Introduction
Arsenic is a naturally occurring metalloid, widely distributed in the environment. Arsenic has been detected in different concentrations in fodder, crops and water in many geographical locations.1 Contamination of drinking water through natural release of arsenic from aquifer rocks is the primary source of exposure in livestock. Apart from this, green fodder grown in arsenic contaminated area is another source of arsenic exposure to livestock.2 Arsenic is very toxic and can cause several health problems in animals and human depending on their concentration and duration of exposure. It can cause dispigmentation, cancers of skin, liver and lungs, hematological, circulatory, reproductive, neurological gastrointestinal and immunological pathologies. However, the contribution of many environmental chemicals including arsenic in the etiology of these diseases is not established till now.3 Exposure to chemical compounds can affect the immunity either by causing hypersentivity and autoimmunity, in which the immune cells fails to differentiate between self and non-self, causing damaging effect on various organs of the body; or by causing immunosuppression in which activity and response of immune system is decreased. Immune complexes or antigen-antibody complexes are persistently formed in the body. Antigen and antibody interacts with each other by non-covalent forces. Presence of Circulatory Immune Complexes (CICs) is an indicator of normal immune response as it is a part of humoral immunity. In case the immune response is effective, phagocytes eliminate the circulatory immune complexes from blood circulation.4 However, during abnormal immune response, circulatory immune complexes can’t be eliminated from the circulation and can cause a variety of systemic disorders. CICs are detected in case of chemical exposure, allergy, autoimmune diseases, rheumatoid arthritis, bacterial, viral, parasitic and cancerous conditions in humans.5 However, effect of metal exposure on circulating level of immune complexes in Kosali cattle remains un-investigated.
Traditionally ethnomedicines are widely used in India for treatment of various disorders due to their easy accessibility, low cost and fewer side effects. In recent years there is increasing demand of plant derived therapeutics. Medicinal herbs have also proven immunoprotective and immunomodulatory potential. Powders and extracts prepared from them are widely used in the treatment of immunological disorders. The bark of Terminalia arjuna, a deciduous tree of the Combretaceae family, has been reported in ancient Indian medicinal literature as well as in current literature for having beneficial effects on various chemical mediated immune disorders.6 Terminalia arjuna bark contains many active constituents, such as tannins, triterpinoid saponins (arjunic acid, arjunolic acid, arjungenin, arjunglycosides), flavonoids, ellagic acid, gallic acid, oligomeric proanthocyanidines, phytosterols, calcium, magnesium, zinc and copper. Metal chelating and immunomodulatory properties of Terminalia arjuna dried bark powder have been established in human and laboratory animals.7
The present study investigated the effect of Terminalia arjuna bark powder on the concentration of circulatory immune complexes and plasma total immunoglobulins in Kosali cattle exposed to arsenic through contaminated drinking water and fodder.
Materials and Methods
Ethical approval
Experimental protocols of this study have been approved by the Institutional Animal Ethical Committee (IAEC). All the experiments were carried out according to the guidelines of the IAEC.
Study area
An arsenic affected area was selected through survey where the drinking water arsenic concentration was above the maximum permissible limit (0.01 µg/ml) described by WHO (2005). Another area approximately 50 km distant from contaminated site without any arsenic contamination problem was taken as the control area. Tube well water, fodder and Kosali cattle blood samples were collected from the dairy farms located in arsenic contaminated and uncontaminated areas. The adult Kosali cattle (3-5 years of age) used in the study were maintained by their owners in organized dairy farms and provided with standard diet and ad libitum water.
Collection of samples
Water
Tube well water samples (50 ml) were collected in duplicate from the dairy farms located in arsenic contaminated (n=10) and control area (n=10). These tube wells were used for irrigation and supply of drinking water to the Kosali cattle. Water samples were collected in polypropylene bottles prewashed with nitric acid (1ml/L). Collected water samples were preserved in concentrated hydrochloric acid (4ml/L) and stored at 4oC in refrigerator till estimation of total arsenic.
Fodder
Fodder samples used for feeding of Kosali cattle were collected in duplicate from the arsenic contaminated (n=50) and control area (n=10). These samples were washed with 2% hydrochloric acid and distilled water to remove all the impurities and dust particles. After removing the extra water with blotting paper, samples were cut into pieces, packed into petridishes, and kept in an oven for drying. The dried samples were grinded and passed through a sieve of 2 mm size and then kept at room temperature till estimation of arsenic.
