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Larva migrans in squirrel monkeys experimentally infected with Baylisascaris potosis
Parasitology International 64 (2015) 284–287
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Larva migrans in squirrel monkeys experimentally infected with Baylisascaris potosis Toshihiro Tokiwa a, Kosuke Tsugo a, Shohei Nakamura b, Kensuke Taira b, Yumi Une a,⁎ a b
Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan Laboratory of Veterinary Parasitology, School of Veterinary Medicine, Azabu University, 1-17-71, Chuo-ku, Sagamihara, Kanagawa 2525-5201, Japan
a r t i c l e
i n f o
Article history: Received 27 December 2014 Received in revised form 8 February 2015 Accepted 10 March 2015 Available online 18 March 2015 Keywords: Baylisascaris potosis Kinkajou roundworm Squirrel monkey Larva migrans Zoonosis Pathogenicity
a b s t r a c t Roundworms of the genus Baylisascaris are natural parasites primarily of wild carnivores, and they can occasionally cause infection in humans and animals. Infection results in visceral larva migrans and/or neural larva migrans, which can be severe or fatal in some animals. Recently, Baylisascaris nematodes isolated from kinkajous (Potos flavus) and previously referred to as Baylisascaris procyonis were renamed as Baylisascaris potosis; however, data regarding the pathogenicity of B. potosis towards animals and humans are lacking. In the present study, we experimentally infected squirrel monkeys (Saimiri sciureus) with B. potosis to determine the suitability of the monkey as a primate model. We used embryonated eggs of B. potosis at two different doses (10,000 eggs and 100,000 eggs) and examined the animals at 30 days post-infection. Histopathological examination showed the presence of B. potosis larvae and infiltration of inflammatory cells around a central B. potosis larvae in the brain, intestines, and liver. Nevertheless, the monkeys showed no clinical signs associated with infection. Parasitological examination revealed the presence of B. potosis larvae in the intestines, liver, lung, muscles, brain, kidney, and diaphragm. Our findings extend the range of species that are susceptible to B. potosis and provide evidence for the zoonotic potential of larva migrans in high dose infections. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Members of the genus Baylisascaris are zoonotic ascarid nematodes that are mainly found in carnivores, with one in rodents. Within this genus, Baylisascaris procyonis, which is also known as the raccoon roundworm, is recognized as a causative agent of severe or fatal larva migrans in a range of mammals, birds, and humans [1–3]. In addition, Baylisascaris melis of European badger (Meles meles), Baylisascaris columnaris of skunks (Mephitis spp., Spilogale spp.), Baylisascaris transfuga of bears (Ursus spp., Thalarctos maritimus), and Baylisascaris schroederi of giant pandas (Ailuropoda melanoleuca) can cause extensive damage in their paratenic hosts, where they sometimes invade the visceral organs, central nervous system, or eyes [4–7]. Baylisascaris potosis was recently discovered in captive kinkajous (Potos flavus) from the co-operative Republic of Guyana [8]. We previously used morphological and molecular analysis to demonstrate that B. potosis was closely related to B. procyonis. Experimental infection of Mongolian gerbils revealed that B. potosis caused visceral larva migrans, but less frequently migrated to the nervous system than did B. procyonis [9]. However, data regarding the susceptibility of other animals to infection with B. potosis are lacking. A considerable number of kinkajous are imported to the USA from South America and are bred in captivity. ⁎ Corresponding author. Tel.: +81 42 769 1628. E-mail address: [email protected] (Y. Une).
http://dx.doi.org/10.1016/j.parint.2015.03.003 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.
