Intranuclear coccidiosis in tortoises: Nine cases

Intranuclear Coccidiosis in Tortoises: Nine Cases

M. M. GARNER, C. H. GARDINER, J. F. X. WELLEHAN, A. J. JOHNSON, T. MCNAMARA, M. LINN,S. P. TERRELL, A. CHILDRESS, AND E. R. JACOBSON

Northwest ZooPath, Monroe, WA (MG); Registry of Veterinary Pathology, Armed Forces Instituteof Pathology, Washington DC (CHG); Wildlife Health Services, Wildlife Conservation Society,Bronx, NY (TM, ML); and College of Veterinary Medicine, University of Florida, Gainesville,

FL (ER, ST, AJ, JW, AC)

Abstract. Chelonian intranuclear coccidiosis has been reported once, in two radiated tortoises(Geochelone radiata), and is apparently rare. We describe intranuclear coccidiosis diagnosedhistologically in two radiated tortoises, three Travancore tortoises (Indotestudo forstenii), two leopardtortoises (Geochelone pardalis), one bowsprit tortoise (Chersina angulata), and one impressed tortoise(Manouria impressa). Infection was systemic and involved alimentary, urogenital, respiratory, lymphoid,endocrine, and integumentary systems. Trophozoites, meronts, merozoites, macrogametocytes,microgametocytes, and nonsporulated oocysts were seen histologically or by electron microscopy.Intracytoplasmic and extracellular stages of parasite development also were identified histologically.Sequencing of a coccidial 18S rRNA consensus polymerase chain reaction (PCR) product revealeda novel sequence that provided phylogenetic information and may be useful for further diagnostic testdesign. Intranuclear coccidiosis was associated with variable degrees of inflammation in all cases, wasconsidered the cause of death in six tortoises, and was a substantial contributing factor to the cause ofdeath in two tortoises.

Key words: Chelonidae; coccidian; coccidiosis; electron microscopy; histopathology; intranuclear;PCR; tortoises.

More than 30 species of coccidian parasites havebeen described in Chelonidae (tortoises and tur-tles).3–6,11 Eimeria spp. are apparently the mostcommon coccidia in chelonians, but Caryosporacheloniae, Isospora spp., and Mantonella sp. alsohave been described.11 Caryospora cheloniae is animportant pathogen of mariculture-reared greenturtles (Chelonia mydas),10 but to the authors’knowledge, there are no reports of pathologicchanges associated with these other coccidia.

The parasitic coccidia of vertebrates mostcommonly have endozoic development (merogenyand gamogeny) within the cytoplasm of entero-cytes. At least 11 species of Eimeria, Isospora, andCyclospora are caryotropic, having intranucleardevelopmental stages. Intranuclear coccidia havebeen described in all classes of vertebrates, in-cluding four species of Isospora that infect lizards.2

Lesions associated with intranuclear coccidiosis areapparently rare, with only a single report of fatalinfection in two radiated tortoises (Geocheloneradiata).9 We describe gross and light and electronmicroscopic findings of fatal intranuclear coccidi-osis in eight additional tortoises representing fourspecies from six sources.

Materials and Methods

Case histories

Case No. 1 was a wild-caught, adult male, impressedtortoise (Manouria impressa) native to Thailand thathad been confiscated by the US Fish and WildlifeService (USFWS) during illegal shipment into the USA.The tortoise was relocated to the Fort Worth Zoo, FortWorth, Texas. On arrival, the tortoise appeared to be ingood physical condition. Over several months, thetortoise became anorectic and emaciated. Anaerobicculture of ascites fluid revealed growth of Clostridiumsp. The tortoise did not respond to a variety ofantibiotics or anthelmintics (fenbendazole or metroni-dazole), and died.

Case No. 2 was a 7-month-old leopard tortoise(Geochelone pardalis) bred at a private facility in BatonRouge, Louisiana. The tortoise died after a 4-weekhistory of lethargy, poor appetite, and apparent icterus.

