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Keywords:

  • AST ;
  • Cardiac markers;
  • Cardiac troponin I;
  • CK-MB;
  • Hemolytic anemia;
  • Theileria annulata

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background

Theileria annulata is a blood parasite affecting ruminants. Hemolytic anemia, secondary hypoxia, and vasculitis are the most important features of tropical theileriosis.

Objectives

Evaluation of electrocardiographic findings and changes in cardiovascular biochemical markers including cTnI concentrations in cattle naturally infected with theileriosis in the absence of acute cardiac failure.

Animals

Ninety adult Holstein cattle (>1 year) with clinical and laboratory evidence of theileriosis and 30 healthy cattle served as controls.

Methods

Case-control study in which blood samples were collected and randomized after clinical, hematologic, parasitologic examination and laboratory confirmation and electrocardiographic recording on all animals, serum cardiac troponin I (cTnI), aspartate aminotransferase (AST), and creatine kinase-MB (CK-MB) were evaluated.

Results

Serum concentration of cTnI was significantly higher in cattle with theileriosis (mean: 0.028 ng/mL; range: 0.005–0.21 ng/mL; control mean: 0.011; range: <0.005–0.09 ng/mL; P = .003). There was significant correlation between serum level of cTnI and PCV (r = −0.257; P < .001) and also between cTnI and parasitemia (r = 0.515; < .001). Mean serum activities of AST and CK-MB were 107 ± 46 and 301 ± 103 U/L in sick animals, which were significantly higher than healthy cattle (P = .002 and P = .041, respectively). There were no pathologic arrhythmias detected in sick animals.

Conclusions and Clinical Importance

Theileriosis is a risk factor for elevation of cardiac biomarkers in naturally infected Holstein cattle. Severity of anemia and parasitemia might contribute to the pathophysiology of myocardial damage. The prognostic significance of increased serum cardiac troponin I concentrations in cattle with hemolytic anemia merits further investigation.

Abbreviations
AST

aspartate aminotransferase

BPM

beat per minute

CK

creatine kinase

CK-MB

creatine kinase-MB

CLIA

chemiluminescent immunoassay

cTnI

cardiac troponin I

DIC

disseminated intravascular coagulation

LD

lactate dehydrogenase

PCV

packed cell volume

Troponins are 3 distinct myofibrillar proteins (I, C, and T) that regulate the calcium-mediated interaction between actin and myosin in both cardiac and skeletal muscle.[1, 2] Cardiac troponin I (cTnI) is the only one that is uniquely expressed in the myocardium. Both cTnI and cardiac troponin T have been widely recognized as highly sensitive and specific blood markers for the noninvasive diagnosis of increased cardiomyocyte permeability, and blood concentrations appear to correlate with the extent of myocardial injury in people and animal.[3-5]

The molecular structure of troponin proteins is highly conserved across species, and some current assays developed for their detection in humans have been used and validated in several other species.[6-8] Bovine cTnI has high amino acid sequence homology (96.4%) with human cTnI.[9] Therefore, it is anticipated that antibodies against human cTnI used in commercially available immunoassays will cross-react with bovine cTnI.[8] Increased blood concentrations of cTnI were reported in cattle with idiopathic pericarditis,[10] traumatic reticuloperitonitis,[11, 12] endocarditis, caudal vena cava thrombosis,[11] monensin toxicosis,[13] in calves with foot and mouth disease,[14, 15] and experimentally induced endotoxemia.[16] In such studies, various immunoassays, developed for use in humans, were used for the measurement of cTnI.

