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*Corresponding author’s e-mail: indu@kvasu.ac.in; 1Department of Veterinary Anatomy, College of Veterinary and Animal Sciences,Pookode-673 576, India, 2Department of Statistics, College of Veterinary and Animal Sciences, Mannuthy- 680 651, India.

Histomorphology and scanning electron microscopy of the pharyngealtonsil in goatsV. R. Indu*, K.M. Lucy, N. Ashok, S. Maya1 and V.L. Gleeja2

Department of Veterinary Anatomy,College of Veterinary and Animal Sciences, Mannuthy- 680 651, Kerala, India.Received: 17-08-2015 Accepted: 06-11-2015 DOI:10.18805/ijar.8428

ABSTRACTGross and histological studies were conducted on the pharyngeal tonsil of six male crossbred goats of six months of age. Inthe nasopharynx, pharyngeal tonsil was located on the caudal part of the pharyngeal septum and was 5.54±1.41cm long and2.19±0.92cm wide. It presented numerous longitudinally arranged primary and secondary folds. Histologically the tonsilwas lined by pseudostratified ciliated columnar epithelium comprising of 8-14 rows of nuclei of three types of cells, viz.basal, supporting and goblet cells. This epithelium was transformed at places into follicle-associated epithelium (FAE) andwas characterized by decreased height of the epithelial cells, absence of cilia and goblet cells and heavy infiltration oflymphocytes through the interrupted basement membrane. The height of surface epithelium was 87.33± 1.20µm and that offollicle-associated epithelium was 52.33± 5.21µm. Propria-submucosa comprised of a central axis of loosely arrangedconnective tissue with dense aggregates of lymphoid tissue, fine blood capillaries and few nerve fibres folded around it.The cryptolymphatic units and tonsillar nodules of varying shape and dimensions constituted the majority of the lymphoidtissue. The average diameter of lymphoid nodules was 921.67±8.72µm and the lymphocyte count per nodule was32233.23±324.24. The average number of lymphatic nodules counted per field under low power magnification of microscopewas 2.5±0.43 and the internodular distance was 29.83±1.40µm. In scanning electron microscopy surface of the pharyngealtonsil was covered by two types of epithelium viz., the ciliated respiratory surface epithelium and the FAE consistingpredominantly of three types of non-ciliated microvillus cells.

Key words: Goats, Histology, Morphology, Pharyngeal tonsil, Scanning Electron Microscopy

INTRODUCTIONPharyngeal tonsils located at the posterior part of

the nasopharynx in animals were ideally placed to sampleantigens passing through the respiratory and digestivesystems (Baykan et al., 2001; Palmer et al., 2011). Thenumber and location of tonsils varies in different animals(Tenorio and Pabst, 2006). A perusal of literature revealedonly few studies on the pharyngeal tonsils in goats and hencethe present work was undertaken.MATERIALS AND METHODS

Six crossbred male goats of six months of age wereused for the present study. The heads collected weresectioned in median plane and rinsed in tap water. One halfof the head was fixed in two per cent acetic acid for 24h tovisualise the morphology of the tonsils clearly. From theother half of head, tissue pieces were collected from theregion of the pharyngeal tonsils.

From median sections of head, the pharyngealtonsils were identified and its length and width weremeasured. Thereafter tissue pieces from the nasopharynxwere collected and fixed in 10 per cent neutral buffered

formalin. The materials were processed routinely to obtain5-6µm thick serial paraffin sections. The sections werestained by Haematoxylin and Eosin (Luna, 1968), Gomori’srapid one step trichrome method for collagen fibres (Luna,1968), Verhoeff’s method for elastic fibres (Singh andSulochana, 1996), Gordon and Sweet’s method for reticularfibres (Bancroft and Gamble, 2003) and Unna’s method formast cells (Luna, 1968).

For scanning electron microscopy, samples ofpharyngeal tonsils were fixed in 2.5 per cent gluteraldehydein 0.1M phosphate buffer (PBS) (pH 7.2) for 24 h at 4ºCand post fixed in two per cent aqueous osmium tetroxide forfour hours. Thereafter the samples were processed andscanned under Scanning Electron Microscope (SEM-Model:JEOL-JSM 5600) at required magnifications at Ruska Labs,College of Veterinary Science, Sri Venkateshwara VeterinaryUniversity, Rajendranagar, Hyderabad, Andhra Pradesh.RESULTS AND DISCUSSION

In the nasopharynx, pharyngeal tonsil was locatedon the caudal part of pharyngeal septum (Fig.1.). Afterfixation in acetic acid, lymphoid nodules could be observed

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macroscopically as small opaque white spots on the surfaceof the tonsils. Similar observations were made by Kumarand Timoney (2001) in horse, Cocquyt et al. (2005) andKumar and Nagpal (2007) in sheep and Liu et al. (2012) inpigs. Pharyngeal tonsils located at the posterior part of thenasopharynx in animals were ideally placed to sampleantigens passing through respiratory and digestive systems(Baykan et al., 2001; Palmer et al., 2011).

