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Immunization of cattle (Bos indicus × Bos taurus) against Hyalomma anatolicum anatolicum using antigens derived from tick salivary gland extracts
RESEARCHNOTE
IMMUNIZATION OF CATTLE (BOS lNDICUS x BOS TAURUS) AGAINST HYALOMMA ANATOLICUM ANATOLICUM USING ANTIGENS DERIVED FROM TICK SALIVARY GLAND EXTRACTS D. P. BANERJEE,R. R. MOMIN* and S. SAMANTARAY~ Department
of Veterinary
Parasitology,
College of Veterinary Hisar- 125 004, Haryana,
Sciences, Haryana India
Agricultural
University,
(Received 9 May 1990; accepted 12 July 1990) A~~aCt-BANE~~~ D. P., MOMIN R. R. and SAMANTARAY S. 1990.Immunization of cattle (Bos jndicu~ x Bos taurus) against Hyalomma anatolicum unatolicum using antigens derived from tick salivary gland extracts. International Journalfor Parasitology 20: 969-972. Studies were conducted on 41 cross-bred (Bos taurus x Bos indicus) calves which were less than 1 year old. Three types of salivary gland antigens (SG Ag), viz. SC Ag-I (whole), SG Ag-II (supernatant) and SG Ag-III (sediment), prepared from the ixodid tick, Hyalomma anarolicum anufol~c~, were used for immunizing these calves. The calves were divided into five groups. The first three groups were immunized with SG Ag-I, -II and -III with Freund’s complete adjuvant (FCA), respectively. The fourth group was immunized with SG Ag-I (without adjuvant), and the fifth group remained as an unimmunized control inoculated with FCA only. In all the groups, immunization was carried out on days 1 and 14 and the animals were challenged on the 21st day. Significant resistance developed in the calves of the SG Ag-I and SG Ag-II (both with FCA) groups. Of all, SG Ag-I with FCA proved the most potent immunogen. Both humoral and cellular immune responses were demonstrated by in vivo and in vitro tests. The capillary tube agglutination test and double diffusion test gave positive reactions 21 days after first immunization. The immunized calves showed a significant increase in the levels of serum gamma globulin and per cent ‘E’ rosettes. Tick salivary gland antigens therefore seem to have promising potentials in inducing resistance in calves. INDEX humoral
KEY WORDS: response; cellular
Hyaiomma anarolicum anatolicum; immunization; response; acquired resistance; resistance to challenge.
tick, Hyalomma anatolicum anatolicum, is the most important vector of pathogens to livestock in India and other tropical countries. The control of cattle ticks by chemical acaricides has become increasingly difficult owing to the emergence of acaricide resistant strains of ticks (Wharton, 1976). As an alternative, immunological control seems to hold considerable promise. Attempts have been made to immunize cattle against the ticks, Bermacentor andersoni (see Allen & Humphreys, 1979), Amblyomma americanum (see McGowan, Barker, Homer, McNew & Holscher, 1981) and Boophilus microplus (see Johnston, Kemp and Pearson, 1986). The present report deals with the attempted immunization of cross-bred [Bos tuurus (Jersey) x Bos THE ixodid
indicus (Hariana)]
calves
against
the tick,
Hyalomma
* Present address: Department of Veterinary Parasitology, Coitege of Veterinary Sciences, Gujarat Agricultural University, Sardar, Krishinagar, Dantiwada-385 506, Gujarat, India. t Present address: Department of Veterinary Parasitology, College of Veterinary Sciences, Rajendra Agricultural University, Patna-800 014, Bihar, India.
anatolicum
salivary
gland
antigen;
anatolicum, using antigens derived from the salivary glands of ticks. Studies were carried out on 41 cross-bred healthy male calves, which were less than 1 year old, and which had no previous exposure to ticks. The calves were divided into five groups. Group I had seven calves which were immunized with the whole salivary gland antigen (SG Ag-I) with Freund’s complete adjuvant (FCA). Group II had 10 calves immunized with the supernatant salivary gland antigen (SG AgII) with FCA. Group III had 10 calves immunized with the sediment salivary gland antigen (SG Ag-III) with FCA. Group IV had seven calves immunized with SG Ag-I without FCA. Group V had seven calves administered with FCA only (control). Harvesting of salivary glands of ticks and antigen preparations were done using the method of Wikel (1981) with some modifications. The salivary glands were removed intact and were placed in 0.1 M-PBS at 4°C and stored at - 20°C for further use. Stored tick salivary glands were thawed and homogenized in a sterile pestle and mortar kept on ice. The homogenate was suspended in 15 mM-sodium desoxycholate (Hi-Media Laboratories Pvt. Ltd, Bombay).