Tropical theileriosis or Mediterranean coast fever—caused by Theileria annulata—is a disease of cattle widely distributed across southern Europe, north Africa, and central Asia. In various surveys, prevalence of T. annulata were reported 4.3 up to 20% in Iran. The bovine mortality rate rises up to 40–60% and even more.[17] It is a progressive lymphoproliferative disease of cattle caused by the protozoan parasite, T. annulata. The parasite acts as a serious constraint to cattle production in endemic areas, causing lethal infections in exotic cattle and considerable mortality in indigenous and crossbred stock.[18] A marked rise in body temperature, reaching 40–41.5°C, is followed by lethargy, lacrimation, nasal discharge, and swelling of the superficial lymph nodes; hemolytic anemia is among the characteristic features of tropical theileriosis. In this disease, hemolytic anemia is caused by an immune-mediated hemolysis, resulting in immune-mediated destruction of affected erythrocytes.[19] Some hematologic changes in erythrocytes, including increased osmotic fragility of erythrocytes, acceleration of clearance, and the presence of hemolytic activity in cattle highly infected with Theileria sergenti, are some of the suggested possible mechanisms in inducing anemia.[20] There is some evidence that oxidative stress and lipid peroxidation are involved in pathogenesis of anemia in theileriosis.[21] As a result, weight loss is rapid, and hemoglobinuria may occur.[22] Because anemic hypoxia and acid-base and electrolyte derangements, diffuse infiltration of inflammatory cells in some organs including heart occur in theileriosis, it is likely that myocardial cell membrane integrity is also compromised in this disease.[23]

The central hypothesis of this study is that anemia associated with theileriosis and secondary hypoxia and vasculitis, leading to some degree of cardiovascular injury. Accordingly, the goals of this study using cattle naturally infected with T. annulata were to (1) quantify the electrocardiographic abnormalities and changes in serum levels of cTnI, aspartate aminotransferase (AST), and creatine kinase-MB (CK-MB); (2) determine if T. annulata is a risk factor of cardiac injury; and (3) evaluate the association of hematologic abnormalities and parasitemia with cardiomyocyte injury in clinically ill cattle.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Animals

Blood samples were collected and randomized from 90 adult Holstein cattle (>1 year) of both sexes (7 males and 83 females) presented to the Veterinary Teaching Hospital, Veterinary Faculty, Islamic Azad University- Tabriz Branch during the April 2008 to July 2009. All females are lactating and males are uncastrated beef cattle. These were clinically sick animals. Cattle with a history of previously diagnosed cardiac disease and cattle treated with antitheilerial drugs up to 30 days before examination were excluded. A total of 30 clinically healthy Holstein adult (4 males and 26 females) cattle from tick-free farms were used as a control group. They were free of any external, blood, and internal parasites. Control cattle were excluded if any abnormality was found. All animal used in this study were aged between 1.5 and 4 years. This study was approved by the Ethics Committee of Faculty of Veterinary Medicine, Islamic Azad University (BNS713/26.03.08).

Clinical and Pathologic Examinations

The conjunctival, nasal, and oral mucous membranes, superficial lymph nodes, rectal temperature, and respiratory and cardiovascular system of the cattle deemed to be undergoing T. annulata infection were examined and clinical and parasitologic observations were recorded. Blood smears were prepared from the ear tip of animals for observation of intraerythrocytic forms of T. annulata. After examining, more than 50 microscopic fields of blood films stained with Giemsa at a magnification of ×1000, the parasitemia in infected animals was quantified in at least 15 microscopic fields and expressed as the percentage of infected erythrocytes.[24] For hematologic and biochemical analysis, approximately 10 mL blood samples were collected from jugular vein into EDTA and serum tubes. Hematologic parameters including red blood cells count (RBCs), white blood cells count (WBCs), and packed cell volume (PCV) measured by routine procedures.[25, 26] Lymph node smears were also prepared from suspected cases of theileriosis (21 cases) by needle biopsy. Smears of lymph node aspirates were stained with Giemsa stain and examined for schizonts (Koch's blue bodies). Infected animals were treated after diagnosis and all of them recovered.[24]

Electrocardiography

After 10–20 minutes, rest and before any treatment, ECGs were recorded for at least 30 seconds using 3 channel electrocardiography and bipolar base apex lead, with a speed 25 mm/s, calibrated for 10 mm/mV. Alligator type electrodes were used for this recording. ECGs were evaluated for heart rate and rhythm, alteration in wave's amplitude and duration, and presence of arrhythmia. Heart rate between 60–80 and 80< beats per minute were assumed normal and tachycardia, respectively.[27]

Biochemical Assay

Serum samples were analyzed for cardiac troponin I (cTnI) by a chemiluminescent method (CLIA) using commercial human system,1 and aspartate aminotransferase (AST), creatine kinase-MB (CK-MB) activities were determined by an automatic analyzer,2 using commercial kits.3 This CLIA assay is able to measure troponin in range 0.005–100 ng/mL. Calibration curves were performed before analysis using human cTnI. Ten cattle with traumatic reticulopericarditis were selected from slaughter to be used as bovine cTnI positive control.