FIG 1: Median section of head showing the location of pharyngealtonsils (Acetic acid fixation)1. Pharyngeal tonsil 2. Nasopharynx 3.Pharyngeal septum

The pharyngeal tonsils were 5.54±1.41cm long and2.19±0.92cm wide and presented numerous longitudinallyarranged primary and secondary folds. Cocquyt et al. (2005)found that the length and width of the tonsil varied from 18to 40mm and 12 to 21mm, respectively in six month-oldsheep. In contrast, Billen et al. (2006) noted that thepharyngeal tonsil of dogs was not easily recognisable andno crypts or folds were present in it. Since the dogs breathedthrough both their nose and mouth as compared to otherdomestic animals which breathed mainly through the nose,contact of the canine nasal and nasopharyngeal mucosa tothe inhaled antigens was comparatively less.

Histologically the pharyngeal tonsil was lined bypseudostratified ciliated columnar epithelium. At variousplaces, the epithelium was modified into stratified cuboidal.The epithelium comprised of 8-14 rows of oval to elongatednuclei of three types of cells, viz. basal, supporting and gobletcells. The basal cells presented lightly stained round to ovalnuclei with two eccentric nucleoli. There were two distincttypes of the tall columnar supporting cells based on thestaining characteristics of their nuclei. Type-I cells had largeand elongated basophilic dark nuclei and were mainly placedtowards the mid zone of the epithelium. The type-II cellspresented round to oval, lightly stained vesicular nuclei andwere distributed above the type-I nuclei. Goblet cells wereseen intermingled between other cell types (Fig.2.). A large

FIG 2: C.S. of pharyngeal tonsil showing surface epithelium(3 month). H&E x 4001. Goblet cell 2. Type II cell 3. Type I cell 4. Basal cell5. Lymphocytes 6. High endothelial venules

number of small to medium sized intraepithelial lymphocyteswere seen in between the cells. These observations confirmedthe earlier descriptions given by Kumar and Timoney (2001)

in equines, Kumar and Kumar (2004) in goats and Kumar etal. (2006) and Kumar et al. (2011) in sheep.

In the present study, the respiratory epithelium ofpharyngeal tonsil showed a large number of primary andsecondary folds that formed crypts within the lymphoidtissue. This epithelium was transformed at places into simplecolumnar or stratified cuboidal reticular epithelium orfollicle-associated epithelium (FAE) (Fig.3). This wascharacterized by decreased height of the epithelial cells,absence of cilia and goblet cells and heavy infiltration oflymphocytes through the interrupted basement membrane.These observations are in accordance with the findings ofKumar and Timoney (2001) in horse, Kumar and Kumar(2004) in goats, Kumar and Nagpal (2007) in sheep andPalmer et al. (2011) in bovines. Absence of mucus helpeddirect contact of microorganisms and their antigens to theFAE in pharyngeal tonsils as reported by Kumar and Timoney(2001) in horse. The height of surface epithelium was 87.33±1.20µm and that of follicle-associated epithelium was 52.33±5.21µm.

The basement membrane in FAE was disrupted dueto infiltration of lymphoid cells in the pharyngeal tonsils.Toppets et al. (2011) suggested that the pharyngeal tonsilswere perfectly adapted to sample foreign antigens due themassive intraepithelial lymphocyte infiltration. Thelymphoepithelial barrier sampled and transferred antigensto the underlying lymphoid tissue (Perry and Whyte, 1998).Propria-submucosa of pharyngeal tonsil comprised of acentral axis of loosely arranged connective tissue with dense

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Fig 3: C.S. of pharyngeal tonsil showing follicle-associatedepithelium and reticular fibers (6 month). Gordon and Sweet silverimpregnation method X 4001. Follicle-associated epithelium 2. Reticular fibers 3. Lymphocytes

aggregates of lymphoid tissue, fine blood capillaries and fewnerve fibres folded around it. This is in accordance with thedescriptions given by Casteleyn et al. (2011) and Toppets etal. (2011) in sheep. Pharyngeal tonsils showed mixeddistribution of collagen and elastic fibres in the subepithelialpart as reported by Toppets et al. (2011) in ovine. Reticularfibres formed a meshwork in which dense distribution oflymphoid tissue in the form of secondary lymphoid nodulesand internodular lymphoid tissue was seen (Fig.3). Mastcells were distributed mainly in the lamina propriaimmediately below the FAE, in the glandular tissue andbetween muscular fibres.

