EFFECT OF PRESCRIBED FIRE ON THE VIABILITY OF BAYLISASCARIS PROCYONIS EGGS

Raccoons are distributed throughout North America, except in Alaska and northern Canada, and have been introduced into central and western Europe, Russia, and Japan (Gehrt 2003). The raccoon roundworm (Baylisascaris procyonis) is a large nematode that resides in the small intestine of raccoons (Procyon lotor) and is a zoonotic parasite that can infect humans, wildlife, and domestic animals (Kazacos 2001). The raccoon roundworm life cycle begins with raccoons shedding eggs via their feces into the environment. Infected raccoon feces can contain >100,000 B. procyonis eggs (Reed et al. 2012). Eggs develop and become infective, which either can be directly transmitted back to raccoons or transmitted to intermediate hosts such as rodents, lagomorphs, other mammals, and birds via ingestion (Kazacos and Boyce 1989). Larvae then migrate and encyst in intermediate hosts causing fatal or detrimental visceral larva migrans, ocular larva migrans, and neural larva migrans, thus making an easy meal for raccoons to complete the indirect infectious cycle, where larvae develop into adults in raccoon small intestine, breed, and produce eggs that are shed in raccoon feces (Kazacos and Boyce 1989).

A single infected raccoon scat upon decay can contaminate an approximately 1-m² area; therefore, a single B. procyonis–infected raccoon could contaminate 0.03±0.01 ha/yr (Ogdee et al. 2017). An infected population of raccoons can deposit millions of eggs in the environment, which can remain viable for years (Kazacos 2001). Ogdee et al. (2016b) found that >92% of B. procyonis eggs remained viable and percolated little within the soil column within 2 yr in the southern Texas environment regardless of soil texture, soil moisture, and sun exposure. Raccoons have a propensity to develop latrines in areas commensal with humans and isolated defecation sites elsewhere (Ogdee et al. 2017). Such behavior creates localized, but heavily contaminated, sites in commensal areas, whereas single defecation sites potentially contaminate a larger area. Thus, the risk of human infection with B. procyonis is potentially great.

Shafir et al. (2011), in a laboratory setting, found that the thermal lethal point is 62 C when water containing B. procyonis eggs is heated, and 57 C when heated water is added directly to eggs. However, Ogdee et al. (2016a) noted that ambient air and soil temperatures rarely, if ever, exceeded the lethal point of common raccoon latrine sites in southern Texas. They speculated that because southern Texas has one of the warmest climates in the US, then it would be unlikely that temperatures of other locations within the US would be hot enough to kill B. procyonis eggs.

Fire disturbances in fire-dependent ecosystems are considered a feasible method of parasite control at the landscape scale (Fuhlendorf and Engle 2004). For example, controlled burns reduced favorable habitat of several parasites, which in turn reduced the prevalence and abundance of gastrointestinal nematodes within sheep (Ovis aries) and goats (Capra aegagrus) in Australia (Hepworth and Hutchens 2011), reduced lungworm (Protostrongylus spp.) intensity in Stone's sheep (Ovis dalli stonei) in British Columbia, Canada (Seip and Bunnell 1985), and disrupted the gastropod hosts of the meningeal brain worm (Parelaphostrongylus tenuis) that infects white-tailed deer (Odocoileus virginianus) of the southeastern US (Weir 2009). Scasta (2015) believes that fire ecology and parasitology should be paired in research to better understand the role that fire can play as a potential regulator of zoonotic parasites.

We hypothesized that prescribed fire may be successful in killing B. procyonis eggs within a location. Grassland prescribed fires of varying fuel loads (1685–7865 kg/ha) ranged from 83 C to 682 C, respectively (Stinson and Wright 1969), which exceed the lethal point for B. procyonis eggs. Therefore, our objectives were to determine the effect of prescribed fire on the viability of B. procyonis eggs at typical raccoon defecation sites and the effect of fuel load, fire intensity, egg depth in soil column, and distance from fire edge on the viability of B. procyonis eggs.

The study was conducted on a 103 ha wildlife research area (27°28′31″N, 97°53′34″W) owned by Texas A&M University–Kingsville in Kleberg County of southern Texas during April–September 2019. The area has clay-loam and clay soils that are characterized with moderately slow draining lower soil layers with moderate shrink-swell potential. The habitat is characterized as a grassland consisting predominantly of Kleberg bluestem (Dichanthium annulatum) and buffel grass (Cenchrus ciliaris) with intermixed shrub-land consisting predominantly of honey mesquite (Prosopis glandulosa) and huisache (Acacia small-ii).

