TOXOCARA MALAYSIENSIS N. SP. (NEMATODA: ASCARIDOIDEA) FROM THE DOMESTIC CAT (FELIS CATUS LINNAEUS, 1758)

J. Parasitol., 87(3), 2001, p 660 665 American Society of Parasitologists 2001 TOXOCARA MALAYSIENSIS N. SP. (NEMATODA: ASCARIDOIDEA) FROM THE DOMESTIC CAT (FELIS CATUS LINNAEUS, 1758) Lynda M. Gibbons, Dennis E. Jacobs*, and Rehana A. Sani Department of Pathology and Infectious Diseases, The Royal Veterinary College (University of London), North Mymms, Hatfield AL9 7TA, United Kingdom ABSTRACT: Toxocara malaysiensis n. sp. from the small intestine of the domestic cat (Felis catus L.) in Malaysia is described and illustrated. This ascaridoid nematode was previously assumed to be Toxocara canis, which it superficially resembles, or designated Toxocara sp. cf. canis. The new species differs from T. canis in the shape of the cervical alae in cross section, spicule length, and the lip structure. It is also distinct from other species assigned to Toxocara. Rhode (1962) and Lee et al. (1993) reported the occurrence of an ascaridoid nematode that they identified as Toxocara canis in Malaysian domestic cats. Examination of an immature male specimen (L. Gibbons, D. Jacobs, and R. Sani, unpubl. obs.) revealed inconsistencies with published descriptions of T. canis, and a collection of ascaridoids from cats in Kuala Lumpur, Malaysia was therefore initiated. Molecular studies on the resultant material indicated that this parasite represented a separate species from T. canis and Toxocara cati (Zhu et al., 1998). The nematodes were assigned to Toxocara Stiles, 1905 and designated Toxocara sp. cf. canis. The present paper reports a detailed morphological examination of the material from Kuala Lumpur and additional specimens from around Ipoh, Perak State, Malaysia. It reveals that the specimens can be distinguished from the other species of the genus (Table I), and a new species Toxocara malaysiensis is established for them. MATERIALS AND METHODS The study is based on 8 males and 7 females, fixed in 70% alcohol or 10% formalin, plus the heads and tails of 4 males and 7 females, fixed in 70% alcohol, from material used in molecular studies (Zhu et al., 1998). En face views and cross sections were cut by hand using a small blade fixed in a needle holder. These were mounted in glycerine jelly. Two males were dissected and mounted in Berlese s fluid to study the spicules. Two females were dissected to study the reproductive system. The dissected females and the remainder of the specimens were cleared in lactic acid or lactoglycerol for examination as temporary mounts. DESCRIPTION Toxocara malaysiensis n. sp. (Figs. 1 17) Description Cuticle distinctly transversally striated. Three well-defined lips each with median cleft. Dentigerous ridge continuous into median cleft of lip. Interlabia absent. Inner circle of papillae present as 2 small sensory organs near outer margin of each lip. Dorsal lip with 2 large papillae of the outer circle, each subventral lip with 1 large, 1 small papillae of the outer circle. Amphid adjacent to small papilla on each subventral lip. Pulp with 2 projecting arms separated by deep cleft (Fig. 1). Cervical alae arise immediately behind lips, just below dorsal margin of subventral lips, gradually increasing in width to approximately mid-length, gradually taper posteriorly, curve ventrally. Sclerotized support of outer margin of alae extends for one-third of width, Received 12 June 2000; revised 20 November 2000; accepted 20 November 2000. * Department of Pathology and Infectious Diseases, The Royal Veterinary College (University of London), North Mymms, Hatfield AL9 7TA, United Kingdom. Faculty of Veterinary Medicine and