Journal of Veterinary Parasitology
Year : 2005, Volume : 19, Issue : 1
First page : ( 1) Last page : ( 8)
Print ISSN : 0971-6157.

Immunity and immunization against Hyalomma tick vectors of bovine tropical theileriosis: A review

Chhabra M.B.¹, Retd. Professor of Veterinary Parasitology, Khurana K.L.², Director

Department of Veterinary Parasitology, CCS Haryana Agricultural University, Hisar-125 004, India

¹Present address: 2518, Sector D-2, Vasant Kunj, New Delhi-110 070

²Present address: Animal Husbandry, Government of NCT, Delhi, Room 101, Old Secretariat, Delhi-110 054

Abstract

Cattle ticks are a major constraint on the livestock industry, because of the diseases transmitted by them and high cost in their control. Problems of resistance, pollution and poisoning due to the widespread use of chemical ectoparasiticides, have led to an emphasis-shift on the need and relevance of alternative tick control measures. A promising new approach is the development of host immunity through administration of tick antigens. It has had a remarkable degree of success against the one-host tick Boophilus microplus. A preponderance of studies has, therefore, emanated from Australia and USA where this tick is the major pest and vector. In the multi-host ticks on the other hand, the scenario so far is not too encouraging. Control of Hyalomma ticks, the vectors of tropical theileriosis, is an economic necessity in India vis-a-vis the cross-breeding programme to upgrade the productivity of its cattle. Thanks to the growth in the fields of immunology and molecular biology and consequent availability of new tools for isolation and fractionation of parasite’s antigens, considerable recent research has addressed itself to host immune-responses to Hyalomma ticks, principally H. anatolicum anatolicum. The objective of this review is to take stock of the research reported on this topic over the last fifteen years. The potential of these approaches to deliver practical results in the foreseeable future will be assessed. The attempt is to collect the available information in a single mass to serve as baseline material and to define the direction and strategies for future research.

Top

Keywords

Immunity, Hyalomma sp., Tick control.

Top

Introduction

Historical aspects and Epidemiology

The ability of vertebrates to acquire resistance to feeding by ticks is well documented. The first published report of bovine resistance against the tick Boophilus microplus on repeated infestation was made in Australia (Johnston and Bancroft, 1918). Apart from outright rejection of ticks with hypersensitivity reaction, there was reduction in the feeding of ticks on resistant animal per-season under natural conditions. The idea of immunological control of ticks was thereby germinated. Trager (1939) successfully immunized guinea pigs by intra-cutaneous inoculation of larval extracts of Dermacentor variabilis. Another landmark was the demonstration of acquired immunity in the bovines using tick organs as antigens (Allen and Humphreys, 1979). This novel approach for control of ticks stimulated extensive studies. Several top-of-the-line reviews provide a comprehensive picture of the progress made over the years (Willadsen, 1980; Wikel and Whelen, 1986; Brown, 1988; Wikel, 1988; De Castro and Newson, 1993; Opdebeeck, 1994; Wikel, 1996). However, most of the compilations relate to the one-host tick Boophilus microplus against which spectacular degree of success has been achieved.

Hyalomma is the predominant one of the tick genera, prevalent in much of Asia. Of the seven species of Hyalomma occurring in India, H. anatolicum anatolicum Koch, 1844 is the most common tick infesting Indian livestock and is most widely distributed (Kaiser and Hoogstraal, 1964). It is the major vector of tropical theileriosis (Theileria annulata) of cattle. Other Hyalomma species are involved to a much lesser extent largely because cattle are not their preferred hosts. Otherwise, their vector potential for transmission of T. annulata may be high (Khurana et al., 1988). A comparison of four species of Hylomma ticks viz. H. a. anatolicum, H.a. excavatum, H. detritum and H. marginatum marginatum (Sayin et al., 2003) regarding experimental infectivity for T. annulata, did not reveal any marked variation, although the female ticks in all four were found to have greater infectivity than the male ticks, as was earlier known for H. a. anatolicum (Sangwan et al., 1989). On the other hand, natural Theileria infection was found highest in H. a. anatolicum, of the four different Hylomma species identified in cattle in Turkey (Aktas et al., 2004). Intraspecies differences of T. annulata transmission by H. a. anatolicum is affected by diverse factors including level of parasitaemia in the host (Khan, 1997; Ram et al., 2004).

