Assessing Potential Environmental Contamination by Baylisascaris procyonis Eggs from Infected Raccoons in Southern Texas

Abstract

Baylisascaris procyonis is a large ascarid of raccoons (Procyon lotor) and is a zoonotic threat. We documented the potential rate a raccoon population can contaminate their environment with B. procyonis eggs. We estimated the population size of raccoons using a 9 × 7 trapping grid of Havahart traps, identified locations of raccoon scats through systematic searches, and enumerated the distance B. procyonis eggs passively travel from site of origin upon scat decay. During an 8-week capture period, the raccoon population was estimated to be 19.6 ± 1.3 raccoons within the 63-ha study area (1 raccoon/3.2 ha). There were 781 defecation sites, of which 744 (95.3%) were isolated sites and 37 (4.7%) were latrine sites. Fifty-three (6.8%) defecation sites occurred in areas associated with human structures (commensal zone). Of the noncommensal sites, 9 (1.2%) and 719 (98.8%) sites were identified as latrine sites and isolated scats, respectively. More latrine sites were located within the commensal zone (p < 0.0001, \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\chi}_1^2 = 292.7$$ \end{document} ) than proportionately available space. Twenty-five raccoon scats containing B. procyonis eggs were allowed to decay on level bare soil by way of simulated rain events, 13 were allowed to desiccate naturally in the environment, and 12 were allowed to desiccate and, subsequently, experience a simulated 1 cm rain event; eggs were found 49 ± 6, 28 ± 8, and 68 ± 8 cm from the initial scat location, respectively. We calculated that a single B. procyonis-infected raccoon could contaminate 0.03 ± 0.01 ha/year with B. procyonis eggs. Our findings indicate that B. procyonis represents a substantial risk to humans in areas where infected raccoons and humans co-occur.

Introduction

B aylisascaris procyonis is a large ascarid of the raccoon (Procyon lotor) definitive host and is a zoonotic threat (Sorvillo et al. 2002). This roundworm has a widespread distribution and can be found in the United States, Canada, Germany, Poland, Czech and Slovak Republics, Japan, and Norway (Kazacos 2001, Davidson et al. 2013).

B. procyonis is transmitted through infected raccoon feces. Raccoons have a communal defecation habit (sites known as latrines; Stains 1956), which plays an important role in the transmission of B. procyonis to definitive and intermediate hosts, and leads to high levels of B. procyonis egg contamination at latrine sites (Page et al. 1999). Raccoons are infected directly by ingestion of embryonated eggs and indirectly when they ingest B. procyonis larvae encysted within intermediate hosts (Kazacos 2001). Intermediate hosts, such as small mammals and birds, accidentally ingest infective eggs while foraging in or around contaminated raccoon latrine sites and can succumb to neural larvae migrans, visceral larvae migrans, and ocular larvae migrans (Kazacos 2001). However, in raccoons, B. procyonis is rarely harmful.

Humans are considered accidental hosts of B. procyonis. In particular, individuals with developmental delays and children have a higher risk of accidentally ingesting infective B. procyonis eggs from contaminated environments (Gavin et al. 2005). At least 20 human cases of neural larvae migrans have been documented in the United States, Canada, Germany, and Brazil (Blizzard 2010) with most clinical cases resulting in severe neurological disease and death (Gavin et al. 2005, Hung et al. 2012). Human exposures may be higher than reported because individuals with low-level infections may not develop clinical manifestations (Kazacos 2001, Hung et al. 2012) and are not diagnosed.

Raccoons can reach high densities near urban and suburban areas where human refuse, pet food, and bird feeders readily provide food resources (Gehrt 2003, Page et al. 2009a, b, Graser et al. 2012). This increases the potential for widespread contamination of human-dominated areas with B. procyonis eggs, which increases the chances for human infection (Strausbaugh et al. 2004, Gavin et al. 2005). As human populations continue to grow, human-raccoon encounters are likely to increase. In addition, B. procyonis eggs are persistent within the environment and can remain viable for years (Kazacos 2001). Therefore, it is important to understand the magnitude of potential environmental contamination from an infected raccoon population. Given the extremely hardy characteristics of B. procyonis eggs (Kazacos and Boyce 1989, Ogdee 2015) and the potential ability of infected raccoons to heavily contaminate latrine sites, the objective of this study was to document the ability and extent to which an infected raccoon population may contaminate their environment.

