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Anticoagulant Exposure and Disease Susceptibility in Bobcats in Southern California Summerlee Foundation Grant: Final Report, December 5, 2011

From: Santa Monica Mountains Fund, Art Eck – Executive Director 401 W. Hillcrest Dr., Thousand Oaks, CA 91360 Background:

Our project addresses how consequences of urban development affect the physiological health and disease susceptibility of bobcat populations near an urban, fragmented landscape in Southern California. Summerlee Funds have specifically supported evaluating how anticoagulant rodenticide exposure may increase disease susceptibility in populations near Los Angeles, CA. Data from longest-running bobcat study generated by the National Park Service (NPS) since 1996 in Santa Monica Mountains National Recreation Area (SMMNRA) have formed the foundation of this project. Our long-term research has included studies on behavior and ecology,

genetic differentiation among populations fragmented by roads, and on anticoagulant and disease exposure in bobcats in the central region of SMMNRA. Results of our foundational research found that coincident with a mange epizootic (Fig. 1) in 2002, the survival rate for radio-collared bobcats fell from a high of 0.847 in 1999 (5-year average, 0.770) to 0.280 in 2003. Anticoagulant exposure is also found to be extremely widespread in the study area:

ninety percent of livers tested from 39 bobcats which died from mange, as well as other causes, tested positively for anticoagulant rodenticides. Necropsy results of bobcats that died with mange revealed that mortality was not due to direct anticoagulant toxicity, but rather from the mange itself. However, mange and anticoagulant exposure were very highly associated and 19 of 19 bobcats with advanced mange tested positive for anticoagulant compounds.

Stated Project Objectives:

Our project seeks to address how sublethal anticoagulant rodenticide exposure is impacting bobcat populations in Southern California. Specifically, we are addressing how widespread anticoagulant rodenticide exposure is in this region. Further, we aim to understand the sublethal physiological consequences of exposure that may lead to decreased immune competence and increased susceptibility to disease in bobcat populations. Our two primary aims for this research include:

1) Assess how widespread exposure to anticoagulant rodenticides is across three

Southern California counties and how exposure varies with proximity to urbanization.

Figure 1. Photos of a bobcat captured in August 2010 (left) before developing severe mange in August 2011 (above) illustrating the severity of this disease for bobcats. He was captured in SMMNRA in area U3 (see Fig. 2).

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Funds provided by Summerlee were half the requested amount and so allowed us to closely examine the widespread nature of anticoagulant rodenticide exposure in only two of the three counties. We were able to test 200 bobcat blood and liver samples procured through field sampling and opportunistic road kill sample collection in Los Angeles and Ventura Counties. However, to date, we have only tested bobcats that have died, using liver tissue. Our hypotheses are that exposure to anticoagulant rodenticides will be widespread across our study area, with prevalence rates highest near urban areas. 2) Understand how the bioaccumulation of toxicants affects physiological health and

contributes to increased disease susceptibility in bobcats in Southern California. Using detailed analyses of blood samples from live-captured animals throughout the study region, as well as necropsy reports from animals that have died from mange, we are assessing the physiological impact these toxicants have on bobcats. Further, we are presently working to construct a hypothesis linking the physiological processes that occur

Figure 2. Map of Santa Monica Mountains National Recreation Area (SMMNRA) where we have conducted intensive field studies. To date, we have collected samples from over 300 bobcats across SMMNRA. Numbered, circled regions represent areas where we focused bobcat sampling. Core areas represent regions where we expected bobcats to be less influenced by urban development and anticoagulant rat poisons. Bobcats in urban areas are expected to have widespread exposure to anticoagulants. However, we have documented anticoagulant exposure in all regions we sampled bobcats. Further, mange is now known to affect or have impacted populations in areas U1, U2, U4, C2, and C3.

