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Serum acute phase proteins in control and Theileria annulata infected water buffaloes (Bubalus bubalis)
Veterinary Parasitology 190 (2012) 12–18
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Serum acute phase proteins in control and Theileria annulata infected water buffaloes (Bubalus bubalis) Wael M. El-Deeb a,b,∗ , Olimpia C. Iacob c,1 a b c
Department of Veterinary Medicine, Infectious Diseases and Fish Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt Department of Clinical Studies, College of Veterinary Medicine and Animal Resources, King Faisal University, P.O. Box 1757, Al-Ahsa 31982, Saudi Arabia Clinics Department, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Iasi, Romania
a r t i c l e
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Article history: Received 20 March 2012 Received in revised form 13 June 2012 Accepted 16 June 2012 Keywords: Water buffaloes Haptoglobin Serum amyloid A ␣1-Acid glycoprotein Theileria
a b s t r a c t This study was carried out to ascertain the changes in acute phase proteins (APPs) and pro-inflammatory cytokines in Theileria annulata infected water buffalo. Thirty infected water buffaloes and 20 parasitologically free were used. In the present study there was significant (P ≤ 0.05) increase in haptoglobin (Hp), serum amyloid A (SAA), ceruloplasmin, ␣1-acid glycoprotein (AGP) and fibrinogen levels (2.18 ± 0.29 g/l, 156.58 ± 3.48 mg/l, 31.23 ± 1.25 mg/dl, 370.23 ± 33.21 mg/l and 16.17 ± 1.18 g/l, respectively) in T. annulata infected water buffaloes when compared to healthy ones (0.13 ± 0.01 g/l, 23.9 ± 0.56 mg/l, 21.23 ± 1.21 mg/dl, 240.53 ± 22.45 mg/l and 4.2 ± 0.16 g/l, respectively). Moreover, there was significant (P ≤ 0.05) increase in the levels of TNF-␣, IL-1␣, IL-6, IL-12, IL-1 and IFN-␥ (2.55 ± 0.12 ng/ml, 98.32 ± 4.21 pg/ml, 152.32 ± 5.62 pg/ml, 26.44 ± 1.43 ng/ml, 240.33 ± 20.45 pg/ml and 123.65 ± 5.67 pg/ml, respectively) in T. annulata infected water buffaloes when compared to healthy ones (0.42 ± 0.04 ng/ml, 55.32 ± 3.21 pg/ml, 88.23 ± 3.21 pg/ml, 7.45 ± 0.67 ng/ml, 98.33 ± 3.45 pg/ml and 34.76 ± 1.56 pg/ml, respectively). There was also significant decrease (P ≤ 0.05) in the Hb content, PCV%, RBCs and WBCs counts in the diseased water buffaloes compared to the control ones. Neutropenia, eosinopenia, lymphopenia, monocytopenia and thrombocytopenia were also recorded. The biochemical changes revealed significant (P ≤ 0.05) elevation in the levels of AST, ALT, ALP, LDL-c, VLDL-c, BHBA and NEFA, with significant (P ≤ 0.05) decrease in the levels of total proteins, albumin, globulins, cholesterol, triglyceride, glucose, G6PD, calcium and phosphorus in T. annulata infected water buffaloes when compared to healthy ones. It could be concluded that APPs and pro-inflammatory cytokines could be used as a valuable biomarkers in T. annulata infected water buffaloes (Bubalus bubalis). © 2012 Elsevier B.V. All rights reserved.
1. Introduction
∗ Correspondingauthor at: Mansoura University, Department of Veterinary Medicine, Infectious Diseases and Fish Diseases, Faculty of Veterinary Medicine, EL-Gomhorya st, Mansoura 35516, Egypt. Tel.: +20 50 2372592/966 050 9296154; fax: +20 50 2379952/966 03 5816635. E-mail addresses: [email protected] (W.M. El-Deeb), [email protected], [email protected] (O.C. Iacob). 1 Tel.: +40 232 407317; fax: +40 232 219113. 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.06.019
Theileria annulata a protozoan parasite of cattle and domestic buffaloes, is transmitted by ticks of the genus Hyalomma, and causes a disease named Mediterranean or tropical theileriosis. It represents a major threat to cattle and buffaloes in many countries, where it causes significant economic losses as well as reduced production. Tropical theileriosis, is present in northern Africa and southern Europe, extending through the Middle East, India, and southern Russia into China (Uilenberg, 1981).
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Many advances in monitoring the acute phase protein (APPs) response in animals for clinical and experimental purposes have been achieved in the last two decades (Gruys et al., 1994; Eckersall, 2000). For instance, researchers have established various quantitative APPs assays, thereby recognizing the species-related difference of the APPs behavior on some key proteins. There is also growing interest in elucidating the mechanisms of the response and standardizing APPs assays, both of which could provide benefits to veterinary medicine (Eckersall et al., 1999; Eckersall and Bell, 2010). The APPs consist of “negative” and “positive” proteins that show a decrease and an increase in levels, respectively, in response to challenge. The negative APPs include albumin, the most abundant constitutive plasma protein, and transferrin. The positive APPs are glycoproteins synthesized mainly by hepatocytes upon stimulation by pro-inflammatory cytokines and released into the bloodstream. The positive APPs include haptoglobin, C-reactive protein, serum amyloid A, ceruloplasmin, fibrinogen and alpha 1-acid glycoprotein (Eckersall and Bell, 2010). Extrahepatic production of APPs is also possible in most of the mammalian species that have been studied (Uhlar and Whitehead, 1999). The interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) are the main mediators of APPs synthesis in the liver (Alsemgeest et al., 1995; Yoshioka et al., 2002). These cytokines are released mainly by macrophages but also by other cells in response to various external or internal stimuli. For instance, IL-6 can be synthesized by Kupffer cells and keratinocytes (Heinrich et al., 1990), in the pituitary (Abraham and Minton, 1997) or in the mucosal epithelium (Pritts et al., 2002). Inflammation, infection or tissue injury will trigger cytokine release by defence-oriented cells, thereby inducing APPs synthesis. The intracellular mechanism for the induction of positive APPs in hepatocytes by cytokines has been reported elsewhere (Jensen and Whitehead, 1998). The induction is associated with a decrease in synthesis of negative APPs such as albumin (Gruys et al., 1994). Monocytes and macrophages produced proinflammatory cytokines (TNF-␣, IL-1, IL-12 and IFN-␥), mediating the effect of APPs, favoring T-helper cell differentiation that construct a bridge between innate resistance and adaptive immunity (Trinchieri et al., 2003). To the best of authors knowledge little is known about APPs and pro-inflammatory cytokines response in T. annulata infected water buffaloes. So, the aim of the present study is to investigate the APPs and cytokines response in T. annulata infected water buffaloes (Bubalus bubalis).