Cattle and laboratory animals exhibit similar immune responses to tick infestations. Resistance was expressed by (a) prolonged period of engorgement; (b) death of ticks on the host; (c) reduced number of engorged ticks; (d) reduction in engorgement weight; (e) inhibition of egg-laying and reduced number and viability of the eggs; (f) reduced hatching per cent, etc.

Host resistance: Acquired immunity/hypersensitivity

Latif (1984) in Sudan evaluated the resistance status of cattle to H. anatolicum anatolicum ticks by releasing 100 nymphs on a resistant animal. Only 7% of them engorged and moulted as against 19–45% on susceptible hosts. There was significant reduction in engorgement weights. A study from Roslin (U.K.) on sequential quantitative histological analysis of feeding sites of adult H. a. anatolicum on rabbits (Gill and Walker, 1985) showed that the cellular reactions at tick feeding sites changed in character and magnitude with time after attachment, and differed considerably between primary infestation and tertiary infestation. Mast cells, basophils and eosinophils appeared to be the major host cells involved in the resistance response, manifested by protracted feeding, reduced engorgement and poor egg-laying. In a later report (Gill, 1986), the cutaneous cellular reactions associated with acquisition of resistance of cattle to H. anatolicum were studied. Resistance to the ixodid was acquired after a single infestation, with its usual expressions (vide supra). Acquisition of resistance corresponded to pronounced epidermal vesiculation and a significant change in nature of the cellular infiltrate at tick feeding sites, indicating cutaneous hypersensitivity. The results suggested that mediators released by the degranulation of mast cells and basophils were major effectors of resistance in cattle to the tick. Subsequently (Gill and Luckins, 1987), it was found that a significant degree of this resistance could be adoptively transferred to naive recipients with immune serum from rabbits repeatedly infested with adult ticks. A direct correlation between resistance and anti-saliva IgG antibodies was indicated by a progressive increase in the degree of resistance and IgG antibody litres following successive infestations. It was concluded that both humoral and cell-mediated mechanisms (CMI) might be involved in acquired host resistance. Banerjee et al. (1992) studied histopathological changes of H. a. anatolicum feeding sites on crossbred calves (Bos taurusxBos indicus) made resistant through repeated infestation or immunization with tick salivary gland antigens. They observed formation of feeding cavities, cellular infiltration and epidermal vesicles at tick attachment sites. Sahibi et al. (1997) observed that resistance to H. marginatum could be induced by repeated infestations in cattle.