These kinkajous are sold as exotic pets [10], implying the potential for accidental infection of domestic animals, animals kept in zoos, and humans. In the present study, we investigated the ability of B. potosis to cause larva migrans in squirrel monkeys (Saimiri sciureus) and evaluated the potential importance of this nematode as a causative agent of disease in primates, including humans. 2. Materials and methods Unembryonated B. potosis eggs were collected from captive kinkajous, incubated for 1 month at 25 °C to produce embryonated eggs, and stored at 10 °C until use. Before inoculation, the eggs were washed twice with tap water to remove formalin. We estimated the number of embryonated eggs for infection by determining the number of eggs containing motile larvae per unit volume. We used adult male squirrel monkeys (n = 2) in our experiments. The monkeys were bred and kept at animal facilities in a roundwormfree environment in Japan. Each animal was housed in a cage in a controlled environment and was provided with a diet of commercial food pellets supplemented with fruit. The monkeys were inoculated with 10,000 or 100,000 embryonated B. potosis eggs by stomach tube under inhalational anesthesia. At 30 days post-infection (PI), one monkey had died and the other was euthanized by using exsanguination under inhalational anesthesia. The brain, heart, spleen, lungs, intestines, liver, kidney, eye, and femoral muscles were individually removed from
T. Tokiwa et al. / Parasitology International 64 (2015) 284–287
each animal. The right side of the brain, right kidney, right eye, right sides of the limb muscles, right lobes of the liver and lungs, right side of the spleen, the diaphragm, and the small and large intestines were weighed and examined for the presence of larvae. These tissue samples were cut with a pair of scissors, ground in a commercial kitchen blender, and digested in a solution of 1% HCl (37%) and 1% pepsin in 40 °C tap water according to Taira et al. (2013) [11]. The remaining tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, cut into 3-μm thick sections, and stained with hematoxylin and eosin. All our experiments adhered to the Guidelines for the Use of Experimental Animals authorized by the Japanese Association for Laboratory Animal Science. The protocol was approved by the Ethics Committee of Animal Experiments at Azabu University (Permit Number 130613-1). 3. Results The monkey inoculated with 10,000 eggs displayed no abnormal behavior and showed no apparent clinical signs during the experiment. The monkey inoculated with 100,000 eggs showed no apparent clinical signs during the experiment, but was found dead in the morning at 30 days PI. At necropsy, we found no gross lesions in the monkey inoculated with 10,000 eggs. However, the monkey inoculated with 100,000 eggs had a slightly enlarged and congested liver, pulmonary edema, and hundreds to thousands of small white nodules on the intestines—especially
285
on the colon (Fig. 1A) and cecum (Fig. 1B). Histopathological examination of both monkeys revealed that numerous lesions were localized in the muscle layers and subserosal tissues of the intestines (Fig. 1C). In the monkey inoculated with 100,000 eggs, we observed similar lesions in the outer layers of the cerebral cortex (Fig. 1D) and liver (Fig. 1E). These lesions were characterized by foamy and often pigmented macrophages and by a few admixed eosinophils and lymphocytes around a central B. potosis larvae. All the larvae observed in these tissues appeared non-degenerate (Fig. 1D, E, Fig. 2). The larvae had an average midbody diameter of 65 μm, platymyarian musculature, prominent single cuticular lateral alae, and round or triangular excretory columns on both sides of the centrally located intestine (Fig. 2). A few large migration tracks with macrophages and lymphocytes were localized throughout the liver (Fig. 1F). We observed no significant abnormalities in deep parts of the brain and the other organs examined. The larval burdens and tissue weights are shown in Table 1. Larvae were recovered from the intestines, liver, lung, brain, kidney, diaphragm, and muscles. The highest larval burden was found in the large intestine. No larvae were recovered from the eye or spleen. The average length of larvae recovered from tissues was 1.5–1.7 mm. 4. Discussion Kazacos et al. (1981) reported that monkeys experimentally infected with 5,000 or 10,500 B. procyonis eggs were less vocal and showed
Fig. 1. Lesions in the squirrel monkey inoculated with 100,000 B. potosis eggs. (A) Colon and (B) cecum showing numerous white nodules. Bar = 2 mm. (C) Larval granuloma in the musculature of the cecum. Hematoxylin and eosin (HE) staining. Bar = 200 μm. (D) Larval granuloma on the surface of the cerebrum. HE staining. Bar = 100 μm. (E) Larval granuloma in the liver. HE staining. Bar = 50 μm. (F) Migration tracks in the liver. HE staining. Bar = 100 μm.
