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. Author manuscript; available in PMC: 2016 Sep 1.
Published in final edited form as: Acta Parasitol. 2015 Sep 1;60(3):466–470. doi: 10.1515/ap-2015-0065

A new species of Isospora (Apicomplexa: Eimeriidae) from eastern coachwhip, Coluber flagellum flagellum (Reptilia: Ophidia) from Oklahoma

Chris T McAllister 1,*, R Scott Seville 2, Matthew B Connior 3
PMCID: PMC4539277  NIHMSID: NIHMS713982  PMID: 26204185

Abstract

Between June 2013 and August 2014, four eastern coachwhips, Coluber flagellum flagellum were collected from Arkansas (n = 2) and Oklahoma (n = 2) and their faeces examined for coccidian parasites. One (25%) harboured an isosporan that we describe here as new. Oocysts of Isospora kiamichiensis sp. n. were spheroidal to subspheroidal with a uni-layered wall and measured (length × width, L × W) 25.0 × 22.2 µm, with an L/W ratio of 1.1. A micropyle, oocyst residuum and polar granule were absent. Sporocysts were ovoidal, 14.2 × 9.8 µm, with an L/W ratio of 1.5. A pronounced, button-like Stieda body was present as well as a substieda body. A sporocyst residuum was present as dispersed granules. This is the first isosporan and fourth coccidian reported from eastern coachwhip snakes. In addition, a single oocyst of an unknown choleoeimerian was recovered from this host.

Keywords: Apicomplexa, Coccidia, taxonomy, oocysts, Reptilia, Ophidia, Isospora kiamichiensis, Oklahoma, Coluber flagellum flagellum

Introduction

The eastern coachwhip, Coluber (=Masticophis) flagellum flagellum Shaw, 1802 is a large colubrid that ranges from North Carolina south to Florida and west to Missouri, Kansas, Arkansas, and eastern Oklahoma and Texas (Conant and Collins 1998). In Oklahoma, M. f. flagellum occurs east of Interstate 35 in open woodlands and prairies where it feeds on insects, rodents, snakes, birds and especially lizards (Sievert and Sievert 2011).

Three coccidians, Eimeria zamenis Phisalix, 1921, Caryospora duszynskii Upton, Current and Barnard, 1984 and Caryospora masticophis Upton, McAllister and Trauth, 1994 have been reported from C. f. flagellum and western coachwhip, C. f. testaceus, all from specimens collected from Arkansas and Texas (see Duszynski and Upton 2009). However, we are not aware of any isosporans reported from C. f. flagellum. Herein, we provide a description of an isosporan from C. f. flagellum from Oklahoma.

Materials and Methods

Between June 2013 and August 2014, four adult C. f. flagellum were collected as fresh road-kills from Union County, Arkansas (n = 2) and Latimer (n = 1) and Pushmataha (n = 1) counties, Oklahoma, and examined for coccidia. Faecal samples from the rectum were placed in individual vials containing 2.5% (w/v) aqueous potassium dichromate (K2Cr2O7). Samples were examined for coccidia by light microscopy after flotation in Sheather’s sugar solution (specific gravity = 1.30). Measurements were taken on 22 sporulated oocysts from a single snake using a calibrated ocular micrometer and reported in micrometres (µm) with means followed by the ranges in parentheses; photographs were taken using Nomarski interference-contrast optics. Oocysts were ~7 days old when measured and photographed. Descriptions of oocysts and sporocysts follow guidelines of Wilber et al. (1998) as follows: oocyst length (L) and width (W), their ranges and ratios (L/W), micropyle (M), oocyst residuum (OR), polar granule(s) (PG), sporocyst length (L) and width (W), their ranges and ratio (L/W), sporocyst (SP), Stieda body (SB), substieda body (SSB), parastieda body (PSB), sporocyst residuum (SR), sporozoites (SZ) anterior (ARB) and posterior (PRB) refractile bodies, and nucleus (N). A host voucher was accessioned into the Arkansas State University Museum of Zoology, Herpetology Collection (ASUMZ), State University, Arkansas, USA. Photosyntypes of sporulated oocysts were accessioned into the Harold W. Manter Laboratory of Parasitology (HWML), Lincoln, Nebraska USA.

Results

One of four (25%) C. f. flagellum were found to be passing coccidian oocysts that we describe as new. In addition, the same snake was concurrently infected with a Choleoeimeria sp.

