Characterization of interdigital glands in the Asian elephant (Elephas maximus).

In the Asian elephant, wetness akin to perspiration is commonly observed on the cuticles and interdigital areas of the feet; this observation has lead to speculation regarding the existence of an interdigital gland. Our goal was to search for interdigital glands and characterise them morphologically, histochemically, and immunohistochemically. Necropsy samples of interdigital areas from two Asian elephants were obtained. Multiple sections were fixed and processed routinely, then stained with hematoxylin/eosin and differential mucin stains. Immunohistochemistry was also performed for cytokeratins 8 and 10. Interdigital glands resembling human eccrine glands were detected deep within the reticular dermis. Histochemical staining indicated neutral mucopolysaccharides and nonsulphated acid mucopolysaccharides in glandular secretions, and the glandular epithelium also showed immunoreactivity to cytokeratins 8 and 10. Both the histochemical and immunohistochemical staining patterns are analogous to human eccrine structures. This study shows with certainty that Asian elephants possess sweat glands as they are defined histologically.     

Characterization of interdigital glands in the Asian elephant (Elephas maximus)

In the Asian elephant, wetness akin to perspiration is commonly observed on the cuticles and interdigital areas of the feet; this observation has lead to speculation regarding the existence of an interdigital gland. Our goal was to search for interdigital glands and characterise them morphologically, histochemically, and immunohistochemically. Necropsy samples of interdigital areas from two Asian elephants were obtained. Multiple sections were fixed and processed routinely, then stained with hematoxylin/eosin and differential mucin stains. Immunohistochemistry was also performed for cytokeratins 8 and 10. Interdigital glands resembling human eccrine glands were detected deep within the reticular dermis. Histochemical staining indicated neutral mucopolysaccharides and nonsulphated acid mucopolysaccharides in glandular secretions, and the glandular epithelium also showed immunoreactivity to cytokeratins 8 and 10. Both the histochemical and immunohistochemical staining patterns are analogous to human eccrine structures. This study shows with certainty that Asian elephants possess sweat glands as they are defined histologically.     

Histochemical detection of the lectin-binding carbohydrates in the zona pellucida during oocyte growth in the wild boar (Sus scrofa scrofa).

The changes that occur in the carbohydrate composition of zona pellucida glycoproteins during oocyte maturation in the wild-boar were studied using periodic-acid Schiff (PAS), High Iron Diamine (HID) and Low Iron Diamine (LID). Lectin staining was performed with a panel of 11 HRP-lectin conjugates combined with neuraminidase digestion and chemical treatments. There were few internal glucidic residues, such as N-acetylglucosamine, in the wild boar zona pellucida but there were many subterminal beta-N-acetylgalactosamine, alpha- and beta-galactose determinants masked by sialic acid. In addition, beta-N-acetylgalactosamine, beta-galactose-(1-3)-N-acetylgalactosamine and beta-galactose-(1-4)-N-acetylglucosamine were detected in the sulphated form in the terminal and/or subterminal position. Some differences in the lectin reactive sites occurred in the zona pellucida, depending on the stage of oocyte maturation.     

Expression of Prostate Glycoconjugates in the Stallion and Castrated Horse

This work was undertaken to determine the glycoconjugates secreted by the epithelium of the prostate in the intact stallion and castrated horse using lectin histochemical procedures in conjunction with enzymatic digestion and deglycosylation treatments. Additionally, anti‐5 and 13‐16‐cytokeratin antibodies were used to localize epithelial basal cells. In the stallion, lectin histochemistry showed the following sugar residues in the Golgi zone of the glandular cells: α‐Glu/Man, α‐Fuc and β‐Gal included in both O‐ and N‐linked oligosaccharides as well as β‐GalNAc, GlcNAc and α‐Gal, which belonged to O‐glycoproteins. β‐Gal and β‐GalNAc moieties were also noted subterminal to sialyl residues. Sialic acid specific lectins identified Neu‐5Ac(α2,3‐6)‐β‐Gal or Neu5Ac(α2,6)‐β‐GalNAc sequences in both N‐ and O‐bound glycoproteins. The prostatic glandular cells of the castrated horse expressed some of the same sugar moieties found in the stallions, such as α‐Glu/Man, α‐Gal and GlcNAc, but significant differences were also noted. In particular, β‐D‐GalNAc was only detected subterminal to sialic acid, β‐D‐Gal‐(1‐3)‐D‐GalNAc was found in N‐linked glycans, whereas β‐D‐Gal‐(1‐4)‐D‐GlcNAc and Neu5Acα2,6Gal/GalNAc were noted only in O‐glycoproteins. These results indicate that the lectin binding patterns in glandular cells may be modified by sex hormones. No specific lectin labelling of basal cells was found in either the stallion or the castrated horse even though they were immunostained with specific anti‐cytokeratin antibodies. These cells stained more strongly in the castrated horse than in the intact stallion suggesting that they are androgen responsive. The glycomolecules detected in the equine prostate secretions may contribute to the remodelling of the sperm surface, which occurs during sperm transit through the male genital tract and also after ejaculation in the seminal plasma. These changes may be important in the understanding of the stallion fertility.     