D. P. BANERJEE,R. R. MOMIN and S. SAMANTARAY
970
The material was further homogenized by an ultrasonicator (Vibrionics Pvt. Ltd, Bombay) at 55,000 cycles per second with simultaneous cooling on ice. The homogenate was then centrifuged at 10,000 g at 5’C for 30 min, the supernatant collected, and this constituted the whole salivary gland antigen (SG AgI). Stored salivary glands were thawed and homogenized in a pestle and mortar kept on ice. The homogenate was centrifuged at 10,000 g at 5°C for 30 min. The supematant was collected as antigen (SG Ag-II). The sediment of antigen-II was suspended in I5 mht-sodium desoxycholate and the resultant preparation was kept as antigen (SG Ag-III). Protein concentration of the antigens was determined using the method of Lowry, Rosebrough, Farr & Randall (1951). Each calf of group I was injected su~utaneously with 43.32 mg (7.22 mg ml-‘) of SG Ag-I with FCA (Difco Laboratories, Detroit, MI, U.S.A.) in a 1:l ratio at a final volume of 12 ml on the first day. A second injection was given 14 days later. Each calf of group II was injected su~utaneously with 42.43 mg (7.07 mg ml-‘) of SG Ag-II with FCA in a 1:l ratio, at a final volume of 12 ml on the first day. A second injection was given 14 days later. Each calf of group III was injected subcutaneously with 22.92 mg (3.82 mg ml-‘) of SG Ag-III with FCA in a 1:l ratio, at a final volume of 12 ml on the first day. A second injection was given 14 days later. Each calf of group IV was injected subcutaneously with 38.32 mg (6.67 mg ml-‘) of SG Ag-I only, at a final volume of 6 ml, on the first day. A second injection was given 14 days later. The calves of group V (control) received an injection of FCA only (but no antigen) at similar times. Seven days after the second injection, all calves were challenged with 50 pairs (50 male and 50 female) of adult i% a. anatolicum ticks on each ear
TABLE I-IMMU~I~TION
OFCALVESWITH if. a. a~urolic~~
(total 200 ticks per calf) (Momin, Banerjee & Samantaray, in press). Blood was collected from these animals on days 0, 7, 14, 21, 28 and 35 for determination of ‘E’ rosettes (Grewal & Babiuk, 1978) with some modifications as described by Madsen & Johnsen (1979), serum gamma globulin estimation (Oser, 1976), capillary tube agglutination (CAT) and double diffusion (DD) tests. For CAT, all antigens (SG Ag-I, -11 and -111) were used. The test was carried out using the method of Ristic (1963) with undiluted sera. CAT was also performed with known negative serum (from a I day old calf) and with PBS to know the specificity of the antigen. Serum and antigen controls were also included. Likewise, for the DD test all three antigens were used and the test procedure of R&tic & Murty (1961) was followed and conducted with the test and control sera as employed in the CAT. The data were analysed by the Student’s ‘t’ test. Results from all the groups of calves have been summarized in Table 1. In the calves of group I, the engorgement period, engorged weight, pre-oviposition period, egg mass weight and number of eggs laid were significantly different (P -c 0.01) between the immunized and control groups, whereas the per cent engorgement, per cent hatch and oviposition period were non-significant (P > 0.05). In the calves of group II, the engorged weight, pre-oviposition period and egg mass weight were significantly different (P < 0.01) between the immunized and control groups; and the number of eggs laid were also significantly different (P < 0.05). However, no significant difference was observed in the per cent engorgement, engorgement period, oviposition period and per cent hatch. The parameters in the calves of group III showed no significant difference between the means of immunized and control animals. The
SALIVARY OLAND ANTIGENS (SG Ag) (MEANf s
E.VALUES)
Parameters
Immunized with SG Ag-I and FCA
Immunized with SG Ag-II and FCA
Immunized with SG Ag-111 and FCAt
Immunized with SG Ag-I only1
Control
9ioengorg~ent Engorgement period (days) Engorged wt (g) Pre-oviposition period (days) Oviposition period (days) Egg mass wt (g) No. eggs laid % hatch
67.6 f 2.90 7.4 f 0.08
66.8 f 3.17 6.9 f 0.12
74.5 f 2.37 6.7 f 0.07
71.3 f 2.85 7.1 f 0.24
74.3 i 2.96 6.4 f 0.05*
0.24 f 0.015 12.1 * 0.67
0.29 f 0.024 I I.8 f 0.46
0.379 f 0.009 10.3 * 0.82
0.363 f 0.015 10.6 f 0.60
0.416 I 0.012t 8.1 i 0.357
18.9 f 0.40
20.1 f 0.33
20.6 f 0.42
18.2 f 0.66
21.4 i: 0.25
0.126 f 0.006 1479 f 81 94.5 rL:2.78
0.164 f 0.015 2045 & 211 94.1 f 2.29
0.219 f 0.009 2753 It 128 98.4 rt 0.56
0.207 f 0.011 2670 f 190 97.2 f 0.76
0.245 + 0.005? 3083 f 112* 98.8 -I: 0.35
* Statistically significant at 1% level of significance (P < 0.01) as observed in Student’s ‘I’ test, when analyzed with the data obtained with SG Ag-I and FCA, and f with the data obtained with SG Ag-II and FCA. t No parameter was statistically significant at 5% level of significance.