Case Nos. 3 and 4 were adult, captive-bred, radiatedtortoises from a facility on St Catherine’s Island,Georgia. Case No. 3 presented with lethargy, weakness,and bilateral ocular and oral mucoid discharges. Thetortoise was treated with antibiotics, but died 11 daysafter presentation. Case No. 4 was an adult maleradiated tortoise that presented with bilateral ocular andnasal mucoid discharges. The tortoise was treated with

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antibiotics, but died 3 days after initial onset of clinicalsigns of disease.

Case Nos. 5–7 were Travancore tortoises (Indotestudoforstenii) native to Celebes that were confiscated by theUSFWS on entry into the USA. The tortoises wererelocated to the Wildlife Conservation Society, Bronx,NY. Case No. 5, an adult female, died after a 3-weekhistory of weakness and dehydration. During that time,it had been treated with antibiotics and fluids admin-istered subcutaneously. Case No. 6 was a male that hadbeen clinically normal until 2 days prior to death, whenit separated from the group and was found dead. CaseNo. 7, a male, had a 3-day history of lethargy, weakness,and red conjunctiva. Despite being treated with anti-biotics, it died.

Case No. 8 was an adult male Bowsprit tortoise,Chersine angulata, from the zoo in central Florida. Ithad a history of anorexia and lethargy. Biopsy speci-mens of liver, kidneys, and small intestine were obtainedduring exploratory coelotomy. Biopsy results indicated

intranuclear coccidia in the renal tubular epithelial cells.Fecal parasite examination revealed coccidian oocysts.The tortoise died 1 day after surgery.

Case No. 9 was an adult female leopard tortoise froma private collection in New York. It presented with a 1-month history of anorexia followed by death. Ancillarydiagnostic procedures were not performed on thistortoise.

All tortoises were necropsied within 24 hours ofdeath. All tissues to be examined microscopically(Table 1) were preserved in neutral-buffered 10%formalin, processed in routine manner, sectioned at 5-to 6-mm thickness, and stained with HE. Select tissuesfrom some cases also were stained by periodic acid–Schiff (PAS) and Fite’s acid-fast techniques. Case Nos. 1,2, and 9 were necropsied on site, and sections of selectedviscera were submitted to Northwest ZooPath forhistologic examination. Sections of kidney from CaseNo. 1 and tympanic scale, and middle and inner ear fromCase No. 2 were further fixed in 4% glutaraldehyde and

Table 1. Distribution of intranuclear coccidia and severity of related lesions in affected tortoises.

Tissue

Tortoise No.

1 2 3 4 5 6 7 8 9

Esophagus ++/mo r/mi +/mi r/mi -/mi -/n r/mi ne ++/moStomach ++/mo ne r/mi ne r/mi -/mi +++/mo r/mo neIntestine a+/mo ne r/mi r/mi r/mo +/mo ++/s +/mi +++/modColon ++/s +/mo ++/mo r/mo r/mo r/mo ++/s -/mi +/modPancreas ne ne ne ne r/s r/mo ++/s r/mo ++/sLiver ++/mi r/mi ++/mi r/mi +/mi -/mi r/mi -/mi -/miKidney +++/s ++/s ++/s +/s -/n -/m r/mi -/mi ++/sSpleen ++/s ne r/mi r/mi -/n -/n r/mi -/n +/miGonad r (f)/mi r(f )/mi +/m/mo +(m)/mi -(f )/n r (m)/mi r/f/mi m/ne f/neEpididymis - - +/mo +mo - -/n - ne -Oviduct -/n ne - - -/n - -/n - -U bladder ++/mi ne ne ne ne ne ne ne neUreter ne ne ++/mo r/mi r/mi ne ne -/mi neEar ne +/mi ne ne ne ne ne -/n neEustach tube ne +/mi ne ne ne ne ne ne neLaryngeal epith -/n +/mi ++/mo ne ne -/mi ne ne neOral epith -/n r/mi r/mi ne ne -/mi ne -/n neNasal epith -/n r/mi r/mi ne ne r/mi ne -/n neConjunctiva -/mi +/mi r/mi r/mi ne -/mi ne -/n neLung +/mi +/mo +/mo +/mi r/mi r/mo +++/mo r/mi neTrachea -/mi -/mi r/mi +/mo -/n r/mo +/mi ne neHeart -/mi -/mi -/s -/mi -/n -/n -/mi -/- neSkin -/n -/n r/mi -/n ne -/n ne -/n neThyroid ne ne ++/mi r/mo -/mi -/n -/n -/n neAdrenal ne ne r/mi ne ne ne ne -/mi neBrain -/n -/mo ne n/mo ne -/mi ne -/mi neCord -/mi -/n -/mo -/mo ne ne ne -/n neGanglia 1/n -/n -/mo -/mi ne -/n -/n ne neSkel Muscle -/n -/n r/mi -/n -/n -/mi -/n -/n -/nCoelom -/n -/n r/mo r/mo -/n -/mi -/mo -/mo -/n

