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Blood smear from an impala Contributed by: Emma Hooijberg, Yolandi Rautenbach Department of Companion Animal Clinical Studies, Faculty of Veterinary Science, University of Pretoria, South Africa Case presentation A three-month old male entire impala (Aepycerus melampus) (Figure 1) was presented to the Production Animal Clinic of the Onderstepoort Veterinary Academic Hospital with a history of weakness and anorexia. The current owners had recently adopted the animal with the intention of nursing it due to its poor condition. Further details concerning the animal’s previous history were unknown. Figure 1: Three-month old impala presented with weakness and anorexia Clinical examination revealed decreased mentation, poor body condition, mild hypothermia, severe tachycardia and mild tachypnoea. The impala was moderately dehydrated, capillary refill time was delayed and mucous membranes were pale. Ticks were noted in both ears. There was also a superficial skin wound around one of the horns, which was thought to be due to an unknown trauma. Blood samples were collected from the jugular vein in EDTA for haematology and in serum for clinical chemistry. Results of interest are presented in Table 1. Our laboratory does not have validated reference intervals for impala. Values considered to be abnormal based on data from goats and healthy impalas from a recent project (unpublished) are indicated. Photomicrographs of the blood smear are shown in figures 2-5. 1 Table 1: Haematology and chemistry results for a juvenile impala buck. Analyte Result Change Haematology (Siemens ADVIA 2120, manual differential white cell count) Haemoglobin (g/L) 45 Red cell count (x 1012/L) 6.3 Haematocrit (%)) 0.15 MCV (fL) 23.4 MCHC (g/dL) 30.3 RDW (%) 38.3 White cell count (x 109/L) 2.50 Seg neutrophils (x 109/L) 1.55 Lymphocytes (x 109/L) 0.90 Monocytes (x 109/L) 0.05 Platelet count (x 109/L) 958 Smear comment: See photomicrographs Chemistry (Roche Cobas Integra 400) Creatine kinase (U/L) 1644 Aspartate aminotransferase (U/L) 488 Creatinine (µmol/L) 67 Urea (mmol/L) 12 Figure 2: Blood smear from an impala (Diff-Quik stain, 500x magnification) 2 Figure 3: Blood smear from an impala (Diff-Quik stain, 1000x magnification) Figure 4: Blood smear from an impala (Diff-Quik stain, 1000x magnification) 3 Figure 5: Blood smear from an impala (Diff-Quik stain, 1000x magnification) 1) Describe the findings in the blood smear. 2) Classify the laboratory abnormalities. 3) Provide a master problem list. 4 Blood smear: Leukon: Leukocyte numbers were low and included reactive lymphocytes and occasional neutrophils showing toxic granulation (not shown). Erythron: Mild polychromasia and marked anisocytosis. There was a moderate poikilocytosis which included a mild acanthocytosis and schistocytosis. Occasional erythrocytes displayed basophilic stippling. Moderate numbers of erythrocytes contained a 1-2 µm in diameter, comma or signet ring-shaped intra-erythrocytic parasite, which was interpreted to be a Theileria spp. Low numbers of erythrocytes contained one to three dark blue coccoid organisms, situated in the cytoplasm away from the cell membrane. These organisms were consistent with Anaplasma centrale. Thrombon: Platelets were increased and showed a marked variation in size. Numerous spirochaetal organisms, measuring up to about 20µm in length, were seen extracellularly. These were consistent with a Borrelia spp. Anaplasma Theileria Laboratory abnormalities: 1) Moderate normocytic normochromic anaemia. No reticulocyte count was performed but the RDW of 38% is slightly higher than that seen in other impala healthy samples in our lab (range 21-37%) and there was a mild 5 polychromasia and basophilic stippling suggesting a mild regenerative response. 2) Moderate leukopenia with lymphopenia and toxic neutrophils. 3) Thrombocytosis with macroplatelets 4) Increased activities of CK and AST indicating a myopathy. Master problems: 1) Moderate (mildly regenerative) normocytic, normochromic anaemia and moderate lymphopenia associated with multiple haemoparasites and spirochaetemia. 2) Clinical signs and laboratory evidence of trauma. Further investigations: The blood sample was submitted for PCR amplification and a reverse line blot (RLB) hybridisation assay in order to identify the intra-erythrocytic parasites. Nucleic acid sequences consistent with Anaplasma centrale, Theileria bicornis, Theileria buffeli and Ehrlichia ruminantium were detected (the latter two organisms showed only a very faint reaction). 