• A=n Univ,,",ity Jounml of Soion" & f } Technology: Biological Sciences Vol. 4 Number I 40-45,2009

Enhancement of Disease Resistance by Indigenous Plants

Vasudeva Rao. yl & Suuil Babu. G2

IDepartment of Biotechnology, Assam Univeristy, Silchar - 788 011, India. 2Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow-226 025, India.

Abstract

Achyranthes aspera seed was incorporated in the test diet (0.5%) and control diet was prepared without plant source. Control and test groups ofCatla catla (20±4 g) were fed with control diet and test diets, respectively. After 4 weeks, the fish were immunized with heat-killed Aeromonas hydrophila, and 4 weeks after immunization animals were experimentally infected with live A. hydrophila. After 7 days after infection, blood and spleen samples were collected from both the groups. Superoxide anion production, bactericidal activity, anti-protease activity, lysozyme, serum protein, albumin, globulin, SGOT and SGPT were determined. Superoxide anion production, serum bactericidal activity, anti-protease activity, lysozyme and serum globulin levels were enhanced in A. aspera treated groups compared to the control group. SGOT and SGPT levels were elevated in control group, but inA. aspera treated groups the levels were similar to the uninfected-control group. 85% mortalities were observed in the control group to day-14 after infection, where as in test group only 30% mortality was observed These results indicate that A. aspera increases resistance to infection.

Keywords: Catla catla; Achyranthes aspera, Aeromonas hydrophila, Disease resistance

Introduction

The modulation of the immune response by various substances has been reported, including synthetic, bacterial, animal and plant products. Several reports are available in which treating animals with immunostimulants increase the resistance and reduce the mortality rates after experimental infections with pathogens. Immunostimulants increase resistance to infectious diseases by enhancing both specific and non-specific defense mechanisms. Glucans derived from Schizophyllum commune and Saccharomyces cerevisiae has enhanced the immunity against various bacterial pathogens (1, 2). Oral administration of peptidoglycan increased resistance to Vibrio anguillarum in Oncorhyncu~ mykiss (3) and oral treatment with glucans increased resistance to V. anguillarum and V. salmonicida in Salmo salar (4). Injection of chitin increased the resistance in brook trout (5) and in rainbow trout (6) and injection of glucans increased phagocytic activity and resistance to Streptococcus sp. in Seriola quinqueradiata (7).

Oral administration of quillaja saponin enhanced the immunity of Seriola quinqueradiata (8) and feeding with Catharanthus roseus plant extract enhanced the immune response of Labeo rohita (9).

Achyranthes aspera, a herb belonging to Amaranthaceae, is widely available and distributed throughout India. The present investigation aims to study the effect of A. aspera on immunity and disease resistance.

Materials and methods

Achyranthes aspera seeds were collected, washed, dried and ground to powder. Artificial diet was prepared using fishmeal (30%), wheat fiour (58%), cod-liver oil (10%) and vitamin­mineral premix (2%). Control diet was prepared using the composition of ingredients, without the plant source. Test diet was prepared by the addition of 0.5% of A. aspera to the normal ingredients.

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Experimentalanimals and feeding: .

Catla catla (20±4 g) were collected from culture ponds, and were acclimatized for 7 days in outdoor conditions. Animals were divided into two groups (20/group) and were fed with control diet during acclimatization. After acclimatization, control and test groups were fed with control diet and test diets, respectively. Feed was given at the rate of3% of body weight. The temperature ranged between 25 and 35°C. Dissolved oxygen was maintained above 5 mg/L throughout the study period, using aerators continually.

Pathogen:

Aeromonas hydrophila was cultured in nutrient broth for 24 h at 37°C. The culture broth was centrifuged at 3 OOOxg for 10 min. The supernatant was discarded and the pellet was washed in phosphate buffered saline (PBS, pH 7.4). Finally bacteria was suspended in PBS and the OD of the solution was adjusted to 1.5 at 456 nm. This bacterial suspension was used for the challenge test. For the immunization, the OD ofthe bacterial suspension was adjusted to 0.5 and kept in a water bath at 60°C for 2 h. Sterility was confirmed by lack of growth on nutrient agar.

Challenge:

After four weeks of feeding with experimental diets, both control and test groups were immunized intraperitoneally with 100 fllofheat­killed A. hydrophila (1 x 1 06 CFU). After four weeks after immunization, both groups were injected intraperitoneally with 100 fll of live A. hydrophila (3xl06 CFU) suspended in PBS. Mortality of the challenged fish was observed daily up to 14 days.

Sampling:

Samples were collected on day 7 after challenge with live A. hydrophila. Blood was drawn from fish using heparin, kept in the refrigerator and assayed on the same day. Blood samples were also collected without heparin and allowed to clot at room temperature. Serum was obtained by centrifuging at 5000xg and was kept in the refrigerator.

