29-kDa: A Potential Candidate for Anti-Tick Vaccine Antigen Source as Immunogenic and Stage Reactive Targeting Hard-Bodied Hyalomma Ticks (Ixodidae)

Pakistan economy is mainly focused in agriculture. Currently its gross domestic product (GDP) growth rate correspond to 4.71%, with an approximate 11.39% livestock contributions as per reports of Ministry of Finance, Pakistan (2016-2017). According to livestock census for 1999 and 2006; horses occupies third place in abundance, 18%, in second place are camels (41%) and sheep (48%) are the most abundant. Horse population in Pakistan have increased by 3.1% over a period of eight years from 0.33 million to 0.34 million and their role is continuously shifting from transport to sports and in recreational activities (Rehman et al., 2017) both in the rural as well in urban areas (Javed et al., 2014).
 
Pakistan lies in the subtropical zone of South Asia, where the hard-bodied ticks genus Hyalomma (Ixodida: Ixodidae) is widely distributed in its different ecological zones. Ticks are non-permanent and obligate hematophagous arthropod. Elevated temperature and the scarcity of water contribute to the rapid growth of tick species, particularly in developing countries including Pakistan. Several Hyalomma ticks are capable of transmitting grievous pathogens such as viral disease and rickettsias (Kernif et al., 2012; England et al., 2016) and have drastic effects both on animal and human health due to their high infestation rate (Asmaa et al., 2014). Special precautionary measures are needed in collection of Hyalomma species because they are responsible for spreading of Crimean-Congo hemorrhagic fever virus in animals and humans (Gargili et al., 2017). Hard-bodied ticks infesting equines in Pakistan has been reported previously. The only tick reported belong to the genus Hyalomma include Hyalomma anatolicum (Kaiser and Hoogstraal 1962; Javed, 2013) and Rhipicephalus turanicus (Ali et al., 2019). Very few studies have been conducted on these species, which were also confined to restricted sites. These studies did not focus on risk factors associated with tick’s infestation (Bisen et al., 2011; Jabbar et al., 2015). 
       
The use of acaricides in different geographical regions is on decline because of its short shelf life, high cost and repeated applications. As ticks have also developed resistance against acaricides, particularly in Hyalomma genus. This has resulted in a decline in the purchase of current acaricides among farmers (Olmeda et al., 2008; Pradeep et al., 2012; Singh et al., 2015). Therefore, there is a need to develop new effective vaccine dealing with ticks. The use of an anti-tick “vaccine” is more practical approach to overcome on the issue of tick resistance.
       
Ticks have salivary glands in their mouth, which are the source of transmission of tick-borne diseases to certain viable livestock (Rizzoli et al., 2014). Salivary glands secrete a proteinaceous matrix called cement cone protein, which helps in blood feeding and immunomodulation of the host responses to the ectoparasite infestation (Sangwan and Sangwan 2016). This unique class of protein could bind with vertebrate immunoglobulins and is crucial for survival of parasite (Steen et al., 2006). In the present work the relationship between the incidences of multi-host tick (i.e. Hyalomma anatolicum anatolicum) and its associated risk factors is explored. Moreover antigen cement cone was inoculated as immunogenic protein emulsified with Montanide (ISA-50) adjuvant, based on this procedure the intensity of humoral responses in terms of hematology and biochemical parameters was quantified. These results are discussed in terms of the zoosanitary pattern of Hyalomma hard-bodied tick infestation and the potential use of tick resistance inducers as vaccines.

Study and sampling
 
To investigate the population density of Hyalomma ticks, a cross-sectional study was designed based on the random three districts namely Quetta, Qilla Abdullah and Nushki (Fig 1). Ticks were identified using taxonomic descriptions (Walker et al., 2014).
 

 
In vitro rearing and tick testing
 
A standard artificial feeding device for ixodid ticks was used in a modified version previously developed by other researchers (Kröber and Guerin 2007). Ten weeks after immunization, each horse was experimentally challenged with total 500 laboratory reared female Hyalomma ticks which were exposed on the flank and ear regions within locally made neoprene chambers. Standard formula was used to calculate the immunization efficacy (Andreotti 2006a).
 