Two 0.5-km² areas of visually different fuel loads (i.e., high and low) were identified that had a 3-m-wide dirt road for a northern boundary. Due to typical prevailing winds for the region that blow from the south-southeast, a northern boundary road provided a natural fire break. Both 0.5-km² areas were divided into 50, 10×10 m plots, of which 10 plots were randomly selected. Small plots were considered adequate for study because Wright and Bailey (1982) found that fire temperatures were similar between fires that burned 16 ha and those that burned 0.001 ha. A 1-m dirt perimeter was established around each of the 10 randomly selected plots. Randomly selected plots were sprayed with Round-up Weed and Grass Killer® (active ingredients: 2% glyphosate as isopropylamine salt and 2% pelargonic acid; Monsanto Company, Marysville, Ohio, USA) as per the manufacturer's recommended dosage. The areas had similar vegetation composition but differed visually in biomass. In addition to the randomly selected plots, five plots within each 0.5 km² area had the vegetation clipped at the soil surface and placed in paper bags. Bags with vegetation were dried for 7 d in a drying chamber maintained at 60 C and <40% relative humidity, dried vegetation was then weighed on a Mettler top loader balance (Mettler-Toledo, LLC, Columbus, Ohio, USA), and the kg/ha vegetation dry weight for each plot was calculated. Average fuel load (kg/ha) and moisture content (%) for the high and low fuel load areas were 4,541±203 kg/ha and 9.5±1.2% and 2,041±86 kg/ha and 6.0±0.7%, respectively. The remaining randomly selected plots of each area were used as prescribed fire plots. Within each of five plots in the high fuel load area, we cleared a 1 m circle of vegetation and debris at the center of the plot that was approximately 8 m from the southern edge of the plot. The cleared circle was representative of a typical raccoon defecation site in southern Texas (Ogdee et al. 2017). We placed eight HOBO 4-channel analog temperature sensors (Model MX1105, Onset Computer Corporation, Bourne, Massachusetts, USA) within each cleared circle of each plot to be burned. Temperature sensors were placed on the soil surface and at 2 cm depth within the vegetation on the leading edge of the fire approximately 20 cm from the edge of the cleared circle, immediately at the leading edge of the circle, at the center of the cleared circle, and at the trailing edge of the fire of the cleared circle (Fig. 1a and Supplementary Materials Fig. 1a). Temperature probes were set to take a temperature reading every 1 s. We inoculated the soil approximately 3 cm to the right of each temperature probe with B. procyonis eggs according to the methods of Ogdee et al. (2016b). We concentrated the eggs of known B. procyonis–positive raccoon scats using a centrifugal sedimentation-floatation method (Kazacos 1983) to yield an average concentration of 1 egg/µL, which was verified by placing a 10 µL aliquot on a hemocytometer slide and quantifying eggs under 100× magnification and checking for larval motility. We pipetted two, 1 mL aliquots of B. procyonis eggs, the first aliquot on the soil surface 3 cm to the right of each temperature sensor and the second aliquot at a depth of 2 cm an additional 3 cm to the right of the first aliquot. Due to soil compaction, a nail was driven into the ground to a depth of 2 cm, the 1 mL aliquot of eggs was pipetted into the nail hole, and a soil plug was pressed into the hole to return soil back to its original state.

Plots were burned between 1100 and 1400 hours with ambient temperatures between 32 C and 36 C and relative humidity between 42% and 50% (Fig. 1b). To control for wind speed and direction, we used a 1.2 m hurricane fan connected to a portable gas generator to produce a constant wind speed of 16 kph and blowing toward the north-northwest. The southern edge of the 10×10 m plot was lit by a hand-held drip torch, and the fire was allowed to sweep across the plot to the northern boundary dirt road. The fire was allowed to extinguish itself by consuming the vegetation within each plot, and the plot was allowed to cool naturally for the subsequent 6 h (Fig. 1c, d).

Soil samples from each plot at the locations of the 2 cm depth aliquots and from the soil surface aliquots were collected with a 2-cm-diameter AMS soil probe (AMS Hammer Head Soil Probe, Forestry Suppliers, Jackson, Mississippi, USA). Soil samples were analyzed according to the methods of Ogdee et al. (2016b). Soil samples were treated with 20% bleach to remove the outer adhesive protein coat (Kazacos 1983), eggs were concentrated by centrifugation, and 200 eggs were quantified with a hemocytometer to determine the fraction of motile larvae in eggs, using motility as a proxy for viability (Shafir et al. 2011).