Materials and Methods

Our study was conducted from April to August 2012 on the Marvin and Marie Bomer Wildlife Research Area (WRA) operated by the Department of Animal, Rangeland, and Wildlife Sciences at Texas A&M University-Kingsville. The Bomer WRA was selected because past research identified the area as having non-Baylisascaris-infected raccoons (Long et al. 2006). A noninfected area was important to our study so we could definitively document movement of B. procyonis eggs from the point of scat placement through wind and rain as the scat decayed.

The Bomer WRA is a 63-ha area (27°25′12″; 98°24′50″) with a three-strand, barbed wire perimeter fence located 19.3 km south of Benavides in Duval County, Texas. The site occurs in the relatively flat coastal region of southern Texas where the climate is subtropical (mean annual temperature 22.1°C) and semiarid (mean annual rainfall 66 cm) (National Oceanic and Atmospheric Administration 1994). The Bomer WRA occurs within the Runge-Delfina-Delmita soil associations, which are well and moderately well drained, loamy fine sands, fine sandy loams, silty loams, and clays with moderately slow draining lower soil layers with moderate shrink–swell potential. The habitat is characterized as grassland consisting predominantly of kleingrass (Panicum coloratum) and buffel grass (Cenchrus ciliaris) with intermixed shrubland predominantly of honey mesquite (Prosopis glandulosa) and huisache (Acacia smallii).

Havahart traps (Forestry Suppliers, Inc., Jackson, MS) were placed in a 7 × 9 grid throughout the Bomer WRA (spaced 100 meters apart; 1 trap/ha density) and baited with sardines and canned cat food. Captured raccoons were tranquilized with 5 mg/kg body weight of ketamine hydrochloride plus 1 mg/kg body weight of xylazine (Nielsen 1999), sexed, and age-classed by tooth wear as either adult or juvenile according to Grau et al. (1970). Stool samples were collected from captured raccoons using a cotton applicator swab, and collected feces were examined by the centrifugal fecal flotation technique (Dryden et al. 2005). Each raccoon was marked with an internal passive integrated transponder (AVID, Norco, CA) and externally with Rhodamine B powder (Sigma Chemical, St. Louis, MO) dissolved in water and sprayed on their fur at the base of their tail, and released at the site of capture when full consciousness returned (i.e., <30 min). Raccoon trapping was daily for 8 weeks with individual raccoon capture history recorded.

Raccoon population estimates were calculated using the Schnabel estimator (Krebs 1989). A regression plot of the proportion of marked raccoons against the number of raccoons previously marked was linear (R ² = 0.94; results not shown), which indicated that assumptions of a closed population and equal catchability were fulfilled. Raccoon density was calculated with program CAPTURE using the removal estimator option (Rexstad and Burnham 1991) as a verification of the Schnabel estimator.

Weekly systematic searches for raccoon scats were conducted for 8 weeks by 10 searchers walking 5 meters apart until the entire study area was searched. The original search collected and removed all encountered scats from the area. Thereafter, the number of raccoon scats per search was enumerated, and the location of each scat was recorded by a GPS unit (GeoExplorer III DGPS; Trimble, Sunnyvale, CA). Scats were untouched so as to not affect future raccoon behavior. Scat identification was determined by the characteristics outlined by Rezendes (1992). We defined a latrine site as where multiple raccoon scats were found (i.e., scats within a 2-m radius of previously located scats or piles of multiple scats). The total number of scats and latrine sites were quantified, and the number of scats was compared to the estimated population size of raccoons to estimate the number of scats produced by a raccoon in one day. In addition, scats located in buildings (i.e., house, barns, storage buildings, pavilions, and trailers) or within 40 meters of such structures were recorded as being commensal with humans, which was ∼2% of the study site. All other locations were considered noncommensal sites. A contingency table analysis (Neu et al. 1974) was conducted to determine if defecation location (i.e., inside or outside the commensal zone) occurred in proportion to the size of the commensal zone. We used ArcMap 10.1 (ESRI^® 2013) to map commensal zones, scat locations, and latrine sites.

