IMPACT OF ANEMIA ON COAGULATION INDICES IN THEILERIOSIS INFECTED CATTLE

The hemostatic mechanism is crucial to stop bleeding while ensuring that tissues receive enough blood flow. This investigation examined selected haematological and coagulation parameters in different degrees of anemic Theileriosis infected cattle and study correlation between RBCs and platelet indices. Haematological analysis indicated substantial declines (p < 0.01) in total RBCs counts, PCV% and (Hb) in different degrees of anemic Theileriosis infected cattle compared with control group. In severe anemic group, MCV showed a substantial rise (p < 0.01) compared with moderate anemic group and (p < 0.05) compared with mild anemic and control groups; while MCH showed a substantial increase (p < 0.05) compared with mild and moderate anemic groups. In the coagulation profile, Platelets count (PLT) and PCT% showed insignificant difference (p > 0.05) among anemic groups compared with control group. MPV and PDW demonstrated substantial decreases (p < 0.01) in all anemic groups when compared with control group. In moderate anemic group, Prothrombin time (PT) exhibited a substantial prolongation (p < 0.01) compared to control group and (p < 0.05) compared to mild anemic group. In severe anemic group, PT was significantly prolonged (p < 0.05) compared with control and mild anemic groups. Correlations between RBCs and platelet indices in mild, moderate and severe anemic Theileriosis infected cattle revealed significant correlations in all blood parameters. It could be concluded that different degrees of anemia caused by T. annulata infection in cattle are associated with marked changes in measured haematological and coagulation parameters.


INTRODUCTION
Anemia is a common abnormal condition in ruminants.Pathological affection of none hemopoietic tissue is the main cause of anemia in cattle, rather than reflecting primary defects in erythropoiesis (Katsogiannou et al., 2018).Anemia associate the later stage of severe diseases (Goddard and Leisewitz, 2010).Blood protozoa and endo-and ectoparasites are typically the cause of it (Radostits et al., 2010 andSingh et al., 2014).In anemic ruminants, pallor mucous membrane, intolerance to exercise, tachypnea or dyspnea, tachycardia and functional murmurs are the most common symptoms (Radostits et al., 2010).
Bovine theileriosis is a tick-borne haemoprotozoan disease transmitted by Hyalomma spp.and caused by Theileria annulata (Sayin et al., 2003).The diseased cow had swelling of the pre-scapular lymph nodes, fever, loss of appetite and lethargy, pale mucous membrane, drop in milk production and abortion in pregnant animals due to high fever (Radostits et al., 2010).Symptoms of acute tropical theileriosis that are most commonly observed include nasal and ocular discharges with congestion of mucous membranes, coughing, and salivation (Osman and Al-Gaabary, 2007).Subcutaneous edema, corneal opacity, and diarrhea could be found in later stages of the disease (Reda, 2012).
Positive and negative regulators play a complicated role in hemostasis; an unbalanced response can result in either hyper coagulation (thrombosis) or hypo coagulation (haemorrhage), or both (Rebar et al., 2005).The use of coagulation and haematological parameters in diagnosis, prognosis, and treatment is beneficial.These factors offer extremely useful information regarding the level of infection (Çöl and Uslu, 2006).Platelets count and coagulation tests are used to evaluate pathological changes of the hemostatic and coagulation systems to direct clinical therapy (Zhao and Lv, 2013).The usual components of a routine hemostasis testing profile are the total number of platelets, activated partial thromboplastin time (APTT), and prothrombin time (PT).
Platelets (PLT) are crucial component of hemostasis, thrombosis, and a variety of bleeding disorders, they are a nuclear cytoplasmic fragments of bone marrow megakaryocytes (Russell, 2010 andBoudreaux et al., 2011).One well-known technique in veterinary diagnostics is platelets quantification in peripheral blood (Souza et al., 2016).Productivity, consumption, sequestration, and loss all have an impact on the total number of platelets (Russell, 2010 andBoudreaux et al., 2011).
The morphology and proliferation kinetics of platelets are correlated with platelets indices (PI), which are indicators of platelets activation.The platelet indicators that are most frequently evaluated are the plateletlarge cell ratio (P-LCR), platelet distribution width (PDW), platelet volume (MPV), and plateletcrit (PCT) (Russell, 2010;Boudreaux et al., 2011 andUstundag Budak et al., 2016).
Cytokines (thrombopoietin, interleukin-6, and interleukin-3) are discovered to be related to the platelet volume (Larsen et al., 2014).MPV levels rise in response to decreasing platelets synthesis because immature platelets grow larger and become more active.Greater MPV is correlated with greater platelet diameter, which is a sign of platelet activation and production rate (Ustundag Budak et al., 2016).Platelet anisocytosis increases PDW, which is a measure of volume variability in platelet size (Ustundag Budak et al., 2016).There is a clear correlation between MPV and PDW under physiological conditions, and both often fluctuate in the same way (Vagdatli et al., 2010).Conflicting reports regarding the link between platelet counts and volume have been found in the literature, indicating that they are affected by different mechanisms (Chandrashekar, 2013 andMariani et al., 2014).
PCT is the proportion of blood volume that platelets occupy (Chandrashekar, 2013).MPV and platelet counts are inversely correlated, although platelet mass is tightly controlled to maintain a consistent level (Margetic, 2012).
A laboratory screening test called prothrombin time is used to assess the coagulation factors of the common and extrinsic pathways to identify conditions affecting the activity of factors I, II, V, VII, and X (Chaudhry and Babiker, 2019).Veterinarians can benefit greatly from knowing the normal prothrombin time.Regarding coagulation tests like PT, APTT, D-Dimer, PLT, and fibrinogen, there is no gold standard in veterinary medicine that is comparable to the scoring system used in human medicine (Taylor et al., 2001).Thus, the primary aims of this investigation were to examine the impact of varying degrees of anemia in cattle infected with theileriosis on prothrombin time and platelets indices, as well as the correlation between red blood cells and platelets indices in cattle that were anemic.

