Enzootic nasal tumor, formerly known as infectious adenopapilloma or infectious nasal adenocarcinoma, is a virus-induced tumor of nasal cavities in small ruminants. The causative agent of ENT is ENTV, a chimeric group B/D retrovirus; ENTV is closely related to exogenous infectious JSRV, the causative agent of jaagsiekte (ie, ovine pulmonary adenocarcinoma), and to endogenous JSRVs (which are present in genomes of all sheep).1
Two variants of ENTV have been identified: ENTV-1 typically infects sheep, and ENTV-2 typically infects goats.2 However, these viruses do not have strict host specificity, and cross-species transmission of these viruses is possible. Enzootic nasal tumor viruses are horizontally transmitted in secretions and can infect a large number of flock animals; animals develop disease after a 1-year virus incubation period.3
Enzootic nasal tumors affect small ruminants worldwide, primarily in countries in which the ovine and caprine industries are well developed. Exceptions include Australia and New Zealand, which have well-developed ovine and caprine industries but in which ENT has not been reported. Prevalence of ENT varies among flocks of sheep and goats. Other authors4 determined that the prevalence of ENT in flocks of sheep is approximately 0.1% to 0.3%, and this value does not change over time. However, authors of another study3 determined that the prevalence of ENT increases over time (from 0.6% to 6.6%). Authors of studies performed in Germany5 and Nigeria6 determined that the prevalence of ENT ranges from 2% to 15% and 0.3% to 2%, respectively. Enzootic nasal tumor has also been detected in goats7 and sheep8 in Italy.
Enzootic nasal tumor is an adenocarcinoma with low malignancy; it is locally aggressive but rarely metastasizes to the regional lymph nodes or lungs.7 Animals with ENTs typically have clinical signs of upper airway disease.8
Both ENTV and JSRV infect secreting epithelial cells, and their oncogenic properties are attributable to a structural protein of the virus envelope. This protein is necessary and sufficient to induce transformation of cells by activation of secondary intracellular transduction pathways; activation of these pathways develops independent from cellular mechanisms responsible for viral replication.1
Typically, ENTV and JSRV do not induce humoral immunity in animals. This may be because sheep have an endogenous retrovirus in their genome that is highly related to ENTV and JSRV; this endogenous retrovirus may block immune system identification of ENTV and JSRV.9 Alternately, JSRV, ENTV-1, and ENTV-2 could be immunosuppressive and may interfere with the development of immune responses by infected lymphoid cells.10
The pathogenesis and epidemiology of ENT have been widely studied; however, to the authors' knowledge, the histogenesis of ENT has not been definitively determined. Results of another study11 in which ENTs of goats were examined histochemically indicated that the papillary component of ENT is derived from serous glands of respiratory mucosa, but the tubular component arises from glands of olfactory mucosa. Determination of the histogenesis of ENT in sheep and comparison with that in goats could further clarify the origin of these tumors. The purpose of the study reported here was to determine glycohistochemical characteristics of ENT of sheep, compare results for ENT with those of histologically normal nasal mucosa of sheep, and identify the histologic origin of ENT.
Materials and Methods
Samples—Tissue samples from five 3-year-old Lacaune sheep euthanized for chronic wasting disease characterized by nasal mucocatarrhal discharge, dyspnea, and sneezing8 were included in the study. Euthanasia was performed by stunning with a nonpenetrating captive bolt followed by exsanguination, in accordance with published guidelines.12,13 Necropsy revealed all 5 sheep had intranasal tumors that were grossly and histologically consistent with ENT. Morphological ultrastructural analysis8 revealed retrovirus-like particles in the tumor tissue, confirming the diagnosis of ENT. Samples of ENT and contiguous grossly normal nasal mucosa from ethmoidal turbinates were collected from sheep with ENT. Samples of grossly normal nasal mucosa were obtained from 5 healthy 3-year-old Lacaune sheep that had been routinely slaughtered at an abattoir.
Tissue sample preparation and histologic evaluation—Samples of ENT and grossly normal nasal mucosa were fixed by 2 methods for determination of morphological and histochemical staining characteristics. Some tissue samples from each sheep were fixed at room temperature (approx 18° to 20°C) in Bouin fluid for 18 hours for determination of tissue morphology. Other samples from each sheep were fixed at room temperature in Carnoy fluid for 24 hours and postfixed in 2% calcium acetate–4% paraformaldehyde solution (1:1) for 3 hours for glycohistochemical procedures. Fixed samples were dehydrated by immersion in a series of progressively more concentrated ethanol solutions, cleared in xylene, embedded in paraffin wax, and cut in 5-μm-thick serial sections.
For determination of morphology, tissue sections were stained with H&E, Mallory azan trichrome, and toluidine blue.14 Carbohydrate characterization was performed via conventional and lectin histochemical analysis15,16 to discriminate glycoconjugates on the basis of their principal chemical groups (-OH, -COOH, or -SO3H−) and principle terminal or internal oligosaccharidic glucidic residues (eg, fucose, sialic acid, mannose, N-acetylglucosamine, N-acetylgalactosamine, and galactose), respectively (Appendix).
