EVALUATION OF SERUM SIALIC ACID LEVEL AND ADENOSINE DEAMINASE ACTIVITY AS A DIAGNOSTIC SIGNIFICAL TEST IN CATTLE NATURALLY INFECTED BY BABESIA. SPP

Evaluation of serum sialic acid level and adenosine deaminase activity as a diagnostic significal test in cattle naturally infected by Babesia. Spp      

F.A. Osman ^* and Gaadee H.I.M ^**

* Parasitological Unit in Animal Healthy Research Institute, New-Valley Lab Branch.

** Biochemistry Unit in Animal Healthy Research Institute, Assuit Lab Branch.               

___________________________________________________________________________

                            Abstract

________________________________________________________________

تقيم مستوى حمض السيالک ونشاط انزيم الادونزين ديميناز فى مصل الدم واهميتة التشخيصية

فى الماشية المصابة طبيعيا بالبابيزيا

فتحى احمد عثمان ، هدى ابراهيم مصطفى جعيدى

استهدفت هذه الدراسة تقدير ترکيز کلا من حمض السيالى الکلى والمرتبط بالدهون ومستوى انزيم الادونزين ديميناز في دم الماشية المصابة طبيعيا بالبابيزياز وقد شملت هذه الدراسة فحص 40 حيوانا من اناث الماشية التي تعانى من البابيزيا وکذلک عدد 10 حيوانا سليما ظاهريا ومعمليا واستخدمت کمجموعة ضابطة وجميعها کانت أعمارها بين 5-7 أعوام. وقد تم تأکيد الإصابة بالبابيزيا بعمل شريحة دموية مصبوغة بالجمسا وفحصها للتعرف على الطفيل وتقسيم الحيوانات المصابة حسب درجة تواجد الطفيلي في الدم إلى خفيفة ومتوسطة وشديدة الإصابة مع نتائج.
ومقارنة الحيوانات السليمة کانت النتائج هى 57,6± 22.88ملجم/ديسلتر و11,07±44,35 وحدة/لتر لکلا من حمض السيالى الکلى وانزيم الادونزين ديميناز کما فى الجدول رقم (2) إما الحيوانات المريضة موضع الدراسة فکانت کالاتى. 102,09± 3,93و 65,18±16.3 على التوالي. من هذة النتائج فقد لوحظ زيادة مطلقة وغير مرتبطة بشدة المرض حيث لوحظ ان معامل ارتباط حمض السيالى وانزيم الادونزين ديميناز اقل من 05,% ,
ومن هذا کله يمکن القول بان الإصابة بالبابيزيا فى الماشية تودى الى زيادة ملحوظة ومطلقة فى مستوى حمض السيالى ومستوى إنزيم الادونزين ديميناز.

___________________________________________________________________________

Key words:  total sialic acid, adenosine deaminase activity, Babesia.cattle naturally infected by Babesia. Spp

__________________________________________________________________________________________

Introduction

           Babesiosis is an emerging, tick- transmitted, zoonotic disease caused by haematotropic parasites of the genus Babesia, Babesial parasites are some of the most widespread blood parasite in the world, and consequently have considerable world wide economic, medical and veterinary impact. The parasite is intraerythrocytic and is commonly called piroplasmosis due to pear-shaped forms found within the infected red blood cells.

Babesia bovis, a hemoprotozoan parasite invading and replicating within bovine erythrocytes, causes serious disease manifestations characterized by fever, anemia, hypertensive shock syndrome and in sever cases a fetal cerebral disease with a high mortality rate (Wright et al., 1988 and Homer et al., 2000). Therefore the spread of infection result in great economic losses all over the world (Brown and Palmer, 1999).

Recently sialic acid (SAs), the terminal or sub terminal non-reducing units attached to the underlying galactose residue of many sialoglycoprotein on the host cell membrane (Varki, 2001) have been shown to play an important role in RBCs invasion by Babesia parasites (Kania et al., 1995 and Zintl et al., 2002), the (SAs) residues' regardless of the type of linkages (α 2-3 andα2-6 linkages) are important in RBCs invasion by Babesia.bovis which can invade both bovin RBCs, human and other animal RBCs in an SA-dependent manner. Gaffar et al. (2003).