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the basis of clinical examination and positive blood and/or lymph node smears. 2.2. Clinical examination Clinical examination was performed on all animals. The signs of T. annulata infection were observed and recorded. Thin blood smears were prepared from the ear veins of all animals. Lymph node aspirates were collected from suspected cases suffered from enlarged superficial lymph nodes. 2.3. Sampling protocol All animals under study were subjected to ear vein puncture and lymph node aspiration. Blood samples were collected from all infected buffaloes and parasitologically free control one through jugular vein puncture, in tubes contaminated with ethylene-diamine-tetraacetic acid dipotassium salt (EDTA-K2) for routine blood tests and into heparinized glass-stoppered tubes for other analysis (Schalm et al., 1986). Thin blood smears were prepared, fixed with absolute methanol (5 min), stained with 10% Giemsa solution (30 min) and examined under oil immersion lens (×1000) to observe intraerythrocytic forms of T. annulata. More than 50 microscopic fields of blood films at a magnification of ×1000 were done. Approximately 20,000 red blood cells (RBCs) were carefully searched per slide. 2.4. Hematological parameters The hematological parameters were determined using electronic cell counter (Vet Scan HM5 Hematology system). 2.5. Biochemical parameters The levels of glucose, triglyceride (TG), cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), total proteins, albumin, globulins and blood urea nitrogen (BUN), as well as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined in serum samples on a Beckman CX-7 autoanalyser using commercial kits (Sigma Chemical Co. Ltd., Poole, Dorset, UK). 2.6. Glucose-6-phosphate dehydrogenase (G6PD) G6PD level was carried out using commercially available test kits supplied by Biodiagnostic-Egypt, according to the methods described by Beutler et al. (1963). 2.7. Serum ˇ-hydroxybutyrate (BHBA)
2. Materials and methods 2.1. Animals This study was carried out on 30 water buffaloes located in small groups and in contact with cattle. In addition, 20 parasitologically free ones located in the same area and under the same levels of nutrition and hygiene were used as a control group. Infected water buffaloes were selected on
Serum concentration of BHBA was determined by a kinetic enzymatic method using a commercially available kit (Ranbut D-3-hydroxybutyrate, Randox, Crumlin Co., Antrim, UK). The assay is based on the reversible reaction between 3-hydroxybutyrate and NAD1 catalyzed by 3hydroxybutyrate dehydrogenase, and the change in NADH concentration is measured by changes in the absorbance at 340 nm.
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Table 1 The mean levels of acute phase proteins in control and Thieleria annulata infected water buffaloes. Variables
Control (n = 20) (mean ± SEa )
Haptoglobin (g/l) Serum amyloid A (mg/l) Fibrinogen (g/l) Ceruloplasmin (mg/dl) ␣1-Acid glycoprotein (mg/l)
0.13 23.9 4.2 21.23 240.53
a *
± ± ± ± ±
Diseased water buffaloes (n = 30) (mean ± SEa )
0.01 0.56 0.16 1.21 22.45
2.18 156.58 16.17 31.23 370.23
± ± ± ± ±
0.29* 3.48* 1.18* 1.25* 33.21*
Standard error. Means are significantly different at the level (P ≤ 0.05).
2.8. Non estrified free fatty acids (NEFA)
TEC, ELX800G, USA). Samples were run in triplicates in all examined cytokines.
Serum concentration of NEFA was carried out using commercially available test kits supplied by Randox Laboratories Ltd.
2.11. Statistical analysis
2.9. Calcium, phosphorus and magnesium levels
The obtained data was analyzed using Student’s ttest according to the method described by Snedecor and Cochran (1989).
Calcium, phosphorus and magnesium levels were carried out using commercial available kits by colorimetric methods using spectrophotometer.
3. Results
2.10. Determination of acute phase proteins and cytokines Serum Hp was determined using the hemoglobin binding assay described by Makimura and Suzuki (1982). SAA was measured with a commercially available ELISA kit (Phase SAA kit, Tridelta Ltd., Ireland), according to the manufacturer’s instructions. Fibrinogen concentration was measured with a commercially available ELISA kit (USCA, Life Science). Ceruloplasmin oxidase activity was measured in duplicate with the use of colorimetric procedures described by Demetriou et al. (1974). All results are expressed as milligram per deciliter with intra-assay and inter-assay variation of ≤5% and 10%, respectively. Serum ␣1-acid glycoprotein (AGP) was analyzed using a commercial radial immunodiffusion kit manufactured by Ecos Institute (Furukawa, Miyagi, Japan). The protocol suggested by the manufacturer was followed and the test result was read after 48 h incubation in a humid chamber at room temperature. The concentration was recorded in mg/l, IL-6, IL-1␣, IL-1, IL-12, TNF-␣, and IFN-␥ levels were determined from undiluted serum samples using commercially available ELISA Kits (Biosurce, Diagnostic Corporation, Belgium). The plates were read at 450 nm on a computerized automated microplate ELISA reader (Bio
The present investigation revealed significant (P ≤ 0.05) increase in Hp, SAA, ceruloplasmin, AGP and fibrinogen levels in T. annulata infected water buffaloes when compared to healthy ones (Table 1). Moreover there was significant (P ≤ 0.05) increase in the levels of TNF␣, IL-1␣, IL-6, IL-12, IL-1 and IFN-␥ in T. annulata infected water buffaloes when compared to healthy ones (Table 2). In the present study the hematological examination (Table 3) revealed significant decrease (P ≤ 0.05) in the Hb content, PCV%, RBCs and TLC in the diseased water buffaloes when compared to control ones. Neutropenia, eosinopenia, lymphopenia, monocytopenia and thrombocytopenia were also recorded in infected buffaloes. The biochemical changes revealed significant (P ≤ 0.05) elevation in the levels of AST, ALT, ALP, LDL-c, VLDL-c, BHBA and NEFA, with significant (P ≤ 0.05) decrease in the levels of total proteins, albumin, globulins, cholesterol, triglyceride, glucose, G6PD, calcium and phosphorus in T. annulata infected water buffaloes when compared to healthy ones (Table 4). 4. Discussion Acute phase proteins (APPs) play an important role in innate defence mechanisms (Eckersall, 2000). In the
Table 2 The mean levels of pro-inflammatory cytokines in control and Thieleria annulata infected water buffaloes. Variables
Control (n = 20) (mean ± SEa )
TNF-␣ (ng/ml) IL-1␣ (pg/ml) IL-1 (pg/ml) IL-6 (pg/ml) IL-12 (ng/ml) IFN-␥ (pg/ml)
0.42 55.32 98.33 88.23 7.45 34.76
a *
± ± ± ± ± ±
0.04 3.21 3.45 3.21 0.67 1.56
Standard error. Means are significantly different at the level (P ≤ 0.05).
Diseased water buffalo (n = 30) (mean ± SEa ) 2.55 98.32 240.33 152.32 26.44 123.65
± ± ± ± ± ±
0.12* 4.21* 20.45* 5.62* 1.43* 5.67*
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Table 3 Hematological parameters in control, and Thieleria annulata infected water buffaloes. Variables
Control (n = 20) (mean ± SEa )
Hb (g/Dl) RBCs (106 /mL) PCV (%) TLC (103 /mm3 ) Neutrophils (103 /mL) Lymphocytes (103 /mL) Eosinophils (103 /mL) Basophils (103 /mL) Monocytes (103 /mL) Thrombocytes (103 /mL)
13.46 9.85 35.23 9.6 2.9 3.8 0.82 0.013 0.42 269.75
a *
± ± ± ± ± ± ± ± ± ±
Diseased water buffaloes (n = 30) (mean ± SEa )
0.39 0.16 0.12 0.18 0.015 0.012 0.01 0.0001 0.001 3.52
6.2 5.23 26.9 6.4 2.3 2.5 0.13 0.0126 0.34 179.35
± ± ± ± ± ± ± ± ± ±
0.17* 0.32* 0.11* 0.22* 0.037* 0.013* 0.001* 0.0001 0.002* 4.52*
Standard error. Means are significantly different at the level (P ≤ 0.05).
