Effect of IDJ on the Immune System of Rabbit
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Effect of IDJ on the Immune System of Rabbits
By Mohammad Ali Kavianpour, M.D
Kermanshah University of Medical Sciences
ABSTRACT
The effect of IDJ on immune responses of Rabbits was investigated. Treatments were: 0, 1400, 2800 and 4200 mg of IDJ given orally at weekly intervals. Rabbits were given ad libitum access to feed and water and were on experiment for 12 weeks .Lymphocyte stimulation indices were significantly higher for Rabbits given the high amount of oral supplementation than for unsupplemented Rabbits.There were no significant differences in the concentrations of immunoglobulins G1 and G2 among treatments. Immunoglobulin M was significantly higher at week 6 in Rabbits given the higher amount of oral supplementation .At week 12, serum from Rabbits given the higher amount of oral supplementation inhibited infectious bovine rhinotracheitis viral replication in tissue cultures as compared with those of unsupplemented Rabbits.
INTRODUCTION
Nutrition plays a significant role in the development and function of the immune system. The level of particular nutrients may affect not only humoral and cell-mediated immune responses but also several nonspecific humoral factors such as C-reactive protein, lysozymes, betalysins, and hormones, which regulate the immune responses. IDJ may affect several humoral factors,for example, serum corticosterone and prostaglandins and thus influence replication of pathogens within the host.
Objectives of Experiment 1 were to study the effect of supplemental IDJ on the immune system of Rabbits for 3 month under normal direct management conditions and to study the effect of serum from supplemented Rabbits on the replication of IBR virus in tissue cultures. Experiment 2 was intended to study the effect of supplemental IDJ on cell-mediated immune response of Rabbits and Experiment 3 to study the effect of in vitro addition of IDJ on lymphocyte blastogenesis.
MATERIALS AND METHODS
Experimental Procedures
Twenty White male 6 months old rabbits (weight 3,350 ± 50 g) were kept in single cages, in two chambers provided with an automatic control of climatic condition. During the 7 days of adaptation before the experimental period , the rabbits were maintained at 22.0°C (relative humidity 65 ± 5%) and had free access to feed and water. Rabbits with a plasma protein (PP) content greater than 5.5 g/dl were selected randomly to one of four treatments: 1) 0 mg, 2) 1400 mg, 3) 2800 mg and 4) 4200 mg of IDJ dissolved in a sufficient quantity of water and given orally with a nipple bottle at weekly intervals, Management was the same for all the Rabbits.
Experiment 1
Analytical Procedures
Plasma Protein and Packed Cell Volume:
Jugular blood obtained into heparinized blood collection tubes was used to determine plasma protein (PP) by refractometry and packed cell volume (PCV)by a microhematocrit centrifuge.
Lymphocyte Blastogenesis Test.
Jugular blood obtained at 2, 4, 6, 8, 10, and 12 week into heparinized blood collection tubes was used within 1 h after collection. Two milliliters of blood mixed with 2 ml of phosphate- buffered saline (PBS) were layered on 4 ml of Histopaque-1077 containing ficoll and sodium diatrizoate, then centrifuged at 400 x g for 30 min. Lymphocytes sedimented at the interface between Histopaque-1077 and blood- PBS mixture were collected with a Pasteur pipette, washed twice with RPMI-1640 medium, and resuspended in RPMI-1640 containing 25 mM Hepes buffer and 10% serum. Cells were counted in a hernocytometer, and the suspension was adjusted to contain 1 x 10 ^5 cells/ml. The cell suspension lacking or containing 5 micro g/ml phytohemagglutinin (PHA), was distributed in 200 micro l/well in quadruplicate in a flat bottom tissue culture plate. This concentration of PHA was predetermined in several experiments in laboratory to give maximum lymphocyte stimulation. Cultures were incubated at 37°C in a humidified carbon dioxide (CO2)incubator. Viability of Iymphocytes was > 95% during the course of the experiment as determined by trypan blue dye exclusion test. After 48 h of incubation, thymidineS (specific activity 6.7 Ci/mM) 0.2 microCi/culture was added. Cultures were harvested 24 h later on glass fiber filters in an automated cell harvester. Filters were counted in a liquid scintillation counter to determine the incorporation of thymidine in lymphocytes. Lymphocyte stimulation indices (LSI) were calculated as:
LSI = Desolution per minute (DPM) of stimulated lymphocyte cultures/dpm of control cultures.