Isospora kiamichiensis sp. nov. (Figs. 1–4)

Figs. 1-3.

Figs. 1-3

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Nomarski interference-contrast photomicrographs of oocysts of Isospora kiamichiensis n. sp. 1. Subspheroidal oocyst and Stieda body (SB). 2. Spheroidal oocyst with point on sporocyst (PT) and substieda body (SSB). 3. Another subspheroidal oocyst showing unilayered oocyst wall (OW) and end view of sporocysts (SP). Scale bars = 10 µm for all figures.

Fig. 4.

Fig. 4

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Composite line drawing of oocyst of Isospora kiamichiensis n. sp. Scale bar = 10 µm.

Description of sporulated oocyst: Oocyst with 2 sporocysts; shape spheroidal to subspheroidal; smooth uni-layered wall, colourless, ~ 0.5 thick, smooth; L × W: 25.0 × 22.2 (20–28 × 19–24); L/W: 1.1 (1.0–1.2); M, OR, PG, all absent.

Description of sporocyst and sporozoites: SP ovoidal with point on end opposite SB; smooth uni–layered wall, ~ 0.5 thick; L × W: 13.9 × 9.4 (11–16 × 8–10); L/W: 1.5 (1.2–1.7); SB pronounced, button-like, SSB body present, PSB body absent; SR: composed of large-sized granules in a compact mass or dispersed along perimeter of SP; SZ: (not measured) sausage-shaped with spheroidal ARB and PRB; single N slightly posterior to midpoint.

Taxonomic summary

Type host: Eastern coachwhip, Coluber flagellum flagellum Shaw, 1802 (Reptilia : Ophidia : Colubridae). Collected 7 August 2014.

Type specimens: Symbiotype host desposited in the ASUMZ as No. 33267. Photosyntype of sporulated oocyst deposited in the HWML as No. 75058.

Type locality: 3.2 km NE of Honobia off Indian Highway, Pushmataha County, Oklahoma, USA (34.550791°N, 94.970198°W, elev. 512 m).

Prevalence: In one of four (25%) of the type host; one of two (50%) Oklahoma.

Sporulation: Exogenous. All oocysts were passed unsporulated or partially sporulated and became fully sporulated within five days at ~23 C.

Prepatent and patent periods: Unknown.

Site of infection: Unknown, oocysts recovered from faeces.

Etymology: The specific epithet is given for the Kiamichi Mountains of southeastern Oklahoma where the type host was collected. Because of the restricted nature of land ownership in the region, these mountains remain rustic, rural and economically undeveloped, with few population centers.

Remarks: When the new species is compared to other isosporans described from related colubrid snakes, there are little or no morphological similarities. Isospora colubris Matuschka, 1986, from the green whip snake, Hierophis (=Coluber) viridiflavus from Italy, possesses spheroidal oocysts that are considerably smaller (17.1 µm) (Matuschka 1986). Another isosporan, Isospora guzarica Davronov, 1985, from the spotted desert racer, Platyceps (=Coluber) karelini from Republic of Uzbekistan (Davronov 1985) is thought to be a Sarcocystis sp. (Upton et al. 1992) and considered a species inquirenda (see Duszynski and Upton 2009). In addition, Isospora wilsoni Upton, McAllister, Trauth and Bibb, 1992 was described from the flathead snake, Tantilla gracilis from adjacent Arkansas. Its subspheroidal oocysts measures 17.2 × 17.0 (15–19 × 15–19) µm, considerably smaller than the new species. The only other valid isosporan from snakes include Isospora natricis Yakimoff and Gousseff, 1935 from the European grass snake, Natrix natrix from Uzbekistan (Yakimoff and Gousseff 1935) which possesses much smaller spheroidal oocysts (14.4 µm). Two other isosporans, Isospora neivai Pinto and Maciel 1929 from the jaracaca, Bothrops jaracaca from Brazil (Pinto and Maciel 1929) and Isospora guersae Yakimoff and Matschoulsky, 1937 from the Levantine viper, Macrovipera lebetina from Uzbekistan (Yakimoff and Matschoulsky, 1937) are considered pseudoparasites, thus species inquirenda (Duszynski and Upton 2009).

