Otterly diverse - A high diversity of Dracunculus species (Spirurida: Dracunculoidea) in North American river otters (Lontra canadensis).

The genus Dracunculus contains numerous species of subcutaneous parasites of mammals and reptiles. In North America, there are at least three mammal-infecting species of Dracunculus. Reports of Dracunculus infections have been reported from river otters (Lontra canadensis) since the early 1900s; however, little is known about the species infecting otters or their ecology. Most reports of Dracunculus do not have a definitive species identified because females, the most common sex found due to their larger size and location in the extremities of the host, lack distinguishing morphological characteristics, and few studies have used molecular methods to confirm identifications. Thus, outside of Ontario, Canada, where both D. insignis and D. lutrae have been confirmed in otters, the species of Dracunculus in river otters is unknown. In the current study, molecular characterization of nematodes from river otters revealed a high diversity of Dracunculus species. In addition to confirming D. insignis infections, two new clades were detected. One clade was a novel species in any host and the other was a clade previously detected in Virginia opossums (Didelphis virginiana) from the USA and a domestic dog from Spain. No infections with D. lutrae were detected and neither new lineage was genetically similar to D. jaguape, which was recently described from a neotropical otter (Lontra longicaudis) from Argentina. These data also indicate that Dracunculus spp. infections in otters are widespread throughout Eastern North America. Currently the life cycles for most of the Dracunculus spp. infecting otters are unknown. Studies on the diversity, life cycle, and natural history of Dracunculidae parasites in wildlife are important because the related parasite, D. medinensis (human Guinea worm) is the subject of an international eradication campaign and there are increasing reports of these parasites in new geographic locations and new hosts, including new species in humans and domestic dogs.

Support for the size-mediated sensitivity hypothesis within a diverse carnivore community.

Carnivore community dynamics are governed by a complex set of often interacting biotic, abiotic and anthropogenic factors that are increasingly volatile as a result of global change. Understanding how these changing conditions influence carnivore communities is urgent because of the important role carnivores play within ecosystems at multiple trophic levels, and the conservation threats that many carnivores face globally. While a great deal of research attention has historically been focused on large carnivores within ecosystems, the size-mediated sensitivity hypothesis has recently been proposed where the smallest carnivore in a system is likely to be the most responsive to the diverse suite of ongoing environmental and anthropogenic changes within ecological communities. We deployed camera traps at 197 sites over 4 years to monitor a diverse suite of mammalian carnivores within the Blue Ridge Mountains of western North Carolina and then used a two-step occupancy modelling-structural equation modelling framework to investigate the relative support for four primary hypothesized drivers (interspecific competition/predation, habitat complexity, food availability and anthropogenic disturbance) on carnivore occurrence. We found that each of the 10 carnivores in our system responded differently to conditions associated with each of these four hypothesized drivers, but that small and medium-sized carnivores had a greater number of significant (p < 0.05) pathways by which these conditions were influencing occupancy relative to large carnivores. In particular, the smallest carnivore observed in our study was the only species for which we found support for each of the four hypothesized drivers influencing occupancy. Collectively, our study supports the size-mediated sensitivity hypothesis and suggests that small carnivores are ideal sentinel species for global change. We echo recent calls for adopting a middle-out approach to investigations into carnivore community dynamics by refocusing sustained monitoring and research efforts on smaller carnivores within systems.

USE OF A POINT OF CARE TEST TO DETERMINE THE PREVALENCE OF ANTIBODIES TO TOXOPLASMA GONDII IN BLACK BEARS FROM NORTH CAROLINA AND PENNSYLVANIA.

Toxoplasma gondii is an important protozoan parasite of humans and animals throughout the world. Black bears are among the animals with the highest seroprevalence of T. gondii in the United States. A rapid point of care (POC) test is commercially available to detect antibodies to T. gondii in humans. We evaluated the utility of the POC test to detect anti-T. gondii antibodies in 100 wild black bears from North Carolina (n = 50) and Pennsylvania (n = 50). In a blind study, sera were tested by the POC test, and results were compared to the modified agglutination test (MAT). Overall, anti-T. gondii antibodies were detected in 76% (76/100) black bears by both MAT and POC tests. One false positive and one false negative result in the POC test were obtained in bears from Pennsylvania. The sensitivity and specificity of the POC test were both 99% when compared to the MAT. Results from our study indicate the POC test could be a useful screening tool for serological surveillance of T. gondii in black bears.

