DISTRIBUTION, PREVALENCE, AND GENETIC CHARACTERIZATION OF BAYLISASCARIS PROCYONIS IN SELECTED AREAS OF GEORGIA AND FLORIDA EMILY LAUREN BLIZZARD

DISTRIBUTION, PREVALENCE, AND GENETIC CHARACTERIZATION OF BAYLISASCARIS PROCYONIS IN SELECTED AREAS OF GEORGIA AND FLORIDA by EMILY LAUREN BLIZZARD (Under the Direction of Michael J. Yabsley) ABSTRACT Baylisascaris procyonis an intestinal nematode commonly found in raccoons (Procyon lotor) can cause fatal larval migrans in numerous species of mammals, birds, and humans. This study investigated the distribution and prevalence of B. procyonis in populations of raccoons in Georgia and Florida. Intestinal tracts of 312 raccoons from 25 Georgia counties and 52 raccoons from three Florida counties were examined for B. procyonis. B. procyonis was detected in Clarke County where 12 of 116 (10.3%) raccoons were infected and 11 B. procyonis worms were collected from northern Florida. Sequence analysis of the 18S and 5.8S rrna genes and the internal transcribed spacer (ITS)-1 and -2 regions confirmed Georgia samples were B. procyonis. These data indicate that the distribution of B. procyonis within Georgia is increasing and now present in Florida. Limited genetic variability was found in the rrna and ITS gene regions among B. procyonis from the southern United States. INDEX WORDS: Baylisascaris procyonis, zoonotic, Georgia, Florida, raccoons

DISTRIBUTION, PREVALENCE, AND GENETIC CHARACTERIZATION OF BAYLISASCARIS PROCYONIS IN SELECTED AREAS OF GEORGIA AND FLORIDA by EMILY LAUREN BLIZZARD B.S., Georgia Southern University, 2006 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2010

2010 Emily Lauren Blizzard All Rights Reserved

DISTRIBUTION, PREVALENCE, AND GENETIC CHARACTERIZATION OF BAYLISASCARIS PROCYONIS IN SELECTED AREAS OF GEORGIA AND FLORIDA by EMILY LAUREN BLIZZARD Major Professor: Committee: Michael J. Yabsley Steven B. Castleberry David E. Stallknecht Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2010

ACKNOWLEDGEMENTS I would like thank my major advisor, Dr. Michael Yabsley, for allowing me the opportunity to work on this project. I would not be completing my Master s degree today if he had not taken a chance and given me the opportunity to work with him. Additionally, this project would not have been possible without his valuable advice, guidance, and encouragement. I would like to thank my committee members, Dr. Steven B. Castleberry and Dr. David E. Stallknecht for their invaluable advice and input. I would also like to thank D. Kavanaugh and J. Smith (APHIS/USDA/WS), C. Groce (WKU), B. Hanson, K. Pederson, B. Wilcox, B. Adler, J. Carroll, G. Doster, S. Ellis-Felege, J. Gonynor, N. Jenkins, J. Slusher, J. Parris, W. Kistler, and B. Shock (UGA) for field assistance and/or permission to collect raccoons. To all the many research technicians, students, and diagnosticians at the S.C.W.D.S your assistance both in the field and in the laboratory was invaluable. Finally, I would like to thank my family, friends, and lab mates for their support, encouragement, patience, friendship, and love. To my lab mates you all are some of the most intelligent, dedicated, passionate, people I have ever met and I have cherished every moment I have been able to spend working with you all. Each of you has become very special to me and I am proud to be considered your friend. I thank you all for your constant encouragement and friendship. You have truly helped to shape the person I have become. Most importantly, I would like to thank my parents for their never wavering faith, encouragement, patience, support, and love. iv

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS... iv LIST OF TABLES... vii LIST OF FIGURES... viii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW...1 Introduction...1 Description and History...3 Distribution and Prevalence...3 Host Range and Life Cycle...5 Clinical Signs and Pathology...12 Potential as a Bioterrorism Agent...12 Diagnosis...13 Treatment...15 Methods of Decontamination...16 Conclusions...17 Literature Cited...18 2 DISTRIBUTION, PREVALENCE, AND GENETIC CHARACTERIZATION OF BAYLISASCARIS PROCYONIS IN SELECTED AREAS OF GEORGIA...36 Abstract...37 v

Introduction...38 Materials and Methods...39 Data Analysis...40 Genetic Characterization...41 Results...42 Discussion...43 Acknowledgements...47 Literature Cited...48 3 GEOGRAPHIC EXPANSION OF BAYLISASCARIS PROCYONIS, FLORIDA...59 Abstract...60 Introduction...60 The Study...61 Conclusions...62 Literature Cited...63 4 CONCLUSIONS...66 Literature Cited...68 vi

LIST OF TABLES Page Table 1.1: Previous studies conducted in various regions of the United States investigating the prevalence of Baylisascaris procyonis in raccoons (Procyon lotor)...28 Table 1.2: Review of human Baylisascaris procyonis infections from 1975 to 2008...29 Table 2.1: Prevalence of Baylisascaris procyonis in raccoons captured from 1997 to 2009 from 25 counties in Georgia...55 Table 2.2: Relationships between Baylisascaris procyonis prevalence and age, sex, season, and land-use in Clarke County, Georgia...56 Table 2.3: Nucleotide sequence variations within the internal transcribed spacer (ITS)-1 region of Baylisascaris procynois from Georgia (GA), Texas (TX), and Kentucky (KY)...57 Table 2.4: Nucleotide sequence variations within the internal transcribed spacer (ITS)-2 region of Baylisascaris procyonis from Georgia (GA), Texas (TX), and Kentucky (KY)...58 vii