16s rRNA PCR amplification and sequencing was carried out in order to identify the spirochaetes. Initial sequencing results showed multiple bacterial species were present and cloning and repeat sequencing were performed. Sequence alignment was performed on the resulting sequences (http://blast.ncbi.nlm.nih.gov) which were found to be most similar to Borrelia theileri (KF569941.1) (100% cover, 99% identity). Case outcome: The impala was treated with intravenous fluid therapy (Lactated Ringers, Fresenius Kabi, South Africa) and an intravenous injection of diminazene (Berenil RTU®, Intervet, South Africa), but died about 18 hours after admission. Unfortunately no post- mortem examination was carried out. Discussion: African wild ruminants are considered to be the ancestral hosts of a wide variety of tick-borne blood parasites and are often infected without showing clinical signs.1 Multiple infections are common and the presence of a variety of blood parasites on blood smears from wild ungulates is not unusual and not usually considered clinically significant. However, when presented with such a blood smear from a sick antelope, a decision must be made as to whether and which organisms seen may be playing a role in the clinical condition of the patient. Treatment options vary. Anaplasma centrale: History: The first rickettsial pathogen was discovered in South Africa in 1910 by Sir Arnold Theiler, in a paper investigating a basophilic, punctate intra-erythrocytic organism in cattle that he named “Anaplasma”. 2 Based on the morphology of the organisms on blood smears, Theiler considered it to be a protozoan with only a 6 nucleus and no cytoplasm, hence the name. During his experiments, he noted that in cows with more severe clinical disease the Anaplasma was situated on the margin of the erythrocyte, and in cases with mild disease, the Anaplasma was situated more centrally. He accordingly named these two variants Anaplasma marginale and Anaplasma marginale variety centrale.3 He went on to effectively demonstrate the use of live A. centrale in 1911 to immunise cattle against A. marginale, a vaccination that is still in use globally today. Subsequent molecular and phyologenetic analyses have shown these organisms are not protozoa, but bacteria belonging to the order of rickettsias. 4 A. marginale and A. centrale are currently classified as separate species. Epidemiology and pathogenicity: Anaplasma marginale occurs worldwide and causes an acute haemolytic anaemia in cattle, as a result of intra- and extravascular (immune-mediated) destruction of parasitised erythrocytes. A centrale, on the other hand, causes only mild disease, characterised by transient pyrexia and no anaemia. A. centrale has been definitively identified in healthy impala. 1,5 A. centrale is transmitted by multi-host ixodid ticks, including Rhipicephalus simus. Blood smear: Anaplasma centrale is seen in peripheral blood smears as small round basophilic organisms situated centrally inside erythrocytes. Treatment: Tetracycline Ehrlichia ruminantium: History: Formerly known as Cowdria ruminantium, E. ruminantium was demonstrated as the causative agent of heartwater in domestic ruminants by Edmund Cowdry in South Africa in 1925.6 Cowdry was invited to investigate the disease by Arnold Theiler who had been working on heartwater since 1904.7 Epidemiology and pathogenicity: This rickettsial organism affects the endothelial cells of the capillaries of the brain, kidneys, spleen and other organs. It causes a vasculitis which leads to wide-spread haemorrhage and oedema (including hydropericardium or “heartwater”). This organism has been detected by PCR in the bone marrow of healthy free-ranging impala.8 Direct inoculation as well as tick transmission of this organism into impalas did not result in clinical signs, and E. ruminantium appears to be non-pathogenic in this species.9 This organism is transmitted by Amblyomma ticks. Blood smear: E. ruminantium is not visible in peripheral blood. Treatment: Tetracycline Theileria: History: Theileria spp. are tick-transmitted piroplasmids that have an initial sporozoite and merozoite stage in leukocytes followed by a piroplasm stage in erythrocytes in 7 their mammalian hosts. The first description of a Theileria piroplasm was made by Robert Koch in 1898 in Dar-es – Salaam.10 This piroplasm, the causative agent of East Coast Fever, was subsequently named Theileria parva in honour of Arnold Theiler, in 1907.10 Epidemiology and pathogenicity: Theileria spp. have a global distribution, and well- known examples include T. equi in horses and T. annae in dogs. When pathogenic, these agents cause either anaemia due to the piroplasm stage in the erythrocytes (e.g. T equi) or lymphadenomegaly and pulmonary oedema related to schizogony in lymphoid organs (e.g. T. parva).11 ‐ Theileria buffeli This theilerial organism occurs in cattle world-wide and has been reported in many wild African ruminants, including healthy impala.1 Although generally considered benign, some strains may be pathogenic
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