Adenosine deaminase (ADA) is an enzyme that catalyses hydrolytic deamination of either adenosine or deoxyadenosine to produce inosine and deoxyinosine respectively (Ungere et al., 1992). In domestic animals. ADA is present in all organs, although the highest activity has been found in lymphoid tissues in animals. ADA and its isoenzyme activities in the spleen, lymph nodes and thymus have been found in high levels and in the brain, adrenal gland, muscles, kidney and liver in low level (Tanabe, 1993). It has been detected in the cell cytoplasm and nucleus (Zuck and Rotzsch, 1990). Furthermore, its enzymatic activity has also been reported in blood cells, serum and plasma (Katopodis and Stock, 1980).

The main biological activity of ADA is to protect lymphocytes from toxic effects of 2-deoxyadenosine, deoxyadenosine triphosphate and deoxyadenosine diphosphate. Which depress immune function. It has been shown that ADA is related to normal condition involving lymphocyte-monocyte proliferation (Kaplan et al., 1985; Baganha et al., 1990).

The aim of the present study was to evaluated the cattle immune response by determine the serum total sialic acid and adenosine deaminase levels in cattle naturally infected with babesiosis.

Materials and methods

 
1- Animals

A total number of 50 female's cattle, 5-7 years old, from private farm in Assuit Governorate were used as subjects for this study. The cattle were divided into two groups according to the clinical and parasitological examinations. 40 female cattle suffering from Babesiosis and used as a diseased group (1). The rest, 10 females were clinical and laboratory healthy cattle and used as control group (2).

2- Clinical examination:

All the selected animals' cattle were subjected to clinical examination. The infected animals showed clinical signs as a high rectal temperature reaching 41.5 ⁰c, anorexia, the mucous membranes' are first injected and reddened, but as erythrocytic lyses occurs, the color changes to the pallor of anemia. This is usually associated with sever hemoglobinuria and hemoglobinemia. Hemolytic anemia and jaundice were evident in advanced stage of infection and beside that all the infected animal are infested with ticks.

3- Samples:

1- Blood samples:

a)  Whole blood samples:

Blood samples were taken into EDTA containing vacationer tubes from the jugular vein for blood film preparation for parasitological examination (Babesia. Spp).

b) Blood samples without anticoagulant:

Serum samples were separated by centrifugation at 3000 rpm at room temperature and stored at -20^oC until biochemical analysis.

2- Fecal samples:

Ten grams of fecal samples were collected from each animal in plastic pages and screened for parasitic infection by concentration flotation technique according to (Soulsby, 1986).

3- Parasitological examination:

EDTA-blood was used to prepare thin and thick blood film form each animal, then fixed in methanol, stained with Giemsa stain and examined by oil immersion lense under microscope. The degree of parasitemia was recorded as the percentage of the infected red blood per 100 cells counted (0.5%, 1%, etc) as in Table (1), according to Saunders (2007). The smear was recorded as negative if no piroplasma bodies were observed in 200 fields/ animal.

Ten grams of fecal sample were screened macroscopically and microscopically for parasitic infestation using standard methods.

4- Biochmical analysis:

Total sialic acid concentration was determined according to Sydow et al. (1988). Briefly, 400 µl of serum were treated with 3ml of 5% perchloric acid for 5minutes at 100 ^oC and centrifuged at 1800 rpm.

Two ml of supernatant were mixed with 400/ul of Echrlich reagent (5g dimethy-laminobenaldehyde /50 ml HCL with 50 ml of distilled water). Incubation for 15 minute at 100c was made. Finally the optical density was read with spectrophotometer (UV-1201.Shimadzu, Japan). While lipid bound sialic acid concentration was determinate according to Katopodis et al. (1980) and protein bound sialic acid were calculate by subtracted lipid bound sialic acid from total sialic acid.

Serum (ADA) activity was determined spectrophotometrically according to. Giusti and Galanti, (1984), where colorimetric method based on the principle of measuring absorbance of color at 628 nm.

5- Statistical analysis.

The result was analyzed by on-way ANOVA followed by Duncan test using computer soft ware. Differences were considered statistically significant when P

Results

Clinical examination:

The most clinical signs of infected cattle with Babesia were hemoglobinuria, increase of rectal temperature (40 ^oc-41.5^o C). Animals commonly develop incoordination and depression and go down with the head extended but later thrown back with involuntary movement of legs. These signs are followed by death.

Parasitological examination:

A- Blood films prepared from diseased cattle indicated the presence of Babesia .Spp in the red blood cells with different degree of parasitism (0.5%- 2%) as in Table 1. While examination of blood films from control group indicated no piroplasma.