LIST OF FIGURES Page Figure 1.1: General distribution and percent of raccoons infected with Baylisascaris procyonis in the United States...34 Figure 1.2: Life cycle of Baylisascaris procyonis...35 viii

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Introduction Baylisascaris procyonis, is an intestinal nematode commonly found in raccoons (Procyon lotor), in some areas of the United States. This parasite is significant because in non-raccoon hosts, larvae undergo extensive migrans through the body which can result in severe disease. These larvae can migrate through any tissue and can cause visceral larval migrans (VLM), ocular larval migrans (OLM), or the most severe form, neural larval migrans (NLM). In addition, this parasite is zoonotic (i.e., it can be transmitted from animals to people). If peridomestic wildlife, such as raccoons, harbor zoonotic diseases, they can pose an important health risk to humans because they potentially could contaminated habitats or items that are utilized by people. As more anthropogenic change occurs, interactions between animals and humans will continue to increase. Combined with increasing animal populations (i.e. raccoon populations taking advantage of readily available food sources such as garbage, pet food, bird feeders) and intentional human feeding, the risk for the transfer of zoonotic diseases could significantly increase. Although B. procyonis infections in humans are relatively rare, this parasite is an important zoonosis because the disease is often severe or fatal, is difficult to diagnosis, lacks an appropriate treatment, and typically occurs in young children or persons with learning disabilities (due to increased likely hood of ingesting fecal contaminated dirt or foreign objects). In addition, this parasite has been suggested as a potential bioterrorist agent because of the high 1

mortality rate, ready availability of infective stages, and the ease of which infective stages can be applied to food and/or water sources (Sorvillo et al., 2002). Landscapes that are altered by human development (e.g., urban and suburban areas) are frequently characterized by increased extinction rates of native species and an influx of more readily adaptable species (e.g., raccoons) (Czech et al., 2000; McKinney, 2002). These anthroprogenic developments can also affect abundance, behavior, and interactions of native wildlife populations (Dickman, 1987; Soule et al., 1988; Crooks and Soule, 1999; Cam et al., 2000). How these wildlife populations respond to these anthropogenic developments also may affect host parasite dynamics (Page et al., 2008). Raccoons are opportunistic omnivores ranging from Canada to South America and are known to exploit readily available food sources such as those found in close proximity to human populations (Lotze and Anderson, 1979). In many urban and suburban areas, raccoon population densities have increased substantially (Page et al., 2008). Raccoons are especially abundant in heterogeneous landscapes especially those closely associate with humans (Riley et al., 1998; Gehrt, 2003). In urban and suburban areas, they have adapted to the use of attics, porches, unused chimneys, and other secluded locations as dens, and regularly forage in garbage cans, landfills, pet food bowls, and bird feeders. For these reasons, raccoons are often characterized as nuisance animals that cause property damage and as potential carriers of zoonotic disease agents (de Almeida, 1987; DeStefano and DeGraag, 2003; Kazacos, 2000; Gompper and Wright, 2005). Raccoon densities in some of these urbanized landscapes can be >100 raccoons per km 2 (Riley et al., 1998; Prange et al., 2003). When you take into account that a single female worm from an infected raccoon can shed as many as 26,000 eggs per gram of feces, this parasite can rapidly contaminate an area. 2

Description and History The first report of a parasite that was later determined to be B. procyonis was from a New York Zoological Park in 1933 (McClure, 1933). In 1938, it was documented on a fur farm in Minnesota (Olsen and Fenstermacher, 1938). Originally believed to be Ascaris columnaris, it was later described as a new species (Ascaris procyonis) from raccoons in Europe (Sprent, 1968). Several common synonyms of the parasite are Ascaris procyonis (Stefanski and Zarnowski, 1951), Toxascaris procyonis (Stefanski and Zarnowski, 1951; Sprehn and Haakh, 1956), and Ascaris columnaris (Leidy, 1856). Sprent defined the genus Baylisascaris in 1968 to include several other ascarids previously categorized as Ascaris or Toxascaris. Baylisascaris species have cervical alae with cuticular bars that extend up to the cuticle and males have a perianal roughened patch (McIntosh, 1939; Sprent, 1952, 1970; Hartwick, 1962). These large nematodes may appear white, beige, or tan in color. Females may reach 12-20 cm and the males are around 9-11 cm (Hartwich, 1962; Sprent, 1968; Overstreet, 1970; Grey 1998). Distribution and Prevalence United States Baylisascaris procyonis is a common parasite of raccoons in several regions of the United States, Europe, and Asia, but is notably absent in raccoon populations in much of the southeastern United States. In the United States, the highest prevalence rates occur in the Midwestern, northeastern, and western states, and in Texas (Figure 1.1). In the Midwest region, the average prevalence was 58% (range 0-100%) among 33 studies which examined 3,967 raccoons (Table 1.1). Among seven studies conducted in the Northeast/mid-Atlantic region, 64% (5-100%) of 476 raccoons were infected (Table 1.1). In the west/south-west region, an average of 49% of 229 raccoons was infected (Table 1.1). In contrast, prevalences in 3

