Optimized immunocytochemistry using leukocyte and tissue markers on Romanowsky-stained slides from dogs and cats.

BACKGROUND: Romanowsky staining is often the initial method used to stain hematologic and cytologic materials. While immunocytochemistry (ICC) is a well-established method on air-dried smears, there are rare veterinary reports of ICC involving Romanowsky-stained slides. OBJECTIVES: This study aimed to compare immunoreactivity of unstained vs Romanowsky-stained specimens, evaluate reactions over time, and assess ICC associations with confirmatory tests of 50 lymphoma cases. Another goal aimed to optimize manual ICC protocols with cellular and tissue immunomarkers to detect CD3ε, CD20, Pax5, MHCII, lysozyme, MUM1, vimentin, cytokeratin, and Melan-A antigens on Romanowsky-stained specimens. MATERIALS AND METHODS: Cytologic specimens from cases of lymphoid and nonlymphoid neoplasms were stained with a methanolic Romanowsky method. Additional unstained slides from these cases were used for comparison with the stained materials. Antigen retrieval involved a citrate buffer pH6 or Tris/EDTA pH9 at 95°C for 25 minutes in a decloaking chamber. Immunocytochemistry used known positive and secondary antibody-only negative cytologic controls. Immunoreactivity of unstained and prestained lymphoma slides was graded by the intensity and percent of stained cells. Signal grading was monitored over time for diagnostic differences. RESULTS: Unstained and Romanowsky-stained slides had similar membrane/cytoplasm graded reactions, but unstained slides produced stronger signals. Romanowsky-stained blood films from B-cell and T-cell leukemias showed minimal loss of signal when monitored over 20 weeks. Signal differences did not change the diagnosis. There was a significant association between ICC and confirmatory tests. Optimization involved antibody dilution and antigen retrieval methodology for each antibody tested. CONCLUSIONS: Immunocytochemistry of Romanowsky-stained material can be successfully performed using antibodies against CD3ε, CD20, cytokeratin, lysozyme, Melan-A, MHCII, MUM1, Pax5, and vimentin.

Histological and anatomical variations of septomarginal trabecula in bovine hearts.

The use of hearts from different animals as models in the experimental pharmacology and surgical clinic has led, in recent years, to an increase on interest of research with this organ. The heart's conducting system, from the septomarginal trabecula, presents several variations, which generates numerous controversies in the literature. So, the objective of the present study is to analyse the morphology of the septomarginal trabecula of bovine hearts, identifying possible macro- and microscopic variations. Thirty-four bovine hearts were analysed. Each trabecula was analysed macroscopically to obtain an anatomical description and measurements of its length and thickness. For histological and morphometric analysis, the samples were fixed in Bouin's solution and then subjected to histological processing. In all the analysed bovine hearts, the septomarginal trabecula presented itself as a smooth, tubular meaty structure of muscular consistency, with variable length and diameter. The anatomical variations observed included a trabecula with forked marginal fixation, and single septal fixation, in addition to a trabecula with extremely reduced or excessively thick caliber. The septomarginal trabecula consists of cardiac muscle fibres, connective tissue, vascular tissue and conduction myofibrils or Purkinje fibres. In the samples of smaller thicknesses, there was a predominance of connective tissue and scarce cardiac muscle tissue, whereas in the thicker samples the predominance was of cardiac striated muscle tissue. Therefore, there are significant macro- and microscopic differences between the bovine septomarginal trabecula concerning their diameter and constituent tissue, and that can lead to possible changes in cardiac physiology.

Changes in adipose cells of Longissimus dorsi muscle in Iberian pigs raised under extensive conditions.