Five raccoons, regardless of sex and age, were captured in Havahart traps after the scat searches on the same study area, maintained separately in 2 × 2 × 6-m cages equipped with boxes for hiding cover and shelter. Each of these raccoons was infected with B. procyonis as outlined by Reed et al. (2012). Briefly, B. procyonis eggs were extracted from adult female nematodes, allowed time to become larvated, treated with 5% bleach solution, and suspended in water at a mean concentration of 400 larvated eggs to 100 μL of solution, and the egg suspension was gavaged to mice (Mus musculus). Mice were maintained in individual cages until central nervous system signs were obvious and then euthanized, and two mice were fed to each raccoon. Raccoons were fed canned dog food (one 340-g can/day/raccoon) and provided water ad libitum. After a 60-day period, feces from each raccoon were checked daily by the flotation method for B. procyonis eggs and the number of eggs/scat quantified as per the methods of Reed et al. (2012).

Fifty fresh B. procyonis-infected scats from the five captive raccoons were collected (i.e., typically 1 scat/raccoon/day for 10 days) and placed in separate freezer bags in a −62°C freezer until all scats were obtained. Embryonated B. procyonis eggs will survive freezing conditions (Kazacos and Boyce 1989). Mean wet weight of raccoon scats was 61 ± 17 g (28 ± 3.4 g dry weight). All scats were placed 20-m apart on level bare soil to negate the influence of slope on movement of B. procyonis eggs from the point of origin at the Bomer WRA. Location of each scat was marked using GPS. Before scat placement, we collected two soil columns with a 2 cm diameter AMS soil probe (Forestry Suppliers, Inc.) and each sample was placed into labeled plastic bags. Soil samples and bags were washed using a centrifugal sedimentation-flotation method (Kazacos 1983), and sediment was examined at 100×magnification to verify the lack of B. procyonis eggs.

Oscillating sprinkler systems (Melnor, Inc., Winchester, VA) simulating 1 cm daily rain events were used to decay and dissolve the scats. Simulated rain events were monitored with an Onset data logging rain gauge (Forestry Suppliers, Inc.) to verify the daily amount of simulated rain. Twenty-five scats were exposed to simulated rain events and the remaining 25 raccoon scats were allowed to weather naturally. Weather parameters of temperature, humidity, rain, and wind speed were recorded daily with a HOBO Weather Station Logger (Forestry Suppliers, Inc.). Scats were monitored daily until they were completely dissolved or desiccated. Half of the desiccated scats (N = 12) then experienced a simulated 1 cm rain event as described above.

Soil samples were then collected with a 2 cm diameter soil probe from the origin of the scat every 2 cm in the eight cardinal directions from the origin. Soil samples were 6 cm in depth and placed in individual bags marked for scat number and distance and direction from scat origin. The magnitude of contamination was considered to be the furthest positive sample from the point of origin that was preceded by five negative soil samples. Soil samples were washed using a centrifugal sedimentation-flotation method outlined by Kazacos (1983). Sediment was then treated with a 20% bleach solution to remove the outer protein coat, making the eggs nonadherent (Kazacos 1983). Eggs from each soil sample were concentrated by centrifugation and quantified with a Beckman Coulter cell counter (Z Series, Indianapolis, IN).

At the completion of the study, B. procyonis eggs and B. procyonis-contaminated soils were burned with a propane torch, sifted, and reburned multiple times to kill B. procyonis eggs. Collection and use of raccoons and postdecontamination of field experimental sites were approved by the Texas A&M University-Kingsville Animal Care and Use Committee (#2010-03-10).

