Morphological characterization of the adherence and invasion of Streptococcus agalactiae to the intestinal mucosa of tilapia Oreochromis sp.: An in vitro model.

Streptococcosis in tilapia Oreochromis sp. is possibly the most important bacterial disease for fish production worldwide. In Colombia, streptococcosis is caused by Streptococcus agalactiae (GBS), but in other countries, Streptococcus iniae is also involved. Prevention of streptococcosis is required and must be addressed for economic, social, international trade and public health reasons. This research used an in vitro culture of tilapia intestine to detail the intestinal mucosal response once the pathogen contacts the epithelium. We show that S. agalactiae sheds off its capsule to adhere to the epithelium. The bacterium adheres as a single individuum, in groups or in chains and is able to divide on the apical border of enterocytes. GBS adheres at and invades exclusively through the apical portion of the intestinal folds, using the transepithelial route. Once within the cytoplasm of enterocytes, the bacteria continue to divide. On the basolateral side of the epithelium, the microorganisms leave the cells to reach the propria and travel through the microcirculation. No evidence of an immuno-inflammatory reaction or goblet cell response in the epithelium or the lamina propria was seen during the process of adherence and invasion of the pathogen.

Histology, histochemistry and fine structure of the lacrimal gland in the one-humped camel (Camelus dromedarius).

Our research aimed to provide complete histological, histochemical and ultrastructural features of the lacrimal gland of the one-humped camel (Camelus dromedarius) as well as novel insights into its adaptability to the Egyptian desert. Our study was applied to 20 fresh lacrimal glands collected from 10 camels instantly after their slaughtering. The results revealed that the gland was a compound tubulo-acinar gland, and its acini were enclosed by a thick connective tissue capsule that was very rich in elastic and collagen fibres. The gland acini had irregular lumens and were composed of conical to pyramidal cells. The nuclei of secretory cells were found in the basal part, and the cytoplasm was eosinophilic and granular. The glandular tissue consisted of serous and mucous acini and seromucous secretory cells. Histochemically, there was a significant amount of neutral mucopolysaccharides in the acini in which mucous cells had a significant periodic acid-Schiff (PAS)-positive reaction, whereas seromucous cells had a mild PAS-positive reaction. Ultrastructurally, the lacrimal cells had numerous secretory vesicles with contents of moderately to highly electron-dense cytoplasm. The nuclear envelope consisted of two prominent membranes surrounding the peri-nuclear cisterna. The acinar cells had numerous electron-lucent and moderately electron-dense secretory granules, mainly situated on the apical surface, and secreted their contents into the lumen. The luminal surface of the mucous secretory cells represents the remains of secretory granules discharged by the merocrine mechanism. In conclusion, the mucous secretion is believed to aid in the washing and moistening of the eyeball, particularly in dry, hot and dusty environments.

Investigations on the endometrial response to intrauterine administration of N-acetylcysteine in oestrous mares.

In mares, mating-induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N-acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid-Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC- and C-mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC-treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki-67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC-treated mares than in C-mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti-inflammatory effect on the equine endometrium was observed.

Infection with the gastrointestinal nematode Ostertagia ostertagi in cattle affects mucus biosynthesis in the abomasum.

The mucus layer in the gastrointestinal (GI) tract is considered to be the first line of defense to the external environment. Alteration in mucus components has been reported to occur during intestinal nematode infection in ruminants, but the role of mucus in response to abomasal parasites remains largely unclear. The aim of the current study was to analyze the effects of an Ostertagia ostertagi infection on the abomasal mucus biosynthesis in cattle. Increased gene expression of MUC1, MUC6 and MUC20 was observed, while MUC5AC did not change during infection. Qualitative changes of mucins, related to sugar composition, were also observed. AB-PAS and HID-AB stainings highlighted a decrease in neutral and an increase in acidic mucins, throughout the infection. Several genes involved in mucin core structure synthesis, branching and oligomerization, such as GCNT3, GCNT4, A4GNT and protein disulphide isomerases were found to be upregulated. Increase in mucin fucosylation was observed using the lectin UEA-I and through the evaluation of fucosyltransferases gene expression levels. Finally, transcription levels of 2 trefoil factors, TFF1 and TFF3, which are co-expressed with mucins in the GI tract, were also found to be significantly upregulated in infected animals. Although the alterations in mucus biosynthesis started early during infection, the biggest effects were found when adult worms were present on the surface of the abomasal mucosa and are likely caused by the alterations in mucosal cell populations, characterized by hyperplasia of mucus secreting cells.