Statistical Analysis

All statistical data were analyzed by Statistical Package for Social Sciences for Windows, version 16.0 (SPSS Inc, Chicago, IL, USA). Data normality was tested by Kolmogorov–Smirnov test. The independent samples t-test was used for comparison of measured factors between two groups (controls and clinical cases). Analysis of variance (ANOVA) test was used for comparison of measured factors in all subgroups with different parasitemias and PCV values. Pearson's correlation coefficient was used for determination of the relationship between parameters at different parasitemias and PCV values. All values were expressed as mean and standard deviation (SD), and < .05 was considered as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

All hematologic parameters showed significant differences between clinically healthy and infected animals (Table 1). In infected cattle, significant negative correlations were observed between parasitemia and RBC count (= −0.88; P = .009), PCV (= −0.92; = .030).

Table 1. Hematologic parameters in clinically healthy and cattle infected with Theileria annulata
Parasitemia (%)Number of CattleRBC (×106/mL)WBC (×103/mL)PCV (%)
  1. Means (±SD) within a column with different superscript letters (a, b, c, d) denote significant differences (P < .05).

Clinically healthy307.94 ± 0.63a7.00 ± 0.52a38.39 ± 0.82a
≤10416.60 ± 0.82b9.97 ± 1.92b31.37 ± 1.54b
11–25325.84 ± 0.19c10.33 ± 1.73b27.20 ± 1.69c
26–40174.70 ± 0.46d6.50 ± 0.85c22.48 ± 2.01d

The mean (±SD) cardiac troponin I concentrations in healthy cattle and theileriosis cases were 0.012 ± 0.009 and 0.028 ± 0.008 ng/mL, respectively, which showed significant increase in sick animals (= .003). cTnI level ranged from 0.005 to 0.21 ng/mL in cattle with theileriosis and <0.005–0.016 ng/mL in healthy ones. There was significant correlation between serum concentration of cTnI and parasitemia (= 0.515; < .001). Also, there was negative correlation between hematocrit and serum concentration of cardiac troponin I (= −0.257; < .001) (Tables 2 and 3).

Table 2. Serum cTnI concentration and AST, CK-MB activities in theileriosis cases based on parasitemia and healthy cattle
Parasitemia (%)Healthy≤1011–2526–40
  1. Means (±SD) within a row with different superscript letters (a, b, c, d) denote significant differences (P < .05).

Number of cattle30413220
cTnI (ng/mL)0.012 ± 0.009a0.012 ± 0.008b0.020 ± 0.013c0.052 ± 0.017d
AST (U/L)79 ± 28a117 ± 74b93 ± 14c83 ± 26c
CK-MB (U/L)249 ± 111a260 ± 110a282 ± 143b237 ± 89a
Table 3. Serum cTnI concentration and AST, CK-MB activities in theileriosis cases based on hematocrit
Hematocrit (%)Healthy31<26–30<25
  1. Means (±SD) within a row with different superscript letters (a, b, c, d) denote significant differences (P < .05).

Number of cattle30364014
cTnI (ng/mL)0.012 ± 0.009a0.022 ± 0.021b0.037 ± 0.026c0.038 ± 0.020d
AST (U/L)79 ± 28a128 ± 56b91 ± 47c139 ± 69d
CK-MB (U/L)249 ± 111a266 ± 101b335 ± 166c328 ± 154c

A reference range for serum cTnI concentrations measured by the LIAISON troponin I immunometric chemiluminescent assay system has not been well-defined in cattle. However, based on this study, 24 of 30 cattle (80%) that were considered clinically normal had serum cTnI concentrations <0.01 ng/mL.

Mean serum activities of AST and CK-MB were 107 ± 46 and 301 ± 103 U/L in the disease group, respectively, which showed significant increase in cattle with theileriosis (= .002 and = .041, respectively).