Animal Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. narrow, pointed at outer margin, divided at base. Cervical alae extend for up to 56% of esophageal length (Figs. 2 4, 8). Male Body length 53 84 mm. Head 0.248 0.288 mm wide. Cervical alae 1.7 2.42 mm long; maximum width 0.18 0.19 mm. Excretory pore opens 0.74 0.8 mm from anterior end. Nerve ring not observed. Esophagus 3.5 4.74 mm long; ventriculus 0.208 0.460 mm long, 0.300 0.392 mm wide. Intestinal cecum absent. Spicules with broad lateral alae, right 0.460 0.680 mm long, left 0.480 0.760 mm long (Fig. 6a, b). Gubernaculum absent. Tail 0.280 0.480 mm long. 18 20 pairs of precloacal papillae, 1 pair double papillae immediately postcloacal, 2 pairs ventral, 2 pairs lateral postcloacal papillae; phasmids between first pair of ventral and lateral postcloacal papillae (Figs. 5, 6b). Female Body length 113 142 mm. Head 0.28 0.36 mm wide. Cervical alae 2.12 3.04 mm long; maximum width 0.2 0.232 mm. Excretory pore opens 0.864 0.920 mm from anterior (Fig. 7). Nerve ring not observed. Nuclei of esophageal glands present in ventriculus (Fig. 12). Esophagus 4.38 5.94 mm long; ventriculus 0.320 0.400 mm long, 0.340 0.576 mm wide (Figs. 7, 8). Intestinal cecum absent. Vulva opens 27 35 mm from anterior end, slitlike opening oriented laterally within small oval area of smooth cuticle (Fig. 10). Proximal end of uterus leads into a modified vagina forming an ovijector (Fig. 13). Undivided uterus (single specimen) 2.4 cm long, divided uterus 2.4 cm long (Figs. 13 17). Eggs 0.060 0.068 mm X 0.068 0.076 mm, with pitted surface; pits 0.002 0.003 mm deep (Fig. 11). Tail 0.280 0.480 mm long, phasmids at level of margin of middle and distal third (Fig. 9). Taxonomic summary Host Domestic cat (Felis catus Linnaeus, 1758). Habitat Small intestine. Type locality Kuala Lumpur, Malaysia. Additional locality Ipoh, Perak State, Malaysia. Deposition of type specimens USDA National Parasite Collection USNPC nos. 090227; M114B (holotype), 090228; M114 C/D (paratypes). Additional paratypes in The Royal Veterinary College Parasite Collection nos. 8022 8023. Etymology The specific name refers to the country of origin. Diagnosis Toxocara malaysiensis n. sp. is assigned to Toxocara on the basis of the presence of an esophagus with a ventriculus without an appendix, the absence of an intestinal cecum, prominent dentigerous ridges on each of the 3 lips, a V-shaped supporting bar in the cervical alae, male with subequal, alate spicules, female with vulva opening in the anterior half of the body and eggs subglobular with a thick pitted shell. The new species differs from the 2 nearest species, T. canis Werner, 1782 and Toxocara pteropodis Baylis, 1936, in the following features: from T. canis in the shape of the cervical alae in cross section (curved in the present specimens, straight in T. canis), spicule length (0.460 0.760 mm in present specimens, 0.91 0.97 mm in T. canis as defined by Warren [1971] in males 199 127 mm long), and the lip structure (deep median notch lined with denticles of the dentigerous ridge, absent in T. canis); from T. pteropodis in the ratio of the divided to undivided uterus (1:1 in present specimens, 1:<0.5 in T. pteropodis), cervical alae width (0.180 0.232 mm in present specimens, 0.070 0.122 mm in T. pteropodis), and the eggs (deeply pitted in the present specimens, finely pitted in T. pteropodis). 660