286
T. Tokiwa et al. / Parasitology International 64 (2015) 284–287
Fig. 2. Cross-section through the midbody of a B. potosis larva in the squirrel monkey liver. Note the prominent and single lateral alae (LC), lateral excretory columns (EC), and intestine with an open lumen and a microvillus border. Bar = 20 μm.
reduced activity and impaired manual dexterity, difficulty in grasping and handling food, loss of balance, intention tremors of the head and forelimbs, head pressing, narcolepsy, and unsolicited vocalization; in addition, by 12–19 days PI, all monkeys were semicomatose and recumbent on the cage floor [12]. Numerous lesions caused by B. procyonis larvae, were visible grossly and microscopically throughout the body, particularly in the brain, head muscles, liver, lungs, and heart, and to a lesser extent in the small intestine and colon. In the present study, neither of the monkeys inoculated with B. potosis eggs showed any neurological signs by 30 days PI, implying lower pathogenicity of B. potosis than B. procyonis. The death of the monkey inoculated with 100,000 eggs was attributed to pulmonary edema; however, no precise cause of death was determined. The distinct pathogenicity of different species of Baylisascaris can be partly explained by variations in somatic migration and invasion of the brain, differing larval activities in the central nervous system, and variations in the growth rate and size of larvae in the host tissues [1,6]. Baylisascaris procyonis larvae are much more pathogenic than B. potosis larvae; hence, the ingestion of fewer infective eggs and the presence of fewer larvae in the brain are associated with severe clinical neurological signs [1]. In the present study, we found B. potosis larvae with associated lesions localized in several organs, but mainly in the intestines and liver. Moreover, all the B. potosis larvae found in the tissues were encapsulated. Baylisascaris potosis larvae in the outer layers of the cerebral cortex doesn't seem to be deep invasion, inflammation there related to migration, and continuing migration of larvae. Our findings suggest that the migration pattern and activity in host primate tissues differ Table 1 Larval burdens of B. potosis in the tissues and organs of squirrel monkeys infected with embryonated B. potosis eggs. Tissue/organ
Cerebrum, brain stem Cerebellum Eye Lung Liver Kidney Muscle of anterior limb Muscle of posterior limb Spleen Diaphragm Small intestine Large intestine a
ND, Not determined.
10,000 egg dose
100,000 egg dose
No. of larvae
Tissue weight (g)
No. of larvae
Tissue weight (g)
0 0 0 0 9 1 0 0 NDa 1 NDa NDa
11.2 1.5 2.3 2.2 5.5 2.4 22.1 42.7 NDa 1.1 NDa NDa
8 0 0 17 9 2 1 21 0 26 38 1347
11.1 1.3 1.9 3.9 5.5 2.0 12.6 16.8 0.1 0.7 4.9 1.2
considerably between B. potosis and B. procyonis larvae and B. potosis larvae primarily having a visceral migration with localization to the gastrointestinal tract. Differentiation of the adult and eggs between B. potosis and other species, including B. procyonis, is difficult because some of these species share morphological characters. We previously observed that the position of phasmidial pores of the adult male was the only differential character between B. procyonis and B. potosis [8]. It is almost impossible to differentiate between the larvae of B. procyonis in tissues of squirrel monkey [12] and spider monkey [13], the larvae of Baylisascaris sp. in rhesus macaque tissues [14] and cynomolgus macaque tissues [15], and the larvae of B. potosis in the squirrel monkey tissues observed in the present study. Molecular genetic techniques using the mitochondrial cytochrome oxidase I and the nuclear internal transcribed spacer 2 and 28S rRNA genes have been proven useful in distinguishing B. potosis from other member of the genus Baylisascaris [8]. We propose these procedures are essential for identification of Baylisascaris larvae in tissues and useful in cases where exposure to particular carnivores is not established. 5. Conclusions In the present study, we have shown that the kinkajou roundworm, B. potosis, mainly causes visceral larva migrans in non-human primates. The pathogenicity of B. potosis towards squirrel monkeys was much lower than that of B. procyonis and most larvae undertook visceral migration with localization along the gastrointestinal tract. Our findings extend the range of species that are susceptible to B. potosis and provide evidence for the zoonotic potential of larva migrans in high infections. Conflict of interest statement None of the authors of this work have financial or personal relationship with other people or organizations that would inappropriate influence or bias the content of this paper. Acknowledgments This work was supported by a Grant-in-Aid for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare of Japan (H24-Shinko-Ippan-006). References [1] Kazacos KR. Baylisascaris procyonis and related species. In: Samuel WM, Pybus MJ, Kocan AA, editors. Parasitic diseases of wild mammals. 