971
Research Note calves of group IV showed no significant resistance to infestation. Only the pre-oviposition period was significantly different (P < 0.05) between the immunized and control animals. No significant difference was observed for other parameters. A positive CAT reaction was recorded in calves immunized with SG Ag-I (with FCA) only from 21 days post-immunization to 70 days. All other animals showed no reaction. A positive DD reaction was observed from 21 days post-immunization until the 70th day in all immunized animals. Furthermore, three calves in the SG Ag-I group showed double precipitin lines. The rest of the animals of all groups showed a single line. Serum gamma globulin levels from 0 to 35 days post-immunization showed a gradual increase (in g per 100 ml) from 1.679 f 0.047 to 2.387 f 0.047 in calves of group I; from 1.561 f 0.078 to 2.521 i 0.092 in calves of group II; from 1,495 ri: 0.057 to 1.960 f 0.064 in calves of group III; from 1.157 f. 0.056 to 1.276 & 0.035 in calves of group IV; and 1.275 i 0.106 to I.322 i 0,090 in control calves. These differences in the calves of groups I, II, III and the control group V, were statistically significant (PC 0.01 for group I and II; P < 0.05 for group III). However, no significant difference was observed between the animals of group IV (SG Ag-I, without FCA) and V (control) calves. Per cent ‘E’ rosette values from 0 to 35 days postimmunization showed a gradual increase from 26.50 & 2.26 to 38.75 f 0.74 in calves of group I; from 25.00 f 2.47 to 36.00 f 1.87 in calves of group II; from 23.75 f 3.64 to 34.50 rt: 2.08 in calves of group III; from 20.25 f 2.77 to 27.50 f 1.68 in calves of group IV: and 24.50 f 1.82 to 25.50 f 1.03 in control calves. Calves of groups I, II and IV showed the difference in values as statistically significant (P < 0.01 to < 0.05) when compared with control calves. The difference in values was non-significant in calves of groups III and V. The use of whole supernatant and sediment fractions of tick salivary gland extract (antigen) to induce an immune response has been described earlier in cattle using B. microplus (see Brossard, 1976), in guinea pigs using D. andersoni (see Wikel, 19X1), in cattle using ~rnb~~~ornrnamacu~af~m (see McGowan et al., 1981) and in guinea pigs using A. americanum (see Brown, Shapiro & Askenase, 1984). Trager (1939) and Boese (1974) reported that ticks, fed on animals immunized with tick salivary gland extract or exposed to previous tick infestations, often failed to engorge completely with consequent reduction in engorgement weight or egg mass weight of the female ticks. Our observations on responses of the adult ticks to immunized calves support these findings. In the present study, incorporation of FCA with SG Ag-I afforded better resistance than when the antigen was used alone. The SG Ag-I with FCA proved most potent as compared to SG Ag-II or SC Ag-III with FCA. All calves showed an antibody response with the DD test, 21 days after first
immunization with all antigenic fractions (with FCA). However, the calves immunized with SG Ag-I (without FCA) showed no precipitin lines. With CAT, the antibody response was observed only in SG Ag-I (with FCA) immunized animals 21 days after first immunization. The acquired resistance to tick infestation by the host after immunization with various tick tissues has been studied in cattle by immunodiffusion (Allen & Humphreys, 1979), passive haemagglutination (McGowan et al., 1981) and gel diffusion (Johnston ef al., 1986) tests. The present study showed that the DD test was probably more sensitive than the CAT and it detected antibodies up to 70 days after first immunization with SG Ag-I (with FCA). The significantly elevated gamma globulin levels in all SG Ag-immunized (with FCA) animals in the present study indicate the possible protective role of gamma globulin. In the present study, ticks from the immunized calves were lighter in weight suggesting an effect of host response or antibody on their feeding. Host immunoglobulin binding to ixodid tissue may interfere with the tick salivation process and gut absorption. Antibodydependent lysis involving complement or antibodydependent cellular cytotoxicity could also affect tick engorgement (Whelan, Richardson & Wikel, 1986). Ackerman, Clare, McGill & Sonenshine (1981) demonstrated the ability of antibody to cross the gut of