* r 5 Rare, + 5 1 intranuclear coccidian/400X field, ++ 5 2/400X field, +++ 5 3/400X field, - 5 no coccidians seen; mi 5 mild,mo 5 moderate, s 5 severe, n 5 no lesions consistent with intranuclear coccidiosis, ne 5 not examined, m 5 male, f 5 female; U5 urinary (bladder), Eustach 5 Eustacian (tube), epith 5 epithelium, skel 5 skeletal (muscle).

312 Garner, Gardiner, Wellehan, Johnson, McNamara, Linn, Terrell, Childress, and Jacobson Vet Pathol 43:3, 2006

examined by electron microscopy at the Armed ForcesInstitute of Pathology, Washington DC (case No. 1), andat the Electron Microscopy Core Laboratory, Universityof Florida (UF) (case No. 2). Case Nos. 3 and 4 werenecropsied on site, and tissues were submitted to theWildlife Conservation Society (WCS), Bronx, NY forhistologic examination. Case Nos. 5–7 were necropsiedon site at the WCS. Necropsy and histologic examinationof Case No. 8 was performed at the UF. All slides oftissues from all tortoises were subsequently reread by onepathologist (M. Gardner).

Polymerase chain reaction (PCR) analysis and sequencing

For each tortoise, DNA was extracted from micro-tome slices of paraffinized, infected tissues using theDNEasy kit (Qiagen, Valencia, CA). Primers weredesigned from conserved regions of various coccidianspecies in various genera targeting an approximately350-basepair segment corresponding to bases 1,018–1,377 of the small subunit ribosomal RNA gene ofToxoplasma gondii (Genbank accession No. L37415).Fifty-microliter reactions were run, with each containing4 ml of extracted DNA, 1 mM forward primer 1135F 59-ACYATAAACTATGCCRACTAGA-39 (Y 5 C or T,R 5 A or G), 1 mM reverse primer 1503R 59-CYTCCYTRCRTTARACACGCAA - 39, 200 mM eachdATP, dCTP, dGTP, and dTTP, 2.5 U of Pwo DNApolymerase (Thermo Hybaid, Franklin, MA), and 4 mlof PCR buffer (Thermo Hybaid). The mixture wasamplified in a thermal cycler (PCR Sprint, ThermoHybaid) with an initial denaturation at 94uC for5 minutes, followed by 40 cycles of denaturation at94uC for 30 seconds; annealing at 48uC for 60 seconds;DNA extension at 72uC for 60 seconds; and a finalextension step at 72uC for 7 minutes.

The PCR products were resolved in 1% agarose gels.Bands were excised and purified using the QIAquick gelextraction kit (Qiagen). Products were sequenced di-rectly in both directions using the Big-Dye TerminatorKit (Perkin-Elmer, Branchburg, NJ) and were analyzedon an ABI 377 automated DNA sequencers at the UFDNA Sequencing Core Laboratory.

