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Anatomical localisation and histology of the ovine tonsils
Veterinary Immunology and Immunopathology 107 (2005) 79–86 www.elsevier.com/locate/vetimm
Anatomical localisation and histology of the ovine tonsils G. Cocquyt *, T. Baten, P. Simoens, W. Van Den Broeck Department of Morphology, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Received 12 November 2004; received in revised form 23 March 2005; accepted 23 March 2005
Abstract The topography and histologic structure of the various tonsils were studied anatomically and microscopically in 15 sheep aged between 9 and 15 months. The palatine, pharyngeal and paraepiglottic tonsils were readily visible macroscopically. They consisted mainly of secondary lymph nodules and were encapsulated in dense connective tissues. The epithelium covering the tonsils and their crypts was frequently infiltrated heavily by lymphocytes. The tubal tonsil and the tonsil of the soft palate were macroscopically visible after fixation in 2% acetic acid. These tonsils consisted of scattered lymph nodules, aggregations of lymphocytes and diffuse lymphoid tissue. They were not encapsulated, and therefore the borders of these tonsils could not be clearly delineated. The lingual tonsil was not macroscopically visible in sheep and consisted of scattered small aggregations of lymphocytes. # 2005 Elsevier B.V. All rights reserved. Keywords: Tonsil; Lymphoid tissue; Sheep
1. Introduction Transmissible spongiform encephalopathies (TSEs), such as bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and Creutzfeldt– Jacob disease in humans, are progressive and fatal neurological diseases which show a typical vacuolisation of neurons and an accumulation of the diseasespecific isoform (PrPSc) of the body-specific prion protein (PrPC). Studies have revealed that the strain of the transmissible agent responsible for the new variant of Creutzfeldt–Jacob disease (nvCJD) is identical to that of the BSE agent (Zeidler and Ironside, 2000). No * Corresponding author. Tel.: +32 92647722; fax: +32 92647790. E-mail address: [email protected] (G. Cocquyt).
cases of natural infection of BSE in sheep have been detected so far, but sheep have been experimentally infected with BSE by oral, intracerebral and intravenous inoculation. Most of the studies to date show little difference between BSE and scrapie in terms of distribution of infection throughout the body, clinical signs and pathology (Baylis et al., 2002; Houston and Gravenor, 2003; Hunter, 2003). In contrast to BSE in cattle where deposition of the PrPSc protein is limited to the central nervous system, it has been shown in sheep that PrPSc is present in nearly all lymphoid tissues (Foster et al., 2001). Furthermore, the gutassociated lymphoid tissue (GALT) of the oropharynx and the gut are believed to be the preferential sites of replication of the scrapie agent in sheep. The palatine tonsils rank among the primary sites where PrPSc
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could be detected after oral infection (van Keulen et al., 2002). Biopsies of tonsils and the conjunctival lymph nodules of the third eyelid can be used in sheep to detect prion protein in the preclinical stage of scrapie (Schreuder et al., 1998). The general distribution of the BSE agent in experimentally infected sheep makes it impossible to remove all the potentially infected tissue from sheep meat intended for human consumption. In order to restrict the risk of BSE-infected sheep entering the food chain, all member countries of the European community are required to examine a fixed number of slaughtered and dead sheep and goats for TSE on an yearly basis (European Commission, 2002). Requirements also order the removal of specified risk material of sheep, consisting of the skull including the brain, the eyes, the tonsils and the spinal cord of animals aged over 12 months, and the spleen of the animals of all age groups (European Commission, 2000). The tonsils (tonsillae or amygdalae) consist of an accumulation of lymphocytes which are usually concentrated in lymph nodules (noduli lymphatici) and are present in the mucosae of the oropharynx, nasopharynx and laryngopharynx. They are a part of the integrated pharyngeal mucosal immune system (Ogra, 2000) and form a ring of lymphoid tissue in the pharyngeal wall which was first described by von Waldeyer-Hartz (1884), and is therefore called the ‘‘Waldeyer ring’’ (Perry and Whyte, 1998). This location involves an important role for tonsils as secondary lymphoid tissue in the immunological response against antigens which enter the body by the oral or nasal route (Brandtzaeg, 1984; Bernstein et al., 1999). Six tonsils are described in sheep: the palatine tonsil (tonsilla palatina), the lingual tonsil (tonsilla lingualis) and the tonsil of the soft palate (tonsilla veli palatini) in the oropharynx, the pharyngeal tonsil (tonsilla pharyngea) and the tubal tonsil (tonsilla tubaria) in the nasopharynx, and the paraepiglottic tonsil (tonsilla paraepiglottica) in the laryngopharynx (Barone, 1997; Thome´ , 1999). The topography and structure of these tonsils in sheep are poorly documented. The palatine tonsil is described as a tonsil with the size of a hazelnut which has three to six crypts (Ellenberger and Illing, 1911). According to Thome´ (1999), the lingual tonsil and the tonsil of the soft palate both consist of a small number of lymph nodules or a small amount of lymphoid