Draft Genome Sequences of 158 Listeria monocytogenes Strains Isolated from Black Bears (Ursus americanus) in the United States.

Listeria monocytogenes is responsible for severe foodborne disease and major economic losses, but its potential reservoirs in natural ecosystems remain poorly understood. Here, we report the draft genome sequences of 158 L. monocytogenes strains isolated from black bears (Ursus americanus) in the southeastern United States between 2014 and 2017.

Genetic architecture and evolution of color variation in American black bears.

Color variation is a frequent evolutionary substrate for camouflage in small mammals, but the underlying genetics and evolutionary forces that drive color variation in natural populations of large mammals are mostly unexplained. The American black bear, Ursus americanus (U. americanus), exhibits a range of colors including the cinnamon morph, which has a similar color to the brown bear, U. arctos, and is found at high frequency in the American southwest. Reflectance and chemical melanin measurements showed little distinction between U. arctos and cinnamon U. americanus individuals. We used a genome-wide association for hair color as a quantitative trait in 151 U. americanus individuals and identified a single major locus (p < 10-13). Additional genomic and functional studies identified a missense alteration (R153C) in Tyrosinase-related protein 1 (TYRP1) that likely affects binding of the zinc cofactor, impairs protein localization, and results in decreased pigment production. Population genetic analyses and demographic modeling indicated that the R153C variant arose 9.36 kya in a southwestern population where it likely provided a selective advantage, spreading both northwards and eastwards by gene flow. A different TYRP1 allele, R114C, contributes to the characteristic brown color of U. arctos but is not fixed across the range.

Prevalence and genetic characterization of a Babesia microti-like species in the North American river otter (Lontra canadensis).

A 4.5-month-old, male, North American river otter (Lontra canadensis) from Athens-Clarke County, Georgia, USA being temporarily housed at a rehabilitation facility, presented with a three-day history of lethargy, anorexia, and severe anemia. Antemortem blood smears revealed intraerythrocytic piroplasms. Supportive care and antiparasitic treatments were initiated, but the animal died three days following presentation. Gross necropsy revealed yellow discoloration of all adipose tissue throughout the carcass and a mildly enlarged, diffusely yellow to pale orange liver. Microscopically, moderate, centrilobular hepatocellular degeneration and necrosis were observed, consistent with hypoxia secondary to apparent hemolytic anemia. Piroplasms were frequently observed in red blood cells in histologic sections. The nearly full-length 18S rRNA gene sequence (1588 bp) was identical to a previously described piroplasm from North American river otters from North Carolina. Phylogenetically, based on the 18S rRNA gene sequence, the otter Babesia sp. was in a sister group with a clade that included several strains of Babesia microti-like species including Babesia sp. from badgers (Meles meles), Babesia vulpes, and Babesia sp. from raccoons (Procyon lotor). To better understand the distribution and genetic variability of this Babesia species, otters from four states in the eastern U.S. and California were tested. Overall, 30 of 57 (53%) otters were positive for Babesia sp. None of four otters from California were positive, but prevalences in eastern states were generally high, 5/9 (55%) in Georgia, 7/14 (50%) in South Carolina, 10/17 (59%) in North Carolina, and 8/13 (62%) in Pennsylvania). Partial 18S rRNA gene sequences from all populations were identical to the clinical case sequence. No Babesia sensu stricto infections were detected. There were six unique COI sequences (937 bp) detected in 18 positive otters. The most common lineage (A) was detected in 12 of 18 (67%) samples from Georgia, North Carolina, South Carolina, and Pennsylvania. Lineage B was found in two otters and the remaining lineage types were found in single otters. These six lineages were 99-99.8% similar to each other and were < 88% similar to related parasites such as B. vulpes, B. microti-like species of raccoons, B. microti, and B. rodhaini. Phylogenetically, the Babesia sp. of otters grouped together in a well-supported clade separate from a sister group including B. vulpes from fox (Vulpes vulpes) and domestic dogs. In conclusion, this report demonstrates that this piroplasm is a potential pathogen of North American river otters and the parasite is widespread in otter populations in the eastern United States.

Carolina critters: a collection of camera-trap data from wildlife surveys across North Carolina.