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as a consequence of sublethal chronic exposure to anticoagulant rodenticides predisposing bobcats to terminal mange infection. Through this work we are increasingly documenting areas where bobcat populations are affected by mange epizootics. Our hypothesis: Anticoagulant rodenticide exposure will be a primary determinant of immune competence and overall physiological health in bobcats, thus increasing their disease susceptibility.

Advancement Toward Project Objectives During the Funding Period:

Fieldwork: The project proposal described fieldwork to take place between June 2010-February 2011. The field season concluded and was highly successful. We captured and collected samples from approximately 50 unique individuals. These numbers are unusually successful for bobcat studies. Combined with banked samples, we have a total of samples from 293 bobcat individuals in Los Angeles and Ventura Counties.

Physiological Assessments: We also conducted physiological assessments on new individuals captured, increasing our understanding of potential modes by which anticoagulants impact the health of bobcat populations in our region. An essential element in understanding the relationship between notoedric mange and anticoagulant rodenticides is procuring samples from individuals with severe mange. We have effectively gathered data from 20 bobcats with mange and are presently in the process of analyzing these data and performing additional tissue histological evaluations from samples collected. However, the data we have gathered and analyzed thus far offer a potential explanation to the relationship between anticoagulant rat poison exposure and notoedric mange.

Additionally, we have collected samples to test the clotting times of individuals using PIVKA (proteins invoked in vitamin K absence) and recently received a small grant from the Audubon Society to test most of the samples collected. The intended physiological mode of action for anticoagulants is to interfere with vitamin K-dependent clotting factors thus leading to death from internal bleeding. However, even for bobcats with severe mange and highly suspected of anticoagulant exposure, we have not detected altered clotting times using this method thus far. These results suggest that anticoagulants affect bobcats in a manner not anticipated given the expected consequences of anticoagulant exposure. However, these results are preliminary, and we have tested fewer than 10 bobcat samples. We look forward to testing our remaining samples to understand better the effects of anticoagulant exposure on bobcat clotting times. Further, by combining clotting time data with the anticoagulant residue data we have collected (funded by Summerlee), we may be able to detect the anticoagulant exposure threshold at which bobcat clotting time is altered.

Anticoagulant Surveys: In addition to measuring clotting times we are also testing samples directly for anticoagulant residues. Until recently, there was a challenge in testing blood samples for residues, but with collaborators at the Center for Animal Health and Food Safety (CAHFS), we have recently increased the sensitivity of the already available detection method to detect the low residue levels in blood samples. We have tested 200 samples using the Summerlee Funds. Data are presented below. Use of Funds Granted:

$20,000 was granted toward this project. Our grant proposal budgeted $40,000 to cover anticoagulant residue testing in samples in Los Angeles, Ventura, Orange and Riverside Counties. Funds were also request to cover student fees for a graduate student completing much of the research associated with this project. Other funding was found to provide support for

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graduate student, Laurel Klein Serieys, and so all of the Summerlee funds are being used for anticoagulant residue analysis. At $100 per anticoagulant survey, we had funds to test 200 samples only from Los Angeles and Ventura Counties. Thus, we have evaluated anticoagulant exposure across two counties rather than four. Results:

Notoedric Mange Observations: Through fieldwork, outreach efforts (see below for more information), and collaborations with biologists in other regions of the State, we have documented mange to be an increasing problem for bobcats across California. In the last year, we have documented an additional 5 counties in California where mange epizootics are now affecting bobcat populations (Fig. 3). Within our own study area (Fig. 2), we have also documented mange epizootics in two additional bobcat populations (areas U2 and U4 [Fig. 1]), as well as sporadic mange cases in areas C3 and C4. Our original work showed mange epizootics to affect bobcats only in area U1- an epizootic that began in 2002.

No new mange cases have been

documented in area U1 since 2007. All of our observations were made through live-capturing of individuals with notoedric mange, radio-collared animals that were captured healthy but subsequently died of mange, evidence provided using remote cameras throughout our study area, and reports (supplemented with photographs) made by the general public and colleagues.

