The Journal of Poultry Science,./ : +,/ῌ +-+ , ,**2 http://www.jstage.jst.go.jp/browse/jpsa Copyright ῌ ,**2, Japan Poultry Science Association. doi:+* . ,+.+ /jpsa. ./ . +,/

Mohammad N. Islam+++ , Mohammad Z.I. Khan , Mir R. Jahan , Mohammad R. Karim+, and Yasuhiro Kon

+ Department of Anatomy and Histology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh,,*, , Bangladesh , Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo*0* , Japan

The aims of this study was to find out the histological di# erences of the mucosa and comparative analysis of Ig-containing plasma cells among the di# erent segments of the gastrointestinal tract of broiler and native chickens of Bangladesh. The conventional histological study revealed that the lining epithelium of the proximal segments (esophagus, crop, and proventriculus) were thicker in the broiler. The esophageal glands were more in the broiler than the native chickens. The villi of the duodenum, jejunum and ileum were slender and longer in the broiler in comparison to the native chickens. The number of goblet cells in the duodenum, jejunum, and ileum was more in the native chickens than the broiler. The indirect immunohistochemistry revealed that very few immunoglobulin (Ig)-containing plasma cells were present in the epithelium and lamina propria of esophagus, crop, and proventriculus of the broiler and native chickens. The frequency of the population of these cells were abundantly located in the lamina propria, around the intestinal gland and in the core of the villi from duodenum to ileum of broiler and native chickens. The intraepithelial IgA-containing epithelium was observed only in the epithelium of the native chickens. Segmental variation of Ig-containing plasma cells was noticed in these two strains of chickens. The IgA-, IgG-, and IgM-containing plasma cells were significantly more in the most of the segments of the small intestine of the native chickens. This suggested that besides the existence of histological variation in the gastrointestinal tract of broiler and native chickens, the Ig-containing plasma cells were significantly more in the di# erent segments of the digestive tract of native chickens possible due to their scavenging.

Key words: broiler, gastrointestinal tract, immunoglobulin cells, mucosa, native chickens J. Poult. Sci.,./ : +,/ῌ +-+ , ,**2

῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍ +313 +32* +322 Introduction Bienenstock, ; Befuset al., ; Arai et al., ). This mucosal immune system undergoes changes with It is well known that the mucosal immune system play aging (Khanet al., +330 a); influenced by sex hormones an important role in the defense against microorganisms (Milicevic and Milicevic,+33- ; Khanet al., +330 b), and because of the presence of migrating immunocompetent antioxidant such as selenium and vitamin E (Khan et al., cells (ICs), such as globule leukocytes (GLs), lympho-,**2 ). cytes, macrophages and plasma cells in this system (Hus- The digestive tract of chickens was the major site of band and Gowans,+312 ; McDonald and Spencer, +33. ). antigenic challenge in the body, being continuously ex- When mucosa was expose to foreign antigens, the contin- posed to antigens and commensal bacteria (Mowat and uous epithelial layer and covering mucous layer function Viney,+331 ). To deal with these challenges the mucosa of as the first line of defense. In addition to the nonspecific the digestive system of chickens are populated by a signifi- defense system, the mucosa-associated lymphoid tissues cant proportion of T lymphocytes, B lymphocytes, plasma (MALT) supply lymphocytes and plasma cells that con- cells, macrophages, dendritic cells and non-professional tribute to the local immune system (McDermott and antigen presenting cells (APCs) (McDonald and Spencer, +33.). These cells principally developed and mobilize from “Gut Associated Lymphoid Tissue (GALT)”, which Received: July-* , ,**1 , Accepted: November ,2 , ,**1 Correspondence: Dr. Md. Z.I. Khan, DepartmentofAnatomy and may include esophageal tonsils, Meckel’s diverticulum, Histology, Faculty of Veterinary Science, BangladeshAgricultural Peyer’s patches, cecal tonsils, and bursa of Fabricius (Arai University, Mymensingh ,,*,, Bangladesh. et al., +322). The plasma cells were the cells of the im- (E-mail: mzikhan+3/1 @yahoo.co.in) mune system that secrete large amounts of antibodies 126 J. Poult. Sci.,./ ( , )