Morphology of the intermandibular gland of the Lesser mouse deer, Tragulus javanicus

The morphology of the intermandibular gland of the Lesser mouse deer (Tragulus javanicus), which plays an important function in marking area and territory and in the reproductive behaviour of the animal, was examined using immunohistochemistry, lectin histochemistry and scanning electron microscopy. The gland was composed of sebaceous and apocrine glandular material. Sebaceous glands occupied a greater area of the total gland and consisted of many large lobules with polyhedral cells having a pale cytoplasm. The sebaceous gland, being holocrine, possessed no special secretory ducts. The apocrine gland was lined by cuboid cells and the secretory products were often seen in the apical portions of the cells. Myoepithelial cells contained actin filaments lining the basal membranes of the apocrine gland and were surrounded by nerve fibres which immunostained with protein gene product 9.5. The secretion of the gland appears to be a mixture of larger amounts of lipid material from sebaceous glands, and glycoconjugates secreted by both sebaceous and apocrine glands. Lectin histochemistry detected these as galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-mannose and D-glucose. The male gland was larger in size and contained more N-acetyl galactosamine and N-acetyl glucosamine in its secretion than the gland of the female. This implied the presence of sexual differences in secretions in the intermandibular gland of the Lesser mouse deer.     

Histochemistry of complex carbohydrates in the horse duodenal gland

Complex carbohydrates were examined in glandular cells of the horse duodenal gland by using lectin histochemical techniques. In the horse, the duodenal gland was distributed in the area from the uppermost part of the small intestine to a point about 6m caudal to the pylorus. It consisted of two types of cells, mucous and serous cells. The former was found in glands distributed almost all over this part, but the latter was present in glands distributed restrictedly to the uppermost part of the small intestine at a point about 10 cm caudal to the pylorus. The cytoplasm of the mucous cell contained neutral glycoproteins with different saccharide residues as alpha-D-mannose, N-acetyl-beta (1----4)-D-glucosamine, galactose, alpha-galactose, alpha-N-acetylglucosamine, beta-D-Gal (1----3)-D-GalNAc, alpha-L-fucose and sialic acid. On the other hand, the serous cell contained neutral and acid glycoproteins with different residues such as alpha-D-mannose, alpha-D-glucose, beta-D-galactose(1----3)-D-N-acetylgalactose, alpha-L-fucose, N-acetyl-alpha-D-galactosamine, N-acetyl-beta (1----4)-D-glucosamine, galactose, alpha-galactose, alpha-N-acetylglucosamine and sialic acid. It is also elucidated in the present study that lipase, an enzyme for digestion, is contained in the serous cell of the equine duodenal gland.     

Histochemical detection of glycoconjugates in the inner perivitelline layer of Japanese quail (Coturnix japonica)

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. In this glycohistochemical study, a panel of fluorescein isothiocyanate (FITC)-labelled lectins was used to characterise and localise the oligosaccharide sequences of the IPVL glycoproteins at different stages of follicular development in the quail ovary. Deacetylation and sialidase digestion were also performed prior to lectin cytochemistry. Contrary to mammals, where the topographical distribution of these carbohydrates is not uniformly distributed throughout the zona pellucida, indicating the regionalisation of oligosaccharide chains, our results demonstrated a homogenous lectin staining of the comparatively thin IPVL. We also found variations in the presence and distribution of the carbohydrate residues in the IPVL during different stages of follicular growth. The IPVL of pre-vitelline follicles distinctly stains with WGA, sWGA and SBA, demonstrating the presence of D-GlcNAc, Neu5Ac and α-D-GalNac in the glycoproteins of the forming IPVL. No staining was found with ConA (specific for α-D-Man, α-D-Glc), LCA (α-D-Man, α-D-Glc), PNA (β-D-Gal-(1-3)-D-GalNAc, VAA (Gal), DBA (α-D-GalNAc(1-3)-GalNAc and UEA-I (α-L-Fuc). With continuing follicular growth of the oocyte and the follicle, this staining pattern changed. LCA and PNA-staining in the IPVL became distinctly positive. As the IPVL of immature oocytes distinctly stains with WGA/sWGA-FITC, but spermatozoa do not bind to immature zona, it appears questionable that carbohydrate residues detected by WGA/sWGA play a major role in sperm-IPVL binding, as suggested in previous investigations.     