Determination of superoxide anion:

Superoxide anion produced by macrophages/ phagocytes present in blood was determined col~rimetrically by using NBT. The blood, collected in heparin, was used in this assay. 50 fll blo~d and 50 fll of RPMI 1640 culture medium was placed in wells of a 96-well flat-bottomed microtitre plate and incubated for 2 h at 37°C in an incubator. After incubation, the non-adherent cells were removed by gently washing the wells with RPMI two times. After washing the wells, 100 fll ofNBT dissolved in RPMI (containing A. hydrophila) was added to the wells and incubated at25°C for 30 min. After incubation, the medium was discarded and the reaction was stopped by adding methanol to the wells. After washing the wells two times with methanol, the formazan formed in each well was dissolved by adding 120 fll of 2M KOH and 140 fll of DMSO. The absorbance was measured in a microplate reader (Biorad-550) at 655 nm.

Serum bactericidal activity:

A. hydrophila bacterial culture was centrifuged and the pellet was washed with PBS and resuspended in PBS. OD of the suspension was adj LIsted to 0.5 at 546 nm. This bacterial sLlspension was serially diluted (1: 10) with PBS five times. Serum bactericidal activity was determined by incubating 10 fll of this diluted bacterial suspension with 50 fll of serum in a micro-vial for 1 h at 37°C. In the bacterial control group, PBS was added instead of the serum. After incubation, these mixtures were cultured on nutrient agar plates separately for 24 h at 37°C. The number of viable bacteria was determined by counting the colonies grown on culture plates.

Determination of anti-protease activity:

Total protease inhibitors serum was performed by incubating 5 ml of serum with trypsin (10).

Determination of serum lysozyme:

Micrococcus lysodeicticus was cultured in Staphylococcus broth. After 24 h incubation, the culture was centrifuged, and the bacterial pellet was washed in PBS. Bacterial cells were again pelleted by centrifugation and subjected to

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lyophilization. Lysozyme was determined by incubating 10 III of serum with 1 ml of M lysodeicticus suspended in acetate buffer (pH S.S) for 60 min atroom temperature. After adding the serum, the initial absorbance was noted immediately at 4S0 nm, and final absorbance was taken after 60 min of incubation.

Determination of total serum protein, albumin and globulin:

Serum globulins were precipitated with saturated ammonium sulphate and desalted by dialysis and resuspended in PBS. Serum total protein, albumin, globulin were determined according to Lowry's method (11).

Determination of SGOT, SGPT:

Serum glutamate oxaloacetate transferase (SGOT), serum glutamate pyruvate transferase (SGPT) were determined using diagnostic kits (Bayer Diagnostics).

Statistical analysis:

All data were statistically analysed by Student's t-test using Microsoft Excel. The level of significance was P<O.OS. .

Results

Serum bactericidal activity:

Serum bactericidal activity was found minimum in the control group and highest in the test group (Fig. 2). The viable bacterial counts were significantly lower in the test group compared with either control group or bacterial control (P<O.OS). The number of bacterial colonies without addition of serum was 573. Incubation with control serum decreased the colonies to 464. The serum of test group individuals decreased the colony numbers to 179.

Anti-proteases:

Inhibition oftrypsin was observed minimum with the serum of control group and higher with test group serum (Fig. 3). This indicates that the anti protease levels are elevated in test group compared to the control group. In test group the anti-protease levels are more than 17% higher than the control group. The difference between both groups were statistically significant (P<0.05).

Serum lysozyme:

Serum lysozyme level was minimum in the control group, lysozyme levels were significantly (P<O.OS) elevated in test group treated with A. aspera (Fig. 3). In the control group, the average serum lysozyme was 20S mg/ml, and in the test group the level was 397 mg/ml.

Superoxide anion production:

Superoxide anion production by the blood leucocytes ofe. catla fed with O.S% A. aspera incorporated diet was significantly higher (P<O.OS) than the control group (Fig. 2). In test group the superoxide anion was produced 40 % higher, compared to the control group.

Serum protein, albumin and globulin:

Though the total protein level was slightly increased in test group compared to the control group, the difference is not significant (P>0.05). Albumin and globulin levels ~ere found to be elevated in test groups compared to the control group (Fig. 5). In test group globulin levels are elevated about 19% compared to the control. Nevertheless the differences were statistically not significant.

SGOT and SGPT:

The level ofSGOT significantly increased in the control group after infection with A. hydrophila compared to the uninfected control fish (*P<0.05). In Achyranthes treated groups, the SGOT levels were found similar to the uninfected control fish (Fig. 6). The SGOT level decreased significantly in test group compared to the infected control group (**P<O.05). The SGPT level was also significantly increased in control group fish after injectingA. hydrophila compared to the uninfected control group (*P<O.05). The SGPT level in treated fish was found similar to the uninfected control group (Fig. 6). In test group, SGPT level was decreased significantly compared with the infected control group (**P<0.05).

Infection and mortality:

After challenging e. catla with A. hydrophila, infection and mortality was recorded for 14 days. Among control and test groups, mortality rate was observed minimum in test group treated with A.

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aspera, and maximum mortality was observed in the control group (Fig. 7). After 14 days after challenging with the pathogen, total accumulated percentages of mortalities were 85% in control, and 30% in the test group.

