1. Introduction

Ticks are obligate hematophagous ectoparasites which rely on host blood to derive nutrition. Ticks are major vectors of a number of pathogens i.e. viruses, rickettsiae, spirochetes, bacteria, fungi, protozoa and filarial nematodes in humans, livestock and wild animals [1] . These affect the health and survival of both domestic and wild animals worldwide [1] . Ticks obtain nutrients from blood meals to fulfill their metabolic requirements [1] . During feeding ticks discharge salivary secretion or cocktail of diverse molecules in the blood of the host. Tick saliva especially secretes anti-coagulatory molecules mainly salivary proteins which inhibit blood clotting and assist in uninterrupting blood feeding by rupturing host skin [2] . Through blood feeding these transfer or inject pathogens into the bloodstream and easily transmit various disease pathogens [3] [4] . Transmission of pathogens results in various diseases and morbidities causing more serious economic losses to humans and livestock [5] . The main factors of tick parasitism are blood-sucking and saliva secretion, host immune status, age, breed and local ecology [6] .

The tick’s fat body plays an essential role in energy storage and utilization. Salivary protein insists ticks have blood meal and the whole process depends upon the injection of tick saliva and its mixing in the blood [7] . Ticks saliva contains so many proteins which display strong pharmacological and immunological activities. These proteins are mainly used in host invasion for blood feeding and are known as evasions. Salivary proteins assist ticks in feeding for more than 8 – 10 days without being noticed by the host animal. These block secretion of the host’s chemokines and prevent painful inflammation. Ticks also generate iron-bound proteins i.e. ferritins during blood feeding [8] [9] . Lipocalins are abundant proteins in the saliva of both soft and hard ticks. Lipocalin isolated from Ixodes ricinus (LIR) was associated with the modulation of inflammation [10] . Tick saliva proteins are involved in several physiological roles, including egg development, transportation of proteins, immunity and anti-microorganism, anti-coagulant, and adhesion. Thus, tick saliva not only controls host hemostasis and wound healing but also subverts the host immune response to avoid tick rejection that creates a favorable niche for the survival and propagation of diverse tick-borne pathogens [1] .

Tick saliva toxins cause significant alterations in various biomolecules after in vivo injection in albino mice. These significantly target various metabolic enzymes and severely affect the host’s physiological and metabolic functions. Similar to other toxic substances these severely affect the activity of various metabolic enzymes mainly acetylcholinesterase [11] , Lactate dehydrogenase (LDH) level [12] and impose cytotoxicity in muscles, serum and liver cells (LDH) level [12] . These cause cell necrosis and death, display hepatoxicity and damage to cardiac muscles and subsequently increase the LDH activity in the serum [13] . Similarly, plasma and blood cholinesterases level also get increased that also displays toxicity in the liver [14] . Two important enzymes glutamic oxaloacetic transaminase (GOT) and serum glutamic pyruvic transaminase (GPT) were found in the liver, heart cells, muscle tissue, pancreas and kidney. Alteration in levels of these enzymes displays damage to these tissues [15] . Herbicides such as atrazine severely affect lipid peroxidation in fish Channa punctatus (Bloch.) that also targets liver hepatocytes [16] . Ammonia poisoning also causes liver damage and alters the levels of these enzymes [17] . Liver is also damaged due to the effect of pollutants like bisphenol A that induces hepatotoxicity and causes severe oxidative stress [18] . In the present study level of various metabolic enzymes was determined after injecting a sub-lethal dose of purified tick saliva toxins in mice models. Most of these enzymes are involved in toxicity reduction and their level displays toxic effects in blood serum, liver, tissue, kidney damage and neuronal effects.

2. Experimental

2.1. Isolation and Purification of Ticks’ Saliva Toxins

The living Rhipicephalus microplus was collected from rural areas of the Gorakhpur district. Living ticks were collected in sterilized plastic vessels and immobilized by quick freezing at −20˚C. Whole body homogenate was prepared in phosphate buffer saline (50 mm, pH 6.9) with the help of a power homogenizer. The homogenate was centrifuged at 24,000 XG at 4˚C for 30 minutes and the supernatant was used as crude saliva toxins.

2.2. Preparation of Homogenate

Rhipicephalus microplus were homogenized properly in a glass-glass homogenizer in 5 ml of different solubilizing buffers such as Triton X-100, PBS buffer (pH 6.9), 10% TCA, Tris-EDTA and absolute ethanol separately. Homogenate was centrifuged at 12,000 rpm in cold for 30 minutes and the supernatant was separated out. Total protein contents were estimated in the different supernatants according to Lowry’s (1951) method [19] .