2nd ed. Iowa State University Press; 2001. p. 301–41. [2] Murray WJ, Kazacos KR. Raccoon roundworm encephalitis. Clin Infect Dis 2004;39: 1087–92. [3] Wise ME, Sorvillo FJ, Shafir SC, Ash LR, Berlin OG. Severe and fatal central nervous system disease in humans caused by Baylisascaris procyonis, the common roundworm of raccoons: a review of current literature. Microbes Infect 2005;7:317–23. [4] Sprent JFA. On the migratory behaviour of larvae of various Ascaris species in white mice. II. Longevity of encapsulated larvae and their resistance to freezing and putrefaction. J Infect Dis 1953;92:114–7. [5] Tiner JD. Fatalities in rodents caused by larval Ascaris in the central nervous system. J Mammal 1953;34:153–67. [6] Sato H, Matsuo H, Osanai H, Kamiya H, Akao N, Owaki S, et al. Larva migrans by Baylisascaris transfuga: fatal neurological diseases in Mongolian jirds, but not in mice. J Parasitol 2004;90:774–81. [7] Bauer C. Baylisascariosis—infections of animals and humans with ‘unusual’ roundworms. Vet Parasitol 2013;193:404–12. [8] Tokiwa T, Nakamura S, Taira K, Une Y. Baylisascaris potosis n. sp., a new ascarid nematode isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana. Parasitol Int 2014;63:591–6. [9] Tokiwa T, Taira K, Une Y. Experimental infection of Mongolian gerbils with Baylisascaris potosis. J Parasitol 2015;101:114–5. [10] Kazacos KR, Kilbane TP, Zimmerman KD, Chavez-Lindell T, Parman B, Carpenter LR, et al. Raccoon roundworms in pet kinkajous—three states, 1999 and 2010. MMWR Morb Mortal Wkly Rep 2011;60:302–5. [11] Taira K, Yanagida T, Akazawa N, Saitoh Y. High infectivity of Toxocara cati larvae from muscles of experimentally infected rats. Vet Parasitol 2013;196:397–400.
T. Tokiwa et al. / Parasitology International 64 (2015) 284–287 [12] Kazacos KR, Wirtz WL, Burger PP, Christmas CS. Raccoon ascarid larvae as a cause of fatal central nervous system disease in subhuman primates. J Am Vet Med Assoc 1981;179:1089–94. [13] Garlick DS, Marcus LC, Pokras M, Schelling SH. Baylisascaris larva migrans in a spider monkey (Ateles sp.). J Med Primatol 1996;25:133–6.
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[14] Gozalo AS, Maximova OA, StClaire MC, Montali RJ, Ward JM, Cheng LI, et al. Visceral and neural larva migrans in rhesus macaques. J Am Assoc Lab Anim Sci 2008;47: 64–7. [15] Shoieb A, Radi ZA. Cerebral Baylisascaris larva migrans in a cynomolgus macaque (Macaca fascicularis). Exp Toxicol Pathol 2014;66:263–5.
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Larva migrans in squirrel monkeys experimentally infected with Baylisascaris potosis Toshihiro Tokiwa a, Kosuke Tsugo a, Shohei Nakamura b, Kensuke Taira b, Yumi Une a,⁎ a b
Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan Laboratory of Veterinary Parasitology, School of Veterinary Medicine, Azabu University, 1-17-71, Chuo-ku, Sagamihara, Kanagawa 2525-5201, Japan
a r t i c l e
i n f o
Article history: Received 27 December 2014 Received in revised form 8 February 2015 Accepted 10 March 2015 Available online 18 March 2015 Keywords: Baylisascaris potosis Kinkajou roundworm Squirrel monkey Larva migrans Zoonosis Pathogenicity
a b s t r a c t Roundworms of the genus Baylisascaris are natural parasites primarily of wild carnivores, and they can occasionally cause infection in humans and animals. Infection results in visceral larva migrans and/or neural larva migrans, which can be severe or fatal in some animals. Recently, Baylisascaris nematodes isolated from kinkajous (Potos flavus) and previously referred to as Baylisascaris procyonis were renamed as Baylisascaris potosis; however, data regarding the pathogenicity of B. potosis towards animals and humans are lacking. In the present study, we experimentally infected squirrel monkeys (Saimiri sciureus) with B. potosis to determine the suitability of the monkey as a primate model. We used embryonated eggs of B. potosis at two different doses (10,000 eggs and 100,000 eggs) and examined the animals at 30 days post-infection. Histopathological examination showed the presence of B. potosis larvae and infiltration of inflammatory cells around a central B. potosis larvae in the brain, intestines, and liver. Nevertheless, the monkeys showed no clinical signs associated with infection. Parasitological examination revealed the presence of B. potosis larvae in the intestines, liver, lung, muscles, brain, kidney, and diaphragm. Our findings extend the range of species that are susceptible to B. potosis and provide evidence for the zoonotic potential of larva migrans in high dose infections. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Members of the genus Baylisascaris are zoonotic ascarid nematodes that are mainly found in carnivores, with one in rodents. Within this genus, Baylisascaris procyonis, which is also known as the raccoon roundworm, is recognized as a causative agent of severe or fatal larva migrans in a range of mammals, birds, and humans [1–3]. In addition, Baylisascaris melis of European badger (Meles meles), Baylisascaris columnaris of skunks (Mephitis spp., Spilogale spp.), Baylisascaris transfuga of bears (Ursus spp., Thalarctos maritimus), and Baylisascaris schroederi of giant pandas (Ailuropoda melanoleuca) can cause extensive damage in their paratenic hosts, where they sometimes invade the visceral organs, central nervous system, or eyes [4–7]. Baylisascaris potosis was recently discovered in captive kinkajous (Potos flavus) from the co-operative Republic of Guyana [8]. We previously used morphological and molecular analysis to demonstrate that B. potosis was closely related to B. procyonis. Experimental infection of Mongolian gerbils revealed that B. potosis caused visceral larva migrans, but less frequently migrated to the nervous system than did B. procyonis [9]. However, data regarding the susceptibility of other animals to infection with B. potosis are lacking. A considerable number of kinkajous are imported to the USA from South America and are bred in captivity. ⁎ Corresponding author. Tel.: +81 42 769 1628. E-mail address: [email protected] (Y. Une).
http://dx.doi.org/10.1016/j.parint.2015.03.003 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.
These kinkajous are sold as exotic pets [10], implying the potential for accidental infection of domestic animals, animals kept in zoos, and humans. In the present study, we investigated the ability of B. potosis to cause larva migrans in squirrel monkeys (Saimiri sciureus) and evaluated the potential importance of this nematode as a causative agent of disease in primates, including humans. 2. Materials and methods Unembryonated B. potosis eggs were collected from captive kinkajous, incubated for 1 month at 25 °C to produce embryonated eggs, and stored at 10 °C until use. Before inoculation, the eggs were washed twice with tap water to remove formalin. We estimated the number of embryonated eggs for infection by determining the number of eggs containing motile larvae per unit volume. We used adult male squirrel monkeys (n = 2) in our experiments. The monkeys were bred and kept at animal facilities in a roundwormfree environment in Japan. Each animal was housed in a cage in a controlled environment and was provided with a diet of commercial food pellets supplemented with fruit. The monkeys were inoculated with 10,000 or 100,000 embryonated B. potosis eggs by stomach tube under inhalational anesthesia. At 30 days post-infection (PI), one monkey had died and the other was euthanized by using exsanguination under inhalational anesthesia. The brain, heart, spleen, lungs, intestines, liver, kidney, eye, and femoral muscles were individually removed from
T. Tokiwa et al. / Parasitology International 64 (2015) 284–287
each animal. The right side of the brain, right kidney, right eye, right sides of the limb muscles, right lobes of the liver and lungs, right side of the spleen, the diaphragm, and the small and large intestines were weighed and examined for the presence of larvae. These tissue samples were cut with a pair of scissors, ground in a commercial kitchen blender, and digested in a solution of 1% HCl (37%) and 1% pepsin in 40 °C tap water according to Taira et al. (2013) [11]. The remaining tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, cut into 3-μm thick sections, and stained with hematoxylin and eosin. All our experiments adhered to the Guidelines for the Use of Experimental Animals authorized by the Japanese Association for Laboratory Animal Science. The protocol was approved by the Ethics Committee of Animal Experiments at Azabu University (Permit Number 130613-1). 3. Results The monkey inoculated with 10,000 eggs displayed no abnormal behavior and showed no apparent clinical signs during the experiment. The monkey inoculated with 100,000 eggs showed no apparent clinical signs during the experiment, but was found dead in the morning at 30 days PI. At necropsy, we found no gross lesions in the monkey inoculated with 10,000 eggs. However, the monkey inoculated with 100,000 eggs had a slightly enlarged and congested liver, pulmonary edema, and hundreds to thousands of small white nodules on the intestines—especially