Camera trap surveys are useful to understand animal species population trends, distribution, habitat preference, behavior, community dynamics, periods of activity, and species associations with environmental conditions. This information is ecologically important, because many species play important roles in local ecosystems as predators, herbivores, seed dispersers, and disease vectors. Additionally, many of the larger wildlife species detected by camera traps are economically important through hunting, trapping, or ecotourism. Here we present a data set of camera-trap surveys from 6,043 locations across all 100 counties of North Carolina, USA from 2009 to 2019. These data come from 26 survey initiatives and contain 215,108 records of 36 mammal species and three species of terrestrial birds. This large data set increases the geographical distribution data for these 39 mammal and bird species by >500% over what is available for North Carolina in the Global Biodiversity Information Facility (GBIF). These data can be used to conduct inquiries about species, populations, communities, or ecosystems, and to produce useful information on wildlife behavior, distribution, and interactions. There are no copyright restrictions. Please cite this paper when using the data for publication.

Seroprevalence of Sarcoptes scabiei in Free-Ranging Black Bears (Ursus americanus) in Eastern North Carolina, USA.

Recent sarcoptic mange epizootics have affected free-ranging black bears (Ursus americanus) in the northeastern US, but not in North Carolina. To determine whether black bears in eastern North Carolina have exposure to Sarcoptes scabiei, serum samples from hunter-harvested black bears (n=45) were collected and evaluated for antibodies using a commercial enzyme-linked immunosorbent assay previously validated in black bears. No dermal lesions consistent with sarcoptic mange were identified in the sampled bears. The seroprevalence among these asymptomatic bears was 18%, with no significant difference between sexes or association with age. This suggests that exposure to Sarcoptes scabiei occurs within the population, and highlights the importance of serosurveys in regions without a history of clinical mange.

LEPTOSPIRA, PARVOVIRUS, AND TOXOPLASMA IN THE NORTH AMERICAN RIVER OTTER (LONTRA CANADENSIS) IN NORTH CAROLINA, USA.

The North American river otter (Lontra canadensis) is the largest mustelid in North Carolina, US, and was once extirpated from the central and western portions of the state. Over time and after a successful reintroduction project, otters are now abundant and occur throughout North Carolina. However, there is a concern that diseases may have an impact on the otter population, as well as on other aquatic mammals, either through exposure to emerging diseases, contact with domestic animals such as domestic cats (Felis catus), or less robust condition of individuals through declines in water quality. We tested brain and kidney tissue from harvested otters for the pathogens that cause leptospirosis, parvovirus, and toxoplasmosis. Leptospirosis and toxoplasmosis are priority zoonoses and are maintained by domestic and wild mammals. Although parvovirus is not zoonotic, it does affect pets, causing mild to fatal symptoms. Across the 2014-15 and 2015-16 trapping seasons, we tested 220 otters (76 females, 144 males) using real-time PCR for Leptospira interrogans, parvovirus, and Toxoplasma gondii. Of the otters tested, 1% (3/220) were positive for L. interrogans, 19% (41/220) were positive for parvovirus, and 24% (53/220) were positive for T. gondii. Although the pathogens for parvovirus and toxoplasmosis are relatively common in North Carolina otters, the otter harvest has remained steady and the population appears to be abundant and self-sustaining. Therefore, parvovirus and toxoplasmosis do not currently appear to be negatively impacting the population. However, subsequent research should examine transmission parameters between domestic and wild species and the sublethal effects of infection.

Metal contamination of river otters in North Carolina.

Aquatic apex predators are vulnerable to environmental contaminants due to biomagnification. North American river otter (Lontra canadensis) populations should be closely monitored across their range due to point and nonpoint pollution sources. Nonetheless, no information exists on environmental contaminants in the North Carolina otter population. Metals and metalloids occur naturally across the landscape, are essential for cellular function, and become toxic when concentrated unnaturally. We conducted our study across the three Furbearer Management Units (FMU) and 14 river basins of North Carolina. We determined the concentrations of arsenic, cadmium, calcium, cobalt, copper, iron, lead, magnesium, manganese, mercury, molybdenum, selenium, thallium, and zinc in liver and kidney samples from 317 otters harvested from 2009 to 2016. Arsenic, lead, and thallium samples were tested at levels below the limit of detection. With the exception of cadmium, we detected all other elements at higher levels in the liver compared with the kidney. Specifically, cadmium, cobalt, copper, iron, magnesium, manganese, mercury, molybdenum, and zinc levels differed by tissue type analyzed. Most element concentrations remained stable or increased with otter age. We detected higher levels of mercury and selenium in the Lower Pee Dee and Cape Fear river basins. River basins within the Mountain FMU were higher in cadmium, copper, iron, lead, and zinc, whereas the Coastal Plain FMU was lower in cobalt and manganese. None of the elements occurred at toxic levels. Our research establishes baseline concentration levels for North Carolina, which will benefit future monitoring efforts and provide insight into future changes in the otter population.