Blood
Jugular vein blood (2ml) was collected from the Kosali cattle of the arsenic contaminated (n=10) and control area (n=10) using a sterile syringe and needle and transferred to a heparinized vial. Blood was transported from the field in an ice box. The whole blood samples were used for estimation of total arsenic.
Determination of arsenic concentration
Processing of samples
Fodder and blood
Fodder samples (1g) and blood samples (3ml) were digested using 15 ml of tri-acid mixture (HNO3, H2SO4, and HClO4 in 10:4:1 ratio) until a transparent solution was obtained. After cooling, the digested sample was filtered using Whatman No. 42 filter paper and final volume of filtrate was made to 10 ml with distilled water.
Procurement of Terminalia arjuna bark powder (TABP)
Terminalia arjuna bark powder used for the treatment of Kosali cattle was obtained from Nature Natural Ayurvedic Life Care.
Quality criteria and selection of dose
Quality of Terminalia arjuna bark powder was maintained by Nature Natural Ayurvedic Life Care; which was congruent to the Ayurvedic Pharmacopoeia of India (API). The dose of Terminalia arjuna bark powder (40mg/kg b. w.) was decided as per the API.
Grouping of Kosali cattle
A total of thirty adult Kosali cattle (same animals used above for determination of blood arsenic level) were divided into following groups:
Control group (n=10): Clinically healthy Kosali cattle selected from the uncontaminated area without any treatment (with blood arsenic level within the normal limit (0-0.05ppm).
Arsenic exposed/ Exposure control group (n=10): Kosali cattle exposed to arsenic through intake of contaminated water and fodder selected from the arsenic contaminated area (with blood arsenic level above the normal limit of 0-0.05ppm).
Treatment group (n=10): Arsenic exposed Kosali cattle treated with Terminalia arjuna bark powder @ 40mg/ kg b. w. for 30 days.
All the cattle were approximately of same age and body weight. The treatment schedule did not cause any change in feed and water intake pattern of animals. Three blood samples (2ml) were collected during the supplementation period i.e. on day 0, 15 and 30 and one blood sample was collected after the withdrawal of supplementation i.e. on 45th day from Kosali cattle of the treatment group. Single blood samples were collected from animals of the control and arsenic exposed groups. The blood samples were centrifuged at 2500 rpm for 10 minutes and plasma was separated.
Circulatory immune complexes
Circulatory immune complexes in plasma were determined by polyethylene glycol (PEG) precipitation method.8 2.7 ml of 0.1 M borate buffer (pH 8.4) and 0.3 ml of plasma were poured into a centrifuge tube and mixed. Then 4.5 ml of 5% PEG solution was added and the mixture was incubated for 2 hours at 4ºC followed by centrifugation at 600 g for 20 minutes. Supernatant was decanted and 3.5 ml of 3% PEG solution was poured over it, stored for 18 hours at 4ºC and centrifuged under the same conditions. The supernatant was discarded, sediment was mixed by beating, 0.3 ml of distilled water and 2.7 ml 0.1N sodium hydroxide was poured over it. Absorbance was determined at 280 nm using UV/VIS spectrophotometer (Lambda 25, Perkin Elmer, Germany). The CIC concentration (mg/mL) was calculated using bovine IgG calibration curve. All determinations were performed in duplicate.
Total immunoglobulins
To 0.1 ml plasma, added 0.1 ml of 90% saturated ammonium sulphate (SAS) to get an overall concentration of 45% saturation. At this concentration of ammonium sulphate, immunoglobulins were precipitated after refrigeration at 4°C overnight followed by centrifugation at 6000 rpm for 15 minutes. The precipitate was washed twice with 45% SAS and pellet was dissolved in 0.1 ml phosphate buffer saline (pH - 7.4).9 0.01ml of this solution was used to estimate the total immunoglobulins(g/dL). To0.01ml of solution (diluted 10 times), added 5ml of alkaline copper solution, mixed well and allowed to stand for 10min. Then added 0.5mL of Folin-Ciocalteau reagent and incubated at room temperature in the dark for 30min. The volume was made up to 1 ml with distilled water. Standard was prepared by taking 20-200µg BSA/ml. Absorbance was recorded at 520 nm against blank using UV/VIS spectrophotometer (Lambda 25, Perkin Elmer, Germany). All determinations were performed in duplicate.