Infectious Bovine Rbinotracbeitis Virus Replication.
The effect of serum collected from experimental Rabbits at O, 2, 4, 6, 8, 10, and 12 week on the in vitro replication of IBR virus was determined. Monolayer cultures of Madin-Darby bovine kidney (MDBK) cells were prepared in 35 × 10-mm tissue culture plastic dishes and maintained in Eagle's minimal essential medium (MEM) with 10% FCS (IBR antibody-free). Cultures were washed twice with PBS and were inoculated with IBR virus at a multiplicity of 0.1 pfu / cell. After 1 h incubation at 37°C in a CO2 incubator, each set of cultures was maintained in MEM containing 10% serum from each experimental rabbit. The infected cultures were harvested 48 h postinoculation and frozen at -70°C. Infectivity titers were determined by tissue culture infective dose (TCID50) method. Frozen cultures were thawed, briefly sonicated, and serial log dilutions were prepared in MEM with 5% FCS (IBR antibody-free). Monolayers of MDBK ceils grown in microtiter plates were infected with 0.2 ml of each serial log dilution (4 wells per dilution). Cells were observed for cytopathic effects 48 h postinfection, and TCID50 was determined by the method of Karber .
Serum Immunoglobulins.
Serum IgG1, IgG2, and lgM were determined at wk 0, 6, and 12 by the single radial immunodiffusion (SRID) procedure with ready-to-use SRID assay kits. Experimental serum samples were diluted to 1:100 for lgG l, 1:15 for lgG2, and 1:3 for IgM, using 0.9% sodium chloride in distilled water before charging wells. Ring diameters were read after 24 h, and sample concentrations were determined by establishing a regression line for the ring diameter and concentration of known standards.
Experiment 2
The objective of this experiment was to study the effect of supplemental IDJ on cell-mediated immune response of Rabbits . Blood samples were obtained just before supplementation and 7 day later to determine LSI. LSI was calculated from the results of the lymphocyte blastogenesis test. Statistical analysis was by paired t test.
Experiment 3
This experiment studied the effect of in vitro addition of IDJ on lymphocyte btastogenesis. Lymphocytes were isolated from blood of 10 unsupplemented Rabbits used in Experiment 1 and the cultures were prepared for the lymphocyte blastogenesis test as described. IDJ was added to lymphocyte cultures in concentrations of O, 1.25, 2.5, 5, 12.5, 25, and 50 micro g/well (4 wells per concentration). The LSI was determined for each concentration of IDJ Statistical analysis of data was by one-way analysis of variance and then means separation by calculating the least significant differences.
RESULTS
None of the Rabbits were sick during the course of the experiment. Growth and metabolic responses were normal. Results of PP and packed cell volume (PCV) determinations and LSI and serum immunoglobulin averaged across weeks are in Table. PP and PCV values were similar in all groups of Rabbits. LSI averaged across weeks was higher in Rabbits given the high oral supplementation than in unsupplemented Rabbits. .
4200 mg Rabbits had significantly higher LSI than unsupplemented Rabbits at week 4 and higher values than all other Rabbits at 8 week. Replication of IBR virus in cultures treated with serum from experimental Rabbits showed a significant treatment × week interaction (P=0.0002), and hence, the overall means and their differences were not calculated. Virus titers at individual weeks were significantly lower with serum from Rabbits given the high oral supplementation at week 12. A trend toward lower virus titers was observed using serum from supplemented Rabbits as compared with serum from unsupplemented Rabbits starting from week 6, except for Rabbits given lower oral supplementation at week 8. In addition, there was a trend toward progessive increase in virus titers with increasing age of Rabbits. Serum IgG1 and lgG2 averaged across weeks were similar in all groups. The IgM were higher in Rabbits given high oral supplementation than in unsupplemented Rabbits. Values were significantly higher at week 6 in Rabbits given the high oral supplementation than in unsupplemented Rabbits.The LSI values in response to in vitro addition of IDJ to lymphocyte cultures with different concentrations of IDJ were not significantly different from control cultures without IDJ .