Antigen preparation and purification
 
Dorsal integument of adult ticks were removed and extracted protein was rinsed and triturated in 0.01 M phosphate-buffered saline solution to avoid desiccation of salivary glands. The mixture vortexes and suspended in 150 μL cold PBS mixture with 1% protease inhibitor. Extracted material was homogenized and sonicated for tissue disruption for 40 s three times on ice bath 40 W. The mixture was then centrifuged at 15000 x g for 20 minutes at 4°C in order to collect the supernatant and decanted the pellet. Centrifuged material was filtered through 0.22 μm non-pyrogenic filter membrane and stored at -20°C.
 
SDS and western blotting
 
Molecular weight of crude extract protein as well as fractions were determined by discontinuous SDS-PAGE (BDH, Poole. England) as per standard procedure (Lämmli 1970). Total protein concentration for extracted acarine saliva was also estimated (Brad, 1976). Electroblotted protein bands obtained from SDS-PAGE were transferred to a nitrocellulose paper using a mini blotter (Bio-Rad). Band intensities were analyzed in ChemiDoc Gel Imaging System (Bio-Rad).
 
Sensitivity and specificity of antigen
 
Specific diagnostic Indirect-Hemagglutination-Assay (IHA) was also performed taking horse red blood cells with 1 mL sample in EDTA tubes, sensitized with 300 μg/ 2 mL antigen (Rehman et al., 1994). Red cell agglutination at ≥ 1:25 was considered positive for the presence of antibody (Harris et al., 2009). White Rabbits weighing 3-4 kg were selected for positive and negative testing groups for the developed vaccine for its possible immunogenic reaction in horses (Kesdangsakonwut et al., 2014).
 
Immunogen preparation
 
Crude protein was emulsified in equal amounts (1:1 ratio) with 1 mL Montanide (ISA-50) and mixed thoroughly on homogenizer before giving it to treatment groups. Three study groups were made comprising of two animals in each group. Group I (IA, IB) received percutaneous, group II (IIA, IIB) intradermal and group III (IIIA, IIIB) for intraperitoneal adjuvants vaccine respectively. Second dose of 500 μg/mL per animal was given after five weeks as described previously (Iqbal et al., 2016). Preparation was made for the second control group by taking 1 mL of PBS (pH 7.4) emulsified in 1 mL of Montanide (ISA-50) adjuvant and administered through intraperitoneal injection.
 
Dynamics of humoral response through ELISA
 
Salivary extract (2 μg/mL) antigen was diluted in carbonate coating buffer (0.1M, pH 9.2). About 100 μL (serially diluted 1:2000) rabbit anti-goat IgG (F9012 Merck, USA) conjugated polyclonal secondary antibodies was added per well and incubated at room temperature for 1 hour. Later on, 100 mL of 3,3', 5,5' -Tetramethylbenzidine peroxidase substrate (T0440, Merck, USA) including horseradish peroxidase (Sigma, H1009) and hydrogen peroxide (0.3%) was dispensed and added on titre plate. After sufficient colour development, the absorbance (450 nm) was monitored using microplate reader (Bio-Rad 680, 168-1000) within 30 minutes at 1.0 Optimal Density (OD) with standard error.
 
Hematological and biochemical parameters
 
We ensured to follow the standard operating procedures for current experiment (Bimerew et al., 2018). Hematological analysis was carried out using automatic hematology analyzer (XS-500i-Sysmex Europe GmBH) and were also compared with standard hematological parameters described in Schalm’s Equine Hematology (Walton 2013). Biochemical levels were determined using OLYMPUS AU 680 analyzer (Beckman Coulter, Japan) and were compared with earlier reported values (Aros et al., 2017; Cywiñska et al., 2015).
 
