Download Full Text - International Journal of Livestock Research

International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
Original Research
Histochemical Localization of Oxidative and Hydrolytic enzymes in the Bursa
of Fabricius in Chicken (Gallus domesticus)
Jeyachandra Kempashi1 , Thandavan Arthanari Kannan2 *, Sabiha Hayath Basha1 ,
Angamuthu Raja3 and Geetha Ramesh1
1
Department of Veterinary Anatomy, Madras Veterinary College, Chennai - 600 007, INDIA
Centre for Stem Cell Research and Regenerative Medicine, Madras Veterinary College, Chennai - 600
007, INDIA
3
Department of Animal Biotechnology, Madras Veterinary College, Chennai - 600 007, INDIA
2
*Corresponding author: [email protected]
Rec. Date:
Jan 25, 2017 10:13
Accept Date:
Feb 15, 2017 04:16
Published Online:
March 02, 2017
DOI
10.5455/ijlr.20170215041648
Abstract
Histochemical localization of oxidative and hydrolytic enzymes was conducted in bursa of Fabricius of
four different age groups of broiler chicken viz., day-old, two, four and six weeks. Epithelium covering the
bursal plicae showed moderate positive reaction and bursal follicles showed mild positive reaction to
alkaline phosphatase (ALP) and acid phosphatase (ACP). However, interfollicular connective tissue,
Tunica serosa and Tunica muscularis showed negative reaction to ALP and ACP activity . Tunica
muscularis, cytoplasm of cortical and medullary lymphocytes, epithelium lining the plicae and in the
cortico-medullary junction and star-shaped cells in the medulla of some follicles showed positive reaction
for all dehydrogenases. Strong α-naphthyl acetate esterase activity was observed in the epithelium
covering the bursal plica. Increased cytochrome oxidase activity was observed as age advanced.
Key words: Chicken, Bursa of Fabricius, Oxidative and Hydrolytic Enzymes, Localization,
Histochemical Methods
How to cite: Kempashi, J., Kannan, T., Basha, S., Raja, A., & Ramesh, G. (2017). Histochemical
Localization of Oxidative and Hydrolytic Enzymes in the Bursa of Fabricius in Chicken (Gallus
domesticus). International
Journal
of
Livestock
Research, 7(3),
165-174.
DOI:
http://dx.doi.org/10.5455/ijlr.20170215041648
thymus in which T-lymphocytes develop and responsible for cell mediated immunity (CMI) (Glick et al.,
1956 and Warner, 1967). The chicken is a foundational model for immunological research and continues
[email protected]
DOI 10.5455/ijlr.20170215041648
Page
Fabricius, the primary site for B-lymphopoiesis which is responsible for humoral immunity (HI) and the
165
Introduction
Unlike other vertebrates, avian species have two discrete primary lymphoid organs, the bursa of
International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
to be a valuable animal model for insights into immune function. In particular, the development of B cells
in this unique organ, the bursa of Fabricius, has provided a novel opportunity to study B cell development
(Funk and Thompson, 1996). The bursa also functions as a peripheral gut-associated lymphoid organ.
Antigens presented via the cloaca and bursal lumen can stimulate specific antibody production by bursal
lymphocytes (Lupetti et al., 1984). Thus, the bursa plays a role in local gut immunologic defence. A
critical component of the local bursal response is the surface epithelium of the bursa overlying the
medullary region of the lymphoid follicles-namely, the follicle-associated epithelium (Houssaint et al.,
1986). There is a paucity of literature about localization of oxidative and hydrolytic enzymes during the
growth phase of the bird. Hence, the present study was designed to localize the oxidative and hydrolytic
enzymes in the bursa of Fabricius of chicken in different age groups.
Materials and Methods
Collection of Bursa
Twenty four birds for the present study were procured from Institute of Poultry Production and
Management, Madhavaram Milk Colony, Chennai. Bursal tissues were collected from four different age
groups viz., day-old, two, four, and six weeks. Six birds each were utilized from each age group for
histochemical study. The bursa was removed immediately after high cervical dislocation and fixed for
enzyme localization and for light microscopy (Kannan et al., 2015). All chemicals, substrates and kits
required for histochemical staining methods were of analytical grade and procured from Himedia®.