I. Animals:
The research study, which took place from May 2021 to September 2022, involved 38 cattle of both sexes with various ages (ranging from 6 months to 5 years, according to the owner's information).On the principle of owner's complaints, animals were divided into two groups.A diseased group, consisting of 27 animals with theileriosis that were clinically anemic and exhibited characteristic symptoms.Animals were brought to the Clinics of Internal Medicine, Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Assiut University, Egypt, from various villages around Assiut city.Clinical examination and laboratory confirmation using Giemsa staining of blood smears were used to diagnose the condition.11 clinically healthy animals were obtained from a private Assiut farm to serve as the control group.

Clinical Examination:
Based on clinical symptoms, tick infestation and blood smear examination, theileriosis was diagnosed.Each animal had a clinical assessment, which includes inspection of skin and mucous membrane of the eye, measuring the body temperature, auscultation of lung, heart and rumen, palpation of superficial lymph nodes and examination of oral cavity.Symptoms of bovine theileriosis are noticed and documented.

Samples: 3.1. Blood samples:
To examine blood films, ear vein punctures were performed on each animal.The jugular vein was used to extract blood samples using an aseptic and dry needle.Two different types of whole blood samples were collected and they were kept cold until examination:

A.
A whole blood sample was obtained using potassium salts of Ethylene Diamine Tetra-Acetic acid (K-EDTA) as an anticoagulant and used for complete blood count (CBC).

B.
Whole blood samples were collected on sodium citrate 3.2% (1:9) for coagulation tests.At the laboratory; centrifugation was performed at 1500 rpm for 10 min for separation of citrated plasma that was used for prothrombin time (PT).

C. Blood films:
Thin blood films were prepared from blood samples obtained from the ear vein of animals.The slide was covered with just a drop of blood, which was then carefully smeared and let to air dry.The blood smear was fixed in absolute methyl alcohol and left to air dry for two minutes.Giemsa stain was applied in a diluted ratio (1:9) and left for 45 minutes, the smear was then rinsed in distilled water and allowed to air dry (Soulsby, 2005).After that, it was examined under a light microscope using an oil immersion lens (x1000).

