Clinical communication — Kliniese mededeling

Putative clinical piroplasmosis in a Burchell’s zebra (Equus quagga burchelli)

F Lampena*, R Bhoorab, N E Collinsb and B L Penzhornb

2008. His estimated weight was 350 kg. ABSTRACT From a distance, he appeared in good A 10-year-old tame zebra gelding was presented after suffering from lethargy,nervousness, condition, slightly irritable, with a dull reported anaemia and icterus as well as a decreased appetite. These symptoms were seen coat. He was still eating selected food over some months, with changing severity. The animal was immobilised, treated, and offered by the owner and could be led blood specimens were submitted for haematology and biochemistry. This report describes around a paddock wearing a harness. molecular characterisation of Theileria equi recovered from this animal, as well as the clinical The owner reported a history of lame- findings, treatment and historical relevance of piroplasmosis in zebra in southern Africa. ness, which had been examined years Keywords: Burchell’s zebra, Equus quagga burchelli, piroplasmosis, Theileria equi, treatment. previously. At that stage no treatment was Lampen F, Bhoora R, Collins N E, Penzhorn B L Putative clinical piroplasmosis in a given, other than his hooves being Burchell’s zebra (Equus quagga burchelli). Journal of the South African Veterinary Association trimmed. The animal managed to cope 80(4): 257–260 (En.). Sindisa Wildlife Clinic and Conservation Centre, PO Box 15718, East with the lameness, but chose to generally Lynn, 0039 South Africa. graze in the vicinity of the farm house. A farrier was standing by to cut the hooves again concurrently with the examination. INTRODUCTION characterisation7,13,22. This is especially To allow closer examination, treatment Piroplasms have been reported from true for the parasites which cause equine and blood collection, the animal was various wild ungulate species; frequently piroplasmosis. Recently 3 genetically dis- immobilised by pole syringe using 5 mg they are documentations of organisms tinct T. equi groups have been identified etorphine (M99®, Novartis South Africa; seen on blood smears randomly taken based on 18S rRNA sequence data; simi- 9.8 mg/m ), combined with 60 mg Azape- from clinically normal animals25. larly 2 B. caballi groups have been charac- rone (Stresnil®, Janssen Pharmaceutica; Equine piroplasmosis is caused by 2 terised4. 40 mg/m ). Induction was very good, with intra-erythrocytic protozoan parasites, The only reported account of clinical sternal recumbancy reached within 3.5 Babesia caballi and Theileria equi (previ- piroplasmosis, attributed to Theileria equi, minutes. The heart rate was 60 beats/min- ously known as Babesia equi)4,11,21,31. These was in newly captured Grévy’s zebras in ute and respiratory rate 9 breaths/minute. parasites are tick-transmitted and infect Kenya9. A zebra owner and a trapper from Temperature was not taken. The mucous horses, mules, donkeys and zebras. Soon Uganda stated that they lost 20–25 % of membranes appeared normal and no after first being described from horses20,28, their animals to the disease, usually 2–3 other noticeable clinical abnormalities Theileria equi was reported from an East weeks after capture9. were found. Blood was collected from the African plains zebra (Equus quagga)in jugular vein into a 10 m serum (BD 190518. Inoculation of blood from this CASE HISTORY Vacutainer® CAT) and 6 m EDTA (BD zebra into horses caused high fever and The behaviour of a 10-year-old tame Vacutainer®, K2E) vacutainer tube and the appearance of piroplasms, leading to Burchell’s zebra (Equus quagga burchelli) the farrier commenced trimming the the conclusion that zebras are carriers gelding, from Cullinan, Gauteng, South hooves. 