Methods
Collected tissue pieces of bursa of Fabricius were rinsed in normal saline and fixed in chilled formolcalcium. Cryosections of 10 µm thickness were obtained from both fresh unfixed tissues and formolcalcium fixed tissues for different histoenzymic studies. Chilled formol calcium fixed frozen sections
were used for localisation of alkaline phosphatase and acid phosphatase. Frozen sections from fresh
unfixed tissue
were
used for localisation of adenosine triphosphatase, α-esterase, glucose
6-phosphate dehydrogenase, lactate dehydrogenase, succinic dehydrogenase, ANAE and cytochrome
[email protected]
DOI 10.5455/ijlr.20170215041648
Page
1. Naphthol AS-BI phosphate method for acid and alkaline phosphatase (Bancroft and Gamble, 2008).
2. Demonstration of succinic dehydrogenase, glucose 6-phosphate dehydrogenase and lactate
dehydrogenase (Bancroft and Gamble, 2008).
3. Demonstration of diaphorase (Bancroft and Gamble, 2008).
4. Demonstration of adenosine triphosphatase (Bancroft and Gamble, 2008).
5. α-naphthyl acetate method for non-specific esterase (Bancroft and Gamble, 2008)
6. Demonstration of cytochrome oxidase (Bancroft and Gamble, 2008).
166
oxidase. The following histochemical staining methods were employed in the present study-
International Journal of Livestock Research
Vol 7 (3) Mar ’17
eISSN : 2277-1964 NAAS Score -5.36
Specific substrate for different enzymes and incubation time employed in histochemical staining methods
are given in Table1.
Table 1: Different enzyme substrates and their incubation time
Sl.
No.
Enzyme
1.
Alkaline phosphatase
2.
Acid phosphatase
3.
Adenosine triphosphatase
Glucose
6-phosphate
dehydrogenase
Succinic dehydrogenase
Lactate dehydrogenase
NADPH diaphorase
α-esterase
Cytochrome oxidase
4.
5.
6.
7.
8.
9.
Substrate
Naphthol
AS-BI
phosphate
Naphthol
AS-BI
phosphate
Adenosine triphosphate
Glucose
6-phosphate
(disodium salt)
Disodium succinate
Sodium DL-lactate
α-naphthyl acetate
Catalase
Incubation
time
Coenzyme
15 min
-
60 min
-
60 min
-
60 min
2 mg NADP
60 min
60 min
60 min
30 min
3 hours
2 mg NAD
2 mg NADPH
-
Results and Discussion
Alkaline phosphatase (ALP)
In the present study, epithelium covering the bursal plicae showed moderate positive reaction and bursal
follicles showed mild positive reaction to ALP (Fig.1). In day-old and two week-old birds, there was
moderate reaction and there after the bursal reaction to alkaline phosphatase enzyme decreased.
Interfollicular connective tissue, tunica serosa and tunica muscularis showed negative reaction to ALP
activity as reported by Hodges (1974) in chicken, Sabiha .H. Basha (1993) in Japanese quail and Indu et
[email protected]
DOI 10.5455/ijlr.20170215041648
Page
Fig. 1: Photomicrograph of the bursa of Fabricius of two week-old chicken showing epithelium (arrow
heads) and lymphoid follicle positive for ALP activity; LF-Lymphoid follicle Naphthol AS-BI
phosphate method x 100
167
al. (2005) in white pekin duck.
International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
Kaneko (1989) stated that ALP was a group of isoforms of non-specific enzymes which hydrolyzed many
types of phosphate esters whose natural substrates were unknown. ALP catalyze the dephosphorylation of
adenosine triphosphatase (ATP), were located in majority of the cells, and had a high specific activity in
the brush borders of secretory epithelium. Their activities were speculated to be a part of the ATP dependent membrane pumps and are believed to be associated with membrane phospholipid synthesis.
Acid phosphatase (ACP)
In all the age groups studied, there was a moderate positive reaction to ACP activity in the bursal
epithelium and mild reaction in the bursal follicles. The tunica serosa and tunica muscular is showed
negative reaction. The bursal epithelium showed stronger reaction to ACP compared to ALP (Fig. 2) as
mentioned by Ruuskanen et al. (1977) and Indu et al. (2005) who reported that the ACP activity was
moderate in the lymphoid follicle compared to the reaction in lining epithelium of bursal mucosa.
Fig.2: Photomicrograph of the bursa of Fabricius of two week-old chicken showing epithelium (arrow
heads) and lymphoid follicle positive for ACP activity; LF-Lymphoid follicle Naphthol AS-BI
phosphate method x 100
Adenosine triphosphatase (ATP-ase)
Epithelium covering the bursal plicae and muscular layer showed positive reaction for ATP -ase, whereas
cytoplasm of cortical and medullary lymphocytes showed negative reaction to ATP-ase activity (Fig. 3)
(Mazzone et al., 2003). However, in day-old birds, epithelium showed moderate positive reaction to ATPase when compared to other age groups and the reaction increased with advancement of age. Compared to
Page
168
follicle associated epithelium, stronger reaction was observed in interfollicular epithelium.