tissue. The tonsil of the soft palate is located at the ventral side of the soft palate (Thome´ , 1999). The pharyngeal tonsil forms an elevation at the caudal part of the pharyngeal septum with several invaginations of the epithelium (Barone, 1997; Thome´ , 1999). The distribution of lymphoid tissue and the epithelium of this tonsil has been examined by Chen et al. (1990, 1991). The tubal tonsil is a slightly elevated, diffuse aggregation of lymph nodules which is restricted to the mucosa at the pharyngeal opening of the auditory tube according to Thome´ (1999), but it can extend to the dorsal surface of the soft palate according to Barone (1997). Koch (1970) mentioned crypts in the tubal tonsil. Chen et al. (1989) described nodular aggregations of lymphoid tissue at the opening of the auditory tube and Stanley et al. (2001) studied the clustered lymph nodules which are concentrated just caudal to the opening of the auditory tube. The paraepiglottic tonsil has been described as a crypt tonsil (Ellenberger and Illing, 1911) which is located ‘‘where the food ball passes after lifting of the soft palate’’ (Koch, 1970). Except for Chen et al. (1989) and Stanley et al. (2001), most authors do not mention the microscopic structure of the ovine tonsils, but refer to data about bovine tonsils. In the present study, we describe the topography and the microscopic structure of the various tonsils in sheep.
2. Materials and methods 2.1. Animals The heads of 15 sheep aged between 9 and 12 months were collected from the slaughterhouse. Ten of these heads were used for the macroscopic examination and the tonsils of the five remaining heads were excised for microscopic examination.
2.2. Macroscopic examination The skin and mandibles were removed from all 10 heads and five heads were sectioned in the median plane. The heads were rinsed with tap water for 2 min and fixed in 2% acetic acid for 24 h to visualise the lymph nodules (Cornes, 1965; Chauhan and Singh, 1970). The location of the lymph nodules was noted,
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the length and width of the tonsils were measured and the number of lymph nodules was counted. 2.3. Microscopic examination The pharyngeal, palatine and paraepiglottic tonsils, the lateral pharyngeal wall surrounding the pharyngeal opening of the auditory tube, the soft palate and the part of the tongue caudal to the lingual torus were collected from five heads. These samples were reduced to blocks of maximum 15 mm 10 mm 5 mm and fixed in 3.5% formaldehyde for at least 2 d. Tissue blocks were processed by routine methods and embedded in paraffin wax. Serial tissue sections (8 mm thick) with a 1000 mm interval were made from all tonsils, except for the paraepiglottic tonsil from which serial sections (8 mm) without intervals were made. The slides were stained with haematoxylin and eosin (HE), and examined for the presence of lymphoid tissue with a motorized microscope (Olympus BX61, Olympus Belgium, Aartselaar, Belgium). Special attention was given to the differentiation of lymph nodules (primary or secondary nodules), the parafollicular tissue, the overlying epithelium and the connective tissue surrounding the lymphoid tissue. Lymph nodules were defined as compact nodular aggregates of lymphocytes surrounded by a diffuse sheet of lymphoid tissue (Thuring et al., 2002).
3. Results
Fig. 1. Median section of a sheep head, (a) palatine tonsil; (b) lingual tonsil; (c) tonsil of the soft palate; (d) pharyngeal tonsil; (e) tubal tonsil; (f) paraepiglottic tonsil.
Fig. 2. Rostral view of the larynx of a sheep. White arrows: palatine tonsil; black arrows: paraepiglottic tonsil.
3.1. The palatine tonsil The palatine tonsil was located bilaterally in the pharynx between the palatoglossal and the palatopharyngeal arch (Fig. 1) and formed an elevation of the pharyngeal mucosae with two or three narrow elongated entrances to the underlying crypts (Figs. 2 and 3). The tonsil was clearly distinguishable from the surrounding tissue by its capsule of dense collagen tissue which invaginated the tonsil once or twice forming two or three lobes. The tonsil approximately had the size of a hazelnut. The squamous epithelium that covered the surface formed tonsillar crypts by invaginating into the underlying connective tissue. These crypts could have a large lumen and were frequently branched. In the walls of these crypts,
Fig. 3. Dorsal view of the larynx and tongue of a sheep. Ellipse: location of the lingual tonsil; white arrows: caudal papilla vallata; black arrows: piriform recess; (a) epiglottis; (b) arytenoid cartilage; (c) soft palate (sectioned); (d) root of tongue; (e) palatine tonsil.
