Protozoologica INTRODUCTION. Acta Protozool. (2008) 47: 69 76

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1 Acta Protozoologica Acta Protozool. (2008) 47: A New Species of Coccidia (Apicomplexa: Sarcocystidae) from the Slendertailed Meerkat Suricata suricatta (Scheber, 1776) from South Africa Amal K. EL-GAYAR 1,6, Patricia J. HOLMAN 1, Thomas M. CRAIG 1, Thomas W. DEMAAR 2, Sandra C. WILSON 3, Peter CHUNG 4, Keith M. WOODS 5, Carrie NORRIS 5 and Steve J. UPTON 5 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University; 2 Gladys Porter Zoo, Brownsville, TX; 3 Sedgwick County Zoo, Wichita; 4 Department of Biology, Pittsburg State University, Pittsburg; 5 Division of Biology, Kansas State University, Manhattan; 6 Department of Parasitology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Summary. A new species of coccidia (Apicomplexa: Sarcocystidae) is described from zoo-housed suricate or slender-tailed meerkat, Suricata suricatta (Scheber 1776). Oocysts of Cystoisospora timoni n. sp. are spherical to subspherical, 25.9 (1.5) 24.8 (1.6) ( ) μm, with a shape index (length/width) of 1.05 ( ). A micropyle, oocyst residuum and polar granule are all absent. Sporocysts are ellipsoidal, 15.3 (1.4) 12.8 (0.8) ( ) μm, with a shape index of 1.20 (1 1.3). Stieda and substieda bodies are absent, but a sporocyst residuum is present and composed as a large cluster of hundreds of small homogeneous granules. Sporozoites are elongate, each with a nucleus slightly posterior to the midpoint of the sporozoite and there is a crystalloid body difficult to discern posterior to the nucleus. Sequence and phylogenetic analysis of the 18S ribosomal RNA gene revealed the coccidian to be a member of the Sarcocystidae. Key words: Coccidia, Cystoisospora timoni n. sp., meerkat, oocyst, Sarcocystidae, Suricata suricatta. Abbreviations: rdna ribosomal DNA, gdna genomic DNA, PCR polymerase chain reaction. INTRODUCTION The slender-tailed meerkat Suricata suricatta (Scheber, 1776), or suricate is a small primarily insectivorous mammal, approximately 1 kg in weight and 50 cm long. Address for correspondence: Amal K. El-Gayar, TAMU MS 4467 RM 215 VMS BLDG, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX Tel.: ; Fax: ; aelgayar@cvm.tamu.edu, amalk18@yahoo.com It belongs to the family Herpestidae, which contains 18 genera and 37 species of carnivore commonly referred to as mongooses, meerkats, suricates, and fossas (Wilson and Reeder 1993, van Staaden 1994). This family is closely related to the family Viveriidae, which contains the civets, genets, and linsangs, but morphologic and genetic evidence supports the separation of the two taxa (Gaubert et al. 2002, 2004). The slender-tailed meerkat (Afrikaans: little lake cat) is a gregarious species that hunts in groups and is protective of their young (Nowak and Paradiso 1983,

2 70 A. K. El-Gayar et al. van Staaden 1994). In the wild, their habitat is the Kalahari Desert and adjacent regions in southern Africa, which includes areas of Angola, Namibia, South Africa, and southern Botswana (Wilson and Reeder 1993). Imported colonies have become favorite exhibits in many zoological gardens in the USA. Little is known about the infectious diseases of meerkats, including parasite infections. However, there are reports of coccidia in related host species from different geographic regions dating back to 1933 (Balozet 1933). A number of Isospora spp. are reported from dwarf mongooses (Helogale parvula and Crossarchus obscurus) in Kenya, the Egyptian mongoose (Herpestes ichneumon), and the Indian grey mongoose (Herpestes edwardsi) (Balozet 1933; Bray 1954, 1959; Pellérdy 1959; Dubey and Pande 1963, 1964; Levine et al. 1975; Bandyopadhyay 1982). Also, Eimeria spp. are reported from genets (Genetta genetta) in the Rome Zoo and from the Indian grey mongoose (Agostinucci and Bronzini 