Discussion

Immunostimulants increase resistance to infectious diseases by enhancing both specific and non-specific defense mechanisms. Theyihcrease the immunity by various mechanisms, such as increasing the number or activation of various cells involved in the immune response, such as phagocytes and lymphocytes. They may also increase protein synthesis to produce more number of molecules involved in immunity, such as complement, lysozyme, anti-proteases, immunoglobulins, chemokines. These earlier results can be correlated with the increased serum protein, lysozyme, anti-proteases, serum bactericidal activity in C. catla treated with A. aspera in the present study. Increase in total serum protein, globulin are good indicators of health status. These results indicate that A. aspera improves the health of fish and keeps them in good condition. Similar results were found with some other immunostimulants. Synthetic oligodeoxynucleotides containing un methylated CpG has enhanced the serum lysozyme activity in Cyprinus carpio, and yeast glucan has enhanced the lysozyme levels in Safrno safar (12, 13). Treatment of C. carpio with aqueous leaf extract of Azadirachta indica significantly increased serum protein levels (14). Protease inhibitors play a role in restricting the ability of bacteria to invade and to grow in the body of the host (15); they defend the body against pathogens by inhibiting their extracellular enzymes. Evidence showed that protease inhibitors can selectively arrest replication of microbial pathogen without untoward toxicity to the host (16). When fed with the A. aspera mixed diet, the protease inhibitor levels were enhanced in C. catla, thus the host can defend more strongly against invading pathogens.

Phagocytosis and killing activity by neutrophils and macrophages is an important defense against pathogenic bacteria. In the present study, superoxide anion production by the leucocytes of

C. carp indicated that treatment with A. aspera either stimulate phagocytes to produce greater amounts of ROIs, or increase the number of phagocytes whereby higher amounts ofROls are produced. Synthetic oligodeoxynucleotides containing unmethylated CpG have enhanced the phagocytic and NBT responses in C. carpio (12). Superoxide anion production and serum lysozyme levels were enhanced in rainbow trout Oncorhynchus mykiss fed with vitamin E and highly unsaturated fatty acids (17).

The increased serum bactericidal activity in A. aspera treated group indicate that various humoral factors involved in innate and/or adaptive immunities are elevated in the serum to protect the host effectively from infection. Similarly, Quit-A, a fraction from Quillaja saponaria Molina, has enhanced serum bacteriCidal activity in Salrno gairdneri (18).

The alkaline phosphatase levels were drastically decreased during the infection in control group, but treatment with A. aspera restored the level to normal as in the un infected control. As a result of infection with A. hydrophila, SGOT and SGPT were increased significantly in the infected control. However, these elevated levels were normalized by treatment with A. aspera and were found similar to the uninfected controls.

These results indicate that treatment of C. catla with A. aspera enhances certain non-specific and specific factors of the imI.Il.une system by enhancing anti-proteases, lysozyme, antibodies, globulins, phagocyte number/activity, which provide elevated defense against invading bacterial pathogens.

Infection and mortality following challenge with A. hydrophila was decreased in the test group treated with A. aspera. Compared with untreated controls, treatment with A. aspera increased the survival rate by 55% in the test group after infection with A. hydrophila. Similarly, dip treatment of C. carpio with aqueous leaf extract of Azadirachta indica significantly protected the fish from A. hydrophila infection (14). In conclusion, the present study has shown that oral treatment with A. aspera increased the immunity and significantly decreased the infection and

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mortality when C. catla were experimentally

700,..---------------,

600 ,; s soo 8 OJ 400

) 300

'5 200

~ 100

o Control Test Bacterial Control

Fig. 1: Effect of A. aspera on serum bactericidal activity· of C. catla. The values represent the mean±SE, n=S. *P<O.OS.

&O~------------~ 450

- 400

~350 .. e 300 ~ 250

~ 200

~ 150 .. ?Jlql

50

0+---Test

Fig. 3: Effect of A. aspera on serum lysozyme levels of C. catla. The values represent the mean±SE, n=S. *P<O.OS.

~,..------------~ 35

30

i: c

"iii

, ~ 15

10

5

o Albumin Globulin T eta! Protein

Fig. 5: Effect of A. aspera on serum albumin, globulin and total protein levels in C. catla. The values represent the mean±SE, n=S.

100

90 so 70 so 50

~

30

20 10 o

UrHnfected control

infected with A. hydrophila, a bacterial pathogen. 85-r--------------, 80

~ 75 .2 :is 70

~ 65 c .8. 60

~ 55

50

Control Test

Fig. 2: Effect ofA. aspera on serum anti-protease levels of C. catla. The values represent the· mean±SE, n=S. *P<O.OS.

0.25,..-------------..,

0.2

E ~ 0.15 to

~ 0.1 o

0.05

0+---' Control Test

Fig. 4: Effect of A. aspera on Superoxide anion production by C. catla blood leucocytes. The val­ues represent the mean±SE, n=S. *P<O.OS.

6~-------------~ DUn-infected conttrol

5 o Control

0+---'---SGOT SGPT

Fig. 6: Effect of A. aspera on SGOT and SGPT levels in C. catla infected withA. hydrophila. The values represent the mean±SE, n=S. *, **p<O.OS.

Control Test

Fig. 7: Effect of A. aspera on survival of C. catla infected with A. hydrophila.

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