Using these data gathered, we examined whether seasonal differences occur between the numbers of notoedric mange cases. Our data (Fig. 4) show that there are more than twice as many mange cases during the dry season (May-October) in Southern California when compared to the number of cases documented during the wet season (November-April). Using a Wilcox Rank Sum Test, we found the difference between groups to be significant with a p-value of

Figure 4. Compiled cases since 2002 of severe mange totaling 75 cases in 6 Southern California Counties. Dry season is from May-October, while wet season is from November to April in Southern California. The difference between the two groups is significant with a p-value of 7.93e-8 using a Wilcoxan Rank Sum Test.

Figure 3. Map of California illustrating the counties where mange epizootics have currently been documented in California. Prior to 2002, notoedric mange was not documented to affect bobcats in California, nor was it documented to cause epizootics or population declines in any wild cat species. Our study was the first to documented it in the starred area between Los Angeles and Ventura Counties. Colleagues subsequently documented it in the second starred area, and now mange epizootics are known to occur in all shaded counties.

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7.93e-08. Additional analyses we plan to perform include determining if bobcat exposure to anticoagulants also varies with season. If anticoagulant exposure and notoedric mange are linked, we may observe a similar pattern in anticoagulant exposure increases during the wet season. However, because some anticoagulant compounds have a half-life of more than 100 days in liver tissue, the samples we have tested have may not be adequate to capture the time of exposure events for our bobcat individuals. Potential explanations for why mange cases are higher during the dry season include several possibilities. First, animals may be stressed during the dry season by reduced water availability and potentially less prey abundance in natural areas, although whether decreased abundance exists during the dry season has not been examined in our study area. However, the stress of reduced water availability, for example, could have physiological consequences on bobcats increasing their susceptibility to notoedric mange. Second, if reduced prey abundance exists in natural areas as a result of the dry season, bobcats may venture more frequently into the urban areas to find prey items. We expect a higher frequency of anticoagulant exposure in the more urban areas. Thus, bobcats may have more anticoagulant exposure events during the dry season, likewise predisposing them to notoedric mange infection. Third, the notoedric mange mites may tend to thrive during the hotter, drier times of the year, although little in known about the biology of the mite. We plan to use data we have already collected to explore further the first two possibilities.

Anticoagulant Rodenticide Exposure: Funds provided by Summerlee allowed us to

expand the geographic range of samples tested, the number of samples tested and finally, the type of sample tested to determine anticoagulant rodenticide exposure prevalence in our study area. Previous anticoagulant rodenticide exposure research has focused on residues found only in liver tissue since previous assays could be applied only to liver tissue. Anticoagulant residues accumulate in the liver, and each type of compound of 7 commercially available compounds has varying half-lives within liver tissue. The half-life data are not available for bobcats, but certain compounds are known to have longer half-lives than others. This assay is limited by the ability to procure liver samples from individuals, thus limiting the testing to animals that die in the study area. However, we recently, in collaboration with UC Davis, have expanded our testing to blood samples. Summerlee funds were used to test 173 blood samples and 27 liver samples from bobcats within the Santa Monica Mountains National Recreations Area (SMMNRA).

Table 2. Wilcox Rank Sum Test p-values calculated to determine whether significant differences exist between urban and core bobcat population anticoagulant exposure results. The highlighted boxes represent those values that are significant after a Bonferroni Correction (Bonferonni corrected p-value= 0.0167).

Table 1. Compiled data for bobcats in SMMNRA grouped by habitat type. “Blood” results are anticoagulant exposure assays performed on blood samples only. “Liver” represents results gained from liver samples. “Combined” represents paired liver and blood samples results for a single individual. Capture or mortality locations are pending for some individuals. “Core Habitat” individuals are those captured or that died more than 0.5km from urban development. Individuals closer than 0.5km to urban development are considered to reside within “Urban Habitat.” Since locations are pending for some individuals, the far right column shows interesting results independent of location, and includes the total number of paired serum and liver results for the study area.