D. variubilis into the haemolymph. They further explained that the antibody could react with internal organs of the feeding tick and inhibit moulting or oviposition processes. The percent ‘E’ rosettes increased significantly after first immunization with SG Ag-I, -11 (both with FCA) and SG Ag-1 (without FCA) indicating the possible participation of cell mediated immune responses (CMIR) in calves against H. a. unatolicum. It has been demonstrated that resistance in guinea pigs to D. andersoni is governed by a CMIR component (Wikel & Allen, 1976; Wikel, Graham & Allen, 1978). In the present study, feeding and reproductive performances of the ticks infesting the immunized calves were significantly reduced, particularly in calves of the SG Ag-I (with FCA) group. Tick salivary gland antigens therefore seem to have promising potential for inducing immunity in calves. REFERENCES ACKERMANS., CURE F. B., MCGILL T. W. & SONEN~HINED. E. 1981. Passage of host serum components, including antibody, across the digestive tract of*Dermacenfor van; abilis. Journal ofParasitology61: 737-740. ALLEN J. R. & &UMPHREYS!% J. 1979. Immunization of guinea pigs and cattle against ticks. Nature (London) 280: 491-493. BOESE J. L. 1974. Rabbit immunity to the rabbit tick Haemaphysalis leporispalustris (Atari, Ixodidae). I. The development of resistance. Journal of Medical Entomology 11: 503-512.
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D. P. BANERJEE, R. R. MOMIN and S. SAMANTARAY
BROSSARDM. 1976. Relations immunologiques entre bovins et tiques, plus particulierement entre bovine et Boophz’lus microplus. Acta Tropica 33: 15-36. BROWN S. J., SHAPIRO S. Z. & ASKENASE P. W. 1984. Characterization of tick antigens inducing host immune resistance. I. Immunization of guinea pigs with Amblyomma americamun derived salivary gland extracts and identification of an important salivary gland protein antigen with guinea pig anti-tick antibodies. Journal of Immunology 133: 3319-3325. GREWAL A. S. & BABIUK L. A. 1978. Bovine lymphocytesan improved technique of ‘E’ rosette formation. Journal of Immunological Methods 24. 355-361. JOHNSTON L. A. Y., KEMP D. H. & PEARSON R. D. 1986. Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: effects of induced immunity on tick populations. International Journal for Parasitology 16:27-34. LOWRY0. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265-275. MADSEN M. & JOHNSENH. E. 1979. A methodological study of E-rosette formation using AET-treated sheep red blood cells. Journal of Immunological Methods 21: 61-64. MCGOWAN M. J., BARKER R. W., HOMER J. T. MCNEW R. W. & HOLSCHER K. H. 1981. Success of tick feeding on calves immunized with Ambl_vomma americanurn (Atari: Ixodidae) extract. Journal of Medical Entomology 18: 328-332. MOMIN R. R., BANERJEED. P. & SAMANTARAYS. (in press) Attempted immunization of cross-bred calves (Bos taurus
x Bos indicus) by repeated natural attachment of ticks, Hyalommn anatolicum anatolicum. Tropical Animal Health and Production. OSER B. L. 1976. Hawk’s Physiological Chemistry, 14th edition, pp. 1099-1100. McGraw Hill Books, New York. RISTIC M. & MURTY D. K. 1961. Characterization of Vibrio foefus antigens. IV. Study of polysaccharide-antibody reactions by a rapid slide gel diffusion technique. American Journal of Veterinary Research 22: 783-789. RISTIC M. 1963. A capillary tube agglutination test for anaplasmosis. A preliminary report. Journal of the American Veterinary Medical Association 141: 588-594. TRACER W. 1939. Acquired immunity to ticks. Journal of Parasitology 25 : 57-8 1. WHARTON R. H. 1976. Tick-borne livestock diseases and their vectors. 5. Acaricide resistance and alternative methods of tick control. World Animal Review 20: S-15. WHELAN A. C., RICHARDSON L. K. & WIKEL S. K. 1986. Dot-Elisa assessment of guinea pig antibody responses to repeated Dermacentor andersoni infestations. Journal of Parasitology 72: 155-l 62. WIKEL S. K. & ALLEN J. R. 1976. Acquired resistance to ticks. I. Passive transfer of resistance. Immunology 30: 311-316. WIKEL S. K., GRAHAM J. E. & ALLEN J. R. 1978. Acquired resistance to ticks. IV. Skin reactivity and in vitro lymphocyte responsiveness to salivary gland antigen. Immunology 82: 257-263. WIKEL S. K. 1981. The induction of host resistance to tick infestation with salivary gland antigen. American Journal of Tropical Medicine and Hygiene 30: 284-288.