Listeria monocytogenes at the human-wildlife interface: black bears (Ursus americanus) as potential vehicles for Listeria.

Listeria monocytogenes is the causative agent of the foodborne illness listeriosis, which can result in severe symptoms and death in susceptible humans and other animals. L. monocytogenes is ubiquitous in the environment and isolates from food and food processing, and clinical sources have been extensively characterized. However, limited information is available on L. monocytogenes from wildlife, especially from urban or suburban settings. As urban and suburban areas are expanding worldwide, humans are increasingly encroaching into wildlife habitats, enhancing the frequency of human-wildlife contacts and associated pathogen transfer events. We investigated the prevalence and characteristics of L. monocytogenes in 231 wild black bear capture events between 2014 and 2017 in urban and suburban sites in North Carolina, Georgia, Virginia and United States, with samples derived from 183 different bears. Of the 231 captures, 105 (45%) yielded L. monocytogenes either alone or together with other Listeria. Analysis of 501 samples, primarily faeces, rectal and nasal swabs for Listeria spp., yielded 777 isolates, of which 537 (70%) were L. monocytogenes. Most L. monocytogenes isolates exhibited serotypes commonly associated with human disease: serotype 1/2a or 3a (57%), followed by the serotype 4b complex (33%). Interestingly, approximately 50% of the serotype 4b isolates had the IVb-v1 profile, associated with emerging clones of L. monocytogenes. Thus, black bears may serve as novel vehicles for L. monocytogenes, including potentially emerging clones. Our results have significant public health implications as they suggest that the ursine host may preferentially select for L. monocytogenes of clinically relevant lineages over the diverse listerial populations in the environment. These findings also help to elucidate the ecology of L. monocytogenes and highlight the public health significance of the human-wildlife interface.

Detection and Prevalence of Babesia spp. in American Black Bears (Ursus americanus) from Eastern and Western North Carolina, USA.

Blood samples collected from American black bears (Ursus americanus) in eastern and western North Carolina, US, were analyzed for piroplasms. Piroplasmids were detected in 17% (23/132) of the animals surveyed. We detected a Babesia spp. previously identified in North American raccoons (Procyon lotor) and a maned wolf (Chrysocyon brachyurus); prevalence was 22% (14/64) and 13% (9/68) in the mountain and coastal black bear populations, respectively. The presence of the same Babesia species in black bears, raccoons, and a maned wolf suggests piroplasms may not be host specific.

LEPTOSPIROSIS IN URBAN AND SUBURBAN AMERICAN BLACK BEARS ( URSUS AMERICANUS) IN WESTERN NORTH CAROLINA, USA.

American black bear ( Ursus americanus) populations in North Carolina, US have recovered significantly in recent decades and now occupy much of western North Carolina, including urban-suburban areas. We used the black bear as a potential sentinel for leptospirosis, a bacterial zoonotic disease caused by Leptospira spp., which is maintained by domestic and wild mammals. We determined whether Leptospira spp. were present across a gradient of housing densities in the urban and suburban black bear population in and around Asheville, North Carolina using serologic and molecular surveys. We collected blood from captured black bears ( n=94) and kidneys and bladders from carcasses ( n=19). We tested a total of 96 (47 females, 47 males, and 2 unknown) serum samples by microscopic agglutination test (MAT) and had positive results (titer >1:100) for L. kirschneri serovar Grippotyphosa (L. Grippotyphosa) in 4 females (8%) and 5 males (10%). No other serovars showed elevated titers in MAT. We tested a total of 125 samples using PCR ( n=96 serum, n=20 kidney, and n=9 bladders) and obtained positive results from one serum (1%), one kidney (5%), and one bladder (11%). The presence of Leptospira spp. in black bears occupying an urban and suburban landscape may indicate a more extensive occurrence of the bacteria among animals in the study region because black bears are the top carnivore in that ecosystem. Potential threats of widespread contamination during natural events such as flood or drought must be considered.

