Relative organ weights
Relative weights of immune organs (bursa of Fabricius and thymus) have been presented in Graph 1.
Bursa of Fabricius
Relative weights of toxin treated groups (A1, A2 and A3) were significantly lower while that of groups fed DS alone (DS1 and DS2) were non-significant when compared with control. A2DS1, A3DS1 and A3DS2 were significantly lower while that of groups DS1 and DS2 along with remaining combination groups were non-significant as compared to that of control.
Thymus
Relative thymus weights of toxin treated groups (A1, A2 and A3) along with group A3DS1 were significantly lower while relative weights of all the remaining groups were non-significant when compared with control.
Histopathological examination of lymphoid organs
Bursa of Fabricius
Histopathological slides of control showed normal cortex and medulla within the bursal follicles and connective tissue was minimum between two adjacent bursal follicles. Superficial pseudostratified epithelium was intact within the folds of bursa and the cell population was thinner in medulla while it was thick within the cortex region. Both compartments of follicles were separated through a thin layer of elongated cells.
Microscopic examination of slides of group A1 showed inta ct, pseudostratified epithelium with increased empty spaces within bursal follicles (both cortex and medulla) due to depletion of lymphocytes. Small cysts like structure were also observed at few places within follicles and the cortical rim of follicles either decreased in thickness or intermingles with the medullary region. Along with this, medullary region of the follicles also presented increased population of macrophages, fibroblasts and undifferentiated cells. These histopathological alterations became more intense in groups A2 and A3 in a dose dependent manner.
Groups A1DS1 and A2DS1 showed normal microscopical picture of bursa which were almost similar to control group. However, group A3DS1 showed mild to moderate congestion within the bursal follicles. An intact superficial pseudo-stratified epithelial layer was present with an increased interfollicular connective tissue leading to a decrease in the diameter of individual follicles. Increased empty spaces were also observed within the bursal follicles due to depletion of lymphocytes. Small cysts like structures were also noticed in both regions while at most of the places, clear demarcation between cortex and medulla was lost. This microscopic picture was however less severe in A3DS1 when compared with individual toxin group (A3).
Histopathological examination of groups A1DS2 and A2DS2 revealed a well-maintained picture of bursal follicles which was similar to that of control. However, in group A3DS2, bursal follicles had smaller diameters and there was increased interfollicular connective tissue between adjacent follicles. Demarcation between cortex and medulla was lost in many of the follicles while superficial pseudo-stratified epithelium was intact. However, there were many empty spaces within cortex and medulla due to depletion of lymphocytes and small cysts like structures were also present at some places within the follicles. Such alterations were however, less severe when compared with toxin treated group (A3).
Thymus
Histopathological examination of thymus of control group revealed few small empty spaces between cortex and medulla and some of the empty spaces has degenerated and pyknotic nuclei. Medullary region was less densely packed with lymphocytes while cortex was highly dense and cells of reticuloendothelial system with clear nuclei and lightly stained cytoplasm were present between cortex and medulla. Hassall’s corpuscles with variable sizes were also found having lightly stained nuclei and pinkish or indistinct cytoplasm.
Microscopic examination of slides of group A1 revealed an increased thickness of the interfollicular connective tissue. Lymphocytic depletion was also noticed as elucidated by increased number of empty spaces and necrotic cells within both cortex and medulla. The diameter of Hassall’s corpuscles was variable throughout the region, and it also had increased number of pyknotic nuclei. Such alterations became more worsen in a dose dependent manner (group A2 and A3).
Histopathological examination of groups A1DS1 and A2DS1 showed a clear demarcation between cortex and medulla which was not observed in most of the places of group A3DS1. Group A3DS1 showed many pyknotic nuclei and Hassall’s corpuscles having variables diameters contained many pyknotic nuclei. However, such alterations were less severe when compared with individual toxin group (A3).
Histological picture of groups A1DS2 and A2DS2 was well maintained, and situation was more or less similar to control group. However, in group A3DS2, clear demarcation between cortex and medulla was lost at most of the places and Hassall’s corpuscles had variable diameters with many pyknotic nuclei. This situation was, however, less severe when comparison was made with individual toxin treated group (A3).
