Histological and Histochemical Studies on the Lingual Tonsil of the Buffalo (Bubalus bubalis)

: The study was conducted on lingual tonsil of six adult buffaloes (5-6 years of age) of the local mixed breed to explore histomorphological characteristics with functional significance. The mucosal surface of the lingual tonsil lined by stratified squamous keratinized epithelium was comprised of strata basale, spinosum, granulosum and corneum whereas towards the deeper folds the epithelium was stratified squamous non-keratinized. The latter epithelium modified into reticular epithelium especially toward the deeper portion and was characterized by a reduced number of epithelial cells, loss of distinct strata and heavy infiltration of lymphoid cells. The propria submucosa had loose irregular connective tissue along with glandular and lymphoid tissue. The lymphoid tissue was mainly distributed in the form of lymphoid follicles and diffused arrangement. The follicles of varying shapes and size showed darkly stained corona and lightly stained germinal centre were surrounded by parafollicular areas. The parafollicular areas possessed high endothelial venules (HEVs) with large sized endothelial cells having round to oval nuclei with distinctly visible centric or eccentric nucleoli. These HEVs are involved in trafficking of lymphocytes by transendothelial and interendothelial migration. The mucous glandular acini presented strong reactions for glycogen, acidic mucopolysaccharides, weakly sulfated mucosubstances, hyaluronic acid, sialomucins and mucins as demonstrated by different histochemical techniques. The secretions of acini also showed the presence of more than 4% content of cysteine. The presence of modified reticular epithelium and associated lymphoid tissue suggested that the lingual tonsil may be involved in sampling of oral antigens and transporting them to the underlying mucosal lymphoid tissue for processing and initiation of immune responses. The future microbiological studies may exploit the tonsil as a targeted organ for improved delivery of existing mucosal vaccines and development of new strategies for oral vaccines.


INTRODUCTION
The tonsils consist of accumulation of lymphocytes which are usually concentrated in the primary and secondary lymph nodules that are surrounded by extranodular zones and covered by reticular epithelium [1]. They constitute part of the integrated pharyngeal mucosal immune system [2] which initiates an immune response to specific antigens encountered along the mucosal surface [3]. The lingual tonsil at the root of the tongue is a component of Waldeyer's ring and an important site for invasion of microbial pathogens and immune surveillance [4]. This location involves an important role for tonsils as secondary lymphoid tissue in the immunological response against antigens which enter the body during feeding or breathing [5,6]. The histomorphological features of the lingual tonsil had earlier been described in the horse [4,7], bovine ( [8,9] and recently the Egyptian buffalo calf [10]. Therefore, the present study was envisaged to explore microscopic features of the lingual tonsil in the buffalo with emphasis on mucosal epithelium along with its modifications and distribution of lymphoid tissue that might contribute a pivotal role in the first line of defence *Address correspondence to this author at the Department of Veterinary Anatomy, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, India; Tel: +919466402637; E-mail: pkumar@luvas.edu.in, pawanrajoria2000@rediffmail.com against ingested or inhaled pathogens entering through mouth cavity.

Histomorphology and Histochemistry
The heads of six adult buffaloes (5-6 years of age) of local mixed breed, irrespective of sex were procured from Municipal Slaughter House, Ghazipur, New Delhi, India after routine decapitation. The tissues were collected from the root of the tongue and fixed in 10% neutral buffered formalin solution for 48 hours. The fixed tissues were processed by routine paraffin technique and sections of 5-6 µ were cut and stained with routine Harris' hematoxylin and eosin stain, Gomori's method for reticulum, Weigert's method for elastic fibres [11], Crossman's trichrome stain for collagen fibres [12], McManus' method for glycogen (PAS), Alcian blue method for mucosubstances (pH 2.5), PAS-Alcian blue method for acidic and neutral mucosubstances (pH 2.5), Meyer's mucicarmine method for mucin, colloidal iron method for acid mucopolysaccharides, diastase digestion method [11], and performic acid-Alcian blue method for proteins [13].