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numerous secondary lymph nodules were located. The stratified epithelium of the crypts was frequently infiltrated heavily by lymphocytes. 3.2. Lingual tonsil The lingual tonsil could not be located by any macroscopic landmarks. Histological examination revealed small concentrations of lymphoid tissue at both sides of the dorsal part of the tongue (dorsum linguae) without reaching the midline. They were located 2–4.5 cm rostral to the base of the epiglottis, extending in the subepithelial connective tissue from 1 cm caudal to 2.5 cm rostral to the most caudal gustatory vallate papilla (papilla vallata) (Fig. 3). The lymphoid cells were mainly organised in small dense aggregations or were scattered in the propria– submucosa layer (Fig. 4). Two primary nodules were observed only in two out of five tongues. The subepithelial connective tissue of several vallate papillae contained lymphoid aggregations. Neither any infiltration of lymphocytes in the stratified squamous epithelium of the tongue nor any tonsillar crypts were seen. 3.3. Tonsil of the soft palate On the ventral (oral) surface of the soft palate, no macroscopic signs of this tonsil were observed, and on histological slides, only few small aggregations were
Fig. 5. Histological section of the tonsil of the soft palate showing two primary nodules in the dorsal (nasal) part of the soft palate with lymphocyte infiltration in the epithelium above the nodules (HE).
found in the subepithelial tissue. However, on the dorsal (nasal) side, scattered nodules of lymphoid tissue varying in number from 7 to 104, were made visible by the acetic acid. The structure of the lymphoid tissue varied from diffuse lymphoid tissue with some lymphoid nodules in the rostral part of the soft palate over an intermediate zone with several primary and secondary lymph nodules and aggregations of lymphocytes (Fig. 5) towards a caudal zone near the arcus veli palatini, where scattered small aggregations were present. The slightly keratinized squamous epithelium of the nasal side of the soft palate was sometimes infiltrated by lymphocytes in the area above the lymph nodules and aggregated lymphocytes. 3.4. Pharyngeal tonsil
Fig. 4. Histological section of the lingual tonsil showing aggregation of lymphocytes (arrows) in the subepithelial connective tissue (HE).
The pharyngeal tonsil was located in the roof of the pharynx on the caudal part of the pharyngeal septum. The length of the tonsil varied from 18 to 40 mm and the total width from 12 to 21 mm. The surface of this tonsil showed several folds which were mostly oriented longitudinally. After fixation in acetic acid, small white spots became visible on the surface of the tonsil. The lymphoid tissue of this tonsil consisted of a layer of secondary nodules lying underneath the pseudostratified ciliated epithelium which was frequently infiltrated heavily by lymphocytes (Fig. 6). At the rostral pole of the tonsil, a gradual transition from nonlymphoid to tonsillar tissue was noticed as the thin layer
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Fig. 6. Histological section of a fold of the pharyngeal tonsil. Note the numerous secondary lymph nodules (arrows) and the central axis of dense connective tissue (asterisks) (HE).
of lymphocytes underlying the epithelium became thicker in caudal direction, and changed progressively into lymphoid tissue organized in secondary nodules. At the dorsal and caudal borders of the tonsil, a more abrupt transition was noticed. In these zones, some scattered secondary nodules were observed. 3.5. Tubal tonsil After fixation in acetic acid, scattered nodules were observed in the lateral pharyngeal wall, ventral, and mainly caudal, to the opening of the auditory tube (Fig. 7). The number of these nodules on each side varied from 41 to 150. The borders of the tubal tonsil could not be clearly distinguished using histology. Lymphoid tissue was found mainly ventral to the pharyngeal opening of the auditory tube, and was distributed variably in rostral to caudal direction in different sheep. The mucosa of the medial nasopharyngeal part of the auditory tube contained several primary and secondary lymph nodules, whereas the lateral mucosa contained less and mostly diffuse lymphoid tissue. The epithelium covering the lymphoid tissue was sometimes infiltrated by lymphocytes.
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Fig. 7. Medial view of the right nasopharyngeal wall after fixation in 2% acetic acid. Note the white-pinhead-sized tonsillar nodules (ellipse) ventral to the opening of the auditory tube (arrow); (a) nasopharynx; (b) tongue; (c) pharyngeal septum after removal of the pharyngeal tonsil; (d) soft palate.
recess (recessus piriformis) (Fig. 2). The tonsil appeared as a cluster of small nodular elevations which became white after fixation in acetic acid. The number of these nodular elevations per tonsil ranged from one to eight. Length of the tonsil varied from 5 to 14 mm and width ranged from 2 to 5 mm. Histology revealed that the elevations consisted of aggregations of lymph nodules surrounded by parafollicular tissue and encapsulated by connective tissue from which septa extended into the larger nodules. Most of the
3.6. Paraepiglottic tonsil The paraepiglottic tonsil was located lateral to the epiglottis in the ventromedial segment of the pharyngeal sidewall, i.e. at the floor of the piriform
Fig. 8. Histological section of the paraepiglottic tonsil showing an aggregation of lymph nodules with a central crypt (arrows). The epithelium of the crypt is infiltrated by numerous lymphocytes (HE).