Tissue sections were stained with the following conventional histochemical stains: PAS, AB (pH, 2.5), AB-PAS, AB (pH, 1), AB (pH, 0.5), low iron diamine, and high iron diamine. Before AB (pH, 2.5) staining, some sections were treated with sialidase or potassium hydroxide and sialidase. For lectin histochemical analysis, sections were immersed in a solution of 3% hydrogen peroxide in absolute methanol for 10 minutes at room temperature to inhibit endogenous peroxidase activity, followed by rinsing in distilled water and 0.1M Na+ and K+ PBS solution (pH, 7.2). Tissue sections were incubated in PBS solution containing 10 to 40 μg of lectin–horseradish peroxidase conjugatea/mL and 0.1mM CaCl2, MnCl2, and MgCl2 for 30 minutes at room temperature. Lectin binding sites were detected by incubating tissue sections with incubation medium containing diaminobenzidine hydrogen peroxide for 15 minutes at room temperature.17 Lectins and their carbohydrate binding specificities were summarized (Appendix).
Tissue sections incubated with lectin solutions to which 0.2M hapten sugars had been added were used as negative control sections for lectin histochemical analysis. Prior to lectin staining, some tissue sections had been incubated at 37°C for 16 hours in a solution of sialidase from Clostridium perfringensa (0.8 U/mL) in 0.1M sodium acetate buffer (pH, 5.5) containing 10mM CaCl2. These sections had been adjacent in tissue samples to sections used for lectin histochemical analysis. Sialic acid residues with O-acetyl substituents at C4, which were not cleaved by sialidase treatment,18,19 were cleaved after removal of acetyl groups via saponification by immersing tissue sections in a 1% solution of potassium hydroxide in 70% ethanol for 15 minutes at room temperature. Negative control sections for sialidase treatment were incubated with sialidase-free buffer at 37°C for 16 hours.
Staining intensities in tissue sections were subjectively scored by 3 authors (PS, FM, and EL) who separately evaluated tissue sections. Tissue sections were evaluated by use of a photomicroscopeb with a 25× objective and imaging software.c
Results
Sections of nasal mucosa included respiratory and olfactory components. Components of respiratory mucosa included cylindrical pseudostratified ciliated epithelium with scattered goblet cells. In the connective tissue, there were numerous glands that comprised mucous-secreting glands in superficial portions and glands with a more typical serous appearance in deep portions (Figure 1). Olfactory mucosa was covered by pseudostratified ciliated epithelium and had numerous serous glands in the connective tissue.
Representative photomicrographic views of histologically normal respiratory (A) and olfactory (B) mucosa and the papillary (C) and tubular (D) portions of ENTs from sheep. Notice that the superficial portion of the respiratory mucosa has pseudostratified ciliated epithelium with scattered goblet cells (arrows), there are glands comprising mucous cells in the connective tissue (arrowhead), and the deep portion has serous glands (A; asterisk). Notice that olfactory mucosa is covered by pseudostratified ciliated epithelium over connective tissue containing serous glands (B; asterisks). Notice that ENT has 2 patterns of growth. Peripherally, ENT has a papillary pattern of growth (C), whereas the deep portion of the tumor has a tubular architecture (D). H&E stain; bars = 50 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory (A) and olfactory (B) mucosa and the papillary (C) and tubular (D) portions of ENTs from sheep. Notice that the superficial portion of the respiratory mucosa has pseudostratified ciliated epithelium with scattered goblet cells (arrows), there are glands comprising mucous cells in the connective tissue (arrowhead), and the deep portion has serous glands (A; asterisk). Notice that olfactory mucosa is covered by pseudostratified ciliated epithelium over connective tissue containing serous glands (B; asterisks). Notice that ENT has 2 patterns of growth. Peripherally, ENT has a papillary pattern of growth (C), whereas the deep portion of the tumor has a tubular architecture (D). H&E stain; bars = 50 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory (A) and olfactory (B) mucosa and the papillary (C) and tubular (D) portions of ENTs from sheep. Notice that the superficial portion of the respiratory mucosa has pseudostratified ciliated epithelium with scattered goblet cells (arrows), there are glands comprising mucous cells in the connective tissue (arrowhead), and the deep portion has serous glands (A; asterisk). Notice that olfactory mucosa is covered by pseudostratified ciliated epithelium over connective tissue containing serous glands (B; asterisks). Notice that ENT has 2 patterns of growth. Peripherally, ENT has a papillary pattern of growth (C), whereas the deep portion of the tumor has a tubular architecture (D). H&E stain; bars = 50 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Grossly, ENTs had a cauliflower-like appearance and extended bilaterally from the ethmoidal region to the middle nasal cavity of sheep. These tumors were soft and covered by thick mucus, which extended to adjacent grossly normal mucosa. Histologically, ENTs were well-differentiated adenocarcinomas with 2 patterns of growth: peripherally, tumors had a papillary pattern (Figure 1), whereas in deep portions, tumors had a compact tubular architecture. Tumor cells were cylindrical to cuboidal, with basally located oval nuclei, irregularly clumped chromatin, and several small nucleoli.