RESULTS
The lingual tonsil was lined by stratified squamous keratinized epithelium towards outer surface. The free surface of the epithelium was uneven, irregular and uniform, whereas the deeper surface presented the papillary pegs (Figures 1, 2). The epithelium was comprised of the different strata having a varying number of rows. The stratum basale, present towards the basement membrane, was having oval to elongated strongly basophilic nuclei which were vertically oriented (Figure 2). These strongly basophilic nuclei contained chromatin material which was aggregated into smaller clumps irregularly throughout the nucleoplasm. These nuclei contained 1-2 nucleoli which were centric/ eccentric in position. These nuclei towards the papillary pegs became narrow cylindrical like structure and were comparatively more basophilic, because of uniform distribution of the chromatin material that masked the presence of the nucleoli. The cytoplasm of these cells was eosinophilic and finely granular (Figure 2). The stratum spinosum was having a varying number of cell layers (16)(17)(18)(19)(20) depending on the thickness of the epithelium. The nuclei of these cells, towards the deeper part close to the stratum basale, were round to oval to elongate in shape (Figure 2). The basophilia of these nuclei was similar to that of stratum basale cells. These nuclei contained the chromatin material which was aggregated into smaller clumps especially toward the outer nuclear membrane. These nuclei were oriented vertically, but towards the superficial layers, these were larger and oriented obliquely or horizontally (Figure 2). These less basophilic nuclei had a fine dusting of chromatin and contained 1-2 centric/eccentric nucleoli. The cytoplasm of these cells was eosinophilic and finely granular and the eosinophilia accentuated as progressed towards the surface of the epithelium. These cells presented spicules or spike-like arrangement because of the tapering ends of the adjacent cells and shrinkage of the cytoplasm (Figure 2).
The stratum granulosum also showed a varying number of rows of nuclei of smaller dimensions. The nuclei were comparatively less basophilic and were oriented horizontally (Figure 2). The cytoplasm of these cells was finely granular and strongly eosinophilic. The cell layers towards the free surface presented a few large-sized nuclei with a vacuolated appearance due to less chromatin material. These cells with fine granular and less eosinophilic cytoplasm presented a groundglass appearance similar to that of stratum lucidum (Figure 2). The stratum corneum was having smallsized, deeply basophilic round to oval to narrow elongated nuclei, some of which presented pyknotic appearance (Figure 2). These strongly basophilic nuclei masked the appearance of nucleoli. The cytoplasm of these cells was also finely granular and comparatively more eosinophilic than that of the deeper cells. The keratinized layer present towards the free surface varied in thickness at different places and it was peeling off at some places. A few concentric whorls-like arrangements were also observed towards the superficial portion of the epithelium (Figure 1). The deeper surface presented the papillary pegs which were of varying shapes and size. Lymphocytes were infiltrated in between the cells of the stratum basale especially in the region of underlying lymphoid tissue.
The stratified squamous non-keratinized epithelium abruptly changed into the reticular epithelium ( Figures  3, 4) towards the deeper part of the folds/crypts because of heavy infiltration in the regions of underlining lymphoid tissue. The different strata of the reticular epithelium were not distinctively visible depending upon infiltration of the lymphoid tissue (Figure 4). At these places, only a few surface epithelial cell layers were present. In the rest of the portion of these patches of the modified epithelium, the lymphoid cells were predominant. At some places, the lymphoid infiltration was so much extensive that it reached to the surface of the epithelium and even the lymphoid cells were also observed towards the groove or the crypt. At some places, the epithelial layer of stratified squamous epithelium presented a spongy appearance, because of large infiltration of lymphoid tissue especially in the region of lymphoid follicles just adjacent to the epithelium. At some places, it was difficult to discern the epithelium and the lymphoid cells. The propria-submucosa was having loose irregular connective tissue comprising of collagen and reticular fibres. The reticular fibres formed the basement membrane which was interrupted in the region of the reticular epithelium. The concentration of the reticular fibres increased towards the periphery of the lymphoid follicles and the interfollicular areas. The collagen fibres were observed intermingled with a fine meshwork of reticular fibers and fine blood capillaries in the subepithelial portion, although the number of collagen fibres was drastically reduced.
