Sheep Naturally Infected with Anaplasma ovis - Evaluation of Cardiac and Inflammatory Biomarkers

Abstract

Background: Anaplasmosis, also called gall sickness or tropical bovine ehrlichiosis, is an infectious disease caused by species belonging to the genus Anaplasma in domestic and wild animals in tropical and subtropical regions. Anaplasma ovis and A. phagocytophilum are important pathogens of sheep. A. ovis is considered the most common species affecting sheep. The infection is usually subclinical and progresses with high fever, anaemia, icterus, weight loss and abortions. This study aimed to investigate changes in cardiac damage markers, oxidative stress and antioxidant status, cytokines, and acute phase proteins in sheep naturally infected with A. ovis.

Materials, Methods & Results: For this purpose, a total of 40 animals, including 20 healthy sheep and 20 sheep infected with anaplasmosis, were used. A. ovis was diagnosed based on clinical findings and peripheral blood smear. Blood smears were prepared from the ear vein. The smears were stained with Giemsa and examined for the presence of Anaplasma spp. Infection was also confirmed by polymerase chain reaction (PCR) analysis. The genomic DNA was isolated from blood, and the MSP-4 gene region was amplified as A. ovis specific target gene. Twenty clinically healthy sheep of the same age group, reared under the same conditions and testing negative in the molecular assessment were used as controls. Blood samples were collected from the cephalic vein and and centrifuged to obtain serum. The serum stored at -20°C until the analysis stage. Serum samples were used for the analysis of cardiac damage markers [troponin I (cTnI), creatine kinase MB (CK-MB), lactate dehydrogenase (LDH) and aspartate transaminase (AST)], oxidative stress parameters [malondialdehyde (MDA), total antioxidant status (TAS), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], cytokines [interleukins IL-6, IL-1β and IL-10, tumour necrosis factor α (TNF-α), and interferon-γ (IFN-γ)] and acute phase proteins [C-reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (Hp)]. cTnI and CK-MB levels were measured using a chemiluminescent immunoassay. MDA, TAS, SOD, CAT,  GPx, TNF-α, IL-1β, IL-6, IL-10, IFN-γ, SAA and Hp levels  were measured by an ELISA reader. LDH, AST and CRP levels were measured in an autoanalyzer. cTnI and LDH levels were significantly increased in the infected animals compared to the healthy ones (P < 0.05). The concentration of AST was decreased in infected animals. MDA, TAS, SOD, CAT and GPx levels were significantly increased in the infected animals compared to the healthy ones (P < 0.05). The levels of the inflammatory parameters such as TNF-α, IL-1β, IL-10 and IFN-γ were significantly increased in the infected animals compared to the healthy ones (P < 0.05). Hp level were significantly increased in the infected group compared with the control group (P < 0.05). However, there was no significant change in CK-MB, SAA and CRP concentrations in the infected animals (P > 0.05).  

Discussion: Ovine anaplasmosis is an obligate intracellular arthropod disease that causes widespread changes in haemato-biochemical, immune response and oxidative stress parameters. Cardiac damage is often overlooked in field conditions due to the lack of adequate knowledge about the pathophysiology of the disease. Our results showed that A. ovis infection leads to significant changes in cardiac biomarkers and that the parasite can cause cardiac dysfunction. This is the first report on cardiac damage markers in Anaplasma-infected sheep. Additionally, the levels of proinflammatory and oxidative stress markers that may cause functional disorders were also found to be increased. Thus, measuring markers of cardiac function, oxidative stress and inflammation can be a useful tool in the early diagnosis of ovine anaplasmosis.

Keywords: anaplasmosis, cardiac damage, acute phase protein, cytokine, oxidative stress, ruminants.