Hematological and blood gas analysis are economically affordable analysis methods that can be easily applied at the bedside in the clinic and provide rapid results ^5,6. The first changes that respond to any disruption in homeostasis related to the blood and hematopoietic system are hematological parameters. Identifying these changes guides the search for diagnosis and plays a key role in monitoring the effectiveness of treatment^7,8. With the developments in medical technologies in recent years, automatic hematology devices analyze many different parameters such as WBC, LYM, MON, HCT, erythrocyte distribution width RDW and MPV^4,9. These parameters are used frequently in the evaluation of the diagnosis and prognosis of different disease conditions in cats and dogs. Unfortunately, no study has evaluated or revealed the specified hematological index in calves with sepsis.

Blood gas analysis is an auxiliary diagnostic method for detecting metabolic or respiratory disorders that cause pH changes in the blood, determining their severity, and facilitating follow-up after treatment¹⁰. Electrolyte and acid-base imbalances due to calf diarrhea are inevitable. For this reason, blood gas analysis are one of the most important auxiliary tools for management of treatment and monitoring of the disease, especially in calves with diarrhea^5,6. In calves with sepsis, the diagnostic significance of hematological index (MLR, RDW, MPV) and blood gas parameters (pO2, pCO2, lactate, Na+, K+, Cl-, HCO3-, pH, AG, and BE) have not been investigated. For this reason, the present study aimed to evaluate hematological and blood gas parameters in calves that developed sepsis due to diarrhea.

Animal Selection and Grouping: It consists of a total of 30 calves aged between 1 and 28 days, brought to Bingöl University Veterinary Faculty Animal Hospital Internal Medicine Department Polyclinic with complaints of diarrhea. The sepsis group consists of 20 calves with acute diarrhea, while 10 calves determined to be healthy constitute the control group. Clinical Examination and Sepsis Diagnosis: After systematic clinical examinations of all animals (rectal temperature, heart rate, and respiratory rate), body temperature was more than 39.5 °C or less than 38.5 °C, heart rate was more than 160/min or less than 100/min, respiratory rate was more than 36/min, and total leukocyte count was more than 12.000/mm3 or less than 4000/mm3. SIRS was considered positive in the presence of at least two of the existing criteria^1,11-13. SIRS is defined as infection or suspected infection leading to the onset of sepsis^11,14. In order to diagnose sepsis, the patient must have at least 2 SIRS criteria and a confirmed etiological factor^11,14,15. Fecal samples taken from calves were analyzed with immunochromatographic test kits (Anigen Rapid BoviD-5 Ag, Bionote, Inc. Korea) for Rotavirus, Coronavirus, Escherichia coli, Cryptosporidium parvum and Giardia lamblia. Calves with SIRS-positive diarrhea were considered to have sepsis in the presence of the agent in the feces and were included in the study. Calves outside the age range of 1-28 days, calves with chronic diarrhea, those receiving antibiotic treatment, those with congenital diseases, those not receiving colostrum, calves with diarrhea but no causative agent identified were excluded from the study. The control group calves were composed of animals that did not have any problems in clinical examination findings, hematological, biochemical and blood gas analyses.

Sample Collection, Hematology and Blood Gas Analysis: From all animals included in the study, 3 mL blood samples were taken into EDTA (BD Vacutainer®, Plymouth, UK) tubes for hematological examinations, and 2 mL blood samples were taken into heparin syringes for blood gas analyses. An automated hematology device (Benesphera H-31, USA) performed WBC, LYM, MON, HCT, RDW, and MPV analyses, while a blood gas device (Wondfo BGA-102) measured the parameters pCO2, lactate, Na+, K+, Cl-, HCO3-, pH, AG, and BE.