Analysis of data

The sequences were compared with known sequencesin GenBank (National Center for Biotechnology In-formation, Bethesda, MD), EMBL (Cambridge, UK),and Data Bank of Japan (Mishima, Shiuoka, Japan)databases using BLASTN.1

Predicted homologous 344–357 nucleotide sequencesof representative coccidial 18S rRNA available fromGenBank were aligned manually. Phylogenetic analysesof the predicted alignment were performed with thePHYLIP (Phylogeny Inference Package, version 3.61)program package.8 A maximum likelihood tree wasgenerated using Dnaml with global rearrangements and10 jumbles. Gaps of all lengths were counted as singleevents. Babesia caballi (GenBank accession No.AY534883), a noncoccidian apicomplexan, was used as

the outgroup. The validity of the tree topology obtainedwas tested by using bootstrap analysis7 starting withSeqboot with 100 resamplings from the alignedsequences, followed by maximum likelihood calcula-tions, and calculation of the most probable (consensus)tree using Consense.

Results

Histopathologic changes

The distribution of intranuclear coccidia andassociated lesions in these tortoises, on the basis oftissues available for microscopic examination, aresummarized in Table 1. Generally, infection in alltissues was accompanied by lymphocytic or lym-phoplasmacytic inflammation and variable degreesof necrosis. Lesions suggestive of coccidiosis werealso frequently detected in heart and centralnervous system (CNS) tissue, but coccidia werenot seen in these tissues. Case Nos. 5 and 7 hadconcurrent amebiasis, and Case Nos. 5, 7, and 8had bacterial septicemia; the lesions associated withthese processes occasionally obscured morphologicfeatures of intranuclear coccidiosis in these ani-mals.

The coccidia were best observed in the HE-stained sections, and selective staining of theorganisms was not observed in histologic sectionsstained by PAS or Fite’s acid-fast techniques.Organisms were most abundant in the kidney andpancreas, but could be readily found in othertissues such as liver, intestine, and spleen. Theorganisms were most often detected in the nucleusof infected cells. Gamete stages were spherical, upto 6 mm in diameter, and had a slightly granularbasophilic cytoplasm and a small, slightly eccentricnucleus. The cytoplasm of the gametes sometimeshad a pink hue in HE-stained specimens. Macro-gametes and microgametes could not be discernedat the light microscopic level. Meronts werespherical, and had a central residual body sur-rounded by up to several (sometimes .16) mer-ozoites. Many infected cells had more than onegamete, and some had as many as four gametes.Rarely, gametes and meronts were detected in thesame nucleus. Infected cells with single smallgametes in the nucleus were difficult to distinguishfrom uninfected cells with hypertrophied nucleoli,especially in partially autolysed tissue. All stages ofthe coccidia also were detected rarely in thecytoplasm of various cell types.

Alimentary tract. Lesions involving the alimen-tary tract were similar at all levels of the tract, butwere most severe in the small intestine and colon.Intranuclear coccidia in various stages of develop-

Vet Pathol 43:3, 2006 Coccidiosis in Tortoises 313

Fig. 1. Small intestine, impressed tortoise, case No. 1. Notice intranuclear gametes (arrows), and intranuclearschizont (arrowhead). Only a few lymphocytes and plasma cells are evident in lamina propria and within theepithelial layers. HE. Bar 5 30 mm.

Fig. 2. Pancreas, leopard tortoise, case No. 9. Notice complete loss of acinar tissue. Few ducts remain (arrows),widely separated by zones of fibrosis and inflammation. HE. Bar 5 230 mm.

Fig. 3. Pancreas, leopard tortoise, case No. 9. Notice two intranuclear gametes (arrow) in an epithelial celllining a degenerative duct (d). Also notice infiltrate of lymphocytes, plasma cells, and heterophils in the adjacentfibrous stroma. HE. Bar 5 28 mm. Inset: higher magnification of inflamed stroma, showing intranuclear schizont(large arrow), extracellular oocyst (small arrow), and intracytoplasmic zoites (arrowheads). Infected cell typescannot be discerned. HE. Bar 5 25 mm.