1955; Dubey and Pande 1963; Patnaik and Ray 1965, 1966; Bandyopadhyay 1982). The discovery of coccidian oocysts during routine meerkat fecal examinations at the Sedgwick County Zoo in Wichita, Kansas and the Gladys Porter Zoo, Brownsville, Texas, prompted shipments of fecal samples to diagnostic laboratories for further evaluation. Upon sporulation, the oocysts proved to represent a previously undescribed species of coccidian. The following paper presents a description of that new species. MATERIALS AND METHODS Coccidian isolates: In October 1999, the Sedgwick County Zoo, Wichita, Kansas acquired a colony of 10 meerkats originating from South Africa. Composite fecal samples were acquired during the routine quarantine procedure and examined microscopically for the presence of parasites. Coccidian oocysts were discovered and samples were shipped to Kansas State University (KSU) for further analyses. Ten weeks later, additional fecal samples were obtained from individual animals during routine veterinary examinations and also shipped to KSU. Similarly, in the spring of 2007, fecal samples obtained from meerkats resident at the Gladys Porter Zoo, Brownsville, Texas were shipped to the Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas for evaluation. Morphological description: On arrival to the diagnostic laboratories, the fecal samples were placed in Petri dishes in a thin layer of 2.5% aqueous potassium dichromate solution and the oocysts were allowed to sporulate for 24 h at room temperature (ca 23 C). Sporulated oocysts were concentrated by flotation in an aqueous sucrose solution (Upton 1997) and examined, measured using a calibrated ocular micrometer, and photographed under Nomarski interference contrast optics or by light microscopy using an Olympus AX 70 microscope (Upton et al. 2001, Dolezalova et al. 2004). Measurements for oocysts and sporocysts are reported in micrometers (μm), followed by the standard deviation with the range in parentheses. Data obtained were subjected to the general linear model (GLM) procedure. Means of oocyst length and width, sporulated oocyst length and width, and shape index of both units were separated by Duncan s difference procedure (Duncan 1955) using one way ANOVA (P 0.05) and were expressed as mean ± standard deviation of mean (SD). Molecular analysis: Feces containing sporulated oocysts from the Kansas meerkats were washed in tap water and centrifuged to pellet the oocysts. The oocysts were surface sterilized by soaking in 10% commercial chlorine bleach in deionized water (v/v) for 10 min. Sporulated oocysts from the Texas meerkats were concentrated by a modified water sedimentation and sugar flotation technique. The sporulated oocysts were washed extensively by centrifugation at 5,000 g for 3 min, then frozen at 80 C and thawed at 37 C a minimum of 2 cycles to rupture the walls. Genomic DNA was extracted using standard protocols (Murray and Thompson 1980; Sambrook and Russell 2001). The 18S rrna genes were amplified from the Kansas oocyst genomic DNA using Expand Hi Fidelity Taq Polymerase and primers 5 -CTGGTTGATCCTGCCAGTAGTCAT-3 (sense) and 5 - GATCCTTCCGCAGGTTCACCTACG-3 (anti-sense), designed from 18S rrna gene sequences of related species in the GenBank database, following manufacturer s instructions (Roche Molecular Biochemicals Inc.). The cycling conditions were 95 C for 5 min initial denaturation, followed by 40 cycles of 1 min at 94 C denaturation, 1 min at 55 C primer annealing, and 2 min at 72 C elongation, with an additional 10 min at 72 C final extension to allow complete elongation of amplification products (PTC-100 DNA thermal controller, MJ Research Inc.). The products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized by ultraviolet transillumination. The appropriately sized amplicons were gel-purified (QIA quick gel extraction