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

Segmental Enteritis Associated with Pythium insidiosum Infection in a Horse

Background: Pythiosis is an infectious disease caused by the oomycete Pythium insidiosum, with higher occurrence in wetlands and hot climate regions. This microorganism develops its cycle in aquatic plants, and most cases happen because of the contact of animals or people with water containing the motile zoospores (infectious form). Horses are the principal species affected and develop principally cutaneous and subcutaneous lesions, but the gastrointestinal tract is seldom affected. Humans develop various forms of pythiosis, such as a vascular form. The objectives of the current study are to describe an unusual case of intestinal pythiosis, its clinical signs, aspects of pathogenesis, and diagnosis.Case: A 13-year-old Crioula mare, from Santa Maria, RS, Brazil, presented with reduced food and water intake, apathy, restlessness, rolling, nasal reflux, firm abdomen upon palpation, and tachypnea during 2 days. The horses of this farm were fed native pasture and horse feed, and they had access to a nearby pond. Two days following the start of the clinical signs, the horse died and was necropsied on the farm. During necropsy, there was around 400 mL of reddish effusion in the abdominal cavity (modified transudate). A 15 cm segment of jejunum was firm upon palpation and had a severe transmural thickening. The wall of the affected area was up to 3 cm in thickness and firm, with small yellowish and irregular masses that stood out and looked friable, interpreted as kunkers. Microscopically, the yellowish masses (kunkers) were characterized by dense accumulations of intact and degenerate eosinophils (eosinophilic necrosis). Within these kunkers, and also on their periphery, there were multiple negatively stained hyphal profiles. Hyphae were also seen on the wall of small arteries inside the kunkers. These hyphae had nearly parallel walls and were occasionally septate, with a diameter of about 10 μm. The hyphae stained black with Grocott’s methenamine silver stain (GMS) and were positive on immunohistochemistry (IHC) using specific anti-P. insidiosum polyclonal antibody.Discussion: Colic is the principal clinical presentation in horses suffering from gastrointestinal tract disorders. In spite of the presence of colic in this horse, intestinal pythiosis was not clinically suspected in this case, principally because of its rare occurrence in horses. Even though it is persistent to observe horses ingesting water with suspected contamination by P. insidiosum, the cutaneous form of pythiosis is a lot more common than the intestinal form. This is the only case of intestinal pythiosis in a horse in 52 years of routine diagnosis in our laboratory. It is suspected that this horse got infected by drinking contaminated water from the nearby pond and microlesions in the intestinal mucosa due to plant material or some unknown pathogen may have favored zoospore adhesion, encysting and starting the colonization of the tissue by emitting a germ tube. A macroscopic observation of kunkers in the intestine is rare, but when observed it is highly indicative of pythiosis. The immunohistochemistry technique using anti-P. insidiosum antibody, in accordance with the literature, validates the diagnosis of pythiosis. In this case, the presence of hyphae in the blood vessel wall inside the kunkers propose that this finding may be involved in the spread of the lesion but needs more detailed studies