Results of conventional and lectin histochemical reactions were summarized (Tables 1 and 2). Sections of histologically normal nasal mucosa from sheep with ENT and sheep unaffected by ENT did not differ with regard to staining; results for these tissues were combined and represented results for histologically normal respiratory and olfactory mucosa. Typically, sections of respiratory mucosa had stronger glycohistochemical staining than did olfactory mucosa in cells of the epithelial lining and glands in the connective tissue. Similarly, sections of papillary portions of ENTs had stronger histochemical staining than tubular portions.
Intensity of conventional histochemical staining in sections of histologically normal respiratory and olfactory mucosa and ENTs obtained from 10 sheep (5 sheep unaffected by ENT and 5 sheep with ENT).
| Histochemical treatment | Respiratory mucosa | Olfactory mucosa | ENT | Sugar moieties detected by stain | |||||
|---|---|---|---|---|---|---|---|---|---|
| Epithelium | Goblet cells | Respiratory glands | |||||||
| Serous glands | Mucous glands | Epithelium | Olfactory glands | Papillary portion | Tubular portion | ||||
| PAS | +a | ++ | ±/+b | ++ | ±b | +/++b | ++ | ±/+ | Vicinal hydroxyls |
| AB (pH, 2.5) | +a | ++ | ±/+b | ++ | − | +/++b | ++ | ±/+ | Acidic groups |
| Sialidase-AB (pH, 2.5) | +a | ++ | ±/+b | ++ | − | +/++b | +/++ | ± | Acidic groups with C4-acetylated SA |
| KOH-sialidase-AB (pH, 2.5) | +a | ++ | ±/+b | ++ | − | +/++b | +/++ | ± | Asialilated acidic groups |
| AB (pH, 1) | ±a | + | −/±a | + | − | +/++b | +/++ | −/± | Sulfated groups |
| AB (pH, 0.5) | ±a | + | −/±a | + | − | +/++b | ±/+ | −/± | Highly sulfated groups |
| AB-PAS | B+/R+a | B++/R+ | B+/R+ | B++/R+ | − | B+/++b | B++/R+ | B+a/R+a,b | Acidic groups and vicinal hydroxyls |
| Low iron diamine | +a | ++ | ±/+a | ++ | − | +/++b | ++ | ±/+ | Acidic groups |
| High iron diamine | ±a | + | −/±a | + | − | +/++b | +/++ | −/± | Sulfated groups |
Intensity of staining in tissue sections was scored as follows: negative (−), weak (±), moderate (+), and strong (++). Slashes indicate that the staining intensity score is between the 2 indicated scores.
Staining detected in cell coats.
Staining detected in the apical aspects of cells. B = Blue stain. R = Red stain. SA = Sialic acid.
Intensity of lectin histochemical staining in sections of histologically normal respiratory and olfactory mucosa and ENTs obtained from the 10 sheep in Table 1.
| Histochemical treatment | Respiratory mucosa | Olfactory mucosa | ENT | Sugar moieties detected by stain | |||||
|---|---|---|---|---|---|---|---|---|---|
| Epithelium | Goblet cells | Respiratory glands | |||||||
| Serous glands | Mucous glands | Epithelium | Olfactory glands | Papillary portion | Tubular portion | ||||
| WGA | +a | + | −/± | ±/+ | +b | + | ±/+c; +a | ± | GlcNac |
| Con A | +b | ± | + | ±/+ | +b | ±/+ | + | ± | D-mannose |
| LTA | +a | + | + | ±/+ | ±/+b | +b | ± | ±/+ | α-L-Fuc |
| UEA I | +a,b | + | + | ±/+ | ±/+b | +b | ±/+ | ± | α-L-Fuc |
| SBA | +a | + | ±/+b | + | ±b | + | ± | ±/+c | α-, β-GalNAc |
| GSA IB4 | + | − | − | − | ±b | + | − | − | α-Gal |
| DBA | ++ | ++ | ++ | −/++ | − | ± | ±; +a | ±/+ | α-GalNAc |
| Sialidase-DBA | ++ | ++ | ++ | −/++ | − | ± | ±; +a | ±/+ | SA (not C4 acetylated)-α-GalNAc |
| KOH-sialidase-DBA | ++ | ++ | ++ | −/++ | − | ± | ±; +a | ±/+ | SA (C4 acetylated or not)-α-GalNAc |
| PNA | − | +/++ | − | − | − | +/++ | −/+b | − | Tβ-Gal(1–3)GalNAc |
| Sialidase-PNA | +c; ++a | ++ | ++b | ++ | +b;+/++a | ++ | +b | ±/+ | β-Gal(1–3)GalNAc T and St to SA (not C4 acetylated) |
| KOH-sialidase-PNA | +c; ++a | ++ | ++b | ++ | +b; +/++a | ++ | +/++b | +/++ | β-Gal(1–3)GalNAc T and St to SA (C4 acetylated or not) |
| RCA I | +b | − | − | − | − | −/±b | ± | ± | T β-Gal(1–4)GlcNAc |
| Sialidase-RCA I | +b | − | − | − | − | −/±b | ± | ± | β-Gal(1–4)GlcNAc T and St to SA (not C4 acetylated) |
Staining detected in the supranuclear portions of cells. α-L-Fuc = α-L-fucose. Gal = Galactose. GalNAc = N-acetylgalactosamine. GlcNac = N-acetylglucosamine. St = Subterminal. T = Terminal.
See Table 1 for remainder of key.