Besides this, proteins (tissue) were solubilized in other solubilizing agents (Triton X-100, PBS, 10% TCA, and EDTA + Tris) in different combinations. Homogenate was centrifuged at 10,000 XG for 30 min and proteins were estimated in supernatant according to Lowry’s (1951) method [19] .

2.3. Purification of Saliva Toxins/Proteins from Rhipicephalus microplus

Proteins were eluted on a Sepharose CL-6B-200 a double cavity gel filtration column with sintered disc filtered in the bottom having a height of 1 meter in 25 mm diameter. A known volume i.e., 5 ml of toxin proteins solubilized in PBS was loaded in the column. The flow rate was maintained between 1 ml/minute by a continuous supply of PBS buffer (pH 6.7) in a cold room. Eluted fractions were collected at a fixed time interval using a Pharmacia fraction collector and the values of protein concentration in different eluted fractions will be plotted on the graph; absorbance in each fraction was determined at 280 nm using Shimadzu spectrophotometer (UV 2001 PC). Further, the absorbance of the same fractions was taken at 640 nm after protein estimation by Lowry’s (1951) method [19] .

2.4. Elution of the Saliva Toxin Protein through Gel Filtration Column

The column was tightly held erect with a stand. Elution of the saliva toxin proteins through the gel filtration column was done at the flow rate of 5 ml/minute.

2.5. Fraction Collection

Eluted fractions of saliva protein/toxins were collected manually at a fixed time interval at a constant flow rate. Total of 140 fractions were collected. The eluted fractions were observed for the detection of the presence of saliva toxin protein at a wavelength of 280 nm. Absorbance was taken on a Shimadzu spectrophotometer (UV 2001 PC) at 640 nm after protein estimation of the eluted fraction by Lowry’s (1951) method [19] . A graph was plotted between absorption at 280 nm and fraction numbers to show the elution pattern of Rhipicephalus microplus saliva toxins/proteins.

2.6. Molecular Weight Determination of Purified Saliva Toxin Proteins

The range of molecular weight of different proteins/toxins in the purified tick saliva toxins/proteins was determined by running the proteins of known molecular weight through the Sepharose CL-6B gel column as done previously at the same flow rate. A calibration curve was drawn between Ve/Vo log M between elution volume in fractions and molecular weight of different known proteins and compared with elution of proteins from Rhipicephalus (Boophilus) microplus saliva proteins/toxins at the same flow rate and same fraction.

2.7. Lyophilization of Eluted Saliva Toxins/Proteins

The eluted fractions of saliva toxin were pooled and lyophilized to get the desired concentration of saliva toxin.

3. Biological Activity of the Purified Saliva Proteins/Toxins

Biological activity testing of Rhipicephalus microplus saliva protein/toxins was determined in albino mice serially known volumes of the purified saliva toxins were injected intra-peritoneal.

3.1. Determination of Lethality of Rhipicephalus microplus Toxins/Proteins

The albino mice were injected subcutaneously with the purified saliva toxin of different serial concentrations and LD₅₀ was determined at the intervals of 24 hours. Deformities such as paralysis and neurotoxic effects were also recorded. Six albino mice were injected with serial concentrations of the saliva toxin to determine LD₅₀. Mortality was determined by using Abbot’s formula. The LD₅₀ values were calculated at which half of the test animals died. The lethal concentration for 40% and 80% of the LD₅₀ was determined with the doses-mortality regression line plotted on the log by using the Probit method (Spier, R.E. 1982) [20] . The confidence limits were calculated at 95% probability levels.

3.2. Dialysis of Lyophilized Saliva Toxin

The dialysis bag of cellulose membrane was boiled for 10 min in a large volume of 2% (w/v) sodium bicarbonate and 1 mM EDTA (pH 8.0) and then the membrane was rinsed thoroughly in distilled water. The membrane was then cooled and stored at 4˚C. The membrane was washed again with distilled water inside and outside before use. The lyophilized saliva toxin protein was filled in the dialyzing bag and dialyzed against three changes of phosphate buffer (50 mM, pH 6.9) to remove the excess salt from the lyophilized saliva toxin protein solution.

3.3. Effect of Purified Rhipicephalus microplus Saliva Protein/Toxins on Certain Serum Enzymes (in Vivo)