Spatial analysis of Yersinia pestis and Bartonella vinsonii subsp. berkhoffii seroprevalence in California coyotes (Canis latrans)

Preventive Veterinary Medicine 56 (2003) 299±311

Spatial analysis of Yersinia pestis and Bartonella vinsonii subsp. berkhof®i seroprevalence in California coyotes (Canis latrans) B.R. Hoara,*, B.B. Chomelb, D.L. Rolfec, C.C. Changb,d, C.L. Fritze, B.N. Sacksf, T.E. Carpentera a

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, One Shields Avenue, Davis, CA 95616, USA b Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA c Animal Health Branch, California Department of Food and Agriculture, 3800 Cornucopia Way, Modesto, CA 95358, USA d Department of Public Health, Graduate Institute of Environmental Health, China Medical College, Taichung, Taiwan e Division of Communicable Disease Control, California Department of Health Services, Sacramento, CA, USA f Wildlife Genetics Section, Veterinary Genetics Laboratory, University of California, Davis, CA 95616, USA Received 26 February 2002; accepted 7 October 2002

Abstract Zoonotic transmission of sylvatic plague caused by Yersinia pestis occurs in California, USA. Human infections with various Bartonella species have been reported recently. Coyotes (Canis latrans) are ubiquitous throughout California and can become infected with both bacterial agents, making the species useful for surveillance purposes. This study examined the geographic distribution of 863 coyotes tested for Y. pestis and Bartonella vinsonii subsp. berkhof®i serologic status to gain insight into the natural history of B. vinsonii subsp. berkhof®i and to characterize the spatial distribution of the two agents. We found 11.7% of specimens positive to Y. pestis and 35.5% positive to B. vinsonii subsp. berkhof®i. The two pathogens had distinct spatial clusters: Y. pestis was more prevalent in eastern portions of the state and B. vinsonii subsp. berkhof®i in coastal regions. Prevalence of Y. pestis increased with increasing elevation, whereas prevalence of B. vinsonii subsp. berkhof®i decreased with increasing elevation. There were differences in the proportions of positive animals on a yearly basis to both pathogens. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Spatial analysis; Yersinia pestis; Bartonella vinsonii subsp. berkhof®i; Coyote * Corresponding author. Tel.: ‡1-530-752-0351; fax: ‡1-530-752-0414. E-mail address: [email protected] (B.R. Hoar).

0167-5877/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 5 8 7 7 ( 0 2 ) 0 0 1 9 4 - 0

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1. Introduction Plague is a ¯ea-borne disease primarily affecting rodents caused by Yersinia pestis (Perry and Fetherston, 1997). Humans are infected by encountering the complex natural plague cycle involving the bacterial agent, an arthropod vector, and a vertebrate host (Poland et al., 1994). Carnivores are involved in the plague cycle as potential carriers of infective ¯eas to other rodent populations (Perry and Fetherston, 1997). Canids appear to be highly resistant to infection with Y. pestis (Perry and Fetherston, 1997; Barnes, 1982), with predation on plague-infected rodents usually causing inapparent-to-mild disease and seroconversion (Smith et al., 1984). This makes canids excellent sentinels of regional plague activity (Willeberg et al., 1979; Thomas and Hughes, 1992). Bartonella is a genus of small Gram-negative bacteria that has been identi®ed in many animal species, including humans (Breitschwerdt and Kordick, 2000). Bartonella vinsonii subsp. berkhof®i (hereinafter, B. berkhof®i) causes endocarditis, myocarditis, and arrhythmia in dogs (Breitschwerdt et al., 1995, 1999) and a case of endocarditis in humans (Roux et al., 2000). A case of a child who developed clinical signs compatible with Bartonella infection following a coyote (Canis latrans) bite in California (Chang et al., 1999) led to a study which found B. berkhof®i in blood of 28% of 109 coyotes sampled from central coastal California (Chang et al., 2000). Because the PCR/RFLP pro®les of B. berkhof®i isolated from several coyotes in Santa Clara County and a domestic-dog isolate were identical (Chang et al., 1999), coyotes might be an important reservoir of B. berkhof®i. Coyotes are an important non-migratory (Shivik, 1995) wildlife species living in close proximity to human populations in many areas of North America (Thomas and Hughes, 1992). In California, coyotes are ubiquitous, occurring in Central Valley grasslands and deserts (Cypher et al., 2000), mountain woodlands and forests of the Coast Ranges (Sacks et al., 1999a,b) and Sierra Nevada (Shivik, 1995), and large urban centers such as Los Angeles (Riley et al., in press) and the San Francisco Bay area (Chang et al., 1999, 2000). Mating occurs in January and February and pups are born in March and April (Sacks and Neale, 2001). Coyotes are mostly full grown by 6 months of age (Sacks and Blejwas, 2000), and may remain in the natal territory as pack associates or leave the natal territory to become transient or to establish a pair-bond and breeding territory of their own (Sacks et al., 1999a). In California, territories of packs or breeding pairs are approximately 5 km2, but transients use much larger areas (Sacks et al., 1999a; Riley et al., in press). Dispersing coyotes in the region have been observed to travel up to 147 km (Hawthorne, 1971). In some exploited populations, coyotes <1 year old compose approximately half of the population (Sacks et al., 1999b). The potential for zoonotic transmission of human plague and bartonellosis is likely to increase as residential development continues and as interest in pursuing outdoor activities increases. Although the ecology of plague and the role played by wild canids in California is well established (Willeberg et al., 1979; Smith et al., 1984; Thomas and Hughes, 1992), that for B. berkhof®i is not. Previous plague surveillance in California focused primarily on county-level antibody prevalence in coyotes. A 9-year study found an overall seroprevalence of 11.6% in sampled carnivores (Smith et al., 1984). Similarly, a previous study of B. berkhof®i antibody prevalence in coyotes was performed at the county level (Chang et al., 1999). We undertook

