Immunohistochemical demonstration of chromogranin A in endocrine organs of the rat and horse by use of region-specific antibodies

Chromogranin A (CgA) is an acidic glycoprotein that is co-stored with hormones or neurotransmitters in granular components of endocrine cells and neurons, and released together with them in response to adequate stimulation. In addition to acting as a packaging protein, CgA functions as a precursor molecule that yields several bioactive peptides by proteolytic cleavage. The purpose of this study is to elucidate how different the processing of CgA is among endocrine tissues by immunostaining using multiple region-specific antisera, and to evaluate the availability of region-specific antisera. When various endocrine organs of rats were immunostained with four region-specific antisera against rat CgA (CgA 1-28, 94-130, 296-314, and 359-389), all amine/peptide-secreting endocrine tissues except the pineal body were stained positively. The adrenal medulla and gastric endocrine cells were equally intensely immunoreactive to all four antisera, while the other endocrine tissues, represented by pancreatic islets, showed different staining patterns depending on the antiserum. These results suggest that the processing of CgA differs from tissue to tissue. An antiserum against horse CgA 335-365, corresponding to rat CgA 359-389 which shows the highest concentration in the plasma and urine of the rat, again stained all endocrine tissues of the horse except the pineal body. Therefore, the anti-horse CgA 335-365 serum is useful for immunohistochemical survey of horse CgA, and may make possible the establishment of a CgA assay system for the measurement of CgA in the plasma, urine and saliva.     

Changing cytokeratin expression patterns in diethylstilbestrol dipropionate-induced metaplastic lesions of the goat prostate.

Five-month-old male goats were treated with 25 mg diethylstilbestrol dipropionate (DES-DP) by a single intramuscular injection, causing characteristic histological alterations in the peripheral glandular epithelium of the prostate, resulting in squamous metaplasia. Using a panel of monoclonal and polyclonal cytokeratin antibodies on frozen tissue sections of control prostates, we were able to immunohistochemically distinguish between the normal secretory cells, which are positive for cytokeratin 18 as detected with the antibody RGE 53, and the scattered basal cells, which could be specifically stained by the antibody RCK 103. Cytokeratins indicating squamous differentiation, i.e., nos 4 and 13, recognised by the antibodies 6B10 and 1C7, respectively, were found in sporadic cells throughout the normal goat prostate. Profound changes in cytokeratin expression were observed in the metaplastic lesions as compared to control peripheral glandular tissue. In this respect three monoclonal antibodies are of special interest. RCK 103 is immunoreactive with resting and all stages of differentiating basal cells. Antibodies 1C7 and 6B10 strongly stain the squamous cells in the metaplastic lesions, with 1C7 staining all the squamous cells in the lesions except the basal cell layer, and 6B10 being immunoreactive with the same suprabasal cells or the more differentiated cells in the upper strata. As a result the number of cytokeratin 18-positive cells is drastically reduced upon metaplasia. The results indicate that the goat system can be used as a suitable model system to further test the applicability of immunohistochemical methods in meat inspection and toxicological pathology.     

Monoclonal antibodies against sheep immunoglobulin light chain, IgM and IgA

Monoclonal antibodies specific for sheep immunoglobulin light chain, IgM and IgA were produced by conventional cell fusion technology. Purified light chain and IgM were used to verify the specificity of anti-light chain and anti-IgM hybridoma supernatants using passive haemagglutination assays, radioimmunoassays and immunoelectrophoresis. In the absence of pure IgA, verification of monoclonal anti-IgA was, based on paired staining of intestinal lymph smears and comparing the percentage of cells stained with hybridoma supernatant with the percentage of cells stained with polyclonal anti-alpha serum.     

An improved immunohistochemical diagnostic technique for canine leptospirosis using antileptospiral antibodies on renal tissue.