Hematological Examination:
Within four hours of the samples being collected, a haematological examination was completed using a three-part diff haematological analyzer model (MTC-3060).In particular, red blood cell count (RBCs), hemoglobin concentration (HGB), haematocrit (HCT), and total and differential leucocytic counts.Red blood cell indices include: main corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and red blood cell distribution width (RDW) were among the haematological variables that were measured.Total platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW), large platelet concentration ratio (LPCR), and plateletcrit (PCT) were measured.

Coagulation profile: 5.1. Prothrombin time (PT):
Using an automated blood coagulation analyzer (TECO coatron® M1 coagulation analyzer, Germany) and a commercial test kit (Siemens healthcare diagnostics products GmbH, Germany), prothrombin time was measured on citrated plasma in accordance with the manufacturer's instructions.

Statistical analysis:
The mean and standard error of the data were shown.To assess statistical significance, an analysis of variance was performed using the statistical software for the Social Sciences for Windows (SPSS, version 10.0, Chicago, IL, USA).The associations between the features of red blood cells and platelets were assessed using Pearson's correlation analysis.Statistically significant differences were defined as p ≤ 0.05 (Borenstein et al., 1997).

A-Clinical findings:
Upon clinical examination, diseased cattle showed symptoms such as fever (39-41°C), enlargement of superficial lymph nodes especially those in the prescapular and prefemoral regions, pale mucous membranes, accelerated heart and respiratory rates, nasal discharges, cough, ruminal atony, corneal opacity in some cases, tearing.Tick infestations in varying degrees were seen around the abdomen, base of the tail, udder, and groin area.In contrast, a clinical assessment revealed that the control group was both clinically and laboratory-healthy.

Blood smear
In addition to the clinical manifestations of theileriosis, the intracellular signet ring of theileria trophozoites in a blood smear was utilized to validate the diagnosis of Theileria infection (Fig. 1).
Table (1) demonstrated substantial declines (p < 0.01) in all anemic groups' packed cell volume (PCV%), haemoglobin (Hb) concentration, and total RBC counts when compared to the control group.Also, among anemic groups, notable declines (p < 0.01) were found except for Hb in moderate anemic, which had a substantial decline (p < 0.05) compared with mild anemic group.
In the severe anemic group, MCV exhibited a substantial increase (p < 0.01) when compared with the moderate anemic group and a substantial increase (p < 0.05) with mild and control groups, while MCH, displayed a substantial increase (p < 0.05) when compared with mild and moderate anemic groups.In mild and moderate anemic groups, MCV and MCH revealed negligible changes (p > 0.05) compared to the control group.MCHC, RDW-CV and RDW-SD in all anemic groups displayed negligible differences (p > 0.05) compared with the control group and between each other.

Total
WBC counts, lymphocytes, monocytes, and granulocytes in the anemic groups didn't differ substantially (p > 0.05) from those in the control group or from one another.In the moderate anemic group, PT exhibited a substantial prolongation (p < 0.01) when compared to the control group and a significant prolongation (p < 0.05) when compared to mild anemic group.In the severe anemic group, there was a significant prolongation (p < 0.05) when compared to the control and mild anemic groups.INR ratio showed that the severe anemic group had a significantly higher increase (p < 0.05) than the moderate anemic group.(3) in the mild anemic group, a notable negative association was found between the total number of RBCs and MCV (r = -0.961*),MCH (r = -0.981*),and MPV (r = -0.998**).MCV was substantially associated positively with MCH (r = 0.990**), RDW (r = 0.992**) and MPV (r = 0.970*).Also, MCH was considerably associated positively with RDW (r = 0.979*) and MPV (r = 0.982*).There was a notable positive association found between PLT count and PCT (r = 0.997**).

DISCUSSION
Data from published works indicates that anemia is typically the primary clinical symptom of theileriosis and the severity of the condition depends on the species of theileria and the degree of parasitemia (Stockham et al., 2000;Omer et al., 2002 andShiono et al., 2004).