Fig. 4. Liver, impressed tortoise, case No. 1. Notice central focus of hepatocellular necrosis admixed with scantinfiltrate of inflammatory cells. Intranuclear and intracytoplasmic coccidia are in the field (arrows), but are difficultto discern at this magnification. HE. Bar 5 60 mm. Inset: Melanomacrophages with intranuclear gamete (smallarrow), intracytoplasmic schizont (S), intranuclear schizont (s), and intracytoplasmic zoites (arrowheads). HE. Bar5 18 mm.


ment were detected in the mucosal epithelium, andrarely in unidentified mononuclear cells resemblingmacrophages in the lamina propria or submucosa.The number of cells containing coccidia variedconsiderably (Table 1), and for some animals,a diligent search at 400X magnification was neededto find the organisms. Lesions associated withcoccidian infection included necrosis of infectedepithelial cells and variable degrees of mucosalepithelial hyperplasia (Fig. 1). The presence ofcoccidia was accompanied by infiltrates of small tolarge numbers of lymphocytes and plasma cells, witha few granulocytes and macrophages distributedthroughout the lamina propria and submucosa andoccasional migration through the overlying mucosalepithelium. Bacterial overgrowth was seen in ulcer-ated portions of the colon in case No. 1, and caseNos. 5 and 7 had concurrent amebic and bacterialinfection in ulcerated portions of the colon. Edemaof the submucosa was prominent in all affectedtortoises.

Coccidia were detected in the nuclei of acinarand ductular epithelium of the pancreas, and a fewintracytoplasmic meronts and merozoites were seenin unidentified cells resembling macrophages anddegenerative ductular epithelial cells. Rarely, ductscontained intraluminal nonsporulated oocysts.Parasitism was accompanied by moderate to severedegeneration and necrosis of infected cells; acinaratrophy and loss; interstitial fibrosis, ductularhyperplasia, and dilatation; and acinar cell necro-sis. Inflammation was interstitial, multifocal, andpredominantly, lymphocytic (Figs. 2, 3).

Intranuclear coccidia were detected in the bileduct epithelium, hepatocytes, and melanomacro-phages of the liver, and intracytoplasmic merontsor merozoites were seen occasionally in themelanomacrophages. Parasitism was accompaniedby small random foci of hepatocellular necrosis andperiportal to random infiltrates of lymphocytes,plasma cells, and heterophils. Rarely, affectedducts had epithelial cell crowding and hypertrophy(Fig. 4).

Respiratory tract. Generally, mucosal lesions in theproximal portion of the respiratory tract were similar tothose seen in the gut mucosa. Lungs had rare tofrequent coccidia in the nuclei of pneumocytes,accompanied by pneumocyte hypertrophy and in-testinal infiltrates of lymphocytes and fewer heterophils.

Urinary tract. Kidney tissue was available fromall tortoises. Coccidia were detected in the nuclei ofcortical and medullary tubules, and intracytoplas-mic meronts and merozoites were detected intubular epithelium. Rare extracellular nonsporu-lated oocysts were noted in the tubular lumina.Parasitism was associated with moderate-to-severerenal tubular necrosis and mild-to-moderate in-terstitial lymphocytic or lymphoplasmacytic in-flammation (Figs. 5, 6). Glomeruli sometimes hadmild mesangioproliferative change. Ureters hadrare to moderate numbers of infected mucosalepithelial cells, with mild mucosal hyperplasia andsubmucosal lymphocyte infiltrates. Case No. 8 hadmild mucosal hyperplasia, but coccidia were notseen. Urinary bladder was available from case No.1, and it had moderate numbers of coccidia in themucosal epithelium, with associated mild epithelialhyperplasia and moderate submucosal lymphocyticinflammation and exocytosis.

Reproductive tract. Ovary tissue had rare coc-cidia in follicular epithelium and unidentifiedinterstitial cells accompanied by mild interstitialinfiltrates of lymphocytes. The ovary from case No.5 did not contain coccidia or lesions. Coccidia orlesions were not detected in oviduct. Testes hadrare to a few infected interstitial cells with mildinterstitial infiltrates of lymphocytes. Epididymishad a few infected epithelial cells and moderatesubmucosal infiltrates of lymphocytes.

Spleen. The spleen had rare to moderate numbersof coccidia in the nuclei of mononuclear cells re-sembling macrophages or lymphocytes in the redpulp. Foci of necrosis and fibrin deposition weredetected adjacent to cells with intranuclear coccidians.

r

Fig. 5. Kidney, leopard tortoise, case No. 9. Notice necrotic tubule (arrowhead) partially delineated by aninfiltrate of mononuclear inflammatory cells (arrow). HE. Bar 5 100 mm.