Prevalence and genetic characterization of Dirofilaria lutrae Orihle, 1965 in North American river otters (Lontra canadensis).

Dirofilaria lutrae Orihle, 1965 is a subcutaneous filarial worm found in North American river otters (Lontra canadensis). Previous studies reported the geographical range of this worm to include Louisiana, Florida, and North Carolina, USA. Although D. lutrae does not cause disease in otters, it must be distinguished from Dirofilaria immitis (Leidy, 1856), which causes heartworm disease in otters. The goal of this study was to determine the prevalence of D. lutrae in several states and to investigate the phylogenetic relationship of D. lutrae and other Dirofilaria species. Adult D. lutrae were collected from 32 of 40 (80%) otters from North Carolina (n = 38), South Carolina (n = 1), and Kentucky (n = 1), whereas all otters from Georgia (n = 1), Pennsylvania (n = 2), Arkansas (n = 184) and California (n = 1) were negative for D. lutrae. A portion of the cytochrome c oxidase I (COI) was amplified from nine North Carolina specimens and one South Carolina specimen. Sequence analysis indicated that all but one were identical to each other and D. lutrae was phylogenetically most closely related to D. immitis; however, few Dirofilaria sequences are available for comparison. Because other subcutaneous parasites may be detected in otters, parasites should be carefully examined to confirm their identity. Finally, given the high prevalence of D. lutrae in otters in some southeastern states, microfilaria found in blood samples of otters must be correctly identified because treatment for D. lutrae is generally not warranted and some pharmaceutical treatments can cause mortality in otters.

PARASITOLOGY AND SEROLOGY OF FREE-RANGING COYOTES (CANIS LATRANS) IN NORTH CAROLINA, USA.

Coyotes (Canis latrans) have expanded recently into the eastern US and can serve as a source of pathogens to domestic dogs (Canis lupus familiaris), livestock, and humans. We examined free-ranging coyotes from central North Carolina, US, for selected parasites and prevalence of antibodies against viral and bacterial agents. We detected ticks on most (81%) coyotes, with Amblyomma americanum detected on 83% of those with ticks. Fifteen (47%) coyotes were positive for heartworms (Dirofilaria immitis), with a greater detection rate in adults (75%) than juveniles (22%). Serology revealed antibodies against canine adenovirus (71%), canine coronavirus (32%), canine distemper virus (17%), canine parvovirus (96%), and Leptospira spp. (7%). We did not detect antibodies against Brucella abortus/suis or Brucella canis. Our results showed that coyotes harbor many common pathogens that present health risks to humans and domestic animals and suggest that continued monitoring of the coyote's role in pathogen transmission is warranted.

Nephrolithiasis in free-ranging North American river otter (Lontra canadensis) in North Carolina, USA.

The North American river otter (Lontra canadensis) serves as an indicator species for environmental monitoring, is prized as a valuable furbearer, and is a popular display animal in zoologic collections. Nephrolithiasis has been reported as a frequent problem in other free-ranging and captive otter species but is rarely reported in North American river otters. In this study, we compared the prevalence of nephrolithiasis diagnosed using routine gross pathologic examination techniques with the use of computed tomography (CT) of excised kidneys. We also evaluated whether otter nephroliths could be accurately classified by their CT densities, and we examined the renal tissue uric acid concentrations in free-ranging otters in North Carolina, USA. Kidneys were collected from carcasses of legally trapped, free-ranging animals. Nephroliths were observed in 16.2% of the individuals (n = 229). Associations were found between age and nephrolith status and between capture location and nephrolith status (P = 0.026 and < 0.001, respectively). Computed tomography Hounsfield unit density measurements were not useful in determining nephrolith chemical composition in this study. Renal tissue uric acid concentrations were similar across genders, age groups, and stone status. The chemical composition of the nephroliths was determined by scanning electron microscopy-energy dispersive X-ray spectroscopy to be calcium phosphate in the carbonate form.

Variation in the ITS-1 and ITS-2 rRNA genomic regions of Cytauxzoon felis from bobcats and pumas in the eastern United States and comparison with sequences from domestic cats.