southeastern states are considerably lower with only an average of 4% of 1,868 raccoons being infected based on 28 studies (Table 1.1). The majority of the positives in the Southeast were from mountainous areas of the Appalachians; therefore, within region differences in prevalence can be significant when making comparison of state-or region-wide prevalences misleading. In enzootic regions, prevalence rates among juveniles can be as high as 90% while prevalence rates in adults tend to be lower (37-55%) (Kazacos, 2001). Outside of the United States Outside of the United States B.s procyonis has also been documented in several Canadian provinces, Poland, Czech and Slovak Republics, Japan, and Germany. In Canada, prevalence rates of B. procyonis documented in several studies have ranged from 2%-61%: British Columbia 61% prevalence (50/82), Nova Scotia 7% prevalence (67/946), Ontario 49% prevalence (41/84), Prince Edward Island 2 % prevalence (1/50), and Quebec 57% prevalence (12/21) (Kazacos, 2001). Prevalence studies have not been conducted in Poland, the Czech Republic, or the Slovak Republic. Although, papers have been published documenting the presence of B. procyonis in at least one free ranging raccoon in each country (Stefanski and Zarnowski, 1951; Tenora et al., 1991; Tenora and Stanek, 1990). One case study has been conducted in Japan by Miyashita in 1993. Miyashita examined 291 raccoon scats from twentyone zoos and documented a 40% prevalence rate in and around these areas (71/178), six animal from dealers had an 8% prevalence rate (3/37), in the pets he examined he found an 8% prevalence rate (3/39), and in wild raccoons no positives were found (0/37) and overall the prevalence rate of B. procyonis in the animals he examined was 27%. Two studies conducted in Germany found a 0% prevalence rate in the German state of Brandenburg and a 71% prevalence rate in Hessen (Lux and Priener, 1995; Gey, 1998). 4

In the early 1930s, raccoons were first released in Germany, primarily for fur farming and hunting (Muller-Using, 1959; Wise et al., 2005). Most introductions, either intentional or accidental, failed; however, a feral population of raccoons did establish and now thrives in the western and middle parts of the European continent (Stubbe, 1999) with an estimated 100,000 raccoons now in Germany. The largest population of wild raccoons inhabits the middle region of Germany in Brandenburgia near the border with Poland (Lutz, 1996; Hohmann, 1999; Wise et al., 2005). B. procyonis has been detected in up to 71% of free-ranging raccoons in Germany (Grey, 1998). The introduction of this parasite has lead to one documented non-fatal case of ocular disease in a German and numerous fatal animal cases (Wise et al., 2005). During the 1970s, a Japanese cartoon Rascal the Raccoon featured a cute and furry caricature of a raccoon. Due to the popularity of Rascal, an estimated 20,000 raccoons were imported to Japan as pets during the 1970s. Many of these pets escaped and have established feral populations in central Honshu and Hokkaido. Research has shown that many of these raccoons are infected with B. procyonis but luckily there have not been any documented human cases to date. However, there have been numerous cases of fatal larval migrans in animals. Control of the raccoon populations in these European and Asian countries has been problematic because the majority of people regard raccoons as welcome peridomestic pets and do not approve of the use of lethal methods to control populations which leads to spread and/or an increase in B. procyonis. Host Range and Life Cycle Definitive hosts The primary definitive host is the raccoon. The parasite can be acquired by naïve raccoons by either ingesting embryonated eggs from the environment or by ingestion of an 5

infected intermediate host (Figure 1.2). Once ingested, the egg hatches and the larvae develop to an adult worm in the small intestine. A single adult female worm, on average,e can shed 20,000 to 26,000 B. procyonis eggs per gram of feces per day (Kazacos, 1982; Snyder and Fitzgerald, 1987). Depending on environmental conditions, eggs can embryonate to the infective stage (L2) in 11 to 14 days (Sakla et al., 1989). The infective eggs can remain viable for years in the environment and for up to 18 years at -20 C (Kazacos and Boyce, 1989). Raccoons appear to have some age resistance and/or they develop immunity that results in clearance of the parasite (Kazacos, 2001). In nearly all surveys, juvenile raccoons have higher worm burdens and prevalence rates compared with adults. This is commonly attributed to their lack of immunity and increased susceptibility to infection from ingestion of embyronated eggs (vs. ingesting of infected intermediate hosts). Patent infections have been documented in kits as young as 3 months of age (Schultz, 1962). The main source of infection for adult raccoons is through ingestion of infected intermediate hosts. Additionally, adults appear to clear infections during the winter months (Kazacos, 2001). Prevalence rates in raccoons tend to peak in the fall (September October) and new infections are frequently documented during late spring and summer (Schultz, 1962; Smith et al., 1985; Kidder et al., 1989; Kazacos, 2001). Several factors can affect the transmission dynamics of this parasite including: land use, latrine densities, host densities, and raccoon behavior (Page et al., 2001). There seems to be a positive correlation between prevalence rates and areas with high intermediate host densities (Page et al., 2001). Interestingly, in some urban landscapes, there seems to be a decline in adult raccoon infections due to the lack of infected intermediate hosts in these areas combined with altered foraging habits. Raccoons in these areas tend to forage among more readily available 6

anthropogenic food sources rather than more common diets which include ingestion of multiple intermediate hosts (Page et al., 2001). While raccoons serve as the primary definitive host species, other wildlife species can serve as accidental definitive hosts including kinkajous (Poto flavus) and olingo (Bassaricyon gabbi)). Experimentally, Virginia opossums (Didelphis virginiana) were susceptible but no natural infection has been reported (Kazacos, 2001). Both ringtails (Bassaricus spp.) and coatimundis (Nasua spp.) are likely susceptible as well since they are close relatives of the raccoon. Domestic dogs can also serve as definitive hosts which is particularly problematic since they reside in domestic situations. There have been over two dozen cases of intestinal B. procyonis infections reported in dogs (Kazacos, 2001); however, numerous other cases have been discussed at meetings suggesting that infection is more common than documented in the literature (MJ Yabsley, personal communication). Often, dogs have mixed infections with B. procyonis and T. canis (Kazacos, 2001); therefore trained personnel are needed to distinguish eggs of these two species. In addition to patent infections, several dogs have developed fatal NLM (Snyder, 1983; Thomas, 1988; Rudmann et al., 1996; Kazacos, 2001). It is not known if dogs with patent infections also have migrating larvae in their tissues (Kazacos, 2001). Because of the severity of VLM in humans, it is important to emphasize the need for regular antihelminthic treatment of dogs. Due to indiscriminate defecation, infected dogs can quickly contaminate broad areas with thousands of eggs (Kazacos, 2001). Intermediate Hosts Over 90 vertebrate species have been identified and confirmed as susceptible intermediate hosts for B. procyonis (Kazacos, 2001). In many species of hosts, B. procyonis 7