Fig. 6. Kidney, leopard tortoise, case No. 9. Degenerative tubules lined by epithelial cells that contain one or moreintranuclear gametes (black arrows), intracytoplasmic protozoa (white arrow), or intranuclear schizont (whitearrowhead). HE. Bar 5 20 mm. The lumen of one tubule contains an oocyst (arrowhead). Inset: Lumen of tubulecontaining three clusters of zoites. HE. Bar 5 24 mm.

Fig. 7. Middle ear, leopard tortoise, case No. 2. Notice intranuclear (arrowheads) and intracytoplasmic (largearrow) protozoa in the epithelium of the mucosa. Also notice the unsporulated oocysts in the lumen (small arrows),admixed with debris and bacteria. HE. Bar 5 20 mm.


Endocrine system, CNS, and special senses. Thethyroid had rare moderate numbers of infectedfollicular epithelial cells with associated mild-to-moderate interstitial lymphocytic inflammation.Tortoise No. 5 had mild lymphocytic inflammationin the interstitium between follicles, but noassociated coccidia. Tortoise Case No. 8 hadfollicular goiter. Both tortoises, for which adrenalgland tissue was available, had mild focal interrenal(cortical) cell necrosis, and case No. 3 had rareinfected interrenal epithelium. The brain had mildmultifocal perivascular lymphocyte infiltrates inthe meninges and neuropile at various levels, witha few microglial nodules, but coccidia or otherorganisms were not seen. The spinal cord had mild-to-moderate perivascular inflammation, and micro-glial nodules were detected in case Nos. 1, 3, and 4,but coccidia were not seen. Eustachian tube andinner/middle ear were available from case No. 2.Frequent coccidia were detected in the nuclei andcytoplasm of mucosal epithelium of the middle earand Eustachian tube, accompanied by mild epithe-lial hyperplasia, exfoliation, and submucosal infil-trates of lymphocytes and plasma cells. Numerousnonsporulated oocysts were seen in the lumen ofthe middle ear (Fig. 7). Middle and inner ear tissuealso was available from case No. 8, but lesions orcoccidia were not detected.

Skin. Rare epithelial cells with intranuclearcoccidia were detected in the epidermis of the skinfrom the head of tortoise 3. Tortoises 3, 6, and 8had small foci of ulceration and epidermal necrosiswith mild, dermal, perivascular lymphocytic in-flammation, and occasional exocytosis.

Musculoskeletal system. Small foci of lympho-cytic inflammation were detected occasionally inskeletal muscle of case Nos. 3 and 6, and rareintranuclear coccidia were detected in mononuclearcells in areas of inflammation in tortoise No. 3.

Ultrastructure. Electron microscopy performedon case Nos. 1 and 2 revealed the microanatomy ofa intranuclear coccidian (Figs. 8–11). Infectednuclei were often enlarged and contained one ormore organisms. Trophozoites measured 2–5 mm indiameter (Fig. 8). Meronts measured up to 7 mm indiameter and contained numerous (.16) mero-zoites (Fig. 9). Merozoites were banana shaped,measured 4.0 3 1.5 mm, and budded from a re-siduum. Macrogametes (Fig. 10) and microgametes(Fig. 11) were numerous, and measured up to 6 mmin diameter. Oocysts were unsporulated, andmeasured 8 mm in greatest diameter.

PCR and sequence analysis. The PCR productsfrom case Nos. 7 and 9 were amplified andsequenced. The sequence from both tortoises wasidentical. This sequence was identical to that fromseveral other cases of intranuclear coccidiosis intortoises (Wellehan and Johnson, unpublisheddata). The TBLASTN results for the sequenceindicated the highest score with the Hyaloklossialieberkuehni 18S small subunit ribosomal RNAgene (GenBank accession No. AF298623).12 Themaximum likelihood phylogenetic tree with boot-strap values is shown (Fig. 12). Sequence data wassubmitted to GenBank; the accession number isAY728896.