Histologically normal respiratory mucosa had positive results for PAS staining in epithelial coats and goblet cells. Strongly positive results for PAS staining were observed in mucous gland cells, whereas in serous glands, there was PAS reactivity in the apical aspects and cell coats of secreting cells. These same sites had positive results for AB-PAS staining; in particular, goblet cells and mucous glands had positive results for AB staining and were strongly reactive to AB (pH, 1). Evaluation of toluidine blue–stained respiratory mucosa indicated there was metachromasia in mucous glands and some goblet cells (Figure 2).
Representative photomicrographic views of histologically normal respiratory (A) and olfactory (B) mucosa and the papillary (C) portion of an ENT from sheep. Notice that only some cells in mucous glands (arrowheads) and a few goblet cells (arrow; inset) in the respiratory mucosa are metachromatic (A). Notice that the apical aspects of serous glands (asterisks) in the olfactory mucosa appear weakly metachromatic (B). Notice that only some cells (arrows) in the papillary portion of the ENT are strongly metachromatic (C). Toluidine blue stain; bars = 50 μm (A, B, and C) and 30 μm (inset).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory (A) and olfactory (B) mucosa and the papillary (C) portion of an ENT from sheep. Notice that only some cells in mucous glands (arrowheads) and a few goblet cells (arrow; inset) in the respiratory mucosa are metachromatic (A). Notice that the apical aspects of serous glands (asterisks) in the olfactory mucosa appear weakly metachromatic (B). Notice that only some cells (arrows) in the papillary portion of the ENT are strongly metachromatic (C). Toluidine blue stain; bars = 50 μm (A, B, and C) and 30 μm (inset).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory (A) and olfactory (B) mucosa and the papillary (C) portion of an ENT from sheep. Notice that only some cells in mucous glands (arrowheads) and a few goblet cells (arrow; inset) in the respiratory mucosa are metachromatic (A). Notice that the apical aspects of serous glands (asterisks) in the olfactory mucosa appear weakly metachromatic (B). Notice that only some cells (arrows) in the papillary portion of the ENT are strongly metachromatic (C). Toluidine blue stain; bars = 50 μm (A, B, and C) and 30 μm (inset).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Sections of histologically normal olfactory mucosa had PAS, AB-PAS, and AB (pH, 1) staining and metachromatic sites in the apical aspects of gland cells and in cell coats of those cells (Figure 2). Strong PAS staining was observed in the apical aspects of cells in the papillary portion of ENTs. Extensive areas of AB reactivity were observed in papillary portions of ENTs stained with AB-PAS; AB reactivity was similar after AB (pH, 1) staining, and only a few cells were metachromatic. Cells in the tubular portion of ENTs had moderate reactivity to PAS and had positive results for AB-PAS staining in cell coats. Metachromasia was not detected in cells in the tubular portion of ENT.
Wheat germ agglutinin binding patterns were detected in cell coats and goblet cells in respiratory epithelium and in mucous glands (Figure 3). In olfactory mucosa, positive results for WGA staining were detected in the apical aspects of epithelial cells and in glands. Moderate WGA reactivity was detected in the supranuclear region of cells in the papillary portion of ENTs, whereas weak reactivity was detected in the tubular portion of ENTs.
Representative photomicrographic views of histologically normal respiratory mucosa (A) and the papillary portion of an ENT (B) from sheep. In the respiratory mucosa, notice WGA binding patterns in the cell coats and goblet cells (arrows); moderate reactivity is also present in mucous glands (A; arrowhead; inset). Notice moderate WGA reactivity in the supranuclear regions of cells of the papillary portion of the ENT (B). WGA stain; bars = 40 μm (A and B) and 20 μm (inset).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory mucosa (A) and the papillary portion of an ENT (B) from sheep. In the respiratory mucosa, notice WGA binding patterns in the cell coats and goblet cells (arrows); moderate reactivity is also present in mucous glands (A; arrowhead; inset). Notice moderate WGA reactivity in the supranuclear regions of cells of the papillary portion of the ENT (B). WGA stain; bars = 40 μm (A and B) and 20 μm (inset).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory mucosa (A) and the papillary portion of an ENT (B) from sheep. In the respiratory mucosa, notice WGA binding patterns in the cell coats and goblet cells (arrows); moderate reactivity is also present in mucous glands (A; arrowhead; inset). Notice moderate WGA reactivity in the supranuclear regions of cells of the papillary portion of the ENT (B). WGA stain; bars = 40 μm (A and B) and 20 μm (inset).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Moderate binding affinity for ConA was detected in the apical aspects of respiratory mucosa epithelial cells and serous glands; ConA binding affinity was lower in mucous glands. In olfactory mucosa, positive results for ConA staining were observed in the apical aspects of epithelial cells and in the glands (Figure 4). Moderate ConA reactivity was detected in the apical aspects of cells in the papillary portion of ENTs, and weak reactivity was detected in the tubular portion of ENTs.