GIBBONS ET AL. T. MALAYSIENSIS N. SP. 661 TABLE I. The species of Toxocara Stiles, 1905. Species Hosts Locality Reference Toxocara canis (Werner, 1782) (=T. Alopex lagopus, Alopex beringensis, Canis Cosmopolitan Sprent, 1958 vulpis (Froelich, 1789), type spe mexicanus, Canis lupus, Canis occidentalis, cies Canis dingo, Canis aureus, Canis familiaris,?canis pallipes, Crocuta crocuta, Fennecus zerda, Lycaon pictus, Meles anakuma, Nyctereutes procyonoides ussuriensis, Procyon lotor, Urocyon cinereoargentatus scotti, Vulpes bengalensis, Vulpes fulva pennyslvanicus, Vulpes fulva regalis, Vulpes vulpes, Vulpes vulpes japonicus Fennucus zerda, Ichneumia albicauda, Panth- Round, 1968 ero leo Herpestes sanguineus Warren, 1972 Toxocara alienata (Rudolphi, 1819) Nasua rufus Brazil Warren, 1971 Toxocara apodemi (Olsen, 1957) Apodemus peninsulae Pup yong-ni, Korea Warren, 1971 Apodemus agrarius Changsa, Hunan, China Asakawa et al. 1994 Toxocara cati (Schrank, 1788) Acinonyx jubilatus, Felis spp., Lynx baileyi, Cosmopolitan Sprent, 1956 (=Toxocara mystax Zeder, 1800) Lynx canadensis, Lynx lynx, Lynx rufus, Lynx rufus texensis Genetta sp., Panthera leo, Panthera pardus Round, 1968 Helogale parvula, Civettictis civetta Warren, 1972 Toxocara canarisi Puylaert, 1967 Felis serval Kenya Puylaert, 1967 Toxocara cynonycterides (Parona, Xantharpyia (Cynonycteris) amplexicaudata Burma Warren, 1971 1889)* Toxocara elephantis (Rudolphi, Elephas maximus Switzerland, U.S.A. Warren, 1971 1819)* known only from animals in captivity Toxocara genettae Warren, 1972 Genetta felina Transkei, South Africa Warren, 1972 Genetta genetta Spain Alverez et al., 1990 Toxocara hippopotami (Canavan, Hippopotamus amphibius Philadelphia Zoo, Uganda Round, 1968 1931) Toxocara indica Naidu, 1981 Bandicota indica Nagpur, India Naidu, 1981 Toxocara mackerrasae (Sprent, Rattus fuscipes, Hydromys chrysogaster SE Queensland, Australia Warren, 1971 1957) Toxocara manzadiensis Vuylsteke, Syncerus caffer The Congo Round, 1968 1956* Toxocara paradoxura Kou, 1958 Paradoxurus minor exitus Kwantung, China Warren, 1971 Toxocara pearsei Chitwood, 1935* Synalpheus brooksi Florida, U.S.A. Warren, 1971 Toxocara pteropodis Baylis, 1936 Pteropus geddei Esperito Santo, New Hebri- Warren, 1971 des Pteropus hypomelanus Florida, U.S.A. Heard et al., 1995 Pteropus conspicullatus, Pteropus poliocephal- Queensland, Australia Prociv, 1990 us Toxocara sprenti Warren, 1972 Viverricula indica Bangkok, Thailand Warren, 1972 Toxocara suricattae (Ortlepp, 1940) Suricata suricatta Caroline, Transvaal, South Mozgovoi, 1953a, War- Africa ren, 1971 Mungos mungos Warren, 1972 Toxocara tanuki Yamaguti, 1940 Nyctereutes procyonoides viverrinus Kyoto, Japan Warren, 1971 Toxocara vajrasthirae Sprent, 1972 Arctonyx collaris Nahkon Nyok, Thailand Sprent, 1972 Toxocara vincenti Puylaert, 1967 Civettictis civetta Brazzaville, the Congo Warren, 1971 Toxocara vitulorum (Goeze, 1782) Bos taurus, Bos indicus, Bubalus bubalis Tropical, subtropical re- Mozgovoi, 1953b gions Bos spp., Capra spp., Ovis spp. Warren, 1971 Toxocara warreni Durette-Desset Hyemoschus aquaticus Booné, Gabon Durette-Desset and and Chabaud, 1974 Chabaud, 1974 * Species inquirendae in Warren (1971). Species inquirendae in Warren (1972). Sprent (1958) records that T. canis has also been recorded in cats, Felis tigris, Acinonyx jubilatus, and Lynx rufa.

662 THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 3, JUNE 2001 0 1mm 1-4,0 2mm 0 5mm I 5 6b FIGURES 1-6. Toxocara malaysiensis n. sp. 1. En face view of female showing the 3 bilobed lips, median notch arrowed. 2. Cross section near anterior end of 1 of the cervical alae of a female. 3. Cross section in anterior third of 1 of the cervical alae of female. 4. Cross section in middle third of 1 of the cervical alae of female. 5. Lateral view of posterior end of male showing caudal papillae. 6a. A single spicule dissected out of the tissues, lateral view. 6b. Lateral view of posterior end of male showing spicules, pre- and postcloacal papillae.