IMMUNIZATION OF CATTLE (BOS lNDICUS x BOS TAURUS) AGAINST HYALOMMA ANATOLICUM ANATOLICUM USING ANTIGENS DERIVED FROM TICK SALIVARY GLAND EXTRACTS D. P. BANERJEE,R. R. MOMIN* and S. SAMANTARAY~ Department
of Veterinary
Parasitology,
College of Veterinary Hisar- 125 004, Haryana,
Sciences, Haryana India
Agricultural
University,
(Received 9 May 1990; accepted 12 July 1990) A~~aCt-BANE~~~ D. P., MOMIN R. R. and SAMANTARAY S. 1990.Immunization of cattle (Bos jndicu~ x Bos taurus) against Hyalomma anatolicum unatolicum using antigens derived from tick salivary gland extracts. International Journalfor Parasitology 20: 969-972. Studies were conducted on 41 cross-bred (Bos taurus x Bos indicus) calves which were less than 1 year old. Three types of salivary gland antigens (SG Ag), viz. SC Ag-I (whole), SG Ag-II (supernatant) and SG Ag-III (sediment), prepared from the ixodid tick, Hyalomma anarolicum anufol~c~, were used for immunizing these calves. The calves were divided into five groups. The first three groups were immunized with SG Ag-I, -II and -III with Freund’s complete adjuvant (FCA), respectively. The fourth group was immunized with SG Ag-I (without adjuvant), and the fifth group remained as an unimmunized control inoculated with FCA only. In all the groups, immunization was carried out on days 1 and 14 and the animals were challenged on the 21st day. Significant resistance developed in the calves of the SG Ag-I and SG Ag-II (both with FCA) groups. Of all, SG Ag-I with FCA proved the most potent immunogen. Both humoral and cellular immune responses were demonstrated by in vivo and in vitro tests. The capillary tube agglutination test and double diffusion test gave positive reactions 21 days after first immunization. The immunized calves showed a significant increase in the levels of serum gamma globulin and per cent ‘E’ rosettes. Tick salivary gland antigens therefore seem to have promising potentials in inducing resistance in calves. INDEX humoral
KEY WORDS: response; cellular
Hyaiomma anarolicum anatolicum; immunization; response; acquired resistance; resistance to challenge.
tick, Hyalomma anatolicum anatolicum, is the most important vector of pathogens to livestock in India and other tropical countries. The control of cattle ticks by chemical acaricides has become increasingly difficult owing to the emergence of acaricide resistant strains of ticks (Wharton, 1976). As an alternative, immunological control seems to hold considerable promise. Attempts have been made to immunize cattle against the ticks, Bermacentor andersoni (see Allen & Humphreys, 1979), Amblyomma americanum (see McGowan, Barker, Homer, McNew & Holscher, 1981) and Boophilus microplus (see Johnston, Kemp and Pearson, 1986). The present report deals with the attempted immunization of cross-bred [Bos tuurus (Jersey) x Bos THE ixodid
indicus (Hariana)]
calves
against
the tick,
Hyalomma
* Present address: Department of Veterinary Parasitology, Coitege of Veterinary Sciences, Gujarat Agricultural University, Sardar, Krishinagar, Dantiwada-385 506, Gujarat, India. t Present address: Department of Veterinary Parasitology, College of Veterinary Sciences, Rajendra Agricultural University, Patna-800 014, Bihar, India.