[email protected]
DOI 10.5455/ijlr.20170215041648
International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
Fig. 3: Photomicrograph of the bursa of Fabricius of four week-old chicken showing ATPase activity in
FAE (arrows) and tunica muscularis (TM) x40
ATP-ases were the good examples of trans-membrane proteins involved in the movement of ions through
the plasma membrane. A number of different ATP-ase enzymes are present in the cell, in which the
hydrolysis of ATP is coupled to transport ions across a membrane. For example ATP -ase (Na +, K+activated) are associated with the plasma membrane, mitochondrial ATP-ase of mitochondrial membrane
are associated with the oxidative phosphorylation (linked to H +-movement) and Ca 2+-activated ATP-ase
are associated with the endoplasmic reticulum and sarcoplasmic reticulum (Price and Stevens, 1996).
Glucose 6-phosphate dehydrogenase (GPDH)
Tunica serosa and muscular layer of the bursa showed mild positive reaction in all the age groups studied.
Inter follicular epithelium (IFE) showed strong positive GPDH activity when compared to Follicle
associated epithelium (FAE). Epithelium between the cortex and medulla of the lymphoid follicle,
cortical and medullary lymphocytes were also positive to GPDH in all the age groups studied (Fig. 4).
However compared to medulla, the cortex showed a stronger reaction to GPDH (Ruuskanen et al., 1977
and Mazzone et al., 2003). There was a strong positive reaction in the bursa of two week-old birds when
compared to day-old, four week and six week-old birds. Epithelium of six week-old birds showed a
strong GPDH activity compared to all other age groups, but the epithelium between cortex and medulla
was weakly positive. In the medulla of some follicles, cytoplasm of star shaped cells showed a strong
positive reaction to GPDH in all age groups studied. These star shaped cells could probably be made up
of reticulo-epithelial cells and macrophages (Mazzone et al., 2003). Endothelium of blood vessels also
Page
169
showed a strong positive reaction to GPDH.
[email protected]
DOI 10.5455/ijlr.20170215041648
International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
Fig.4: Photomicrograph of the bursa of Fabricius of six week-old chicken showing GPDH Activity; EFollicular epithelium e-Epithelium in the cortico-medullary junction Nitro BT method x 400
GPDH catalyses the first step in the pentose phosphate pathway, which produces NADPH. This reductant
is essential in many biosynthetic pathways and also protects the cells from oxidative damage by hydrogen
peroxide (H2 O2 ) and superoxide free radicles and highly reactive oxidants generated as metabolic
byproducts (Nelson and Cox, 2005). This indicated that in the present study, the interfollicular epithelial
cells were metabolically more active than follicle associated epithelial cells.
Lactate dehydrogenase (LDH)
Epithelium, basement membrane and tunica muscularis of the bursa showed intense reaction to LDH
enzyme when compared to the tunica serosa, cortical and medullary lymphocytes. Star shaped cells
present in the medulla of some follicles showed intense positive reaction to LDH (Fig.5).
blood vessels showed mild positive reaction to LDH. As age advanced, enzyme activity also increased
from day-old to six week-old birds as reported by Ruuskanen et al. (1977) and Mazzone et al. (2003).
[email protected]
DOI 10.5455/ijlr.20170215041648
Page
Compared to FAE, IFE showed strong positive reaction to LDH. Interfollicular connective tissue and
170
Fig. 5: Photomicrograph of the bursa of Fabricius of day-old chicken showing LDH activity in the FAE
(arrow heads) and in the cortico-medullary junction (arrows); M-Medulla C-Cortex
S- Star shaped cells; Nitro BT method x 100
International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
NADPH Diaphorase
In all the age groups studied, IFE and FAE showed strong positive reaction to NADPH diaphorase.
Cytoplasm of cortical and medullary lymphocytes of the lymphoid follicle was moderately positive.
Epithelium between cortex and medulla showed mild positive reaction to the enzyme. The intensity of
reaction increased as age advanced. These findings were in accordance with Mohamed Ali et al. (1996).