GIBBONS ET Al.-T. MALAYS/ENS/SN. SP. 663 8 : -:::. -. - - \ - l - I - 10 1mm 11 11 10.smm 9,10 1 0mm 7,8 FIGURES 7-10. Toxocara malaysiensis n. sp. 7. Lateral view of anterior end of female showing esophagus, ventriculus, and position of cervical alae (dotted lines). 8. Dorsal view of anterior end of female, body dorsoventrally flattened to show shape of cervical alae. 9. Lateral view of female tail. 10. Undivided uterus dissected out showing slitlike vulva and surrounding oval area of smooth cuticle attached to section of body wall; arrow shows direction of anterior end of body. 11. Eggs shown with (a) external surface and (b) in optical section.

664 THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 3, JUNE 2001 a;f 12,14,0 4mm 15,16 0 2mm 13 1cm 17 FIGURES 12-17. Toxocara malaysiensis n. sp. 12. Ventriculus showing nuclei of the esophageal glands from dissected female. 13. An enlargement of region a in Figure 17 showing the vagina leading to vulva (v), arrow indicates direction of lateral field of body. 14. An enlargement of region b in Figure 17 showing a section of the undivided uterus. 15. An enlargement of region d of Figure 17 showing the oviduct linking the seminal receptacle and ovary to the divided uterus. 16. An enlargement of region c of Figure 17 showing the junction of the undivided and divided uterus. 17. A diagrammatic drawing to scale of part of the female reproductive system.

GIBBONS ET AL. T. MALAYSIENSIS N. SP. 665 DISCUSSION The diagnostic keys published for Toxocara by Warren (1971, 1972) indicate that the specimens described in this paper are morphologically close to T. canis Werner, 1782 and T. pteropodis Baylis, 1936. They can be distinguished from these species on the basis of lip morphology, cervical alae, spicule length, and features of the female reproductive system, all criteria used by Warren (1971, 1972) in the separation of species of the genus. Since the publication of these 2 papers, 3 species have been added to the genus: Toxocara vajrasthirae Sprent, 1972, Toxocara warreni Durette-Desset and Chabaud, 1974, and Toxocara indica Naidu, 1981. The present specimens differ from T. vajrasthirae in the width of the cervical alae (0.180 0.232 mm in the present specimens, 0.08 0.10 mm in T. vajrasthirae) and the spicule length (0.460 0.760 mm in the present specimens, 1.7 2.2 mm in T. vajrasthirae). The other species have no cervical alae. The conclusion that T. malaysiensis is a valid species distinct from T. canis, which it superficially resembles, is supported by the molecular work of Zhu et al. (1998). These authors compared the nuclear ribosomal DNA sequences of the first internal transcribed spacer (ITS-1), the 5.8S gene, and the second internal transcribed spacer (ITS-2) of Toxocara sp. cf. canis (now named T. malaysiensis), T. cati, and T. canis. Differences in the ITS-1 and ITS-2 sequences among the taxa (9.4 26.1%) were markedly higher than variation between samples within T. cati and T. canis (0 2.9%) (Jacobs et al., 1997). Phenograms derived from pairwise sequence difference data for these and other ascaridoids (Zhu et al., 1998, 2000) showed that Toxocara sp. cf. canis (T. malaysiensis) clustered with the other Toxocara species examined (viz. T. canis, T. cati, and Toxocara vitulorum) and that, when ITS-2 alone or combined ITS-2, 5.8S, and ITS-1 data were used, Toxocara sp. cf. canis (T. malaysiensis) was genetically more similar to T. cati than it was to T. canis. For the ITS-1 data alone, T. canis and T. cati were more similar to each other than either was to Toxocara sp. cf. canis (T. malaysiensis). Thus, morphological and molecular evidence converge to indicate that T. malaysiensis is a valid species. ACKNOWLEDGMENTS Sincere gratitude is due to N. A. B. Y. Cheng and C. Panchadcharam for collecting the specimens. J. F. A. Sprent and R. B. Gasser are thanked for their support and encouragement. LITERATURE CITED ALVAREZ, F., R. IGLESIAS, J. BOS, J.TOJO, AND M. L. SANMARTIN. 1990. New findings on the helminth fauna of the common European genet (Genetta genetta L.): First record of Toxocara genettae Warren, 1972 (Ascarididae) in Europe. Annales de Parasitologie Humaine et Comparee 65: 244 248. ASAKAWA, M., J. F. 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