Statistical analysis
 
ArcGIS software was used to map tick collection sites. Data were analyzed in three ways: (i) Data related to the vaccine efficacy were analyzed by applying Student t-test (p (one tail) <0.05, considered significant), using PAST 3.2 (Øyvind Hammer, Natural History Museum, University of Oslo) (ii) Data on possible risk factors of hard-bodied tick burden and associated aspects in farmland were assessed by applying Mantel-Haenszel analysis based on odds ratio (OR) at 95% confidence intervals using WinEpi-info^® 7.0 (CSELS) (iii) Mean values of hematological and biochemical parameters were evaluated on ANOVA using SPSS® 20.0 (Inc., Chicago, Illinois, USA).

Purification of antigen
 
A broad protein profile shows seven immunopositively bands of molecular weight (Fig 2) with few impurities. Among these immunogenic fractions, only 29-kDa remained consistent throughout feeding stages showing common stage reactivity. Crude cement cone protein was selected due to its easy handling, cost effectiveness and the fact that its purification allow it to be used as source of vaccine. It also helps to run anti-tick vaccination campaigns in underdeveloped countries (Iqbal et al., 2016). It is difficult to draw any conclusion for this entire work due to the distribution of ticks over the different host species, differences in extraction preparation salivary protein and variation in effectiveness of the developed vaccine. Therefore, it is justified to say that currently developed vaccine can specifically work on horse ticks.
 

 
Vaccine efficacy
 
An overall 99.98% vaccine efficacy was achieved after 15 days of Hyalomma tick test. The current findings show that engorged ticks were detached after 13 days from post tick infestation in all vaccinated groups. All these observations show a direct effect on the fertility index and subsequently an increase in the overall vaccine efficacy (Table 1). A significant increase in the tick rejection was indicated by our entomological data. Longer feeding period, oviposition and reduced reproductive performance were observed in those ticks that were recovered from immunized host. It was observed that the results were independent of the weights of the vaccinated horses. This suggests that there are no obvious negative effects of vaccination on the target host.
 

       
Our sample of animals in this study was small owing to cost constraints. It was not possible to use a large sample in the absence of any financial support. The experiments involving vaccine were initially conducted on rabbits to minimize the cost of animals under test i.e. horse. The rabbits used in preliminary trials did not show any clinical symptoms. During the study no animal died as a result of ticks and tick-borne diseases. The results of current research were in agreements with results of other researchers (Andreotti et al., 2013b; Miller et al., 2012).
 
Clinical aspect of experimental animals
 
Six horses were used and none of them showed any clinical symptoms throughout the experiments except that a slight increase in temperature in one horse inoculated through intraperitoneal route. This increase in temperature lasted only for a maximum of 4 days on second vaccination dose due to antigenic response. It is evident from the results of Table 2 that total leucocyte counts was higher in all experimental groups than in control group. No specific change in any other hematological parameters was observed except neutrophil. All biochemical parameters remained fairly in the normal range as reported by other researchers (Cywiñska et al., 2015; Aros et al., 2017).
 

 
Indirect ELISA based humoral response
 
Antibody titre experiments showed predominantly higher antibody production in those animals that were inoculated intradermally. Intradermal vaccination has potential to induce more antibodies titre and is significantly more effective in elderly animals, which respond less to standard vaccination routes (Arnou et al., 2009). Elicited antibody titre results showed an increase in antibody level after one week of immunization and maximum peak was achieved after seven weeks of testing (Fig 3). Raised antibody titre confidently reflects the failure of the attachment of ticks on horse skin during this course of research.
 

 
Potential risk factor assessment
 
Table 3 shows the results of Mantel-Haenszel test applied on given parameters. Prevalence of Hyalomma tick burden was found higher on those horses which were reared in muddy farmland (OR = 4.07, Cl = 0.59-27.87). It has been observed that animal travel history is another important factor in the possible spread of ticks (OR = 2.80, Cl = 0.63-12.27) and has not been reported earlier from this region. In our analysis, high prevalence of ticks was strongly associated with the animals that were moving across the border. During handling of these animals, it is essential to adopt personal protective measures (Mead et al., 2018). It may be stressed that human behavior is directly linked with prevalence of ticks in domestic animals.