Total Antioxidant Capacity (TAC)
Values of Total Antioxidant Capacity (TAC) for various organs in control and different treatment groups are presented as Graph 2.
In liver samples, TAC values of aflatoxin treated groups (A1, A2 and A3) along with groups A3DS1 and A3DS2 were significantly lower compared to those of control group while there was an insignificant difference in TAC values of DS treated groups (DS1 and DS2) and remaining treatment groups.
TAC values of kidney samples were non-significant in DS treated groups (DS1 and DS2) and groups A1DS1, A2DS1, A1DS2 and A2DS2, while aflatoxin treated groups (A1, A2 and A3) and treatment groups A3DS1 and A3DS2 had significantly lower TAC values compared to that of control. Similar trend was observed in TAC values for muscles and plasma samples.
Antibody Response to Sheep RBCs
The values of Hemagglutination inhibition (HI) titers in response to intravenous injection of sheep RBCs are presented as Table 3.
Table 3
HI titers (log2) induced by Sheep RBCs in Control and Treatment Groups (Mean ± SD)
Groups
|
Antibody titers 7 day after primary injection
|
Antibody titers 14 day after primary injection
|
Antibody titers 7 day after secondary injection
|
Antibody titers 14 day after secondary injection
|
Total Ig
|
IgG
|
IgM
|
Total Ig
|
IgG
|
IgM
|
Total Ig
|
IgG
|
IgM
|
Total Ig
|
IgG
|
IgM
|
Control
|
4.7 ± 0.5
|
1.7 ± 0.5
|
3.0 ± 0.6
|
2.8 ± 0.4
|
1.8 ± 0.4
|
1.0 ± 0.0
|
6.7 ± 0.5
|
4.3 ± 0.5
|
2.3 ± 0.5
|
5.3 ± 0.5
|
3.5 ± 0.5
|
1.8 ± 0.4
|
A1
|
3.5 ± 0.5*
|
0.5 ± 0.5*
|
3.0 ± 0.6
|
2.2 ± 0.4*
|
0.8 ± 0.4*
|
1.3 ± 0.5
|
4.8 ± 0.4*
|
3.2 ± 0.4*
|
1.7 ± 0.8
|
4.2 ± 0.4*
|
2.3 ± 0.5*
|
1.8 ± 0.4
|
A2
|
2.3 ± 0.5*
|
0.3 ± 0.5*
|
2.0 ± 0.9*
|
1.5 ± 0.5*
|
0.2 ± 0.4*
|
1.3 ± 0.5
|
3.8 ± 0.4*
|
2.2 ± 0.4*
|
1.7 ± 0.5
|
3.5 ± 0.5*
|
2.2 ± 0.4*
|
1.3 ± 0.5
|
A3
|
1.2 ± 0.4*
|
0.2 ± 0.4*
|
1.0 ± 0.6*
|
0.3 ± 0.5*
|
0.2 ± 0.4*
|
0.2 ± 0.4*
|
2.7 ± 0.5*
|
1.2 ± 0.4*
|
1.5 ± 0.5*
|
2.7 ± 0.5*
|
1.2 ± 0.4*
|
1.5 ± 0.5
|
DS1
|
5.0 ± 0.6
|
1.7 ± 0.5
|
3.3 ± 0.5
|
2.7 ± 0.5
|
1.7 ± 0.5
|
1.0 ± 0.6
|
6.8 ± 0.4
|
5.3 ± 0.5
|
1.5 ± 0.5*
|
5.5 ± 0.5
|
3.3 ± 0.5
|
2.2 ± 0.4
|
DS2
|
5.2 ± 0.4
|
1.8 ± 0.4
|
3.3 ± 0.5
|
3.2 ± 0.4
|
2.3 ± 0.5
|