infections results in high morbidity or mortality. In these hosts, the larvae aggressively migrate through the visceral and neural tissues. Typical intermediate hosts include various rodent species, dogs, guinea pigs, chickens, pheasant, swine, nonhuman primates, prairie dogs, and other mammalian, avian, and marsupial species (Wise et al., 2005). Once an egg is ingested by a intermediate host, the egg hatches in the small intestine and the larvae burrow through the intestinal wall and migrate to the liver and the lungs. Within 12-24 hours, pulmonary hemorrhages caused by larvae breaking out of capillaries in the lungs can be observed in these intermediate hosts (Sprent, 1952, 1953, 1955; Kazacos, 1986). After exiting the capillaries, the larvae migrate to the left heart ventrical via the pulmonary veins and eventually disseminate throughout the body. Numerous experimental infections conducted with primates and rodents have documented the predilection of B. procyonis larvae to migrate to the CNS. In the majority of these experiments, larvae migrate anteriorly up the spinal cord and into the brain. In experimental infections using white laboratory mice, neurologic abnormalities and ocular larval migrans occurred three days post-infection and clinical central nervous system disease was evident after 9-10 days (Tiner, 1953; Kazacos et al., 1985; Kazacos, 1986). Experimental infections in birds and mice documented mortality in some hosts after a single larvae migrated into the brain (Tiner, 1953; Sheppard and Kazacos, 1997). In naturally-infected rodents and avian species, as few as five larvae have been documented in the brain of fatal cases (Tiner, 1953; Armstrong et al., 1989; Van Andel et al., 1995). Intermediate hosts often become infected by foraging among raccoon latrine sites (Giles, 1939; Stains, 1956; Yeager and Rennels, 1943). These sites serve as communal defecation sites and serve as readily available food source for many grainivorous birds and mammals. For 8

example, Page (1998) documented 16 different species of mammals and 15 different species of birds foraging among raccoon latrine sites. These latrine sites also serve as a ready source of B. procyonis eggs because millions of eggs can accumulate at these locations over several years (Page et al., 2008). Due to their diet and foraging behavior, grainivorous hosts are most likely to acquire infection (Tiner,1952; Wirtz, 1982; Kazacos and Boyce, 1989; Sheppard and Kazacos, 1997; Page, 1998; Page et al., 1999). In addition, B. procyonis eggs are sticky and can adhere to fur and be ingested while grooming (Kazacos and Boyce, 1989; Yeitz et al., 2009). In addition to natural infections of free-ranging wildlife, there have been numerous fatal NLM infections among captive animals in rehabilitation centers and zoological parks. These infections presumable come from animals being housed in enclosures which previously contained infected raccoons (resulting in contamination of the cage with eggs, by ingestion of food that is contaminated with raccoon feces, or by raccoons visiting the enclosures and defecating in areas where the captive animals forage (Kidder et al., 1989; Stringfield and Sedgwick, 1997; Kazacos, 2001). Case Study on Wood Rat Extirpation Raccoons consume a wide variety of wild fruits and at times their feces can contain up to 35% or more of undigested seeds by volume (Giles, 1940). Many small mammals have been shown to frequently forage for these undigested seeds among raccoon latrines including: Allegheny woodrats (Neotoma magister), white-footed mice (Peromyscys leucopus), eastern chipmunks (Tamias striatus), and gray and fox squirrels (Sciurus carolinensis and S. niger) (Logiuduce, 2001). This foraging behavior among Allegheny woodrats is especially important as this behavior predisposes these animals to B. procyonis infections, most of which are fatal in this host. Over the last 20 years, Allegheny woodrat populations have continued to decline in the 9

northern parts of their range and infection with B. procyonis has been implicated as a potential cause for these dramatic declines (McGowan, 1993; Birchet al., 1994; Balcom and Yahner, 1996; LoGindice, 2000; Kazacos, 2001). Field and laboratory based studies of Allegheny woodrats and white-footed mice demonstrated species specific behaviors which explained differential infection risks for B. procyonis infections (Logiudice, 2001). The Allegheny woodrat s foraging behavior predisposed them to fatal B. procyonis rates more so than white-footed mice because they tended to forage through latrine sites after the feces had dried (average of 21 days which results in the eggs being embronated and infectious) while white-footed mice tended to forage through fresh feces (which would contain non-infective eggs) (Logiudice, 2001). Mice also tended to tear the fecal piles apart consuming seeds or removing them and caching them (Logiudice, 2001). Page (1998) documented white-footed mice caching fragments of feces but this behavior was rare compared to the fecal caching among the woodrats as whole raccoon scats are commonly found in woodrat food caches (Logiudice, 2001). The collection of dried feces which could contain embryonated eggs combined with the high prevalence of B. procyonis in northern populations of raccoons could be contributing to Allegheny woodrats declines (McGowan, 1993; LoGiudice, 2000). Humans Similar to many other animals, humans are susceptible to infection. Although there have been fewer than 30 documented human cases of Baylisascaris procyonis infections since the 1980 s (Table 1.2), many cases are likely not diagnosed. The majority of human cases have been diagnosed in young children, patients with learning disabilities, and/or patients with a history of pica or geophagia. 10