Succinic dehydrogenase (SDH)
Tunica serosa and muscularis layer of the bursa showed mild positive reaction to SDH in all the age
groups studied. There was a mild reaction in the epithelium covering the plicae and basement membrane
between the cortex and medulla compared to LDH and GPDH. The cytoplasm of cortical and medullary
lymphocytes showed mild positive reaction to SDH in the present study. However compared to medulla,
the lymphocytes in cortex showed stronger enzyme reaction. FAE showed mild positive reaction to SDH
when compared to IFE (Fig. 6). The basal part of the epithelium showed strong positive reaction.
Fig. 6: Photomicrograph of the bursa of Fabricius of two week-old chicken showing SDH activity in
tunica muscularis (TM), FAE (arrow heads) and in the cortico-medullary; M-Medulla
C-Cortex; Nitro BT method x 40
The bursal reaction to SDH enzyme, increased as age advanced from day-old to six week-old birds.
Endothelium of capillaries present in the interfollicular area showed moderate positive reaction to SDH.
The tip of the mucosal epithelium showed strong positive reaction when compared to base of the
epithelium in the bursa of two, four and six week-old birds. This increase in enzyme activity could be due
to the increased functional activity of the organ because SDH is the only membrane bound enzyme in the
In all the age groups studied, epithelium covering the bursal plicae showed strong positive reaction to αnaphthyl acetate esterase (ANAE) and in some part of the epithelium showed spot-like esterase activity
(Fig. 7). Tunica serosa, muscularis and lymphoid follicles showed negative reaction to ANAE activity. T
[email protected]
DOI 10.5455/ijlr.20170215041648
Page
Non-specific Esterase
171
citric acid cycle (Nelson and Cox, 2005).
International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
lymphocytes in interfollicular connective tissue and subepithelial region showed strong reaction to ANAE
as reported by Dolfi et al. (1988) in chicken and Saifuddin et al. (1988) in Shaver cockerels.
M-Medulla, C-Cortex, Nitro BT method x 40
Fig. 7: Photomicrograph of the bursa of Fabricius of two week-old chicken showing ANAE
activity in FAE (arrow heads); Ic - Interfollicular connective tissue, α-naphthyl acetate method x 40
Cytochrome oxidase
The capsule and interfollicular connective tissue of bursa showed mild positive reaction to cytochrome
oxidase. In four week-old and six week-old birds, the cortical and medullary lymphocytes showed a
moderate positive reaction, when compared to day-old and two week-old birds. In two week-old birds,
apical surface of the epithelium covering the plicae showed intense positive reaction to cytochrome
oxidase (Fig. 8). These results were in contrary to the findings of Ruuskanen et al. (1977) who reported
that faint activity was seen in all tissues with no specific appearance in the bursa of chicken. Cytochrome
oxidase and NADPH helps in the synthesis of glucocorticoids (GC) and these GC have long been shown
to induce rapid apoptosis in immature thymocytes and B cells (De and Guha, 1987 and Oskar et al.,
[email protected]
DOI 10.5455/ijlr.20170215041648
Page
Fig. 8: Photomicrograph of the bursa of Fabricius of two week-old chicken showing Cytochrome oxidase
activity in FAE (arrow heads), tunica muscularis (TM) and lymphoid follicle (LF); DAB x 40
172
2001).
International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
Conclusion
Histochemical localisation of oxidative and hydrolytic enzymes was observed in bursa of Fabricius in
different age groups of Chicken. The epithelium covering the bursal plicae and lymphoid follicles showed
mild to moderate ALP abd ACP activity. The ALP started declining as age advanced. Epithelium
covering the bursal plicae, muscular layer and cytoplasm of cortical and medullary lymphocytes of
lymphoid follicle showed negative reaction to adenosine triphosphatase activity. In all the age groups
studied, there was mild positive reaction to GPDH in tunica serosa and muscular layer. IFE showed
strong positive GPDH activity when compared FAE. Epithelium between cortex and medulla and star
shaped cells in the medulla of some follicles showed strong positive reaction to GPDH. As age advanc ed
the intensity of enzyme also increased. Compared to all other dehydrogenases, there was a mild reaction
to SDH in the bursal tissue. In all the age groups studied, positive reaction was noticed for NADPH
diaphorase, ANAE and cytochrome oxidase activity.
Acknowledgment
Authors are highly thankful to the Dean, Madras Veterinary College, Chennai – 600 007, India, for
providing necessary financial support.
[email protected]
DOI 10.5455/ijlr.20170215041648
Page
1. Bancroft, J.D. and M. Gamble, 2008. Theory and Practice of Histological Techniques. 6th Edn.
Churchill Livingstone Publishing Co. Ltd., Honkong.
