Cutaneous Squamous Cell Carcinoma with Mucinous Metaplasia in Dogs

Background: Squamous cell carcinoma (SCC) is one of the most common malignant skin tumors in domestic animals. Histologically, they are characterized by a proliferation of neoplastic keratinocytes with varied keratin production. Some SCCs have peculiar histological characteristics that permit them to be classified into uncommon to rare histological subtypes, reported in animals and humans. However, according to the authors' knowledge, the mucin-producing subtype described in humans has not yet been reported in animals. In this study, we report the occurrence of two mucin-producing SCCs in dogs, a histological presentation similar to that seen in cutaneous SCCs with mucinous metaplasia in humans.Cases: Two dogs, a 5-year-old Yorkshire female and a 17-year-old Dachshund male, had a skin nodule near the tail and on the right eyelid. The nodules varied from 1 to 5 cm in diameter, were firm and covered with skin and hair. The cut surface was firm and white. Histological findings were compatible with squamous cell carcinoma, characterized by a neoplastic proliferation of keratinocytes originating in the epidermis and infiltrating the dermis. The keratinocytes were arranged in islands and occasional anastomosed cords, supported by a fibrous stroma. The formation of pearls varied from moderate to sparse. The nuclear and cellular pleomorphism was accentuated in case two and moderate in case one. Mitosis figures ranged from two to five in a high magnification field. Within the neoplasm, there were large vacuolated neoplastic cells with slightly fibrillar intracytoplasmic basophilic content. This content has been rarely observed in an extracellular medium. The presence of mucin was confirmed by positive Alcian Blue (AA) staining. In immunohistochemistry (IHC), tumor cells showed strong immunostaining for pancitokeratin, and in areas with marked mucin deposition, immunostaining was predominantly moderate to weak. No tumor cells were immunostained for CD34 and Blc-2 antibodies. Compared to AA and Harris' hematoxylin, it was possible to demonstrate the presence of mucin in the cytoplasm of neoplastic keratinocytes using IHC. No vascular or lymphatic invasion by neoplastic cells was observed. The average cell proliferation index assessed by counting the nucleolar argyrophilic organizing regions (AgNOR) was 3.4 in case 1 and 4.5 in case 2.Discussion: Although the SCC routinely does not present a diagnostic challenge in veterinary practice, the histological presentation of the reported cases does not fit the current classification available in veterinary medicine. The histological presentation observed in these two dogs is similar to that described for cutaneous SCCs with mucinous metaplasia in humans, and so far not described in animals. The observation of intracytoplasmic mucin in humans is an essential finding for the diagnosis of SCC with mucin metaplasia. In the present cases, we observed a slightly basophilic amorphous substance in the cytoplasm of proliferated neoplastic keratinocytes, which stained strongly in blue when applied the Alcian Blue (AA) histochemical technique. This observation became more evident when using IHC counterstained with AA and Harris hematoxylin. In the histological analysis, the absence of an adenoid growth pattern or glandular formation amid neoplastic proliferation ruled out the possibility of a mucinous adenocarcinoma or a mucoepidermoid carcinoma. In addition, we could rule out a follicular neoplasia, including keratinizing infundibular acanthoma due to the absence of a central pore and the absence of immunostaining for CD34 and Bcl-2. These immunohistochemical findings, together with histological findings, reinforce the diagnosis of SCC with mucinous metaplasia in our dogs