Depending on the number of larvated eggs ingested, the symptoms may range from asymptomatic to fatal with a broad range of CNS disease. When few eggs are ingested, or if few larvae migrate to the CNS, clinical signs will be minimal or absent. Many of these mild cases are likely never diagnosed. However, if large numbers of larvated eggs are ingested, larvae migrating through the CNS will cause mild to severe CNS disease. Similarly, larvae migrating through the eye can cause blindness. The lack of a commercially available serologic test for large-scale epidemiologic studies has limited our understanding on how common mild cases are within the human population (Wise et al., 2005). The first documented human case was a fatal eosinophilic meningoencephalitis in a 10- month old boy from Pennsylvania during 1980 (Huff et al., 1984.) The next case was diagnosed in 1984 and was a fatal NLM in an 18-month-old boy from Illinois (Fox et al., 1985). Details on other human cases are shown in Table 1.2. The two most recent cases were diagnosed in Brooklyn, New York in 2008. One Brooklyn case was an infant with a history of geophagia who presented with acute developmental regression, seizures, eosinophilic meningoencephalitis, postural deficits, and irritability. Antibodies to B. procyonis were detected and anthelmintic and steroid therapy was administered. After treatment, the patient did not improve and to date remains hospitalized with permanent brain damage due to NLM. The second most recent case from Brooklyn was a 17-year-old teenager with no travel history outside of New York City who developed ocular larval migrans in the right eye. The larva was successfully destroyed using laser photocoagulation and steroid therapy was administered. Despite treatment, the child remains blind in the right eye. 11

Clinical Signs and Pathology Definitive hosts rarely develop disease. The only two cases of disease in raccoons (both juveniles) were due to intestinal obstruction due to extremely high nematode burdens. These two raccoons had burdens of 636 and 1,321 nematodes. No clinical signs have been reported in domestic dogs with patent infections (Kazacos, 2001). Infected intermediate hosts often develop CNS signs that cause significant morbidity or mortality. The development of neurologic signs is adaptive for the parasite and results in increased transmission to raccoons that can more easily prey on neurologic intermediate hosts. Similar to humans, clinical signs in intermediate hosts varies considerably based on number of larvae ingested, type of tissues that are damaged by migrating larvae, and species of host. Depending on host species, the larvae exhibit differences in migration patterns, as larval migrate far more aggressively in some species of intermediate hosts vs. other species which results in more substantial tissue damage in CNS (Wise et al., 2005). Additionally, the longer larvae migrate in a host, they increase in size and it is more likely that these larvae will migrate though a vital organ (Wise et al., 2005). Smaller host species are also more likely to develop severe disease compared with large animals because fewer numbers of larvae can cause more significant damage. These combined factors typically result in severe debilitating CNS disease which often results in death (Wise et al., 2005). Potential as a Bioterrorism Agent In light of recent events involving terrorism and increased security precautions taken into affect after September 11, 2001, numerous agencies have evaluated a plethora of organisms for their bioterrorism risk. Baylisascaris procyonis has been suggested as a possible agent for bioterrorism (Sorville et al., 2002). Several factors make this organism a feasible bioterrorist 12

agent: 1) eggs can easily be obtained (either from the environment or from naturally- or experimentally-infected raccoons), 2) eggs are viable for years, 3) eggs would be relatively easy to add to food sources, 4) very few eggs would be necessary to cause clinical disease in humans, and 5) a lack of effective treatment. However, if embroynated eggs were added to the municipal water system, they would be easily filtered out; therefore, it would be more likely that eggs would have to be added to a food source. Although this scenario sounds highly unlikely, a similar situation has occurred before on a smaller scale. In 1972, four university students were hospitalized after ingesting a meal intentionally contaminated with large amounts of Ascaris suum eggs (Phills et al., 1972). Diagnosis Definitive hosts Recovery of characteristic eggs in fecal samples of raccoons or dogs can be accomplished by fecal floatation using standard parasite recovery sugar or salt solutions. These eggs must be differentiated from related species of ascarids such as Toxocara canis and Toxascaris leonina. Additionally, adult nematodes can be recovered from the small intestine of infected raccoons and dogs at necropsy. Intermediate hosts The majority of intermediate hosts die as a result of infection due to severe neurologic disease that leads to death or predation. Gross lesions are rarely observed. Depending on the number of migrating larvae, microscopic lesions may be rare to common. The ascarids cause severe inflammatory reactions (primarily eosinophils) and necrosis, but cross sections of larvae are rarely observed. If a cross section of the larvae is found it is diagnostic for an ascarid and can often be determined to be Baylisascaris based on size (~60 μm in diameter), but the species 13