A-Clinical findings:
The  (2020).The unchecked growth and spread of lymphoid cells infected with schizont, as well as the increased production of pro-inflammatory cytokines by parasitized monocytes, may be the cause of tissue damage and unfavorable clinical findings in cases of bovine theilerosis (Glass et al., 2003 andRadostits et al., 2010).
Cattle with bovine theilerosis may have fever as a result of activation of the thermoregulatory centre triggered by endogenous pyrogens released from cell lysis and parasitemia (Glass et al., 2003).According to Radostits et al. (2010), anorexia may develop as a result of a persistently high fever during the acute phases of the disease.uncontrolled hyperplasia of T-lymphocytes infected with schizonts was the source of the generalized lymphoid proliferation that resulted in the enlargement of superficial lymph nodes (Radostits et al., 2010).Leukocytic infiltration as a result of white blood cell infiltration may be the cause of corneal opacity in advanced cases (Hussein et al., 2007 andOsman &Al-Gaabary, 2007).According to Muraguri et al. (2006) and Radostits et al. (2010), severe pulmonary edema that develops in advanced cases due to the release of vasoactive chemicals from disintegrating lymphocytes in the lungs may be the cause of the respiratory indications that have been noticed, such as coughing, respiratory distress, and nasal discharge.
The etiology of anemia in theileriosis was thought to be extravascular hemolysis brought on by enzymatic, immune-mediated, mechanical, toxic, erythroid hyperplasia, and enhanced hemagglutinin processes (Stockham et al., 2000 andShiono et al., 2003).Additional causes could be protease enzymes (Shiono et al., 2004), oxygen radicals, elevated oxidative proteins in the membranes of red blood cells, and oxidative damage (Shiono et al., 2003).Anemia and low haemoglobin concentration were indicated by pale mucous membranes as infected erythrocytes were distracted and removed by the reticuloendothelial system (Singh et al., 2001).

B-Hematological findings:
According to reports from Singh et al. (2001) anemia can develop in the advanced stage of theileriosis after parasitaemia.Haematological results in this study demonstrated substantial declines in all anemic groups' packed cell volume (PCV%), haemoglobin (Hb) concentration, and total RBC counts compared to the healthy control group and amongst each other.The breakdown of erythrocytes by macrophages in the monocyte-macrophage system organs may be the cause of a drop in RBCs, PCV%, and Hb content (Singh et al., 2001).According to Al-Emarah et al. (2012), the primary cause of the defects in erythrocytes that arise from parasite infections is their toxic activity.Anemia was reported by Haron et al. (2014) and might have been caused by erythrocyte lysis brought on by the parasite within the cells (Ghanem et al., 2013 andTemiz et al., 2014) The severe anemic group demonstrated a substantial increase (p < 0.01) in MCV and a negligible difference (p > 0.05) in MCHC compared to the control, mild and moderate anemic groups; this resulted in macrocytic normochromic anemia, which indicated a regenerative response consistent with a hemolytic anemia (Stockham et al., 2000).The same was reported by Haron et al. (2014) and Devadevi et al. (2018).
The leucogram results showed that both relative to the control group and within the anemic groups, there were hardly detectable differences (p > 0.05) in the total WBC counts.The findings corroborated those of Ramin et al. (2011) andTemiz et al. (2014) who noted a negligible association between WBCs count and the severity of anemia.Also, the same was mentioned by Ganguly et al. (2015)  The chronic detrimental effects of Theileria's toxic metabolites on hemopoietic organs, particularly the bone marrow, and their disruption of the leucogenesis process may be the cause of changes in leucograms (Sarma et al., 2016).
The current investigation found that the anemic groups' lymphocyte, monocyte, and granulocyte counts didn't have detectable variations (p > 0.05) relative to the control group or one another.The outcomes were comparable to those of Ramin et al. (2011) and Patel et al. (2017), who noted that the TLC and DLC (Neutrophil, Lymphocyte, Eosinophils and Monocyte) values fell within the standard range.Furthermore, Al-Emarah et al. (2012) noted that basophiles, eosinophils, and monocytes didn't exhibit a substantial statistical shift.In contrast, some studies referred to leucopenia and lymphopenia (Sandhu et al., 1998) and others to lymphocytosis and leukocytosis (Stockham et al., 2000 andRadostits et al., 2010).According to Yamaguchi et al. (2010), lymphocytosis results from intralymphocytic theilerial parasites developing into host cells and clonally growing lymphocytes.According to Ganguly et al. (2015), there was a noteworthy rise in both monocytes and eosinophilic counts.This could perhaps be attributed to the multiplication of these cells as a host defense mechanism against infection, as documented by Modi et al. (2015) and Gunes et al. (2017).The leucocytic response is highest during acute infection and gradually declines during chronic and persistent infection, potentially explaining the variations in leucocytic profiles observed across different studies (Haron et al., 2014).