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Fig. 9. Histological section of the paraepiglottic tonsil showing an aggregation of lymph nodules without crypt. Note the septum of dense connective tissue (asterisks) and the invagination (arrows) of the epithelium next to the aggregation (HE).
lymph nodules were secondary nodules. Primary lymph nodules were the most obvious at the periphery of the aggregations. Crypts were present in 6 out of the 27 aggregations which were observed in the 10 tonsils examined (Fig. 8). Epithelial invaginations were frequently observed near the aggregations without crypts (Fig. 9). The stratified squamous pharyngeal epithelium covering the lymphoid tissue was frequently infiltrated by numerous lymphocytes. In the aryepiglottic fold (plica aryepiglottica), varying numbers (0–15) of lymph nodules could be determined.
4. Discussion Three out of the six tonsils present in sheep are clearly distinguishable from the surrounding tissue, even without fixation in 2% acetic acid, viz. the palatine, the pharyngeal and the paraepiglottic tonsil. The present findings about the palatine and pharyngeal tonsils match the data mentioned in previous studies (Ellenberger and Illing, 1911; Barone, 1997; Thome´ , 1999), and are similar to those given in other species. In contrast, novel data are given about the paraepiglottic tonsil which were previously described only briefly (Ellenberger and Illing, 1911; Koch, 1970). As crypts could not be clearly demonstrated in a preliminary study of serial sections of the para-
epiglottic tonsil with intervals of 500 mm (unpublished data), we performed serial sections without intervals in the present study, and we observed crypts in 6 out of 27 aggregations (22%). Consequently, the ovine paraepiglottic tonsil is composed of some tonsillar follicles (folliculi tonsillares) that contain crypts, and numerous aggregations without crypts. For the latter type of aggregations, we could not find a term in the literature. Therefore, we suggest to introduce the term ‘‘tonsillar nodules’’ (noduli tonsillares) for naming the clearly distinguishable aggregations of lymph nodules and paranodular tissue which are surrounded by connective tissue but lack epithelial crypts. The tubal tonsil and the tonsil of the soft palate of sheep are not macroscopically distinguishable from the surrounding tissue, but they can be visualized by fixation in 2% acetic acid, although even then, their borders are not very clearly visible. Both tonsils consist of scattered lymph nodules, aggregations of lymphocytes and diffuse lymphoid tissue, and they are not encapsulated. In contrast to the description of Thome´ (1999), we found almost no lymphoid tissue on the oral side of the soft palate. The dorsal surface of the soft palate, on the other hand, contains a large amount of lymphoid tissue. Therefore, we concluded that the tonsil of the soft palate of sheep is located on the dorsal surface of the soft palate and faces the nasopharynx instead of the oropharynx. The lingual tonsil cannot be determined macroscopically in sheep. In contrast to the bovine lingual tonsil which is clearly macroscopically visible and consists of numerous tonsillar follicles (Manesse et al., 1995), only a few scattered aggregations of lymphoid cells are present in the lingual tonsil of sheep. However, as this lymphoid tissue has a typical distribution on the lingual surface and is more abundant at this location than in the other parts of the tongue, we think that it is justified to use the term ‘‘lingual tonsil’’ also in sheep. Several issues about the lymphoid tissue in sheep need to be further elucidated to understand better the invasion and spreading of scrapie and other TSE. Membranous epithelial cells (M-cells) or cells with feature characteristics of M-cells were demonstrated in human, canine, porcine, equine and rabbit tonsils (Belz and Trevor, 1995, 1996; Claeys et al., 1996; Gebert et al., 1995; Gebert, 1997; Kumar and
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Timoney, 2001) and in ovine pharyngeal and tubal tonsils (Chen et al., 1990, 1991; Stanley et al., 2001). They are considered as specialized cells for transepithelial transport, delivering the foreign antigens and microorganisms to the organized subepithelial lymphoid tissues (Kraehenbuhl and Neutra, 2000). Gonzalez et al. (2001) and Stanley et al. (2001) studied the distribution of the B and T subtypes of lymphocytes in ovine lymph nodes and pharyngeal tonsils, respectively. However, no research on the distribution of B and T subtypes, macrophages, dendritic and follicular dendritic cells in the different tonsils of sheep have been performed. Special attention needs to be given to the distribution of follicular dendritic cells, because it has been proved that the replication of the BSE and scrapie agents takes place in these cells (Brown et al., 1999, 2000). Furthermore, the innervation of the tonsils should also be elucidated better in order to understand the possible link with neuroinvasion in tonsils.