ECG interpretations revealed that 62 infected cattle (62/90) had tachycardia (HR 80 <bpm) and 28 cattle had normal heart rate. There is no case of bradycardia. Average heart rate in sick cattle calculated 88.59 ± 15.74 beats per minute, which was higher than control group (P = .002). All animals in control group had a heart rate at reference range (60–80 bpm) (Table 4). Based on electrocardiographic findings, 62 cases of sinus tachycardia, 15 cases of sinus arrhythmia, 9 cases of concurrent occurrence of sinus tachycardia and sinus arrhythmia, 6 cases of first degree atrioventricular block, and 1 case of atrial fibrillation were observed. These arrhythmias are common in cattle and were considered nonpathologic. No differences in cTnI concentrations were detected between cattle with and without dysrhythmias (= .576).

Table 4. Heart rate in affected cattle with theileriosis
RangeNormal (60–80 beat/min)Abnormal (>80 beat/min)
<6060–7071–8081–9091–100>100
Number of cattle2818242216
Percent2.28.82026.724.517.8
Total28 (31%)62 (69%)

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Analysis of serum cTnI has been shown to be more sensitive in detecting myocardial abnormalities than determination of lactate dehydrogenase (LD), creatine kinase (CK), MB isoenzyme of CK (CK-MB), or ECG.[1-3, 28] Aspartate aminotransferase (AST), myoglobin, LD, and CK are very sensitive markers of myocardial cell damage, but they lack cardiac specificity.[2, 28, 29] Cardiac troponins are a more sensitive and persistent indicator of cardiac injury, with high tissue specificity, than other markers in the presence of marked skeletal muscle injury, liver disease, and chronic renal failure.[6, 9] The level of serum AST in infected cattle was higher as compared with that of the healthy ones (Tables 2 and 3). Similar results had been reported previously.[22, 26] Increase in AST levels in infected animals compared with healthy animals might indicate hepatic dysfunction in parasite-positive animals. The MB isoenzyme of creatine kinase is normally present in the skeletal muscle in low amounts and substantial injury to skeletal muscle can increase CK-MB activity and substantially elevate values to abnormal levels.[1] To our knowledge, no cases considering CK activity in cattle with theileriosis have been reported. This is an important consideration in theileriosis, in which liver impairment and secondary rhabdomyolysis as a result of recumbency can occur. Also hemoglobinuria, which occurred in theileriosis, may lead to ischemic and toxic nephrosis.[27] Thus in theileriosis, serum enzyme activities such as AST, CK-MB could also be falsely high because of both muscle and liver disease.[23] Unfortunately, renal and liver specific function tests were not performed in this study.

Cardiac troponin I is released from myocytes in both reversible and irreversible myocardial injury. The changes in myocyte membrane permeability resulting from the injury could be enough for the release of cardiac troponins from the free cytosolic pool of myocytes without structural damage.[30] The exact mechanism of myocardial injury in an apparently normal heart is unclear.[31] It may be speculated that increased oxygen consumption by the myocardium during a prolonged period of tachycardia is combined with a reduced oxygen supply to the myocardium because of the shortened diastole during tachycardia.[31, 32] There are few published reports on human patients with high troponin levels secondary to supraventricular arrhythmias.[30-32] There is no case of supraventricular tachycardia in this study, but sinus tachycardia were diagnosed in 68.8% of infected cattle. Although, one of presumptive reasons of increased serum cTnI concentration in cattle with theileriosis may be attributed to occurrence of tachycardia secondary because of anemia in cattle, but its significance is unclear.

The mechanism of myocyte damage in cattle with theileriosis is unclear. However, in cattle with theileriosis, in addition to typical signs of disease including fever, anorexia, rumination ceases, decrease in milk production, enlargement of superficial lymph nodes, tachycardia, anemia and jaundice, hemorrhages on visible mucous membranes and sometime on skin, respiratory distress and pulmonary edema[27, 33]; cardiac lesions, such as excessive pericardial fluid, ecchymotic and petechial hemorrhages on the epicardium, myocardium, and endocardium,[23, 33] degeneration of myocardial fibers, and diffuse infiltration of inflammatory cells have been described. Severe myocarditis combined with intense inflammatory cell infiltration (predominantly macrophages and lymphocytes), myocytes degeneration and necrosis, infarcted areas, interstitial tissue edema, hemorrhages, and fibrin material in the myocardium, presence of fibrin and thrombi in myocardial vessels in calves with Theileria spp. infection have been reported.[23, 34] Hydropericardium and edema of the heart valves in postmortem examination of a Friesian calf with concurrent infection with T. annulata and bovine leukemia virus have been reported previously.[35] In cattle with cerebral theileriosis (turning sickness) parasitized lymphoblasts accumulate in cerebral blood vessels, with resultant thrombosis and infarction of affected organs.[27] Perhaps, similar events occur in other viscera, such as heart, in noncerebral theileriosis. Atypical location of the cells containing the parasite may be because of an embolism or sequestration in myocardial vessels.[35] Also in a pathologic study of cattle naturally infected with T. annulata, the heart had petechial and ecchymotic hemorrhages on the outer and inner surface of auricles.[33] Necrotic and hemorrhagic nodules were seen on those surfaces. In microscopic examination, degenerated cardiac muscle, intense lymphocytic infiltration, and hemorrhage had been seen.[33, 36] Myocyte damages could be because of the endothelial damages and thromobosis combined with cytotoxic mechanism associated with the presence of an intense inflammatory infiltrate.[34] However, there is no evidence of acute cardiac failure or systemic inflammatory disease in animals under study, which would cause severe elevations in cTnI.