Twenty-four Iberian castrated male pigs were used to characterize and evaluate the effect of the duration of "Montanera" in the adipocytes size and its relation with the backfat thickness and intramuscular fat. The animals were fed under extensive conditions during 30, 60 and 90 days in the "Dehesa" before slaughtered. Carcass weight, percentage of intramuscular fat, thickness of backfat and its three layers and adipocytes size of the intramuscular fat were obtained. The group which expended 90 days on fattening obtained the highest adipocytes, with an area higher by a 50% than those that only expended 30 days. The differences in diameter and perimeter adipocyte were not as marked as area. A significant positive correlation between the diameter, area and perimeter of adipocyte with the backfat thickness were found. The fat cells in Iberian pig hypertrophy during the "montanera stage", being this increase significant from month to month in this period of fattening. Also, this adipocyte increase is correlated with the animal weight. The correlation between adipocyte size and inner layer of backfat shows that the inner layer obtained in live pig by ultrasound techniques could be a good marker of fat infiltration in pigs fattening in "montanera" system.

Biochemical and biomechanical characterisation of equine cervical facet joint cartilage.

BACKGROUND: The equine cervical facet joint is a site of significant pathology. Located bilaterally on the dorsal spine, these diarthrodial joints work in conjunction with the intervertebral disc to facilitate appropriate spinal motion. Despite the high prevalence of pathology in this joint, the facet joint is understudied and thus lacking in viable treatment options. OBJECTIVE: The goal of this study was to characterise equine facet joint cartilage and provide a comprehensive database describing the morphological, histological, biochemical and biomechanical properties of this tissue. STUDY DESIGN: Descriptive cadaver studies. METHODS: A total of 132 facet joint surfaces were harvested from the cervical spines of six skeletally mature horses (11 surfaces per animal) for compiling biomechanical and biochemical properties of hyaline cartilage of the equine cervical facet joints. Gross morphometric measurements and histological staining were performed on facet joint cartilage. Creep indentation and uniaxial strain-to-failure testing were used to determine the biomechanical compressive and tensile properties. Biochemical assays included quantification of total collagen, sulfated glycosaminoglycan and DNA content. RESULTS: The facet joint surfaces were ovoid in shape with a flat articular surface. Histological analyses highlighted structures akin to articular cartilage of other synovial joints. In general, biomechanical and biochemical properties did not differ significantly between the inferior and superior joint surfaces as well as among spinal levels. Interestingly, compressive and tensile properties of cervical facet articular cartilage were lower than those of articular cartilage from other previously characterised equine joints. Removal of the superficial zone reduced the tissue's tensile strength, suggesting that this zone is important for the tensile integrity of the tissue. MAIN LIMITATIONS: Facet surfaces were sampled at a single, central location and do not capture the potential topographic variation in cartilage properties. CONCLUSIONS: This is the first study to report the properties of equine cervical facet joint cartilage and may serve as the foundation for the development of future tissue-engineered replacements as well as other treatment strategies.

MICROCOMPUTED TOMOGRAPHIC, MORPHOMETRIC, AND HISTOPATHOLOGIC ASSESSMENT OF CONGENITAL BONE MALFORMATIONS IN TWO NEOTROPICAL VIPERIDS.

Congenital malformations have been reported in all classes of vertebrates and may be a determinant of life span and survival. In reptiles, the incidence of congenital malformations can be associated with genetic and environmental causes, including pollution. The characterization of pathological processes involved in the development of congenital malformations of bone in snakes is rare in the literature, but is of great relevance in the field of reptile conservation and environmental health. We describe congenital bone lesions in 50 newborn jararaca (Bothrops jararaca) and 26 South American rattlesnakes (Crotalus durissus terrificus) born from wild-caught pregnant females in Southeastern Brazil. Lesions were evaluated by morphometric quantitative analysis, x-ray microtomography, and histopathologic descriptive analysis. Morphometric analysis showed that jararaca presented more severe axial lesions (kyphosis, scoliosis, and kyphoscoliosis) than rattlesnakes. Female rattlesnakes presented more severe axial lesions than did males. In rattlesnakes, spinal deformities were more frequently diagnosed in the caudal segment of the body. We present x-ray microtomographic assessments and images of malformed snakes (n=9) and characterized novel malformations, such as the agenesis of frontal, parietal, and supraoccipital bones in a jararaca specimen. Histopathologic findings included vertebral body fusion, myositis, coagulation necrosis, and disorganization of periaxial muscle fibers. The new methods and results presented in this study will be useful and informative for future research in pathology, teratology, embryology, and ecotoxicology in snakes.