Acknowledgments The authors thank L. Standaert and L. De Bels for their excellent technical support. This work was supported by BOF-grant #011B4101 (Ghent University).
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Anatomical localisation and histology of the ovine tonsils G. Cocquyt *, T. Baten, P. Simoens, W. Van Den Broeck Department of Morphology, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Received 12 November 2004; received in revised form 23 March 2005; accepted 23 March 2005
Abstract The topography and histologic structure of the various tonsils were studied anatomically and microscopically in 15 sheep aged between 9 and 15 months. The palatine, pharyngeal and paraepiglottic tonsils were readily visible macroscopically. They consisted mainly of secondary lymph nodules and were encapsulated in dense connective tissues. The epithelium covering the tonsils and their crypts was frequently infiltrated heavily by lymphocytes. The tubal tonsil and the tonsil of the soft palate were macroscopically visible after fixation in 2% acetic acid. These tonsils consisted of scattered lymph nodules, aggregations of lymphocytes and diffuse lymphoid tissue. They were not encapsulated, and therefore the borders of these tonsils could not be clearly delineated. The lingual tonsil was not macroscopically visible in sheep and consisted of scattered small aggregations of lymphocytes. # 2005 Elsevier B.V. All rights reserved. Keywords: Tonsil; Lymphoid tissue; Sheep
1. Introduction Transmissible spongiform encephalopathies (TSEs), such as bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and Creutzfeldt– Jacob disease in humans, are progressive and fatal neurological diseases which show a typical vacuolisation of neurons and an accumulation of the diseasespecific isoform (PrPSc) of the body-specific prion protein (PrPC). Studies have revealed that the strain of the transmissible agent responsible for the new variant of Creutzfeldt–Jacob disease (nvCJD) is identical to that of the BSE agent (Zeidler and Ironside, 2000). No * Corresponding author. Tel.: +32 92647722; fax: +32 92647790. E-mail address: [email protected] (G. Cocquyt).
cases of natural infection of BSE in sheep have been detected so far, but sheep have been experimentally infected with BSE by oral, intracerebral and intravenous inoculation. Most of the studies to date show little difference between BSE and scrapie in terms of distribution of infection throughout the body, clinical signs and pathology (Baylis et al., 2002; Houston and Gravenor, 2003; Hunter, 2003). In contrast to BSE in cattle where deposition of the PrPSc protein is limited to the central nervous system, it has been shown in sheep that PrPSc is present in nearly all lymphoid tissues (Foster et al., 2001). Furthermore, the gutassociated lymphoid tissue (GALT) of the oropharynx and the gut are believed to be the preferential sites of replication of the scrapie agent in sheep. The palatine tonsils rank among the primary sites where PrPSc
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could be detected after oral infection (van Keulen et al., 2002). Biopsies of tonsils and the conjunctival lymph nodules of the third eyelid can be used in sheep to detect prion protein in the preclinical stage of scrapie (Schreuder et al., 1998). The general distribution of the BSE agent in experimentally infected sheep makes it impossible to remove all the potentially infected tissue from sheep meat intended for human consumption. In order to restrict the risk of BSE-infected sheep entering the food chain, all member countries of the European community are required to examine a fixed number of slaughtered and dead sheep and goats for TSE on an yearly basis (European Commission, 2002). Requirements also order the removal of specified risk material of sheep, consisting of the skull including the brain, the eyes, the tonsils and the spinal cord of animals aged over 12 months, and the spleen of the animals of all age groups (European Commission, 2000). The tonsils (tonsillae or amygdalae) consist of an accumulation of lymphocytes which are usually concentrated in lymph nodules (noduli lymphatici) and are present in the mucosae of the oropharynx, nasopharynx and laryngopharynx. They are a part of the integrated pharyngeal mucosal immune system (Ogra, 2000) and form a ring of lymphoid tissue in the pharyngeal wall which was first described by von Waldeyer-Hartz (1884), and is therefore called the ‘‘Waldeyer ring’’ (Perry and Whyte, 1998). This location involves an important role for tonsils as secondary lymphoid tissue in the immunological response against antigens which enter the body by the oral or nasal route (Brandtzaeg, 1984; Bernstein et al., 1999). Six tonsils are described in sheep: the palatine tonsil (tonsilla palatina), the lingual tonsil (tonsilla lingualis) and the tonsil of the soft palate (tonsilla veli palatini) in the oropharynx, the pharyngeal tonsil (tonsilla pharyngea) and the tubal tonsil (tonsilla tubaria) in the nasopharynx, and the paraepiglottic tonsil (tonsilla paraepiglottica) in the laryngopharynx (Barone, 1997; Thome´ , 1999). The topography and structure of these tonsils in sheep are poorly documented. The palatine tonsil is described as a tonsil with the size of a hazelnut which has three to six crypts (Ellenberger and Illing, 1911). According to Thome´ (1999), the lingual tonsil and the tonsil of the soft palate both consist of a small number of lymph nodules or a small amount of lymphoid
tissue. The tonsil of the soft palate is located at the ventral side of the soft palate (Thome´ , 1999). The pharyngeal tonsil forms an elevation at the caudal part of the pharyngeal septum with several invaginations of the epithelium (Barone, 1997; Thome´ , 1999). The distribution of lymphoid tissue and the epithelium of this tonsil has been examined by Chen et al. (1990, 1991). The tubal tonsil is a slightly elevated, diffuse aggregation of lymph nodules which is restricted to the mucosa at the pharyngeal opening of the auditory tube according to Thome´ (1999), but it can extend to the dorsal surface of the soft palate according to Barone (1997). Koch (1970) mentioned crypts in the tubal tonsil. Chen et al. (1989) described nodular aggregations of lymphoid tissue at the opening of the auditory tube and Stanley et al. (2001) studied the clustered lymph nodules which are concentrated just caudal to the opening of the auditory tube. The paraepiglottic tonsil has been described as a crypt tonsil (Ellenberger and Illing, 1911) which is located ‘‘where the food ball passes after lifting of the soft palate’’ (Koch, 1970). Except for Chen et al. (1989) and Stanley et al. (2001), most authors do not mention the microscopic structure of the ovine tonsils, but refer to data about bovine tonsils. In the present study, we describe the topography and the microscopic structure of the various tonsils in sheep.
