Determination of Vitamin D and D-Dimer Levels in Dogs with Ehrlichia canis Infection

       
Vitamin D is a fat-soluble molecule with a steroid structure that increases the absorption of phosphate and calcium from the intestine and also plays a role in bone formation by providing the development of osteoid (Turgut, 2000). Requirement of Vitamin D is dependent on the P and Ca levels in the diet (Dede et al., 2018). Besides, vitamin D deficiency causes microbiological changes in the body (Holick and Chen, 2008). Vitamin D was also found to be effective on intestinal microbiota balance. It has been reported that it prevents invasion of pathological bacteria, reduces inflammation and provides cellular integrity (Asi, 1999).
       
D-dimer occurs by plasmin destruction when coagulation system for any reason caused by the activation of the cross-linked fibrin coagulation (Blomback et al., 1978). Among the clinical conditions leading to the increase of D-dimer in plasma; advancing age, neonatal period, pregnancy, hospitalization, infection, tumor, recent surgical interventions, trauma, burn, DIC, venous thromboembolism, ischemic cardiopathy, paralysis, peripheral cardiopathy, aneurysm, congestive heart failure, hemolysis, bleeding, acute respiratory syndrome, liver and kidney disease, inflammatory bowel disease, thrombolytic treatment and aortic rupture (Stein et al., 2006; Di Nisio et al., 2007).
       
The aim of this study was to determine the serum vitamin D levels and D-dimer levels in in dogs with Ehrlichiosis and relationship between different stages of the disease. It was also aimed to determine the levels of D-dimer and vitamin D at different stages of Ehrlichiosis infection for evaluating the alterations causing predisposition to thrombosis.

A total of 33 dogs were enrolled to those of different breeds, ages and sexes. This research was carried out at Adnan Menderes University, Faculty of Veterinary Medicine, between the period of August 2018-January 2019. Tentative and differential diagnosis was made by clinical examination and laboratory tests in 33 dogs with thrombocytopenia with or without clinical findings. All patients were evaluated for Ehrlichiosis with rapid Snap 4DX test kit and PCR as well. The study group dogs without any prior treatment application and demographic information involving physical examination findings, laboratory analyzes were obtained. Out of total 33 dogs screened, 27 were diagnosed with Ehrlichiosis by both rapid test kits and PCR method; and they were divided into four different groups (Table 1).
 

       
Descriptive statistics of the obtained data were performed by determining the mean and standard error values. As a result of normality test, the data of both parameters did not show normal distribution according to Kolmogorov-Simirnov analysis in some groups. After the transformation operations according to Log10 base, the data in these groups still did not constitute a normal distribution then non-parametric tests were used. Statistical analysis of the data was performed with the help of independent samples Kruskall Wallis test and comparisons were made between the groups. SPSS 21.0 (IBM, USA) program was used for all statistical analyzes and p <0.05 was considered statistically significant.
 
Molecular analysis
 
Blood samples from all subjects were examined by commercial test kits (Snap 4DX) and evaluated for Ehrlichiosis. Fresh whole blood samples with EDTA were used in all cases.
       
DNA; 200 µl of peripheral samples blood and centrifuged infected and non-infected DH82 cell cultures were isolated using DNAeasy Tissue Kit (Qiagen, Germany). DNA; The Milli-Q Integral System (Merck Millipore, USA) was washed with distilled water and then measured with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA).
 
Vitamin D analysis
 
Commercially produced test kits were used to quantitatively determine 25 (OH) D levels in plasma, serum or whole blood samples. Standard packages of 50 tests were used for evaluation. Fluorescent immuno-chromatography was performed using Savant fluorescent immunoassay (RDA Grup Istanbul, Turkey). The evaluation was based on competitive reaction principle. The reagent placement provided by coating the previously labeled 25 (OH) D complete antigen on the nitrocellulose membrane was performed according to the test kits. Then, the formation of anti-rabit immunoglobulin G monoclonal antibodies on the nitrocellulose membrane was performed as a control line. In the next step, mouse anti 25 (OH) D monoclonal antibody and rabbit Ig Gs were placed on the latex fluorescent microsphere. The pre-labeled antibody contained in the assay assembled with the 25 (OH) D antigen present in the donor’s blood to form the competitive envelope antigen. The biomarkers combined with the test line and control indicates the optical signals at a certain intensity. Concentration T / C (ratio of test line signal to control line) of the sample was determined by forming the standard curve with negative correlation with the sample concentration.
 