Statistical analysis
Data was analyzed using Statistical package for Social Sciences (SPSS) software (Version 16.0). Multiple comparisons of data were carried out by one way analysis of variance (ANOVA) and the group means were compared by Duncan’s Multiple Range Test (DMRT). Additional statistical comparisons between means of different groups were carried out using independent t-tests. Correlation was determined by Karl Pearson’s coefficient of correlation.
Results
Arsenic concentration in tube well water, fodder and cattle blood
In the current study, arsenic concentration was reported to be significantly (p<0.05) increased in tube well water, fodder and Kosali cattle blood samples (Table 1) collected from arsenic contaminated area compared to the control area. Significant positive relationship (r = 0.516, p<0.05) was observed between arsenic concentration in tube well water and fodder samples from the arsenic contaminated area. Arsenic levels in the tube well water and fodder samples from arsenic contaminated area exhibited positive correlation (p<0.05) with blood arsenic level of untreated cattle reared in these areas (r = 0.821 and 0.672 respectively) (Table 2).
Table 1
Arsenic concentration (mean ± S.E.) in tube well water (µg/ml), fodder (µg/g) and Kosali cattle blood (µg/ml) samples of control and arsenic contaminated area
|
Samples |
Arsenic Concentration |
|
|
Control Area (n=10) |
Arsenic Contaminated Area (n=10) |
|
|
Tube well water |
0.004±0.001 |
0.09±0.011* |
|
Fodder |
0.019±0.005 |
0.64±0.03* |
|
Kosali cattle blood |
0.016±0.007 |
0.35±0.005* |
[i] *Indicates significant difference at p<0.05. Maximum permissible limit of arsenic in drinking water in view of animal health is 0.01µg/ml (WHO, 2005).10 Phyto-toxicity limit of arsenic is 1 µg/g dry weight basis (Sidhu et al., 2012).
Environmental arsenic contamination problem is increasing year by year in different parts of the world. Large number of animals in different parts of globe especially of India is reported to be affected with arsenic toxicity through drinking water and consumption of contaminated fodder.1 Dairy animals are mostly supplied with the tube well water for drinking. Frequent use of underground water has lowered the water level; contaminating it with arsenic containing salts and minerals. Increased concentrations of sulfate, phosphate and hydroxyl ions along with an increased pH (>8.0) in the ground water aquifers are responsible for the increased release of arsenic.11 Higher level of arsenic in tube well water samples indicates that source of arsenic in water is natural rather than human activities. Dairy animals are mainly fed with chopped green fodder. Arsenic concentration in fodder was reported to be higher in the arsenic contaminated area but the level was below the maximum permissible limit for fodders.12 Fodder seldom accumulates arsenic at concentrations fatal to animal because phytotoxicity results before such threshold concentrations are reached in plants.11
Table 2
Relationship between arsenic concentrations in tube well water, fodder and Kosali cattle blood samples of arsenic exposed area (before Terminalia arjuna bark powder treatment)
Higher level of arsenic in fodder samples can be attributed to the increased use of arsenal pesticides in the agricultural fields and irrigation with the arsenic contaminated water. Increased fodder arsenic level could be a threat to the Kosali cattle because they are mainly fed with the green fodder. Higher arsenic level observed in the Kosali cattle blood samples collected from the arsenic contaminated area could be due to the intake of arsenic mainly through contaminated drinking water and fodder which was supported by the close relationship observed between arsenic concentrations in tube well water, fodder and cattle blood samples in the present study.
Circulatory immune complexes and total immunoglobulins
Level of circulatory immune complexes in Kosali cattle of the arsenic contaminated area was significantly (p<0.05) elevated with a decline (p<0.05) in the level of plasma total immunoglobulins compared to control area (Figure 1). Plasma CICs concentration was 2.96 fold increased whereas total immunoglobulins level was observed to be decreased by 2.22 fold in Kosali cattle of the arsenic contaminated area in comparison to control area. Blood arsenic level in untreated Kosali cattle was significantly correlated with circulatory immune complexes (r = 0.212, p<0.05) and plasma total immunoglobulins level (r = -0.468, p<0.05) in arsenic contaminated area (Table 3).