The purpose of this study was to compare the immunoreactivity in canine renal tissues stained with antisera specific for 3 leptospiral antigens and those processed with traditional staining methods. In addition, immunoglobulin staining was done on tissues with immunoreactivity to leptospiral antigens. Formalin-fixed renal sections from 12 dogs with chronic interstitial nephritis suspected or proven to have leptospirosis (6 dogs with silver-stained leptospires and 6 dogs in which silver-stained leptospires were not detected) were used. Antibodies consisted of a monoclonal antibody to Leptospira kirschneri serovar grippotyphosa lipopolysaccharide (LPS) and 2 polyclonal antibodies to outer membrane proteins, including OmpL1, a leptospiral porin, and LipL41, an outer membrane lipoprotein. The murine monoclonal antisera against LPS (F71C2-1) had the most abundant and consistent immunoreactivity. Immunoreactive areas were present in 6 of 6 sections positive by silver staining and included extracellular granular debris in intertubular areas, debris in macrophages, organisms in tubular lumina, and cytoplasmic granules in tubular epithelia. Antisera with specificity for the outer membrane proteins OmpL1 and LipL41 detected only intact organisms in tubular lumina. Immunoreactivity to OmpL1 (polyclonal 338) occurred in 4 of 5 sections positive by silver staining, but immunoreactivity to LipL41 (polyclonal 813) occurred in only 1 of 6 silver-positive sections. Each of the kidney sections in which leptospiral antigens were detected by immunohistochemistry also was positive by silver staining. Sections negative by silver staining were also negative by immunostaining. Although immunohistochemistry did not enhance sensitivity, amplification of signal by secondary antibody and hematoxylin counterstaining improved the ease of diagnosis and allowed better evaluation of tissue morphology than did silver staining methods. IgG was the most abundant immunoglobulin. IgG immunoreactivity occurred predominantly in plasma cells within interstitial infiltrates. Interstitial infiltrates contained abundant immunoreactivity to LPS, but immunoreactivity to OmpL1 and LipL41 was not noted.     

Identification of monoclonal antibodies for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed paraffin-embedded tissues.

Commercially-available monoclonal antibodies to B lymphocytes were evaluated for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed, paraffin-embedded tissues using an avidin biotin complex immunoperoxidase immunohistochemical technique. Three monoclonal antibodies: F46A and F72A raised to "carnivore" B lymphocytes and RA3.6B2 raised to murine B lymphocytes, stained B lymphocyte-dependent areas of frozen feline lymphoid tissue. In addition, antibody RA3.6B2 stained B lymphocyte dependent areas in formalin-fixed, paraffin-embedded feline tissues. There was no staining of T lymphocyte-dependent areas in either frozen or formalin-fixed tissues. Dual parameter flow cytometry, using an anti-pan-T lymphocyte antibody, revealed that greater than 99% of the cells stained by RA3.6B2 are a population distinct from T lymphocytes. F46A was shown to stain a sub-population of those cells stained with RA3.6B2. These antibodies may be useful in the identification of feline B lymphocytes using immunohistochemistry and flow cytometry and thereby provide additional tools to study B lymphocyte ontogeny and the significance of lymphocyte phenotype in lymphoid neoplasia in cats.     

Immunohistochemical and ultrastructural investigation of granular cell tumours in dog, cat, and horse.

Six canine, one feline and one equine granular cell tumours (GCTs) were investigated electron microscopically and immunohistochemically. The tumours were tested for reactivity with monoclonal antibodies against vimentin and desmin and with polyclonal antibodies against cytokeratin, S-100 protein, glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE). All GCTs were characterized by their PAS positive cytoplasmic granules in light microscopy, which in electron microscopy appeared as lysosome-like granules. In each case two canine GCTs were stained by the antibody against cytokeratin, vimentin and S-100 protein. Cells of the equine GCT showed reactivity with the antiserum against S-100 protein. In the feline GCT no reactivity with any of the antibodies tested was observed. These differences of the immunohistochemical reactions of GCTs suggest a nonuniform histogenesis of GCTs in domestic animals. The reactivity of the tumour cells with the antiserum against NSE is discussed.     

Genital bovine papillomavirus infection in Saudi Arabia.

Genital bovine papillomavirus infection was observed for the first time in the Al-Ahsa region of Saudi Arabia. The disease involved 1 female and 2 male 2-4-year-old crossbred cattle. Fibropapillomas (warts) were limited to the prepuce and vulva. Electron micrographs of thin sections of the lesions revealed the presence of intranuclear viruslike particles. Using a broadly cross-reactive rabbit polyclonal antiserum directed against papillomavirus group-specific antigens, the infection was confirmed by immunohistochemical staining of paraffin-embedded tissues to be due to a papillomavirus. Staining with a series of monoclonal antibodies of various specificities indicated that the virus was bovine papillomavirus type 1. Attempts to propagate the virus by inoculation of tumor homogenates onto chorioallantoic membranes of chicken embryos were unsuccessful.     

