Ascaridiosis is one of the most important zoonotic diseases worldwide³. The disease is most common in dogs younger than 6 months old⁴. The disease is transmitted by the fecal-oral and placental routes⁵. Ascaridiosis causes, vomiting, growth retardation, immunosuppression, tympani, constipation, and mechanical obstruction in the digestive tract in puppies⁶. The mechanism of the development of anemia is due to the absorption capacity violation of the mucous membrane invaded with toxocara. Long-term B12 deficiency is a serious disease. It appears within the development of megaloblastic anemia and/or neurological disorders³. Toxocara canis causes paralysis of motor neurons. The probable cause of mental stagnation and hind leg paralysis is paralysis of motor neurons⁴.

Fecal examination plays an important role in the diagnosis of the disease⁷. There are biomarkers that have recently been used in disease diagnosis and provide information about disease severity ⁶. In recent years, studies on biomarkers have been emphasized in order to evaluate the severity of disease in digestive system diseases caused by various reasons in dogs^8-10. Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker released by monocytes, neutrophils, macrophages, and T cells that is involved in a variety of immunological reactions, including migration, adhesion, differentiation, and proliferation ¹¹. It plays an important role in the assembly of inflammatory cells by showing chemotaxic properties¹². As a result, serum suPAR levels are important in determining both the severity of inflammation and the infected animals prognosis¹³. The polypeptide-structured liposaccharide binding protein (LBP) is an acute phase reactivator produced by the liver and muscles¹⁴. The molecular weight of this protein is 58 kDa. Its primary function is to bind endotoxins and form the LBP-endotoxin complex, which then stimulates Toll-like receptors and initiates the inflammatory response via cytokine release¹⁵. An acute phase response occurs with the occurrence of damage in living tissues. The primary goal is to eliminate both tissue damage and pathogens that cause damage ¹⁶. The liver produces acute-phase proteins during the inflammatory response, and their levels change before clinical signs of the disease appear. Ceruloplasmin and haptoglobin are among the important acute-phase proteins¹⁷.

The assessment of biomarkers, acute phase proteins, parasitological examination clinical and haematological findings provides critical information for confirming the diagnosis and determining the severity of the disease. The aim of this study was to determine the changes in serum suPAR, LBP, ceruloplasmin, haptoglobin levels in dogs infected with Toxocara canis.

Animals: Animal material of this study consisted of 30 dogs of different breeds and genders at the age of 1-6 months that were diagnosed with T. canis based on stool examination and brought to the Department of Internal Medicine of the Faculty of Veterinary Medicine of Kafkas University with complaints of anorexia, diarrhea, and vomiting. Twenty dogs with healthy clinical and laboratory findings formed the control group. Clinical and vital signs were examined to determine the control group. However, microscopic fecal examination was performed. Those with normal clinical and vital signs and no ascarid eggs in their stool were included in the control group. Age of the control group was less than 6 months. The sick dogs were brought to our clinic within 24 hours after they started to show clinical symptoms without any other treatment or medication. Clinical examinations of the animals in the infected animal and control groups were performed and rectal body temperature, pulse rate per minute, and respiratory rate were determined.

Collection of Blood Samples: Blood samples were collected from sick dogs before treatment and from healthy dogs once with the help of holder and compatible sterile needle tip (Vacuette®, Greiner Bio-One GmbH, Austria) into vacuum gel serum tubes (BD Vacutainer®, BD, UK) and vacuum EDTA blood tubes (BD Vacutainer®, BD, UK) V. cephalica taken from antebrachi. To obtain serum, the blood samples were centrifuged at 3000 rpm for 10 minutes (Hettich Rotina 380R®, Hettich, Germany). The serum samples were kept at -20⁰C until used for analysis

Biochemical and Hematological Analyses: Total leukocyte count (WBC), erythrocyte count (RBC), haematocrit percentage (Hct), haemoglobin concentration (Hb), lymphocyte count (Lym), monocyte count (Mon), granulocyte count (Gra) were determined from whole blood samples using a complete blood counting device (VG-MS4e®, Melet Schloesing, France), were measured.

The serum samples were analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP) enzyme activities while glucose, urea and total bilirubin (TBIL) levels using a fully automatic biochemistry device (Mindray BS120®, Mindray Medical Technology, Istanbul, Türkiye).

