Characterization of serum protein electrophoresis patterns and C-reactive protein in canine tick-borne diseases

Abstract Background and Aim: Canine tick-borne diseases are important diseases with a worldwide distribution. In Thailand, the most important canine tick-borne diseases are ehrlichiosis, babesiosis, and hepatozoonosis. This study aimed to determine the serum protein electrophoresis patterns (SPEPs) and C-reactive protein (CRP) levels associated with Ehrlichia canis, Babesia canis, or Hepatozoon canis single infections. Materials and Methods: A total of 650 canine blood samples were collected from animal hospitals and clinics in Bangkok and its vicinity to examine health status and blood parasite infection. Suspected blood parasite infections were examined by buffy coat thin blood smear and confirmed by polymerase chain reaction. Normal dog and positive E. canis, B. canis, and H. canis single infections and serum protein profiles were determined by agarose gel electrophoresis. CRP concentration was measured by fluorescent immunoassay. Results: In dogs infected with E. canis, B. canis, and H. canis single infections, albumin levels and A/G ratios significantly decreased, whereas β2-globulin levels increased (p<0.05). The γ-globulin level significantly increased in E. canis and H. canis infections (p<0.05). A monoclonal gammopathy pattern wasi observed in E. canis and B. canis sngle infections, whereas β-γ bridging patterns and increased β- and γ-globulin fractions were found in H. canis single infections. The CRP level increased in dogs with blood parasite single infections and may be related to the pathogenesis of the infection. Conclusion: SPEPs and CRP levels can be used to monitor health status and blood parasite problems in infected dogs.


Introduction
Canine tick-borne diseases are transmitted by hard tick species, such as the brown dog tick (Rhipicephalus sanguineus), which is common in Thailand [1] and is distributed worldwide [2]. Ticks not only feed on blood cells but also transmit different types of pathogens, such as protozoa, viruses, rickettsia, and bacteria [3], causing both morbidity and mortality. Ehrlichia canis, Babesia canis vogeli, and Hepatozoon canis are commonly found in Thailand [4,5]. Ehrlichiosis and babesiosis can develop when a dog is bitten by an infected tick, but hepatozoonosis is caused by its ingestion. B. canis subspecies vogeli is the main species causing canine babesiosis in Thailand [5]. B. canis is the large form of Babesia spp. Babesia organisms enter and multiply in the host erythrocytes. Clinical manifestations are anorexia, lethargy, pale mucous membranes, fever, jaundice, chronic nephropathy, and glomerulonephritis [6]. The severity of infection depends on the subspecies of canine babesiosis. B. canis causes a subclinical to a mild degree of infection [6,7]. E. canis is a Gram-negative intracellular rickettsia that can infect monocytes and lymphocytes in dogs [8]. Canine ehrlichiosis can be classified into three stages according to clinical signs: Acute, subclinical, and chronic. In the acute stage, clinical signs appear at 1-3 weeks after infection and include fever, weakness, lethargy, depression, lack of appetite, and limb edema. In the subclinical stage, the organism may be present for months to years without clinical symptoms. In the chronic stage, the infected dog has abnormal bleeding due to thrombocytopenia, severe weight loss, fever, difficulty in breathing due to lung inflammation, joint pain, seizures in some cases, lack of coordination, anemia, and kidney failure [9]. H. canis is an apicomplexan parasite that belongs to the family Hepatozoidae. The clinical signs of H. canis infection can vary from subclinical to severe and life-threatening. The most frequently observed clinical signs are anemia, extreme lethargy, intermittent fever, and emaciation [7].
Copyright: Asawakarn and Taweethavonsawat. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons. org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Available at www.veterinaryworld.org/Vol.14/August-2021/22.pdf Serum protein electrophoresis patterns (SPEPs) show fractions of two major types of protein: Albumin and globulin. Albumin is the single most abundant protein in the serum and is synthesized by the liver. Globulins are also synthesized by the liver, except for immunoglobulins. In dogs, globulin fractions can be separated into five fractions: α1, α2, β1, β2, and γ. The measurement of serum protein may help detect and monitor various diseases and pathological processes. SPEPs can be used as a diagnostic tool in a wide spectrum of diseases, including infectious and inflammatory diseases, renal, hepatic, and gastrointestinal disorders, immunodeficiency status, and paraproteinemia, caused by plasma cell neoplasia [10]. The acute-phase response is considered part of the innate host defense system, and the systemic effects include leukocytosis, fever, and increased blood cortisol. C-reactive protein (CRP) is one of the acute-phase proteins and is synthesized by hepatocytes, smooth muscle cells, macrophages, endothelial cells, lymphocytes, and adipocytes. It is a major acute-phase protein in dogs and is part of the γ-globulin fraction. Its concentration increases dramatically in response to inflammation, infection from pathogens (including bacteria and parasites), and injury and has been used as a predictive marker for disease risk and to monitor the response to treatment [11].
This study aimed to investigate the SPEPs and CRP concentrations associated with single infections of E. canis, B. canis, and H. canis.

