Efficacy of Purified Glutathione -S- Transferee in Providing Protection against Haemonchus Contortus Infection in Sheep

Abstract

The procedures in all experimental native breed animals were performed in agreement with the recommendations and guidelines stated by the ethical Committee of the National Research Centre under certificate number (16050). Ethical approval Material and MethodsAdult worms of H. contortus were collected from all abomasal content of infected sheep at El-monib slaughter house, Giza, Egypt according to [23]. Intact washed worms were incubated at 37 ºC for 2-3hr to obtain eggs. Eggs were then used in fecal culture to recover third larval stage. H. contortus isolateThe crude adult antigen was prepared from the collected worm as stated by [24]. The purification of H. contortus adult GST was done as reported by [25]. Briefly, the sepharose 4B column (GE Healthcare., UK) was applied for GST purification. Equilibration of glutathione sepharose was conducted through 4B packed column using Tris buffer (Tris-Hcl pH 8). The crude extract of adult worms was dispensed onto the column. Then it was washed to remove unbound fractions by equilibration buffer. The elution buffer (50 MmTris-Hcl, 10 mM Glutathione reduced pH 9.6) was used on the column to elute the glutathione bound fractions in 1 ml fractions. The latter was pooled and subjected to further dialysis concentration using polyethylene glycol (MW 8000). The specific activity of purified GST was determined according to using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate [26]. The CDNB molar extinction is 0.0096µM-1 /cm. The purified GST was stored at -20 ºC until use. The protein amount in the CA and GST antigens was detected according to [27]. Crude adult and GST antigens preparationNine healthy white New Zealand male rabbits were classified into three groups, each group contained three rabbits; first group was inoculated with CA antigen and second group was injected with GST according to and the third one used as control negative rabbit [28]. Briefly, 200 µg of each prepared antigens emulsified with an equal amount of mineral oil was injected subcutaneously in each rabbit. After two weeks, two booster dosages were injected subcutaneously with a week interval. After one week from the last booster dose, sera samples were collected and stored at -20 ºC.