Lectin Histochemistry of Glycoconjugates in Horse Salivary Glands

The glycoconjugate content of major horse salivary glands was investigated by means of horseradish peroxidase-conjugated lectins. Qualitative differences were observed in the terminal sugar residues of secretory glycoproteins and glycoconjugates linked to the apical surface of excretory duct epithelial cells. Mucous acinar cells in mandibular and sublingual glands contained oligosaccharides with D-galactose, α- and β-N-acetylgalactosamine, N-acetylglucosamine and fucose residues, whereas mandibular, sublingual and parotid serous cells contained only oligosaccharides with terminal α- and β-N-acetylgalactosamine residues. The apical portion of striated and interlobular duct lining cells of mandibular and sublingual glands stained for α- and β-N-acetylgalactosamine and for N-acetylglucosamine. In parotid gland the cytoplasm of intercalated duct cells and the apical surface of striated duct epithelial cells stained for α-N-acetylgalactosamine.     

Detection and molecular characterization of Theileria sp. in fallow deer (Dama dama) and ticks from an Italian natural preserve

The prevalence of piroplasms in a closed population of fallow deer (Dama dama L.) living in the Italian preserve of “Bosco della Mesola” - Ferrara (Mesola wood) was investigated. Blood samples and ticks were collected from 62 fallow deer. On microscopic observation, 28 (45.0%) blood samples were positive for piroplasms while PCR provided evidence for piroplasms infection in 47 (75.8%) fallow deer. The 67 ticks, collected from positive and negative animals, were identified as Ixodesricinus L., 1758 (89.6%) and Haemaphysalisconcinna Koch, 1844 (10.4%). At the PCR, four samples of I. ricinus were positive for piroplasms. The sequences of the 18S rRNA gene from both blood and ticks were identical and showed high identity (99.6%) with Theileria sp. 3185/02 (DQ866842) and Theileria capreoli (AY726011) from roe deer. Interestingly, the phylogenetical analyses evidenced differences between the Theileria strain from Mesola wood and the ones isolated in fallow deer from other Italian areas.     

Basic and Lectin Histochemical Characterization of Bovine Gustatory (von Ebner's) Glands

The Bovine tongue possesses numerous circumvallate papillae (8–16) each side). The troughs around the papillae are the openings of the ducts of the gustatory (von Ebner's) glands. In this study, we have characterized in situ the glycosidic composition of the secretion of bovine gustatory glands using traditional histochemical methods and lectin histochemistry with and without prior neuraminidase (sialidase) digestion. The lectin-horseradish peroxidase conjugates employed were: PNA, DBA, SBA, WGA, LTA, UEA I and ConA. Acinar cells show a diffuse positivity towards PAS and Alcian blue at pH 2.5 and the most intense and homogeneous lectin staining was obtained with PNA. This indicates that bovine gustatory glands secrete glycoproteins with 1,2-glycol containing hexoses and carboxyl-rich glycoconjugates and that galactosyl (β1 → 3) Nacetylgalactosamine is the most frequent sugar residue present in these glycoproteins. Results were compared with data reported in the literature on the same glands of other species.     

Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta

Background

This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDS-PAGE and N-terminal microsequencing.

Results

Four distinct fallow deer PAG (fdPAG) sequences were identified and submitted to Swiss-Prot database. Comparison of fdPAG with PAG sequences identified in other ruminant species exhibited 64 to 83% identity. Additionally, alpha-fetoprotein was identified in fetal and maternal tissues.

Conclusion

Our results demonstrate the efficacy of VVA and bovine PAG-2 affinity chromatographies for the isolation of PAG molecules expressed in deer placenta. This is the first report giving four specific amino acid sequences of PAG isolated from feto-maternal junction (FCT and MCT) in the Cervidae family.     