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this study to determine whether available surveillance data could provide indirect evidence for similar ecologies, based on ®ner scales of spatial mapping and temporal trends, of Y. pestis and B. berkhof®i. Spatial statistics might provide insight into the transmission dynamics of the two organisms. 2. Materials and methods Blood specimens (serum or Nobuto ®lter strips) were collected by convenience sampling of coyotes from 34 of the 58 counties in California from 1994 to 1998. Samples were obtained by United States Department of Agriculture/Wildlife Services specialists and other contractors as part of livestock-depredation control and the statewide plaguesurveillance program overseen by the California Department of Health Services/Vectorborne Disease Section. No coyotes were killed expressly for this study. Antibodies to Y. pestis were tested using a passive hemagglutination (PHA) paper-strip blood-sampling technique (Nobuto) with hemagglutination inhibition (HI) controls (World Health Organization, 1970; Wolff and Hudson, 1974). A serum antibody titer of 1:32 was considered positive. Serum determined to be positive by the PHA test was tested with the HI test. If the HI titer were below the PHA titer by less than twofold, the positive PHA test was considered non-speci®c and the sample called negative. Samples were tested for antibodies to B. berkhof®i using an enzyme-linked immunosorbent assay (ELISA) (Chang et al., 1999). The cut-off value (optical density (OD) >0.2) for seropositivity was determined by the average OD plus three standard deviations (S.D.) of 76 Nobuto strips from counties where all OD values were <0.190. Statistically, animals with OD values >0.2 can be considered to be seropositive with 99% con®dence (Chang et al., 1999). Location, elevation, and date of sample collection were recorded. The percent of coyotes testing positive to each pathogen was calculated by year and elevation of collection. Chisquare tests of homogeneity (Sokal and Rohlf, 1995) were performed to determine whether signi®cant differences occurred within each variable. A randomness-of-runs test (Sokal and Rohlf, 1995) was performed for each pathogen to determine if there was clustering along the elevation of collection. In this test, a run is a sequence of like events (e.g., a number of consecutive negative test results) and if there are fewer than expected runs, a grouping of like events (clustering) due to lack of independence could be occurring. The expected number of runs given a random distribution was compared to the observed number of runs. Results with P < 0:05 were considered signi®cant. The expected number of coyote specimens testing positive to both agents if serologic status were independent was calculated and compared to the observed number testing positive to both agents. A McNemar's paired-sample test (Sokal and Rohlf, 1995) was performed to determine whether these two values were signi®cantly different. Each sample was assigned a locational longitude and latitude, based on descriptions of distances and directions from landmarks where the animal was sampled. These locations were plotted using ArcView GIS 3.2a (Environmental Systems Research Institute, Inc., Redlands, CA). Maps were prepared showing the distribution of all animals sampled and of animals seropositive to Y. pestis and B. berkhof®i within California geographic subdivisions (``Jepson regions''), which incorporate natural landscape features including