Immune responses in laboratory animals and the bovine host

Rahman (1984) studied the effect of host immunity on the life cycle of H. marginatum ruflpes following feeding on immunized rabbits and observed significant reduction in tick yield, engorged weight; number of eggs laid and egg viability. Banerjee et al. (1990) immunized crossbred calves against H. a. anatolicum with three types of salivary gland antigens and got significant response with whole and sediment antigens rather than the supernatant. A different approach (Banerjee and Manohar, 1990) relied on the use of culture of embryonic H. a. anatolicum for inducing tick resistance in rabbits through three successive subcutaneous injections 10 days apart. Increase in engorgement period of female challenge ticks as well as significant decrease in mean egg weight were recorded. Repeated infestation of crossbred calves with H. a. anatolicum ticks was also found to stimulate resistance acquired between 1st and 10th infestation (Momin et al., 1991). Even a single natural infestation with adudlt ticks on crossbred calves resulted in significant (65 % or more) rejection of the tick larvae and nymphs (Singh et al., 1991). Four antigenic preparations viz., salivary gland antigen, whole tick extract antigen (WTE Ag) and 30,000 g supernatant fraction and pellet of WTE Ag, prepared from partially fed adult females of H. a. anatolicum (Manohar and Banerjee, 1992a), were used for immunizing groups of rabbits. Of these, WTE Ag was found the most effective in altering tick performance while the pellet fraction was least effective. In another study (Manohar and Banerjee, 1992b), humoral and CMI responses were investigated in rabbits immunized with WTE Ag prepared from semi-engorged female ticks. ELISA detected antibody on day 14 and the titre thereafter gradually increased up to day 28, remaining significantly higher than the control rabbits throughout (63 days post immunization). Double diffusion test also detected antibody from 14 to 63 dpi. Lymphocyte stimulation index was observed from 21 to 42 days and leucocyte migration index in immunized rabbits was lower on 28, 35 and 42 dpi indicating enhanced CMI. Singh (1991) found no adverse effect on the moulting behaviour of H. a. anatolicum ticks fed on calves immunized with antigens extract from midgut. However, Thakur et al. (1992). reported successfull experimental immunization of rabbits with midgut antigens of H. a. anatolicum mixed with saponin. Immunity was assessed by challenging with unfed larvae and adults. Reduced feeding rate and poor reproductive performance in these ticks was indicative of level of protection in the immunized rabbits. Furthermore, extensive damage to the gut lining with resultant high death rate was observed in adult ticks fed on immunized rabbits. WTE antigen of H. a. anatolicum was also used for immunizing crossbred cattle in another study (Banerjee and Manohar, 1992) and effect on performance of female ticks challenge fed on them, was assessed. Cutaneous response to H. a. anatolicum antigens in crossbred calves was the subject of a study at Ludhiana (Kumar and Singh, 1994). Delayed hypersensitivity (18–24h) reaction of macroscopic oedematous swelling at the inoculation site was more in response to pellet fraction of whole ground tick antigen than the gut or salivary gland antigens.

Inoculations of rabbits with H. dromedarii tick derived antigens with Freund’s complete antigen (FCA) induced immunity in terms of reduced feeding and reproductive performance of the challenge ticks (Kumar, 1993; Kumar and Kumar, 1995). The gut supernatant fraction gave the best protection. Enhanced immunity to H. a. anatolicum ticks could be induced in crossbred calves by using Ascaris extract (AE) immunomodulator with the tick salivary gland extract (SGE) antigens (Singh, 1993; Sran et al., 1996). x003BInterestingly, apart from increased rejection of nymphs, incorporation of the immunomodulator AEd also resulted in a higher percentage of abnormally fed larvae and nymphs. Kumar and Kumar (1996a) demonstrated the presence of serum antibodies in rabbits after their immunization with partially fed H. dromedarii derived midgut antigens (supernatant and pellet fractions). Humoral response was measured with double diffusion and capillary tube agglutination tests. Both the tests were positive up to 84 days in response to supernatant with FCA but only up to 63 days without FCA. A comparison of three immunizing modes viz., repeated infestations, salivary extracts and intestinal extracts (IE) ofH. marginatum marginatum (Sahibi et al., 1997) indicated that IE was not effective. Kumar and Kumar (1997a) reported the adverse effect on the feeding and moulting performance of H. dromedarii developmental stages when fed on the rabbits immunized with the partly fed adult tick midgut concealed antigens. Sangwan et al. (1998) reported immunization of cattle with partially engorged nymphal H. a. anatolicum extracts. Crossbred calves immunized with whole nymphal extract (WNE) showed significantly greater rejection of challenge ticks as compared to those immunized with either nymphal membrane antigens (NMA) or nymphal soluble antigens (NSA). Ghosh et al. (1998) achieved similar results with antigens prepared from homogenized unfed larvae (HL Ag) and nymphs (HN Ag). Following immunization, rabbits developed a significant level of protective immunity to infestation with adult H. a. anatolicum. Female ticks fed on immunized rabbits were influenced in their reproductive parameters. Additionally, elevated antibody levels were recorded in the immunized animals (up to 126 days recorded with HL Ag and 112 days with HN Ag). Later, this group of workers (Ghosh et al., 1999) used larval antigens purified by immunoaffinity chromatography. Affinity purified total larval extract (Aff-TLE) immunized crossbred animals had higher rejection of larvae and nymphs, and also significantly decreased resultant nymphs and adults. Extending these findings, Ghosh and Khan (1999) reported that enhanced anti-tick immunity resulted from the incorporation of FCA with the larval antigens of H. a. anatolicum in crossbred calves. Khurana (1999) used whole tick extract (WTE) and whole tick extract fractionated antigens (WTEF) of partially fed adult females of H. a. anatolicum for immunizing groups of rabbits. The immunized rabbits developed ELISA litres (higher in WTEF group). Intradermal inoculation of antigens in the immunized animals depicted mild immediate reaction and a very pronounced delayed type of hypersensitivity reaction. Both the antigens induced significant resistance in rabbits to a challenge infestation in terms of feeding and reproductive parameters. More recently, purification of unfed larval extract antigens by immuno-affinity chromatography using anti-gut IgG as ligand, achieved by IVRI group (Das et al., 2000) resulted in high rejection rates viz., 70.6% larvae, 54.5% nymphs and 61.9% adult ticks in immunized crossbred calves. In trials with soluble nymphal antigens purified by immuno-affinity chromatography using CNBr-activated Sepharose 4B coupled with immunoglobulin ligands (Sharma et al., 2001), significant rejections of larvae (P<; 0.001, 84.2%), nymphs (P<0.05, 61.4%) and adults (P<0.05, 58.7%) were recorded in crossbred calves.