of Baylisascaris species cannot be determined. Baylisascaris larvae have prominent lateral alae that may also be observed. To increase the chances of detecting a migrating Baylisascaris larvae, large numbers of histologic sections of CNS tissues should be examined. Additionally, rabies, and other diseases that can cause neurologic signs, must be ruled out as possible causes of morbidity or mortality. Humans Diagnosis of baylisascariasis in humans is difficult. Infections should be suspected in cases of acute neurologic signs and/or vision impairment that develop in people with possible exposure to raccoons feces in endemic areas. A complete history should be taken; however, parents often fail to recall instances of geophagia or pica and are unaware of all of the objects that their children may have put into their mouth. Complete blood workups should be conducted to look for eosinophilia (increased numbers of eosinophils). Additionally, many physicians rarely include baylisascariasis as a potential differential diagnosis. Other possible parasitic differential diagnoses for neurologic symptoms and eosinophilia include Toxocara canis, Toxocara cati, Angiostrongylus cantonensis, Gnathostoma spinigerum, and free-living amoeba (Wise et al., 2005). Of these agents, specific serologic testing is only available for T. canis. Two serologic tests (ELISA and IFA) for B. procyonis have been developed by Dr. Kazacos at Purdue University (Kazacos 2001). These assays utilize Baylisascaris-specific excretory/secretory antigens (Wise et al., 2005). However, the availability of these antigens is very limited, thus serologic testing is only carried out for highly suspect cases. Use of MRIs to diagnose Baylisascaris NLM is complicated because white matter changes in the brain are frequently not evident at the onset of CNS disease (Wise et al., 2005). In some cases of NLM, brain biopsies have been conducted to sample for migrating larvae (Wise 14

et al., 2005); however, when low numbers of migrating larvae are present, they would be frequently missed by this technique. Unfortunately, infections are most often diagnosed by postmortem by histologic examination of tissues. Ocular larval migrans are easier to diagnose as ophthalmologists can often see migrating larvae with the aid of microscopic equipment. Treatment Currently there is no highly effective treatment for larval migrans associated with B. procyonis in humans (Muarry, 2002). Treatment of NLM is complicated because the majority of antihelminths do not cross the blood- brain barrier; however, treatment with anthelmiths, even if they do not penetrate the blood-brain barrier, may be useful since it would result in a reduction in the number of larvae migrating through other organ systems. However, any damage that has occurred from larvae migrating into and through the brain prior to treatment may lead to permanent damage. Recently, one drug (albendazole) which will cross the blood-brain barrier, was successfully used to treat a single case (Pai et al., 2007). It is recommended that treatment with albendazole should be paired with corticosteroids to reduce inflammation caused by albendazole and the host s reaction to dead and dying parasites (Wise et al., 2005). Treatment of OLM involves using a laser to kill the larvae in the eye followed by antiinflammatory drugs and steroids to aid in the possible recovery of any remaining visual acuity. The majority of diagnosed patients that survived remained comatose, in a vegetative state, or had severe mental disabilities. However, recently a 4-year-old child from New Orleans, Louisiana, recovered after presenting with a headache, right arm pain and emeis (Pai et al., 2007). Because a helminth infection was suspected, albendazole (10 mg/kg every 12 hours for 5 days) and corticosteroids was administered. The patient began to show marked improvement after three days of treatment. Sixteen days post treatment the patient had mild dysmetria of his 15

right hand (Pai et al., 2007) but by one month after discharge, he was asymptomatic. The patient had no residual deficits after 24 months (Pai et al., 2007). The remarkable recovery by this patient may have been due to low numbers of migrating larvae or because an early diagnosis (this patient was regularly seen by doctors as he had a history of sickle cell anemia and splenectomy). Methods of Decontamination Infected raccoons can quickly contaminate an area with embryonated eggs. Importantly, peridomestic raccoons will often defecate in areas that may increase the risk of human exposure (e.g., porches, roofs, stone walls, wooden decks, open chimneys, etc). To decrease risk of infection, these areas should be cleaned immediately to avoid accidental ingestion of eggs by children or pets. Because the eggs are not immediately infectious and must develop in the environment for a period of time before becoming infective, frequent sanitation will limit the buildup of eggs on these surfaces. However, eggs will continue to accumulate in the surrounding environment and once the eggs embryonate, they can remain viable for several years. The eggs are also sticky and adhere to surfaces. Currently only two methods have shown successful in decontaminating areas infested with B. procyonis eggs. One method involves using highly concentrated caustic chemicals such as a 50/50 mixture of xylene and absolute alcohol, boiling lye, or boiling Lysol. An alternative method to using chemicals to decontaminate an area is to use burn the areas contaminated by raccoon feces. Although burning is the most effective way to kill eggs, it is not useful for flammable areas such as roofs, decks, etc. 16

Conclusions Although B. procyonis is relatively common in raccoons in some regions of the northeastern, midwestern, and mid-atlantic states and along coastal areas of Texas, California, Washington, and Oregon, it has been historically absent from much of the southeastern United States. The first report, outside of the Appalachian regions of Kentucky and Virginia, occurred in Dekalb County, GA in 2002 (Eberhard et al., 2003). During the same year a wildlife rehabilitator in Athens, Georgia reported finding B. procyonis in a single raccoon; however, the history of this raccoon was not available (county of origin is unknown) (Eberhard et al., 2003). In 2006, the parasite was detected in two raccoons from Athens, Georgia submitted to the Southeastern Cooperative Wildlife Disease Study at the University of Georgia. Because of the zoonotic and wildlife health implications of this parasite we iniated this study to determine the prevalence and distribution of B. procyonis in selected counties of Georgia and Florida. Acknowledgements I would like to thank D. Kavanaugh and J. Smith (APHIS/USDA/WS), C. Groce (WKU), B. Hanson, K. Pederson, B. Wilcox, B. Adler, J. Carroll, G. Doster, S. Ellis-Felege, J. Gonynor,, N. Jenkins, J. Slusher, J. Parris, W. Kistler, and B. Shock (UGA) for field assistance and/or permission to collect raccoons. I would also like to thank the many research technicians at the Southeastern Cooperative Wildlife Disease Study who were of great assistance both in the field and in the laboratory. Additionally, I would like to thank the Southeast Center for Emerging Biologic Threats and The Centers for Disease Control and Prevention. I would also like to thank the various diagnosticians at SCWDS who conducted necropsies on clinical cases. 17