Ethical approval
The research protocol was approved by the Institutional Review Board of Chulalongkorn University Animal Committee (approval no. 1931052). All methods were performed in accordance with relevant guidelines and regulations.

Study period and location
Blood samples were collected from Bangkok and its vicinity area during January-December 2020.

Sample collection
A total of 650 canine (Canis familiaris) blood samples were collected and examined for health status and blood parasite infection. Some cases showed clinical signs of blood parasite infection, including anorexia, lethargy, pale mucous membranes, and fever. Only a few cases exhibited jaundice. Samples were collected in EDTA tubes and serum collection tubes and suspected blood parasite infections were examined by buffy coat thin blood smear and confirmed by polymerase chain reaction according to Rucksaken et al. [8]. The numbers of dogs with B. canis, E. canis, or H. canis single infections were 13, 20, and 14, respectively. The criteria for normality were healthy dogs with no clinical signs of blood parasite infection and no history of ectoparasite infestation. Blood chemistry profiles of normal dogs were in the normal range [12]. All serum samples were kept at −20°C until analysis.

Serum protein profile determination and electrophoresis
Serum from normal (n=9), B. canis (n=13), E. canis (n=20), and H. canis (n=14) single infections was analyzed for total serum protein and SPEPs. Total serum protein was measured by the Biuret colorimetric test (Human ® , Wiesbaden, Germany). The serum protein samples were separated by agarose gel electrophoresis (SPIFE ® split beta SPE kit; Helena Laboratories, TX, USA). Fifteen microliters (1.3 mg) of the serum protein sample were placed in each well, and electrophoresis was performed at 400 V for 6 min. The gel was pre-dried at 53°C for 12 min, stained with acid blue staining solution, and destained in citric acid destaining solution. All steps were carried out in an automated machine (Spife ® 3000; Helena Laboratories). The density of each serum protein band in the electrophoresis pattern was measured and analyzed using QuickScan Touch (Helena Laboratories).

Measurements of CRP concentration
CRP concentrations of blood parasite-positive serum samples were measured by fluorescent immunoassay (Vcheck Canine CRP 2.0 Test kit; Bionote, South Korea). About 5 µL of each sample was diluted in 4 mL diluent buffer from the test kit. About 100 µL of the diluted sample was mixed and added to the test device. The CRP concentration was displayed on the screen after 5 min. A CRP concentration above 30 mg/L was considered abnormal.

Statistical analysis
SPEP data for blood parasite single infections were tested using analysis of variance. Tukey's test was used for pairwise comparisons between single infections and normal dogs. Significance was set at p<0.05. The normal distribution of SPEP data was tested using GraphPad Software, (GraphPad, San Diego, CA, USA).

Results
SPEPs were determined by agarose gel electrophoresis ( Figure-1). Tables-1 and 2 show the total protein, albumin, and globulin fractions and A/G ratios in single blood parasite infections. Comparisons were made among single infections of B. canis, E. canis, or H. canis in normal dogs and between groups. There were no significant differences among the three groups of blood parasite infections. In this study, the monoclonal gammopathy was 39% (5 of 13) of B. canis and 35% (7 of 20) of E. canis single infections. In dogs with H. canis single infection, the serum protein pattern showed β-γ bridging and an increase in βand γ-globulin peaks in 43% (6 of 14) of the cases.