285
on the colon (Fig. 1A) and cecum (Fig. 1B). Histopathological examination of both monkeys revealed that numerous lesions were localized in the muscle layers and subserosal tissues of the intestines (Fig. 1C). In the monkey inoculated with 100,000 eggs, we observed similar lesions in the outer layers of the cerebral cortex (Fig. 1D) and liver (Fig. 1E). These lesions were characterized by foamy and often pigmented macrophages and by a few admixed eosinophils and lymphocytes around a central B. potosis larvae. All the larvae observed in these tissues appeared non-degenerate (Fig. 1D, E, Fig. 2). The larvae had an average midbody diameter of 65 μm, platymyarian musculature, prominent single cuticular lateral alae, and round or triangular excretory columns on both sides of the centrally located intestine (Fig. 2). A few large migration tracks with macrophages and lymphocytes were localized throughout the liver (Fig. 1F). We observed no significant abnormalities in deep parts of the brain and the other organs examined. The larval burdens and tissue weights are shown in Table 1. Larvae were recovered from the intestines, liver, lung, brain, kidney, diaphragm, and muscles. The highest larval burden was found in the large intestine. No larvae were recovered from the eye or spleen. The average length of larvae recovered from tissues was 1.5–1.7 mm. 4. Discussion Kazacos et al. (1981) reported that monkeys experimentally infected with 5,000 or 10,500 B. procyonis eggs were less vocal and showed
Fig. 1. Lesions in the squirrel monkey inoculated with 100,000 B. potosis eggs. (A) Colon and (B) cecum showing numerous white nodules. Bar = 2 mm. (C) Larval granuloma in the musculature of the cecum. Hematoxylin and eosin (HE) staining. Bar = 200 μm. (D) Larval granuloma on the surface of the cerebrum. HE staining. Bar = 100 μm. (E) Larval granuloma in the liver. HE staining. Bar = 50 μm. (F) Migration tracks in the liver. HE staining. Bar = 100 μm.
286
T. Tokiwa et al. / Parasitology International 64 (2015) 284–287
Fig. 2. Cross-section through the midbody of a B. potosis larva in the squirrel monkey liver. Note the prominent and single lateral alae (LC), lateral excretory columns (EC), and intestine with an open lumen and a microvillus border. Bar = 20 μm.
reduced activity and impaired manual dexterity, difficulty in grasping and handling food, loss of balance, intention tremors of the head and forelimbs, head pressing, narcolepsy, and unsolicited vocalization; in addition, by 12–19 days PI, all monkeys were semicomatose and recumbent on the cage floor [12]. Numerous lesions caused by B. procyonis larvae, were visible grossly and microscopically throughout the body, particularly in the brain, head muscles, liver, lungs, and heart, and to a lesser extent in the small intestine and colon. In the present study, neither of the monkeys inoculated with B. potosis eggs showed any neurological signs by 30 days PI, implying lower pathogenicity of B. potosis than B. procyonis. The death of the monkey inoculated with 100,000 eggs was attributed to pulmonary edema; however, no precise cause of death was determined. The distinct pathogenicity of different species of Baylisascaris can be partly explained by variations in somatic migration and invasion of the brain, differing larval activities in the central nervous system, and variations in the growth rate and size of larvae in the host tissues [1,6]. Baylisascaris procyonis larvae are much more pathogenic than B. potosis larvae; hence, the ingestion of fewer infective eggs and the presence of fewer larvae in the brain are associated with severe clinical neurological signs [1]. In the present study, we found B. potosis larvae with associated lesions localized in several organs, but mainly in the intestines and liver. Moreover, all the B. potosis larvae found in the tissues were encapsulated. Baylisascaris potosis larvae in the outer layers of the cerebral cortex doesn't seem to be deep invasion, inflammation there related to migration, and continuing migration of larvae. Our findings suggest that the migration pattern and activity in host primate tissues differ Table 1 Larval burdens of B. potosis in the tissues and organs of squirrel monkeys infected with embryonated B. potosis eggs. Tissue/organ
Cerebrum, brain stem Cerebellum Eye Lung Liver Kidney Muscle of anterior limb Muscle of posterior limb Spleen Diaphragm Small intestine Large intestine a
ND, Not determined.