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Table 1.1. Previous studies conducted in various regions of the United States investigating the prevalence of Baylisascaris procyonis in raccoons (Procyon lotor) (Kazacos, 2001). Midwest N Number Positive (%) Illinois 439 296 (67) Indiana 2,377 1,440 (60) Iowa 82 34 (41) Kansas 136 61 (45) Michigan 275 173 (63) Minnesota 213 109 (51) Nebraska 4 3/4 Ohio 29 8 (28) South Dakota 250 30 (12) Wisconsin 213 109 (51) Northeast/Middle N Number Positive (%) Atlantic Connecticut 1 1/1 Maryland 323 94 (29) New Jersey 158 52 (33) New York 758 369 (49) Pennsylvania 1 1/1 Washington, DC 44 19 (43) Southeast N Number Positive (%) Alabama 371 0 (0) Arkansas 30 0 (0) Florida 160 0 (0) Georgia 310* 12* (4) Kentucky 70 21 (30) North Carolina 219 0 (0) South Carolina 208 0 (0) Tennessee 253 20 (8) Virginia 95 24 (25) West/Southwest N Number Positive (%) California 109 77 (71) Nevada 16 2 (13) Oklahoma 1 1/1 Oregon 69 40 (58) Texas 112 22 (20) Washington 91 50 (55) * not including the animals in this study 28

Table 1.2. Review of human Baylisascaris procyonis infections from 1975 to 2008. Developmental Status Female Missouri unknown Normal Right side weakness, irritability Year Age Sex Location Risky Behaviors 1975 18 months 1980 10 months 1984 18 months Male Pennsylvania Pica Normal Loss of movement, eosinophilic meningoencephalitis Male Illinois Pica Down s Syndrome Lethargy, eosinophilia, encephalitis None Thiabendazole (50mg/kg/d for 5 d Symptoms Treatment Diagnostic testing Piperazine citrate Serologic (65mg/kg/d for 2 d) (crossreacting) Autopsy, serologic Autopsy, serologic Outcome Weakness, Spastic right arm and leg Reference 17,18,23,1, 20 Death 20,22,18, 13,11,23, 17 Death 10,8,17,20, 22,8,11,13, 23 1986 21 yr Male Oregon Pica/geophagia Down s Syndrome Developmental delays None Mentally disabled 10,23,18,7 1990 13 Male New York Pica Normal Refused to walk, Serologic 7,10,17,20, months eosinophilia 23,22,18 1991 48 yr Female 1992 29 yr Male 1993 9 months Male 1993 13 months Germany California Diffuse unilateral subacute neuroretinitis Diffuse unilateral subacute neuroretinitis Pica Unknown Eosinophilic encephalitis, Michigan seizures, blindness, hepatomegaly Male California Pica/geophagia Normal Lethargy, speech deterioration, stagger, eosinophillia 1995 10 yr Male Massachusetts unknown Mild Mental delays Abdominal pain, eosinophilia, unresponsiveness 1995 15 yr Male Brazil Diffuse unilateral subacute neuroetinitis 1996 2 yr Male Michigan Eosinophilic meningoencephalitis 1996 6 yr Male Illinois Pica/geophagia Developmental Developmental delay, Delays Diffuse unilateral subacute neuroretinitis 1996 13 months 1997 13 months Male Minnesota Unknown Normal Irritability, weakness, eosinophilia Male California Eosinophilic meningoencephalitis Thiabendazole (50 mg/kg/d for 7 d), ivermectin (175 ug/kg for 1 d), and prednisone (2 mg/kg/d for 7 d) Neurologic deficits, cortical blindness, brain atrophy Visual Ocular deficits 22,12 Visual Ocular deficits 22,4 Not recorded Serologic Neurologic deficits, cortical blindness Solumedrol and prednisolone Serologic Sezures, blindness, neurolofic deficits VLM? Fatal due to cardiac pseudotumor Argon laser treatment Visual Acute visual impairment left eye Severe neurologic deficits Albendazole and prednisone Methylprednisolone, vincristine, and thioguanine 10,17,20,16 10,19,17,20,22,18,11 20,22,4,7,2 3 6 22,14 Serologic Epilepsy, Visual impairments, Severe residual deficits 10,11 Serologic Death 10,15,17,11,15 None Severe neurologic deficits 22,23,18,19,11 29

