/leptospira. Accessed Apr 20, 2020.
R, version 3.3.2, R Foundation for Statistical Computing, Vienna, Austria. Available at: www.R-project.org. Accessed Apr 20, 2020.
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