2. Materials and methods 2.1. Animals The heads of 15 sheep aged between 9 and 12 months were collected from the slaughterhouse. Ten of these heads were used for the macroscopic examination and the tonsils of the five remaining heads were excised for microscopic examination.
2.2. Macroscopic examination The skin and mandibles were removed from all 10 heads and five heads were sectioned in the median plane. The heads were rinsed with tap water for 2 min and fixed in 2% acetic acid for 24 h to visualise the lymph nodules (Cornes, 1965; Chauhan and Singh, 1970). The location of the lymph nodules was noted,
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the length and width of the tonsils were measured and the number of lymph nodules was counted. 2.3. Microscopic examination The pharyngeal, palatine and paraepiglottic tonsils, the lateral pharyngeal wall surrounding the pharyngeal opening of the auditory tube, the soft palate and the part of the tongue caudal to the lingual torus were collected from five heads. These samples were reduced to blocks of maximum 15 mm 10 mm 5 mm and fixed in 3.5% formaldehyde for at least 2 d. Tissue blocks were processed by routine methods and embedded in paraffin wax. Serial tissue sections (8 mm thick) with a 1000 mm interval were made from all tonsils, except for the paraepiglottic tonsil from which serial sections (8 mm) without intervals were made. The slides were stained with haematoxylin and eosin (HE), and examined for the presence of lymphoid tissue with a motorized microscope (Olympus BX61, Olympus Belgium, Aartselaar, Belgium). Special attention was given to the differentiation of lymph nodules (primary or secondary nodules), the parafollicular tissue, the overlying epithelium and the connective tissue surrounding the lymphoid tissue. Lymph nodules were defined as compact nodular aggregates of lymphocytes surrounded by a diffuse sheet of lymphoid tissue (Thuring et al., 2002).
3. Results
Fig. 1. Median section of a sheep head, (a) palatine tonsil; (b) lingual tonsil; (c) tonsil of the soft palate; (d) pharyngeal tonsil; (e) tubal tonsil; (f) paraepiglottic tonsil.
Fig. 2. Rostral view of the larynx of a sheep. White arrows: palatine tonsil; black arrows: paraepiglottic tonsil.
3.1. The palatine tonsil The palatine tonsil was located bilaterally in the pharynx between the palatoglossal and the palatopharyngeal arch (Fig. 1) and formed an elevation of the pharyngeal mucosae with two or three narrow elongated entrances to the underlying crypts (Figs. 2 and 3). The tonsil was clearly distinguishable from the surrounding tissue by its capsule of dense collagen tissue which invaginated the tonsil once or twice forming two or three lobes. The tonsil approximately had the size of a hazelnut. The squamous epithelium that covered the surface formed tonsillar crypts by invaginating into the underlying connective tissue. These crypts could have a large lumen and were frequently branched. In the walls of these crypts,
Fig. 3. Dorsal view of the larynx and tongue of a sheep. Ellipse: location of the lingual tonsil; white arrows: caudal papilla vallata; black arrows: piriform recess; (a) epiglottis; (b) arytenoid cartilage; (c) soft palate (sectioned); (d) root of tongue; (e) palatine tonsil.