Results

During the 8-week capture period, 77 raccoons were captured, 56 of which constituted recaptures. Raccoon demographics were six and four for adult and juvenile males, respectively, and four and seven for adult and juvenile females, respectively. Raccoon sex and age structure did not deviate from a 1:1 relationship (p > 0.3, \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\chi}_1^2 = 1.1$$ \end{document} ). The raccoon population was estimated to be 19.57 ± 1.3 raccoons within the 63-ha area (1 raccoon/3.2 ha). Raccoon density, calculated by program CAPTURE, was estimated to be 0.3 ± 0.05 raccoons/ha.

We found 884 raccoon scats at 781 defecation sites (110.5 ± 17.5 scats/week, average number of scats by an individual raccoon 0.81 scats/day) by the end of the 8-week survey period (Fig. 1). Of these, 744 (84.2%) scats occurred singly and 140 (15.8%) occurred at latrine sites. Of the 781 defecation sites, 744 (95.3%) were isolated defecation sites and 37 sites (4.7%) were latrine sites. Of the 781 defecation sites, 53 occurred within the commensal zone, of which 25 and 28 sites were identified as latrine sites and isolated scats, respectively. Of the remaining 728 defecation sites that were located in the noncommensal zone, 9 and 719 sites were identified as latrine sites and isolated scats, respectively. The probability of a defecation site being a latrine site (rather than an isolated scat) was higher (p < 0.0001, \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\chi}_1^2 = 292.7$$ \end{document} ) when sites were in the commensal zone than when sites were in the noncommensal zone. In addition, while the commensal zone comprised only 2% of the acreage in the study area, 6.7% (95% CI: 5.3–8.3) of the 781 defecation sites were located in this zone, indicating a higher use of the commensal zone (p < 0.0001, \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\chi}_1^2 = 91.3$$ \end{document} ) than in proportion to its availability.

FIG. 1.

Location of individual raccoon (Procyon lotor) scats, latrine sites, and commensal sites on the Bomer Wildlife Research Area in Duval County, Texas, during April–August 2012.

The average number of B. procyonis eggs per gram of wet feces from our experimentally-infected captive raccoons was 17,338 ± 4213 ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${\bar {\rm {x}}} \pm {\rm {SE}}$$ \end{document} ). Mean temperature, relative humidity, and wind speed during the scat decay portion of the study were 29°C (range: 23–36°C), 83% relative humidity (RH) (range: 47–97% RH), and 10 kph (range: 0–35 kph), respectively. A mild mist occurred on July 3, 2012 but accumulated <2 mm during a 3-h period and was considered to have no effect on the simulated rain events designed within the study. Raccoon scat decay was enhanced more by rain rather than time of exposure or other associated weather parameters. Scats completely dissolved after receiving ∼20 cm of simulated rain. B. procyonis eggs were found in soil samples after simulated rain events 49 ± 6 cm from where the scat was placed and 28 ± 8 cm in soil samples where scats were decayed by the sun only; whereas B. procyonis eggs were found in soil samples 68 ± 8 cm from scats that first desiccated in the sun and then received a 1 cm rain event. The number of B. procyonis eggs in soil samples, regardless of method of decay or distance from scat origin, ranged from 4323 to 18,796 eggs per gram of soil. Using the data from this study (i.e., number of scats produced on average/day by a raccoon, % single defecation and % latrine sites, and distance that eggs can be found from scat site upon decay from various weather parameters), we calculated that a B. procyonis-infected raccoon, on average, potentially can contaminate 0.03 ± 0.01 ha/year with B. procyonis eggs.

Discussion

Raccoons infected with B. procyonis have the potential to shed millions of eggs per day [present study, Reed et al. (2012)]. In a companion study, Ogdee (2015) determined that a minimum of 30% of B. procyonis eggs remain on the soil surface after 2 years, of which >92% were viable (i.e., motile larvae in eggs). This constitutes ∼300,000 viable eggs/scat on the soil surface. Therefore, communal defecation sites can become heavily contaminated with B. procyonis eggs, thereby increasing the probability of B. procyonis transmission to potential hosts (Hirsch et al. 2014).