Commercial canine specific ELISA kits were used to determine serum suPAR (Dog suPAR ELISA Kit®, Cat No: ELK9083, ELK Biotechnology, China) and LBP (Dog LBP ELISA Kit®, Cat No: ELK9084, ELK Biotechnology, China) concentrations. ELISA tests were carried out according to the manufacturer's instructions, and optical density was determined using an ELISA reader (Epoch®, Biotech, USA) at 450 nm. Regression analysis was used to read the suPAR and LBP values. Haptoglobin and ceruloplasmin levels were determined using the colorimetric method^18,19. Başbug et al.^20, reported that the specificity of this ELISA Kits was 93.3% in their study on dogs.

Fecal Examination: The fecal samples from 1-6 months old dogs with diarrhea were collected at the Kafkas University, Veterinary Faculty Animal Hospital and were transferred in sterile fecal sample containers with animal information recorded and kept at 4⁰C until examination. Firstly, the adult forms of the helminths (T. canis) were screened in the macroscopically examined samples and then all the fecal samples were examined under a light microscope by fulleborn flotation technique using saturated saline solution (specific gravity: 1.45) for the identification of egg forms. Species identifications were made using the relevant literature based on the morphological characteristics of the detected helminth eggs^21,22. Fecal samples taken from dogs with diarrhea were used with triple rapid test kits (Canine Parvovirus, Giardia, Coronavirus Antigen-Ag Test, Asan Easy Test, China) and those with negative test results were included in the study.

Statistical Analysis: Statistical analysis of the data was performed using SPSS® (SPSS 26.0, Chicago, IL, USA) software. The statistical differences between the groups with normal distributions according to the Shapiro-Wilk test were compared by the independent sample t-test. The obtained results were given as the mean ± standard error of the mean (SEM). P<0.05 was considered statistically significant in the evaluation of the results ²³.

Büyütmek İçin Tıklayın

Table 1: Mean and standard error values of hematological and vital signs in patient and control dog

Clinical and hematological findings of sick and healthy dogs are given in Table 1. A comparison was made between the infected animal group and the control group. Among the haematological parameters; RBC (x10⁶/μL), Hct (%), and Hb (g/dL) (P<0.001), Mon (x10³/μL) count (P<0.05) showed statistically significant differences when compared to the control group. Since the animals in the infected animal group had anemia, RBC, Hb and Hct levels decreased significantly. In addition, WBC (x10³/μL), LYM (x10³/μL), Gra (x103/μL) counts among haematological parameters were not statistically different (P>0.05) in the infected animal group compared to the control group.

Biochemical findings of sick and healthy dogs are given in Table 2. In serum biochemistry, statistically significant results were found according to ALT (P<0.05), AST (P<0.05), GGT (P<0.05), ALP (P<0.001) and TBIL (P<0.05) levels in the infected animal and control groups. Urea, TBIL and Glucose levels showed no statistical difference between the patient and control groups (Table 2). Serum suPAR (P<0.001), LBP (P<0.001), ceruloplasmin (P<0.001) and haptoglobin (P<0.05) concentrations were significantly higher in the infected animal group compared to the control group (Figure 1A, 1B, 1C, 1D).

Büyütmek İçin Tıklayın

Table 2: Mean and standard error values of biochemical findings in patient and control dogs

Büyütmek İçin Tıklayın

Figure 1: Comparison of suPAR, LBP, ceruloplasmin (CP) and haptoglobin (Hp) concentrations in the patient and healthy group. A. Comparison of suPAR concentrations in the patient and healthy group (P<0.001). B. Comparison of LBP concentrations in the patient and healthy group (P<0.001). C. Comparison of ceruloplasmin concentrations in the patient and healthy group (P<0.001). D.Comparison of haptoglobin concentrations in the patient and healthy group (P=0.003).

All of the sick dogs in our study had a tense abdominal wall, retarded growth and a depressed state. The agent causes internal bleeding as a result of intestinal perforation, which causes anaemia in puppies infected with T. canis. Hb, Hct, and RBC, which are used to evaluate anemia in sick dogs, were found to be statistically low in the current study compared to the control, most likely since the causative agent caused internal bleeding by causing intestinal perforation²⁶. In this manuscript, necropsy was performed on two of the dogs in infected animal group that died. As a result of necropsy, it was observed that there was bleeding due to perforation in the intestines, consistent with the literature.