(McGheeet al., +331 ). They di # erentiate from B cells finally the sections were cut at 0 -m m thickness using sliding upon stimulation by an antigen and were the principal microtome (MIC/*3 , Euromex, Japan). The sections e# ector cells involved in humoral immunity. These im- were then stained with Hematoxylin and Eosin staining munoglobulin (Ig)-containing plasma cells are distributed method (Gridley,+30* ). in the lamina propria of the respiratory tract (Bienenstock Antibodies et al.,+33-), digestive tract (Jeurissen et al., +323 ), genital The antibodies for detecting Igs-containing plasma cells tract (Khanet al.,+331 ), lymphoid organs (Khan et al., used in this experiment were normal rabbit serum (Bio- +332), and Harderian gland (Khanet al., ,**1 ). In the source, Camarillo, California, USA), goat anti-chicken chickens, particularly in the digestive tract, the Ig-con- IgA (Bethyl Lab, USA), goat anti-chicken IgG (Bethyl taining plasma cells have been reported in the esophageal Lab, USA), goat anti-chicken IgM (Bethyl Lab, USA), tonsil, proventriculus and Payer’s patches (Araiet al., and HRP-conjugated rabbit anti-goat IgG (Bethyl Lab, +322; Jeurissenet al., +323 ). USA). In Bangladesh, most of the farmers rear Kasilla broiler Immunohistochemical staining method and native chickens (Gallus domesticus ). Both of the Indirect immunoperoxidase staining method was done chicken breeds attain a weight around+*** gm at + month for the study of the distributional pattern and frequency of and0 month of their ages, respectively. The broilers the Ig-containing plasma cells in the di# erent segments of reared in well hygienic condition, in contrast, the native digestive tract of Kasilla broilers and native chickens. chickens are scavenger in nature (Rahamanet al., ,**- ) The tissues were fixed in ice-cold PLP (Periodate-lysine- fed by kitchen by-products, seeds and grains, garden left- paraformaldehyde), dehydrated in a series of graded alco- over, insect, green grasses and all other human refusal that hol, cleared in xylene, and embedded in para$$ n. Para n would otherwise go to waste. These two strains of chick- sections,0 -m m thickness, were immunostained by the in- ens reared in two di# erent conditions totally. Therefore, direct immunoperoxidase method as described earlier the present research work was designed to understand the (Khanet al., +331 , ,**1 ). histological variation of the mucosa, and the frequency of Histoplanimetry Ig-containing plasma cells in the gastrointestinal tract Only lamina propria and core of the villi of the di# erent (esophagus to ileum) of broiler and native chickens of segments of gastrointestinal tract were selected for count- Bangladesh. ing of Ig-containing plasma cells, because the frequencies Materials and Methods of immune cells were more and homogenously distributed in these regions. The immunopositive cells in the di# erent Chickens segments of gastrointestinal tract of broilers and native A total of ten adult chickens, five from Kasilla broiler chickens were counted in,* fields using ocular microme- (-* days old) and five from native chickens (Gallus do- ter at a magnification of.* , where Ig-containing plasma mesticus) (0# months old) irrespective of sex were pur- cells were evenly and di usely distributed and their rela- chased from a local market of Bangladesh Agricultural tive frequency per*+ . mm, was calculated according to University. The broilers were reared by the commercial Weibel (+303 ). The areas containing the nodular aggre- poultry farmers with commercial feed (Aftab poultry gates of lymphocytes and plasma cells were excluded from feed) and waterad libitum before selling these chickens in counting. The goblet cells also counted in the similar way the market, in contrast, the native chickens were fed by in the mucosa of duodenum, jejunum, and ileum of broiler kitchen by product, garden left over and green grasses. and native chickens. Tissues used for the study Statistical Analysis The birds were killed by cervical sub-luxation method. The number of Ig-containing plasma cells in the lamina After exsanguinations di# erent segment of gastrointestinal propria and core of the villi, and goblets cells in the tract e.g., esophagus, proventriculus, crop, duodenum, epithelium of gastrointestinal tract was counted and com- jejunum, and ileum of both the breeds of chickens were pared between broiler and native chickens and the data used for study purpose which were free from pathological were evaluated by Student’st - test (Zar,+330 ). lesions. The cecal tonsil is the lymphoid organ, and the Results colorectum is the terminal part of the digestive tract which has less importance in the digestion of food, therefore we Histological studies of the mucosa of gastrointestinal tract omitted these two parts of the digestive tract from the of broiler and native chickens present study. The mucosa of the digestive tract of chickens was Preparation of samples for histological studies consisted of lamina epithelia, lamina propria, and lamina For histological studies all the samples were cut into muscularis. The lining epithelium of the esophagus, crop pieces and then fixed in the “Bouin’s fluid” (Gridley, and proventriculus was lined by stratified squamous epi- +30*), dehydrated in a series of ascending grades of thelium which was thickened in the broilers than the alcohol, cleared in several changes of xylene, and in- native chickens in the present study (Fig.+ a-b). The filtrated with di#$ erent grades of melted para n in the mucous type of glandular cells was located in the lamina oven. The tissues were then embedded in para$ n and propria of the esophagus of these chickens. These es- Islamet al. : Mucosa and Ig-Cells in Chickens 127

Fig.+ . Histological sections of the esoph- agus of broiler (+ a) and native chickens (b).+ The epithelium (E) of the broiler is thicker than the native chickens. The mu- cous glands (asterisks) located in the lam- ina propria (LP) of the esophagus is com- paratively showing more in the broiler chickens. HE staining. Scale bars repre- sent+2* m m.