Discussion
Serum protein profile is one of the standard tests used to monitor health and disease status, such as infections and acute and chronic inflammatory responses. In this study, the average relative concentration of serum protein fraction albumin levels and A/G ratios significantly decreased, whereas the absolute total protein of H. canis significantly increased. Decreased albumin concentrations are usually caused by acute inflammation, liver damage, starvation or cachexia, digestive disorders, or kidney diseases [13] .  However, the pathogenesis of B. canis, H. canis, and E. canis infections is usually caused by acute inflammation and/or liver damage [9,14]. Decreased A/G ratios result from a reduction in albumin concentrations and an increase in globulin concentrations. In the previous report, ehrlichiosis (in subclinical and chronic diseases) was associated with significant hypoalbuminemia, hyperglobulinemia, and hypergammaglobulinemia [15]. The serum protein profile of B. canis-infected dogs showed decreased albumin concentrations and A/G ratios but increased αand β-globulin concentrations. The abnormalities in β-globulin concentrations might be due to hypertransferrinemia [16,17]. The total protein of H. canis increased, probably due to hyperglobulinemia caused by the stimulation of the humoral response of the organism to severe chronic inflammation [18,19].
For the globulin fraction, the relative concentrations and absolute values of β2-globulin protein levels significantly increased in B. canis, H. canis, and E.  infections. An increased β2-globulin level may result from increased C3a (complement) protein concentrations. Complements are involved in the regulation of inflammatory processes, and this complement protein plays a role in the development of intravascular hemolysis, especially in babesiosis [16,17]. The γ-globulin fractions increased in all three groups, with significantly increased absolute protein values in E. canis and H. canis infections. The γ-globulin fraction consists of various classes of immunoglobulins, and an increase in the fraction could lead to monoclonal gammopathy (narrow peak) or polyclonal gammopathy (broad peak). Monoclonal gammopathies result from a single line of B lymphocytes or plasma cells, whereas polyclonal gammopathies are usually an indication of chronic inflammation and chronic liver damage [13]. In this study, monoclonal gammopathy was found in 35% of E. canis and 39% of B. canis single infections. In a previous study, polyclonal and benign monoclonal gammopathies were observed in E. canis-infected dogs [20]. In dogs with H. canis single infection, the serum protein pattern showed β-γ bridging and an increase in the βand γ-globulin peaks in 43% of the cases. The β-γ bridging pattern is usually found in liver damage [21]. The γ-globulin concentrations of H. canis single infection differed significantly from other blood parasite infections because hepatozoonosis has a longer life cycle than others and produces a chronic immune response [14].
CRP is a major acute-phase protein in dogs, mostly synthesized in the liver after tissue damage caused by infection, inflammation, or trauma. The acute-phase response is an innate host defense mechanism during tissue injury or immunological disorders and in the early stage of blood parasite infections. It is responsible for the accumulation and activation of granulocytes and mononuclear cells, releasing cytokines, interleukin (IL)-1, IL-16, and tumor necrosis factor-α [22]. In this study, the average CRP concentrations in dogs with E. canis, B. canis, and H. canis single infections were higher than the reference range (>30 mg/L). The average CRP concentration in B. canis single infection was higher than E. canis and H. canis single infections. In canine hepatozoonosis, most dogs were within normal levels, and a few increased. In a previous study, the CRP concentration in dogs infected with E. canis increased during the acute stage of infection; this increase could eliminate E. canis in macrophages of infected dogs. The CRP level might help assess the severity of inflammatory damage in E. canis-infected dogs, and veterinarians may use this information to choose inflammatory therapy [23]. The typical hematological abnormality of blood parasite infections, including ehrlichiosis, babesiosis, and hepatozoonosis, is anemia. Ehrlichiosis and babesiosis might be caused by canine immune-mediated anemia (IMHA) [24,25]. Serum CRP concentrations in canine autoimmune hemolytic anemia and primary IMHA are increased [26,27].

Conclusion
SPEPs in a blood parasite single infection mostly showed decreased albumin levels and A/G ratios and increased β2and γ-globulin levels. CRP concentrations extremely increased in dogs with all blood parasite infections. SPEPs and CRP levels can help veterinarians monitor health status and blood parasite problems in sick dogs during the treatment process.

Authors' Contributions
SA and PT: Designed the study, corrected data, data analysis and interpretation, and drafted the manuscript. The authors have read and approved the final manuscript.