In infected dogs elevations in liver enzymes might be observed during the migration of the larva the difference here was found to be significant. During the migration of the larvae, mechanical damage occurs in the liver. There is an increase in liver enzyme activity due to this damage²⁶. Despite the statistical difference, GGT, ALP, ALT, and AST enzyme activities remained within normal reference values in the current study (Table 2). It is stated in the literature that liver enzyme activity will not increase unless 75% damage occurs in the liver²⁷. We think that the reason why ALT and GGT levels, which are important indicators of liver damage, are low in the infected animal group may be due to the fact that liver damage is less than 75%.

The level of suPAR increases in body fluids in infectious and tumoral diseases and in cases where immune response develops. Serum suPAR levels, as a result, provide information about the degree of immune response²⁸. Serum suPAR is released by monocytes, neutrophils, macrophages, and T cells and is involved in a variety of immunological functions, including migration, adhesion, differentiation, and proliferation. Its amount in leukocytes increases in inflammatory conditions¹¹. In our study, the infected animal group had a statistically higher number of monocytes and serum suPAR levels than the control group, which could be the cause of increased as a result of inflammation. The correlation table has been omitted from the manuscript to avoid ambiguity. suPAR is released from monocytes. Depending on the inflammation, there is an increase in the number and activation of monocytes. Depending on the activation in monocytes, the serum suPAR level increases. We think that an immune response is formed against parasitic invasion and thus contributes to the increase in suPAR.

Gucsav and Akyuz²⁹ found that increased suPAR levels in dogs with parvoviral enteritis were associated with the development of systemic inflammatory response syndrome. The reason for the increase in suPAR level in the infected animal group, similar to our study, could be the development of severe inflammation as a result of T. canis damage in the intestines. An increase in serum suPAR indicates activation of the immune system and inflammatory response. It provides information on both the severity of inflammation (which increases in inflammatory and infectious diseases) and the disease prognosis^13,30,31. Infected animals with high suPAR levels have a poor prognosis^28,32,33.

Lipopolysaccharide binding protein is an acute phase protein produced by the liver for the organism's immune response to endotoxins^14,15. It has a very important role in the differentiation of systemic inflammatory response syndromes (SIRS) of infectious and noninfectious origin^15,34. The possibility of SIRS development in dogs in our study may have increased the LBP levels in the infected animal group compared to the control group. Furthermore, haptoglobin and ceruloplasmin levels were found to be statistically higher in the infected animal group compared to the control group, indicating that the inflammation was severe. Given that LBP induction is slow and elimination is rapid, it should be used in conjunction with other biomarkers to determine the severity of an inflammation¹⁵. Odabaşı and Bülbül³⁵ report that an increase in LBP levels occurs 6-8 hours after infection. The fact that the sick dogs were brought in within a short period of time (within 24 hours of the disease onset) was found to be consistent with the fact that LBP increased significantly more than other acute-phase proteins. LBP quickly reflects the severity of the inflammation.

Acute phase proteins derived from the liver are produced in response to acute phase stimuli such as inflammation, tissue damage, and infection^36,37. Haptoglobin is another acute-phase protein concentration of which rises during acute infection, inflammation, and trauma and provides information about disease severity^29,38.

In a study on puppies with parvoviral enteritis, haptoglobin concentration increased due to perforation and inflammation in the intestine caused by gastroenteritis²⁹. The statistically higher haptoglobin concentration in the infected animal group compared to the control group in our study is most likely the result of intestinal destruction and severe inflammation. T. canis larval liver migration may also stimulate haptoglobin synthesis. Ceruloplasmin protects cells against oxidative damage and has cytoprotective activity³⁹. Ceruloplasmin can also be used to detect infection and inflammation⁴⁰. In many studies, ceruloplasmin concentration increased due to infection^37,39,41. Increased ceruloplasmin concentrations in the infected animal group compared to the control group in our study could be the result of inflammation, similar to haptoglobin.