18 of the parasite . Piroplasms were also Africa, changed markedly over the course Treatment, by intramuscular injection, reported as an incidental finding from of a few months. The animal had recently consisted of 20 m long-acting penicillin plains zebras from Umfolozi in Kwa- been separated from a zebra mare and (Peni LA® , Virbac), 12 m sulpha- 23 Zulu-Natal, South Africa, in 1931 . Babesia foal due to uncharacteristic and severe diazine/trimethoprim (Norotrim 24®, spp. were reported from both plains and aggression, and now grazed with the Norbrook; Sulphadiazine 200 mg/m and Grévy’s zebras (Equus grevyi) from East local horses on a 100-ha farm. Grazing Trimethoprim 400 mg/m ), 10 m VitBCo 6 Africa . Theileria equi has also been reported consisted mostly of a mixture of sweet injection (Oberon Pharma) and 5 m from Cape mountain zebras (Equus zebra and sour veld grass, but the animal also non-steroidal anti-inflammatory drug 30,32 zebra) in South Africa . received additional pelleted horse food. (Ketofen®, Merial; 0.1 g/m ). An anthel- In many cases, identification of piro- He was regularly treated for ticks and mintic (5 m Ivermectin, MDB Iver 1 %, plasms based on morphology in a blood none could be seen on the animal at the CEVA) was also administered. smear is no longer appropriate; more time of examination. The owner noticed In spite of the gelding having a normal specific definition requires molecular that he started behaving in an increas- mucous membrane colour, he was treated ingly abnormal and irritable manner and for piroplasmosis based on the history of aSindisa Wildlife Clinic and Conservation Centre, PO Box that his appetite gradually decreased. His grey and at times ‘orange’ mucous mem- 15718, East Lynn, 0039 South Africa. bDepartment of Veterinary Tropical Diseases, Faculty of coat became very dull, and his gingivae branes, lethargy, decreased appetite, a Veterinary Science, University of Pretoria, Private Bag were orange-yellow, later turning grey- waxing and waning condition and other X04, Onderstepoort, 0110 South Africa. ish. He showed pronounced nervous- non-specific symptoms. Eight m imido- *Author for correspondence. ® E-mail: [email protected] ness, which was against his nature. carb dipropionate (Forray 65 , Schering- Received: May 2009. Accepted: October 2009. The animal was examined in November Plough Animal Health), which is a similar

0038-2809 Jl S.Afr.vet.Ass. (2009) 80(4): 257–260 257 dose as given to that for a horse of 400 kg, Table 1: Clinical chemistry and haematology readings for a Burchell’s zebra gelding, was administered intramuscularly. compared to normal values. The general anaesthetic was reversed Normal Mean* (SD)** Unit by administering 1.5 m diprenorphine ® hydrochloride (M5050 , Novartis South Clinical chemistry Africa; 12 mg/m ) intravenously. Recovery Urea 4.3 6.4261 (1.428) mmol/ was prolonged, and the gelding struggled Creatinine 126 167.96 (35.36) µmol/ to stand up. Even after half an hour he Total serum protein 75.3 64 (6) g/ appeared sedated, with his head hang- Albumin 34.7 31 (4) g/ ing. The owner reported that he looked Globulin 40.6 35 (3) g/ listless for the next 3 days, but then picked Albumin/globulin 0.85 up, and recovered well, until he appeared Aspartate aminotransferase 226 334 (88) U/ , 37°C to have returned to his normal demeanour Gamma-glutamyl transferase 51 53 (23) U/ , 37°C and appetite. Na+ 138.2 137 (3) mmol/ The blood samples were stored in a cooler K+ 3.81 3.9 (0.4) mmol/ ++ box after collection and, on completion of Ca (total) 2.78 2.65 (0.13) mmol/ + the procedure, were taken to the Clinical mg 0.72 0.66 (0.18) mmol/ – Pathology Laboratory, Department of Cl 103.1 97 (3) mmol/ Companion Animal Clinical Studies, Haematology Faculty of Veterinary Science, University Haemoglobin 148 150 (12) g/ of Pretoria, for further analysis. The labo- Red cell count 9.81 9.59 (1.15) 1012/ ratory