Razmi et al. (2003). immunized Holstein-Freisian male calves with the supernatant fraction extracted from the midgut of H. a. anatolicum. The calves were challenged with fifteen pairs of homologous adult ticks. A significant decrease in engorgement, engorged weight, feeding index, percentage of oviposition, egg mass and fertility index were observed in the ticks fed on the immunized calves. Immunization of crossbred calves by affinity-purified concealed antigens adjuvanted with FCA/IFA animal (Das et al., 2003) decreased 74.4% and 52.2% population of nymphs and adults, respectively, besides significant percentages of abnormally fed larvae and nymphs. Banerjee et al. (2003) reported significant response in immunization of crossbred calves with fractionated midgut supernate antigen of H. a. anatolicum. Three inocula of 1 mg antigen protein were given (with FCA i/m on day 0; with IF A s/c on day 14; and without adjuvant s/c on day 35). Midgut supernate fraction was found to be the most immunogenic and immunoprotective in rabbits immunized with midgut-derived antigens (Kumar et al., 2003).

Cross-protection: Heterologous challenge

Miranpuri (1989) studied the effect of repeated “pure” infestations of B. microplus and H. a. anatolicum on subsequent challenge with mixed infestations of both the ticks in crossbreed calves. He concluded that host responses elicited to one species did not provide cross-resistance to the second species in his study. Subsequent studies by various investigators made observations to the contrary. Cross-resistance to phylogenetically related ticks (same genera) was shown by several investigators. Manohar and Banerjee (1992c) found that rabbits immunized with whole tick antigen of H. a. anatolicum were resistant to challenge of H. marginatum isaaci, but not of Rhipicephalus haemaphysaloides. Thakur et al. (1992) reported that rabbits immunized with midgut antigen of H. a. anatolicum or B. microplus mixed with saponin evinced equal levels of protection against challenge of H. a. anatolicum adults. Immunological cross-reactivity was established between salivary gland extract antigens of H. a. anatolicum and B. microplus (Parmar et al., 1996) using native-PAGE immunoblotting, Dot-enzyme immunoassay (Dot-EIA) and in vivo intradermal skin test showing immediate type hypersensitivity swelling reaction in crossbred calves (immunized to H. a. a. or B. m. ticks and inoculated intradermally with SGE antigens of either species). Kumar and Kumar (1996b) reported cross-resistance to H. a. anatolicum in rabbits immunized with midgut antigens of H. dromedarii. In other report (Kumar and Kumar, 1997b), rabbits immunized with H. dromedarii tick derived midgut antigens showed protection (estimated as per cent cumulative rejection of ticks) to a maximum of 71 % to homologous challenge and 40.5% to heterologous challenge of H. a. anatolicum. Sangwan et al. (1998), using three types of nymphal antigens viz., WNE, NMA and NSA of H. a. anatolicum for immunizing crossbred calves found that only calves immunized with NSA showed some cross-protection against H. dromedarii larvae. Ghosh and Khan (1998) reported a strong inter-generic reactivity between Boophilus, Hylomma and Rhipicephalus ticks by Ouchterlony double immunodiffusion (DID) and ELISA tests. Khurana (1999) reported that groups of rabbits immunized with WTEF and WTE H. a. anatolicum antigens gave protection against homologous as well as heterologous (H. dromedarii) challenges. The BM 86 antigen, as originally identified in B. microplus, is the basis of commercial tick vaccine against this tick species. The potential for using this antigen or its homologues in vaccination against other tick species has been assessed (Vos et al., 2001) in vaccine trials in calves. The result not only revealed high degree of protection viz, reduction in reproductive capacity of homologous challenge (74% for B. microplus and 70% for B. decolorants) but also against H. a. anatolicum (50%) and interestingly, highest (95%) against H. dromedarii. This was highly significant in terms of strategy for development of multi-valent anti-tick vaccines. Banerjee et al. (2003) also found that a fractionated mid-gut supernate antigen from H. a. anatolicum induced a considerable degree of cross-protection in calves against H. dromedarii larval ticks. Serological cross-reactivity of mid-gut antigens of several ticks was established by DID test recently (Latha et al., 2003).

