IMMUNOGENICITY OF SALIVARY GLAND AND MIDGUT HOMOGENATE ANTIGENS AGAINST CAMEL TICK HYALOMMA DROMEDARII (IXODOIDEA: IXODIDAE) AND RELATED BIOLOGICAL IMPACTS AND CONTROL POTENTIAL : IN VIVO STUDY

Walaa Ahmed Moselhy 1 , Aly Fahmy El-Sayed 2 , Fatma El-Sayed Ali, Eman Ahmed El-Kelesh, Wesam Soliman Ebrahim 1 and Abd El-Baset Badr El-Din Zayed 1 . 1. Zoology DepartmentFaculty of Science Al-Azhar University (Girls branch) 2. VACSERAEgypt 3. Parasitology Department, Animal Health Research Institute, Dokki, Giza, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Preparation of salivary gland and midgut antigens:-
Salivary gland and midgut tissues from approximately 200 semi-fed H. dromedarii females were prepared. Living semi-fed females were washed with 0.01 M Phosphate-buffered saline (PBS, pH 7.2) and embedded in a Petri-dish containing a mixture of paraffin wax and charcoal. The dorsal integument of the tick was removed and the internal organs were immersed with PBS. Salivary glands and midgut were removed, placed in cold PBS (4⁰C) and stored at -20⁰C. They were subsequently thawed and sonicated by an ultra-sonicator in ice bath at 55,000 cycle/sec five times, one minute each, followed by one minute as cooling interval. The extracts were centrifuged at 4⁰C for 1 h at 14000 rpm, using high speed cooling centrifuge, according to Heller-Haupt et al. (1996). The resulting supernatant of each tissue was separated and used as antigenic material. The protein content of SgAg and MgAg, was measured using the micro total protein (MT-P) Pyrogallol-Red kit (Egyptian company for Biotechnology (S.A.E).
Immunization:-Rabbits were artificially immunized against H. dromedarii tick infestation according to the protocol of artificial immunization (Sran et al., 1996). Rabbits were divided into 4 groups,( 3/ each). Group I was kept as negative control, group II inoculated with aluminium hydroxide adjuvant (Alum) while groups III and IV inoculated with MgAg and SgAg, respectively. Rabbits of groups III and IV received three subcutaneous inoculations of 100 µg/kg protein (Abdel-Shafy et al., 2008) emulsified with Alum in equal volumes. The first immunization dose was administered on day 0 post emulsification with Alum. The second and third doses were given on days 14 and 28 respectively same as the first dose. On the other hand, group II received three inoculations of 1ml PBS with an equal volume of Alum at the same days as groups III and IV. On day 36 post-immunization, rabbits were infested with adult unfed H. dromedarii ticks. The infestation was carried out using 10 males and 10 females per rabbit. Daily observation was performed to detect some biological parameters of females.

Biological parameters:-
The biological performance of females was examined by determination of their feeding percent and period, oviposition, hatchability and fertility. Feeding % was determined by dividing the number of fed females / total number of females x 100. Feeding period is the time taken from starting feeding to engorgement. The amount of blood ingested by females was determined by the weight prior to feeding subtracted from the weight directly post feeding. Oviposition was represented by oviposition percent (number of ovipositing females / total number of 940 females x100), pre-oviposition period (days from engorgement to onset of egg laying), and the number of eggs for the oviposited female. Hatchability was represented by the pre-hatching period (days from onset of egg laying to onset of hatching) and hatching percent (number of hatched eggs / total number of eggs X100). Fertility is the weight of egg mass divided by the weight of replete female (Khalil et al., 1984) in addition to the weight of one egg (Weight of egg mass/Total number of eggs). The efficiency of the tested vaccines was assessed by determining the number of engorged females (%DT), the egg laying capacity (%DO) and the efficacy (%E) (Galaï et al., 2012) where %DT = 100 [1 − ] (NTV is the mean number of engorged females collected from vaccinated groups and NTC is the mean number of engorged females collected from the control group), %DO = 100 [1 − ] (PATV is the average weight of eggs per replete female of the vaccinated groups and PATC is the average weight of eggs per replete female of the control group) and %E = 100[1 − ] [CRT is the reduction in the mean number of females in vaccinated groups compared to the control group (NTV/NTC) and CRO is the reduction in the egg laying capacity of vaccinated females compared to the control group (PATV/PATC)].
