Mukhtar Ahmad

In-Vetro And In-Vivo Anti-Theilerial Activity Of Medicianal Plants - 2013

In vitro study was carried out in order to estimate the anti-theileria effect of Calotropis procera and Peganum harmala. Water and chloroform extracts of each plant were used in-vitro study along with standard drug Buparvaqoune (Butalex®). For this four concentrations i.e.4, 8, 12 and 16mg/ tested solutions of each extracts of each plant were applied on cultured lymphocytes exposed to theileria parasite infection. ELISA reader findings showed that the mean OD were found less in treated theileria infected lymphocytes cell culture as compared to untreated culture wells mean OD values. The highest cell reduction (94.36%) with C. procera chloroform extract treatment was observed at a concentration of 16 mg/ml solution. Lowest concentration (1mg/ml solution) of C. procera chloroform extract reduced non-significant (P>0.05) lymphocytes cell proliferation (40.97%) as compare to control negative group. P. harmala water extract was effective against the theileria parasite as significantly lower (P<0.05) mean OD value (1.802 ±0.341) was measured at a concentration of 4mg/ml solution and maximum inhibitory effect (92.20) was seen at a concentration of 16 mg/ml solution. ELISA reader findings showed that P. harmala chloroform extract treatment failed to inhibit lymphocytes cells propagation even at highest concentration. The highest inhibitory effect (85.33%) against theileria infected lymphocytes propagation was seen at a concentration of 16 mg/ml solution.
Plant extract was evaluated in respect of feed intake in rabbits. It showed that when administered extracts of C. procera in rabbits at dose of 3 mg and 5 mg/kg body weight, did not affect on feed intake in rabbits. However the chloroform and water extracts of both plant i.e. C. procera and P. harmala when were administered in rabbits parentally at dose 10 of mg/kg body. It showed that the feed intake of rabbits was non-significantly reduced as compared to other treatments groups. Hematological parameters such as WBC X103 count, RBC X 106 count and Hb g/dl values were measured at various days. Findings showed that significantly lesser RBC X 106 count was in group A3 and D3 than control at day 30 of experiment. A non-significant difference (P>0.05) was seen in RBC X 106 count and Hb g/dl measurements in all treatments groups. Kidney and liver functions were evaluated by measuring biochemical parameters, uric acid, creatinine and ALT at 0 days, 9 days and 30 days. Findings showed that serum creatinine and urea enzyme levels were significantly higher (P<0.05) in group A3 as compare to control group at day 30 of experiment. Serum level of urea was also significantly higher (P<0.05) in group B3 and D3 at day 30 of experiment. A non-significant difference (P>0.05) was seen in ALT in all treatment groups at day 30 than control. Post-mortem was performed at day 30 of experiment. Gross lesions consisting of hemorrhages, congestion, and lung emphysema were seen in rabbits treated with high dose i.e. 10 mg/kg of both extract of C. procera. Rabbits treated with P. harmala chloroform extract at dose 10 mg/kg showed moderate gross lesions. Histopathology of organs such as lungs, kidney, liver and heart was performed. Toxicity lesions were seen in rabbits treated with high dose i.e. 10 mg/kg of both extract of C. procera. Rabbits treated with P. harmala chloroform extract at dose 10 mg/kg showed histopathological lesions in lungs, liver and kidney.
Theileria infection was studied in vivo by developing through theileria infected Hayalomma ticks in crossbred calves (n=30) through. At day 15 of infection maximum increase in mean rectal temperature (105.24 ± 0.46F) was observed, twenty four calves had pyrexia (104.1- 105.6 F) and six claves were showing pyrexia > 105.6 F. A significant increase (P<0.05) in pre-scapular lymph node enlargement score of challenged calves was seen by day 7 of infection and maximum lymph node score (grossly enlarged size) was noticed in twenty calves (Table 4.14 , Plate 4.16) with peak mean score (2.73±0.44) on day 13 of infection. The piroplasm peak score (3.80±0.83) was observed in challenged calves at 22 day of infection and remained significantly higher (p<0.05) (2.60±0.54) in untreated calves until the 36 day of infection (Fig.4.29 and Table 4.16). A significant increase (P<0.05) in mean schizonts was observed in pre-scapular lymph node biopsy smear from day 7 of infection to onwards. Blood samples of challenged calves (n =30) were confirmed theileria positive through PCR test. The amplification of Theileria species were amplified at 1098 bp (Plate 4.20 and Theileria annulata was amplified at 721 bp (Plate 4.21).
In order to estimate the pattern of disease severity, severity score was measured by summation of mean score of piroplasms, schizonts, lymph node swelling and rectal temperature. From day 7, mild response (3-5 score) was seen in infected calves (n=10).
With increase in the severity of disease a significant decrease (P<0.05) was observed in mean values of the Hb g/dL amount, WBC and RBC count, Hct (%) concentration and lymphocytes percentage from day15 of infection onward to 36 day of infection. A non-significant decrease (P>0.05) in the mean values of MCH pg was seen throughout the experiment. A significant decrease (P<0.05) in mean values of MCHC g/dL along with significant increase (P<0.05) in the mean measurement of MCV fL (64.14±3.53) values was seen at day 36 of infection as compare to day 0 values, indicating macrocytic hypochromic anemia in challenged calves. These findings showed a significant increase (P<0.05) in excretory products (uric acid and creatinine) from day 15 of infection and onward as compared to day 0 values, indicating damaged kidney in infected calves. Biochemical analysis showed the significant increase (P<0.05) in liver enzymes (ALT, AST) from day 15 infection and onward.
Anti-Theileria activities of drugs were estimated by evaluating clinical manifestation of the disease and parasitological findings. Beside this treatment effect on hematological and biochemical reactions of liver and kidney functions was determined. A significant difference (P<0.05) in rectal temperature of calves groups (B and E) was observed than control positive (group F) at day 21 of post-treatment. On other hand calves treated with treatments A, C and D had a non-significant difference (P>0.05) in rectal temperature compared with untreated calves (group F). It was found that calves (n=5) dosed with C. procera chloroform extract (group A) had rectal temperature in normal range by the day 7 of post-treatment. Similarly calves (n=5) treated with Butalex were found with normal rectal temperature from the day 7 of pos-treatment. On other hand, at day 21 of treatment 40%, 20%, 40% and 80% calves were found with pyrexia in treatments groups B, C, D and F, respectively (Table 4. 46).
By the day 14 of treatment, calves of treatment groups B and E showed no parasitemia (piroplams ?1). Disease severity was estimated on accumulative score of rectal temperature, lymph node swelling and parasitological findings (piroplasms and schizonts score). It was found a significant decrease (P<0.05) occurred in the disease severity of score of disease in calves of groups B and E as compare to A, C and F at day 3 of post-treatment. At day 21 of treatment all treated calves were recovered from anemia.

Department of Clinical Medicine & Surgery


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