Cytauxzoon felis, a tick-borne protozoan parasite, is the causative agent of cytauxzoonosis in domestic cats in the United States. The natural reservoir for this parasite is the bobcat (Lynx rufus), which typically does not develop clinical signs. Although not likely important reservoirs, C. felis has also been detected in pumas (Puma concolor) in Florida and Louisiana. Recent studies suggest that specific genotypes of C. felis that circulate in domestic cats may be associated with variable clinical outcomes and specific spatial locations. In the current study, we investigated the intraspecific variation of the C. felis internal transcribed spacer (ITS)-1 and ITS-2 rRNA regions from 145 wild felids (139 bobcats and six pumas) from 11 states (Florida, Georgia, Kansas, Kentucky, Louisiana, Missouri, North Carolina, North Dakota, South Carolina, Oklahoma, and Pennsylvania). Unambiguous ITS-1 and ITS-2 data were obtained for 144 and 112 samples, respectively, and both ITS-1 and ITS-2 sequences were obtained for 111 (77%) samples. For the ITS-1 region, sequences from 65 samples collected from wild felids were identical to those previously reported in domestic cats, while the other 79 sequences were unique. C. felis from 45 bobcats and one puma had ITS-1 sequences identical to the most common sequence reported from domestic cats. Within the ITS-2 region, sequences from 49 bobcats were identical to those previously reported in domestic cats and 63 sequences were unique (with some occurring in more than one bobcat). The most common ITS-2 sequence from domestic cats was also common in wild felids (31 bobcats and a puma). Samples from three pumas from Florida and two bobcats from Missouri had a 40- or 41-bp insert in the ITS-2 similar to one described previously in a domestic cat from Arkansas. Additionally, a previously undescribed 198- or 199-bp insert was detected in the ITS-2 sequence from four bobcats. Collectively, based on combined ITS-1 and ITS-2 sequences, five different genotypes were detected in the wild felids. Genotype ITSa was the most common genotype (11 bobcats and one puma) and fewer numbers of ITSb, ITSe, ITSg, and ITSi were detected in bobcats. These data indicate that, based on ITS-1 and ITS-2 sequences, numerous C. felis strains may circulate in wild felids.

Distribution and prevalence of Cytauxzoon felis in bobcats (Lynx rufus), the natural reservoir, and other wild felids in thirteen states.

Cytauxzoon felis, a protozoan parasite of wild and domestic felids, is the causative agent of cytauxzoonosis in domestic and some exotic felids in the United States. The bobcat (Lynx rufus) is the natural reservoir for this parasite, but other felids such as Florida panthers (Puma concolor coryii) and domestic cats may maintain long-term parasitemias and serve as reservoirs. Experimentally, two tick species, Dermacentor variabilis and Amblyomma americanum, have demonstrated the ability to transmit C. felis. These two tick species have overlapping distributions throughout much of the southeastern United States. The objective of the current study was to determine the distribution and prevalence of C. felis in free-ranging bobcat populations from 13 states including California, Colorado, Florida, Georgia, Kansas, Kentucky, Missouri, North Carolina, North Dakota, Ohio, Oklahoma, South Carolina, and West Virginia. These states were selected because of differential vector presence; D. variabilis is present in each of these states except for the region of Colorado sampled and A. americanum is currently known to be present only in a subset of these states. Blood or spleen samples from 696 bobcats were tested for C. felis infection by a polymerase chain reaction (PCR) assay which targeted the first ribosomal internal transcribed spacer region (ITS-1). Significantly higher prevalences of C. felis were detected from Missouri (79%, n=39), North Carolina (63%, n=8), Oklahoma (60%, n=20), South Carolina (57%, n=7), Kentucky (55%, n=74), Florida (44%, n=45), and Kansas (27%, n=41) compared with Georgia (9%, n=159), North Dakota (2.4%, n=124), Ohio (0%, n=19), West Virginia (0%, n=37), California (0%, n=26), and Colorado (0%, n=67). In addition to bobcats, seven cougars (Puma concolor) from Georgia, Louisiana, and North Dakota and one serval (Leptailurus serval) from Louisiana were tested for C. felis. Only one cougar from Louisiana was PCR positive, which represents the first report of an infected cougar outside of the Florida panther population. These data also indicate that C. felis is present in North Dakota where infection has not been reported in domestic cats. Based on a nonparametric analysis, prevalence rates were significantly higher in states where there are established populations of A. americanum, which supports recent data on the experimental transmission of C. felis by A. americanum and the fact that domestic cat clinical cases are temporally associated with A. americanum activity. Collectively, these data confirm that bobcats are a common reservoir for C. felis and that A. americanum is likely an epidemiologically important vector.