Cytochemical characterisation of the leucocytes and thrombocytes from Murray cod (Maccullochella peelii peelii, Mitchell).

Cytochemistry has proven effective in differentiating specific cell lineages and elucidating their functional properties. This study utilised a range of cytochemical techniques to further investigate the leucocyte populations from Murray cod, an iconic Australian teleost fish species. This analysis provided clear insight into the structure and function of the leucocytes from this fish, which were found to be broadly similar to those of other fish species. However, some important differences were identified in Murray cod, such as the presence of naphthol AS chloroacetate esterase activity in the heterophil population, positive staining for periodic acid Schiff's, alkaline phosphatase and Sudan black B in the lymphocyte population, and a prevalent population of myeloid precursor cells.

Cervical chondroid chordoma in a Shetland sheep dog.

A spayed female Shetland sheep dog aged 12 years was presented for examination with ataxia and hindlimb paralysis. Extradural spinal cord compression was found at the level of vertebrae C6-C7 by radiography and myelocomputed tomography. A jelly-like mass (0.6 x 1.3 cm) was removed surgically. Histopathological findings were characterized by proliferation of vacuolated polygonal cells (physaliphorous cells) in a mucinous matrix and the presence of chondroid tissue shown immunohistochemically to express S-100. The physaliphorous cells were immunolabelled strongly for vimentin and S-100, and weakly for cytokeratin. A diagnosis of canine cervical chondroid chordoma was made. This is considered to be the first report of a chondroid chordoma originating from the cervical region of the spine in the dog.

Tonsillar lymphangiomatous polyp in an adult dog.

A female spayed cocker spaniel dog, aged 8 years, developed a large, smooth pedunculated mass arising from the right palatine tonsil. Histology revealed that the mass was composed of many, variably dilated, thin-walled lymphatic channels filled with pale eosinophilic fluid lacking red blood cells and embedded in a dense, fibrovascular stroma. The dilated lymphatic channels were lined by a single layer of flattened, discontinuous endothelium with scattered intraluminal valves. Periodic acid-Schiff staining highlighted the discontinuous basement membrane and immunohistochemistry revealed strong cell membrane and cytoplasmic immunoreactivity for CD31 and von Willebrand factor, respectively. The clinical and histological findings were consistent with a tonsillar lymphangiomatous polyp - an uncommon, benign tumour of the tonsil in man that has previously been unrecognized in veterinary medicine.

A case of pulmonary cryptococcosis in a free-living toad (Bufo bufo).

Pulmonary cryptococcosis was observed in a free-living adult female common toad (Bufo bufo) that was killed by a vehicle. Both lungs had various eosinophilic, monomorphic, and spherical to elliptical organisms identified as Cryptoccocus spp. The yeasts were demonstrated by Grocott's silver method and the periodic acid-Schiff reaction and the capsule was positive for mucin with a mucicarmine stain. The agent was confirmed by immunohistochemistry, using the monoclonal antibody anti-Cryptococcus neoformans, and by a polymerase chain reaction-based method using a C. neoformans-specific primer. This report, to the best of our knowledge, represents the first case of cryptococcosis in a common toad.

Comparison of goblet cell staining methods in jejunal mucosa of turkey poults.

This study compared the intestinal goblet cell density of turkey poults at 2 different ages using Alcian blue-periodic acid-Shiff (AB-PAS) and mucicarmine stains. Neutral mucins are stained with periodic acid-Shiff whereas acidic mucins are stained with Alcian blue. Mucicarmine and AB-PAS are specific to the mucins of epithelial origin. Mucicarmine has only been used for the assessment of goblet cells in human specimens, and it may have advantages for use in animals as a result of the methodological simplicity of staining as compared to AB-PAS. A mid-section of jejunum was taken from 80 turkey poults at 21 and 28 d, and fixed in 10% buffered formalin for 48 h. Each fixed tissue was dehydrated with ethanol, cleared with Sub-X, placed in paraffin wax, prepared on 2 slides, cleared and hydrated. The 2 slides were randomly assigned to 2 treatments which consisted of AB-PAS and mucicarmine stains in a completely randomized design. Goblet cell counts were taken from four villi per slide and the villus height was measured and averaged. There was no difference in the goblet cell density between the staining methods AB-PAS and mucicarmine at 21 or 28 d posthatch. These results show that both staining methods are viable for assessment of goblet cell density in turkey poults.