Representative photomicrographic views of histologically normal respiratory epithelium (A) and the tubular portion of an ENT (B) from sheep. Notice that the supranuclear region of the respiratory epithelium has moderate ConA reactivity (A; asterisks). Notice the tubular portion of the ENT has weak ConA reactivity (B). ConA stain; bars = 40 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory epithelium (A) and the tubular portion of an ENT (B) from sheep. Notice that the supranuclear region of the respiratory epithelium has moderate ConA reactivity (A; asterisks). Notice the tubular portion of the ENT has weak ConA reactivity (B). ConA stain; bars = 40 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory epithelium (A) and the tubular portion of an ENT (B) from sheep. Notice that the supranuclear region of the respiratory epithelium has moderate ConA reactivity (A; asterisks). Notice the tubular portion of the ENT has weak ConA reactivity (B). ConA stain; bars = 40 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
All structures in sections of histologically normal respiratory mucosa had LTA (Figure 5) and UEA I binding sites, although in some instances, the staining intensity was different. Respiratory mucous glands, goblet cells, and olfactory glands had moderately positive results for SBA staining, whereas other sites were weakly reactive.
Representative photomicrographic view of histologically normal respiratory mucosa from a sheep. Notice LTA reactivity in goblet cells (arrows) and mucous glands (arrowheads). LTA stain; bar = 40 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic view of histologically normal respiratory mucosa from a sheep. Notice LTA reactivity in goblet cells (arrows) and mucous glands (arrowheads). LTA stain; bar = 40 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic view of histologically normal respiratory mucosa from a sheep. Notice LTA reactivity in goblet cells (arrows) and mucous glands (arrowheads). LTA stain; bar = 40 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Only weakly reactive GSA IB4 binding sites were detected in epithelium of histologically normal respiratory and olfactory mucosae. Strong DBA reactivity was detected in respiratory mucosa (Figure 6), and no reactivity was detected in olfactory mucosa. Dolichos biflorus agglutinin–reactive binding sites were observed in apical aspects of cells of both papillary and tubular portions of ENTs.
Representative photomicrographic views of histologically normal respiratory mucosa (A) and the papillary (B) and tubular (C) portions of ENT from sheep. Notice the epithelial lining of respiratory mucosa and serous glands (A; asterisks) have strong DBA reactivity, whereas mucous glands have 2 types of cells with strong reactivity (arrows) or without reactivity (arrowheads). Notice cell coats in the papillary portion of the ENT (B) have DBA reactivity, and there is moderate reactivity in the tubular portion of the ENT (C). DBA stain; bars = 50 μm (A) and 30 μm (B and C).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory mucosa (A) and the papillary (B) and tubular (C) portions of ENT from sheep. Notice the epithelial lining of respiratory mucosa and serous glands (A; asterisks) have strong DBA reactivity, whereas mucous glands have 2 types of cells with strong reactivity (arrows) or without reactivity (arrowheads). Notice cell coats in the papillary portion of the ENT (B) have DBA reactivity, and there is moderate reactivity in the tubular portion of the ENT (C). DBA stain; bars = 50 μm (A) and 30 μm (B and C).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory mucosa (A) and the papillary (B) and tubular (C) portions of ENT from sheep. Notice the epithelial lining of respiratory mucosa and serous glands (A; asterisks) have strong DBA reactivity, whereas mucous glands have 2 types of cells with strong reactivity (arrows) or without reactivity (arrowheads). Notice cell coats in the papillary portion of the ENT (B) have DBA reactivity, and there is moderate reactivity in the tubular portion of the ENT (C). DBA stain; bars = 50 μm (A) and 30 μm (B and C).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
In sections of histologically normal respiratory mucosa, PNA binding sites were detected in goblet cells (Figure 7); sialidase treatment enhanced this PNA reactivity and enabled PNA reactivity in epithelial cell coats, in apical aspects of serous gland cells, and in mucous glands. Olfactory glands had PNA reactivity; sialidase treatment enhanced this PNA reactivity and enabled PNA reactivity in cell coats. Saponification did not have any effect on PNA reactivity. In sections of the papillary portion of ENTs, there was moderate PNA staining in the apical aspects of a few cells; PNA binding was enhanced in ENT sections by sialidase treatment (Figure 8), after which almost all cells had PNA-reactive sites. Peanut agglutinin reactivity was further enhanced by potassium hydroxide treatment. Peanut agglutinin reactivity was not detected in cells of the tubular portion of ENTs; however, moderate PNA reactivity was observed in cells in this portion of ENTs after sialidase treatment. These cells had strong PNA reactivity in ENT sections treated with potassium hydroxide and sialidase.
