PREVALENCE AND MOLECULAR CHARACTERISATION OF HAEMOGREGARINES IN AFRICAN HINGE-BACK TORTOISES (KINIXYS BELLIANA AND KINIXYS HOMEANA) IN IBADAN, NIGERIA

Abstract

Kinixystortoises are one of the most vulnerable of all vertebrates, with about 61% reportedly threatened or becoming extinct. They represent a global biodiversity and therefore require urgent conservation attention.Kinixys tortoises are also sentinels of ecosystem degradation. They are an endangered speciesdue to human over-exploitation and infectious diseases including those caused by haemogregarines. Information on the prevalence, health indices, biodiversity and host-specificity of haemogregarines in African Hinge-back Tortoises (AHT) in Nigeria is scarce.This study was therefore designed to determine the prevalence of and characterise haemogregarines in AHT (Kinixys belliana and Kinixyshomeana) in Ibadan, Nigeria. One hundred and twenty AHT (K. belliana, n=70;K. homeana, n=50)captured from the wild were sourced from Bode Market in Ibadan, during the rainy (K. belliana, n=36;K. homeana, n=24) and dry (K. belliana, n=34;K. homeana, n=26) seasons between April, 2016 and December, 2017. Blood samples were collected to determine the prevalence of haemogregarines by light microscopy and confirmed using Polymerase Chain Reaction (PCR)with 18S RNA-specific primers. Purified amplicons were sequenced bi-directionally using a genetic analyser. Sequences obtained were aligned and compared with those in the GenBank. Phylogenetic analyses of the sequenced genes were performed using software. Haematology, plasma proteins and enzyme activities were evaluated in haemogregarine-positive and negative tortoises. The tortoises were examined for the presenceand quantity of vectors using standard morphological keys. Data were analysed using descriptive statistics, Student's t-test and correlation coefficient test at α0.05. Overall, haemogregarine prevalence was 53.3% and 75.8% by light microcopy and PCR, respectively. Generally, higher prevalence of haemogregarine (82.9%) was recorded in K. belliana compared to K. homeana (66.0%). Seasonal prevalence in K. homeana was significantly higher during rainy (91.7%) than dry season (42.3%). However, higher prevalence was recorded during dry (85.3%) than rainy (80.6%) seasons in K.belliana. Sequences (590 bp) generated had 100% similarity with Hepatozoon fitzsimonsi ex zobensis (KR069084) isolated from South African hinge back tortoise (K. zobensis). There was a significant difference between the counts of white blood cells in haemogregarine-positive tortoises (7.26+0.99109/L) than haemogregarine-negative (5.58+1.18 109/L) as well as for eosinophils in haemogregarine-positive (40.41+2.22%) than haemogregarine-negative (29.79+3.76%). Haemogregarine-positive recorded lower values of haematocrits (22.75+2.56%), total protein (3.97+0.87 g/dL), Albumin (1.26+0.29 mg/dL) and globulin (2.71+0.58 mg/dL), when compared with 32.79+2.68%, 5.33+0.93g/dL, 1.67+0.51mg/dL and 3.66+0.42mg/dL, respectively for haemogregarine-negative. However, haemogregarine-positive had higher values for ALT (33.91+14.42U/L), ALP (179.27+92.52 U/L) than 8.21+2.21 U/L and 147.93+10.51 U/L, respectively in haemogregarine-negative. Ticks of the genus Amblyomma were the only vectors found on thehaemogregarine-positive tortoises. There was a moderate positive correlation (r = 0.3758) between tick infestation and parasitaemia in the haemogregarine-positive tortoises. A high prevalence of haemogregarines in African hinge-back tortoisesin Ibadan was established, with Amblyommaticks as possible vectors. The identifiedhaemogregarine, Hepatozoonfitzsimonsi was closely related to that of South African origin. Routine screening of Kinixys tortoises for haemogregarines and ticks vectors is therefore recommended to promote their conservation.