Spontaneous subcutaneous myxosarcoma in a captive European hedgehog (Erinsceus europaeus).

A surgically excised biopsy representing a subcutaneous mass on the left side of the neck from a 3-year-old female European hedgehog (Erinsceus europaeus) was presented. Spontaneous myxosarcoma was diagnosed based on histological, immunohistochemical, and ultrastructural characteristics. The neoplasm grossly consisted of a firm, pale, multilobulated mass with a characteristic clear gelatinous fluid. Histologically, the neoplasm was nonencapsulated and composed of pleomorphic stellate or spindle-shaped vimentin and periodic acid-Schiff-positive cells arranged in loose sheets and occasionally whorls. The neoplastic cells were suspended in Alcian blue-positive stroma and contained infrequent mitotic figures. Evidence of a viral etiology was not detected using electron microscopy and polymerase chain reaction. This is the first case report of a myxosarcoma in a captive European hedgehog.

Cryptosporidium infection in domestic geese (Anser anser f. domestica) detected by in-situ hybridization.

An in-situ hybridization (ISH) procedure was developed for the detection of Cryptosporidium sp. in paraffin wax-embedded tissues with a digoxigenin-labelled probe targeting the 18S rRNA. This technique was used in addition to traditional methods, such as haematoxylin and eosin staining, periodic acid-Schiff reaction, transmission electron microscopy and the polymerase chain reaction, to examine the bursa of Fabricius (BF), conjunctiva and other tissues from 20 domestic geese aged 16-36 days for the presence of cryptosporidia. Positive signals were found to a moderate or marked extent in both conjunctival samples (89%) and BF samples (88%) but not in other tissues. Sequencing of the PCR amplification product revealed identity with Cryptosporidium baileyi. The infected geese showed no clinical signs and only scanty histological lesions. These results confirm reports showing that young waterfowl are especially vulnerable to cryptosporidium infection and indicate that the BF and conjunctiva are the preferred sites for the presence of the protozoon. ISH proved a good method for detecting and identifying even small numbers of cryptosporidia in tissue sections.

Detection of Batrachochytrium dendrobatidis in Eleutherodactylus fitzingeri: effects of skin sample location and histologic stain.

Batrachochytrium dendrobatidis is a fungal pathogen that has been implicated in amphibian declines worldwide. Histopathologic techniques have been used to diagnose the disease, but their sensitivity has not been determined. It is also unclear whether the probability of detection varies between skin samples derived from different body parts. We examined 24 Fitzinger's rainfrogs (Eleutherodactylus fitzingeri) with chytridiomycosis. This is a common frog species with a broad range and high abundance throughout most of Costa Rica. We sampled 12 different body parts from each animal, and alternated the staining between a routinely used stain (hematoxylin and eosin [H&E]), and a more fungus-specific stain (periodic acid-Schiff [PAS]). The pelvic patch and the innermost finger of the hand were consistently the best places to detect the disease, although significant differences were found only with the gular area, the abdomen, and toes four and five. We found more positive samples using PAS than using H and E in all body parts, although significant differences were detected only in samples derived from the pelvic patch. Using the best combination of factors (stain and body part) and animals with the lightest infections (to test the sensitivity of the technique), we calculated that at least 17 sections are needed in order to reach 95% confidence that a frog is or is not infected. We conclude that the choice of stain and body part can significantly alter estimates of prevalence of B. dendrobatidis.

"Carry-on" dermal baggage: a nodule from a dog. Pyogranulomatous inflammation with intralesional fungal agents.

A 2-year-old intact female Australian Cattle Dog presented with a 1-cm diameter nonexudative dermal nodule on the medial aspect of the right thigh. Fine-needle aspiration revealed pyogranulomatous inflammation and many ovoid, 2-4 microm diameter, thin-capsulated, basophilic bodies that appeared to be fungal spores or yeast. Results of CBC, serum chemistry panel, lymph node palpation, and radiographs were unremarkable. Excisional biopsy and histopathology revealed pyogranulomatous folliculitis, furunculosis, and perifolliculitis. Rare fungal hyphae and spore forms were intimately associated with, and occasionally within, hair shafts. A morphologic diagnosis of dermatophytosis was made and Microsporum canis infection was confirmed by fungal culture. M canis is a common infectious agent found within the follicles and superficial keratin layers of canine skin. The kerion-type clinical presentation observed in the dog of this case is uncommonly observed with M canis. Additionally, the cytologic finding of multiple arthroconidia without hyphae is unusual. In the absence of hyphae, M canis arthroconidia may be confused with other fungal yeast bodies; therefore close scrutiny of a cytologic sample for arthroconidia associated with keratin, hair fragments, or hyphal structures is recommended.