present study there was a significant (P ≤ 0.05) increase in Hp, SAA, ceruloplasmin, AGP and fibrinogen levels in T. annulata infected water buffaloes when compared to healthy ones. These results are consistent with Glass et al. (2003) and Nazifi et al. (2010) in cattle. The authors reported that T. annulata causes severe pathology in susceptible cattle by inducing high levels of APPs. Following experimental infection, SAA appeared first, followed by a rise in ␣1-acid glycoprotein in all cattle, whereas Hp which is a major APPs in cattle only appeared in some of the animals and generally at a low level. SAA, Hp and ␣1-acid glycoprotein only elevated around or after the appearance of schizonts and after the first rise in temperature. Increased ␣1-acid glycoprotein level coincided with the appearance of piroplasms. The production of SAA and ␣1-acid glycoprotein correlated strongly with each other and also with some clinical features of disease severity including the time of fever, development of leukopenia, parasitemia rate and mortality. The obtained results are also in consistent with those reported by Glass and Jensen (2007) in cattle. The primary trail leading to significant elevation in APPs in infected water buffaloes may involve initial release of pro-inflammatory cytokines by macrophages at the site
of infection or inflammation. This results in a cascade of further release of cytokines by macrophages and other cells. The most important inducers of APPs are cytokines of the IL-1, TNF and IL-6 families (Glass et al., 2003). If these pro-inflammatory cytokines spill into the circulation, they induce release of APPs by the liver into the circulation (Baumann and Gauldie, 1994). The utility of these significant elevation of APPs is not well established but they appear to be involved in controlling inflammation and contribute to innate host defences against a wide range of pathogens (Glass et al., 2003). Their circulating levels may also be related to the severity of the response to infection, and thus may provide valuable quantifiable biochemical indicators of the inflammatory response. Bacterial infections induce strong acute phase responses whereas viral infections generally lead to weak or non-detectable acute phase reactions. There are relatively few reports of APPs induction by parasite infections. However, infections with Plasmodium falciparum (Graninger et al., 1992), Babesia canis (Matijatko et al., 2007), Leishmania infantum (Martinez-Subiela et al., 2002) and Trypanosoma brucei (Shapiro and Black, 1992; Eckersall et al., 2001) might be accompanied by increased levels of APPs.
Table 4 Biochemical parameters in control and Thieleria annulata infected water buffaloes. Variables AST (U/l) ALT (U/l) ALP (U/l) Total proteins (g/dl) Albumin (g/dl) Globulins (g/dl) TG (mg/dl) Cholesterol (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL-c (mg/dl) Glucose (mg/dl) BHBA (mmol/l) NEFA (mmol/l) G6PD (IU/g Hb) Calcium (mg/dl) Phosphorus (mg/dl) Magnesium (mg/dl) a *
Control (n = 20) (mean ± SEa ) 88.8 44.2 121.8 6.3 3.55 2.8.35 29.2 69.0 26.3 22.8 6.1 63.0 0.45 363.6 22.45 10.54 5.56 2.63
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Standard error. Means are significantly different at the level (P ≤ 0.05).
4.54 0.36 4.37 0.13 0.13 0.23 0.53 2.60 0.26 0.57 0.50 0.6 0.03 5.5 0.15 0.32 0.21 0.21
Diseased water buffaloes (n = 30) (mean ± SEa ) 322.0 229.8 239.0 4.51 2.4 1.45 17.8 39.5 16.5 30.9 7.8 37.2 1.9 536 17.28 7.89 3.42 2.72
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.68* 8.55* 6.20* 0.12* 0.12* 0.23* 0.87* 0.52* 0.27* 0.89* 0.30* 2.1* 0.01* 10.8a 0.29* 0.23* 0.21* 0.45
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Inflammation or tissue injury causes the release of pro-inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor (TNF) which alter the blood concentration of a variety of proteins that are produced primarily in the liver (Nazifi et al., 2009). Increases in these proteins are recognized by elevations in the ␣- and/or -regions by serum protein electrophoresis. The concentration of these proteins is generally low to non-detectable in healthy animals and elevations are used to diagnose and monitor inflammatory diseases (Glass et al., 2003; Nazifi et al., 2010). The specific type of APPs and the time course for alterations in these proteins vary in different species on the basis of the initiating disorder or underlying inflammatory process (Feldman et al., 2000). In the present study there was significant (P ≤ 0.05) increase in the levels of TNF-␣, IL-1␣, IL-6, IL-12, IL-1 and IFN-␥ in T. annulata infected water buffaloes when compared to healthy ones. The elevated levels of selected cytokines could be attributed to hemorrhages, lymphocytic infiltration and necrosis in abomasum, liver, kidney, intestine, lung and bone marrow which indicate a widespread inflammatory reaction in bovine theilerisosis. These lesions induce the release of cytokines such as IL-1, IL-6 and TNF under the effect of which, APPs are synthesized in liver (Jubb et al., 1991; Radostits et al., 2007). In the same concern Brown et al. (1995) and Forsyth et al. (1999) reported that schizontinfected macrophages express mRNA for pro-inflammatory cytokines (interleukin-1 (IL-1), IL-6 and tumor necrosis factor (TNF-␣)) and contain intracellular TNF-␣ protein. In addition, uninfected macrophages from animals recovering from or immune to tropical theileriosis secrete TNF-␣, at least in vitro (Preston et al., 1993). This would suggest that pro-inflammatory cytokines are released systemically. If so, they probably play a major role in pathology and indeed many of the clinical signs of tropical theileriosis resemble those induced by experimental TNF-␣ administration to cattle (Bielefeldt Ohmann et al., 1989). Hematological examination revealed significant decrease (P ≤ 0.05) in the Hb content, PCV%, RBCs and TLC in the diseased water buffaloes compared to the control ones. Neutropenia, eosinopenia, lymphopenia, monocytopenia and thrombocytopenia were also recorded. These results are in agreement with those obtained by Osman and AL-Gaabary (2007) and El-Deeb and Younis (2009). The decrease in RBC counts could be due to increase in the levels of activated complement products (Omer et al., 2002) and erythrophagocytosis (Yagi et al., 2002). In addition, pro-inflammatory cytokines, particularly TNF-␣, have been implicated in mediating anemia associated with tropical theileriosis (Graham et al., 2001). This decrease is also related to the destruction of lymphocytes in lymphoid organs and infiltration of these cells into various organs (Sandhu et al., 1998). However, no significant difference in absolute basophile and monocyte counts between healthy and infected cattle was recorded by Omer et al. (2002). This variation could be attributed to differences in the stage and severity of the disease. The decline in RBC, PCV and Hb may be attributed to destruction of erythrocytes by macrophages in the lymph nodes, spleen and other organs of monocyte–macrophage system (Singh et al., 2001). Sandhu et al. (1998) evaluated hematological and