anatolicum
salivary
gland
antigen;
anatolicum, using antigens derived from the salivary glands of ticks. Studies were carried out on 41 cross-bred healthy male calves, which were less than 1 year old, and which had no previous exposure to ticks. The calves were divided into five groups. Group I had seven calves which were immunized with the whole salivary gland antigen (SG Ag-I) with Freund’s complete adjuvant (FCA). Group II had 10 calves immunized with the supernatant salivary gland antigen (SG AgII) with FCA. Group III had 10 calves immunized with the sediment salivary gland antigen (SG Ag-III) with FCA. Group IV had seven calves immunized with SG Ag-I without FCA. Group V had seven calves administered with FCA only (control). Harvesting of salivary glands of ticks and antigen preparations were done using the method of Wikel (1981) with some modifications. The salivary glands were removed intact and were placed in 0.1 M-PBS at 4°C and stored at - 20°C for further use. Stored tick salivary glands were thawed and homogenized in a sterile pestle and mortar kept on ice. The homogenate was suspended in 15 mM-sodium desoxycholate (Hi-Media Laboratories Pvt. Ltd, Bombay).
D. P. BANERJEE,R. R. MOMIN and S. SAMANTARAY
970
The material was further homogenized by an ultrasonicator (Vibrionics Pvt. Ltd, Bombay) at 55,000 cycles per second with simultaneous cooling on ice. The homogenate was then centrifuged at 10,000 g at 5’C for 30 min, the supernatant collected, and this constituted the whole salivary gland antigen (SG AgI). Stored salivary glands were thawed and homogenized in a pestle and mortar kept on ice. The homogenate was centrifuged at 10,000 g at 5°C for 30 min. The supematant was collected as antigen (SG Ag-II). The sediment of antigen-II was suspended in I5 mht-sodium desoxycholate and the resultant preparation was kept as antigen (SG Ag-III). Protein concentration of the antigens was determined using the method of Lowry, Rosebrough, Farr & Randall (1951). Each calf of group I was injected su~utaneously with 43.32 mg (7.22 mg ml-‘) of SG Ag-I with FCA (Difco Laboratories, Detroit, MI, U.S.A.) in a 1:l ratio at a final volume of 12 ml on the first day. A second injection was given 14 days later. Each calf of group II was injected su~utaneously with 42.43 mg (7.07 mg ml-‘) of SG Ag-II with FCA in a 1:l ratio, at a final volume of 12 ml on the first day. A second injection was given 14 days later. Each calf of group III was injected subcutaneously with 22.92 mg (3.82 mg ml-‘) of SG Ag-III with FCA in a 1:l ratio, at a final volume of 12 ml on the first day. A second injection was given 14 days later. Each calf of group IV was injected subcutaneously with 38.32 mg (6.67 mg ml-‘) of SG Ag-I only, at a final volume of 6 ml, on the first day. A second injection was given 14 days later. The calves of group V (control) received an injection of FCA only (but no antigen) at similar times. Seven days after the second injection, all calves were challenged with 50 pairs (50 male and 50 female) of adult i% a. anatolicum ticks on each ear
TABLE I-IMMU~I~TION
OFCALVESWITH if. a. a~urolic~~
(total 200 ticks per calf) (Momin, Banerjee & Samantaray, in press). Blood was collected from these animals on days 0, 7, 14, 21, 28 and 35 for determination of ‘E’ rosettes (Grewal & Babiuk, 1978) with some modifications as described by Madsen & Johnsen (1979), serum gamma globulin estimation (Oser, 1976), capillary tube agglutination (CAT) and double diffusion (DD) tests. For CAT, all antigens (SG Ag-I, -11 and -111) were used. The test was carried out using the method of Ristic (1963) with undiluted sera. CAT was also performed with known negative serum (from a I day old calf) and with PBS to know the specificity of the antigen. Serum and antigen controls were also included. Likewise, for the DD test all three antigens were used and the test procedure of R&tic & Murty (1961) was followed and conducted with the test and control sera as employed in the CAT. The data were analysed by the Student’s ‘t’ test. Results from all the groups of calves have been summarized in Table 1. In the calves of group I, the engorgement period, engorged weight, pre-oviposition period, egg mass weight and number of eggs laid were significantly different (P -c 0.01) between the immunized and control groups, whereas the per cent engorgement, per cent hatch and oviposition period were non-significant (P > 0.05). In the calves of group II, the engorged weight, pre-oviposition period and egg mass weight were significantly different (P < 0.01) between the immunized and control groups; and the number of eggs laid were also significantly different (P < 0.05). However, no significant difference was observed in the per cent engorgement, engorgement period, oviposition period and per cent hatch. The parameters in the calves of group III showed no significant difference between the means of immunized and control animals. The