Year Age Sex Location Risky Behaviors 1997 19 Male Minnesota Klinefelter months syndrome 1998 11 months Developmental Status Developmental delays Male California Pica/ stones Normal Irritability, behavioral regression, ocular symptoms 2000 17 yr Male California Developmental delays, geophagia, pica Developmental delays Symptoms Treatment Diagnostic testing Ataxia, unresponsiveness, Prednisone, vincristine, Serologic eosinophilia and thioguanine Drowsiness, eosinophilic meningoencephalitis 2000 2.5 yr Male Illinois Pica/geophagia Normal Enfephalopathy, fever, lethargy, eosinophilia Albendazole (40 mg/kg/d for 28 d) and methylprednisolone Albendazole and antiinflammatories Albendazole, and solumedrol Serologic Brain biopsy, serologic Serologic Outcome Severe residual mental deficits, visual impairment and epilepsy Seizures, Neurologic deficits, serious visual imparement Reference 10,18,17,20,1115,23,7 10,18,17,20,1123 Death 10,9,17,20, 22,23 Neurologic deficits, blindness, spasticity 10,9,11,17, 20,23 2002 11 months Male California Pica/geophagia Unkown Eosinophillic meningoencephalitis 2004 4 yr Male Louisiana Unknown Normal CSF, mild dysmetria, mild ataxia 2005 7 yr Male Toronto Autism, Autism, Mental Decreased food intake ADHD, mental retardation and level of retardation consciousness, neck stiffness 2008 Infant New York geophagia Normal Eosinophilic meningoencephalitis neural migrans Albendazole and antiinflammatories Serologic Residual neurologic deficits, cortical blindness, and epilepsy 10,16,2,17, 20,23 Corticosteroid therapy Serologic full recovery 17?? Stable with neurologic impairment Anthelminthic and steroid therapy 2008 17 yr. New York? Normal Ocular larva migrans Laser photoccoafulation and steroid therapy Serologic Ocular Migrans 20,5 Hospitalized, 6 neurologic deficits, seizures Blind in right eye 6 Table Literature Cited 1. Anderson, D. C., R. Greenwood, M. Fishman, I. G. Kagan. 1975. Acute infantile hemiplegia with cerebrospinal fluid eosinophilic pleocytosis: an unusual case of visceral larva migrans. Journal of Pediatrics 86: 247-9. 30

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9. From the Centers for Disease Control and Prevention, Raccoon roundworm encephalitis-chicago, Illinois, and Los Angeles, California. 2000. Journal of the American Medical Association 287: 580-1. 10. Gavin, P. J., K. R. Kazacos, and S. T. Shulman. 2005. Baylisascariasis. Clinical Microbiology Reviews 18:4: 703-18. 11. Gavin, P. J., K. R. Kazacos, T. Q. Tan, W. B. Brinkman, S. E. Byrd, A. T. Davis, M. B. Mets, S. T. Shulman. 2002. Neural larva migrans caused by the raccoon roundworm Baylisascaris procyonis. Journal of Pediatric Infectious Diseases 21: 971-5. 12. Goldberg, M. A., K. R. Kazacos, W. M. Boyce, E. Ali, B. Katz. 1993. Diffuse unilateral subacute neuroretinitis. Morphometic, serologic, and epidemiologic support for Baylisascaris as a causative agent. Opthalmology 100: 1695-701. 13. Huff, D. S., R. C. Neafie, M. J. Binder, G. A. De Leon, L. W. Brown, K. R. Kazacos.1984. The first fatal Baylisascaris infection in humans: an infant with eosinophillic meningoencephalitis. Pediatric Pathology 2: 345-52. 14. Kuchle, M., H. L. Knorr, S. Medenblik-Frysch, A. Weber, C. Bauer, G. O. Naumann. 1993. Diffuse unilateral subacute neuroretinitis syndrome in a German most likely caused by the raccoon roundworm Baylisascaris procyonis. Graefes Arch Clinical Experimental Ophthalmology 231: 48-51. 15. Moertel, C. L., K. R. Kazacos, J. H. Butterfield, H. Kita, J. Watterson, and G. J. Gleich. 2001. Eosinophil-associated inflammation and elaboration of eosinophil-derived proteins in 2 children with raccoon roundworm (Baylisascaris procyonis) encephalitis. Pediatrics 108: E93. 16. Murray, W. J. and K. R. Kazacos. 2004. Raccoon roundworm encephalitis. Clinical Infectious Diseases 39: 1484-92. 32

17. Pai, J. P., B. G. Blackburn, K. R. Kazacos, R. P. Warrier, and R. E. Begue. 2004. Full Recovery from Baylisascaris procyonis Eosinophilic Meningitis. Emerging Infectious Diseases 13:6: 928-30. 18. Park, S. Y., C. Glaser, W. J. Murray, K. R. Kazacos, H. A. Rowley, D. R. Fredrick, and N. Bass. 2000. Raccoon Roundworm (Baylisascaris procyonis) Encephalitis: Case Report and Field Investigation. Pediatrics 106: E56. 19. Rowley, H. A., R. M. Uht, K. R. Kazacos, J. Sakanari, W. V. Wheaton, A. J. Barkovich, and A. W. Bollen. 2000. Radiologic- Pathologic Findings in Raccoon Roundworm (Baylisascaris procyonis) Encephalitis. American Society og Neuroradiology 21:415-20. 20. Shafir, C. S., M. E. Wise, F. J. Sorvillo, and L. R. Ash. 2006. Central Nervous System and Eye Manifestations of Infection with Baylisascaris procyonis. Current Infectious Disease Reports 8:307-13. 21. Slavinski, S. and Fine, M. Baylisascaris USA: (NY). [ProMED-mail, April 19, 2009.] http://www.promedmail.org/pls/otn/f?p=2400:1202:7594744946315556::no::f2400_p1202_check_display,f2400_p12 02_PUB_MAIL_ID:X,76989. 22. Sorvillo, F., A. R. Lawrence, J. Yatabe, C. Degiorgio, and S. A. Morse. 2002. Baylisascaris procyonis: An Emerging Helminthic Zoonosis. Emerging Infectious Diseases 8:4: 355-9. 23. Wise, M. E., F. J. Sorvillo, S. C. Shafir, L. R. Ash, O. G. Berlin. 2005. Severe and fatal central nervous system disease in humans caused by Baylisascaris procyonis, the common roundworm of raccoons: a review of current literature. Microbes and Infection 7: 317-23. 33

Figure 1.1. General distribution and percent of raccoons infected with Baylisascaris procyonis in the United States. 34

Figure 1.2. Life cycle of Baylisascaris procyonis (Kazacos, 2001). 35