0.8 ± 0.4
|
7.2 ± 0.4
|
5.5 ± 0.8
|
1.7 ± 0.5
|
5.7 ± 0.5
|
3.8 ± 0.4
|
1.8 ± 0.4
|
A1DS1
|
4.8 ± 0.4
|
1.5 ± 0.5
|
3.3 ± 0.8
|
2.5 ± 0.5
|
1.5 ± 0.5
|
1.0 ± 0.9
|
6.5 ± 0.5
|
4.8 ± 0.4
|
1.7 ± 0.5
|
5.0 ± 0.6
|
3.0 ± 0.6
|
2.0 ± 0.0
|
A2DS1
|
3.2 ± 0.4*
|
0.5 ± 0.5*
|
2.7 ± 0.8
|
1.7 ± 0.5*
|
0.8 ± 0.4*
|
0.8 ± 0.4
|
5.2 ± 0.4*
|
3.2 ± 0.4*
|
2.0 ± 0.0
|
4.2 ± 0.4*
|
2.3 ± 0.5*
|
1.8 ± 0.4
|
A3DS1
|
2.7 ± 0.5*
|
0.5 ± 0.5*
|
2.2 ± 0.4*
|
1.5 ± 0.5*
|
0.8 ± 0.4*
|
0.7 ± 0.5
|
4.5 ± 0.5*
|
3.2 ± 0.4*
|
1.3 ± 0.8*
|
3.3 ± 0.5*
|
2.3 ± 0.5*
|
1.0 ± 0.0*
|
A1DS2
|
5.0 ± 0.6
|
1.5 ± 0.5
|
3.5 ± 0.5
|
3.0 ± 0.6
|
2.3 ± 0.5
|
0.7 ± 0.5
|
6.7 ± 0.5
|
4.8 ± 0.4
|
1.8 ± 0.4
|
5.3 ± 0.5
|
3.8 ± 0.4
|
1.5 ± 05
|
A2DS2
|
4.7 ± 0.5
|
1.3 ± 0.5
|
3.3 ± 0.5
|
2.7 ± 0.5
|
2.3 ± 0.5
|
0.3 ± 0.5
|
6.5 ± 0.5
|
4.8 ± 0.4
|
1.7 ± 0.5*
|
5.0 ± 0.6
|
3.8 ± 0.4
|
1.2 ± 0.4
|
A3DS2
|
3.0 ± 0.6*
|
0.5 ± 0.5*
|
2.5 ± 0.8
|
1.5 ± 0.5*
|
0.8 ± 0.4*
|
0.7 ± 0.5
|
5.3 ± 0.5*
|
3.2 ± 0.4*
|
2.2 ± 0.8
|
4.5 ± 0.8*
|
2.2 ± 0.4*
|
2.3 ± 0.8
|
Results with (*) are significantly different as compared to control (p ≤ 0.05) |
Description of abbreviations: A1= 100 µg AflatoxinB1/kg feed, A2= 200 µg AflatoxinB1/kg feed, A3= 600 µg AflatoxinB1/kg feed, DS1=5g Distillery Sludge/kg feed, DS2=10 g Distillery Sludge/kg feed, Ig= immunoglobulin
Table 4
Lymphoproliferative response (in mm) to subcutaneous injection of PHA-P in Control and Treatment Groups (Mean ± SD)
Groups
|
Response at 24 hrs
|
Response at 48 hrs
|
Control
|
0.38 ± 0.06
|
0.34 ± 0.05
|
A1
|
0.28 ± 0.02*
|
0.22 ± 0.03*
|
A2
|
0.21 ± 0.02*
|
0.16 ± 0.01*
|
A3
|
0.15 ± 0.02*
|
0.11 ± 0.01*
|
DS1
|
0.38 ± 0.05
|
0.34 ± 0.05
|
DS2
|
0.37 ± 0.06
|
0.32 ± 0.04
|
A1DS1
|
0.37 ± 0.07
|
0.34 ± 0.04
|
A2DS1
|
0.38 ± 0.05
|
0.32 ± 0.03
|
A3DS1
|
0.19 ± 0.02*
|
0.14 ± 0.02*
|
A1DS2
|
0.38 ± 0.06
|
0.34 ± 0.04
|
A2DS2
|
0.38 ± 0.04
|
0.33 ± 0.03
|
A3DS2
|
0.21 ± 0.02*
|
0.17 ± 0.02*
|
Results with (*) are significantly different as compared to control (p ≤ 0.05) |
Description of abbreviations: A1 = 100 µg AflatoxinB1/kg feed, A2 = 200 µg AflatoxinB1/kg feed, A3 = 600 µg AflatoxinB1/kg feed, DS1=5g Distillery Sludge/kg feed, DS2=10 g Distillery Sludge/kg feed.