SALIVARY OLAND ANTIGENS (SG Ag) (MEANf s
E.VALUES)
Parameters
Immunized with SG Ag-I and FCA
Immunized with SG Ag-II and FCA
Immunized with SG Ag-111 and FCAt
Immunized with SG Ag-I only1
Control
9ioengorg~ent Engorgement period (days) Engorged wt (g) Pre-oviposition period (days) Oviposition period (days) Egg mass wt (g) No. eggs laid % hatch
67.6 f 2.90 7.4 f 0.08
66.8 f 3.17 6.9 f 0.12
74.5 f 2.37 6.7 f 0.07
71.3 f 2.85 7.1 f 0.24
74.3 i 2.96 6.4 f 0.05*
0.24 f 0.015 12.1 * 0.67
0.29 f 0.024 I I.8 f 0.46
0.379 f 0.009 10.3 * 0.82
0.363 f 0.015 10.6 f 0.60
0.416 I 0.012t 8.1 i 0.357
18.9 f 0.40
20.1 f 0.33
20.6 f 0.42
18.2 f 0.66
21.4 i: 0.25
0.126 f 0.006 1479 f 81 94.5 rL:2.78
0.164 f 0.015 2045 & 211 94.1 f 2.29
0.219 f 0.009 2753 It 128 98.4 rt 0.56
0.207 f 0.011 2670 f 190 97.2 f 0.76
0.245 + 0.005? 3083 f 112* 98.8 -I: 0.35
* Statistically significant at 1% level of significance (P < 0.01) as observed in Student’s ‘I’ test, when analyzed with the data obtained with SG Ag-I and FCA, and f with the data obtained with SG Ag-II and FCA. t No parameter was statistically significant at 5% level of significance.
971
Research Note calves of group IV showed no significant resistance to infestation. Only the pre-oviposition period was significantly different (P < 0.05) between the immunized and control animals. No significant difference was observed for other parameters. A positive CAT reaction was recorded in calves immunized with SG Ag-I (with FCA) only from 21 days post-immunization to 70 days. All other animals showed no reaction. A positive DD reaction was observed from 21 days post-immunization until the 70th day in all immunized animals. Furthermore, three calves in the SG Ag-I group showed double precipitin lines. The rest of the animals of all groups showed a single line. Serum gamma globulin levels from 0 to 35 days post-immunization showed a gradual increase (in g per 100 ml) from 1.679 f 0.047 to 2.387 f 0.047 in calves of group I; from 1.561 f 0.078 to 2.521 i 0.092 in calves of group II; from 1,495 ri: 0.057 to 1.960 f 0.064 in calves of group III; from 1.157 f. 0.056 to 1.276 & 0.035 in calves of group IV; and 1.275 i 0.106 to I.322 i 0,090 in control calves. These differences in the calves of groups I, II, III and the control group V, were statistically significant (PC 0.01 for group I and II; P < 0.05 for group III). However, no significant difference was observed between the animals of group IV (SG Ag-I, without FCA) and V (control) calves. Per cent ‘E’ rosette values from 0 to 35 days postimmunization showed a gradual increase from 26.50 & 2.26 to 38.75 f 0.74 in calves of group I; from 25.00 f 2.47 to 36.00 f 1.87 in calves of group II; from 23.75 f 3.64 to 34.50 rt: 2.08 in calves of group III; from 20.25 f 2.77 to 27.50 f 1.68 in calves of group IV: and 24.50 f 1.82 to 25.50 f 1.03 in control calves. Calves of groups I, II and IV showed the difference in values as statistically significant (P < 0.01 to < 0.05) when compared with control calves. The difference in values was non-significant in calves of groups III and V. The use of whole supernatant and sediment fractions of tick salivary gland extract (antigen) to induce an immune response has been described earlier in cattle using B. microplus (see Brossard, 1976), in guinea pigs using D. andersoni (see Wikel, 19X1), in cattle using ~rnb~~~ornrnamacu~af~m (see McGowan et al., 1981) and in guinea pigs using A. americanum (see Brown, Shapiro & Askenase, 1984). Trager (1939) and Boese (1974) reported that ticks, fed on animals immunized with tick salivary gland extract or exposed to previous tick infestations, often failed to engorge completely with consequent reduction in engorgement weight or egg mass weight of the female ticks. Our observations on responses of the adult ticks to immunized calves support these findings. In the present study, incorporation of FCA with SG Ag-I afforded better resistance than when the antigen was used alone. The SG Ag-I with FCA proved most potent as compared to SG Ag-II or SC Ag-III with FCA. All calves showed an antibody response with the DD test, 21 days after first