C-Coagulation profile findings:
Platelets (PLT) and platelet indices are used in human medicine to diagnose a wide range of diseases and assess prognoses.However, the veterinary literature still has little to say about their clinical usefulness (Koenhemsi, 2019).There is evidence to suggest that in patients with thrombocytopenia, PCT, not platelet counts, predicts the risk of bleeding (Mohr et al. 1986).According to Bath and Butterworth (1996) In a moderate anemic group of the current investigation, prothrombin time (PT) revealed substantial prolongation (p < 0.01) relative to the control group and (p < 0.05) relative to the mild anemic group; while in severe anemic group, there was a substantial prolongation (p < 0.05) relative to the control and mild anemic groups.INR ratio showed a substantial rise (p < 0.05) in the severe anemic group when compared with the mild anemic group.Hepatic damage, including localized necrosis, sinusoidal dilatation and disruption with haemorrhage and mononuclear cell infiltration on histological examination, can be used as an explanation for the prolongation of PT (Singh, 1998).These results are in line with those of Çöl and Uslu (2006) and Kilinc et al. (2018) who found that the prothrombin time of cattle infected with T. annulata was significantly longer (P < 0.05) than that of the control group.The coagulation abnormalities found in animals infected with theileria were also documented by Maxie et al. (1982) andSingh et al. (2001).The findings were in direct opposition to those of Gunes et al. (2017), who reported that there was no significant difference in (PT) between the control and infected groups in cattle having natural theileriosis.According to Hosny et al. (2010), the presence of parasites causes multiple petechial haemorrhages, lesions in the endothelium lining of blood vessels, and tissue damage in organs such as the liver, kidney, and lung.These outcomes are important because they contribute to the development of coagulation defects (Levi et al., 1997).
In this investigation, the total number of RBCs displayed a notable opposite association with MCV (r = -0.961*,r = -0.703**and r = -0.895*) in mild, moderate and severe anemic groups, respectively, and with RDW (r = -0.497*) in the moderate anemic group.There was a considerable positive association between MCV and RDW (r = 0.992** and r = 0.737**) in mild and moderate anemic groups, respectively.Also, a notable positive association between PLT count and PCT (r = 0.997**, r = 0.986** and r = 1.000**) in mild, moderate and severe anemic groups, respectively was observed.Abd Ellah (2015) reported the same outcomes.

CONCLUSION
Notable alterations in the coagulation and haematological indicators have been linked with varying degrees of anemia in cattle caused by Theileria infection.

Table 1 :
Hematological parameters in control and anemic cattle with theileriosis (Mean ± SE).
Data expressed as mean ± SE, means of various superscript letters a, b, c, d in the same raw are substantially different at (p < 0.05), RBCs= Red blood cells, Hb=Hemoglobin, PCV=Packed cell volume, MCV=Mean corpuscular volume, MCH=Mean corpuscular haemoglobin, MCHC=Mean corpuscular hemoglobin concentration, WBCs= white blood cells, Lymph=Lymphocytes, Mono= Monocytes, Gran=Granulocytes C-Coagulation profile findings:

Table 2 :
Coagulation parameters in control and anemic cattle with theileriosis (Mean ± SE).
a Data expressed as mean ± SE, means of various superscript letters a, b, c, d in the same raw are substantially different at (p < 0.05), PLT= platelets count, PCT = plateletcrit, MPV= mean platelet volume, PDW= platelets distribution width, P-LCC= platelet-large cell count, P-LCR= platelet-large cell ratio, PT= Prothrombin time, INR= International Normalized Ratio.

Table 3 :
Association between platelets and RBCs indices in mild anemic cattle with theileriosis.

Table 4 :
Association between platelets and RBCs indices in moderate anemic cattle with theileriosis.

:
Haematobiochemical Alterations in CattleSuffering from Anaemia and Their Effect on Quality of Some Meat.