biochemical factors in experimental T. annulata infection in crossbred calves. They reported a significant progressive decrease in Hb concentration, PCV and RBC, whereas the total leukocytes count showed an initial non-significant leukocytosis followed by a significant leukopenia. Dhar and Gautam (1979), Sharma (1979), Mehta et al. (1988) and Rayula and Hafeez (1995) reported a progressive decrease in Hb, PCV and RBC in acute T. annulata infection. A progressive decrease in the Hb and PCV, along with a marked reticulocytosis was also reported by Singh et al. (2001). Concerning the biochemical changes in T. annulata infected water buffaloes, there was significant (P ≤ 0.05) elevation in the levels of AST, ALT, ALP, LDL-c, VLDL-c, BHBA and NEFA, with significant (P ≤ 0.05) decrease in the levels of total proteins, albumin, globulins, cholesterol, TG, glucose, G6PD, calcium and phosphorus in T. annulata infected water buffaloes when compared to healthy ones. The obtained results are in concern with those reported by El-Deeb and Younis (2009) in water buffaloes and Khan et al. (2011) in cattle. The findings were also in line with the findings of Col and Uslu (2006) who reported hypoproteinemia and hypoalbuminemia in tropical theileriosis in cattle. The results were in agreement with the findings of Yadav and Sharma (1986) and Singh et al. (2001) in cattle and crossbred calves respectively. The decreased serum proteins concentrations may be attributed to extra-vascular proteinaceous fluid in body cavities due to diseased lymph nodes resulting edema. The decrease in total serum proteins was attributed to low albumin and globulin concentrations as a result of liver failure (Omer et al., 2003). The elevated concentrations of the examined enzymes are indicators of hepatic function (Forsyth et al., 1999). Similar findings were observed by Col and Uslu (2006) who reported an increase in ALT concentration in cattle. The rise in serum ALT concentration may be due to muscular trauma as a result of prolonged recumbency due to bovine theileriosis (Col and Uslu, 2006). In the present study, there was significant decrease in serum glucose and G6PD concentrations with significant increase in the levels of BHBA and NEFA in T. annulata infected water buffaloes when compared to healthy controls. These findings were in line with the findings of Col and Uslu (2006). They reported that, the decreased glucose serum concentration could be due to utilization of glucose by Theileria in the blood and hepatic dysfunction as a result of Theileria infection. Similar findings were obtained by El-Deeb and Younis (2009) in T. annulata infected water buffaloes. They reported that T. annulata could act as ketotic stressor in infected buffaloes. The findings were in contrast with those obtained by Sandhu et al. (1998) who reported a non-significant decrease in serum glucose in calves affected with theileriosis. The significant decrease in the activity of G6PD in infected water buffaloes suffering from severe anemia is an indicator of a metabolic disturbance in the erythrocytes and associated with increased RBCs hemolysis in theileriosis (Singari et al., 1991) and increased oxidative stress in endothelial cells (Leopold et al., 2003 and El-Deeb and Younis, 2009). The obtained results were different from that reported by Grewal et al.
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(2005) who reported significant increase in the activity of G6PD in cattle naturally infected with T. annulata. The variation of G6PD activities might be related to the severity of the anemia. Interestingly there was decreased serum calcium and phosphorus concentrations in T. annulata infected water buffaloes when compared to healthy ones. Similar findings were reported by Omer et al. (2003) and Khan et al. (2011) in infected cattle. Hypocalcaemia could be due to hypoproteinemia, decreased dietary intake, intestinal malfunction and kidney damage and the decreased phosphorus concentration due to diarrhea and renal wasting (Col and Uslu, 2006). Non-significant differences was observed in magnesium concentrations in infected and healthy controls. These results are consistent with those obtained by Khan et al. (2011) in cattle. There were significant decreases in serum cholesterol, triglycerides, HDL-c concentrations with significant increase in the levels of LDL-c and in T. annulata infected water buffaloes. These results are in agreement with those reported by Singh et al. (2001) in cattle. These decreases in cholesterol and triglyceride levels may be ascribed to anorexia and diarrhea (Col and Uslu, 2006). The findings were otherwise than the findings of Yadav and Sharma (1986) who recorded a marked increase in serum cholesterol concentration in experimentally T. annulata infected cattle from day 0 to day 15, followed by a sudden fall, reaching values below the pre-infection level by day 40. They reported that, this could be due to liver damage that results in a concurrent increase in the level of fats with the reduction of sugar and protein. 5. Conclusion In the present study the APPs and pro-inflammatory cytokines could be used as a valuable biomarkers in T. annulata infected water buffaloes (B. bubalis). Moreover T. annulata may be considered as a cofactor induces detectable changes in lipoproteins profile, calcium and phosphorus levels in water buffaloes (B. bubalis). References Abraham, E.J., Minton, J.E., 1997. Cytokines in the hypophysis: a comparative look at interleukin-6 in the porcine anterior pituitary gland. Comp. Biochem. Physiol. A: Physiol. 116A, 203–207. Alsemgeest, S.P., Lambooy, I.E., Wierenga, H.K., Dieleman, S.J., Meerkerk, B., Van Edern, A.M., Niewold, T.A., 1995. Influence of physical stress on the plasma concentration of serum amyloid-A (SAA) and haptoglobin (Hp) in calves. Vet. Q. 17 (1), 9–12. Baumann, H., Gauldie, J., 1994. The acute phase response. Immunol. Today 15, 74–80. Beutler, E., Duron, O., Kelly, M.B., 1963. J. Lab. Clin. Med. 61, 882. Bielefeldt Ohmann, H., Campos, M., Snider, M., Rapin, N., Beskorwayne, T., Popowych, Y., Lawman, M.J., Rossi, A., Babiuk, L.A., 1989. Effect of chronic administration of recombinant bovine tumor necrosis factor to cattle. Vet. Pathol. 26, 462–472. Brown, D., Campbell, J.D.M., Russell, G.C., Hopkins, J., Glass, E.J., 1995. T cell activation by Theileria annulata-infected macrophages correlates with cytokine production. Clin. Exp. Immunol. 102, 507–514. Col, R., Uslu, U., 2006. Haematological and coagulation profiles during severe tropical theileriosis in cattle. Turk. J. Vet. Anim. Sci. 30, 577–582. Demetriou, J.A., Drewes, P.A., Gin, J.B., 1974. Ceruloplasmin. In: Cannon, D.C., Winkelman (Eds.), Clinical Chemistry. Principles and Techniques. , 2nd ed. Harper and Row, Hagerstown, MD, pp. 857–864.