Localization and Coexistence of Calcium-Binding Proteins and Neuropeptides in the Vagal Ganglia of the Goat

This study was performed to investigate the neurochemical characteristics of the vagal ganglia of the goat by immunohistochemical methods using calbindin D-28k (CB), calretinin (CR). parvalbumin (PA), substance P (SP). calcitonin generelated peptide (CGRP) and galanin (GAL) antibodies. In the proximal vagal ganglia (jugular ganglia), CGRP- (57.1%), SP- (48.2%), GAL- (8.6%), PA- (8.7%), CB- (8.5%) and CR-like (5.3%) immunoreactive cells were observed. In the distal vagal ganglia (nodose ganglia), CGRP- (40.5%), SP- (30.20%), CB- (22.0%) and CR-like (18.10%) immunoreactive cells were present. The double immunohistochemical study showed, that in the proximal vagal ganglia, CGRP immunoreactivity was co-localized in SP- (84.8%), GAL-(100%), CB- (5.6%) and CR- (5.7%) immunoreactive cells: SP immunoreactivity was co-localized in the CGRP- (80.0%), GAL- (100%). CB- (5.3%) and CR- (5.6%) immunoreactive cells; GAL immunoreactivity coexisted in the CGRP- (4.4%) and SP- (19.8%) immunoreactive cells, but not in calcium-binding proteins (CBP)-immunoreactive cells; PA immunoreactivity was absent in the CGRP- and SP-immunoreactive cells; CB and CR immunoreactivities were seen in the CGRP-(0.8%) and SP-immunoreactive (0.9%) cells. On the other hand, in the distal vagal ganglia, CGRP immunoreactivity appeared in SP- (66.6%), CB- (1.0%) and CR- (1.2%) immunoreactive cells; SP immunoreactivities were observed in the CGRP- (44.1%), CB- (1.0%) and CR- (1.2%) immunoreactive cells; CB immunoreactivities were present in the CGRP- (0.5%) and SP- (0.8%) immunoreactive cells; CR immunoreactivities were contained in the CGRP- (0.5%) and SP- (0.8%) immunoreactive cells. These findings indicate that the goat is distinct from other mammalian species in the distribution and localization of neurochemical substances in the vagal ganglia. and suggest that these differences may be related to physiological characteristics, particular those of the ruminant digestive system.     

Identification of guinea pig gammadelta T cells and characterization during pulmonary tuberculosis

Guinea pigs are an alternative small animal model for many disease studies. Here we describe a pan-gammadelta monoclonal antibody (anti-TCRdelta1) specific for the constant region of human T cell receptor delta chains that cross-reacts with a subpopulation of guinea pig (Cavia porcellus) lymphocytes. The phenotype and distribution of this subpopulation is consistent with the guinea pig gammadelta T cell subset. FACS analysis of fresh PBMC and splenocytes from na?ve guinea pigs revealed the presence of a subset of cells that stained with the anti-TCRdelta1 mAb. The relative percentage of anti-TCRdelta1 positive cells in PBMC and tissues is similar to that described for gammadelta T cells in other species. Immunohistochemistry of tissues also revealed a distribution of anti-TCRdelta1 positive cells consistent with gammadelta T cells. These data are further supported by staining of a polyclonal guinea pig T cell line that became progressively CD4 and CD8 negative in long-term culture. Analysis of PBMC from guinea pigs following aerosol infection with virulent Mycobacterium tuberculosis revealed no apparent changes in the steady-state percentage of blood gammadelta+ T cells. Taken together, these data suggest that the anti-TCRdelta1 antibody recognizes the gammadelta T cell subset in guinea pigs. This reagent may be useful for examining gammadelta T cells in various disease models where the guinea pig is a more desirable model for study.     

Update on drugs used to treat endocrine diseases in small animals.

Drug therapy for the endocrine system is implemented to replace a hormone deficiency or to prevent or reduce the formation or effects of excess hormone. Treatment of endocrine disorders covers diseases of the pituitary, adrenal, parathyroid, and thyroid glands as well as the endocrine pancreas. This article focuses on new therapies currently available for specific diseases. Administration of trilostane for treatment of hyperadrenocorticism and use of insulin glargine, protamine zinc insulin (PZI), and porcine Lente insulin for diabetes mellitus are discussed. In addition, transdermal methimazole therapy for treatment of feline hyperthyroidism and administration of progestins for pituitary dwarfism are considered.     