In addition to the above mentioned isosporans, there have been two other species of Isospora previously described from snakes: Isospora cenchoae Asmundsson, Upton and Freed, 2001 from Amazon basin tree snake (Imantodes lentiferus) and Isospora dipsasi Asmundsson, Upton and Freed, 2001 from vermiculate snail-eater (Dipsas vermiculata), both from Ecuador (Asmundsson et al. 2001). Neither of these have any morphological similarities with the new species.

We also report that the same host was concurrently infected with a choleoeimerian (Fig. 5) having a single elongate oocyst that measured 28.4 × 15.1 µm (L/W = 1.9) and sporocysts that were 9.1 × 7.7 µm (L/W = 1.2). Our measurement for oocyst length is smaller than those for E. zamenis previously reported from M. f. flagellum from an unknown locality (Roudabush 1937) but fall within those for kingsnakes, Lampropeltis spp. from Illinois (Anderson et al. 1968) and racers, Coluber spp. from Iowa (Wacha and Christiansen 1974); however, E. zamenis is likely composed of multiple species (Duszynski and Upton 2009), and further study with additional oocysts and possibly DNA sequencing will be necessary to help unravel this enigma.

Fig. 5.

Fig. 5

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Nomarski interference-contrast photomicrograph of oocyst of Choleoeimeria sp. Scale bar = 10 µm.

Discussion

We here describe a new species of Isospora from an eastern coachwhip snake, but with some caution because (1) it was found in only one snake, (2) we do not have tissues for study of endogenous stages, and (3) there is the possibility of pseudoparasitism. Unfortunately, tissues were not available from this road-killed snake because they were in a state of decomposition when examined. In addition, because coachwhips are known to feed on a variety of reptiles, particularly lizards (Trauth and McAllister, 1995), this isosporan could be a pseudoparasite. And, only was snake was found to be passing oocysts and it would have been ideal to have additional infected snakes. Given this latter limitation, however, it is particularly noteworthy that in our previous studies on coccidia of coachwhip snakes from Arkansas, Oklahoma and Texas (Upton and McAllister, 1990; Upton et al., 1994; McAllister et al. 1995) only two of 40 individuals were found to be infected with three different species of coccidia, so prevalence may be quite low in these snakes.

Although no previous isosporans have been are reported from Oklahoma snakes, there is potential for additional coccidians to be described from the state. In fact, McAllister et al. (2014) recently described a new species of Isospora from the ground skink, Scincella lateralis from adjacent Arkansas. In addition, McAllister et al. (2012) described a new species of Caryospora from T. gracilis from Oklahoma.

The state of Oklahoma supports 99 species of reptiles in a wide variety of diverse habitats ranging from coastal plains to high mesas (Sievert and Sievert 2011). It is noteworthy that about 90% of the reptiles in the state have not yet been surveyed for coccidia and what already has been reported about the other 10% is few and only includes snakes as hosts (Table 1). Indeed, we have just scratched the surface of the known coccidians of the state’s herpetofauna and it is very likely that species new to science will be discovered in the course of future surveys, expanding our knowledge of the coccidia of Oklahoma’s reptiles.

Table 1.

Coccidia reported from Oklahoma snakes.

Coccidian and host County locale Prevalence* Reference
Caryospora duszynskii
 Lampropeltis calligaster not given 1/1 (100%) McAllister et al. (2011)
 Pantherophis emoryi Greer 2/2 (100%) McAllister et al. (1995, 2011)
Eimeria choctawensis
 Tantilla gracilis Choctaw 1/15 (7%) McAllister et al. (2012)
Eimeria striatula
 Virginia striatula McCurtain 2/2 (100%) McAllister et al. (1995)
Isospora kiamichiensis
 Coluber flagellum Pushmataha 1/4 (25%) This study
Sarcocystis sp.
 L. calligaster McCurtain 2/4 (50%) McAllister et al. (2013)
Pantherophis obsoletus Le Flore 2/13 (15%) McAllister et al. (2013)
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*

Number infected/number examined (%).

Acknowledgements

We thank Drs. S.L. Gardner (HWML) and S.E. Trauth (ASUMZ) for expert curatorial assistance. This study was supported, in part, by a grant from the National Institute of General Medical Sciences (8P20GM103432-12), National Institutes of Health to R.S. Seville. Scientific collecting permits were provided to CTM and MBC by the Arkansas Game and Fish Commission and the Oklahoma Department of Wildlife Conservation.

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