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Nazifi, S., Razavi, S.M., Esmailnejad, Z., Gheisari, H., 2009. Study on acute phase proteins (haptoglobin, serum amyloid A, fibrinogen, and ceruloplasmin) changes and their diagnostic values in bovine tropical theileriosis. Parasitol. Res. 105, 41–46. Nazifi, S., Razavi, S.M., Reiszadeh, M., Esmailnezhad, Z., Ansari-Lari, M., Hasanshahi, F., 2010. Evaluation of the resistance of indigenous Iranian cattle to Theileria annulata compared with Holstein cattle by measurement of acute phase proteins. Comp. Clin. Pathol. 19, 155–161. Omer, O.H., El-Malik, K.H., Magzoub, M., Mahmoud, O.M., Haroun, E.M., Hawas, A., Omar, H.M., 2003. Biochemical profiles in Friesian cattle naturally infected with Theileria annulata in Saudi Arabia. Vet. Res. Commun. 27, 15–25. Omer, O.H., El-Malik, K.H., Mahmoud, O.M., Haroun, E.M., Hawas, A., Sweeney, D., Magzoub, M., 2002. Haematological profiles in pure bred cattle naturally infected with Theileria annulata in Saudi Arabia. Vet. Parasitol. 107, 161–168. Osman, S.A., AL-Gaabary, M.H., 2007. Clinical, haematological and therapeutic studies on tropical theileriosis in water buffaloes (Bubalus bubalis) in Egypt. Vet. Parasitol. 146, 337–340. Preston, P.M., Brown, C.G.D., Entrican, G., Richardson, W., Boid, R., 1993. Synthesis of tumour necrosis factor alpha and interferons by mononuclear cells from Theileria annulata infected cattle. Parasite Immunol. 15, 525–534. Pritts, T., Hungnese, E., Wang, Q., Robb, B., Hershko, D., Hasselgren, P.O., 2002. Mucosal and enterocyte IL-6 production during sepsis and endotoxemia – role of transcription factors and regulation by the stress response. Am. J. Surg. 183, 372–383. Radostits, O.M., Gay, C.C., Hinchcliff, K.W., Constable, P.D., 2007. Veterinary Medicine, 10th ed, p. 1529. Rayula, V., Hafeez, M.D., 1995. Haematological values in cattle infected with Theileria annulata. Indian J. Anim. Sci. 65, 1202–1203. Sandhu, G.S., Grewal, A.S., Singh, A., Kondal, J.K., Singh, J., Brar, R.S., 1998. Haematological and biochemical studies on experimental Theileria annulata infection in crossbred calves. Vet. Res. Commun. 22, 347–354.
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Serum acute phase proteins in control and Theileria annulata infected water buffaloes (Bubalus bubalis) Wael M. El-Deeb a,b,∗ , Olimpia C. Iacob c,1 a b c
Department of Veterinary Medicine, Infectious Diseases and Fish Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt Department of Clinical Studies, College of Veterinary Medicine and Animal Resources, King Faisal University, P.O. Box 1757, Al-Ahsa 31982, Saudi Arabia Clinics Department, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Iasi, Romania
a r t i c l e
i n f o
Article history: Received 20 March 2012 Received in revised form 13 June 2012 Accepted 16 June 2012 Keywords: Water buffaloes Haptoglobin Serum amyloid A ␣1-Acid glycoprotein Theileria
a b s t r a c t This study was carried out to ascertain the changes in acute phase proteins (APPs) and pro-inflammatory cytokines in Theileria annulata infected water buffalo. Thirty infected water buffaloes and 20 parasitologically free were used. In the present study there was significant (P ≤ 0.05) increase in haptoglobin (Hp), serum amyloid A (SAA), ceruloplasmin, ␣1-acid glycoprotein (AGP) and fibrinogen levels (2.18 ± 0.29 g/l, 156.58 ± 3.48 mg/l, 31.23 ± 1.25 mg/dl, 370.23 ± 33.21 mg/l and 16.17 ± 1.18 g/l, respectively) in T. annulata infected water buffaloes when compared to healthy ones (0.13 ± 0.01 g/l, 23.9 ± 0.56 mg/l, 21.23 ± 1.21 mg/dl, 240.53 ± 22.45 mg/l and 4.2 ± 0.16 g/l, respectively). Moreover, there was significant (P ≤ 0.05) increase in the levels of TNF-␣, IL-1␣, IL-6, IL-12, IL-1 and IFN-␥ (2.55 ± 0.12 ng/ml, 98.32 ± 4.21 pg/ml, 152.32 ± 5.62 pg/ml, 26.44 ± 1.43 ng/ml, 240.33 ± 20.45 pg/ml and 123.65 ± 5.67 pg/ml, respectively) in T. annulata infected water buffaloes when compared to healthy ones (0.42 ± 0.04 ng/ml, 55.32 ± 3.21 pg/ml, 88.23 ± 3.21 pg/ml, 7.45 ± 0.67 ng/ml, 98.33 ± 3.45 pg/ml and 34.76 ± 1.56 pg/ml, respectively). There was also significant decrease (P ≤ 0.05) in the Hb content, PCV%, RBCs and WBCs counts in the diseased water buffaloes compared to the control ones. Neutropenia, eosinopenia, lymphopenia, monocytopenia and thrombocytopenia were also recorded. The biochemical changes revealed significant (P ≤ 0.05) elevation in the levels of AST, ALT, ALP, LDL-c, VLDL-c, BHBA and NEFA, with significant (P ≤ 0.05) decrease in the levels of total proteins, albumin, globulins, cholesterol, triglyceride, glucose, G6PD, calcium and phosphorus in T. annulata infected water buffaloes when compared to healthy ones. It could be concluded that APPs and pro-inflammatory cytokines could be used as a valuable biomarkers in T. annulata infected water buffaloes (Bubalus bubalis). © 2012 Elsevier B.V. All rights reserved.
1. Introduction
∗ Correspondingauthor at: Mansoura University, Department of Veterinary Medicine, Infectious Diseases and Fish Diseases, Faculty of Veterinary Medicine, EL-Gomhorya st, Mansoura 35516, Egypt. Tel.: +20 50 2372592/966 050 9296154; fax: +20 50 2379952/966 03 5816635. E-mail addresses: [email protected] (W.M. El-Deeb), [email protected], [email protected] (O.C. Iacob). 1 Tel.: +40 232 407317; fax: +40 232 219113. 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.06.019
Theileria annulata a protozoan parasite of cattle and domestic buffaloes, is transmitted by ticks of the genus Hyalomma, and causes a disease named Mediterranean or tropical theileriosis. It represents a major threat to cattle and buffaloes in many countries, where it causes significant economic losses as well as reduced production. Tropical theileriosis, is present in northern Africa and southern Europe, extending through the Middle East, India, and southern Russia into China (Uilenberg, 1981).
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Many advances in monitoring the acute phase protein (APPs) response in animals for clinical and experimental purposes have been achieved in the last two decades (Gruys et al., 1994; Eckersall, 2000). For instance, researchers have established various quantitative APPs assays, thereby recognizing the species-related difference of the APPs behavior on some key proteins. There is also growing interest in elucidating the mechanisms of the response and standardizing APPs assays, both of which could provide benefits to veterinary medicine (Eckersall et al., 1999; Eckersall and Bell, 2010). The APPs consist of “negative” and “positive” proteins that show a decrease and an increase in levels, respectively, in response to challenge. The negative APPs include albumin, the most abundant constitutive plasma protein, and transferrin. The positive APPs are glycoproteins synthesized mainly by hepatocytes upon stimulation by pro-inflammatory cytokines and released into the bloodstream. The positive APPs include haptoglobin, C-reactive protein, serum amyloid A, ceruloplasmin, fibrinogen and alpha 1-acid glycoprotein (Eckersall and Bell, 2010). Extrahepatic production of APPs is also possible in most of the mammalian species that have been studied (Uhlar and Whitehead, 1999). The interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) are the main mediators of APPs synthesis in the liver (Alsemgeest et al., 1995; Yoshioka et al., 2002). These cytokines are released mainly by macrophages but also by other cells in response to various external or internal stimuli. For instance, IL-6 can be synthesized by Kupffer cells and keratinocytes (Heinrich et al., 1990), in the pituitary (Abraham and Minton, 1997) or in the mucosal epithelium (Pritts et al., 2002). Inflammation, infection or tissue injury will trigger cytokine release by defence-oriented cells, thereby inducing APPs synthesis. The intracellular mechanism for the induction of positive APPs in hepatocytes by cytokines has been reported elsewhere (Jensen and Whitehead, 1998). The induction is associated with a decrease in synthesis of negative APPs such as albumin (Gruys et al., 1994). Monocytes and macrophages produced proinflammatory cytokines (TNF-␣, IL-1, IL-12 and IFN-␥), mediating the effect of APPs, favoring T-helper cell differentiation that construct a bridge between innate resistance and adaptive immunity (Trinchieri et al., 2003). To the best of authors knowledge little is known about APPs and pro-inflammatory cytokines response in T. annulata infected water buffaloes. So, the aim of the present study is to investigate the APPs and cytokines response in T. annulata infected water buffaloes (Bubalus bubalis).