Glomerulocystic kidney in a domestic dog.

Glomerulocystic kidney was diagnosed in a 5-year-old female Shiba dog, which died from chronic renal failure with convulsions, vomiting and diarrhoea. Haematological examination revealed non-regenerating anaemia, azotaemia and high serum creatinine. Grossly, both kidneys were mildly atrophic with multiple small cysts in the cortex. Histopathological examination revealed marked dilatation of Bowman's space, often with glomerular atrophy or loss, and mild interstitial fibrosis. Bowman's basement membranes (BMs) were tortuous and thickened, with patchy calcification. Glomerulo-tubular junctions in the urinary pole side of the kidneys had a stenotic appearance associated with thickening of Bowman's BMs and calcification. Focal interstitial fibrosis around the glomerulo-tubular junction was also found. Continuity with the proximal tubule was evident in cystic glomeruli. Ultrastructurally, marked thickening of Bowman's BMs with many granular deposits in the urinary pole side was observed. The findings indicate that glomerular cystic changes may have developed as a consequence of glomerulo-tubular junctional stenosis due to thickened Bowman's BMs and focal periglomerular fibrosis in the urinary pole side of the kidneys.

Orbital meningioma with a granular cell component in a dog, with extracranial metastasis.

A meningioma with a significant component of granular cells is described in the left ocular orbit of a 5-year-old male Golden retriever dog that presented with exophthalmos. The neoplastic mass surrounded the optic nerve. Microscopically, the tumour was characterized by nests of large, round to polygonal cells, variable in size, with eccentric nuclei and abundant eosinophilic cytoplasm, containing PAS-positive granules. In focal areas, cells were smaller and occasionally formed whorl-like structures. Immunohistochemical analysis revealed that neoplastic cells reacted positively for vimentin and, with less intensity, for neuron specific enolase and S-100, whereas they were negative for glial fibrillary acidic protein and cytokeratins. Metastatic growths, formed by similar cells, were seen in the lung and heart.

Histopathologic lesions of diabetes mellitus in a domestic ferret.

A 2-year-did ferret was referred with a diagnosis of diabetes mellitus. The animal died despite insulin therapy and was submitted for necropsy. Light microscopic examination revealed a mild hepatic lipidosis and pancreatic lesions consisting of a diffuse vacuolation of the Langerhans' islet cells with a periodic acid-Schiff positive material compatible with glycogen.

Parotid salivary mucocele in a companion rabbit (Oryctolagus cuniculus): a report on the clinical and laboratory characteristics and surgical treatment - case report / [Mucocele salivar parotídea em coelho doméstico (Oryctolagus cuniculus): relato de características clínico-laboratoriais e tratamento cirúrgico - relato de caso]

The present report aimed to describe the main characteristics of the parotid salivary mucocele in an adult male rabbit, which presented with a painless fluctuating mass with fluid content. Owing to the need for repeated fluid drainage, surgical excision was the chosen mode of treatment. Preoperative evaluation included radiography and blood analysis. The patient underwent surgical excision of the affected gland. Inhalation anesthesia was maintained by means of isoflurane and the cardio-respiratory parameters were monitored throughout the surgery. A bipolar electrocautery was used to assist in the incision and polyglactin 910 and nylon were the suture materials used in the wound closure technique. Post-operative medications included analgesics, anti-inflammatory agents and antibiotics. Histopathology of the excised glandular tissue revealed a mixed inflammatory infiltrate, fibroblasts, hemorrhage and hyperemia. The observations in this case suggest that surgical excision of the affected gland is the treatment of choice for the management of mucocele in companion rabbits, which has already been identified for other animals. Periodic acid-reactive Schiff staining confirmed the diagnosis of salivary mucocele. The complementary investigations helped to rule out the differential diagnosis and indicated a favorable prognosis, in view of the fact that the case was successfully resolved without complications or recurrence.(AU)