Equine Podotrochlear Apparatus - Histologic Characterization

Background: The navicular syndrome may be associated with alterations in other podotrochlear apparatus components, such as the deep digital flexor tendon, collareral sesamoid and distal sesamoid ligaments, podotrochlear bursa and distal sesamoid bone. However, the clinical significance and nature of these changes are not well understood, many of descriptive reports about distal sesamoid bone lesions are rarely accompanied by a complete and comprehensive comparison with animals of the control group. The aim of this study was to described histologically findings of the podrotrochlear apparatus components, allowing the understanding of the inserts and their microscopic appearance, thus providing the future recognize of their alterations.Materials, Methods & Results: Fourteen samples of the podotrochlear apparatus were taken out of 44 equine thoracic limbs specimens, separated at the radiocarpal joint of Crioulo and Thoroughbred horses, with an average age of 6.0-year-old, coming from a private clinic in southern Brazil. The thoracic limbs specimens were refrigerated at 4ºC at the clinic and then they were sent to the University Federal of Santa Maria (UFSM). Once at the University laboratory, the specimens were dissected to isolate the podotrochlear apparatus from each one.  Subsequently, transversal and longitudinal samples from the distal sesamoid bone, deep digital flexor tendo,             distal sesamoid ligament, colateral sesamoid ligament, were collected and podotrochlear            bursa which were processed at the Veterinary Pathology Laboratory of the UFSM and University Federal of Santa Catarina (UFSC). The tissues samples were fixed in a 10% formaldehyde solution for 14 days and routinely processed for histology. The samples were sectioned at 3 µm and stained using the hematoxylin-eosin (H-E) routine method. The bone samples, after fixation, underwent a decalcified process in a formic acid-sodium citrate aqueous solution and routinely processed for histopathology. Histologic tendons evaluation showed that it is arranged in honeycombs fascicles in a transverse section observation. The tendinous fibers have a more compact and aligned collagen fibers arrangement due to the dense connective tissue. The synovial membrane of the podotrochlear bursa is composed of a cubic pseudo conjunctival epithelium arranged with a layer of synoviocytes forming villi towards the lumen. This characterization is very similar to that of the synovial joint membrane. The collateral sesamoid ligament fibers are not perfectly aligned, occurring at their confluence in some areas accompanied by the dissection of adipose tissue. The distal sesamoide ligament fibers, composed by of loose connective tissue associated with synoviocytes and vascular stroma. The articular surface of the distal sesamoid bone is arranged in 3 layers of chondrocytes embedded in a hyaline matrix. There was a clear differentiation between the subchondral bone area and the fibrocartilage tissue in the palmar aspect of the distal sesamoid bone.Discussion: The morphophysiological characterization of the podotrochlear structures inside the hoof capsule it is important for the future recognition of abnormalities and the possible hypothesis that originates the podotrochlear syndrome. These changes have great clinical relevance and very often associated of distal limb lameness in horses. The normal macroscopic, ultrasonographic and radiographic images along with the histomorphometric study of the podotrochlear structures have contributed for the morphophysiological comprehension and consequently future interpretation of the podotrochlear structures diseases.Keywords: podotrochlear apparatus, distal sesamoide bone, deep digital flexor tendon, navicular syndrome, histological findings

Histochemistry of Equine Damaged Tendons, Ligaments and Articular Cartilage

Background: The injury repair process in tendons and ligaments includes different phases such as inflammation, neovascularization, fibroblast proliferation and fibrosis. Collagen type and tissue characteristics of tendon and ligament repair are described such as type collagen differentiation and properties of the scars tissue. The degeneration of articular cartilage when, characterized by loss of the articular layers associated of the decreased of proteoglycans. The aim of this study is to describe by histochemistry techniques the characteristics of tissue scar, collagen type in the repair process of tendons and ligaments, as well as articular cartilage degeneration.Materials, Methods & Results: Tissue samples of equine tendons, ligaments and articular cartilage of the metacarpophalangeal joint region were evaluated by ultrasonography, macroscopically and prepared for routine histopathology (H&E staining). The inclusion criterion of the samples in this study was based on the presence of lesions characterized in H&E stain as fibroplasia, neovascularization, collagenolysis, chondroid metaplasia in tendons and ligaments and fibrillation and cartilaginous eburnation lesions in the articular cartilage samples. The Masson’s trichrome, Picrosirius red and Alcian blue staining techniques were also performed in addition to H&E. Pathologic findings in the tendons and ligaments included fibroplasia, collagenolysis, chondroid metaplasia and lymphohistioplasmacytic inflammation. Tendons and ligaments scars were composed of type III collagen but there was also some type I collagen. Fiber alignment of tendons and ligaments in the reorganization tissue was not flawless and the fiber appearance was characterized by a lack of the fiber crimp and parallelism. The fibroplasia was characterized by endotendinous tickening areas associated with the presence of loose connective tissue. In the areas of loose connective tissue substitution, collagen type fibers are intercalated to a lesser extent by type-III collagen fibers. In the Alcian blue stained samples of articular cartilage observed the surface layer and the matrix zone of calcified cartilage were weakly stained in blue.Discussion: Three special stains were utilized in this study along with the H&E evaluation elucidating the behavior tendons, ligaments and articular cartilage injury. The important observation in this study was fibroplasia in tendons and ligaments seems to be composed by abundant of loose connective tissue, chondrocytes and intermingled collagen type I and III fibers associated with lack of crimps alignment of the fibers. The fragile structure suggested by the Masson’s trichrome stain results (presence of the loose connective tissue) in this study perhaps make the tendons and ligaments receptive to other lesions. The characteristic blue discoloration of collagen fibers was only observed in the loose connective tissue may be because the dye penetration becomes easier when compared to the dense connective tissue (stained in red). The Masson’s trichrome made possible the differentiated the dense connective tissue of the loose connective tissue. The combined histochemistry staining technics allowed an improved characterization of fiber alignment, collagen type, inflammatory cell infiltration and neovascularization, which happens during the repair process of tendons and ligaments. The fibrillation and eburnation of the articular cartilage were associated with the decrease Alcian Blue staining characterized by degeneration process of articular cartilage