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the basis of clinical examination and positive blood and/or lymph node smears. 2.2. Clinical examination Clinical examination was performed on all animals. The signs of T. annulata infection were observed and recorded. Thin blood smears were prepared from the ear veins of all animals. Lymph node aspirates were collected from suspected cases suffered from enlarged superficial lymph nodes. 2.3. Sampling protocol All animals under study were subjected to ear vein puncture and lymph node aspiration. Blood samples were collected from all infected buffaloes and parasitologically free control one through jugular vein puncture, in tubes contaminated with ethylene-diamine-tetraacetic acid dipotassium salt (EDTA-K2) for routine blood tests and into heparinized glass-stoppered tubes for other analysis (Schalm et al., 1986). Thin blood smears were prepared, fixed with absolute methanol (5 min), stained with 10% Giemsa solution (30 min) and examined under oil immersion lens (×1000) to observe intraerythrocytic forms of T. annulata. More than 50 microscopic fields of blood films at a magnification of ×1000 were done. Approximately 20,000 red blood cells (RBCs) were carefully searched per slide. 2.4. Hematological parameters The hematological parameters were determined using electronic cell counter (Vet Scan HM5 Hematology system). 2.5. Biochemical parameters The levels of glucose, triglyceride (TG), cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), total proteins, albumin, globulins and blood urea nitrogen (BUN), as well as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined in serum samples on a Beckman CX-7 autoanalyser using commercial kits (Sigma Chemical Co. Ltd., Poole, Dorset, UK). 2.6. Glucose-6-phosphate dehydrogenase (G6PD) G6PD level was carried out using commercially available test kits supplied by Biodiagnostic-Egypt, according to the methods described by Beutler et al. (1963). 2.7. Serum ˇ-hydroxybutyrate (BHBA)
2. Materials and methods 2.1. Animals This study was carried out on 30 water buffaloes located in small groups and in contact with cattle. In addition, 20 parasitologically free ones located in the same area and under the same levels of nutrition and hygiene were used as a control group. Infected water buffaloes were selected on
Serum concentration of BHBA was determined by a kinetic enzymatic method using a commercially available kit (Ranbut D-3-hydroxybutyrate, Randox, Crumlin Co., Antrim, UK). The assay is based on the reversible reaction between 3-hydroxybutyrate and NAD1 catalyzed by 3hydroxybutyrate dehydrogenase, and the change in NADH concentration is measured by changes in the absorbance at 340 nm.
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Table 1 The mean levels of acute phase proteins in control and Thieleria annulata infected water buffaloes. Variables
Control (n = 20) (mean ± SEa )
Haptoglobin (g/l) Serum amyloid A (mg/l) Fibrinogen (g/l) Ceruloplasmin (mg/dl) ␣1-Acid glycoprotein (mg/l)
0.13 23.9 4.2 21.23 240.53
a *
± ± ± ± ±
Diseased water buffaloes (n = 30) (mean ± SEa )
0.01 0.56 0.16 1.21 22.45
2.18 156.58 16.17 31.23 370.23
± ± ± ± ±
0.29* 3.48* 1.18* 1.25* 33.21*
Standard error. Means are significantly different at the level (P ≤ 0.05).
2.8. Non estrified free fatty acids (NEFA)
TEC, ELX800G, USA). Samples were run in triplicates in all examined cytokines.
Serum concentration of NEFA was carried out using commercially available test kits supplied by Randox Laboratories Ltd.
2.11. Statistical analysis
2.9. Calcium, phosphorus and magnesium levels
The obtained data was analyzed using Student’s ttest according to the method described by Snedecor and Cochran (1989).
Calcium, phosphorus and magnesium levels were carried out using commercial available kits by colorimetric methods using spectrophotometer.
3. Results
2.10. Determination of acute phase proteins and cytokines Serum Hp was determined using the hemoglobin binding assay described by Makimura and Suzuki (1982). SAA was measured with a commercially available ELISA kit (Phase SAA kit, Tridelta Ltd., Ireland), according to the manufacturer’s instructions. Fibrinogen concentration was measured with a commercially available ELISA kit (USCA, Life Science). Ceruloplasmin oxidase activity was measured in duplicate with the use of colorimetric procedures described by Demetriou et al. (1974). All results are expressed as milligram per deciliter with intra-assay and inter-assay variation of ≤5% and 10%, respectively. Serum ␣1-acid glycoprotein (AGP) was analyzed using a commercial radial immunodiffusion kit manufactured by Ecos Institute (Furukawa, Miyagi, Japan). The protocol suggested by the manufacturer was followed and the test result was read after 48 h incubation in a humid chamber at room temperature. The concentration was recorded in mg/l, IL-6, IL-1␣, IL-1, IL-12, TNF-␣, and IFN-␥ levels were determined from undiluted serum samples using commercially available ELISA Kits (Biosurce, Diagnostic Corporation, Belgium). The plates were read at 450 nm on a computerized automated microplate ELISA reader (Bio
The present investigation revealed significant (P ≤ 0.05) increase in Hp, SAA, ceruloplasmin, AGP and fibrinogen levels in T. annulata infected water buffaloes when compared to healthy ones (Table 1). Moreover there was significant (P ≤ 0.05) increase in the levels of TNF␣, IL-1␣, IL-6, IL-12, IL-1 and IFN-␥ in T. annulata infected water buffaloes when compared to healthy ones (Table 2). In the present study the hematological examination (Table 3) revealed significant decrease (P ≤ 0.05) in the Hb content, PCV%, RBCs and TLC in the diseased water buffaloes when compared to control ones. Neutropenia, eosinopenia, lymphopenia, monocytopenia and thrombocytopenia were also recorded in infected buffaloes. The biochemical changes revealed significant (P ≤ 0.05) elevation in the levels of AST, ALT, ALP, LDL-c, VLDL-c, BHBA and NEFA, with significant (P ≤ 0.05) decrease in the levels of total proteins, albumin, globulins, cholesterol, triglyceride, glucose, G6PD, calcium and phosphorus in T. annulata infected water buffaloes when compared to healthy ones (Table 4). 4. Discussion Acute phase proteins (APPs) play an important role in innate defence mechanisms (Eckersall, 2000). In the
Table 2 The mean levels of pro-inflammatory cytokines in control and Thieleria annulata infected water buffaloes. Variables
Control (n = 20) (mean ± SEa )
TNF-␣ (ng/ml) IL-1␣ (pg/ml) IL-1 (pg/ml) IL-6 (pg/ml) IL-12 (ng/ml) IFN-␥ (pg/ml)
0.42 55.32 98.33 88.23 7.45 34.76
a *
± ± ± ± ± ±
0.04 3.21 3.45 3.21 0.67 1.56
Standard error. Means are significantly different at the level (P ≤ 0.05).