However, raccoons at our study site in southern Texas did not display the common communal defecation habits described in the literature (Giles 1939, Yeager and Rennels 1943, Stains 1956, Page et al. 1998, Hirsch et al. 2014). Two findings in our study are important with respect to better understanding defecation habits of raccoons and implications for potential spread of B. procyonis. First, while it is commonly thought that raccoons use latrine sites, our results suggest that this habit is not independent of potential raccoon–human interaction—in particular, whereas 53% of defecation sites were latrine sites in the commensal zone, only 1.2% of defecation sites were latrine sites in the noncommensal zone: apparently, raccoons modified their defecation behavior in the commensal zone. Second, raccoons preferentially used the commensal zone for defecation: while only 2% of our study area was classified as commensal, nearly 7% of the defecation sites were located in the commensal zone.

Habitats within commensal and noncommensal zones were similar with respect to plant species composition, diversity, structure, and water sources. In addition, raccoon demographics were consistent across the study area so it was unlikely that habitats were used disproportionately by subpopulations of raccoons. However, it is possible that grassland habitat within the noncommensal zone was used as travel corridors by raccoons, although home range and movement data would be required to verify this presumption. Several commensally located raccoon scats were located in, on, and around equipment such as tractors and farm implements. Users of such equipment often would brush or kick away the scats, thus increasing their potential exposure risk to B. procyonis compared to those who did not use the equipment.

A trade-off occurs between latrine sites and noncommunal defecation sites when considering the spread of B. procyonis eggs into the environment. Latrine sites concentrate B. procyonis eggs, creating a heavily contaminated, but a more localized area, whereas raccoons that defecate at single isolated locations can potentially contaminate a larger area. Experimentally infected raccoon scats in our study contained >1,000,000 B. procyonis eggs/scat; therefore, a single scat contains a sufficient number of eggs to transmit the parasite to a variety of potential intermediate hosts.

Because raccoons that were commensal with humans in our study selected to defecate more so at latrine locations, such sites can become highly contaminated with B. procyonis eggs and can be major foci for the infection and spread of B. procyonis as hypothesized by Hirsch et al. (2014). Evans (2002) documented an extreme case in which 100% of raccoon scats were positive for B. procyonis throughout a residential area of Orange County, California. Such a scenario highlights the public health implications when given a high-density raccoon population infected with B. procyonis and their potential to contaminate environments associated with humans. Our calculated estimate of a B. procyonis-infected raccoon contaminating 0.03 ha/year is conservative because it assumes that every square meter within the contaminated area becomes infested. However, such a scenario is unlikely because raccoons do not defecate in an evenly distributed and systematic approach. As Figure 1 highlights within our study, if our raccoon population was infected with B. procyonis, then a substantially more widespread area would become contaminated, thus increasing the likelihood of a potential intermediate host coming into contact with infective B. procyonis eggs.

Conclusions

It is essential for wildlife managers, city employees, and public health officials to recognize the potential level of contamination in their area from an infected raccoon population, particularly in areas of high human density. Decontamination efforts should take into account the potential of B. procyonis eggs to spread and the degree to which raccoons use latrines, which serve to concentrate B. procyonis eggs, and the apparent adoption of more common latrine-defecation habits in commensal areas. Our calculated estimate of a single raccoon to contaminate 0.03 ha/year with B. procyonis eggs should be considered conservative and was based on simulating light to moderate rain events in a subtropical and semiarid environment. Heavy rain and wind events, particularly in areas of varying topography, will likely facilitate transport of B. procyonis eggs further from scat deposition sites (single or latrines), thereby increasing the area of contamination by B. procyonis eggs within the environment.

Footnotes

Acknowledgments

The authors thank the Harry L. Willet Foundation for their generous support of this research. The authors also thank the Caesar Kleberg Wildlife Research Institute and Texas A&M University-Kingsville for providing the facilities and wildlife research areas. This is contribution 16-124 of the Caesar Kleberg Wildlife Research Institute.

Author Disclosure Statement

No competing financial interests exist.

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