Detection of Babesia spp by using of Acridine Orange and Giemsa Stain with Study of its Effect on Blood Picture in Local Breed Cows

ةيلحملا راقبلاا يف ةيومدلا ةروصلا ىلع هريثات ةسارد عم ازميك ةغبصو ةيلاقتربلا نيدركلاا ةغبص مادختساب ايزيبابلا يليفط نع فشكلا

Babesia bigemina appears within the red blood cells in the form of double pear shape, with a sharp angle appear between them and is characterized by its large size and can also appear in the form of a single pear in addition to the oval shape and irregular shape (4).
While Babesia bovis appears inside the red blood cells in the form of double pear, between them a separate angle and its located near to the surface of the red blood cells and has a nuclear mass in one of the poles and is characterized by small in size and it is more virulent than Babesia divergens, Babesia bigemina. Giemsa stain has been used since a long period of time in detection of Babesia spp in blood smear , Despite the efficiency of the Giemsa stain in the diagnosis of the parasite where reliable in determining the form of the parasite such as pears or oval, However the acridine orange stain is simply applied and takes about 2-4 minutes in the diagnosis of Babesia spp using a fluorescent microscope compared to Giemsa stain which process of pigmentation takes more time (30-60) minutes using a normal light microscope (5).
Acridine orange stain is one of Fluorescent dyes which can be penetrate the cell wall and binds to DNA and RNA and this binding lead to emitting an orange color under the Fluorescent microscope So the parasite appears orange or yellow inside the red blood cell, while the red blood cell appears pale color because it does not contain DNA (5). Fever , pale or icteric mucus membrane, Hemoglobinuria and sometime abortion were the main characteristic signs showed by diseased cows (6).
The Babesia sp after entering the blood stream through the tick bite begins to attack red blood cells and then begins to divide and multiply within them, which increases the pressure inside the red blood cell and then break down of red blood cells, then parasite is released into the plasma to attack new erythrocytes and thus break down or lysis a large number of erythrocytes, causing a decrease in the number of erythrocytes and release of Hemoglobin into the blood (hemoglobinemia), which finally leads to hemoglobinuria (7). decrease in RBC, HB, PCV are recorded by (4).
The type of anemia in infected cows depends on the severity of infection and virulence of the parasite, some studies shows Normocytic Normochromic Anemia with non-significant changes in Mean corpuscular volume (MCV), Mean corpuscular hemoglobin concentration (MCHC) (8), and another study shows microcytic hypochromic anemia (9). The parasite also has an effect on the number of white blood cells, leukocytosis with lymphocytosis and monocytosis are recorded by (10), while leukopenia are seen by (11).

Materials and Methods
The study was conducted on 60 local breed cows, with different ages from many districts of Mosul city (Hawi Al-Kanesa ,ALGawssiat ,AL Quba, Telkif) . The period of study was from the beginning of February 2018 until the end of July 2018. Seventeen out of sixty local cows were infected with Babesia spp. The infection was confirmed by using blood smears staining by Giemsa and acridine orange stain, and 10 cows clinically intact served as control.

Blood sample collection:
Blood were drained from The infected and control cows by using a sterile syringe from the jugular vein .3ml of blood are drained and mixed with anticoagulant(EDTA) to evaluating of Hemogram (erythrocyte count (RBC), hemoglobin (HB), packed cell volume (PCV), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin concentration (MCHC), total leukocyte counts (TLC) by using Beckman (automatic digital cell counter), differential leukocyte counts (DLC) are estimated by using blood smears stained by Giemsa stain (12).

Detection of Babesia spp in blood smears:
blood smears were prepared by puncture of ear vein and a drop of blood was placed on glass slide then spread by other slide, blood smear was dried and fixed by a solute methanol (3-5 minute). Then blood smears were stained with Giemsa stain and acridine orange stain.

Staining of blood smears by Giemsa stain:
Giemsa stain was added on fixed blood smears and left for 30 minute, blood smears were washed with tap water and examined under light microscope, the percentage of parasitemia in red blood cells was determined according to the following equation (13):

Staining of blood smears by acridine orange stain:
The acridine orange stain were prepared by dissolving 25 mg of stain in 5 ml of distilled water and then placed in an opaque glass bottle and kept in the refrigerator for four weeks, 1ml of stain were mixed with 0.5ml of glacial acetic acid and 50ml of distal water, then the stain become ready to use, the stain was added to fixed blood smears for 2 minute and blood smears were washed with tap water and examined under fluorescent microscope (14).
Statistical analysis: Results were statistically analyzed using SPSS statistical program and T test when comparing between diseased and healthy cows (15).

Results and Discussion
The diseased cows showed several clinical signs (fever, loss of appetite, pale and in other cows icteric mucus membrane, hemoglobinuria (coffee color like urine), and the infection was confirmed by using Giemsa and Acridine orange staining blood smears, Where the parasite appeared inside red blood cells in various forms, including the single pear shape and double pear shape between them sharp or obtuse angle in addition to irregular forms, and the parasite appeared in different sizes ( Figure 1,2). It was noted that Giemsa Stain was more accurate in the diagnosis of the parasite depending on the form, but the continuous filtering of the stain to prevent the stain deposits on the surface of red blood cells was one of the disadvantages of this Stain, in addition the staining time lasted 45 minutes, while the staining method with acridine orange characterized by quickly and easily and the period of staining was only 2 minutes and The uninfected erythrocytes was observed in a pale color ,while the parasiteinfected erythrocytes appeared in bright orange or yellow, (Figure 3). The study also showed that 17 of the 60 cows were infected with Babesia spp, percentage of infection was (28.33) Table (1) . The results of the study showed significant decrease (P < 0.05) in the number of erythrocytes, hemoglobin concentration and the volume of packed cell volume in infected cows compared with healthy cows. The decrease of erythrocytes may be due to the direct effect of the parasite to the infected erythrocytes and decrease life span of RBCs in addition to suppression of hemopoitic system and the result was agreement with (7)(16), Auto immunity and the anti erythrocytic auto antibodies lead to increasing of erythrophagocytosis and bone marrow depression was recorded by (11) as the main causes of anemia and decrease of erythrocytes.

Table (1) Total percentage of infection in cows
No significant changes was observed in Mean corpuscular volume (MCV), Mean corpuscular hemoglobin concentration between infected cows and control group, So the type of anemia was Normocytic Normochromic anemia, the result was agreement with (16). The percentage of parasitemia ranged between (5-23%) with mean of (12.57%), while The parasitemia of control cows was 0% Table (2). A significant decrease was showed in total leukocytes count as well as in neutrophils count in infected cows, some studies were recorded decrease of Total leukocytes count and another studies were showed increase of its counts and this may be depending on the stage of disease, where decrease of total leukocytes count was reported by (17) (18) in early stage of disease, on the other hand increase of its counts were showed by (16) in advance stage of disease. Total leukocytes count was decreased in current study and the cause may be due to the direct effect of parasite on bone marrow and inhibit its production, similar result was seen by (11)(17) , While the study showed a significant increase (P <0.05) in the number of lymphocytes in infected cows compared with the control cows and this may be attributed to the response of the immune system to produce antibodies against of babesia spp through the increase of lymphocytes, and the results were agreed with (9), non-significant changes in Eosinophil ,Basophile and Monocyte numbers were seen in this study Table(3).