The lymphoid aggregations were present in the form of lymphoid follicles or the diffused form (Figures 1, 3 -5). The lymphoid follicles of varying shapes and size generally showed darkly stained corona and lightly stained central portion (Figures 3, 5). The lymphoid follicles were surrounded by parafollicular areas, which were separated from those of the adjacent ones by interfollicular areas. The parafollicular areas showed the presence of the high endothelial venules (Figure 6). These venules presented the endothelial cells, having round to oval nuclei with distinctly visible centric or eccentric nucleoli. The cytoplasm of these endothelial cells was finely granular and eosinophilic. In most of the cases, the lymphocytes were observed in these venules. The diffuse arrangement of the lymphoid tissue was also present in the subepithelial portion of the propria-submucosa which was separated from the deeper part by the dense arrangement of the collagen bundles, reticular fibres and glandular tissue. The mucous glands and striated muscles were observed towards the deeper part. The mucous glands were also observed in the superficial part where the lymphoid tissue was absent. These mucous acini presented vacuolated appearance because of washing of the mucous during the processing of the tissues.   The glandular ducts with varying types of epithelia coursed towards the surface of the epithelium. In the deeper part, the fatty tissue was also observed in between the fasciculi of the muscles and in between the clusters of the glandular tissue. Very fine elastic fibres were present in the subepithelial portion of propria and in between clusters of the glandular acini. These were well demonstrated in the tunica intima of the blood vessels.
The surface and reticular epithelial cells were devoid of any PAS activity, whereas the basement membrane showed slight PAS-positive reaction. The glandular acini showed a strong positive reaction for acidic mucopolysaccharides and glycogen as demonstrated by PAS-AB and McManus' PAS methods, respectively (Figures 7-8). The neutral mucopolysaccharides were negligible. The glandular  acini also showed strong positive reaction for Alcian blue indicating the presence of weakly sulfated mucosubstances, sialomucins and hyaluronic acid (Figure 9). The glandular acini were also positive for the presence of mucin as well as acidic mucopolysaccharides, as demonstrated by Meyer's mucicarmine and colloidal iron method, respectively (Figures 10-11). The intra and inter-glandular ducts except at few places did not exhibit any PAS-positive reaction by any of the technique employed during the present study. Fine blood capillaries present in the lymphoid tissue showed a mild positive reaction. The glandular acini exhibited a strong reaction for performic acid-Alcian blue indicating the presence of more than 4% cysteine. The deepest part of the glandular tissue exhibited comparatively lower concentration of the cysteine as compared to that of the superficial part (Figure 12).

DISCUSSION
The mucosal surface of the lingual tonsil was lined by stratified squamous keratinized epithelium as reported in the camel [14], and the goat [15] in contrast to the non-keratinized epithelium in Egyptian buffalo calf [10]. A review on the tonsils of domestic animals reported that lingual tonsil in sheep and goat was entirely covered by keratinized stratified squamous epithelium [16], a finding that contrasted previous studies in the goat [17] where stratified squamous nonkeratinized epithelium was described as observed in the horse [4]; however, in pig, it was stratified squamous epithelium with varying degrees of keratinization [16,18]. The free surface of the epithelium was uneven and irregular whereas the deeper surface presented the papillary pegs, as reported in the horse [4] and the goat [17]. This epithelium was comprised of the strata basale, spinosum, granulosum and corneum as described in the keratinized epithelial lining of the lingual tonsil in the pig [18]. However, the strata comprised of basale, spinosum and superficial in the buffalo calf [10] similar to those of horse [4], goat [17], and the non-keratinized epithelial portion in the pig [18]. The outer stratified squamous epithelium was infiltrated by occasional CD4 positive and CD8+ T cells in the horse [7].
The strata had a varying number of cell layers except for stratum basale depending on the thickness of the epithelium as described in the horse [4]. The stratum basale present towards the basement membrane was having a single row of the vertically oriented oval to elongated strongly basophilic nuclei with chromatin material irregularly aggregated into smaller clumps as reported in the buffalo calf [10], however, the reaction was comparatively less in the horse [4] and the pig [18]. The cell layers of stratum spinosum were 16-20 in contrast to 8-12 in the goat [17] and 3-4 in the pig [18]. These cells presented spicule like the arrangement as reported in goat [17] and pig [18], usually the spikes contained cytokeratin filaments [19]. The nuclei of these layers presented different shapes, size and distribution of chromatin material. The cell layers of stratum granulosum towards the surface presented a few large-sized nuclei having a vacuolated appearance and their cytoplasm revealed ground-glass appearance similar to that of stratum lucidum. The stratum corneum presented small-sized deeply basophilic, round to oval to narrow elongated nuclei. Some of these nuclei exhibited degenerative changes as observed in the goat [15], whereas; such changes were reported in the outermost stratum superficial layer in the horse [4] and the pig [18]. The free surface presented the keratinized layer which varied in thickness at different places. Keratins are intermediate protein filament constituting major components of the vertebrate epithelial cytoskeleton [20] mainly responsible for barrier functionality [19]. The keratinization of the overlying epithelium suggested that lingual tonsil was effector rather than an inductive site based on their immunological roles [21].