Statistical analysis: Statistical analysis of the data was performed using SPSS 26 (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp.). Data are presented as mean ± standard deviation, minimum-maximum values. Whether the data had a normal distribution was evaluated with the Shapiro-Wilk test. To determine the differences between the sepsis group and the control group, the Mann-Whitney U test was applied for data showing a nonparametric distribution, and the independent sample T test was applied for data showing a parametric distribution. Receiver Operating Characteristic (ROC) analysis was performed to determine under the curve (AUC), sensitivity, specificity and cut-off values of the parameters. According to the AUC values of the parameters, 0.500 was considered non-diagnostic, 0.600-0.700 was considered to have poor diagnostic performance, 0.700-0.800 was considered to be in the acceptable diagnostic range, 0.800-0.900 was considered good, and above 0.900 was considered to have very good diagnostic performance (16). The statistical significance level between groups was accepted as P value <0.05.

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Table 1: Etiological factors detected in calves with sepsis

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Table 2: Clinical examination findings and statistical significance of calves with sepsis and control groups

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Table 3: Descriptive statistics and significance levels of hematology analyzes in sepsis and control group calves

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Table 4: Descriptive statistics and significance levels of blood gas analyzes in sepsis and control group calves

The ROC analysis of hematological parameters is presented in Table 5 and Figure 1. The AUC value of WBC in calves with sepsis was determined to be 0.793, with a with a sensitivity 70%, a specificity 70%, and a cut-off 11.75.In the diagnosis of sepsis, the MON AUC value was determined to be 0.895, the sensitivity was 80%, the specificity was 75%, and the cut-off was 1.65. The AUC of RDW in the diagnosis of sepsis was determined as 0.840, sensitivity 80%, specificity 75%, and cut-off 41.05. In the HCT diagnosis of sepsis, the AUC was determined to be 0.698, the sensitivity was 60%, the specificity was 70%, and the cut-off was 28.00. An AUC of 0.915, 100% sensitivity, 90% specificity, and a cut-off value of 9.35 were determined for MPV in the diagnosis of sepsis. In the diagnosis of sepsis, MLR, 0.910 AUC, 80% sensitivity, 80% specificity, and 0.37 cut-off values were determined.

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Table 5: ROC analysis of hematological parameters

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Figure 1: ROC analysis of hematological parameters

Monocytes are a subset of circulating white blood cells that can differentiate into macrophages and dendritic cells¹⁹. Monocytes and macrophages are known to play an important role in the immune response against microorganisms as well as in the pathogenesis of sepsis²⁰. Studies have also demonstrated that monocytes actively initiate cellular immune responses and participate in the humoral response in hosts infected with enteric pathogens^21,22. Activation of different receptors (Toll-like receptors 1-2) that play a part the inflammation of monocytes in sepsis has also been seen in humans^19,23. In the presented study, it was determined that the number of monocytes in the sepsis group was significantly high and showed good diagnostic performance (AUC = 0.895, sensitivity = 80%, specificity = 75%) in the recognition of sepsis. The monocyte values obtained in this study are similar to the results of Chae et al.^24, Naseri et al.²⁵ and Kim and al.²² and are different from those of Naseri and İder²⁶ and Akyuz et al. ²⁷. The reason for the differences between studies may be related to the immune system of the animals and the variable course of monocyte numbers in cattle¹⁸.

RDW is the coefficient of variation of erythrocyte (RBC) volume and reflects the heterogeneity of RBC size²⁸. Increased RDW in cattle is associated with other trace mineral deficiencies associated with iron deficiency and macrocytic/microcytic anemia ¹⁸. However, studies have shown that the RDW value indicates systemic inflammation in horses^28, dogs^29, and humans³⁰ and can be used diagnostically in determining infectious conditions. In recent years, studies have been conducted on the RDW parameter in calves with diarrhea, but the change in RDW concentration in calves developing sepsis due to diarrhea has not been evaluated. In studies conducted on calves with diarrhea by many researchers^31,32,33, it has been reported that the RDW value does not show a statistically significant difference between groups. However, in this study, it was determined that the RDW value of the sepsis group was significantly higher than the control group. Additionally, the RDW variable showed good diagnostic properties (AUC = 0.840, sensitivity = 80%, specificity = 75%) in determining sepsis. Proinflammatory cytokines suppress erythropoietin-induced erythrocyte maturation and proliferation and downregulate erythropoietin receptor expression, which may be the cause of the increase in RDW in sepsis (30, 34). Therefore, in the presented study, it is thought that the increase in RDW is due to the inflammatory response caused by sepsis.