Representative photomicrographic views of histologically normal respiratory (A and B) and olfactory (C and D) mucosa from sheep; tissue sections were not treated (A and C) or were treated (B and D) with sialidase. Notice there is PNA binding in goblet cells in respiratory mucosa (A; arrows). Notice that sialidase digestion enhances goblet cell PNA reactivity (B; arrows). Notice sialidase treatment promotes PNA reactivity in epithelial cell coats and mucous glands (arrowhead) and in the apical aspects of serous glands (B; asterisks). Notice PNA reactivity in serous glands of olfactory mucosa (C; asterisks). Notice sialidase treatment enhances PNA reactivity (asterisks) and enables strong reactivity in binding sites in epithelial cell coats (D; arrows). PNA stain; bars = 50 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory (A and B) and olfactory (C and D) mucosa from sheep; tissue sections were not treated (A and C) or were treated (B and D) with sialidase. Notice there is PNA binding in goblet cells in respiratory mucosa (A; arrows). Notice that sialidase digestion enhances goblet cell PNA reactivity (B; arrows). Notice sialidase treatment promotes PNA reactivity in epithelial cell coats and mucous glands (arrowhead) and in the apical aspects of serous glands (B; asterisks). Notice PNA reactivity in serous glands of olfactory mucosa (C; asterisks). Notice sialidase treatment enhances PNA reactivity (asterisks) and enables strong reactivity in binding sites in epithelial cell coats (D; arrows). PNA stain; bars = 50 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of histologically normal respiratory (A and B) and olfactory (C and D) mucosa from sheep; tissue sections were not treated (A and C) or were treated (B and D) with sialidase. Notice there is PNA binding in goblet cells in respiratory mucosa (A; arrows). Notice that sialidase digestion enhances goblet cell PNA reactivity (B; arrows). Notice sialidase treatment promotes PNA reactivity in epithelial cell coats and mucous glands (arrowhead) and in the apical aspects of serous glands (B; asterisks). Notice PNA reactivity in serous glands of olfactory mucosa (C; asterisks). Notice sialidase treatment enhances PNA reactivity (asterisks) and enables strong reactivity in binding sites in epithelial cell coats (D; arrows). PNA stain; bars = 50 μm.
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of sections of papillary (A) and tubular (B) portions of ENT from sheep. Notice that all cells of the papillary portion of ENT are moderately reactive to PNA after sialidase treatment (A) and this reactivity is stronger than it is in sections not treated with sialidase (left inset), in which binding is restricted to the apical aspects of some cells; notice saponification via potassium hydroxide treatment (right inset) enhances PNA reactivity. Notice the tubular portion of ENT has moderate PNA reactivity after sialidase digestion (B), which is enhanced by saponification (right inset); the tubular portion of ENT sections not treated with sialidase do not have PNA reactivity (left inset). Sialidase treatment and PNA stain; bars = 50 μm (panels A and B) and 20 μm (insets in panels A and B).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of sections of papillary (A) and tubular (B) portions of ENT from sheep. Notice that all cells of the papillary portion of ENT are moderately reactive to PNA after sialidase treatment (A) and this reactivity is stronger than it is in sections not treated with sialidase (left inset), in which binding is restricted to the apical aspects of some cells; notice saponification via potassium hydroxide treatment (right inset) enhances PNA reactivity. Notice the tubular portion of ENT has moderate PNA reactivity after sialidase digestion (B), which is enhanced by saponification (right inset); the tubular portion of ENT sections not treated with sialidase do not have PNA reactivity (left inset). Sialidase treatment and PNA stain; bars = 50 μm (panels A and B) and 20 μm (insets in panels A and B).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Representative photomicrographic views of sections of papillary (A) and tubular (B) portions of ENT from sheep. Notice that all cells of the papillary portion of ENT are moderately reactive to PNA after sialidase treatment (A) and this reactivity is stronger than it is in sections not treated with sialidase (left inset), in which binding is restricted to the apical aspects of some cells; notice saponification via potassium hydroxide treatment (right inset) enhances PNA reactivity. Notice the tubular portion of ENT has moderate PNA reactivity after sialidase digestion (B), which is enhanced by saponification (right inset); the tubular portion of ENT sections not treated with sialidase do not have PNA reactivity (left inset). Sialidase treatment and PNA stain; bars = 50 μm (panels A and B) and 20 μm (insets in panels A and B).
Citation: American Journal of Veterinary Research 73, 8; 10.2460/ajvr.73.8.1128
Strong reactivity to RCA I was observed in the apical zone of the epithelial lining of respiratory mucosa. Weakly reactive RCA I binding sites were detected in olfactory glands and in ENTs.
No staining was observed in tissue sections treated with unconjugated lectins or after incubation with hapten sugar in lectin–horseradish peroxidase conjugate solution. Immersion of tissue sections in enzyme-free buffer solution resulted in unmodified lectin binding.
Discussion
Analysis of conventional histochemical results of the present study enabled us to distinguish between neutral and acid glycoproteins and carboxylated and sulfated glycosaminoglycans on the basis of the chemical groups expressed by cells in tissue sections. We were also able to discriminate secretion and surface glycoconjugates on the basis of their locations in tissue sections. Neutral and acid glycoproteins were detected in respiratory serous glands and in papillary and tubular portions of ENTs, whereas neutral surface glycoproteins were prevalent in nasal mucosal epithelium and olfactory glands. The weak AB (pH, 1), AB (pH, 0.5), and high iron diamine reactivities, observed in cell coats in both the epithelial lining and serous glands of respiratory mucosa, indicated that sulfated groups pertaining to glycoproteins were in these structures. Indeed, sulfated groups are often linked to galactose residues,11,20,21 which were detected at those sites in both terminal and subterminal side chain positions (as indicated by PNA and RCA I reactivities).
Comparison among results for AB (pH, 2.5), AB (pH, 1), and AB (pH, 0.5) reactivities, and between results for low iron diamine and high iron diamine reactivities, indicated expression of secretion glycosaminoglycans (both hyaluronic acid and chondroitin sulfated type-like) in goblet cells and cells of mucous glands of respiratory mucosa. Olfactory glands had small amounts of chondroitin sulfated type–like material. Chondroitin sulfated type–like glycosaminoglycans were prevalent in the papillary portion of ENTs, whereas the tubular portion of ENTs did not produce glycosaminoglycans (Table 3).