In the present study, concentric whorl-like arrangements were occasionally observed towards the superficial portion of the epithelium. Lamellated structures resembling Hassall's corpuscle were also identified towards the outer surface epithelium in the horse [4]. Degeneration of epithelial cells with subsequent formation of epithelial corpuscles has been associated with reticulation of the tonsillar and thymic epithelium [22]. It has been proposed that occlusion and interruption of crypts led to the isolation of islands of epithelial cells, which lose their polarization and later degenerate to form onion-like corpuscles [4]. The epithelium towards the deeper part of the fold was stratified squamous non-keratinized as reported in the horse [4]. Small crypt-like structures were observed at some places in the present study. The tonsillar crypts reported in bovine lingual tonsil [9,23] have not been identified in sheep [24] and camel [14]. The latter studies in camel, however, differed with the recent findings [25] which reported poorly noticeable crypts. Tonsillar crypts were narrow epithelial diverticula which greatly increased the available surface area for accumulation of foreign antigen and subsequent antigenic stimulation [26].
The epithelial lining of the crypt was modified into the reticular epithelium as previously described in goat [17] and horse [4]. Reticular epithelium had been implied in initiating and maintaining immune responses to incoming infectious agents and antigens and could serve as a potential route of entry for pathogens to the lymphoid tissue underneath [4,27]. The lining epithelium was stratified squamous keratinized in ox, non-keratinized in sheep and slightly keratinized in the pig [16]. In most of these species, there was a variable degree of infiltrations by lymphoid cells [16] except in sheep [24,28] and camel [14] where such infiltrations have not been observed. In the horse, the number of positive T-cell along with IgA, IgG (T), IgGb+ cells, B cells, macrophages and some Langerhans cells were variably present in the crypt reticular epithelium [7].
In the present study, the underlined lymphoid tissue deeply infiltrated the reticular epithelium and the different strata were not distinctively visible as reported in the horse [4]. However, such infiltrations were not observed in the sheep [28] and camel [14], except in the horse where these were numerous especially Bcells [7]. The degree of reticulation and infiltration varied considerably [29] and was thought to be initiated by infiltration of immune-related cells [30] including the lymphocytes, macrophages, plasma cells and dendritic cells [31,32]. The presence of these cells as early as the 15 th week of gestation in the human suggested that reticulation along with the formation of primary follicles, interfollicular areas and high endothelial venules [33,34] was a usual developmental process. T and B cells in the reticular epithelium indicated its participation in both cellular and humoral immune responses [7].
Only a few surface epithelial cells related to stratum corneum were observed in the present study. Reticulated patches often showed desquamation of the upper cell layers and were associated with disruptions in the continuity of the underlying basement membrane [29]. These have been attributed to mechanical rupture, because of the force of deglutition or micro-ulceration associated with bacterial activity [35]. These patches may be repaired under the influence of bacterial activity with reciprocal formation of germinal centres [36]. In the rest of the portion of these patches of the modified epithelium, there was a predominance of the lymphoid cells as reported in Egyptian buffalo calf [10]. A feature of the reticulated epithelium was the coexistence of epithelial and non-epithelial cells with the predominance of the latter as reported in the horse [4]. At some places, the lymphoid infiltration was so extensive that it reached to the surface of the epithelium and even the lymphoid cells were also observed towards the groove or the crypts as observed in the horse [4] and the pig [18].
The propria-submucosa was comprised of loose irregular connective, lymphoid, glandular, muscular and adipose tissue as reported in the horse [4], sheep [9,24] and the pig [18]. The reticular fibres formed the basement membrane which was interrupted in the region of the reticular epithelium as reported in the horse [4], goat [17] and the pig [18]. The increased number of blood capillaries in the reticular epithelial region may compensate for the increased metabolic demands of the epithelium and the subsequent interaction between the endothelial cells and leucocytes [4]. Numerous irregularly distributed mast cells in the vicinity of blood vessel reported in the horse [4] could not be observed during the present study.