Diseased water buffalo (n = 30) (mean ± SEa ) 2.55 98.32 240.33 152.32 26.44 123.65
± ± ± ± ± ±
0.12* 4.21* 20.45* 5.62* 1.43* 5.67*
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Table 3 Hematological parameters in control, and Thieleria annulata infected water buffaloes. Variables
Control (n = 20) (mean ± SEa )
Hb (g/Dl) RBCs (106 /mL) PCV (%) TLC (103 /mm3 ) Neutrophils (103 /mL) Lymphocytes (103 /mL) Eosinophils (103 /mL) Basophils (103 /mL) Monocytes (103 /mL) Thrombocytes (103 /mL)
13.46 9.85 35.23 9.6 2.9 3.8 0.82 0.013 0.42 269.75
a *
± ± ± ± ± ± ± ± ± ±
Diseased water buffaloes (n = 30) (mean ± SEa )
0.39 0.16 0.12 0.18 0.015 0.012 0.01 0.0001 0.001 3.52
6.2 5.23 26.9 6.4 2.3 2.5 0.13 0.0126 0.34 179.35
± ± ± ± ± ± ± ± ± ±
0.17* 0.32* 0.11* 0.22* 0.037* 0.013* 0.001* 0.0001 0.002* 4.52*
Standard error. Means are significantly different at the level (P ≤ 0.05).
present study there was a significant (P ≤ 0.05) increase in Hp, SAA, ceruloplasmin, AGP and fibrinogen levels in T. annulata infected water buffaloes when compared to healthy ones. These results are consistent with Glass et al. (2003) and Nazifi et al. (2010) in cattle. The authors reported that T. annulata causes severe pathology in susceptible cattle by inducing high levels of APPs. Following experimental infection, SAA appeared first, followed by a rise in ␣1-acid glycoprotein in all cattle, whereas Hp which is a major APPs in cattle only appeared in some of the animals and generally at a low level. SAA, Hp and ␣1-acid glycoprotein only elevated around or after the appearance of schizonts and after the first rise in temperature. Increased ␣1-acid glycoprotein level coincided with the appearance of piroplasms. The production of SAA and ␣1-acid glycoprotein correlated strongly with each other and also with some clinical features of disease severity including the time of fever, development of leukopenia, parasitemia rate and mortality. The obtained results are also in consistent with those reported by Glass and Jensen (2007) in cattle. The primary trail leading to significant elevation in APPs in infected water buffaloes may involve initial release of pro-inflammatory cytokines by macrophages at the site
of infection or inflammation. This results in a cascade of further release of cytokines by macrophages and other cells. The most important inducers of APPs are cytokines of the IL-1, TNF and IL-6 families (Glass et al., 2003). If these pro-inflammatory cytokines spill into the circulation, they induce release of APPs by the liver into the circulation (Baumann and Gauldie, 1994). The utility of these significant elevation of APPs is not well established but they appear to be involved in controlling inflammation and contribute to innate host defences against a wide range of pathogens (Glass et al., 2003). Their circulating levels may also be related to the severity of the response to infection, and thus may provide valuable quantifiable biochemical indicators of the inflammatory response. Bacterial infections induce strong acute phase responses whereas viral infections generally lead to weak or non-detectable acute phase reactions. There are relatively few reports of APPs induction by parasite infections. However, infections with Plasmodium falciparum (Graninger et al., 1992), Babesia canis (Matijatko et al., 2007), Leishmania infantum (Martinez-Subiela et al., 2002) and Trypanosoma brucei (Shapiro and Black, 1992; Eckersall et al., 2001) might be accompanied by increased levels of APPs.
Table 4 Biochemical parameters in control and Thieleria annulata infected water buffaloes. Variables AST (U/l) ALT (U/l) ALP (U/l) Total proteins (g/dl) Albumin (g/dl) Globulins (g/dl) TG (mg/dl) Cholesterol (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL-c (mg/dl) Glucose (mg/dl) BHBA (mmol/l) NEFA (mmol/l) G6PD (IU/g Hb) Calcium (mg/dl) Phosphorus (mg/dl) Magnesium (mg/dl) a *
Control (n = 20) (mean ± SEa ) 88.8 44.2 121.8 6.3 3.55 2.8.35 29.2 69.0 26.3 22.8 6.1 63.0 0.45 363.6 22.45 10.54 5.56 2.63
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Standard error. Means are significantly different at the level (P ≤ 0.05).
4.54 0.36 4.37 0.13 0.13 0.23 0.53 2.60 0.26 0.57 0.50 0.6 0.03 5.5 0.15 0.32 0.21 0.21
Diseased water buffaloes (n = 30) (mean ± SEa ) 322.0 229.8 239.0 4.51 2.4 1.45 17.8 39.5 16.5 30.9 7.8 37.2 1.9 536 17.28 7.89 3.42 2.72
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.68* 8.55* 6.20* 0.12* 0.12* 0.23* 0.87* 0.52* 0.27* 0.89* 0.30* 2.1* 0.01* 10.8a 0.29* 0.23* 0.21* 0.45
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Inflammation or tissue injury causes the release of pro-inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor (TNF) which alter the blood concentration of a variety of proteins that are produced primarily in the liver (Nazifi et al., 2009). Increases in these proteins are recognized by elevations in the ␣- and/or -regions by serum protein electrophoresis. The concentration of these proteins is generally low to non-detectable in healthy animals and elevations are used to diagnose and monitor inflammatory diseases (Glass et al., 2003; Nazifi et al., 2010). The specific type of APPs and the time course for alterations in these proteins vary in different species on the basis of the initiating disorder or underlying inflammatory process (Feldman et al., 2000). In the present study there was significant (P ≤ 0.05) increase in the levels of TNF-␣, IL-1␣, IL-6, IL-12, IL-1 and IFN-␥ in T. annulata infected water buffaloes when compared to healthy ones. The elevated levels of selected cytokines could be attributed to hemorrhages, lymphocytic infiltration and necrosis in abomasum, liver, kidney, intestine, lung and bone marrow which indicate a widespread inflammatory reaction in bovine theilerisosis. These lesions induce the release of cytokines such as IL-1, IL-6 and TNF under the effect of which, APPs are synthesized in liver (Jubb et al., 1991; Radostits et al., 2007). In the same concern Brown et al. (1995) and Forsyth et al. (1999) reported that schizontinfected macrophages express mRNA for pro-inflammatory cytokines (interleukin-1 (IL-1), IL-6 and tumor necrosis factor (TNF-␣)) and contain intracellular TNF-␣ protein. In addition, uninfected macrophages from animals recovering from or immune to tropical theileriosis secrete TNF-␣, at least in vitro (Preston et al., 1993). This would suggest that pro-inflammatory cytokines are released systemically. If so, they probably play a major role in pathology and indeed many of the clinical signs of tropical theileriosis resemble those induced by experimental TNF-␣ administration to cattle (Bielefeldt Ohmann et al., 1989). Hematological examination revealed significant decrease (P ≤ 0.05) in the Hb content, PCV%, RBCs and TLC in the diseased water buffaloes compared to the control ones. Neutropenia, eosinopenia, lymphopenia, monocytopenia and thrombocytopenia were also recorded. These results are in agreement with those obtained by Osman and AL-Gaabary (2007) and El-Deeb and Younis (2009). The decrease in RBC counts could be due to increase in the levels of activated complement products (Omer et al., 2002) and erythrophagocytosis (Yagi et al., 2002). In addition, pro-inflammatory cytokines, particularly TNF-␣, have been implicated in mediating anemia associated with tropical theileriosis (Graham et al., 2001). This decrease is also related to the destruction of lymphocytes in lymphoid organs and infiltration of these cells into various organs (Sandhu et al., 1998). However, no significant difference in absolute basophile and monocyte counts between healthy and infected cattle was recorded by Omer et al. (2002). This variation could be attributed to differences in the stage and severity of the disease. The decline in RBC, PCV and Hb may be attributed to destruction of erythrocytes by macrophages in the lymph nodes, spleen and other organs of monocyte–macrophage system (Singh et al., 2001). Sandhu et al. (1998) evaluated hematological and