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vegetative, geologic, topographic, and climatic variation (Hickman, 1993). The median value of longitude was used to classify samples as relatively east or west. Similarly, the median value of latitude was used to classify samples as relatively north or south. Chisquare tests of homogeneity (Sokal and Rohlf, 1995) were performed to determine if the proportion of samples positive and negative to each pathogen was signi®cantly different by either longitude or latitude. Clustering of seropositive animals was assessed by two methods. First, a global test for clustering, the Cuzick±Edwards' test (Cuzick and Edwards, 1990), was applied to detect any overall clustering of cases. This test determines the number of kth-order nearestneighbor (NN) case pairs and compares that to the number expected if the distribution of cases and non-cases were random; clustering is indicated by more than expected kth-order NN case pairs. The spreadsheet add-in Spatial Statistics (University of California, 1998), written by one of us (Carpenter), was used for this analysis. Second, the spatial scan test (Kulldorff and Nagarwalla, 1995) was used to test whether the cases were distributed randomly over space and, if not, to evaluate any identi®ed spatial disease cluster(s) for statistical signi®cance. The SaTScan software program, version 2.1, using a Bernoulli model was used (Kulldorff et al., 1998). This test compares the composition of points inside a circular window to the remainder of the population. The window moves over the area, and varies in size from zero to a maximum radius (never including >50% of the total population). The choice of 50% of the population at risk as the maximum circle size allows both small and large clusters to be detected, while ignoring those `clusters' that contain more than half the population at risk (these would be more suitably interpreted as a `negative cluster' of lower than expected risk outside the circle; Kulldorff, 2001). For each window of varying position and size, the software tested the risk of seropositivity within and outside the window, with the null hypothesis of equal risk (Kulldorff and Nagarwalla, 1995). 3. Results A total of 869 coyote blood samples was obtained, of which 863 had complete information and were analyzed. IgG antibodies to Y. pestis were detected in 101 (11.7%) specimens and to B. berkhof®i in 306 (35.5%) specimens. Coyotes were sampled from a wide range of habitats throughout California (Fig. 1). However, limited numbers were sampled from East of Sierra Nevada (SNE), Mojave Desert (DMoj) or Sonoran Desert (DSon) (sparsely populated areas), and relatively few samples were obtained from the Great Central Valley (GV) (major agricultural region). The locations of animals seropositive to Y. pestis and to B. berkhof®i are shown in Figs. 2 and 3, respectively. For Y. pestis, positive animals were most frequently observed in the Modoc Plateau (MP) bioregion and southwestern Sierra Nevada (SN) bioregion, with occasional positive animals from northern SN, Northwestern California (NW), and southern sections of Central Western California (CW). The largest grouping of animals positive for B. berkhof®i was found in the northern CW bioregion. Seropositive animals also were identi®ed in the southern sections of the CW bioregion and the western slope of the SN bioregion. Overall, B. berkhof®i positive coyotes were observed by inspection to be found in more diverse regions of the state than were Y. pestis positive coyotes.

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Fig. 1. Location of coyotes sampled for exposure to Y. pestis and B. vinsonii subsp. berkhoffii, California, 1994± 1998. : Location where coyote sample obtained, NW: Northwestern California, CaR: cascade ranges, MP: Modoc Plateau, SN: Sierra Nevada, SNE: East of Sierra Nevada, GV: Great Central Valley, CW: Central Western California, Dmoj: Mojave Desert, Dson: Sonoran Desert, SW: Southwestern California.

There was both a North/South and East/West separation of B. berkhof®i serologic status, with greater proportions of positive animals from South of the median latitude (chisquare ˆ 15:0, d:f: ˆ 1, P < 0:001) and West of the median longitude (chi-square ˆ 33:9, d:f: ˆ 1, P < 0:001) than negative animals. For Y. pestis, there was an East/West separation, with a greater proportion of positive animals from East of the median longitude (chisquare ˆ 69:0, d:f: ˆ 1, P < 0:001) than negative animals. Climate in California is extremely variable; however, in general, regions to the west (especially along the coast) typically receive more precipitation than regions to the east, while southern regions are hotter and drier than northern regions.