The presence of androgen receptors in the epididymis and prostate of the stallion and cryptorchid horse--a preliminary study

Distribution of androgen receptors (ARs) in the epididymal duct and prostate of three entire stallions and one bilaterally cryptorchid horse was studied immunohistochemically using a polyclonal rabbit antiserum against the ARs. In both the healthy stallions and the cryptorchid, the epithelial cells of the epididymides showed nuclear staining for ARs. The intensity of AR-staining in the principal cells of the epididymis was stronger than that of the basal cells. In the prostate, the glandular secretory cells were moderately stained whereas the basal cells expressed weak AR-staining. Immunostaining for ARs in the reproductive tissues of the cryptorchid horse was always stronger than in those of the stallions. Our results demonstrate for the first time the AR localisation to equine epididymal and prostatic cells, which are directly regulated by androgens.     

Preparation and characterization of polyclonal and monoclonal antibodies against Lawsonia intracellularis.

Proliferative enteropathy is an intestinal infectious disease caused by the obligate intracellular bacterium Lawsonia intracellularis. Immunohistochemistry staining has superior sensitivity over hematoxylin and eosin and silver staining for detecting L. intracellularis in histological sections. A L. intracellularis-specific monoclonal antibody (MAb) produced in the UK (IG4 MAb) has been described in the literature. However, no monoclonal or polyclonal antibodies are commercially available. Therefore, the objective of this study was to produce and characterize new polyclonal and monoclonal antibodies against L. intracellularis that are suitable for diagnostic use. The new monoclonal (2001 MAb) and polyclonal antibodies (1999 PAb) were compared with the IG4 MAb using Western blot analysis of outer membrane proteins (OMPs) of 6 L. intracellularis isolates, Bilophila wadsworthia and Brachyspira hyodysenteriae and using immunohistochemistry of known positive and negative histologic samples and pure cultures of L. intracellularis, B. wadsworthia, B. hyodysenteriae, Salmonella choleraesuis, S. typhimurium, and Escherichia coli K88. Immunogold staining using 2001 MAb was performed to show the specificity of the antibody against an L. intracellularis surface protein. Western blot analysis showed that the 2001 MAb targeted an OMP of 77 kD, which made it different from the IG4 MAb that targeted an 18-kD OMP. The immunogold stain demonstrated the specificity of the 2001 MAb to a surface protein of L. intracellularis. The polyclonal antibody (1999 PAb) targeted 5 OMPs (77, 69, 54, 42, and 36 kD). Both the 2001 MAb and 1999 PAb stained known positive, but not negative, histologic samples. Both the 2001 MAb and 1999 PAb reacted with a pure culture of L. intracellularis but not with any other common enteric pathogens. These two new antibodies will be useful for immunodiagnosis of L. intracellularis.     

Immunohistochemistry for parathyroid hormone-related protein (PTHrP) in benign and malignant mammary mixed tumors of dogs with and without hypercalcemia

We evaluated the expression of parathyroid hormone-related protein (PTHrP) by immunohistochemistry in eight benign and malignant mammary mixed tumors of dogs with (n = 4) and without (n = 4) hypercalcemia. Positive immunoreactive staining for PTHrP was observed in all four tumors from hypercalcemic dogs. The mammary tumors from 2 of the 4 normocalcemic dogs stained positively for PTHrP, but the numbers of immunoreactive cells and intensity of the immunoreaction were less than in the hypercalcemic dogs. In the other 2 tumors without hypercalcemia, the tissue samples were negative for PTHrP.     

Immunohistochemical identification of generalized AA-amyloidosis in a mountain gazelle (Gazella gazella)

Generalized amyloidosis was diagnosed post-mortem in a mountain gazelle (Gazella gazella). To test whether the amyloid deposits consisted of amyloid-A fibril protein a series of monoclonal and polyclonal antibodies directed against amyloid-A fibril protein of different species was applied to formalin-fixed paraffin sections using the indirect immunoperoxidase technique. The immunohistochemical results showed a moderate cross-reaction of gazelle amyloid with human, murine, hamster, and canine amyloid-A fibril protein. A strong cross-reaction, however, was found with one of two monoclonal anti-human amyloid-A antibodies and with an antiserum against bovine amyloid-A fibril protein, the amyloid fibril protein of another ungulate. These results demonstrate the presence of amyloid-A fibril protein in the gazelle amyloid and illustrate the diagnostic value of cross-reacting anti-amyloid-A antibodies for the identification of amyloid-A-amyloidosis in species and in individuals in which amyloid has not yet been examined.     

The detection of Bacillus anthracis protective antigens by enzyme immunoassay (EIA) using polyclonal and monoclonal antibodies

An Enzyme-Immunoassay (EIA) for the detection of Bacillus anthracis-protective antigen (PA) within one hour was developed. If the rabbit antiserum was used, 15 ng PA/ml could be detected and with the monoclonal antibody, the detection limit was 60 ng PA/ml. With respect to the higher specificity and with regard to the aspects of animalcare, monoclonal antibodies should be used in the test instead of the polyclonal antiserum.     