Times and temperatures were downloaded from HOBO temperature probes, and the data were used to calculate base temperature (C), time (s) required to reach maximum temperature, maximum temperature (C), temperature (C) rise, rate of temperature increase (C/s), time (s) temperature was above 63 C, time (s) temperature was above 49 C, temperature decrease (C), rate of temperature decrease (C/s), and the number of viable B. procyonis eggs from a sample of 200 eggs. Base temperature was defined as the ground surface temperature (C) immediately prior to the fire. Time required to reach maximum temp was the number of seconds required to reach the maximum temperature reading during the fire. Maximum temperature equaled the maximum temperature reading (C) obtained during fire. Temperature rise was defined as the difference in temperatures from the maximum temperature reading and the ground surface temperature immediately prior to the fire. Rate of increase was the rate at which the maximum temperature was reached (i.e., Maximum temperature/Time required to reach the maximum temp). Time above 63 C was the time in seconds that temperature remained above 63 C, which was the lethal temperature for 100% eggs. Time above 49 C was the time in seconds that temperature remained above 49 C, which was the LD50 temperature for B. procyonis eggs. Temperature decrease was defined as the temperature difference between the maximum temperature reading and 49 C. Rate of decrease was the rate at which maximum temperature cooled below 49 C (i.e., Maximum temperature/Time above 49 C).

We used five study plots as blocks. At each study plot, we recorded responses at four distances from the fire leading edge and at two soil burial depths for each distance. Thus, the experimental design was a randomized block design with five blocks and four distance treatments; sampling depth was a subplot factor in a split plot arrangement. Data were analyzed with a linear mixed model that included distance from fire leading edge, depth, and their interaction as fixed effects and the crossed interaction between block and distance as a random effect. The depth effect was modeled as a split plot factor (two depth data points were collected at each distance). We modeled several variance-covariance structures for the depth effect (first-order autoregressive, compound symmetry, Toeplitz and heterogeneous versions of these patterns, as well as variance-components, first-order autoregressive moving-average, and an unstructured pattern) and used information-theoretic comparisons (corrected Akaike information criterion) to select the most appropriate pattern.

In a second series of experiments, we established and randomly selected similar 10×10 m plots within the high and low fuel load areas. We placed HOBO temperature sensors on the soil surface at 0, 0.7, 1.2, and 1.8 m from the vegetation edge for each plot (Fig. 1b). In addition, aliquots of B. procyonis eggs were placed near each temperature sensor as previously described. Plots were burned, soil samples collected, and temperature sensors handled as described for the first experiment.

For the second experiment, we used five replications of two levels of fuel in a completely randomized design; each experimental unit also yielded data on all four distances from the fire front. Data were analyzed with a linear mixed model that included fuel load, distance from fire front, and their interaction as fixed effects and replication nested within fuel load as a random effect. The distance was modeled as a split plot factor. We modeled the same variance-covariance structures for the distance effect as for the first experiment and used corrected Akaike information criterion to select the most appropriate patterns. For both experiments, significant interactions were followed by tests of simple main effects and (when appropriate) simple effect tests (Kirk 2013). All analyses were completed with SAS version 9.4. (SAS Institute 2012).

Location of temperature sensors relative to fire and depth of their placement interacted (F_{3, 26.6}=1685, P<0.001) in their effects on heat intensity of prescribed fire at typical raccoon defecation sites (Table 1). Prescribed fire had no effect on maximum soil temperature (F_{3, 15.2}=0.19, P=0.911) at any placement within the 1 m² circle at 2 cm soil depth; maximum soil surface temperatures varied only between 29.4 C and 37.2 C (Fig. 2). Additionally, viability of B. procyonis eggs ranged from 99.8% to 100% at the 2-cm depth at the four locations. Although statistically a difference occurred (F_{3, 12.7}=13.39, P=0.001) in placement of sensors at the 2 cm soil depth for base temperature, the difference was only 2.5 C (Fig. 2 and Table 1). Maximum temperature (F_{3, 15.2}=1869.15, P<0.001), temperature rise (F_{3, 16.2}=671.41, P<0.001), and temperature decrease (F_{3, 12}=1347.46, P<0.001) were affected by placement of sensors on the soil surface (Fig. 2). These three temperature responses were each highest in grass and lowest in the plot center; additionally, they were higher at the trailing edge of the fire than at the leading edge of the fire. Base temperatures at the soil surface also differed (F_{3, 12.7}=258.03, P > 0.001) among placement locations, where, however, temperatures were highest in the plot center, lowest in grass, and not different between leading and trailing edges of the fire. Time above 49 C (F_{3, 12}=365.82, P<0.001) and 63 C (F_{3, 12}=126.96, P<0.001) differed among locations at the soil surface, with the longest times recorded in grass locations; although differences in times among the other locations were statistically significant, they were small in comparison with times recorded in grass (Fig. 3). Eggs of B. procyonis experienced 100% mortality on the soil surface within the 1 m² circle (Table 1).