2. De, M. and B. Guha, 1987. The role of glucocorticoids on histomorphology, nucleic acid and protein
level of bursa of Fabricius of chicken. Gegenbaurs, Morphol. Jahrb., 133: 353.
3. Dolfi, A., F. Giannessi, F. Bianchi and M. Lupetti, 1988. Distribution of esterase activity at the level
of the epithelium of the diffusely infiltrated area (DIA) and of the cloaca in the Gallus domesticus: An
ultrastructural study. Anat. Rec., 221 (1): 469–474.
4. Funk, P.E. and C.B. Thompson, 1996. Current concepts in chicken B cell development. Curr. Top.
Microbiol. Immunol, 212: 17-28.
5. Glick, B., T. S. Chang and R. G. Jaap, 1956. The bursa of Fabricius and antibody production. Poult.
Sci., 35:224–225.
6. Hodges,
R.D.,
1974.
The
Histology
of
Fowl.
Academic
Press,
London.
pp: 213-221.
7. Houssaint, E., E. Diex and M.M. Hallet, 1986. The bursal microenvironment: phenotype
characterization of the epithelial component of the bursa of Fabricius with the use of monoclonal
antibodies. Immunol, 58: 43-49.
8. Indu, V.R., J.J. Chungath, K.R. Harshan and N. Ashok, 2005. Morphology and histochemistry of the
bursa of Fabricius in White Pekin ducks. Indian. J. Anim. Sci., 75 (6): 637-639.
9. Kaneko, J.J., 1989. Clinical biochemistry of domestic animals. 4th edition, Academic
Press,
California.
10. Kannan,T.A., Geetha Ramesh, S.Venkatesan, S.Ushakumari and Sabiha Hayath Basha, 2015.
Cytoarchitecture of Periarterial lymphatic sheath (PALS) in Chicken Spleen – Light and Transmission
electronmicroscopic study. Int.J. of Adv.Res., 3:11, 1167 – 1172.
173
References
International Journal of Livestock Research
eISSN : 2277-1964 NAAS Score -5.36
Vol 7 (3) Mar ’17
Page
174
11. Lupetti, M., A. Dolfi, T. Malatesta and S. Michelucci, 1984. A contribution to the study of the
regulatory system of local immune response in “Gallus domesticus.” Dev. Comp. Immunol., 8: 663.
12. Mazzone, A.M., M. Aita, F. Gabrielli, E. Moriconi and D. De Orsi, 2003. Identification of cells
secreting a thymostimulin-like substance and examination of some histoenzymatic pathways in aging
avian primary lymphatic organs: II. Bursa of Fabricius. Eur. J. Histochem., 47 (4):
325-338.
13. Mohamed Ali., A. Chan and S. Leong, 1996. Histochemical and immunohistochemical localisation of
nitrergic neuronal and non-neuronal cells in the bursa of Fabricius of the chicken. Cell. Tissue. Res.,
285: 273–279.
14. Nelson, D.L. and M.M. Cox, 2005. Lehinger Principles of biochemistry. 4th edition, W.H. Freeman
and Company, England.
15. Oskar, L., D. Hermann, G. Jan Wiegers, V. Melanic and W. Georg, 2001. Glucocorticoid production
in the chicken bursa and thymus. Int. Immunol., 13 (6): 769-776.
16. Price, N.C. and L. Stevens, 1996. Fundamentals of enzymology. 2nd Edition, Oxford University press.
New York. pp: 400.
17. Ruuskanen, O., A. Toivanen and J. Raekallio, 1977. Histochemical characterization of chicken
lymphoid tissues. Dev. Comp. Immunol., 1 (3): 231-240.
18. Sabiha .H. Basha, 1993. Histomorphology and histochemistry of the thymus and the bursa of Fabricius
in Japanese Quail (Coturnix coturnix japonica). M.V.Sc thesis submitted to Tamil Nadu Veterinary
and Animal Sciences University.
19. Saifuddin, M., B.W. Manktelow, K.M. Moriarty, N.H. Christensen and M.J. Birtles, 1988. Age-related
functional changes in the follicle-associated epithelium of the bursa of Fabricius in Shaver cockerels.
NZ. Vet. J., 36 (3):108-111.
20. Warner, N.L., 1967. Immunological role of avian thymus and bursa of Fabricius. Folia. Biol., 13:1-17
[email protected]
DOI 10.5455/ijlr.20170215041648