kit, Qiagen) and ng DNA was ligated into pgemt (pgemt-ez vector kit, Promega). Competent XL1-blue Escherichia coli were transformed with the ligation product, the resulting colonies were subcloned, and plasmid DNA was purified (QIA-prep Spin Miniprep Kit; Qiagen). Plasmid DNA was verified to contain the desired inserts by PCR conducted as above. Both strands were sequenced using primers T7 and SP6, which flank the insert in the pgemt-ez vector, by PE Biosystems Big Dye and drhodamine chemistries on an ABI 3700 DNA analyzer. To complete the sequencing of the full length DNA insert, inner primers were constructed to overlap the boundaries of the internal sequences. Both complementary strands from all clones were sequenced a minimum of 4 times each to ensure accuracy. The 18S rrna gene was amplified from Texas oocyst genomic DNA using primers 5 -TCTGGTTGATCCTGCC-3 (sense) and 5 - GATCCTTCCGCAGGTT-3 (anti-sense) according to manufacturer s instructions (Advantage 2 PCR System, BD Biosciences, Palo Alto, California). The cycling parameters were 94 C for 2 min, followed by 45 rounds of 10 sec at 94 C, 10 sec at 55 C, and 2 min at 72 C, then 10 min at 72 C and hold at 4 C (PCR Express thermocycler; Hybaid, Ashford, UK). Nested PCR was conducted as above, but with the product as template and with nested primers 5 - GCCATGCATGTCTAAGTATAAG-3 (sense) and 5 -ACCTACG-

3 New Coccidia from Slender-Tailed Meerkat 71 GAAACCGTG-3 (anti-sense). The cycling conditions were 96 C for 3 min, then 35 cycles of 10 sec at 96 C, 10 sec at 50 C and 2 min at 72 C, followed by 10 min at 72 C and a hold at 4 C. Resulting PCR products were cloned, sequenced, and analyzed as previously described (Schoelkopf et al. 2005). The meerkat 18S rrna gene sequence was aligned (Clustal W 1.8 Program, with additional 18S rrna gene sequences from the Genbank database from Cryptosporidium parvum, Cyclospora cayetanensis, Cystoisospora (Isospora) belli, Cystoisospora (Isospora) ohioensis, Cystoisospora (Isospora) suis, Eimeria falciformis, Eimeria nieschulz, Isospora robini, Sarcocystis mucosa, and Toxoplasma gondii. Cladograms of the resulting alignment were constructed by parsimony and maximum-likelihood algorithms using Phylogenetic Analysis using Parsimony (PAUP) 4.0 beta version 8 (Swofford 1998). RESULTS Microscopic analysis of intact, sporulated oocysts (Figs 1, 2) revealed the meerkat coccidian to represent a previously undescribed species. Sporulated oocysts from the two isolates were morphologically indistinguishable (Fig. 2). Oocyst and sporocyst measurements of the Kansas (n = 25) and Texas (n = 20) isolates (Table 1) show that the mean values obtained by the two different laboratories are significantly different, but there is overlap in the range of values obtained. Thus, the measurements are given below as means for the total 45 oocyst and for the total 45 sporocyst values. An identical 18S rrna gene sequence was shared between the Kansas and Texas isolates (GenBank Accession Nos. AY and EU200792, respectively), although microheterogeneity was noted among the three sequenced clones of the Texas isolate (not shown). A BLAST search showed 100% identity between the Texas isolate consensus 18S rrna gene sequence and the sequence from the Kansas parasite, and this sequence shared 99% identity with that of Isospora belli. Phylogenetic analysis of the 18S rrna gene clearly places this coccidian within the family Sarcocystidae, genus Cystoisospora Frenkel 1977 rather than the Eimeriidae (Fig. 3). Fig. 1. Nomarski interference contrast photomicrograph of the sporulated oocyst of Cystoisospora timoni n. sp., showing refractile body (RB) and sporocyst residuum (SR) (Kansas isolate, Genbank Accession No. AY279205). Fig. 2. Composite line drawing of Cystoisospora timoni sporulated oocyst derived from the Kansas isolate (Genbank Accession No. AY279205).