In conclusion, serum suPAR, LBP, ceruloplazmin and haptoglobin levels changed in T. canis-infected puppies with diarrhea. The parameters examined showed a significant increase. Furthermore, given the fact that there are few studies on suPAR and LBP in veterinary medicine, this study will be a source of new research in this field.

1) Nind F. Small animal gastroenterology. In: Chandler M, Nind F. (Editors). Solutions in Veterinary Practice. 1 st Edition, St. Louis Missouri: Elsevier Saunder 2011: 85-87.

2) Willard MD. Small Animal Internal Medicine. St. Louis: Elsevier, 2013.

3) Macpherson CN. Dog zoonoses and human health: A global perspective. CAB Mini Rev 2013; 8: 1-2.

4) Overgaauw PAM, Van Knapen F. Veterinary and public health aspects of Toxocara canis. Vet Parasitol 2013; 193: 398-403.

5) Rostami A, Riahi SM, Fallah Omrani V, et al. Global prevalence estimates of Toxascaris leonina infection in dogs and cats. Pathogens 2020; 9: 503.

6) Epe C. Intestinal nematodes: biology and control. Vet Clin North Am-Small Anim Pract 2009; 39: 1091-1107.

7) Dryden MW, Payne PA, Ridley R, Smith V. Comparison of common fecalflotation techniques for the recovery of parasite eggs and oocysts. Vet Ther 2005; 6: 15-28.

8) Heilmann RM. Evaluation of Canine S100A12 and sRAGE as Novel Disease Markers in Dogs with Inflammatory Bowel Disease. PhD Thesis, Texas: A&M University, 2015.

9) Heilmann RM, Steiner JM. Clinical utility of currently available biomarkers in inflammatory enteropathies of dogs. JVIM 2018; 32: 1495-1508.

10) Akyuz E, Gokce G. Neopterin, procalcitonin, clinical biochemistry, and hematology in calves with neonatal sepsis. Trop Anim Health Prod 2021; 53: 354.

11) Bilgili B, Cinel I. The significance of soluble urokinase plasminogen activator receptor (suPAR) in ICU patients. Türk Yoğun Bakım Derneği Dergisi 2013; 11: 33-39.

12) Backes Y, Van Der Sluijs K, Mackie DP, et al. Usefulness of suPAR as a biological marker in patients with systemic inflammation or infection: A systematic review. Intensive Care Med 2012; 38: 1418-1428.

13) Matsuyama A, Wood GA, Speare R, Schott CR, Mutsaers AJ. Prognostic significance of the urokinase plasminogen activator system in tissue and serum of dogs with appendicular osteosarcoma. PLoS ONE 2022; 17: e0273811.

14) Jerala, R. Structural biology of the LPS recognition. Int J Med Microbiol 2007; 297: 353-363.

15) Sonmezer MÇ, Tulek N. Biomarkers in bacterial infections and sepsis. Klimik Derg 2015; 28: 96-102.

16) Milanovic Z, Vekic J, Radonjic V, et al. Association of acute Babesia canis infection and serum lipid, lipoprotein, and apoprotein concentrations in dogs. J Vet Intern Med 2018; 33: 1686-1694.

17) Gokce Hİ, Bozukluhan K. Important acute phase proteins in livestock and their use in veterinary medicine. Dicle Üniv Vet Fak Derg 2009; 1: 1-14.

18) Colombo JP, Richterich R. On the determination of ceruloplasmin in plasma. Schweiz Med Wochenschr 1964; 23: 715-720.

19) Batchelor J, Fuller JC, Woodman DD. A simple method for measurement of the haemoglobin binding capacity of canine haptoglobin. Lab Anim 1989; 23: 365-369.

20) Basbug O, Aydogdu U, Agaoglu Z. Neopterin and soluble urokinase type plasminogen activator receptor as biomarkers in dogs with systemic inflammatory response syndrome. J Hell Vet Med Soc 2020; 71: 1945-1952.

21) Urquhart GM, Armour J, Duncan JL, Dunn AM, Jennings FW. Veterinary Parasitology. 2nd Edition, Great Britain: Blackwell Science Ltd, 1996.