Fig., . Histological sections of the jeju- num and ileum of broiler (,, a, c) and native chickens (,, b, d). The villi (v) of the jejunum of broiler (, a) are longer and the apex is blunt. In comparison, the villi (v) of the jejunum of native chickens (, b) are shorter in their length. The villi (v) of the ileum of broiler (, c) are longer and pointed at their apex, whereas, the villi (v) of the ileum of the native chickens (, d) are shorter and their apex is wider. HE stain- ing. Scale bars represent+2* m m.

Table+ . The segmental comparison of the frequencies the lamina propria of the duodenum in both the strains of of goblet cells in the gastrointestinal tract of broiler the chickens. and native chickens (Duodenum to ileum). In the present study, the jejunal villi of the broiler were Chickens Duodenum Jejunum Ileum longer, its apical part was blunt (Fig., a). The villi of the Broiler /04,/ῌῌ +,4,+ +//4,/ ++4,1 +*1- ῌ -04/3 jejunum of the native chickens, in contrast, were shorter than broiler (Fig., b). The number of goblet cells was 2241/ῌῌ 042. -** +14+3 +//.41/ ῌ ++04-0 Native * * more in the jejunum of native chickens than that of the The present table is showing that the goblet cells are more in broilers (Table+ ). The common sites for the presence of their frequencies in the di# erent segments of gastrointestinal plasma cells in the jejunum were the core of the villi and tract of native chickens than the broilers. the lamina propria in these two strains of chickens. The villi of the ileum were long and slender, and, the goblet cells were more in comparison to the other seg- ophageal glands were more in the broiler than those of ments of the digestive tract. In the broiler, most of the native chickens (Fig.+ a-b). In the present study there villi of the ileum had pointed apical part and wide basal were01ῌ glandular units around an esophageal crypt of part (Fig. , c). In contrast, the villi of the native chickens the broiler, and in contrast, around the crypt of the were shorter and its apex was wider (Fig., d). The esophagus of native chicken there were-/ῌ or less glandu- presences of goblet cells were more in the ileum of the lar units. native chickens than those of the broilers (Table+ ). The distal part of the segments of the digestive tract of Distribution and frequency of immunoglobulin (Ig)-con- the chickens was lined by simple columnar epithelium. In taining plasma cells in the mucosa of di# erent segments of broiler, the apical parts of the villi of the duodenum were the digestive tract of broiler and native chickens slightly pointed, and the basal parts were wider. In In the present study, the immunoglobulin (Ig)-con- contrast, the apical part of the villi was blunt and the base taining plasma cells were not stained abundantly in the was also wider in the duodenum of the native chickens. lamina propria of the esophagus, crop, and proventriculus The number of goblet cells was more in the duodenum of of the broiler and native chickens; however, few cells of the native chickens (Table+ ). The most common sites for the stratum spinosum of broiler esophagus (Fig.- a) and the presence of plasma cells were the core of the villi and few cells of the lamina propria of the esophagus of native 128 J. Poult. Sci.,./ ( , )