Discussion

Lesions associated with intranuclear coccidiosiswere considered the principal cause of death in caseNos. 1–4, 6, and 9. The death of case Nos. 5 and 7was attributable to intranuclear coccidiosis andconcurrent bacterial infection and amoebiasisoriginating in the intestinal tract of these tortoises.Intranuclear coccidiosis may have been an in-cidental finding in case No. 8.

Intranuclear coccidiosis in these tortoises wasa systemic event, involving alimentary, respiratory,urogenital, integumentary, lymphoid, and endo-crine systems. Organisms were present in pancreas,lung, and small intestine of all tortoises for whichthese tissues were available. Lesion severity invarious affected tissues generally was proportionalto density of infected cells in the tissue. Lesionswere most severe in pancreas, colon, kidney, andlung. Lesions were judged to be subacute to chronicin most sites, especially the alimentary tract.Lesions in the pancreas were consistently severe,and loss of functional pancreatic tissue may havecontributed substantially to the wasting detected incase Nos. 5, 8, and 9. Chronic inflammation andmucosal alterations associated with intestinalcoccidia may have contributed to malabsorptionand dysbiosis, and in case Nos. 5 and 7, to bacterialand amebic enteritis and septicemia. Lesions in thekidney were usually severe, and likely wereassociated with appreciable renal dysfunction, asreported in radiated tortoises.9 On the basis ofmorphologic alterations in the renal parenchyma,the renal lesions were less chronic than those in thegastrointestinal tract and pancreas. The tubularnecrosis, absence of tubular regeneration, andmild-to-moderate inflammatory response in thisorgan indicated a shorter course of infection, andsuggest that infected tortoises may not survive longafter the kidney becomes involved. The inflamma-


Fig. 8. Kidney, impressed tortoise, case No. 1. Electron micrograph of intranuclear trophozoites in a tubularepithelial cell. Uranyl acetate and lead citrate. Bar 5 3 mm.

Fig. 9. Kidney, impressed tortoise, case No. 1. Electron micrograph of a meront in nucleus of renal tubularepithelial cell. Notice that merozoites have a polar ring (p), micronemes (m), and a large nucleus (n). Also notice theresidual body (r) from which the merozoites budded. Uranyl acetate and lead citrate. Bar 5 4 mm.

Fig. 10. Kidney, impressed tortoise, case No. 1. Electron micrograph of the nucleus of a tubular epithelial cellcontaining a microgametocyte. Notice the flagella (arrows) of the numerous microgametes. Uranyl acetate and leadcitrate, bar 5 2.4 mm.

Fig. 11. Kidney, impressed tortoise, case No. 1. Electron micrograph of the nucleus of a tubular epithelial cellcontaining a macrogametocyte (arrow). Uranyl acetate and lead citrate. Bar 5 2.1 mm.


Fig. 12. Maximum likelihood phylogenetic tree of partial coccidial 18S rRNA nucleotide sequences. Babesiacaballi was used as the outgroup (GenBank accession No. AY534883). The validity of the tree topology obtainedwas tested by using bootstrap analysis with 100 resamplings. Branchings with bootstrap values ,50 are not shown,and areas where these branchings occurred are checkered. Intranuclear coccidia are in bold. Other sequences wereretrieved from GenBank: Adelina bambarooniae (AF494059), Besnoitia besnoiti (AF109678), Caryospora bigenetica(AF060975), Choleoeimeria sp. (AY043207), Cryptosporidium andersoni (AB089285), Cryptosporidium baileyi(AJ276096), Cryptosporidium serpentis (AF151376), Cryptosporidium wrairi (AF115378), Cyclospora colobi(AF111186), Eimeria bovis (U77084), Eimeria dipidomys (AF339490), Eimeria necatrix (U67119), Goussia janae


tory and proliferative changes in the respiratorytract and lung also seemed more acute than those inthe gastrointestinal tract and pancreas. Lunglesions were considered sufficiently severe tocontribute substantially to the death of affectedtortoises.