B- Fecal examination (Gross and microscopically) in all animals included in these study indicated that all animals were free from internal parasites to avoid the cross reaction which may be occur with the results of babesiosis.

Biochemical analysis:

Mean serum sialic acid concentration of these study were summarized in Table 2, which indicated marked significantly (p>0.05) increase in the concentration of sialic acid in the diseased cattle than in the control. However the individual rates of parasitemia were not correlated with sialic acid concentration(r =0.027p>0.05).

Mean value of serum (ADA) activity are presented in Table 2, where significant increase (p> 0.05) in (ADA) levels were detected in diseased group than the control group.

Table 1: Degree of parasitism in the animals of the present study.

Table 2: Statistical analysis of serum total sialic acid and adenosine deaminase enzyme levels in cattle naturally infected with Babesia. Spp and healthy control group.

Discussion

The present study was conducted to examine the effect of Babesia infection on serum sialic acid concentration and adenosine deaminase enzyme activity in cattle.

Our finding indicated that Babesia infection induced significant (p>0.05) increase in total sialic acid and adenosine deaminase activity.

It is currently unknown how babesia infection lead to the increase in sialic acid content. However. Eslevo et al. (1982) and Ertekin et al. (2000) reported significant increase of serum sialic acid in cattle infected with Thieleria or anaplasma. In addition Olaniyi et al. (2001) reported an elevation in serum sialic acid concentration in trypanosomes. Conversely sialic acid deficiency in the erythrocytes of infected cattle with babesia bovis has been reported by Gaffar et al. (2003).

The increase in sialic acid may be attributed to the fact that sialic acid could modulate biological cell-cell interaction in two non –mutually exclusive ways, sialic acid could mask the underlying suger chain (i.e lactosaminic sequma), hindering them from interactinge with galactose-specific lectins (Galectins) as recorded by Razi and Varki (1998). Second way, sialic acid would directly interact with specific sialic acid binding lectins (Sigles). Therefore increase content of sialic acid would interfere with attachment of sporozoites on host cells or promote the invasion of erythrocytes by merozoites (Dallolio, 2000).

Host sialic acid plays an important role in erythrocytes invasion by babesia parasite. Afact that agree with Yokpyama et al. (2006), where sialic acid play as a host receptor in the erythrocytes invasion by Babesia bovis. Treatment prevented the increase of sialic acid (Gaffar et al., 2003). In addition, Okamura et al. (2005), observed that sialic acid residues on host RBCs play an important role in the erythrocyte infection by Babesia Caballi and Babesia equi and cause significant increase in its concentration.

Adenosine deaminase enzyme level is increased with many diseases due to the stimulation of cellular immunity. In the present study serum ADA was significantly increased (p<0.05) in the diseased group in contrast to the control group. The increase in serum (ADA) came in agreement with Kontas and Salmonglu (2006).

The increase of (ADA) may be attributed to erythrocytic damage caused by the parasite and/or the phagocytic activity of macrophages (Ustundage et al., 1999).

Finally we can be conclude that Babesia infection in cattle induced marked increase of serum sialic acid concentration and adenosine deaminase enzyme level, suggesting that these indicators would indirectly promote the invasion of the parasite in host.

Reference

Baganha, M.F.; Pego, A.; Lima, M.A.; Gaspar, E.V. and Cordeiro, A.R. (1990): Serum and pleural adenosine deaminase correlation with lymphocytic population. Chest., 97: 605-610.

Brown, W.C. and Palmer, G.H. (1999): Designing blood stage vaccines against Babesia bovis and B.bigemina. Parasitol. Today, 275-281.

Dallolio, F. (2000): The sialy 1-2, 6 lactosaminy1-structrue. Biosynthesis and functional role. Glycoconjugate, J. 17: 669-676.

Ertken, A.; Keles, I.; Ekin, S.; Karaca, M. and Atkan, H.A. (2000): An investigation sialic acid and lipid bound sialic acid in animals with blood parasites. Yyu. Vet. Fak. Derg. 11: 34-35.

Eslevo, K.; Sacar, D.I.; Ilemobade, A.A. and Hallawy, M.H. (1982): Variation in erythrocytes surface and free serum sialic acid concentration during experimental trypanosome vivex infection in cattle Res. Vet. Sci. 32: 1-5.

Gaffar, F.R.; Franssen, F.F. and Devries, E. (2003):Babesia bovis merozoities invade human. Ovin. Equine. Porcine and Caprine erythrocytes by a sialic acid dependent mechanism fallowed by development arrest after a single round of cell fission. Int. J. Parasitol, 33: 1595-1603.