D-dimer analysis
 
Measurement of D-dimer concentrations made by fluorescence immunoassay rapid tests (Finecar A, Wondfo Biotech Co. Ltd., Finecar A, Atateknik Turkey). FIA meter commercial test kits (CTnI test, Finecare, Wondfo Biotech Co. Ltd) were used to measure serum CTnI concentration. Serum samples were transferred to the relevant laboratory at 20°C until the analysis period without hemolysis.

The D-Dimer and Vitamin D levels of the Study and Control Groups were shown in Table 1. Difference between the groups were found significant (p = 0.04). Regarding differences between the acute infected group and the control group a statistical significance was also evident (p = 0.23). Besides among the exposed group and the control group significance was presented (p = 0.004) for vitamin D analysis (Fig 1) (Table 2).
 

 

       
The difference between the general groups for D-dimer was found to be significant (p = 0.01: p = 0.022) between the control group and the active infected groups. On the other hand significance (p = 0.004) between the control group and the acute infected group were presented (Fig 2) (Table 2).
 

       
Ehrlichiosis is a vector-borne disease in dogs with anemia, thrombocytopenia and various systemic findings. Thromboembolism is one of the important factors affecting mortality levels in patients with bleeding disorders. It is known that deep vein thrombosis and thromboembolism might exist related to diseases causing bleeding disorder such as Ehrlichiosis (Griffin et al., 2003). In this study, after clinical and laboratory evaluations of the dogs with high fever, lymphadenopathy and anorexia which Ehrlichiosis was diagnosed in association to the usage of Snap 4Dx test (n = 27) and healthy dogs (n = 6) were also enrolled. Sera D-dimer (3059.0 ± 1074.4 ng / ml) levels of Ehrlichiosis-infected dogs were statistically higher than healthy dogs (p= 0.011). In dogs with Ehrlichiosis, D-dimer might be combined with advanced diagnostic techniques and even if thromboembolism exist, should be considered as a biomarker to shed light on the diagnostic approach in the veterinary field.
       
In recent studies, vitamin D is thought to be a hormone-like sterol rather than a fat-soluble vitamin and may serve as a key element in vital phenomena in addition to its actual metabolism (Dusso et al., 2005; Holick and Chen., 2008; Jussilaa et al., 2012). Vitamin D, which is suggested to be associated with various diseases, might have relationship with inflammation. The determination of VDR in inflammatory cells in peripheral blood points out that the vitamin may also be effective in the immune system. Vitamin D deficiency causes a decreased T cell response in immune reactions (Nicholson et al., 2012). Vitamin D suppresses Th1 cell proliferation, thereby suppressing the occurrence of interferon gamma and interleukin-2-like proinflammatory cytokines (Lim et al., 2005; Nerich et al., 2011). Proinflammatory cytokines, which are activated in vitamin D deficiency and are closely related to Th1 response, persist in the formation of several diseases (Cantorna et al., 2008; Hassan et al., 2013). In Ehrlichiosis, especially vitamin D levels were found to be very low when compared with control group, especially in the acute infected form and it was concluded that an effective immune response could not develop and facilitated the emergence of the disease.

This study was derived from the master thesis of Gozde Atikyilmaz (Thesis No: 2019-012/ Afyon Kocatepe University Health Science Institute). This research was supported by  Afyon Kocatepe University Scientific Research Projects (BAP) Coordinating Office (Project No; 18. Sag. Bil. 09). We thank Prof. Dr. Kerem URAL (Adnan Menderes University) for technical contribution and for comments on the manuscript.