Platelets are important for stopping bleeding, but they are also inflammatory cells that can connect the immune system's humoral and cellular responses with molecular pathways and synthetic abilities that have not been known before³⁵. The MPV value obtained from the platelet histogram shows the average volume of platelets. It is known that in cases where platelet production decreases, such as sepsis, younger platelets, which are larger and more active, enter the circulation, and therefore MPV levels increase³⁶. Significant increases in MPV levels have been in dogs with various inflammatory diseases^37, horses³⁸ and humans³⁹. In cattle infected with the Bovine viral diarrhea virus⁴⁰ and sepsis infected cattle⁴¹ MPV is said to be used as a diagnostic and prognostic marker. In this study, consistent with previous studies, it was determined that the MPV level was significantly higher in the sepsis group and that MPV showed very good diagnostic performance (AUC = 0.925, sensitivity = 100%, specificity = 90%) in the diagnosis of sepsis.

In inflammatory diseases, a variety of hematological rates can be used both for diagnosis and prognosis. (41). An increase in the thrombocyte/lymphocyte ratio (PLR) in cats and dogs with acute pancreatitis has been attributed to the ability of the platelets to modulate phagocytosis and leukocyte function.⁴². By Gavazza et al.⁴³ in lymphoma dogs, neutrophil-lymphocyte ratio (NLR), thrombocytes/neutrophils (PNR), MLR, thrombocyte volume/thrombocyte (MPV/PLT) ratio have been to be useful parameters in the early diagnosis of a subclinical condition.

Aydın⁹ reported that NLR, lymphocyte/monocyte ratio (LMR) in calves with diarrhea, Yanar et al.⁴¹ evaluated NLR and PLR values may have potentials as indicators of inflammation in calves with sepsis. Likewise, in the presented study, it was determined that the MLR value of the sepsis group was significantly higher and showed very good diagnostic performance (AUC = 0.910, sensitivity = 80%, and specificity = 80%) in the recognition of sepsis.

Determination of acid-base balance is determined by changes in pH, HCO3-, BE and AG in plasma^44-46. Metabolic acidosis classically results from an initial loss of intestinal HCO3- as well as a decrease in glomerular filtration rate in response to severe dehydration^6,45,47. In the presented study, consistent with the data in the literature^6,46,48, pH, HCO3- and BE concentrations were found to be low and AG levels were high in the sepsis group compared to the control group. It is stated that changes in these blood gas parameters are caused by changes in dehydration and acidemia⁶. In calves with diarrhea, changes in electrolyte concentrations occur, as well as acidemia and dehydration^6,44. Although potassium loss occurs due to the increased amount of feces as a result of diarrhea, calves may be hyperkalemic, normokalemic or hypokalemic in this study, K+ concentrations were found to be significantly higher in the sepsis group compared to the control group. It is reported that the increase in K+ concentrations in the blood during metabolic acidosis is due to the buffering mechanism of hydrogen ions entering the cells⁴⁶. In addition, it is stated that hyperphosphatemia and the decrease in kidney function due to dehydration also contribute to hyperkalemia⁴⁸.

As a result, in this study, changes in hematological and blood gas analyzes in diarrhea calves that developed sepsis were determined. In the diagnosis of sepsis, it was determined that WBC had moderate diagnostic properties, monocyte and RDW had good diagnostic properties, and MPV and MLR had very good diagnostic properties. In future studies, it would be useful to classify hematological indices according to the etiology of sepsis and evaluate them in studies with a large population.

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