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numerous secondary lymph nodules were located. The stratified epithelium of the crypts was frequently infiltrated heavily by lymphocytes. 3.2. Lingual tonsil The lingual tonsil could not be located by any macroscopic landmarks. Histological examination revealed small concentrations of lymphoid tissue at both sides of the dorsal part of the tongue (dorsum linguae) without reaching the midline. They were located 2–4.5 cm rostral to the base of the epiglottis, extending in the subepithelial connective tissue from 1 cm caudal to 2.5 cm rostral to the most caudal gustatory vallate papilla (papilla vallata) (Fig. 3). The lymphoid cells were mainly organised in small dense aggregations or were scattered in the propria– submucosa layer (Fig. 4). Two primary nodules were observed only in two out of five tongues. The subepithelial connective tissue of several vallate papillae contained lymphoid aggregations. Neither any infiltration of lymphocytes in the stratified squamous epithelium of the tongue nor any tonsillar crypts were seen. 3.3. Tonsil of the soft palate On the ventral (oral) surface of the soft palate, no macroscopic signs of this tonsil were observed, and on histological slides, only few small aggregations were
Fig. 5. Histological section of the tonsil of the soft palate showing two primary nodules in the dorsal (nasal) part of the soft palate with lymphocyte infiltration in the epithelium above the nodules (HE).
found in the subepithelial tissue. However, on the dorsal (nasal) side, scattered nodules of lymphoid tissue varying in number from 7 to 104, were made visible by the acetic acid. The structure of the lymphoid tissue varied from diffuse lymphoid tissue with some lymphoid nodules in the rostral part of the soft palate over an intermediate zone with several primary and secondary lymph nodules and aggregations of lymphocytes (Fig. 5) towards a caudal zone near the arcus veli palatini, where scattered small aggregations were present. The slightly keratinized squamous epithelium of the nasal side of the soft palate was sometimes infiltrated by lymphocytes in the area above the lymph nodules and aggregated lymphocytes. 3.4. Pharyngeal tonsil
Fig. 4. Histological section of the lingual tonsil showing aggregation of lymphocytes (arrows) in the subepithelial connective tissue (HE).
The pharyngeal tonsil was located in the roof of the pharynx on the caudal part of the pharyngeal septum. The length of the tonsil varied from 18 to 40 mm and the total width from 12 to 21 mm. The surface of this tonsil showed several folds which were mostly oriented longitudinally. After fixation in acetic acid, small white spots became visible on the surface of the tonsil. The lymphoid tissue of this tonsil consisted of a layer of secondary nodules lying underneath the pseudostratified ciliated epithelium which was frequently infiltrated heavily by lymphocytes (Fig. 6). At the rostral pole of the tonsil, a gradual transition from nonlymphoid to tonsillar tissue was noticed as the thin layer
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Fig. 6. Histological section of a fold of the pharyngeal tonsil. Note the numerous secondary lymph nodules (arrows) and the central axis of dense connective tissue (asterisks) (HE).
of lymphocytes underlying the epithelium became thicker in caudal direction, and changed progressively into lymphoid tissue organized in secondary nodules. At the dorsal and caudal borders of the tonsil, a more abrupt transition was noticed. In these zones, some scattered secondary nodules were observed. 3.5. Tubal tonsil After fixation in acetic acid, scattered nodules were observed in the lateral pharyngeal wall, ventral, and mainly caudal, to the opening of the auditory tube (Fig. 7). The number of these nodules on each side varied from 41 to 150. The borders of the tubal tonsil could not be clearly distinguished using histology. Lymphoid tissue was found mainly ventral to the pharyngeal opening of the auditory tube, and was distributed variably in rostral to caudal direction in different sheep. The mucosa of the medial nasopharyngeal part of the auditory tube contained several primary and secondary lymph nodules, whereas the lateral mucosa contained less and mostly diffuse lymphoid tissue. The epithelium covering the lymphoid tissue was sometimes infiltrated by lymphocytes.
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Fig. 7. Medial view of the right nasopharyngeal wall after fixation in 2% acetic acid. Note the white-pinhead-sized tonsillar nodules (ellipse) ventral to the opening of the auditory tube (arrow); (a) nasopharynx; (b) tongue; (c) pharyngeal septum after removal of the pharyngeal tonsil; (d) soft palate.
recess (recessus piriformis) (Fig. 2). The tonsil appeared as a cluster of small nodular elevations which became white after fixation in acetic acid. The number of these nodular elevations per tonsil ranged from one to eight. Length of the tonsil varied from 5 to 14 mm and width ranged from 2 to 5 mm. Histology revealed that the elevations consisted of aggregations of lymph nodules surrounded by parafollicular tissue and encapsulated by connective tissue from which septa extended into the larger nodules. Most of the
3.6. Paraepiglottic tonsil The paraepiglottic tonsil was located lateral to the epiglottis in the ventromedial segment of the pharyngeal sidewall, i.e. at the floor of the piriform
Fig. 8. Histological section of the paraepiglottic tonsil showing an aggregation of lymph nodules with a central crypt (arrows). The epithelium of the crypt is infiltrated by numerous lymphocytes (HE).