Individual oocysts of Isospora (Apicomplexa: Coccidia) parasites from avian feces: from photo to sequence.

Numerous microscopic studies of coccidian oocysts from avian feces have become the basis for species identification. In contrast, molecular studies of wild birds' Coccidia are still in their infancy and are mostly based on DNA extracted from the blood stages of these parasites. Linking microscopic and molecular data requires a method that reliably extracts DNA from single oocysts with parallel detailed morphological examination of the same cell. We offer a thorough manual of isolating, photographing, and trapping single oocysts from avian feces, followed by extraction of parasite DNA and amplification of mitochondrial DNA from the same cells. In 39 single oocysts from 6 wild blackcaps, we combined microscopic studies of individual cells with studies on their mitochondrial haplotype. In 72% of the single oocysts sampled, we detected unambiguous sequences. From feces and blood of investigated birds, we obtained 6 different haplotypes of Isospora sp. (iSAT1-iSAT 6), finding both the same haplotype in different host individuals and various haplotypes in the same host individual. Our described methodology enables linking the huge amount of morphological data with innovative gene analysis. This method expands the scope of genetic studies conducted on Isospora species, including routine molecular analysis of single oocysts isolated from fecal samples.

CD20 expression in normal canine B cells and in canine non-Hodgkin lymphoma.

We examined the expression of CD20 in normal canine peripheral blood mononuclear cells, normal canine spleen, and canine non-Hodgkin lymphoma (NHL) to determine the feasibility of using this antigen as a diagnostic aid and as a possible target for therapy. An antibody generated against a C-terminal (intracytoplasmic) epitope of human CD20 recognized proteins of 32-36 kd in normal and malignant canine lymphocytes. This antibody showed restricted membrane binding in a subset of lymphocytes in peripheral blood, in the B-cell regions from a normal canine spleen and lymph node, and in malignant cells from 19 dogs with B-cell NHL, but not from 15 dogs with T-cell NHL. The patterns of CD20 reactivity in these samples overlapped those seen using an antibody that recognizes canine CD79a. This anti-CD20 antibody is therefore suitable as an aid to phenotype canine NHL. In contrast, normal canine B cells were not recognized by any of 28 antibodies directed against the extracellular domains of human CD20 (including the chimeric mouse-human antibody Rituximab) or by any of 12 antibodies directed against the extracellular domains of mouse CD20. Thus, the use of CD20 as a therapeutic target will require the generation of specific antibodies against the extracellular domains of canine CD20.

Estudio de las repercusiones arteriales de la deprivación y aporte de hormonas femeninas. Estudio experimental / A study of the repercussions of the deprivation and supply of female hormones on arteries. An experimental study

Introducción. Se ha considerado de una forma generalizada que las hormonas femeninas protegen el desarrollo de enfermedades cardiovasculares y muy especialmente el desarrollo de las alteraciones degenerativas de las arterias. Objetivo. El estudio trata de evidenciar las relaciones que el componente hormonal tiene sobre la pared arterial, valoradas desde el punto de vista histológico y morfométrico. Materiales y métodos. Se realiza un trabajo experimental en la rata con la utilización de diferentes grupos de animales, soportado el estudio en un modelo animal para evidenciar las repercusiones arterialmente de las diferentes situaciones hormonales. El diseño experimental se estructura en diferentes grupos de estudio sobre la base de una deprivación hormonal, posterior suplencia, ya sea farmacológica o a través del trasplante ovárico sobre la pared arterial. Resultados y conclusiones. Las repercusiones de la deprivación hormonal no han sido muy evidentes, pero sí detectables mediante métodos morfométricos. Los resultados demuestran que la suplencia hormonal neutraliza los posibles efectos degenerativos derivados de la deprivación hormonal. Se discute la interacción de las hormonas estrogénicas y su implicación en el desarrollo de patología arterial y las evidencias existentes al respecto, bibliográficamente (AU)

Functional loss of p21/Waf-1 in a case of benign canine multicentric melanoma.