immunization with all antigenic fractions (with FCA). However, the calves immunized with SG Ag-I (without FCA) showed no precipitin lines. With CAT, the antibody response was observed only in SG Ag-I (with FCA) immunized animals 21 days after first immunization. The acquired resistance to tick infestation by the host after immunization with various tick tissues has been studied in cattle by immunodiffusion (Allen & Humphreys, 1979), passive haemagglutination (McGowan et al., 1981) and gel diffusion (Johnston ef al., 1986) tests. The present study showed that the DD test was probably more sensitive than the CAT and it detected antibodies up to 70 days after first immunization with SG Ag-I (with FCA). The significantly elevated gamma globulin levels in all SG Ag-immunized (with FCA) animals in the present study indicate the possible protective role of gamma globulin. In the present study, ticks from the immunized calves were lighter in weight suggesting an effect of host response or antibody on their feeding. Host immunoglobulin binding to ixodid tissue may interfere with the tick salivation process and gut absorption. Antibodydependent lysis involving complement or antibodydependent cellular cytotoxicity could also affect tick engorgement (Whelan, Richardson & Wikel, 1986). Ackerman, Clare, McGill & Sonenshine (1981) demonstrated the ability of antibody to cross the gut of D. variubilis into the haemolymph. They further explained that the antibody could react with internal organs of the feeding tick and inhibit moulting or oviposition processes. The percent ‘E’ rosettes increased significantly after first immunization with SG Ag-I, -11 (both with FCA) and SG Ag-1 (without FCA) indicating the possible participation of cell mediated immune responses (CMIR) in calves against H. a. unatolicum. It has been demonstrated that resistance in guinea pigs to D. andersoni is governed by a CMIR component (Wikel & Allen, 1976; Wikel, Graham & Allen, 1978). In the present study, feeding and reproductive performances of the ticks infesting the immunized calves were significantly reduced, particularly in calves of the SG Ag-I (with FCA) group. Tick salivary gland antigens therefore seem to have promising potential for inducing immunity in calves. REFERENCES ACKERMANS., CURE F. B., MCGILL T. W. & SONEN~HINED. E. 1981. Passage of host serum components, including antibody, across the digestive tract of*Dermacenfor van; abilis. Journal ofParasitology61: 737-740. ALLEN J. R. & &UMPHREYS!% J. 1979. Immunization of guinea pigs and cattle against ticks. Nature (London) 280: 491-493. BOESE J. L. 1974. Rabbit immunity to the rabbit tick Haemaphysalis leporispalustris (Atari, Ixodidae). I. The development of resistance. Journal of Medical Entomology 11: 503-512.
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BROSSARDM. 1976. Relations immunologiques entre bovins et tiques, plus particulierement entre bovine et Boophz’lus microplus. Acta Tropica 33: 15-36. BROWN S. J., SHAPIRO S. Z. & ASKENASE P. W. 1984. Characterization of tick antigens inducing host immune resistance. I. Immunization of guinea pigs with Amblyomma americamun derived salivary gland extracts and identification of an important salivary gland protein antigen with guinea pig anti-tick antibodies. Journal of Immunology 133: 3319-3325. GREWAL A. S. & BABIUK L. A. 1978. Bovine lymphocytesan improved technique of ‘E’ rosette formation. Journal of Immunological Methods 24. 355-361. JOHNSTON L. A. Y., KEMP D. H. & PEARSON R. D. 1986. Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: effects of induced immunity on tick populations. International Journal for Parasitology 16:27-34. LOWRY0. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265-275. MADSEN M. & JOHNSENH. E. 1979. A methodological study of E-rosette formation using AET-treated sheep red blood cells. Journal of Immunological Methods 21: 61-64. MCGOWAN M. J., BARKER R. W., HOMER J. T. MCNEW R. W. & HOLSCHER K. H. 1981. Success of tick feeding on calves immunized with Ambl_vomma americanurn (Atari: Ixodidae) extract. Journal of Medical Entomology 18: 328-332. MOMIN R. R., BANERJEED. P. & SAMANTARAYS. (in press) Attempted immunization of cross-bred calves (Bos taurus
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