Glycoconjugates detected on the basis of results of conventional and lectin histochemistry in sections of histologically normal respiratory and olfactory mucosa and ENTs obtained from the 10 sheep in Table 1.
| Tissue | Tissue component | Complex carbohydrates detected with conventional histochemical analysis | Glucidic residues or sequences pertaining to oligosaccharidic chains detected with lectin histochemical analysis |
|---|---|---|---|
| Respiratory mucosa | Epithelium | Prevalently neutral surface GPs; sulfated groups not pertaining to GAGs | β-Gal(1–4)GlcNAc, α-L-Fuc, α-D-GalNAc, and SA-β-Gal(1–3) GalNAc |
| Goblet cells | Secretion GAGs of both HyAc and ChSC type-like | β-Gal(1–3)GalNAc, SA-β-Gal(1–3)GalNAc, α-D-GalNAc, and α-L-Fuc | |
| Serous respiratory glands | Neutral and acid surface GPs; sulfated groups not pertaining to GAGs | α-L-Fuc, α-D-GalNAc, and SA-β-Gal(1–3)GalNAc | |
| Mucous respiratory glands | Secretion GAGs of both HyAc and ChSC type-like; secretion acid GPs | SA-β-Gal(1–3)GalNAc, α-D-GalNAc, α-L-Fuc | |
| Olfactory mucosa | Epithelium | Prevalently neutral surface GPs | α-L-Fuc, SA-β-Gal(1–3)GalNAc, α-Gal, β-D-GalNAc |
| Olfactory glands | Prevalently neutral surface GPs; secretion GAGs of ChSC type-like | α-Gal, β-Gal-([1–3]GalNAc; [1–4]GlcNAc), α-D-GalNAc, α-L-Fuc, and SA-β-Gal(1–3)GalNAc | |
| ENT | Papillary portion | Secretion GAGs of ChSC type-like; neutral and acid surface GPs | α-L-Fuc, α-D-GalNAc, β-Gal(1–3)GalNAc, and SA C4-acetylated-β-Gal(1–3)GalNAc |
| Tubular portion | Neutral and acid surface GPs | α-L-Fuc, α-D-GalNAc, β-Gal(1–4)GlcNAc, and SA C4-acetylated-β-Gal(1–3)GalNAc |
Lectin histochemical results of the present study indicated glycoproteins with both O- and N-glycosidically linked oligosaccharides were present in tissue sections because binding sites for ConA, WGA, LTA, and UEA I were detected.17,22,23 Respiratory mucosa and the papillary portion of ENTs were more reactive to LTA and UEA I staining than were olfactory mucosa and the tubular portion of ENTs. These 2 lectins had similar binding sites, although in some instances the intensity of the reaction was different. This finding was not unusual for these 2 lectins that have the same nominal specificity24 and could be attributable to efficiency of internalization of these 2 lectins (that have different molecular weights [UEA I, 68 kDa; LTA, 120 kDa]) or to the fact that UEA I seems to preferentially bind α-L-fucose linked to galactose,25,26 whereas LTA preferentially binds α-L-fucose linked to N-acetylglucosamine.22,27 Fucoglycoconjugates were primarily localized in cell coats or apical aspects of cells, supporting the role of α-L-fucose in the morphogenetic process of plasma membranes undergoing rapid turnover.28 Moreover, secretion fucoglycoproteins were observed in goblet cells and mucous glands of respiratory mucosa in the present study.
α-D-N-acetylgalactosamine terminal residues were observed in all examined structures, with the exception of the epithelial lining of olfactory mucosa, which instead expressed β-D-N-acetylgalactosamine in addition to α-galactose. However, low amounts of this residue were detected.
β-Galactose residues were linked to -(1–3)N-acetylgalactosamine and -(1–4)N-acetylglucosamine, as demonstrated by PNA and RCA I reactivities. β-Galactose also acted as a hapten sugar for sialic acid residues, but only when it was bound to -(1–3)N-acetylgalactosamine. Indeed, sialidase treatment affected only PNA reactivity, whereas RCA I reactivity was unchanged by this type of enzymatic digestion. The terminal sequence sialic acid-β-galactose (1–3)N-acetylgalactosamine was found in all examined structures. However, regarding ENTs, sialic acid residues were C4-acetylated, as indicated by the increase in PNA reactivity when sialidase treatment was preceded by potassium hydroxide treatment (ie, saponification).
An interesting finding of the present study was that C4-acetylated sialoderivatives were expressed by cells of both papillary and tubular portions of ENTs; C4-acetylated sialic acid residues were not detected in histologically normal mucosa. Presence of these sialoderivatives may increase resistance of ENT cells to pathogens versus non-ENT cells. In fact, C4-acetylated sialic acid could protect ENT cells from bacteria because this sialoderivative is resistant to cleavage by bacterial sialidase.26 Expression of multiple types of sialoderivatives by cells could provide multiple receptor sites for microorganisms; however, these sialoderivatives may mask cell recognition sites29–32 and indirectly enhance defense of cells against microorganisms.