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Fig. 9. Histological section of the paraepiglottic tonsil showing an aggregation of lymph nodules without crypt. Note the septum of dense connective tissue (asterisks) and the invagination (arrows) of the epithelium next to the aggregation (HE).
lymph nodules were secondary nodules. Primary lymph nodules were the most obvious at the periphery of the aggregations. Crypts were present in 6 out of the 27 aggregations which were observed in the 10 tonsils examined (Fig. 8). Epithelial invaginations were frequently observed near the aggregations without crypts (Fig. 9). The stratified squamous pharyngeal epithelium covering the lymphoid tissue was frequently infiltrated by numerous lymphocytes. In the aryepiglottic fold (plica aryepiglottica), varying numbers (0–15) of lymph nodules could be determined.
4. Discussion Three out of the six tonsils present in sheep are clearly distinguishable from the surrounding tissue, even without fixation in 2% acetic acid, viz. the palatine, the pharyngeal and the paraepiglottic tonsil. The present findings about the palatine and pharyngeal tonsils match the data mentioned in previous studies (Ellenberger and Illing, 1911; Barone, 1997; Thome´ , 1999), and are similar to those given in other species. In contrast, novel data are given about the paraepiglottic tonsil which were previously described only briefly (Ellenberger and Illing, 1911; Koch, 1970). As crypts could not be clearly demonstrated in a preliminary study of serial sections of the para-
epiglottic tonsil with intervals of 500 mm (unpublished data), we performed serial sections without intervals in the present study, and we observed crypts in 6 out of 27 aggregations (22%). Consequently, the ovine paraepiglottic tonsil is composed of some tonsillar follicles (folliculi tonsillares) that contain crypts, and numerous aggregations without crypts. For the latter type of aggregations, we could not find a term in the literature. Therefore, we suggest to introduce the term ‘‘tonsillar nodules’’ (noduli tonsillares) for naming the clearly distinguishable aggregations of lymph nodules and paranodular tissue which are surrounded by connective tissue but lack epithelial crypts. The tubal tonsil and the tonsil of the soft palate of sheep are not macroscopically distinguishable from the surrounding tissue, but they can be visualized by fixation in 2% acetic acid, although even then, their borders are not very clearly visible. Both tonsils consist of scattered lymph nodules, aggregations of lymphocytes and diffuse lymphoid tissue, and they are not encapsulated. In contrast to the description of Thome´ (1999), we found almost no lymphoid tissue on the oral side of the soft palate. The dorsal surface of the soft palate, on the other hand, contains a large amount of lymphoid tissue. Therefore, we concluded that the tonsil of the soft palate of sheep is located on the dorsal surface of the soft palate and faces the nasopharynx instead of the oropharynx. The lingual tonsil cannot be determined macroscopically in sheep. In contrast to the bovine lingual tonsil which is clearly macroscopically visible and consists of numerous tonsillar follicles (Manesse et al., 1995), only a few scattered aggregations of lymphoid cells are present in the lingual tonsil of sheep. However, as this lymphoid tissue has a typical distribution on the lingual surface and is more abundant at this location than in the other parts of the tongue, we think that it is justified to use the term ‘‘lingual tonsil’’ also in sheep. Several issues about the lymphoid tissue in sheep need to be further elucidated to understand better the invasion and spreading of scrapie and other TSE. Membranous epithelial cells (M-cells) or cells with feature characteristics of M-cells were demonstrated in human, canine, porcine, equine and rabbit tonsils (Belz and Trevor, 1995, 1996; Claeys et al., 1996; Gebert et al., 1995; Gebert, 1997; Kumar and
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Timoney, 2001) and in ovine pharyngeal and tubal tonsils (Chen et al., 1990, 1991; Stanley et al., 2001). They are considered as specialized cells for transepithelial transport, delivering the foreign antigens and microorganisms to the organized subepithelial lymphoid tissues (Kraehenbuhl and Neutra, 2000). Gonzalez et al. (2001) and Stanley et al. (2001) studied the distribution of the B and T subtypes of lymphocytes in ovine lymph nodes and pharyngeal tonsils, respectively. However, no research on the distribution of B and T subtypes, macrophages, dendritic and follicular dendritic cells in the different tonsils of sheep have been performed. Special attention needs to be given to the distribution of follicular dendritic cells, because it has been proved that the replication of the BSE and scrapie agents takes place in these cells (Brown et al., 1999, 2000). Furthermore, the innervation of the tonsils should also be elucidated better in order to understand the possible link with neuroinvasion in tonsils.
Acknowledgments The authors thank L. Standaert and L. De Bels for their excellent technical support. This work was supported by BOF-grant #011B4101 (Ghent University).
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