prepared a blood smear stained Haematocrit 0.41 0.428 (0.059) / with a Diff-Quick Romanowsky stain. Mean corpuscular volume 42.2 44.6 (4.2) fl Intra-erythrocytic piroplasms were iden- Mean corpuscular haemoglobin 15.1 15.7 (1.7) g/d cells tified under the microscope. A full blood Mean corpuscular haemoglobin concentration 35.6 35.2 (3.5) g/d cells Red blood cell distribution width 16.9 % count and biochemistry analysis was also 9 performed (Table 1). The sample was then White cell count 9.73 7.8 2(2.6) x10 / Neutrophils (total)*** 7.59 x109/ sent on to the Department of Veterinary Neutrophils (mature)*** 7.59 5.19 (2.47) x109/ Tropical Diseases for molecular charac- Neutrophils (immature)*** 0 0.16 (0) x109/ terisation of the piroplasm. 9 Lymphocytes*** 1.95 2.37 (0.94) x10 / Haematology findings (Table 1) were Monocytes*** 0.19 0.276 (0.16) x109/ within the normal range (mean ± 1 stan- Eosinophils*** 0 0.22 (0.22) x109/ dard deviation) given for this species. Basophils*** 0 – x109/ Among clinical chemistry findings, urea, Platelet Count 219 256 (84) x109/ creatinine and AST levels were reduced, Anisocytosis 1+ while TSP and chloride levels were ele- Acanthocytes 1+ vated (Table 1). Three months after treatment the zebra *International Species Information System Physiological Data Reference Values 2002: Common zebra (Equus again started showing similar clinical signs. burchellii), male, 3–50 >20 year old, http://www.isis.org/cmsHome/ ** Standard deviation. The animal was once more treated with ***Absolute count. 8m imidocarb dipropionate, this time without requiring general anaesthesia. detection of B. caballi parasites, a recently were adjusted manually using BioEdit He made an uneventful recovery, and has developed TaqMan MGB real-time PCR (version 7.0.5.2)14. Searches of databases since then not shown any further clinical assay was used4. for homologous sequences were per- signs. A fragment of the parasite 18S rRNA formed using BLASTN3. gene was subsequently amplified using 3 The zebra sample tested negative for MOLECULAR DIAGNOSIS different primer sets. Primers NBabesia1F the presence of parasite DNA when using Genomic DNA was extracted from and 18SRev-TB were used in a primary the RLB assay. However, the presence of 200 µ of EDTA-treated blood using the PCR reaction to amplify a fragment of T.equi parasite DNA was confirmed using QIAamp DNA Blood Mini Kit (Qiagen, approximately 1600 bp. Two subsequent the real-time PCR assay developed for the Hilden, Germany) according to the nested PCR reactions were performed detection of T. equi parasites17. Parasite manufacturer’s instructions. Primers using primers NBabesia1F and BT18S3R, DNA could be detected at a cycle thresh- RLB-F2 (5’-GAC ACA GGG AGG TAG and primers 18SRev-TB and BT18S3F. All old (Ct) value of 30 which indicated the TGA CAA G-3’) and RLB-R2 (Biotin-5’- reactions were performed as previously presence of low parasitaemia. The fact CTA AGA ATT TCA CCT CTG ACA GT-3’) described4. Amplicons were purified using that T. equi parasite DNA could not be specific for Theileria and Babesia species, the Qiagen PCR purification kit (Qiagen, detected using the RLB was probably due were used to amplify the V4 hyper- Hilden, Germany) and samples were to the low parasitaemia in the sample. No variable region of the 18S rRNA gene of sequenced using BigDye chemistry (v.3.1, B. caballi DNA was detected using the the parasite/s present in the sample, as Applied Biosystems) in a 3130XL sequen- TaqMan MGB real-time PCR assay. described previously24. PCR products cer (Applied Biosystems). A fragment of the 18S rRNA gene were subjected to reverse line blot (RLB) Sequences were assembled and edited (~1170 bp) was sequenced and BLASTN hybridisation as previously described4. using gap4 of the Staden software suite26,27. analysis revealed that the sequence The piroplasm status of the animal was Multiple sequence alignments were showed 98 % identity to the 18S rRNA further confirmed using a TaqMan performed using the MAFFT alignment gene sequence of a T.equi-like isolate from real-time PCR assay developed for the (Multiple Sequence Alignment) employing a horse from Spain (strain ET1, accession detection of T. equi parasites17, and for the the FFT-NS-1 algorithm15,16. The alignments number AY534882)22 and 99 % identity to