Intestinal candidiasis in a loggerhead sea turtle (Caretta caretta): an immunohistochemical study

Post mortem examination of a juvenile loggerhead sea turtle (Caretta caretta) stranded in the Canary Islands revealed a fishing-line in the small intestine. Histologically, severe necrotic enteritis, multiple haemorrhages, and marked oedema of the intestinal submucosa were observed. Yeast cells and fungal hyphae were seen in the lamina propria of the intestinal mucosa and in the connective tissue of the submucosa. Because fungal cultures were not taken at the time of necropsy, an immunohistochemical study was performed in order to identify the fungus involved. Specific monoclonal and heterologously absorbed polyclonal antibodies served as the primary reagents for identification of aspergillosis, candidiasis, fusariosis, geotricosis, scedosporiosis, and zygomycosis, using an indirect immunofluorescence staining technique. The fungal elements were strongly stained only by a polyclonal antibody against Candida albicans and a monoclonal antibody against C. albicans. There are no known previous reports of Candida sp. causing skin disease or systemic mycotic infection in sea turtles.     

Endocrine tumors.

Because of the diverse nature of endocrine organs, and their vast range of physiologic functions, endocrine tumors encompass a wide range of origination sites and disease entities. The clinical picture of affected individuals is highly dependent on the tissue of origin, and the presence or absence of functional hormone secretions. Identification, localization, and therapeutic strategies, as well as prognosis can vary greatly. Many endocrine tumors have been described in human as well as veterinary patients. This article focuses on endocrine tumors of dogs and cats. Various tumors affecting the pancreas, thyroid, parathyroid, adrenal and pituitary glands are described, including insulinoma, gastrinoma, glucagonoma, and thyroid carcinoma, as well as parathyroid hormone- and growth hormone-secreting tumors. The syndrome of multiple endocrine neoplasia is also described.     

Application of the peroxidase-antiperoxidase procedure to the localization of pituitary hormones and calcitonin in various domestic animals and human beings

Specific cell populations in the pituitary glands of the rat, cat, pig, and human being were positive for thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). When reacted with prediluted rabbit anti-human TSH, LH, and FSH, antisera were not positive for the demonstration of these hormones in the horse, cow, or dog. Immunocytochemical staining was obtained in the horse, cow, and dog by the use of a primary antiserum against a specific beta-subunit of bovine TSH. The immunocytochemical staining of TSH, LH, FSH, adrenocorticotropic hormone, growth hormone, prolactin, and calcitonin was examined by the peroxidase-antiperoxidase method, using standard commercially available kits. All species examined had a strong positive reaction in specific pituitary cell populations for adrenocorticotropic hormone, growth hormone, and prolactin. Sections of normal thyroid gland tissue had positive staining of C cells containing calcitonin at the dilution of 1:100 of the primary antibody in the rat, horse, cow, dog, cat, pig, and human being.     

Characterization of monoclonal antibodies recognizing immunoglobulin kappa and lambda chains in pigs by flow cytometry.

The existence of two types of the immunoglobulin (Ig) light chain in pigs was documented>30 years ago and has been confirmed by the cloning of porcine light chain genes homologous to human and murine Ig kappa (Igkappa) and Ig lambda (Iglambda). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igkappa and Iglambda and certain anti-porcine light chain monoclonal antibodies (mAb) are useful in distinguishing light chain isotypes by flow cytometry (FCM). Porcine B cell lines L23 and L35 stained positive only with anti-human Iglambda antiserum and were negative when tested using anti-human Igkappa antiserum. While mAbs K139.3E1, 1G6 and 27.7.1 also tested positive on these cell lines, mAb 27.2.1 did not. Therefore, FCM was used to examine the hypothesis that K139.3E1, 1G6 and 27.7.1 are Iglambda-specific whereas mAb 27.2.1 recognizes the Igkappa chain in pigs. Double staining of peripheral blood mononuclear cells (PBMC) with pairs of anti-light chain mAbs and using cocktails of anti-light chain mAbs and anti-human polyclonal antiserum, confirmed this hypothesis with the exception that mAb K139.3E1 appears to recognize only a subset of Iglambda(+) B cells in most pigs. In summary, we identified two pan-specific anti-pig Iglambda mAbs, one anti-lambda mAb that recognizes a lambda-light chain subset and one anti-pig Igkappa mAb.     

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus)

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.