Comparison of results of the present study with those of another study11 indicated there are differences between sheep and goats with regard to glycohistochemical characteristics of ENT and histologically normal nasal mucosa. In tissues from sheep in the present study, greater lectin reactivity was detected in respiratory mucosa versus olfactory mucosa. In addition, the papillary portion of ENTs had greater lectin reactivity than the tubular portion of ENTs, whereas the opposite was reported for goats.11 In tissue samples from sheep in the present study, the dimer β-Gal (1–4)N-acetylglucosamine was detected in olfactory mucosa and the tubular portion of ENTs, whereas it is not detected in these tissues of goats.11 In tissue samples from sheep in the present study, the terminal sequence sialic acid-β-galactose (1–3)N-acetylgalactosamine was detected in respiratory mucosa and the papillary portion of ENTs, whereas in goats, it is expressed by cells in all histochemically examined structures.11 The C4-acetylated sialic acid residues detected in ENTs from sheep in the present study were not detected in ENTs of goats.11
Authors of another study11 have suggested that the papillary portion of ENTs originates from respiratory glands and the tubular portion originates from olfactory glands in goats. This assumption was based on the finding that respiratory glands primarily produced secretion neutral and sulfated glycoconjugates, as did the papillary portion of ENTs, whereas olfactory glands primarily produced surface neutral and, to a lesser extent, carboxylated glycoconjugates, as did the tubular portion of ENTs. Results of lectin histochemical analysis in that study11 indicated the strong presence of neutral glucidic residues in ENTs of goats. By comparing the glycohistochemical characteristics of histologically normal nasal mucosa with those of ENTs of goats, those authors11 determined the papillary portion of ENTs originates from respiratory mucosa (because of the presence of glycoconjugates characterized by the terminal sequence sialic acid-N-acetylgalactosamine); similarly, the tubular portion of ENTs was determined to originate from olfactory mucosa (because of the presence of fucoglycoproteins).
Findings of the present study indicated that the glycohistochemical characteristics of histologically normal mucosa and ENTs in sheep were more similar than are those in goats.11 There were some similarities between results for histologically normal nasal mucosa and ENTs in sheep, such as the predominant expression of secreting glycoconjugates (ie, highly sulfated glycosaminoglycans and acid glycoproteins characterized by the sialic acid-β-galactose[1–3] N-acetylgalactosamine terminal sequence) in the papillary portion of ENT; that finding indicated a respiratory gland origin of this portion of ENTs, with likely involvement of goblet cells. Another similarity was the predominant expression of neutral surface glycoproteins (with terminal α-L-fucose, α-D-N-acetylgalactosamine, and β-galactose[1–4]N-acetylglucosamine) in the tubular portion of ENTs, indicating an olfactory gland origin.
Despite differences between results for histologically normal nasal mucosa from sheep in the present study and goats in another study,11 findings of the present study and those of that other study11 support the suggestion that the tubular portion of ENTs originates from olfactory glands, whereas the papillary portion of ENTs originates from respiratory mucous glands. Results of the present study also indicated that goblet cells may have a role in the histogenesis of the papillary portion of ENTs in sheep.
ABBREVIATIONS
| AB | Alcian blue |
| ConA | Concanavalin A agglutinin |
| DBA | Dolichos biflorus agglutinin |
| ENT | Enzootic nasal tumor |
| ENTV | Enzootic nasal tumor virus |
| GSA IB4 | Griffonia simplicifolia agglutinin B4 isolectin |
| JSRV | Jaagsiekte sheep retrovirus |
| LTA | Lotus tetragonolobus agglutinin |
| PAS | Periodic acid–Schiff |
| PNA | Peanut agglutinin |
| RCA | Ricinus communis agglutinin |
| SBA | Soybean agglutinin |
| UEA | Ulex europaeus agglutinin |
| WGA | Wheat germ agglutinin |
Sigma, St Louis, Mo.
Nikon Eclipse E800, Nikon Corp, Tokyo, Japan.
Act1, Nikon Corp, Tokyo, Japan.
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Appendix
Names, sources, and carbohydrate binding specificities of lectins used to stain sections of histologically normal respiratory and olfactory mucosa and ENTs from 5 sheep unaffected by ENT and 5 sheep with ENT.
| Name of lectin | Source of lectin | Carbohydrate binding specificity |
|---|---|---|
| WGA* | Triticum vulgare | N-acetylglucosamine |
| Con A* | Canavalia ensiformis | D-mannose and D-glucose† |
| LTA | Lotus tetragonolobus | α-L-fucose |
| UEA I | Ulex europaeus | α-L-fucose |
| SBA | Glycine max | α-and β-N-acetylgalactosamine |
| GSA IB4 | Griffonia simplicifolia | α-Galactose |
| DBA | Dolichos biflorus | α-N-acetylgalactosamine |
| PNA | Arachis hypogea | Galactosyl (β1–3)N-acetylgalactosamine |
| RCA I | Ricinus communis | Galactosyl (β1–4)N-acetylglucosamine |
Lectins bind only terminal carbohydrate residues unless otherwise indicated.
Lectin binds to residues in both terminal and internal positions of carbohydrates.
Binding to D-mannose > binding to D-glucose.