The lymphoid aggregations were present in the form of lymphoid follicles and diffused forms in the subepithelial portion of the propria-submucosa as reported in the bovine [37]. The lymphoid tissue is involved in local immune induction and effector responses including generation of effector and memory lymphocytes, with the potential to migrate to other mucosal sites [38]. The lymphoid follicles of varying shapes and size were comprised of the lymphocytes of varying size as reported in the horse [4,7]. Some of these follicles were either oriented towards the epithelial crypts in the horse [4], buffalo calf [10] or not associated with crypts at all in the bovine [8].
These follicles showed darkly stained corona and lightly stained central portion, surrounded by parafollicular areas, which were separated from those of the adjacent ones by interfollicular areas. The germinal centre was comprised of small and large lymphocytes, mature plasma cells, macrophages, follicular, and dendritic cells [4]. T-cells, CD4+ and CD8+, predominated in the parafollicular area along with a few IgA+, B-cells and macrophages [7]. The parafollicular area may, therefore, be functionally similar to the thymus-dependent areas of other lymphoid organs [7]. The interfollicular zone was rich in macrophages, lymphocytes, plasma cells, dendritic cells, reticular cells, blood capillaries, and high endothelial venules (HEVs) in a fine meshwork of reticular fibers as described in different species [7,8,39].
The HEVs were first identified in lymph nodes and their role in the recirculation of lymphocytes has been elucidated [3,40]. Since afferent lymph vessels are lacking, HEVs are the only trafficking routes of lymphocytes from the blood vascular system to these tonsils [41]. B cells must migrate through areas rich in T cells, which explain the interspersed B-lymphocytes [7]. These HEVs presented the endothelial cells, having round to oval nuclei with distinctly visible nucleoli. In most of the cases, the lymphocytes were observed in these venules as reported in buffalo calf [10]. The height of the epithelial cells of HEVs and the lymphocyte numbers in the afferent and the efferent lymphatics differed significantly in different species [42]. The lymphoid tissue was separated from the deeper part of the propria-submucosa by the dense arrangement of the collagen bundles and fine reticular fibres as reported in the horses [4]. In the deeper part, the mucous glands, striated muscles arranged in different directions, small nerve bundles and fatty tissue were observed as described in the goat [17]. The intra-glandular ducts coursed towards the surface of the epithelium and showed varying types of epithelia as reported in the horse [4], and goat [17].
The stratified squamous epithelium and the propriasubmucosa showed negative reaction by McManus' PAS method as reported in the goat [17] whereas in the horse [4] very faint reaction was reported and was related to the degree of stratification [32] of the epithelium. A PAS-positive reaction was observed in the basement membrane. The mucous glandular acini were positive for glycogen, weakly sulfated mucosubstances, sialomucins, hyaluronic acids and acidic mucopolysaccharides as reported in the goat [17] and the pig [18]. The neutral mucopolysaccharides were negligible but were more abundant in the horse [4] and the pig [18]. Fine blood capillaries present in the lymphoid tissue, showed a mild positive reaction. The intra and inter-glandular ducts except a few did not exhibit positive reaction for any of the special stains used in the study. The glandular acini also showed a strong reaction for performic acid-Alcian blue, showing the presence of more than 4% cysteine.

CONCLUSION
The lingual tonsil of the adult buffaloes lined by stratified squamous keratinised epithelium towards outer surface showed modifications towards crypt in the form of non-keratinised, reticular and the spongy epithelia. The reticular epithelium characterized by infiltration of lymphoid cells and an interrupted basement membrane presented a varying number of rows of epithelial cells which were difficult to discern at places. The features of the reticular epithelium may be considered equivalent to follicle associated epithelium present in the nasopharyngeal tonsil. The reticular epithelium and arrangement of lymphoid follicles are suggestive of components of the adaptive immune protection and may explain mysteries of early pathogenesis of some bacterial or viral diseases of buffaloes by microbiologists.

ACKNOWLEDGEMENT
The Indian Council for Cultural Relation (ICCR, African Scholarship Section) is gratefully acknowledged for the sponsorship of PhD program of Dr. Ibrahim Alhaji Girgiri.