biochemical factors in experimental T. annulata infection in crossbred calves. They reported a significant progressive decrease in Hb concentration, PCV and RBC, whereas the total leukocytes count showed an initial non-significant leukocytosis followed by a significant leukopenia. Dhar and Gautam (1979), Sharma (1979), Mehta et al. (1988) and Rayula and Hafeez (1995) reported a progressive decrease in Hb, PCV and RBC in acute T. annulata infection. A progressive decrease in the Hb and PCV, along with a marked reticulocytosis was also reported by Singh et al. (2001). Concerning the biochemical changes in T. annulata infected water buffaloes, there was significant (P ≤ 0.05) elevation in the levels of AST, ALT, ALP, LDL-c, VLDL-c, BHBA and NEFA, with significant (P ≤ 0.05) decrease in the levels of total proteins, albumin, globulins, cholesterol, TG, glucose, G6PD, calcium and phosphorus in T. annulata infected water buffaloes when compared to healthy ones. The obtained results are in concern with those reported by El-Deeb and Younis (2009) in water buffaloes and Khan et al. (2011) in cattle. The findings were also in line with the findings of Col and Uslu (2006) who reported hypoproteinemia and hypoalbuminemia in tropical theileriosis in cattle. The results were in agreement with the findings of Yadav and Sharma (1986) and Singh et al. (2001) in cattle and crossbred calves respectively. The decreased serum proteins concentrations may be attributed to extra-vascular proteinaceous fluid in body cavities due to diseased lymph nodes resulting edema. The decrease in total serum proteins was attributed to low albumin and globulin concentrations as a result of liver failure (Omer et al., 2003). The elevated concentrations of the examined enzymes are indicators of hepatic function (Forsyth et al., 1999). Similar findings were observed by Col and Uslu (2006) who reported an increase in ALT concentration in cattle. The rise in serum ALT concentration may be due to muscular trauma as a result of prolonged recumbency due to bovine theileriosis (Col and Uslu, 2006). In the present study, there was significant decrease in serum glucose and G6PD concentrations with significant increase in the levels of BHBA and NEFA in T. annulata infected water buffaloes when compared to healthy controls. These findings were in line with the findings of Col and Uslu (2006). They reported that, the decreased glucose serum concentration could be due to utilization of glucose by Theileria in the blood and hepatic dysfunction as a result of Theileria infection. Similar findings were obtained by El-Deeb and Younis (2009) in T. annulata infected water buffaloes. They reported that T. annulata could act as ketotic stressor in infected buffaloes. The findings were in contrast with those obtained by Sandhu et al. (1998) who reported a non-significant decrease in serum glucose in calves affected with theileriosis. The significant decrease in the activity of G6PD in infected water buffaloes suffering from severe anemia is an indicator of a metabolic disturbance in the erythrocytes and associated with increased RBCs hemolysis in theileriosis (Singari et al., 1991) and increased oxidative stress in endothelial cells (Leopold et al., 2003 and El-Deeb and Younis, 2009). The obtained results were different from that reported by Grewal et al.
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(2005) who reported significant increase in the activity of G6PD in cattle naturally infected with T. annulata. The variation of G6PD activities might be related to the severity of the anemia. Interestingly there was decreased serum calcium and phosphorus concentrations in T. annulata infected water buffaloes when compared to healthy ones. Similar findings were reported by Omer et al. (2003) and Khan et al. (2011) in infected cattle. Hypocalcaemia could be due to hypoproteinemia, decreased dietary intake, intestinal malfunction and kidney damage and the decreased phosphorus concentration due to diarrhea and renal wasting (Col and Uslu, 2006). Non-significant differences was observed in magnesium concentrations in infected and healthy controls. These results are consistent with those obtained by Khan et al. (2011) in cattle. There were significant decreases in serum cholesterol, triglycerides, HDL-c concentrations with significant increase in the levels of LDL-c and in T. annulata infected water buffaloes. These results are in agreement with those reported by Singh et al. (2001) in cattle. These decreases in cholesterol and triglyceride levels may be ascribed to anorexia and diarrhea (Col and Uslu, 2006). The findings were otherwise than the findings of Yadav and Sharma (1986) who recorded a marked increase in serum cholesterol concentration in experimentally T. annulata infected cattle from day 0 to day 15, followed by a sudden fall, reaching values below the pre-infection level by day 40. They reported that, this could be due to liver damage that results in a concurrent increase in the level of fats with the reduction of sugar and protein. 5. Conclusion In the present study the APPs and pro-inflammatory cytokines could be used as a valuable biomarkers in T. annulata infected water buffaloes (B. bubalis). Moreover T. annulata may be considered as a cofactor induces detectable changes in lipoproteins profile, calcium and phosphorus levels in water buffaloes (B. bubalis). References Abraham, E.J., Minton, J.E., 1997. Cytokines in the hypophysis: a comparative look at interleukin-6 in the porcine anterior pituitary gland. Comp. Biochem. Physiol. A: Physiol. 116A, 203–207. Alsemgeest, S.P., Lambooy, I.E., Wierenga, H.K., Dieleman, S.J., Meerkerk, B., Van Edern, A.M., Niewold, T.A., 1995. Influence of physical stress on the plasma concentration of serum amyloid-A (SAA) and haptoglobin (Hp) in calves. Vet. Q. 17 (1), 9–12. Baumann, H., Gauldie, J., 1994. The acute phase response. Immunol. Today 15, 74–80. Beutler, E., Duron, O., Kelly, M.B., 1963. J. Lab. Clin. Med. 61, 882. Bielefeldt Ohmann, H., Campos, M., Snider, M., Rapin, N., Beskorwayne, T., Popowych, Y., Lawman, M.J., Rossi, A., Babiuk, L.A., 1989. Effect of chronic administration of recombinant bovine tumor necrosis factor to cattle. Vet. Pathol. 26, 462–472. Brown, D., Campbell, J.D.M., Russell, G.C., Hopkins, J., Glass, E.J., 1995. T cell activation by Theileria annulata-infected macrophages correlates with cytokine production. Clin. Exp. Immunol. 102, 507–514. Col, R., Uslu, U., 2006. Haematological and coagulation profiles during severe tropical theileriosis in cattle. Turk. J. Vet. Anim. Sci. 30, 577–582. Demetriou, J.A., Drewes, P.A., Gin, J.B., 1974. Ceruloplasmin. In: Cannon, D.C., Winkelman (Eds.), Clinical Chemistry. Principles and Techniques. , 2nd ed. Harper and Row, Hagerstown, MD, pp. 857–864.
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