Immunoprophylaxis Against Haemaphysalid Ticks In Sheep
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Date
2004
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Tamil Nadu Veterinary and Animal Sciences University
Abstract
The membrane midgut antigen of Haemaphysalis bispinosa (HbGMAg)
characterized by SDS-PAGE revealed nine polypeptide bands with Coomassie blue
staining ranging from 127.0 to 22.0 kDa molecular weight. Western blot analysis of
HbGMAg revealed five immunogenic polypeptide bands at 87.5, 47.0, 36.0, 29.0 and
22.0 kDa. Rabbits were immunized with immunogenic polypeptide fractions separated
from SDS-polyacrylamide gels. Sera collected on 35 DPI showed positive precipitin
reaction by AGID test.
Rabbits were challenged with 10 pairs of H. bispinosa ticks on 35 days post
immunization (DPI) to identify the effective immunoprotective polypeptide based on
feeding and fertility parameters of ticks. Rabbits immunized with 29 kDa polypeptide
showed highest percent reduction of adult female ticks (29.41), weight of engorged
female (67.04) and offered a vaccine efficacy of 75.56 percent.Immunoglobulin G was purified from the serum collected from 29 kDa
polypeptide using caprylic acid method and the purified IgG were used as ligands in
immunoaffinity chromatography column. Purification yielded 3.67 percent of bound
and eluted Aff-29 kDa antigen from the total 179 mg of HbGMAg loaded in batches.
Sheep were immunized with 500 g of Aff-29 kDa protein alongwith Saponin
or Montanide ISA 25 as adjuvants. Immune response was assessed by evaluating the
humoral, cell mediated immunity and tick challenge studies. Sera collected on 30, 60,
and 90 DPI from Saponin and Montanide groups showed a reactive band at 29 kDa
molecular weight on EITB analysis. The ELISA absorbance values were significantly
higher up to 105 DPI in Saponin and 120 DPI in Montanide group. Lymphocyte
stimulation indices (SI) were significantly higher (above 2) during 28 to 56 DPI in
Saponin and 28 to 70 DPI in Montanide groups in response to Aff-29 kDa antigen in
vitro. The skinfold thickness was significantly increased between 24 and 72 hours Post
intradermal injection indicating that the immunogen elicited delayed type of
hypersensitivity reaction.
Indirect peroxidase test (IPT) to demonstrate the in situ localization of antigen
determinants in midgut showed positive brown colour reaction on the entire surface of
digestive cells and midgut caecal epithelial membrane. Histological examination of
H&E stained sections of ticks fed on immunized animals revealed extensive damage of
gut lining and complete detachment of midgut epithelial membrane. Sheep were
challenged with 20 pairs of Haemaphysalis bispinosa and 10 pairs of Hyalomma
anatolicum anatolicum on 45 and 95 DPI.
Highest percent reductions in mean engorgement weight of H. bispinosa ticks
were recorded in Montanide (primary 49.60 % and secondary 32.63%) groupcompared to Saponin (primary 42.64% and secondary 30.14%) group. The H. a.
anatolicum ticks recovered from immunized sheep showed highest percent reduction
in engorged weights during primary challenge (Saponin 33.28% and Montanide
34.67%) compared to secondary challenge (Saponin 4.06% and Montanide 6.36%).
The percent reduction in mean weight of egg mass (DO%) varied between
29.75 to 41.24 in immunized groups during H. bispinosa challenges, while H. a.
anatolicum showed 2.83 to 23.41 percent reduction in egg mass weight. The percent
reduction of fertility (DF%) varied between 58.35 and 37.31 percent in H. bispinosa
and 51.76 to 5.70 percent in H. a. anatolicum ticks collected from immunized groups
during primary and secondary challenge.
Sheep immunized with Aff-29 kDa along with Montanide ISA 25 showed
highest efficacy (primary 80.69%; secondary 66.66%) compared to Saponin (primary
75.63%; secondary 60.96%) groups during different challenges with H. bispinosa.
Whereas cross protection studies conducted with H. a. anatolicum challenge offered
66.52 and 32.15 percent in Saponin, 68.49 and 40.86 percent in Montanide groups
during primary and secondary challenges respectively.
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Keywords
biological phenomena, livestock, carbohydrates, eggs, proteins, adjuvants, research methods, extraction, inorganic acid salts, electrophoresis