Cerebral Candida albicans Infection in Two dogs

Background: Candida spp. are pleomorphic fungi that are commensal inhabitants of the oral, gastrointestinal, upper respiratory and urogenital mucosa of mammals. Candida albicans is described as the most important species. This opportunistic pathogen may produce local or systemic infections in dogs. Local infections have been reported in several tissues and systemic infection is rare in dogs with few reports in the literature describing this presentation. The aim of the present study was to report two cases of cerebral Candida albicans infection in dogs in Brazil.Case: Two cases of cerebral Candida albicans infection in dogs that showed nervous signs are described. In both cases, the brain showed marked asymmetry of the telencephalic hemispheres with multifocal to coalescing yellowish or reddish areas and a partial loss of distinction between gray and white matter. In Case 1, the mediastinal, tracheobronchial and mesenteric lymph nodes, as well as the right kidney and adrenal gland, showed altered architecture due to numerous whitish gray nodules. Histological lesions were characterized by granulomatous (case 1) or pyogranulomatous (case 2) necrotizing meningoencephalitis with intralesional fungal organisms. In case 1, similar granulomatous infiltrate with intralesional fungal organisms was also seen in the lymph nodes, kidney and adrenal gland. In case 2, there was evidence consistent with an underlying infection of canine distemper virus. Were observed lymphoplasmocytic interstitial pneumonia, lymphoid rarefaction in lymph nodes, and viral intracytoplasmic inclusion bodies in the epithelial cells of the stomach and vesical urothelium. In these two cases, the fungal organisms displayed three different morphological patterns. The first pattern was characterized by delicate tubular structures with thin parallel walls that were rarely septate and tended to undulate slightly, and measured approximately 4-20 µm (true hyphae). The second pattern was characterized by chains of elongated yeast, separated by constrictions at septal sites, and measured approximately 4-10 µm (pseudohyphae). The other morphological pattern, which was rarely observed, was characterized by round budding yeast cells that measured 3-4 µm in diameter and were often elongated by the germ tube. Sections were also stained using immunohistochemical antibodies against Candida albicans. The fungi revealed strong immunolabelling of the cytoplasm and wall for Candida albicans.Discussion: The diagnosis was based on the histomorphological and immunohistochemical characteristics of the agent, which were consistent with Candida albicans. In these two cases, immunocompromise appeared to have been an important factor in the progression of the infection. In case 1, the dog was senile and treated with corticosteroids, conditions that could effectively limit innate, humoral and cell-mediated immune response to infection. In case 2, the dog displayed cachexia and was infected by canine distemper virus, evidenced by the presence of viral inclusion bodies. The inflammatory reactions in both cases were characteristic of fungal infections, but were distinct due to differences in the infectious process. In case 1, there was a subacute to chronic progression; while in case 2 neutrophilic infiltrate predominated, suggesting an acute progression. The different morphological patterns of the fungi suggested a pleomorphic fungi and the immunohistochemistry allowed us to identify the infectious agent. In conclusion, opportunistic infections caused by Candida albicans should be considered when diagnosing diseases that affect the central nervous system of dogs, particularly in immunosuppressed animals

Efficacy of azithromycin and miltefosine in experimental systemic pythiosis in immunosuppressed mice

We evaluated the efficacy of azithromycin (50 mg/kg, every 12 h [q12h] orally) and miltefosine (25 mg/kg, q24h orally) treatments in an experimental model of vascular/disseminated pythiosis in immunosuppressed mice. Azithromycin was the only treatment able to reduce mortality. The histopathological findings showed acute vascular inflammation, pathogen dissemination, necrotizing myositis, neuritis, and arteritis. The results suggest that azithromycin, but not miltefosine, may have clinical relevance in the treatment of vascular/disseminated pythiosis

Bronchopulmonary Aspergillosis Associated with Acute Myocardial Infarction in Dog