22) Bozukluhan K, Merhan O, Kiziltepe S. Determination of haptoglobin, some biochemical and oxidative stress parameters in calves with pneumonia. Fresenius Environ Bull 2021; 30: 9492-9496.

23) Tekin ME. Veterinerlik Biyoistatistik. Konya: Atlas Nobel Yayınevi, 2021.

24) Sarchahi AA, Borji H. Generalized lower motor neuron paralysis associated with Toxocara canis in a husky puppy. Comp Clin Pathol 2017; 22: 993-996.

25) Schnieder T, Laabs EM, Welz C. Larval development of Toxocara canis in dogs. Vet Parasitol 2011; 175: 193-206.

26) Kocademir S, Yildiz K. Toxocara canis ve visceral larva migrans. Bull Vet Pharm Tox 2022; 13: 47-54.

27) Turgut K. Veterinary Clinical Laboratory Diagnosis. Ankara: Bahçıvanlar Press Industry, 2000.

28) Erkiliç EE, Merhan O, Kirmizigul AH. Salivary and serum levels of serum amyloid a, haptoglobin, ceruloplasmin and albumin in neonatal calves with diarrhoea. Kafkas Üniv Vet Fak Derg 2019; 25: 583-586.

29) Gucsav AB, Akyuz E. Diagnostic and prognostic value of lipopolysaccharide binding protein and soluble urokinase plasminogen activator receptor in dogs with parvoviral enteritis. Eurasian J Vet Sci 2023; 39: 37-45.

30) Ostergaard C, Benfield T, Lundgren JD, Eugen-Olsen J. Soluble urokinase receptor is elevated in cerebrospinal fluid from patients with purulent meningitis and is associated with fatal outcome. Scand J Infect Dis 2004; 36: 14-19.

31) Wittenhagen P, Kronborg G, Weis N. The plasma level of soluble urokinase receptor is elevated in patients with streptococcus pneumoniae bacteraemia and predicts mortality. Clin Microbiol Infect 2004; 10: 409-415.

32) Lawn SD, Myer L, Bangani N, Vogt M, Wood R. Plasma levels of soluble urokinase-type plasminogen activator receptor (suPAR) and early mortality risk among patients enrolling for antiretroviral treatment in South Africa. BMC Infect Dis 2007; 7: 41.

33) Sidenius N, Sier CF, Ullum H. Serum level of soluble urokinase-type plasminogen activator receptor is a strong and independent predictor of survival in human immunodeficiency virus infection. Blood 2000; 96: 4091-4905.

34) Gaini S, Koldkjaer OG, Pedersen C, Pedersen SS. Procalcitonin, lipopolysaccharide-binding protein, interleukin-6 and C-reactive protein in community-acquired infections and sepsis: A prospective study. Crit Care 2006; 10: R53.

35) Odabaşi İÖ, Bülbül A. Neonatal sepsis. Med Bull Sisli Etfal Hosp 2020; 54: 142-158.

36) Petersen HH, Nielsen JP, Heegaard PM. Application of acute phase protein measurements in veterinary clinical chemistry. Vet Res 2004; 35: 163-187.

37) Akyuz E, Aydin U. Prognostic value of haptoglobin and ceruloplasmin levels determined in cows with adhesive and non-adhesive traumatic reticuloperitonitis. Kafkas Üniv Vet Fak Derg 2022; 28: 337-344.

38) Bowman DD, Lynn RC. Georgis Parasitology for Veterinarians. 1st Edition, USA: WB Saunders Company, 1995.

39) Eugen-Olsen J, Gustafson P, Sidenius N. The serum level of soluble urokinase receptor is elevated in tuberculosis patients and predicts mortality during treatment: A community study from Guinea-Bissau. Int J Tuberc Lung Dis 2002; 6: 686-692.

40) Bozukluhan K, Merhan O, Buyuk F, Çelebi Ö, Gokce G. Determination of the level of some acute phase proteins in cattle with brucellosis. Ankara Üniv Vet Fak Derg 2016; 63: 13-16.

41) İssi M, Gül Y, Başbuğ O, Ulutaş PA. Haptoglobin, serum amyloid A and ceruloplasmin concentrations incattle with suspicion of coryza gangrenosa bovum. Veterinarski Arhiv 2017; 87: 703-712.