Glycoconjugate histochemistry of the seminal vesicles of the Japanese miniature (Shiba) goat

The present study localizes and characterizes complex glycoconjugates in the seminal vesicles of the Japanese Shiba goat, using several carbohydrate histochemical procedures, including lectin techniques at light and electron microscopic levels. Glandular epithelial cells and luminal secretions were shown to contain neutral and acidic glycoconjugates with various saccharide residues, such as alpha-D-Man, alpha-D-Glc, alpha-L-Fuc, beta-D-Gal, GalNAc, GlcNAc, and NeuAc (sialic acid). The terminal oligosaccharide chains of sialoglycoconjugates present in the seminal vesicles were NeuAc alpha 2-3Gal beta 1-3GalNAc and NeuAc alpha 2-3Gal beta 1-4GlcNAc. In addition, in lysosomes of the glandular epithelial cells alpha-D-Man, alpha-D-Glc, GlcNAc and NeuAc (sialic acid) residues could be detected, the secretory vesicles contained alpha-L-Fuc, and the endoplasmic reticulum exhibited alpha-D-Man and alpha-D-Glc residues. The complex glycoconjugates with various sugar residues found in the seminal vesicles of the goat may be involved in various fertilization-related events.     

Glycoconjugate Histochemistry in the Duodenum of Fetal and Adult Fallow Deer

This work aimed to characterize the complex carbohydrates in the duodenum of fetal and adult fallow deer. The proximal parts of the duodenum were removed from the intestine of 12- and 16-week-old fetuses and of adult fallow deer. Sections were assessed by conventional carbohydrate histochemistry combined with glycosidase digestions and treatment with KOH. During development, there was an increase in the acid carboxylated components of the glycoconjugates in goblet cells, while the duodenal glands expressed high quantities of sulphated glycoconjugates at the beginning of development but, in the adult, mainly secreted neutral and carboxylated glycoconjugates containing sialic acid. Sulphated components, probably represented by chondroitin sulphate B-like and heparan sulphate-like glycosaminoglycans may play a role in the morphofunctional differentiation of the duodenum.     

Histochemical Analyses of Anti‐Microbial Substances in Canine Perianal Skin with Special Reference to Glandular Structures

Circumanal glands are prominent features of the canine perianal skin, which are often located near to the sebaceous glands and apocrine glands. As the functional relevance of circumanal glands is yet unknown, we studied the localisation of sialic acids and anti‐microbial substances (lysozyme, immunoglobulin A, lactoferrin, β‐defensin) in these glandular structures by lectin histochemistry and immunohistochemistry. The glands exhibited a number of sialic acids that were linked to α2‐6Gal/GalNAc and α2‐3Galβ1‐4GlcNAc. Additionally, lysozyme, lactoferrin and β‐defensin could be demonstrated in the three types of skin glands, whereas IgA was only detectable in the apocrine glands. The results of the study suggest the specific significance of the circumanal glands. Independent of a certain endocrine role, their products may mainly function as protective agents to preserve the integrity of the anal region, considering that sialic acids and anti‐microbial substances are important in defence mechanisms.     

Prevalence of Anaplasma phagocytophilum in fallow deer (Dama dama) and feeding ticks from an Italy preserve

Up to date, information concerning the Anaplasma phagocytophilum infection in fallow deer is scant, therefore, to verify its prevalence in these ungulates serological and PCR screenings were performed on blood of 72 fallow deer hunted in a Central-Northern Italian preserve. Molecular analyses were also performed on 90 ticks removed from the animals.A. phagocytophilum infection in fallow deer was confirmed in 20 out 72 by IFA assay and in 11 out 72 by PCR. The sequence obtained revealed a complete genetic homology among the blood samples and strong degrees of homology with other European isolates. Considering the 90 ticks collected we found that 7.3% of Ixodes ricinus harboured A. phagocytophilum specific DNA. The data obtained confirmed that fallow deer can be a competent host for A. phagocytophilum and, therefore, that may represent a biological reservoir playing an important role in the epidemiological scenarios of the infection, in the geographical areas where is widespread.     