4 72 A. K. El-Gayar et al. Morphological description Cystoisospora timoni n. sp. (Figs 1, 2) The morphology of sporulated oocysts is spherical to sub-spherical, 25.9 ± 1.5 ( ) μm 24.8 ± 1.6 ( ) μm (n = 45). Shape index (length/width), 1.05 ( ). Wall smooth, ca 1.5 thick, two layers of equal thickness; micropyle absent; oocyst residuum absent; polar granule absent; sporocysts ellipsoidal, 15.3 ± 1.4 ( ) μm 12.8 ± 0.8 (10 14) μm (n = 45); Fig. 3. Cladogram constructed using Parsimony (PAUP 4) showing relationships among 18S rrna gene sequences of 12 different species of coccidia with bootstrap support for each branch shown. Table 1. Oocyst and sporocyst measurements from the Texas (TX) and Kansas (KS) coccidian isolates showing mean and standard deviation (range in parentheses) and the shape index. Oocyst Sporocyst Isolate Length (µm) Width (µm) Shape Index Length (µm) Width (µm) Shape Index TX ±1.44 b ± 1.5 b 1.07 a 14.5 ± 1.5 b 12.4 ± 0.6 b 1.3 (n = 20) ( ) ( ) ( ) ( ) ( ) ( ) KS 26.6 ± 1.26 a 25.9 ± 0.71 a 1.03 b 16.0 ± 0.54 a 13.3 ± 0.43 a 1.2 (n = 25) ( ) ( ) ( ) ( ) ( ) ( ) Mean 25.9 ± ± ± ± (n = 45) ( ) ( ) ( ) ( ) ( ) ( ) a b Means (± SD) within columns that do not share a common superscript are significantly different (p 0.05).

5 New Coccidia from Slender-Tailed Meerkat 73 shape index 1.2 ( ) (n = 45), wall smooth, ca 0.5 thick, single-layered; Stieda body absent; substieda body absent; parastieda body absent; sporocyst residuum present as a large cluster of hundreds of small homogeneous granules; sporozoites elongate, nucleus slightly posterior to midpoint of sporozoite; crystalloid body present, often difficult to discern, posterior to nucleus, in diameter. Type host: Suricata suricatta (Scheber, 1776) suricate or slender-tailed meerkat (Carnivora: Herpestidae). Type specimens: Phototypes are deposited at the U.S. National Parasite Museum, Beltsville, MD as USNM collection number Sporulation: Exogenous. Oocysts were all unsporulated upon arrival at KSU or TAMU, but became fully sporulated within 24 h at room temperature (ca 23ºC). The extent of parasite maturation while in the mail was not determined. Prevalence: Unknown. Composite fecal samples obtained during quarantine at Sedgwick County Zoo in Wichita, Kansas contained numerous oocysts. Follow up fecal samples collected from individual animals 10 wk later showed only 2/10 (20%) of the meerkats to be passing oocysts. A history of coccidiosis in meerkats, including mortalities, at the Gladys Porter Zoo in Brownsville, Texas dates back to the early 1990 s. Etymology: The trivial name is derived from the Greek philosopher Timon of Philius ( BC), who promoted skepticism though satire. DISCUSSION A 2002 survey of North American institutions holding meerkats found three reporting coccidiosis as a health concern of 33 total respondents (Manharth 2004). Follow-up of this information revealed that coccidia are sporadically found in meerkats during parasite screening (unpublished). None of the interviewed institutions considered coccidia to be a pathogen nor was treatment prescribed. While collecting samples for this study at the Gladys Porter Zoo, it was observed that not all fecal samples from the 18 resident meerkats contained coccidia, nor would coccidia be found regularly in successive samples from an individual earlier identified as infected. Previously, coccidiosis was associated with the death