Immune response:-Blood samples were collected weekly from the ear vein of rabbits from all groups till the 7 th week postimmunization. Sera were separated, aliquoted and stored at -20˚C till use. The antibody levels against SgAg and MgAg were measured using indirect ELISA technique according to Voller et al. (1976) with some modifications. Initially, a checkerboard titration was used to optimize the reaction conditions regarding the sensitizing antigen concentration, antibody and conjugate dilutions. Post optimization, the tested antigens were applied in a concentration of 20 µg/ml coating buffer (0.1 M carbonate-bicarbonate buffer, pH 9.6), and 4% bovine serum albumin (BSA) in PBS buffer was used to block nonreactive sites on the microtiter plates. Serum samples were dispensed in triplicates and the starting dilution of sera samples was 1:100 prepared in 1% BSA/PBS, while anti rabbit conjugate was used as 1/1000 in 1% BSA/PBS. Ortho-phenylene diamine (OPD) was used as a substrate and allowed to react in the dark at room temperature for 15-20 minutes. The reaction was stopped with 1N H 2 SO 4 and optical density (OD) was measured at 450 nm by ELISA reader (BioRad, USA).

Polyacrylamide Gel Electrophoresis (SDS-PAGE):-
Electrophoretic analysis of SgAg and MgAg was performed using the Mini-Protein II Dual-Slab Cell (BioRad, USA) and 10% SDS-PAGE under reducing conditions according to the method described by Laemmli, 1970. Protein bands were visualized by Commassie blue stain.

Western blot analysis:-
The profile of reactive bands of the tested antigens was recognized by hyperimmune sera collected from rabbits on day 36-post primary immunization. The electrophoresed antigens were transferred from gels to nitrocellulose membranes for immunoblotting using a Bio-Rad Semi-dry transfer cell according to the manufactures protocol. Membranes were cut into 0.5 cm stripes, blocked with 5% skim milk/PBS-Tween and probed with rabbit anti-sera 1:100 diluted for 1 1 2 h over a shaker at room temperature. After four wash cycles with hot PBS-Tween (65˚C), membranes were incubated with anti-rabbit IgG peroxidase conjugate at a dilution of 1:500 in 5% skim milk/PBS-Tween. After three wash cycles with hot PBS-Tween (65˚C) and two times with PBS alone, chromogen DAB/PBS-30% H 2 O 2 was added to the nitrocellulose stripes for 10-15 minutes allowing the colorimetric reaction to develop. The reaction was stopped using distilled H 2 O and visualized by the naked eye.
Statistical analysis:-Data were statistically analyzed by SAS (2004) using general linear model procedure (GLM) classification, followed by Duncan Multiple Range Test to examine the significance between means.