O presente relato teve como objetivo descrever as principais características da mucocele salivar parotídea em um coelho macho adulto, que apresentou uma massa flutuante indolor e com um conteúdo líquido em seu interior. Devido à necessidade de repetidas punções deste fluido, optou-se pela excisão cirúrgica como forma de tratamento. A avaliação pré-operatória incluiu radiografias e análises sanguíneas. O paciente foi submetido ao tratamento cirúrgico com excisão da glândula afetada. A anestesia inalatória foi mantida com isoflurano e os parâmetros cardio-respiratórios foram monitorados durante a cirurgia. Um eletrocautério bipolar foi utilizado para auxiliar na incisão e os fios poliglactina 910 e nylon foram utilizados na técnica de sutura. As medicações pós-operatórias incluíram agentes analgésicos, anti-inflamatórios e antibióticos. A histopatologia do tecido glandular removido revelou infiltrado inflamatório misto, fibroblastos, hemorragia e hiperemia. As observações neste caso sugerem que a remoção cirúrgica da glândula afetada seja o tratamento de escolha no manejo da mucocele em coelhos de companhia, o que já vem sendo identificado para outros animais. A coloração do ácido periódico reativo de Schiff confirmou o diagnóstico da mucocele salivar. Os exames complementares ajudaram a excluir as doenças que fazem parte do diagnóstico diferencial e indicaram um prognóstico favorável, tendo em vista o fato que este caso foi resolvido com sucesso, sem complicações ou recorrência.(AU)

Adrenal cortical epithelial cysts in two saddleback tamarins (Saguinus fuscicollis).

Adrenal cysts of epithelial origin were found incidentally in the adrenal cortical tissues of two adult female saddleback tamarins. In one case, a small cluster of minor cysts was located at the cortico-medullary border. The cysts were filled with a periodic acid-Schiff (PAS)-positive, amorphous substance and lined by a cuboidal PAS-negative epithelium, which resembled morphologically the ascending Henle's loops of the kidney. These microcysts were thought to be mesonephric remnants. In the second case, two large cysts of 1 to 3 mm diameter extended throughout the entire cortex. The cysts were filled by a watery, PAS-negative fluid and lined by a bi- to multi-layered, cytokeratin-positive epithelium. The basal epithelial layer consisted of cuboidal cells, which became cylindrical to drop-like in appearance towards the cyst lumen. The cysts closely resembled mesothelium-derived adrenal cysts in man. This is the first report of adrenal cysts in non-human primates.

Hepatocellular endoplasmic reticulum storage disease in an African elephant (Loxodonta africana).

Large intracytoplasmic inclusions were observed in hepatocytes of a 7-year-old African elephant (Loxodonta africana). The inclusions were oval to polyhedral with either a homogeneous glassy or a granular appearance. They were positive for the periodic acid-Schiff (PAS) reaction. Electron microscopical examination revealed that the inclusions consisted of granular material with moderate electron-density and were membrane-bounded. The findings suggested that the inclusions were derived from endoplasmic reticulum. The light and electron microscopical features were similar to those of endoplasmic reticulum storage disease of the liver in man. Such inclusions have not previously been reported in animals.

Histochemical and immunohistochemical characterization of canine mammary mucinous carcinoma.

Mucinous carcinoma is a rare mammary tumour, characterized by intracellular and extracellular mucin. It is still uncertain whether the origin of the mucin is epithelial, myoepithelial or fibroblastic. Eleven canine cases originally classified as mucinous carcinomas were reassessed and compared with myoepithelial nests of mixed tumours. All samples were examined (1) histochemically by the periodic acid-Schiff (PAS) and PAS-diastase methods, and with alcian blue (pH 2.5 and pH 1.0), mucicarmine and Grimelius silver stain, and (2) immunohistochemically for cytokeratin 19, vimentin, alpha-actin and chromogranin A. This examination revealed that only five of the 11 tumours were genuine mucinous carcinomas. In these five tumours the mucus-secreting cells showed cytoplasmic cytokeratin 19 positivity; the mucus showed PAS-diastase and mucicarmine positivity, and alcianophilia which was stronger at pH 2.5 than at 1.0. The remaining six cases were re-classified as mixed tumours because both mucus and mucus-producing cells shared the following similarities with myoepithelial nests of mixed tumours: vimentin and alpha-actin cytoplasmic positivity, PAS negativity, alcianophilia both at pH 2.5 and 1.0, and mucicarmine positivity.