of two weanling meerkat kits at another zoological institution when an unidentified coccidian was recovered at necropsy (unpublished). However, it is unclear if death was solely the result of the coccidia infection. This report presents the first description of a coccidian from the slender-tailed meerkat. The meerkat coccidian, C. timoni n. sp. possesses the definitive structural features of oocysts (two sporocysts each containing four sporozoites with no Steida bodies present) that place it in the genus Cystoisospora (Barta et al. 2005). This taxonomic positioning is further supported by molecular analysis based on the 18S rrna gene that places the meerkat coccidian, C. timoni, in the monophyletic Cystoisospora cluster, on a well-supported branch separate from the other Cystoisospora species (Fig. 3). The parasite is morphologically distinct from previously described species found in related hosts (Table 2). Cystoisospora timoni oocysts differ from Isospora dasuguptai from the Indian grey mongoose (Herpestes edwardsi) and the small Indian mongoose (Herpestes javanicus). Cystoisospora timoni oocysts are spherical to subspherical, whereas I. dasuguptai oocysts are ellipsoidal or ovoid and smaller in size (Dubey and Pande 1963, Knowles and Das Gupta 1931, Levine et al. 1975). Isospora garnhami oocysts from the dwarf mongoose (Helogale parvula and Crossarchus obscurus) in Kenya possess a thin fragile outer layer and thick, tough, elastic inner layer and the sporozoite nucleus is central (Bray 1954, 1959; Pellèrdy 1959), whereas the two layers of the C. timoni oocyst wall are smooth and equal in thickness, and the sporozoite nucleus is slightly posterior to the midpoint of the sporozoite. Isospora herpestei oocysts from the small Indian mongoose (Herpestes javanicus) in India, I. hoarei from the dwarf mongoose (Helogale parvula) in Kenya, and the oocysts of I. ichneumonis from the Egyptian mongoose (Herpestes ichneumon) in Egypt all tend to be smaller than those of C. timoni, while those of I. mungoi from the Indian grey mongoose (H. edwardsi) in India tend to be larger (Balozet 1933, Bray 1954, Dubey and Pande 1963, Levine et al. 1975). Isospora pellerdyi oocysts from the Indian grey mongoose in India are ovoid with an outer, thicker, and yellowish to orange layer with a darker inner layer (Dubey and Pande 1963, 1964; Patnaik and Ray 1965; Levine et al. 1975). Isospora viverrae oocysts from the civet cat (Civettictis civetta) in Sierra Leone and in South Africa tend to be smaller in size (Adler 1924, Bray 1964). The Isospora-type coccidia, i.e., those with oocysts containing two sporocysts each containing four sporozoites, are parasites of importance in human and veterinary medicine. These coccidia historically were as-

6 74 A. K. El-Gayar et al. Table 2. Comparative descriptive measurements of coccidia from members of the Herpestidae (mongooses, meerkats, suricates and fossas) and Viveriidae (civets, genets, and linsangs) families; mean (range) measurements are in µm. Coccidian Host species (Synonym) Locality Cystoisospora timoni Slender-tailed meerkat, Suricata suricatta USA (Africa) Oocyst Sporocyst Length Width Length Width 25.9 ± 1.5 ( ) 24.8 ± 1.6 (20 7.2) 15.3 ± 1.4 ( ) 12.8 ± 0.8 ( ) Reference This study Coccidium Civet cat, Civettictis civetta Liberia Bray 1954 Common genet, Genetta genetta (Genetta Agostinucci and Bronzini 1955 Eimeria genettae