Chemical toxins can form immune complexes and have suppressive effect on the immune system. CICs have profound effect on humoral and cellular immune response through the receptors of immunocompetent cells. Permanent presence of CICs indicates abnormal immune regulation of an organism. Phagocytic cells such as neutrophils and macrophages remove the CICs from the organism. Persistent presence of CICs exhausts the neutrophils losingits function for a short period. Activation of glycolysis in neutrophils in an alkaline medium observed in exhausted neutrophils restores their function again. Erythrocyte complement receptor (E-CR1)-C3b complex can bind to CICs which safely deliver these complexes to monocyte-phagocytic and reticuloendothelial system. This process prevents deposition of CICs in tissues.4 The increased concentration of immune complexes observed in Kosali cattle of arsenic contaminated area in present study could be a result of constant intake of arsenic through drinking water and fodder. Arsenic can form complexes with antibodies and deposited in the tissues.
Figure 1
Concentration (mean ± S.E.) of plasma total immunoglobulins and circulatory immune complexes (CIC) in Kosali cattle of control and arsenic contaminated area*Indicates significant (p<0.05) difference from corresponding controls
Table 3
Relationship between blood arsenic level and immunological indices in Kosali cattle of arsenic contaminated area (before Terminalia arjuna bark powder treatment)
|
Parameters |
Pearson’s correlation coefficient (r) |
|
Arsenic (Kosali cattle blood) Vs. Plasma CICs |
0.172* |
|
Arsenic (Kosali cattle blood)Vs. Total plasma Igs |
- 0.453* |
The tissue injury caused due to free radical damage by arsenic releases the CICs to plasma as observed in the present study. CICs formation has also been reported in Kosali cattle exposed to environmental pollutant which is in accordance with the present study.8 The formation of CICs might be the reason for systematic antigen stimulation and reduction in activity of immune system. However, the mechanism of formation of CICs due to arsenic exposure is not clearly understood.4 Immunoglobulins are produced by plasma cells and act as a critical part of immune response by binding with specific antigens. The decreased total immunoglobulins observed in Kosali cattle of arsenic contaminated area in present study might be a consequence of disturbances in immune regulation of the organism and abnormal function of white blood cells due to formation of CICs. Significant correlation of blood arsenic level with total immunoglobulins and CICs in untreated Kosali cattle of arsenic contaminated area provided indication about the probable influence of this toxic metalloid on humoral and cellular immune response.
Effect of Terminalia arjuna bark powder on the concentration of CICs and total immunoglobulins
Treatment of arsenic exposed Kosali cattle with Terminalia arjuna bark powder @ 40mg/kg b. w. for 30 days resulted in significant (p<0.05) decrease in circulatory immune complexes in arsenic exposed cattle compared to control (Figure 2 A). However, concentration of plasma total immunoglobulins was significantly (p<0.05) enhanced to the level compared to that of control after 15 days of treatment with Terminalia arjuna bark powder @ 40mg/kg b. w. (Figure 2 B).
Figure 2
Concentration (mean ± S.E.) of (A) plasma total immunoglobulins and (B) circulatory immune complexes in arsenic exposed Kosali cattle supplemented with Terminalia arjuna bark powder. Columns superscripted with different letters are significantly different at p<0.05
The effect of Terminalia arjuna bark powder on the level of CICs and total immunoglobulins could be due to its immunomodulatory activity. Terminalia arjuna bark extract has been reported to improve the neutrophil function in humans.6, 13 Previous research on mice and rats suggested that Terminalia arjuna bark powder has immunomodulatory potential.14 Saponins present in Terminalia arjuna bark have been reported to possess immuomodulatory activity and stimulate the lymphocytes in vitro.6 The exact mechanism by which the compounds present in Terminalia arjuna bark eliminate the CICs is still not clear. The decrease in concentration of CICs followed by Terminalia arjuna bark powder treatment might be due to activation of phagocytic activity, macrophages or erythrocyte complement receptor which is in line with the results of the present study.4 Improved plasma cell function and efficient immune regulation by the host treated with Terminalia arjuna bark powder might be the reason for improve in plasma total immunoglobulins which has been observed in the present study.
Conclusion
Exposure to arsenic contaminated water and fodder altered the levels of circulatory immune complexes and plasma total immunoglobulins in Kosali cattle. Treatment with Terminalia arjuna bark powder @ 40mg/ kg. b.w. reduced the level of circulatory immune complexes with enhancement of plasma total immunoglobulins concentration in arsenic exposed Kosali cattle towards the value compared to control. The bark of this plant could be very useful for improving the immune status of animals. However, more studies are required to identify and characterize the compounds present in Terminalia arjuna bark that is responsible for these curative effects.