Results:-
Immunization of rabbits using MgAg and SgAg affected markedly the ability of females to attach and feed. It resulted in a substantial reduction (P > 0.01) in the feeding percentage to 83.33% and 70%, respectively, versus 100% and 95% of negative control and Alum-inoculated groups [ Table 1]. On the other hand, the time required for full engorgement was significantly reduced (P > 0.05) post immunization. The shortest feeding period was observed in group IV being 6.21 days compared with the other groups recording 6.94, 6.73 and 6.44 days for groups I, II and III, respectively. The amount of blood ingested by females fed on immunized rabbits exhibited a significant decrease (P > 0.01) compared to those fed on unvaccinated one. Females of groups III and IV ingested 633.50 and 572.00 941 mg, respectively, whereas those of the control ones (I and II) consumed 868.63 and 774.33 mg respectively [ Table  1]. Fully engorged females were collected, weighed and put individually in glass vials, then placed in an incubator and checked daily for egg laying. The ratio of ovipositing females was 100% in groups I and II, which decreased significantly (P > 0.01) to 76.6% and 60% in groups III and IV, respectively. No significant difference was observed in the preoviposition period among the different groups. Salivary gland antigen-immunized group only exhibited a significant prolongation (P > 0.05) in their prehatching period being 23.5 days compared with the negative control group which recorded 22.17 days. On the other hand, females fed on rabbits immunized with SgAg have the most prolonged oviposition time being 24.62 days which decreased to 23.55, 22.87 and 22.33 days in MgAg-immunized, Alum-inoculated and negative control groups, respectively. Meanwhile, the number of eggs laid by females and the hatching percent decreased significantly (P > 0.01) as a result of immunization by both antigens [ Table 2]. The mean weights of replete females, egg masses laid and fertility were presented in table (3). Data revealed that all these parameters decreased significantly as a result of immunization of rabbits with the tested antigens. The mean weight of replete females of group III (754.55 mg) was statistically similar (P > 0.05) to those of group II (782.67 mg). On the other hand, it differed significantly (P > 0.01) when compared to the negative control which recorded 877.50 mg. The lowest mean weight (651.25 mg) of replete females was observed among group IV compared to all other groups. Egg mass laid by females of group I presented the maximum weight being 623.33 mg which significantly decreased to 533.33, 457.27 and 343.75 mg in groups II, III and IV, respectively. Fertility (weight of egg mass/weight of replete female) was reduced significantly (P > 0.01) from 0.71 and 0.68 in groups I and II, respectively, which are insignificantly differ; to 0.59 and 0.52 in groups III and IV, respectively. On the other hand, the mean weight of one egg showed a diverse trend since it is increased significantally (P > 0.01) from 0.06649 and 0.06670 mg in control groups I and II respectively, to 0.06721 mg in SgAg-immunized group. Whereas, MgAgimmunized group which is statistically similar to the Alum-inoculated and SgAg-immunized group, exhibited a significant increase when compared with the negative control since it was 0.06686 mg [ Table 3].   Fig. 1].  at high levels throughout the experiment. No significant differences between the antibody levels of the two antigens except on the days 21 and 28 (P > 0.01). Also, no specific IgG antibodies were detected in the pre-immunization sera or sera from the negative control group.      al. (1996), it was reported that salivary gland antigens are involved in the acquisition of resistance to H. anatolicum as an important vector of bovine tropical theileriosis. The gut antigen also induced the best protection in terms of reduced feeding and reproductive performance of ticks (Kumar & Kumar,  1995). The significantly decreased percentage of fed females observed in the present study may be attributed to the ability of ticks to manipulate the hemostatic effects of the mammalian hosts. Attenuation of fibrinolytic or antihemostatic agents of tick saliva causes disruption of blood flow and blocks successful feeding of the blood meal of ticks (Maritz-Olivier et al., 2007). Also, the moderate reduction in the feeding percentage of females fed on SgAgimmunized rabbits was recorded, which was in agreement with the results obtained by Kumar and Kumar (1995) and found that immunization of cattle with salivary gland extract against H. marginatum reduced the percentage of attachment by 47%, while intestinal extractgenerated immunity had no effect (0%) on the attachment percentage. In contrary, Szabó and Bechara (1997) reported that the lowest tick recovery was obtained from guinea pigs immunized with gut extract and from dogs immunized with the gut extract emulsified in Freund's adjuvant instead of saponin adjuvant. Moreover, the attachment percent was found to be similar among rabbits immunized with both salivary gland and midgut antigens against Boophilus microplus, with no significant difference compared to control group (86.67%, 83.34% and 85%, respectively) (Asif et al., 2011). The variable reduction percentages may be attributed to the tick species, host variation and even variable tolerance among individuals of the same species. It was found that, rejection of large ticks represents a lower percentage than that of small ones as observed by Retarded feeding or complete rejection of ticks from immunized hosts was attributed to the influence of anti-saliva response not to the response against gut components that have been regurgitated during blood meal (Connat, 1991). In the present study, the number of females that have the ability to oviposit was greatly reduced by 40% and 23% for SgAg-and MgAg-immunized groups, respectively, and those that did, took a considerably longer time to complete oviposition with no significant change in the pre-oviposition period. In ticks, the actual volume or weight of condensed blood meal exerts its effects on reproductive development (Diehl et al.,  1982). However, at suboptimal levels, oogenesis may not be initiated or, if it begins, will not be sustained. A further general reduction in the number of eggs produced may be due to prolongation of the period between feeding and oviposition. In the present study, ticks fed on SgAg-and MgAg-vaccinated rabbits and succeeded in laying eggs, produced few number that took longer time before the hatched larvae emerged when compared to control groups.