IgM-positive cells were more in the proventriculus, duode- num, and ileum (Fig.2+* and ); IgG-positive cells were more in the proventriculus and ileum (Fig.3 ). In the broiler, however, the IgA- and IgM-positive cells were more in the jejunum (Fig.2+* and ); and IgG-positive cells were more in the duodenum and jejunum (Fig.3 ). Discussion The various system of chickens, such as, digestive, re- spiratory and reproductive system are exposed frequently too many microorganism both externally and internally. Among them the digestive system of broiler and native chickens are more frequently exposed to microorganisms because feed is one of the main source of microorganism in chickens. Moreover, the native chickens are scavenger animal, and in most of the small house hold farms the quality feed are not supplied to the broilers in Bangladesh. The mucosal lining barrier and the immunocompetent cells (plasma cells containing di# erent classes of immu- noglobulins) of the mucosa of digestive tract play a defen- sive role normally. Therefore, the present research fo- cused on these two points only. In the present study, the mucosal fold of esophagus, crop, and proventriculus was thicker in broiler in compar- ison to the native chickens, and esophageal glands were Fig.- . Immunostained sections of the esophagus of more in the broiler than the native chickens. The apical broiler (-- a) and native chickens ( b) showing anti parts of the villi of the duodenum of broiler in the present IgA-positive cells (arrow heads) in the epithelium (E) study were slightly pointed and the basal part of the villi of the broiler and in the lamina propria (LP) of the was wider. On the other hand, the base of the villi was 1/ native chickens. Scale bars represent .m m. wider and the apex of the villi was blunt in the duodenum of the native chickens. The villi of the jejunum and ileum of broiler were longer in comparison to the native chickens chickens were stained for IgA-containing plasma cells in the present study. Reports in this regard were not (Fig.- b). found in the available literature. The population of Ig-containing plasma cells was ab- In the present study, the immunoglobulin (Ig)-con- undantly located in the lamina propria interglandular taining plasma cells were not stained abundantly in the space and in the core of the villi of the di# erent segments lamina propria of the esophagus and crop of the broiler of intestine of the digestive tract of both the broiler and and native chickens, however, scattered few Ig-containing native chickens in the present study (Fig../ῌ ), and their plasma cells were stained in the epithelium and lamina frequency of population were more from duodenum to propria these two segments of broiler and native chickens. ileum in both the strains of chickens (Fig,0+*ῌ ). In the The frequencies of the population of Ig-containing plasma proventriculus, however, very few Ig-containing plasma cells were abundantly present from proventriculus to the cells were located in both the strains of the chickens (Fig. ileum of broiler and native chickens. In the broiler and 0+*ῌ ). In the present study it was found that, IgA-con- native chickens the IgA-containing plasma cells were more taining plasma cells were more followed by IgM- and IgG- followed by IgM- and IgG-positive cells. This observation containing plasma cells from proventriculus to ileum in was similar with the statement of McDermott and Bien- broiler; and in native chickens, the frequency of these cells enstock (+313 ) in rat. were more from proventriculus to jejunum (Fig.01ῌ ). In the present study segmental variation in the presence The Ig-containing plasma cells were also intraepithelial in of Ig-containing plasma cells was observed. The frequen- the intestinal glands of the jejunum of native chickens in cies of occurrences of IgA-, IgG-, and IgM-containing the present study (Fig./ b). The glandular cells of this plasma cells were significantly more from the duodenum segment of the native chickens were reactive for only anti- to ileum in both the broilers and native chickens in the IgA (Fig./ b). present study. In the lamina propria of the White leghorn The frequencies of occurrences of Ig-containing plasma strain A (WLA), Suzanet al. (+323 ) recognized some cells, in the present study, were varied among the seg- IgA-, and IgM-containing plasma cells in the di# erent ments of the digestive tract of the broiler and native segments of intestines, and all three types of Igs-con- chickens (Fig.2+*ῌ ). In the native chickens, the IgA- and taining plasma cell in the proventriculus only. In contrast, Islamet al. : Mucosa and Ig-Cells in Chickens 129

Fig../ . Immunostained sections of the Fig. . Immunostained sections of the jejunum of broiler (./ a) and native chick- ileum of broiler ( a) and native chickens ens (./ b) showing IgG cells in the core of ( b) showing IgA cells in the core of the the villi (CV) and around the intestinal villi (CV) and around the intestinal gland (IG) of the lamina propria.The gland (IG) of broiler and native chick- IgG-positive cells are more in the jeju-ens. In the native chickens the intra- num of broiler than the native chickens. epithelial IgA cells (arrow heads) are Scale bars represent1/ .mm m. stained. Scale bars represent 1/ . m.

Fig.01 . Frequencies of Ig-containing plasma cells in Fig. . Frequencies of Ig-containing plasma cells in the di## erent segments of the gastrointestinal tract of the di erent segments of the gastrointestinal tract of broiler.The present graph is showing that the fre- native chickens. The present graph is showing that the quency of population of IgA is higher in all the frequency of IgA-positive cells are higher in the pro- segments of broiler followed by IgM and IgG cells. ventriculus, duodenum, and jejunum followed by IgM These Ig-containing plasma cells are more in the duo- and IgG cells. The frequencies of occurrence of these denum and jejunum followed by ileum and proven- Ig-positive cells are more in the duodenum and ileum triculus. Values are given as the meanῌ῍ SEM (n. ). followed by jejunum and proventriculus. Values are given as the meanῌ῍ SEM (n. ). 130 J. Poult. Sci.,./ ( , )

Fig.2+* . Comparison of frequencies of IgA-containing Fig. . Comparison of frequencies of IgM-containing plasma cells among di## erent segments of the gastro- plasma cells among di erent segments of the gastro- intestinal tract in between broiler and native chick- intestinal tract in between broiler and native chick- ens.The present graph is showing that the frequen- ens. The present graph is showing that the frequen- cy of IgA-positive cells is higher in the proventricul- cy of IgM-positive cells is more in the proventricul- us, duodenum, and ileum of native chickens; in con- us, duodenum, and ileum of native chickens; in con- trast, IgA-positive cells are more in the jejunum of the trast, IgM-positive cells are more in the jejunum of the broiler. Values are given as the meanῌ῍ SEM (n.. ). broiler. Values are given as the mean ῌ῍ SEM (n ).

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