Fuel load and distance to fire interacted in their effects on intensity of prescribed fire (F_{3, 29.5}=35.1, P<0.001), time to maximum temperature (F_{3, 24}=19.22, P<0.001), temperature rise (F_{3, 7.59}=231, P<0.001), and time above 49 C (F_{3, 29.4}=44, P<0.001) and above 63 C F_{3, 28}=134.93, P<0.001 (Table 2). In general, greater fuel load resulted in hotter temperatures that were sustained for a longer time up to 1.2 m from the fire's edge, after which, distance from fire was similar between fuel loads (Table 2 and Figs. 4, 5). Within high fuel load plots, the general pattern of each fire parameter was that fires were hottest and temperatures sustained for longer periods at the edge of the fire and were less intense at each subsequent distance from the fire (Figs. 4, 5). This pattern held true for all fire parameters except base temperature (Fig. 3a), and time temperature was above 63 C (Fig. 4b). Soil base temperatures increased with increasing distance, a response that was consistent at both fuel loads (Fig 4a). Fire temperatures did not exceed 63 C at or beyond 1.2 (Fig. 4b). Similar patterns were observed for the lower fuel load, except the patterns were less pronounced and differences at each subsequent distance were not always observed (Figs. 4, 5).

Prescribed fire did not kill 100% of B. procyonis eggs at any distance or fuel load (Fig. 5d). However, within the high fuel load area, the majority of B. procyonis eggs were killed up to 0.7 m from the fire's edge, whereas <20% of B. procyonis eggs were killed beyond 1.2 m from the fire (Fig. 5d). Areas of lower fuel load were less successful in killing B. procyonis eggs, where <50% of the eggs died at the fire's edge and nearly all eggs survived at subsequent distances from the fire (Fig. 5d).

Prescribed fire can be a useful management tool in reducing the quantity of B. procyonis eggs that contaminate an environment; however, it must be realized that fire will not eliminate all eggs from the environment and that the reduction of eggs from the environment may not necessarily be a long-term solution. Fuel loads need to be sufficient (e.g., >4,000 kg/ha) to produce the heat intensity to kill at least 98% of the B. procyonis eggs on the soil surface of an area that is free of sparse vegetation. Therefore, areas to be burned need to be rested from grazing to achieve such fuel loads (Wright and Bailey 1982). Because raccoons often use areas of sparse vegetation or bare spots to defecate (Ogdee et al. 2017), such areas may not burn during a prescribed fire and may need to be spot-treated and burned with a propane torch in order to kill B. procyonis eggs on the soil surface. Also, spot treatment may be needed in commensal areas where raccoons have a propensity to produced latrine sites (Ogdee et al. 2017) because prescribed fire is not feasible in areas near human habitation.

Prescribed fire will be successful in killing only B. procyonis eggs that occur on the soil surface. Soil has insulative properties that keeps heat from penetrating into the soil column (Wright and Bailey 1982), thus, protecting the eggs from desiccation. However, burrowing animals, such as pocket gophers (Geomys spp.) and grubbing wildlife species such as striped skunks (Mephitis mephitis) and feral hogs (Sus scrofa), can potentially bring B. procyonis eggs to the surface by their burrowing and foraging behaviors, thus, potentially increasing exposure to other species.

Infected raccoons through defecation can recontaminate an area; however, recontamination can be slowed through the use of prescribed fire. Jones et al. (2004) demonstrated that prescribed fire resulted in a 62% reduction in probability of use by raccoons. However, raccoons are known to use habitat features that offer the least path of resistance as travel corridors (Barding and Nelson 2008), which a recently burned area would present. Therefore, to reduce environmental contamination by B. procyonis eggs, prescribed fire in combination with raccoon depopulation may be needed. However, raccoon depopulation as a management plan for parasite reduction could backfire. Henke (2001) found that the raccoon population on a southern Texas ranch doubled after a removal program was implemented because younger raccoons that maintained half the home range size of their predecessors immigrated into the removal area. If a similar outcome would occur and the raccoons that immigrate into the removal area were infected with B. procyonis, then the potential rate of contamination would be exacerbated.

Our study involved controlled conditions that were replicated. Previous studies concerning the relationship between helminths and fire exist (Fuentes et al. 2007; Hossack et al. 2013; Torre et al. 2013); however, those studies assessed the effect of wildfires, which differed in fire propagation and heterogeneity of landscape features.

Our study supports the results of Ogdee et al. (2016a) that even though the semiarid climate of southern Texas is one of the warmest areas within North America (Fulbright and Bryant 2002), it is not hot enough to kill B. procyonis eggs. Soil temperatures prior to the initiation of prescribed fires were warmest in sun-exposed areas and were cooler in areas of vegetative shade but did not approach the required LD50 temperature suggested by Shafir et al. (2011). Prescribed fire can be a management tool to exceed the needed temperature to kill B. procyonis eggs, but it is not a remedy to this zoonotic parasite problem.

This is manuscript 20-115 of the Caesar Kleberg Wildlife Research Institute.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-20-00078.