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The prehatching period of MgAg-immunized group was similar to that of the Alum-inoculated one but slightly longer than that of the negative control group. Meanwhile, the egg laying capacity, hatchability and fertility of both vaccinated groups were significantly reduced. microplus. It seems clear that, the reduction in the production indices may be attributed simply to impairment in meal processing or to the host immune response which affects directly the physiology of ticks in some permanent way that persists even after the parasite detachment. Ben-Yakir et al. (1986) and Wang and Nuttall (1994) cleared that midgut is permeable to host immunoglobulins, and that these antibodies enter the tick hemolymph and can bind to antigens associated with other internal organs of the tick. These host immune effectors present in the blood meal not only destroying the candidate cells as the damage caused to the gut cells and leakage of gut contents into the haemocoel in response to immunization with gut antigens against B. microplus (Agbede and Kemp, 1986), but also could bind to antigens associated with the vital organs such as the reproductive organs and could have detrimental effects on the oogenesis and egg hatchability of ticks. This can be explained on the basis of the presence of common antigens in different organs of the ticks (Wang and Nuttal, 1999).
The drastic effects of immunizing hosts with SgAg-and MgAg on the feeding and reproductive performance of the tick H. dromedarii observed in the present study may based on the concept that ticks feeding on appropriately immunized hosts might ingest antibodies specific for target antigens within the tick. This suggests that these antibodies may apply their effect through certain routes, possibly the nervous system and specifically via the neuroendocrine or endocrine mechanism essential in the regulation of feeding and consequently oocyte differentiation and/or vitellogenesis (Pound & Oliver, 1979). In the present study, a strong antibody response was rapidly detected in vaccinated rabbits. The immunogenic capacity of these antigens and their characterized specific proteins has been extensively documented through a high number of immunization trials ( . This protective efficacy was observed as a physical damage to ticks (Imamura et al., 2006) and as a reduction of the biotic potential (Barriga, 1999) which would likely result in progressive reduction of tick populations, while in turn can enhance reduction of acaricides use. Also, MgAg-immunized group had a slightly higher antibody titer than SgAg-immunized one. In the present study, western blotting analysis revealed that SgAg and MgAg could induced polyclonal antibodies against the two Ags polypeptides, and MgAg was more immunogenic. This may be attributed to the gut proteins content that enhance random stimulation of immune system than that of the SgAg, which was supported by the reactivity to 10 and 7 bands of proteins, respectively. The protective efficacy, impairment of feeding and reproduction were influenced by vaccination. This may be attributed to the different concentrations, antigens content and feeding duration. Also, the possible reasons could be due to some proteases are abundant in the various tissues of the tick and that they exist as multiple isoenzymes, therefore a high concentration of antibodies directed against them could be necessary to induce a significant injury to the tick (Imamura et al., 2009). Additionally, some saliva proteolytic enzyme inhibitors such as Iris was found to be upregulated during the blood meal and their concentration increased at the end of feeding (Leboulle et al., 2002) when tick ingest the great amount of blood (Sonenshine, 1991), suggesting its particular usefulness in this late feeding step as it could be ingested with the blood meal to the midgut and could facilitate both blood intake and protection of the midgut walls (Prevot et al., 2007). Kemp et al. (1989) suggested that B. microplus can, to a significant extent, repair immunologically induced damage to its gut. Perhaps the balance between the damage and repair will determine the outcome of vaccination, which may differ from species to species. The degree of damage and oviposition capacity may depends also on the amount of blood ingested, and hence the amount of antibodies developed post vaccination. Finally it can be concluded that experimentally immunization of rabbits with salivary gland extract of H. dromedarii homogenates effectively impaired tick feeding and reproduction much better than vaccination with midgut homogenate suggesting further investigations to characterize the protective antigens involved in the protection process. 946