At 07 days post primary dose, Total Antibody (Ig) Titers in aflatoxin treated groups (A1, A2 and A3) and treatment groups A2DS1, A3DS1 and A3DS2 were significantly lower, however, values in other treatment groups including DS1 and DS2 were non-significant compared to control. Similar trend was also observed in case of IgG titers. At 14 days post primary dose, Total Antibody Titers in treatment groups DS1, DS2, A1DS1, A1DS2 and A2DS2 were non-significant while those of groups A1, A2, A3 and remaining treatment groups were significantly lower when compared to those of control. Similar pattern was also noticed in IgG titers.
At 07 days post booster dose, Total Antibody Titers in aflatoxin treated groups (A1, A2 and A3) and treatment groups A2DS1, A3DS1 and A3DS2 were significantly lower while those of all other treatment groups including DS1 and DS2 were non-significant as compared to those of control. Similar trend was observed in case of IgG titers. At 14 days post booster dose, similar pattern of total Ig and IgG titers were observed as noticed on 07 days post booster injection.
In situ Lymphoproliferative Response to PHA-P
At 24 hours post PHA-P injection, lymphoproliferative response of aflatoxin treated groups (A1, A2 and A3) and treatment groups A3DS1 and A3DS2 were significantly lower; however, groups DS1, DS2 and other treatment groups exhibited non-significant results compared to control. Similar trend was noticed at 48 hours post PHA-P injection.
Mononuclear Phagocytic System Function Assay (Carbon Clearance Assay)
Phagocytic index elucidated by Carbon Clearance Assay in chicks fed different combinations of AFB1 and DS has been presented in Table 5.
Table 5
Phagocytic index determined by Carbon Clearance Assay in Control and Treatment Groups (Mean ± SD)
Groups
|
Response at 3 min
|
Response at 15min
|
Control
|
269.2 ± 4.8
|
31.2 ± 3.8
|
A1
|
356.5 ± 7.0*
|
78.0 ± 6.6*
|
A2
|
427.2 ± 7.9*
|
131.7 ± 5.8*
|
A3
|
504.5 ± 11.4*
|
211.2 ± 8.5*
|
DS1
|
267.8 ± 6.5
|
30.2 ± 5.2
|
DS2
|
264.1 ± 8.3
|
29.2 ± 4.3
|
A1DS1
|
270.8 ± 8.1
|
30.9 ± 5.3
|
A2DS1
|
271.6 ± 6.3
|
33.6 ± 5.7
|
A3DS1
|
481.5 ± 7.8*
|
180.6 ± 5.3*
|
A1DS2
|
276.1 ± 12.6
|
33.7 ± 6.8
|
A2DS2
|
275.7 ± 9.0
|
32.2 ± 7.2
|
A3DS2
|
277.1 ± 9.3
|
34.6 ± 8.2
|
Results with (*) are significantly different as compared to control (p ≤ 0.05) |
Values were determined as K = (logn OD1 − logn OD2)/(T2 − T1), where OD1 and OD2 are optical densities (640 nm) at times T1 and T2, respectively. Absorbance from a ‘0 min’ sample was considered as the ‘zero’ value for each respective sample. |
Description of abbreviations: A1 = 100 µg AflatoxinB1/kg feed, A2 = 200 µg AflatoxinB1/kg feed, A3 = 600 µg AflatoxinB1/kg feed, DS1=5g Distillery Sludge/kg feed, DS2=10 g Distillery Sludge/kg feed.
At 3 minutes post injection, the response of groups A1, A2, A3 and A3DS1 was significantly higher while that of groups DS1, DS2 and all other combination groups was non-significant when compared to that of control. Similar pattern of phagocytic index was also observed at 15 minutes post intravenous injection of carbon particles.