Characterization of tick antigens

Development of an effective vaccine against Hyalomma ticks hinges on the purification and characterization of protective antigens isolated from the tissues of the adult and developmental instars of the ticks. Accordingly, this is one of the current priority areas of research. Early on, Gill (1984) had documented 9 and 17 antigenic glycoproteins, respectively in saliva and salivary gland extract from 96 h-fed females of H. a. anatolicum, to sera from hypersensitized rabbits. Intradermal inoculations of certain antigens, saliva or salivary gland extract into tick-exposed rabbits, elicited both immediate and delayed hypersensitivity reactions. The salivary antigens had molecular weights in the range of 1,44,000 to 1,30,000 (Gill et al., 1986). Antigen 1 (MW, 1,30,000) showed acid phosphatase activity and antigen III (MW, 96,000) both non-specific esterase and aminopeptidase activity.

Parmar and Grewal (1996) used native PAGE and immunoblotting assay to characterize the immunogenic protein of salivary gland extract (SGE) and the saliva of H. a. anatolicum. Native PAGE of the SGE revealed 10 proteins of mol. wts. 56, 60, 64, 66, 120, 148, 220, 264, 300 and > 300 kDa. In the immunoblot assay of the tick SGE, mice hyperimmune serum reacted with only the 66 kDa protein. In contrast to the SGE, the tick saliva revealed only two proteins of 264 and 66 kDA. The 66 kDa protein was also observed in the tick larval extract. In another report, Parmar et al. (1996)., it was recorded that only four of these viz., molecular weights 56, 64, 120 and 220 kDa were exclusive to H. a. anatolicum, while the other six proteins were common to H. a. anatolicum and B. microplus. Characterization of immunogenic fractions of salivary gland extract of H. a. anatolicum (Kumar, 1998) by SDS-PAGE and western blotting revealed seven immunogenic fractions. Ghosh et al. (1998) found that antisera to homogenized unfed larval antigens and homogenized common antigen bands of 97.4, 85, 66, 47.3, 42 and 31 kDa in the homogenates of larvae, nymphs and adults. Khurana (1999) observed 45 protein bands with molecular range (Mr) of 11 to 219 kDa on SDS-PAGE of three antigens prepared from WTE of partially fed adult females of H. a. anatolicum. Of these, 34 bands with Mr of 11 to 210 kDa and 19 bands with Mr of 11 to 169 kDa were present in the supernatant and pellet fractions, respectively.