258 0038-2809 Tydskr.S.Afr.vet.Ver. (2009) 80(4): 257–260 that of a sample from a mountain zebra immunity1. It has been shown that Rome, 24–26 September 1964: 262–263 from the Bontebok National Park (RBE intrauterine infection with T. equi in 10. de Vos A J 1979 Epidemiology and control of 4 bovine babesiosis in South Africa. Journal of 101, accession number EU642507) . horses may be the rule rather than the 2 the South African Veterinary Association 50: exception . In cattle, passive immunity, 357–362 DISCUSSION acquired from the dam, will protect new- 11. de Waal D T, van Heerden J 2004 Equine In horses, clinical signs of equine born calves. Subsequent to this, any infec- piroplasmosis. In CoetzerJAW,Thomson piroplasmosis range from acute fever, tion before 9 months will stimulate a solid G R (eds) Infectious diseases on livestock, Vol.1 (2nd edn). Oxford University Press, Cape immunity without the animal showing inappetence and malaise, to anaemia and Town: 425–434 jaundice, sudden death, or chronic weight any clinical signs. This stable situation 12. Frerichs W M, Allen P C, Holbrook A A 1973 11,31 loss and poor exercise tolerance . The requires a high prevalence of infection in Equine piroplasmosis (Babesia equi): thera- clinical signs seen in this zebra were very cattle, as well as a large enough tick popu- peutic trials of imidocarb dihydrochloride non-specific, but similar to those found in lation to ensure that calves become in horses and donkeys. Veterinary Record 93: 73–75 horses. The gelding’s response following infected during the critical period. There 13. Geysen D, Delespaux V, Geerts S 2003 treatment further substantiates that he are indications that in wildlife popula- PCR-RFLP using Ssu-rDNA amplification was showing clinical signs attributable to tions a similar endemically stable situa- as an easy method for species-specific the parasite. Treatment can suppress tion may exist25. It is speculated that any diagnosis of Trypanosoma species in cattle. clinical signs, but the currently available disruption to such stability may then Veterinary Parasitology 110: 171–180 treatments are ineffective in clearing result in the animal not building up the 14. Hall T A 1999 BioEdit: a user-friendly 11,29 25 biological sequence alignment editor and T. equi from carriers . Early claims of necessary immunity , leading to a analysis program for Windows 95/98/NT. successful clearing of T. equi infections by flare-up of the infection. Nucleic Acids Symposium Series 41: 95–98 means of an intensive imidocarb treat- During the 2nd consultation, the owner 15. Katoh K, Kuma K, Toh H, Miyata T 2005 ment regimen8,12 (4 treatments of 4 mg/kg stated that the gelding was treated for MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic at 72 h intervals) have not been substanti- ticks on a monthly basis, a protocol also 5,19 Acids Research 33: 511–518 ated by subsequent research . followed with the horses on the property. 16. Katoh K, Misawa K, Kuma K, Miyata T 2002 While the RLB assay failed to detect It was recommended that the owner MAFFT: a novel method for rapid multiple parasite DNA in a blood sample from this enforces a less stringent tick control sequence alignment based on fast Fourier zebra, a quantitative real-time PCR assay programme to enable the animal to build transform. 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