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Fig. 2. Location of coyotes testing positive for exposure to Y. pestis, and location of spatial clusters of Y. pestis seropositive coyotes, California, 1994±1998. : Location of seropositive coyote, : location of spatial cluster, NW: Northwestern California, CaR: Cascade Ranges, MP: Modoc Plateau, SN: Sierra Nevada, SNE: East of Sierra Nevada, GV: Great Central Valley, CW: Central Western California, Dmoj: Mojave Desert, Dson: Sonoran Desert, SW: Southwestern California.

Antibodies to both agents were identi®ed in 19 (2.2%) specimens. Thirteen (68.4%) of these 19 coyote specimens were found in the southwestern SN bioregion. Other dually seropositive coyotes were found in the northeastern SN bioregion (four coyotes, 21.1%), and the central section of the Southwestern California (SW) bioregion (two coyotes, 10.5%). With 11.7% seropositive to Y. pestis and 35.5% seropositive to B. berkhof®i, 4.2% seropositive to both pathogens would be expected if infection were independent. This is signi®cantly greater than the 2.2% observed (McNemar's paired-sample test P < 0:001). The proportion of coyote specimens positive for Y. pestis ranged from a low of 5% in 1996 to a high of 20% in 1995 (Fig. 4; chi-square ˆ 16:2, d:f: ˆ 4, P ˆ 0:003). The

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Fig. 3. Location of coyotes testing positive for exposure to B. vinsonii subsp. berkhoffii, and location of a spatial cluster of B. vinsonii subsp. berkhoffii positive coyotes, California, 1994±1998. : Location of seropositive coyote, : location of spatial cluster, NW: Northwestern California, CaR: Cascade Ranges, MP: Modoc Plateau, SN: Sierra Nevada, SNE: East of Sierra Nevada, GV: Great Central Valley, CW: Central Western California, Dmoj: Mojave Desert, Dson: Sonoran Desert, SW: Southwestern California.

proportion of specimens positive for B. berkhof®i ranged from 28% in 1998 to 42% in 1997 (chi-square ˆ 10:2, d:f: ˆ 4, P ˆ 0:04). Randomness-of-runs tests indicated signi®cant clustering of positive samples by elevation for each pathogen (P < 0:001). Of 562 coyotes sampled below 1000 m elevation, 269 (48%) were positive for B. berkhof®i compared to 37 (12%) of 301 sampled at or above 1000 m (chi-square ˆ 108:4, d:f: ˆ 1, P < 0:001). In contrast, 17 (3%) of 562 coyotes sampled below 1000 m were positive for Y. pestis, compared to 84 (28%) of 301 sampled at or above 1000 m (chi-square ˆ 117:4, d:f: ˆ 1, P < 0:001) (Fig. 5).

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Fig. 4. Percent of coyotes seropositive for Y. pestis and B. vinsonii subsp. berkhoffii by year of sample collection, California, 1994±1998.

Fig. 5. Percent of coyotes seropositive for Y. pestis and B. vinsonii subsp. berkhoffii by elevation of sample collection, California, 1994±1998.

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Table 1 Cuzick±Edwards' test results for Y. pestis and B. vinsonii subsp. berkhoffii in coyotes in California, USA (1994± 1998) Organism