On SDS-PAGE analysis of affinity purified antigens of total larval extract of H. a. anatolicum, Ghosh et al. (1999) identified a 39 kDa protein reported for the first time as the antigen responsible for the induction of resistance in the host. Later, the same (39 kDa) protein was also isolated from soluble nymphal antigens purified by immunoaffinity chromatography (Sharma et al., 2000) and found immunogenic. This led the investigators to conclude that the same antigens were responsible for the induction of resistance against all the stages of the tick. However, unfed larval extract of H. a. anatolicum purified by a slightly modified process, had revealed on SDS-PAGE analysis, three antigenic proteins of 100, 59.4 and 37 kDa (Das et al., 2000). High rejection rates achieved by immunization of crossbred calves with this antigen have already been mentioned in the foregoing. Kumar et al. (2002). reported that SDS-PAGE of gut supernatant antigen derived from partly fed H. a. anatolicum adult females revealed 26 discrete polypeptide bands with Mr from 25 to 208 kDa. Of these, seven bands were of major polypeptides with molecular weights between 25.2 and 185.8 kDa. On immunoblotting with antisera raised in rabbits against gut supernatant (crude extract) eight immunogenic polypeptides with mol. wts. between 51.7 and 185.8 kDa were identified. Das et al. (2003) isolated concealed antigens from the adult of H. a. anatolicum. These bound antigens on characterization by SDS-PAGE revealed two proteins of 106.7 and 68 kDa. Western blot analysis detected the latter as the immunogenic protein band responsible for the reported resistance (high per cent rejections) in the host.

Effect of host immunity on vectorial capacity of ticks

Rubaire-Akiki (1990) made the observation that high level of bovine tick resistance rendered the H. a. anatolicum ticks feeding on them, significantly less susceptible to infection by T. annulata. Such compromising of the vectorial capacity of H. a. anatolicum ticks may be of epidemiological significance in areas endemic for tropical theileriosis. Contrary to this, Ramachandra and Wikel (1992) reported that ixodid tick infestation reduced cytokine levels and produced immunomodulation of the host that may facilitate pathogen transmission. Banerjee and Manohar (1992) detected schizonts of T. annulata in lymph node, spleen, liver as well as piroplasms in peripheral blood smears of immunized and unimmunized cattle indicating no effect on transmission of Theileria. Rahman et al. (1993) reported that calves experimentally infested with H. a. anatolicum and vaccinated with tissue culture rinderpest virus (TCRPV) responded normally to vaccination. The level of neutralizing antibodies to TCRPV was the same in calves with or without the Hyalomma infestation. Furthermore, infection with Babesia, Theileria or Anaplasma did not alter the vaccine response to the virus. Sran et al. (1996). had postulated that the high percentage of abnormally fed nymphal H. a. anatolicum in their immunity study could differ in their disease transmission potential. However, Sangwan et al. (1998) observed that nymphs, which engorged on the immunized calves in their experiment, were fully susceptible to infection by T. annulata as indicated by the intensity and abundance of infected acini in the resulting adults ticks comparable to those from unimmunised infected cattle.

Conclusion and Future prospects

Risk-benefit considerations have stimulated efforts to develop technology to find alternatives to the excessive use of acaricides for the control of vector ticks of bovine theileriosis. In spite of impressive volume of work reported and definite strides in understanding the modes of expression, mechanism of acquired resistance and characterization of antigens, we are far from the prospects of a workable vaccine against multi-host ticks. Another noteworthy aspect is that in multi-host tick infestation, detection of cross-reactive antigen between different tick species may facilitate development of a single vaccine against all ruminant ticks (Latha et al., 2004). The potential of Bm 86 antigen of B. microplus origin for protection against intergeneric challenge (Vos et al., 2001) also merits further exploitation. This requires further exploitation of molecular cloning techniques for isolating the most immunogenic fractions of tick antigens. There is also a growing school of thought that the selection and use of breeds of cattle with a natural propensity for developing acquired resistance to ticks is possibly the best option till date (George, 1992). High host resistance is the key to effective long term tick control and can make acaricides and vaccination more effective (Frisch, 1999). Resistance is as heritable as milk yield or growth and in tropical breeds, can be increased to very high levels by selection (Frisch et al., 2000). In this perspective, perhaps there is need for our policy planners to reorient our cattle breeding programme.

Top