In the deeper part of the propria-submucosa thatformed the central axis of the tonsil, loosely arrangedconnective tissue, mucus glandular acini, adipose tissue,nerve fibres and blood vessels could be noted. Thecryptolymphatic units and tonsillar nodules of varyingshape and dimensions constituted the majority of thelymphoid tissue. The lymphoid nodules were dome shapedtowards the epithelium and consisted of a parafolliculararea and central nodular area. These nodules were separatedfrom each other by internodular areas. Most of the noduleshad darkly stained corona formed by large number of smalllymphocytes and a germinal centre (Fig.4). Theseobservations confirmed the reports of Kumar et al. (2001)in horse, Kumar and Kumar (2004) in goats, Billen et al.(2006) in dogs, Kumar et al. (2011) in sheep and Liu et al.(2012) in pigs.

The lymphoid tissue comprised of small, mediumand large lymphocytes, plasma cells and macrophages. Theaverage diameter of lymphoid nodules was 921.67±8.72µmand the lymphocyte count per nodule was32233.23±324.24. The average number of lymphaticnodules counted per field under low power magnification

FIG 4: C.S. of pharyngeal tonsil showing lymphoid tissue(5 month). H&E x 1001. Epithelium 2. Germinal centre 3. Corona 4. Parafolliculararea 5. Internodular area 6. Lymph vessel

of microscope was 2.5±0.43 and the internodular distancewas 29.83±1.40µm. Similar reports on the micrometry oflymphoid tissue of pharyngeal tonsils are not available forcomparison. According to Anderson (1974), the lymphoidtissue in nasopharyngeal tonsil of cattle was an importantsource of immunoglobulin production for other mucosalsites like tracheo-bronchial tree. Maximum presence oflymphoid tissue in nasopharyngeal tonsil was probablybecause of increased deposition of air-borne particles, dueto bending of air stream from the nasal passages andconvergence of multiple streams of mucus carrying particlesfrom all parts of nasal passages in the nasopharynx (Morganet al., 1984).

Large number of plasma cells was seen beneath theepithelium, in both follicular and diffuse lymphoid tissueand in the glandular tissue. The average number of plasmacells counted per field under high power magnification withinthe pharyngeal tonsils was 63.50±2.89. A few bloodcapillaries, high endothelial venules (HEVs) and venuleswere distributed in the interfollicular area. Hafeez et al.(2009) noticed that higher count of HEVs beneath thenasopharyngeal tonsils contributed towards higher level ofimmune response.

In scanning electron microscopy, surface of thepharyngeal tonsil was covered by two types of epitheliumviz., the respiratory epithelium and the epithelium overlyingthe area of lymphoid nodules (FAE). The respiratoryepithelium consisted of chiefly ciliated cells with a few gobletcells and squamous cells dispersed in between. The FAEwas disrupted and consisted predominantly of three types ofnon-ciliated microvillus cells. The large sized type-Imicrovillus cells were most numerous and possessed smallmicrovilli which were uniformly distributed and denselypopulated. The round to oval type-II microvillus cells were

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smaller with mixed distribution of small and large sizedmicrovilli. The type-III microvillus cells were membranous/microfold (M) cells with wide membranous folds instead ofmicrovilli (Fig.5). Reports on ultramicroscopic studies byKumar et al. (2001) in horse, Kumar and Kumar (2004) ingoats, Casteleyn et al. (2010) in sheep and Palmer et al.(2011) in bovines confirmed the observations made in thepresent study. The topography of FAE on the surface abovethe lymphoid nodules was on the basis of the suggestion thatthey acted as mechanism for entrapping and samplingantigens in the airstream as reported by Mair et al. (1987) inequine.CONCLUSION

In goats, lymphoid tissue was well developed inthe pharyngeal tonsils suggesting that they could be exploitedas targets for nasal vaccines for the induction of mucosalimmune response in this species.

FIG 5: Scanning electron micrograph of pharyngeal tonsil showingfollicle associated epithelium (6 month). SEM x 50001. Type I microvillus cell 2. Type II microvillus cell 3. Type IIImicrovillus cell

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