Giusti, G. and Galanti, B. (1984): Colorimetric method of enzymatic Analysis. Verlog chemic. Weinheim in Bergmeyer. H.U 2^nd Ed. 315-323.

Homer, M.J.; Aguilar-Delfin, I.; Telford, S.R.3^rd; Krause, P.J. and Persing, D.H. (2000): Babesiosis. clin. Microbial. Rev. 13: 451-469.

Kania, S.A.; Allred, D.R. and Barbet, A.F. (1995): Babesia bigemina host factors affecting the invasion of erythrocytes Exp. Parasitol. 80: 76-84.

Kaplan, A.M.; Gerrard, T.L.; Strawson, J. and Squire, C. (1985): Aultman. M., Role of adenosine deaminase in human monocytes deffernatiation and tumor cell cytotoxicity, Ann. N.Y. Acad. Sci., 451: 264-278.

Katopodis, N. and Stock, C. (1980): Improved method to determine lipid bound sialic acid in plasma or serum. Res. Commun. chem. Pathol. pharmacol., 30: 171-180.

Kontas, T and Salmanoglu, B. (2006): Tumor necrosis factor-α adenosine deaminase and nitric oxide levels in cattle babesiosis before and after treatment. Bull. Vet. Inst. Pulawy 50: 485-487.

Okamura, M.; Yakoyama, N.; Wiekrama Thilakanp, Takabatake, N.; Ikehara, Y. and Igarashi, I. (2005): Babesia caballi and Babesia equi. Implotion of host sialic acid in erythrocyte infection. Exp. parasitol. 110: 406-411.

Olaniyi, M.O.; Taiwo, V.O. and Ogunsanmi, A.O. (2001): Hematology and dynamics of erythrocytes membrane sialic acid concentration during experimental trypanosome Congolese and Trypanosome Brucei infection of sheep. J. Appl. Anim. Res. 20: 57-64.

Razin, M. and Varkia, H. (1998): Masking and unmasking of the sialic acid-binding lectin activity of the CD 22 (siglee-2) on B. lymphocytes Proc. Nat. Acad. Sci. USA. 95: 7469-7474.

Saunders, (2007): Dorland's Medical Dictionary for Health consumers, an imprint of Elsevier. Inc. All right reserved.

Solusby, E.J.L. (1986): Helminthes, Arthropods and Protozoa of Domestic Animals. 7^th Ed. Blackwell scientific publication.

Sydow, G.; Wittmann, W.; Bender, E. and Starick, E. (1988): The sialic acid content of the serum of cattle infected with bovine leukosis virus. Arch Exp Veterinar. Med, 42: 194-197.

Tanabe, T. (1993): Adenosine deaminase activities in the sera and tissues of animals and their clinical significance. Jpn. j. Vet. Res., 41-52.

Ungere, J.P.J.; Oasthuizen, H.M.; BissBORT, S.h. and Vermaak, W.J.H. (1992): Serum adenosine deaminase isoenzymes and diagnostic application. Clin. chem., 38: 1322-1326.

Ustundage, B.; Canatan, H.; Bahcecioglu, I.H.; Ozercan, I.H. and Cinkililinic, N. (1999): Serum adenosine deaminase activity levels in rats with experimentally-induced cirrhosis. Turk. J. Biocheh, 24: 16-22.

Varki, A. (2001): N.glycolylneuraminic acid deficiency in humans. Biochimie. 83: 615-1585.

Wright, I.G.; Goodeger, B.V. and Clark, I.A. (1988): Immunopathophysiology of Babesia bovis and plasmodium falciparum infection. Parasitol. Today. 4: 214-218.

Yokoyama, N.; Okamura, M. and Igarashi, I. (2006): Erythrocytes invasion by Babesia parasites. Current advances in the elucidation of the molecular interaction between the protozoon ligand and host receptor in the invasion stage. Vet Parasitol. 138: 22-32.

Zintl, A.; Westbrook, C.; Skerrett, H.E.; Gray, J.S. and Mulcahy, G. (2002): Chymotrypsine and neuroamindase treatment inhibites host cell invasion by babesia divergen Parasitology. J. 125: 45-50.

Zuck, V.U. and Rotzsch, W. (1990): Adenosine deaminase-biologic and medical diagnostic significance. Z. Med. Lab. Diagn., 31: 231-238. (article in German with an abstract in English).