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The landscape within SMMNRA is a mixture of highly fragmented, urban-associated habitat fragments (‘Urban’ habitat) and more continuous, natural regions with little fragmentation or urban influence (‘Core’ habitat). Prior to beginning this study, we expected to find bobcats in core areas to have no exposure to anticoagulants. We expected that urban bobcats would have more exposure to anticoagulants, and thus the number of compounds, as well as the concentrations of the compounds, would be greater in urban versus core areas (see Fig. 2). Our findings are shown in Tables 1-2 and Figure 4. The upper limit of the ranges of total compound concentrations and number of compounds detected are greater in urban areas compared to core areas (Table 1). By comparing these data and calculating whether the differences between groups are significant, we find that the total number of compounds detected in blood and the total concentrations of combined, paired (for a single individual) liver and blood samples are significantly different (Table 2) between urban and core areas.

By examining all of our anticoagulant exposure results (Fig. 4), we observe that liver samples are a better detector of anticoagulant exposure than blood. However, we have also observed that certain compounds (such as diphacinone) are more frequently detected in blood samples than in the liver samples. Diphacinone has a shorter liver half-life compared to compounds such as bromadiolone and brodifacoum, the two compounds most frequently detected in liver tissue. Thus, combining paired liver and blood samples for a single individual, when possible, more accurately detects multiple compound anticoagulant exposures than either method alone. Accordingly, we find that when we combine paired liver and blood samples for individuals, the most frequent number of compounds individuals are exposed to is 3 (Table 1, Fig. 4), rather than the previously documented 2 compounds. Further, our exposure rate is observed to be 95% using these combined data (Fig. 4). Physiological Assessments (Preliminary Results): To evaluate the potential link between anticoagulant rodenticides and notoedric mange, we aimed to evaluate the physiological effects of anticoagulant exposure. We are measuring a variety of parameters, including 16 blood chemistry values. Blood chemistry values can be indicative of a variety of physiological and environmental conditions within individuals. For this reason, they are typical diagnostic tests performed by veterinarians or medical doctors to diagnose and determine health of individuals.

Figure 4. Chart illustrating the percentage of individuals exposed to a range of anticoagulant rat poisons compounds according to the sample type tested. After combining paired blood and liver samples for 39 individuals, we see that individuals in our study area are most commonly exposed to at least 3 anticoagulant rodenticide compounds. We previously reported the most common number (according to liver results only), to be 2 compounds.

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Thus far, we have focused on determining to physiological status of bobcats with mange and anticoagulant exposure. We have compared preliminary data from bobcats with mange to two healthy, control bobcat groups. We successfully collected these data from 20 mangy bobcats exposed to anticoagulants in addition to data from 2 control groups: a wild healthy bobcat group (captured in area C1 [Fig. 1]), and a captive bobcat group (Table 3).

After comparing each parameter between the three groups, we found abnormalities in the serum chemistries of bobcats for multiple parameters (Table 3-4, Fig. 5). Elevations in liver

enzyme, AST) were detected in both bobcats with mange as well as those from the healthy wild control group but not the captive controls, suggesting that these findings may be due to the stress of capture. Serum electrolyte abnormalities were observed in some bobcats suggesting dehydration. Bobcats with severe mange are often severely dehydrated. Bobcats with mange had low levels of cholesterol, albumin, globulin, and total protein possibly due to protein loss secondary to the observed skin disease and/or a primary gastrointestinal loss. No evidence of an additional route for protein loss has been noted on necropsy necropsy of bobcats with mange, and a urinalysis was not performed on these animals so a urinary loss cannot be entirely ruled out. Further, a gastrointestinal loss of albumin could account for the

significantly low values of calcium that were observed in some bobcats with mange. The increased blood urea nitrogen in the absence of increased serum creatinine values suggests gastrointestinal bleeding. Gastrointestinal bleeding secondary to gastrointestinal disease, such as inflammatory bowel disease, could potentially explain the increased blood urea nitrogen, as well as the decreased albumin, globulin, and cholesterol. Hypoglycemia was observed in some

Table 4. Results of Wilcoxon Rank-Sum Tests for values found to be signficant using Kruskal-Wallis analyses. The Bonferroni corrected p-value for each value compared between the three groups was 0.017. Those that are significant are highlighted in yellow. WH=wild healthy (n=9), C=captive (n=18), and M=mange (n=20).