Statistic

kth NN

Y. pestis

Observed NN pairs Expected NN pairs z P

B. vinsonii subsp. berkhoffii

Observed NN pairs

164

340

Expected NN pairs z P

108.3 4.4 <0.001

216.5 7.0 <0.001

1 22 11.7 2.4 0.007

2 53 23.4 4.9 <0.001

3

4

5

126 46.9 9.2 <0.001

164 58.6 10.9 <0.001

499

665

828

324.8 8.1 <0.001

433.1 9.4 <0.001

541.4 10.4 <0.001

92 35.2 7.6 <0.001

6 210 70.3 13.1 <0.001 1007 649.6 11.8 <0.001

Results of the Cuzick±Edwards' test (Table 1) indicate signi®cant spatial clustering for each pathogen at all kth-order NN calculations. For Y. pestis, the spatial scan test detected two statistically signi®cant clusters of cases (Fig. 2). The ®rst was located at 35.12 latitude, 118.45 longitude (southwestern SN bioregion; P < 0:001) with a radius of 0.188 (18 km). Of 12 coyotes within this circle, 10 (83.3%) tested positive; if the distribution were random, 1.7 cases would be expected. There was a second cluster at 40.30 latitude, 120.37 longitude (MP bioregion; P < 0:001) with a radius of 1.498 (147 km). At this cluster there were 88 animals, 29 (33.0%) testing positive; 12.2 cases would be expected if the distribution were random. For B. berkhof®i, the spatial scan test detected one statistically signi®cant (P < 0:001) cluster at 37.15 latitude, 121.61 longitude (north central CW bioregion) with a radius of 1.718 (170 km). Of a population of 109 coyotes in this area, 77 (70.6%) were seropositive, while 41.4 were expected if there were no clustering (Fig. 3). 4. Discussion The observed distributions of positive and negative coyotes, combined with the results of the spatial clustering tests and the differences in seroprevalence by elevation for the two agents, indicated that the two pathogens infect coyotes in different regions of the state. We interpret this as evidence that for coyotes in California, B. berkhof®i is not transmitted by the same vector as Y. pestis. The Cuzick±Edwards' test indicates that seropositive coyotes tend to be closer to other seropositive coyotes than to seronegative coyotes for both pathogens. We interpret this to indicate that coyotes are exposed to the two bacterial agents in geographically localized areas, rather than randomly throughout the state. This is true for the ®rst through sixth NN. We did not examine beyond the sixth NN because the z-score used in the test is cumulative and, therefore, the level of clustering observed when k ˆ 7 includes the contribution of clusters of the ®rst to sixth NNs. Thus, if there were signi®cant clustering in a pack of, e.g., four coyotes, as k increases, this impact will likely continue indicating higher-level

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clustering, even though the pack does not exceed four animals. In this study, samples were not randomly obtained; however, an important feature of the Cuzick±Edwards' test is that it adjusts for bias that might arise if a population is inhomogeneously distributed (Cuzick and Edwards, 1990). The spatial scan test revealed the location of speci®c clusters of seropositive animals. This cluster analysis was performed by using the default maximum spatial cluster size of 50% of the total population. Using this default method minimizes pre-selection bias of cluster size. We found that coyotes positive to Y. pestis were clustered in the Modoc Plateau bioregion (high desert, with hot dry summers and cold moist winters) and southwestern Sierra Nevada bioregion (variable elevation with dry mild summers and rainy to snowy winters). A cluster of B. berkhof®i positive coyotes was identi®ed in the Central Western California bioregion (Mediterranean climate with cool summers, mild falls, and chilly, rainy winters). Differences in cluster locations could be related to a different vector being involved in transmission of the two disease agents, or that infection rates of the two pathogens in host species differ in their distribution, or that different animal species can serve as reservoirs. Although plague is maintained in nature through a complex cycle involving ¯eas (Poland et al., 1994), the transmission of B. berkhof®i is not clearly established. Many species of Bartonella have been isolated from rodents, but there are no reports of B. berkhof®i being isolated from rodents (Breitschwerdt and Kordick, 2000). Although B. henselae is transmitted by cat ¯eas (Chomel et al., 1996), the route of transmission of B. berkhof®i might involve ticks (Pappalardo et al., 1997; Chang et al., 1999; Breitschwerdt and Kordick, 2000). Molecular evidence of Bartonella spp. in questing adult Ixodes paci®cus (Chang et al., 2001), in nymphal I. paci®cus and Dermacentor sp. ticks (Chang et al., 2002), I. scapularis (Eskow et al., 2001) and I. ricinus ticks (Schouls et al., 1999) favors such an hypothesis. In this study, the observed distribution of coyotes seropositive to B. berkhof®i mimicked the known distribution of many tick species in California, including I. paci®cus and D. variablilis (Furman and Loomis, 1984). To our knowledge, there is no evidence of cross-protective immunity existing between these two agents; therefore, observing fewer coyotes seropositive to both than expected if infection were independent supports the hypothesis that the two agents are likely to have different vectors or reservoirs that have different habitat requirements. The several coinfected animals in the southwestern Sierra bioregion suggests that this geographic area represents a junction of two different ecosystems where the two arthropod-borne bacteria cycles intersect. The area is immediately west of the Tehachapi Mountains. Coyotes in this region can have home ranges that encompass a large variation in elevation thereby exposing them to a variety of ecosystems and local vectors. The cyclic pattern noticed in the annual distribution of positive Y. pestis samples could re¯ect ¯uctuations in the population of reservoir rodents, either through die-offs and repopulation by naõÈve animals, or population responses to variable environmental factors such as abundance of food and harborage. Above-average winter±spring precipitation has been associated with increased incidence of human plague in New Mexico (possibly due to increased small mammal food resources leading to an increase in endemic hosts during wet years) (Parmenter et al., 1999). Although signi®cant differences in the annual proportion positive were also obtained for B. berkhof®i, there was no apparent pattern. In general, it appears that the proportion testing positive for this pathogen remained relatively stable