Rome Zoo dongolona) (20 30) (13 25) (6 13) (5 8) Eimeria Indian grey mongoose, Herpestes edwardsi Bandyopadhyay 1982 India jalpaiguriensis (Herpestes mungo) (21 23) (18 21) (9 11) (6 8) Eimeria newalai Indian grey mongoose, H. edwardsi India Eimeria pandei Indian grey mongoose, H. edwardsi India Indian grey mongoose, H. edwardsi and Small Indian mongoose, Herpestes Isospora dasguptai 1 javanicus (H. auropunctatus) Isospora garnhami Dwarf mongoose, Helogale parvula (Helogale undulata rufula) and Common India Kusimanse, Crossarchus obscurus 2 Kenya 17 (16 19) 23.0 (23 24) 20.6 (19 22) 28.6 (26 32) 14 (13 16) 18.6 (17 19) 17.2 (16 19) 24.5 (22 28) (12 15) 11.5 (11 12) Dubey and Pande 1963, Patnaik and Ray 1965 Patnaik and Ray 1965, 1966 Dubey and Pande 1963, Knowles and Das Gupta 1931, Levine et al Bray 1954, 1959; Pellérdy 1959 Isospora herpestei Small Indian mongoose H. javanicus (H. auropunctatus) India Isospora hoarei Dwarf mongoose, Helogale parvula Kenya Isospora Egyptian mongoose Herpestes ichneumon Eygpt ichneumonis 3 Isospora mungoi 4 Indian grey mongoose, Herpestes edwardsi India Indian grey mongoose, Isospora pellerdyi 5 H. edwardsi India 19.9 (18 24) 17.3 (16 19) 22 (19 26) 34 (27 34) 28 (27 30) 15.4 (13 18) 14.8 (13 17) 19 (16 20) 27 (23 27) 23 (20 25) 12.9 (11 15) 9.1 (9 10) 9.3 (8 11) 8.6 (8 9) 21 (19 21) 15 (17 19) 14 (12 14) 11 (12 14) Levine et al Bray 1954, Levine et al Balozet 1933, Levine et al Balozet 1933, Bray 1954, Dubey and Pande 1963, Levine et al Dubey and Pande 1963, 1964; Levine et al. 1975; Patnaik and Ray 1965 Isospora sp. Yellow mongoose, Cynictis pennicillata South Africa Markus 1972 Isospora viverrae Civet cat, C. civetta Adler 1924, Bray 1964 Sierra Leone; South Africa 22.8 (19 28) 19.0 (15 25) 1 Synonyms: Isospora garnhami (Bray 1954) of Dubey and Pande (1963), pro parte (small form); Isospora rivolta (Grassi 1879) of Knowles and Das Gupta (1931); non Isospora garnhami Bray (1954). 2 Bray (1959) reported oocysts from the Kusimanse as Isospora garnhami. It is unknown whether this represents the same or simply a morphologically similar coccidian. 3 Synonym: Isospora rivolta (Grassi 1879) of Balozet (1933). 4 Synonym: Isospora garnhami (Bray 1954) of Dubey and Pande (1963), pro parte (large form); non Isospora garnhami Bray (1954). 5 Synonym: Isospora knowlesi (Ray and Das Gupta 1936); Isospora knowlesi Dubey and Pande (1963); Isospora dubeyi (Patnaik and Ray 1965).

7 New Coccidia from Slender-Tailed Meerkat 75 signed to the genus Isospora, which traditionally has been classified in the family Eimeriidae (Carreno et al. 1998). Both morphological and molecular characterization now differentiate the Isospora-type coccidia into two apparently monophyletic groups of parasites, the Isospora (Eimeriidae) and Cystoisospora (Sarcocystidae) (Schneider 1881, Frenkel 1977, Barta et al. 2005). Isospora includes coccidia with tetrasporozoic, diplosporocystic oocysts possessing sporocysts with Stiedae bodies, whereas Cystoisospora includes coccidia with tetrasporozoic, diplosporocystic oocysts with sporocysts lacking Stiedae bodies. This recently identified species of Cystoisospora, C. timoni, was found in two different populations of meerkats, one that was recently imported and one that has been resident in the USA since at least 1993 (Tom Alvarado, Dallas Zoo, Dallas, TX, pers. comm.). 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