Nucleic acid sequences were obtained from onlytwo of nine cases. Possible reasons for failure toobtain sequences from the other seven cases includedegradation of DNA over time, cross-linkingduring formalin fixation of tissue, handling loss inthe laboratory, PCR failure, or failure of theprimers to bind. As the primers are designedtoward conserved areas and have successfullyamplified those of other coccidian species, webelieve that the last possibility is unlikely, and thatthe first two possibilities are the most probableexplanation.

Sequence data obtained from these isolatesprovides information for the design of nucleic-acidbased diagnostics. Previous phylogenetic analysisof coccidial 18S rRNA found two main clusters,which correspond roughly to the families Sarco-cystidae and Eimeriinae,12,13 and our results are inagreement. Our results also indicated that themonophyly of the genus Isospora is not supported,and that Goussia is not found within the familiesSarcocystidae and Eimeriinae. This is in agreementwith other nucleic acid-based coccidial phyloge-nies,12,13 providing support for the usefulness of thissubregion of 18S rRNA for limited phylogeneticanalysis. Phylogenetic analysis of the intranuclearcoccidial sequence does not provide support forinclusion in either the Sarcocystidae or theEimeriinae, and this organism may be paraphyleticto these families. Although TBLASTN resultsindicated the highest score with Hyaloklossialieberkuehni, more rigorous examination in thecontext of other sequences confuses this relation-ship. Although there is strong support for cluster-ing of this organism with other coccidia, additionalsequences of 18S rRNA and other genes from thisorganism and others are needed to more firmlyresolve the phylogeny of this organism.

To our knowledge, intranuclear coccidia havebeen reported from tortoises only once previously.9

The organisms reported here are morphologicallydistinct from those reported in radiated tortoises.In our tortoises, the meronts were 7 mm in greatestdiameter, whereas those in the previously describedcases were 15 mm in diameter. Oocysts in ourtortoises measured 8 mm in greatest diameter,whereas those in the previously reported tortoiseswere 12 mm in diameter. Additionally, our merontscontained over 16 merozoites, whereas those of theprevious report contained up to 16 merozoites. Theroute of oocyst shedding is unknown, but ispresumed to be the gastrointestinal tract on thebasis of presence of gametes and oocysts in themucosal epithelium; however, the presence ofcoccidia in epithelium of the urinary tract, re-spiratory tract, conjunctiva, male reproductivetract, and skin suggest that transmission may alsooccur after contact with secretions or excretionsfrom these sites. Oocyst isolation and sporulationstudies would provide additional useful informa-tion on the taxonomic status of the organism.Experimental infectivity studies would be needed toelucidate the complete pathogenesis of the in-fection.

Acknowledgements

The authors thank Drs. Bonnie Raphael, NancyLung, Donna Kleinpeter, Terry Norton, and Paul Raitifor submission of cases, and Leroy Brown of HistologyConsulting Service for technical assistance. We alsothank Jamie Kinion, Christie Buie and HannahThompson-Garner for data retrieval. This study waspartially funded by Northwest ZooPath.

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r

(AJ276096), Cryptosporidium serpentis (AF151376), Cryptosporidium wrairi (AF115378), Cyclospora colobi(AF111186), Eimeria bovis (U77084), Eimeria dipidomys (AF339490), Eimeria necatrix (U67119), Goussia janae(AY043206), Hepatozoon americanum (AF176836), Hepatozoon sp. (Boiga host), Hyaloklossia lieberkuehni(AF298623), Isospora robini (AF080612), Isospora suis (U97523), Lankesterella minima (AF080611), Sarcocystishominis (AF176945), Sarcocystis lacertae (AY015113), Sarcocystis tenella (L24383), and Toxoplasmagondii (L37415).


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13 Morrison DA, Bornstein S, Thebo P, WerneryU, Kinne J, Mattson JG: The current status ofthe small subunit rRNA phylogeny of thecoccidian (Sporozoa). Int J Parasitol 34:501–514,2004

Request reprints from Dr. Michael M. Garner, Northwest Zoopath, 654 W. Main, Monroe, WA 98272 (USA).E-mail: [email protected].


Intranuclear coccidiosis in tortoises: Nine cases

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