Table 3. Means, standard deviations (s.d.), and medians for blood chemistry values for each bobcat group. Cho=cholesterol, Glu= glucose, CA=calcium, P=phosphorus, BUN=blood urea nitrogen, Creat=creatinine, T.P.=total protein, Glob=globulin, ALB=albumin, AKT= Alkaline phosphatase, ALT= Alanine aminotransferase, AST= Aspartate aminotransferase, T.B.= Total bilirubin, Cl=chloride, K=potassium, NA=sodium. Values found to be statistically different between groups are highlighted in yellow (Bonferoni corected p-value= 0.003).

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bobcats with mange, suggestive of either starvation, septicemia or possible liver function insufficiency.

Altogether, these data are suggest a multifactorial process that may predispose certain bobcats to the development of severe mange with secondary dermatopathology. Primary predisposing disease considerations would include gastrointestinal hemorrhage associated with intestinal parasitism, inflammatory bowel disease, or potentially a coagulopathy subsequent to sublethal rodenticide ingestion. Chronic gastrointestinal disease could contribute to a nutritional compromise and partial immunosuppression such that infestation with the mange mite would be particularly severe. Ultimately severe dermatopathy and secondary septicemia could cause

dehydration, hypoglycemia, and death. Although to date no significant association or correlation between the clinical blood chemistries and the residue concentrations found in the livers or blood of bobcats with mange, we cannot exclude an association between gastrointestinal disease and sublethal, chronic anticoagulant rat poison exposure. Further examination of gastrointestinal tract of these bobcats is warranted. We are thus following up with our data gathered so far by performing tissue histologies on gastrointestinal tract and liver tissue of bobcats with notoedric mange to determine if those exposed to anticoagulants have tissue damage associated with exposure. Outreach Efforts:

Figure 5. Dot plots of raw data values for each bobcat group found to be signficantly different using Kruskal-Wallis analyses. The plots illustrate non-normality of the data warranting the use of nonparametric analyses.

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Understanding human impact on wildlife is an important part of conservation efforts. Another key element to wildlife conservation is public outreach and education. The source of anticoagulant poisons for our bobcat and mountain lion populations are from human use in urban areas. We hope that by educating the public about the consequences of anticoagulant use in these urban areas, that fewer people will use these common poisons. Accordingly, we have collaborated with graduate student Laurel Klein Serieys to create a website (urbancarnivores.com) to serve this important purpose. The website is still in development, but already we are making available what we are learning about our local native carnivores through our extensive research efforts. Surprisingly, many people living near park regions are unaware that bobcats and mountain lions roam our local mountains. We hope that by publicizing what we are learning, we will cultivate interest in the posterity of our native wildlife. Second, we are able to provide information to the general public about the prevalence and consequences of anticoagulant rodenticides, with creative tools such as our hypothetical food web we designed (Fig. 5). This food web shows that when we use poisons around our homes, many more species than the target pests are affective. We hope that education about these issues will encourage people to decrease their use of these common poisons. This website receives unique visitors daily and since it was launched in March has received more than 5,000 unique visitors. As we increase the information on the website, those figures increase and we hope that this will be an important awareness and conservation tool for our local bobcat and mountain lion populations.

Figure 6. Hypothetical food web illustrated by UCLA Environmental Studies undergraduate student, Christine Danner, illustrating the multitude of species potentially affected when a single home chooses to use anticoagulant rat poisons.