10,000 egg dose
100,000 egg dose
No. of larvae
Tissue weight (g)
No. of larvae
Tissue weight (g)
0 0 0 0 9 1 0 0 NDa 1 NDa NDa
11.2 1.5 2.3 2.2 5.5 2.4 22.1 42.7 NDa 1.1 NDa NDa
8 0 0 17 9 2 1 21 0 26 38 1347
11.1 1.3 1.9 3.9 5.5 2.0 12.6 16.8 0.1 0.7 4.9 1.2
considerably between B. potosis and B. procyonis larvae and B. potosis larvae primarily having a visceral migration with localization to the gastrointestinal tract. Differentiation of the adult and eggs between B. potosis and other species, including B. procyonis, is difficult because some of these species share morphological characters. We previously observed that the position of phasmidial pores of the adult male was the only differential character between B. procyonis and B. potosis [8]. It is almost impossible to differentiate between the larvae of B. procyonis in tissues of squirrel monkey [12] and spider monkey [13], the larvae of Baylisascaris sp. in rhesus macaque tissues [14] and cynomolgus macaque tissues [15], and the larvae of B. potosis in the squirrel monkey tissues observed in the present study. Molecular genetic techniques using the mitochondrial cytochrome oxidase I and the nuclear internal transcribed spacer 2 and 28S rRNA genes have been proven useful in distinguishing B. potosis from other member of the genus Baylisascaris [8]. We propose these procedures are essential for identification of Baylisascaris larvae in tissues and useful in cases where exposure to particular carnivores is not established. 5. Conclusions In the present study, we have shown that the kinkajou roundworm, B. potosis, mainly causes visceral larva migrans in non-human primates. The pathogenicity of B. potosis towards squirrel monkeys was much lower than that of B. procyonis and most larvae undertook visceral migration with localization along the gastrointestinal tract. Our findings extend the range of species that are susceptible to B. potosis and provide evidence for the zoonotic potential of larva migrans in high infections. Conflict of interest statement None of the authors of this work have financial or personal relationship with other people or organizations that would inappropriate influence or bias the content of this paper. Acknowledgments This work was supported by a Grant-in-Aid for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare of Japan (H24-Shinko-Ippan-006). References [1] Kazacos KR. Baylisascaris procyonis and related species. In: Samuel WM, Pybus MJ, Kocan AA, editors. Parasitic diseases of wild mammals. 2nd ed. Iowa State University Press; 2001. p. 301–41. [2] Murray WJ, Kazacos KR. Raccoon roundworm encephalitis. Clin Infect Dis 2004;39: 1087–92. [3] Wise ME, Sorvillo FJ, Shafir SC, Ash LR, Berlin OG. Severe and fatal central nervous system disease in humans caused by Baylisascaris procyonis, the common roundworm of raccoons: a review of current literature. Microbes Infect 2005;7:317–23. [4] Sprent JFA. On the migratory behaviour of larvae of various Ascaris species in white mice. II. Longevity of encapsulated larvae and their resistance to freezing and putrefaction. J Infect Dis 1953;92:114–7. [5] Tiner JD. Fatalities in rodents caused by larval Ascaris in the central nervous system. J Mammal 1953;34:153–67. [6] Sato H, Matsuo H, Osanai H, Kamiya H, Akao N, Owaki S, et al. Larva migrans by Baylisascaris transfuga: fatal neurological diseases in Mongolian jirds, but not in mice. J Parasitol 2004;90:774–81. [7] Bauer C. Baylisascariosis—infections of animals and humans with ‘unusual’ roundworms. Vet Parasitol 2013;193:404–12. [8] Tokiwa T, Nakamura S, Taira K, Une Y. Baylisascaris potosis n. sp., a new ascarid nematode isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana. Parasitol Int 2014;63:591–6. [9] Tokiwa T, Taira K, Une Y. Experimental infection of Mongolian gerbils with Baylisascaris potosis. J Parasitol 2015;101:114–5. [10] Kazacos KR, Kilbane TP, Zimmerman KD, Chavez-Lindell T, Parman B, Carpenter LR, et al. Raccoon roundworms in pet kinkajous—three states, 1999 and 2010. MMWR Morb Mortal Wkly Rep 2011;60:302–5. [11] Taira K, Yanagida T, Akazawa N, Saitoh Y. High infectivity of Toxocara cati larvae from muscles of experimentally infected rats. Vet Parasitol 2013;196:397–400.
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