Our result mainly demonstrated that cattle diagnosed with theileriosis had significantly higher cTnI concentrations in comparison with the healthy ones. This may substantiate higher incidence of myocyte damages in cattle with hemolytic anemia because of theileriosis. The current result may confirm our hypothesis that anemia followed by secondary hypoxia and vasculitis possibly will result in some degrees of cardiovascular damages in cattle with theileriosis. Although, no cases of disease led to recumbency, severe disturbances in vital signs, and death were observed, but it seems that decreased hematocrit may lead to an increase in serum cTnI levels. Hemolysis, which plays a role in the pathogenesis of the anemia with the tropical theileriosis caused by T. annulata, is extravascular type and its severity increases as well as the disease progress. In this regard, the highest mean cardiac troponin I concentrations occurred in cattle with hematocrit <25 and sever parasitemia (26–40%). Similar result obtained from a cohort study in human.[37] The proportion of patients with detectable cTnI was higher in anemic compared with nonanemic patients.[37] Anemia has been associated with hemolytic diseases such as theileriosis.[26, 27, 38] Cardiac troponin I (cTnI) reflects hemodynamic stress and cardiac myocyte injury. The release of cTnI in the serum may reflect a predisposition to cardiac ischemia, necrosis, repetitive stunning, and general cardiac myocyte injury in the setting of anemia.[37, 39]

Myocardial affectation in protozoan diseases is very rare.[34] However, cardiac involvement of some hemolytic infectious diseases such as equine piroplasmosis,[40] babesiosis,[28, 41] and trypanosomiasis in dog,[42] primary immune-mediated hemolytic anemia[43] and ehrlichiosis[44] in dog had been studied. However, in these studies there is clear potential for the infectious agent to cause direct myocyte damage and thereby making it difficult to objectively establish whether anemia per se is inducing cardiac myocyte damage or not. In a horse with piroplasmosis, cardiac arrhythmias and increase in serum cTnI had been attributed to an electrolyte and acid–base imbalance, hypoxia, endothelial injury, DIC, and inflammatory process.[40] A similar pathophysiologic mechanism for myocardial damage may have occurred in the cattle with theileriosis.

The prognostic value of cTnI concentrations in cattle with hemolytic anemia because of theileriosis deserves further investigations. Studies on the prognostic significance of cTnI concentrations in human patients with nonprimary cardiac disorders have also found that cTnI can predict disease outcome.[45, 46] Long-term follow-up of serum cTnI concentrations in cattle with hemolytic anemia would also be valuable in assessing the relationship between anemia and myocyte damage.

In conclusion, this study has demonstrated that anemic cattle with theileriosis have higher serum cTnI concentration compared with clinically healthy cattle. The prognostic significance of cTnI concentrations in cattle with clinical sings of theileriosis deserves further study.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors thank Dr Hassanpour, Dr Davood Nazmi, Shahram Pourhassan, and Sayyed Razi Bahavarnia for technical assistance.

Conflict of Interest Declaration: Authors disclose no conflict of interest.

Footnotes
  1. 1

    LIAISON® Troponin I, DiaSorin, Saluggia (Vercelli), Italy

  2. 2

    WS-ROCHE912, Roche Hitachi, Tokyo, Japan

  3. 3

    Pars Azmoon Co Inc, Karadj, Iran

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References