Background: Fungi of the genus Aspergillus are ubiquitous microorganisms of opportunistic character, of which animals and humans are continually exposed. Are described three main forms of aspergillosis in dogs: nasal, disseminated and bronchopulmonary. This form which is restricted to the lung parenchyma is considered rare in dogs. The aim of this paper is to report a case of pulmonary aspergillosis associated to acute myocardial infarction in a dog, focusing on epidemiological, clinical, pathological and immunohistochemical characteristics. Case: A male dog, 4-year-old, Pinscher, clinically presented dyspnea, anorexia, vomiting, concentrated urine and dark and mollifed faeces. On physical examination, there were congested oral and conjunctival mucosas, dehydration and petechiae in the outer ear. The animal died fve days after the clinical attendance and was sent for necropsy. The macroscopic lesions were restricted to the lung, heart and liver. There were multifocal areas of consolidation in the lung parenchyma and yellowish content in the trachea and bronchi lumen. The heart was rounded and with pale areas in the myocardium. Liver was discreet accentuation of the lobular pattern. Histologically the lesion was characterized by pyogranulomatous and necrotizing bronchopneumonia, multifocal to coalescing, moderate, associated with myriads of intralesional hyphae consistente with Aspergillus spp., associated with acute myocardial infarction. The hyphae weakly stained basophilic by hematoxylin and eosin were strongly impregnated by Grocott’s methenamine silver nitrate and showed strong immunolabelling of the cytoplasm, which were evidenced in brown. There were areas of acute myocardial infarction and hepatocellular degeneration and congestion in the liver, although fungal hyphae were not observed in these tissues. Discussion: The diagnosis of pulmonary aspergillosis was established based on the morphological and tinctorial characteristics of the agent and confrmed by immunohistochemistry. In this case, is not included in the clinical history any conditions that could promote immunosuppression, neither were observed morphological changes in the pathological examination which suggested other intercurrent diseases. It is likely that the dog has been exposed to the inhalation of large amounts of conidia for a long period of time, condition that favors the occurrence of the disease, even in immunocompetent animals. Probably were not evidenced severe respiratory clinical signs, that characterize the pulmonary impairment, due to the shortcourse of the disease. The pyogranulomatous and necrotizing inflammatory reaction is typical of fungal infection and was restricted to the lung parenchyma. The areas of coagulation necrosis observed in the myocardium are probably secondary to vascular injury caused by the fungi, with subsequent thromboembolism, ischemia and infarction. In Brazil, there are few reports of aspergillosis in dogs, have been reported the nasal and systemic forms. In the world literature, there are few reports about this bronchopulmonary form of the disease. Although uncommon, pulmonary aspergillosis should be considered in the differential diagnosis of diseases that affect the lower respiratory tract of dogs, as well as it’s systemic complications resulting from angioinvasive characteristic of the fungus, which in this case was determinant to the death of the animal. Keywords: Aspergillus, fungal disease, respiratory trac

Mastitis by Pythium insidiosum in Mares

Background: Pythiosis is a chronic inflammatory disease caused by the oomycete Pythium insidiosum. This disease has been reported in several species, but most cases occur in horses. In this species, the cutaneous form is more prevalent, with occasional involvement of adjacent lymph nodes and bone tissue. The involvement of the mammary glands was not properly characterized. Therefore, the aim of this study is to describe cases of pythiosis with the involvement of the mammary glands in mares.Case: A retrospective study of all biopsy samples and necropsy examinations of horses, performed at the Animal Pathology Laboratory of the Federal University of Campina Grande from January 2000 to December 2018, was carried out. During the study period, 142 cases of cutaneous pythiosis were identified. Of these, in three the lesions extended to the mammary glands. Mares with 3-, 7- and 12-year-old, mongrel, from the backlands of the Paraiba were affected. On physical examination, the mares presented swollen mammary glands and multifocal wounds, which discharge serosanguinolent secretion. Clinical evolution ranged from 15 to 30 days. The animals had been medicated, but the wounds did not heal and mastectomies were performed. Macroscopically, the mammary glands were enlarged and with multifocal areas of ulceration measuring 1 to 3.5 cm in diameter. At the cut surface, they exhibited fistulous tracts containing kunkers. In the histopathological evaluation, was observed diffuse and marked inflammatory infiltrate of eosinophils extending through the deep dermis and interlobular mammary stroma; and multifocal areas, sometimes focally extensive, of eosinophilic necrosis with intralesional negative images of hyphae. In histological sections stained by periodic acid–Schiff, the walls of the hyphae were weakly stained in blue; but when impregnated by Grocott methenamine silver, they were strongly marked in black and were characterized by semi-parallel walls, rare septations and branching, and a diameter varying from 3-8μm. At immunohistochemistry, the hyphae showed strong cytoplasmic and wall immunostaining for the anti-P. insidiosum antibody.Discussion: The diagnosis of mastitis by P. insidiosum was established based on the epidemiological, clinical, anatomopathological and immunohistochemical findings. Lesions in horses are more frequently located at the extremities of the limbs and the ventral portion of the thoracoabdominal wall. In these cases, it is likely that the lesions have started on the skin adjacent to the udder and have extended to the mammary parenchyma. The intralesional structures known as kunkers have been described in horses, mules, donkeys, and camels, being absent in the other animal species affected. The kunkers are always present in equine pythiosis and allow the establishment of an accurate presumptive diagnosis. Clinically important findings include wounds that discharge serosanguineous secretions and do not heal through conventional treatments. Histological lesions characterized by areas of eosinophilic necrosis with intralesional hyphae, which are weakly stained by PAS and strongly impregnated by GMS, are highly suggestive of pythiosis. Pythiosis is relatively frequent in horses in the backlands of Paraiba, predominantly in the cutaneous form. The involvement of the mammary glands is an uncommon presentation that should be included as a differential diagnosis of mastitis in mares