Mutations of tumor suppressor genes remove mechanisms that normally arrest proliferation of transformed cells, resulting in tumor formation. The p53 gene product functions as a tumor suppressor that induces p21/Waf-1, the 21-kDa product of the waf-1/cip-1/mda-6 gene. p21/Waf-1 is a pan-cyclin-dependent kinase inhibitor that arrests cell cycle progression under a variety of circumstances. We examined tissues from a dog with multiple primary pigmented proliferative lesions (benign, multicentric melanoma consisting of three distinct dermal lesions and a matrical cyst) for p21/Waf-1 and p53 expression by immunohistochemistry and immunoblotting. p21/Waf-1 and p-53 proteins were undetectable in the tumor cells and in the cyst but were present in adjacent normal tissues. Abundant cyclin-dependent kinase 4 (Cdk4), a protein related functionally to p21/Waf-1, also was present in the cyst. A somatic mutation of the waf-1 gene or of the p53 gene may have resulted in the loss of p21/Waf-1 expression in a common precursor of pigment-producing cells from the affected dog. Furthermore, this functional loss of p21/Waf-1 may play an important role in the genesis of canine benign melanoma.

Experimental Clostridium perfringens type D enterotoxemia in goats.

The effects of intraduodenal administration of Clostridium perfringens cultures and culture products in goats were evaluated to develop a reliable experimental model of enterotoxemia in this species. Five conventionally reared, 11-16-week-old Angora goat kids were dosed intraduodenally with whole cultures of C. perfringens type D; five similar animals were dosed with C. perfringens type D filtered culture supernatant; and a third group of five kids was dosed with C. perfringens type D washed cells. Two kids were used as controls and received sterile, nontoxic culture medium intraduodenally. All animals received starch solution into the abomasum. All five kids inoculated with whole culture and three of five dosed with culture supernatant and with washed cells developed central nervous system signs. Diarrhea was observed in two of five kids inoculated with whole culture, in all five of those dosed with culture supernatant, and in three of five of those that received washed cells. The most striking postmortem findings consisted of lung edema, necrotizing pseudomembranous colitis, and cerebral vasogenic edema. The protocol thus provided a reasonable model of naturally occurring enterotoxemia in goats, producing a range of clinical signs and postmortem changes similar to those observed in the natural disease.

Type, diameter and distribution of fibres in some respiratory and abdominal muscles of the goat.

This study was designed to determine the histochemical properties, size and composition of fibres in the diaphragm, intercostal and abdominal muscles of goats to clarify whether reported similarities in respiratory muscle physiology between goats and humans have a structural basis. Serial sections (10 microns) of muscular tissue from adult female goats were stained for myosin adenosine triphosphatase and reduced nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase activities; the fibres were classified into type I, IIA and IIB; and their mean diameter and composition were determined. Abdominal and intercostal muscles contained types I, IIA and IIB fibres in the ratio 1:1:1, and the mean diameter of the fibres ranged from 49.2 to 62.2 microns. In contrast, the diaphragm contained 58.9% type I and 41.1% type II fibres, and the latter could not be differentiated into types IIA and IIB. Diaphragmatic fibres were also smaller (36.9-40.9 microns). These findings contrast with those in humans, where the diaphragm, intercostal and abdominal muscles contain > 50% type I fibres and have fibres of identical diameter. The differences in fibre characteristics between the diaphragm, intercostal and abdominal muscles of goats and the differences between goats and humans need to be taken into consideration in interpreting the results from studies in respiratory muscle physiology.