DISCUSSION
Mean PP concentrations in week 1 were similar in all groups, suggesting that all Rabbits received similar and adequate amounts of colostrum. The immunological factors obtained through colostrum are Ig, complement, and lymphocytes.Very little is known about the mechanism of transport and functional significance of complement and lymphocytes present in colostrum. There is no placental transfer of Ig, dependent upon colostrum for passive immunity . The Ig of Rabbits appeared to be positively correlated with PP . PP should be above 5.5% for satisfactory performance of Rabbits. Supplemental IDJ for Rabbits on typical diets enhanced their cell-mediated immunity, as suggested by higher LSI for Rabbits given the high oral supplementation and in animals in both Experiment 1 and 2. Phytohemagglutinin is a specific mitogen for thymus-derived (T)dymphocytes and, hence, LSI mainly represents the cell-mediated immune response
There are several possible reasons for higher blastogenic response to PHA in supplemented animals. The antioxidant action and prevention of peroxidative damage to the membranes of the cells and subcellular organelles such as mitochondria, microsomes, and lysosomes may be the most important mechanism. Lymphocytes normally have a higher concentration of free fatty acids (FFA) than other cells. Lipid peroxidation and free radical attack may alter the membrane fluidity and result in changes in cell to cell and cell to substrate interaction and subsequent metabolic processes in lymphoid tissue and may be responsible for observed immunosupression. Another mechanism by which IDJ may exert its effect on the immune system would be by controlling the synthesis of prostaglandins . The prostaglandins derived from dihomogammalinolenic acid play a major role in the regulation of thymus development and T-lymphocyte function . Some of the lymphocyte functions inhibited by prostaglandins include mitogen responsiveness , cell-mediated cytotoxicity , and lymphokine production In vitro blastogenic response of lymphocytes to PHA was depressed when prostaglandin was added in concentrations ranging from 10^ -4 to 10^ -7 M. Lower serum corticosterone in Rabbits fed a high IDJ diet may explain some of the observations of enhanced Tlymphocyte activity, such as PHA-induced mitogenesis and antibody dependent cellmediated cytotoxicity.
Future research with Rabbits should monitor the prostaglandin and serum corticosterone levels to determine possible reasons for higher PHA-induced lymphocyte blastogenesis.Serum samples obtained after 6 week from Rabbits given supplemental IDJ showed a trend toward inhibiting IBR virus replication. At week 12, with serum from Rabbits given the high oral supplementation , the inhibition was significant as compared to that of unsupplemented Rabbits. Viral titers were similar for all groups of Rabbits until week 6, probably because passive transfer of antibodies through colostrum was similar in all Rabbits or because IDJ did not alter other serum factors until week 6. IDJ affects the levels of several enzymes and hormones in the body . However, research is needed to study the effect of supplemental IDJ on nonspecific factors of resistance such as interferon, C-reactive protein, betalysins, lysozymes, and serum transferrins. Our results suggest that supplemental IDJ may be useful for Rabbits on conventional diets, at a stage when they are most vulnerable to respiratory diseases.
Although there is no clear information on age-specific infection rate due to IBR virus, protective effect of maternally acquired antibody in Rabbits affected the age distribution of infection rate. The persistence of colostrally acquired immunity varies from Rabbits to Rabbits; some Rabbits lose it as early , but a few may have detectable maternal antibody at 6 mo of age . Results of the present experiment suggest that during this transition period between the loss of maternal antibody and the production of antibodies by rabbits Supplemental IDJ alters serum factors in such a way that there may be less morbidity due to a pathogen. Thus, in general, our results support that supplemental IDJ may not only enhance humoral immune response but also possibly reduce morbidity and mortality due to pathogens. Although there were no significant differences in IgG1 and IgG2 among treatments, there was a trend toward higher IgG1 concentrations in supplemented Rabbits. It is not known whether this trend was due to less metabolic degradation of passively acquired antibody or enhanced synthesis by supplemented Rabbits. Rabbits given the high oral supplementation showed higher serum IgM concentrations than unsupplemented Rabbits, suggesting their capability for a higher primary immune response, as IgM is the first class of antibodies to appear in the immune response to most antigens .
Conclusions
This study suggested that supplemental IDJ enhance both cell-mediated and humoral immune responses. It also altered serum factors in other ways, which may increase the possibility for protection against pathogens. Possibly these beneficial effects will be greater under more intensive management conditions. Further research with graded amounts of different forms of IDJ given on a daily basis should determine the requirements of Rabbits. Our data on immune responses of Rabbits suggest that criteria for minimum requirements should not be based entirely on growth rate studies or on the amounts required to prevent clinical malnutrition deficiencies but also on the amounts needed to attain optimal health and immune competence.
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About the Author
Mohammad Ali Kavianpour, M.D Kermanshah University of Medical Sciences
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