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throughout the duration of the study (one-third testing positive each year). With only 5 years of data, the apparent annual trends and cycles need to be followed for several more years before de®nitive conclusions can be reached. Also, in coyotes, hemagglutinating antibodies to Y. pestis become detectable in 8±14 days, and diminish to undetectable levels within 6±8 months (Barnes, 1982). Persistence of serum antibody titers might depend on the number of organisms initially ingested and the cumulative effects of repeated infection (Messick et al., 1983). Although duration of antibody persistence to B. berkhof®i in coyotes has not been determined, in experimentally infected beagles, IgG antibodies to B. berkhof®i were detectable for the entire duration of a 149-day study period (Pappalardo et al., 2001). Therefore, coyotes seropositive to either agent might represent a combination of incident and prevalent cases. The effect of antibodies that persist from year-to-year would be to reduce the observed variation in annual proportions positive to the agents (thereby potentially masking substantial changes in incidence). Seroprevalence of B. berkhof®i decreased with increasing elevation, whereas seroprevalence of Y. pestis increased with increasing elevation. Such differences might re¯ect the distribution of reservoir hosts, the activity of various vectors, or climatic conditions. Lowelevation coastal regions of California are typically cooler and moister than foothill and mountain locations, which might allow for increased coastal tick activity (Furman and Loomis, 1984). Fleas are essentially nest-dwelling insects; nests moderate the temperature and relative humidity (Perry and Fetherston, 1997), allowing for survival in more extreme climates. Seroprevalence to Y. pestis apparently is endemic in coyotes in California. Plague surveillance performed throughout California from 1974 to 1982 using the same diagnostic test and a similar sampling scheme found 10.6% of 2617 sampled coyotes seropositive (Smith et al., 1984), which is similar to our ®ndings >12 years later. Our study adds to previous surveillance studies by providing spatial statistical analysis and speci®cally determined the number and location of plague clusters. It also con®rms the continued presence of sylvatic plague in many regions of the state. Conclusions from this study must be tempered by the understanding that coyote samples were not randomly collected. The USDA Wildlife Services depredation control is done on an as-needed basis. Therefore, the sample of coyotes obtained for this study might not be representative of California coyotes in general. However, we believe that there would not be signi®cant observer bias introduced by this sampling method. As far as can be determined, neither agent causes signi®cant clinical disease in coyotes (Smith et al., 1984; Chang et al., 2000); therefore, seronegative and seropositive coyotes should be equally available for capture. Wildlife-control specialists would have no advance knowledge of the serologic status of coyotes they sampled. Misclassi®cation with respect to location of sampling is also possible. Given that the home range of coyotes can be up to 80 km2 (Nowak, 1999), we believe that the precision of locational data is adequate for this study. The exact location of sampling might not indicate the exact location where the animal was infected. The effect of such imprecision would be to reduce the degree of clustering calculated because a coyote might be infected at a particular nidus of infection then travel a long distance before being sampled. This study illustrates that, while coyotes in California are commonly exposed to Y. pestis and B. berkhof®i, infection is geographically non-uniform and discontinuous throughout

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