αv β3 Integrin may participate in conceptus attachment by regulating morphologic changes in the endometrium during peri-implantation in ovine

The objective of this study was to determine expression and potential functions of α(v) and β(3) integrin subunits in ovine endometrium during the peri-implantation period (days 8-17 after fertilization). The morphologic changes in the endometrium were observed histochemically following haematoxylin/eosin (HE) staining, whereas the expressions of α(v) and β(3) integrin subunits were analysed by RT-PCR, immunohistochemistry and Western blot. The filamentous conceptus attached to the luminal epithelium (LE) on day 17 of pregnancy, with no differences in endometrial morphology between days 8-12 of pregnancy. However, endometrial glands in the endometrial stroma (S) underwent extensive hyperplasia from day 14 to day 17, increased reductus of the LE with an obvious proliferation of caruncles, and an increased number and diameter of blood vessels (V) in the endometrium. The relative expression levels of α(v) and β(3) integrin subunits mRNA gradually increased until day 16, but sharply declined on day 17. Western blot analysis revealed that the expression pattern of α(v) and β(3) integrin subunit proteins paralleled that of the corresponding mRNA. In addition, immunohistochemical localization of α(v) and β(3) integrin subunits confirmed their presence in the glandular epithelium (GE), LE and endometrial stroma. Immunostaining on LE and stroma varied with the increasing days of pregnancy, with the strongest immunostaining on days 16 and 17. In conclusion, expression of α(V) and β(3) integrin subunits was closely related to the early progression of pregnancy and conceptus attachment; therefore, we inferred that α(v) β(3) integrin may participate in conceptus attachment by the regulation of endometrial morphology during peri-implantation in ovine.     

Glycoproteins and glycosidases of the cervix during the periestrous period in cattle

The cervix and its secretions undergo biochemical and physical changes under the differential influences of estrogen and progesterone. These include changes in the glycoprotein profile of the endocervix and its secretions. A comprehensive survey of such changes in cervical epithelium and cervical secretions was performed on bovine samples throughout the periestrous period. Cervical tissue samples and swabs were collected from synchronized beef heifers that were slaughtered 1) 12 h after controlled intravaginal progesterone-releasing device (CIDR) removal, 2) 24 h after CIDR removal, 3) at the onset of estrus, 4) 12 h after the onset of estrus, 5) 48 h after the onset of estrus, and 6) 7 d after the onset of estrus. Histological staining with hematoxylin and eosin, periodic acid Schiff, Alcian blue, and high-iron diamine was carried out to map overall patterns of stored glycoproteins and tissue structure. Biotinylated lectins were also used to detect the presence and distribution of a range of saccharide structures. The activities of β-galactosidase, α-L-fucosidase, β-N-acetyl-hexosaminidase, and sialidase were measured in cervical swabs using specific substrates. The epithelial layer of the cervix exhibited dynamic changes in cellular hypertrophy and amounts of stored glycoprotein. The greatest content of neutral and acidic mucins was observed 48 h after onset of estrus (P < 0.05). Sialylated mucins predominated at the bases of cervical folds, whereas sulfated mucins were more abundant (P < 0.05) at their apices. The stained area of core mucin glycans changed (P < 0.05) in association with follicular versus luteal phases, whereas terminal glycans changed (P < 0.05) mainly at the time of estrus and shortly thereafter. The greatest activity of β-galactosidase and sialidase was observed 12 h after onset of estrus, whereas β-hexosaminidase and α-fucosidase peaked at the luteal time point (P < 0.05). Taken together, we suggest that the well-known changes in the endocervix and its secretions that are associated with the physiological modulation of sperm transport and function of the cervical barrier are, in part, driven by glycosylation changes.     

Immunolocalization of inhibin/activin subunit proteins during the breeding season in testes and scented glands of muskrats (Ondatra zibethicus)

The objective of this study was to investigate the cellular immunolocalization of inhibin a and inhibin/activin (β(A) and β(B)) subunits in the muskrat testes and scented glands during the breeding season. Inhibin α and inhibin/activin (β(A) and β(B)) subunits were expressed in Sertoli cells and Leydig cells of testes and glandular cells of scented glands, respectively. Also, positive signals of inhibin α and inhibin/activin (β(A) and β(B)) subunits by Western blotting were both observed in testicular and scented glandular tissues. These results suggested that the testes and scented glands of the muskrats had the ability to synthesize inhibins and activins and that activins and inhibins might play an important role in testicular and scented glandular function in muskrats.     

A study of antibody levels in wild ruminants vaccinated against rabies

The authors have vaccinated 22 fallow deer (Dama dama) and 10 mouflons (Ovis ammon musimon) against rabies with an inactivated vaccine: 4 fallow deer with 1 ml, 14 fallow deer and 10 mouflons with 2 ml, 4 animals were kept as controls (fallow deer). The antibody responses were checked by fluorescent foci inhibition carried out on blood samples collected during a two-year period. All the animals developed antibody titres and were still protected after 24 months.