Recommended Guidelines for Submission, Trimming, Margin Evaluation, and Reporting of Tumor Biopsy Specimens in Veterinary Surgical Pathology

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

Histopathological Changes in the Placentas and Fetuses of Mice Infected with Trypanosoma cruzi Isolated from the Myotis nigricans nigricans Bat

Histopathological changes and placental transmission were studied in the late stages of pregnancy in mice infected with a strain of Trypanosoma cruzi, isolated from a Myolis nigricans nigricans bat. Large amastigote nests were observed in uterine muscles, as well as in decidual and endothelial placental cells. In addition, persistent coagulative and fibrotic Vascular degeneration was observed. Large amastigote burdens were found in giant cells, spongioblasts and endothelial cells within the labyrinthine layer. Transplacental transmission was confirmed in 30% of the fetuses examined, in which amastrigote nests were seen only in striated muscle. During tire acute phase, intrauterine development was impaired as the result of parasitic invasion of the placenta, and fetal mortality rose to 10%. (C) 2008 Elsevier Ltd. All rights reserved.

Lesions caused by cardiovascular flukes (Digenea : Spirorchidae) in stranded green turtles (Chelonia mydas)

Evidence of infection with spirorchid flukes (Digenea: Spirorchidae) was sought at necropsy of 96 stranded green turtles, Chelonia mydas, that were examined during the course of a survey of marine turtle mortality in southeastern Queensland, Australia. Three species of spirorchid (Hapalotrema mehrai, H. postorchis, and Neospirorchis schistosomatoides) were identified. Severe disease due to spirorchid fluke infection (spirorchidiasis) was implicated as the principal cause of mortality in 10 turtles (10%), and appeared to be one of multiple severe problems in an additional 29 turtles (30%). Although flukes were observed in only 45% of stranded C. mydas in this study, presumed spirorchid fluke infection was diagnosed in an additional 53% of turtles, based principally on characteristic necropsy lesions and to a lesser extent on the histopathological detection of spirorchid eggs. Characteristic necropsy lesions included miliary spirorchid egg granulomas, which were observed most readily on serosal surfaces, particularly of the small intestine. Cardiovascular lesions included mural endocarditis, arteritis, and thrombosis, frequently accompanied by aneurysm formation. Resolution of thrombi was observed to occur via a combination of granuloma formation about indigestible components (spirorchid fluke egg shells) and exteriorization through the vessel wall, which resulted in granulomatous nodules on the adventitial surface. Septic aortic thrombosis complicated by disseminated bacterial infection, observed in five turtles, was recorded for the first time. Egg granulomas were ubiquitous in turtle tissues throughout this study. Although they generally appeared to be mild or incidental lesions, they were occasionally associated with severe multifocal granulomatous pneumonia or meningitis.

Functional and structural characterization of isolated perfused stingray liver including effects of ischaemia/reperfusion

The morphological and functional characteristics of stingray liver were studied, including the effect of ischaemia/reperfusion. With an isolated perfused model, it was shown that the stingray liver was more resistant than the rat liver to ischaemia/reperfusion injury; this was consistent with the differing partial oxygen tensions usually present in the two species. This study confirmed that whereas stingray hepatocytes form tubules with central bile canaliculi as in other fish, the stingray liver has portal triads and a lobular architecture as in mammals. Apoptosis of hepatocytes, demonstrated in the normal liver, was only marginally enhanced by ischaemia/reperfusion. Resulting apoptotic bodies were phagocytized by macrophage-like cells in hepatocyte tubules. In contrast to rat liver, the stingray liver showed no necrosis after ischaemia-reperfusion. (C) 1998 W.B. Saunders Company Limited.

Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 X 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. Al; the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

Effects of intravenous injection of Clostridium perfringens type D epsilon toxin in calves

In cattle, a neurological lesion similar to that produced in sheep and goats by Clostridium perfringens type D enterotoxaemia has been reported. However, no causal relationship has been established between this disease and the lesion in cattle. The effects of single and multiple intravenous injections of epsilon toxin in three calves aged 6 months were studied. A further calf was inoculated intravenously with saline solution and used as a control. Epsilon toxin invariably produced neurological signs within 2-60 min of the end of the injection process. Clinical signs consisted of loss of consciousness, recumbency, convulsions, paddling, opisthotonus, hyperaesthesia and dyspnoea. Gross changes consisted of severe acute pulmonary oedema, which was particularly marked in the interlobular septa. The histological lesions consisted of intra-alveolar and interstitial oedema of the lung and variable degrees of perivascular proteinaceous oedema in the internal capsule, thalamus and cerebellar white matter. No clinical or post-mortem changes were observed in the control calf. These results show that calves are susceptible to the intravenous injection of epsilon toxin, and that they can show at least some of the histological lesions produced in sheep and goats by this toxin. (C) 2002 Harcourt Publishers Ltd.

Thermolysin activates equine lamellar hoof matrix metalloproteinases

Cultured equine lamellar hoof explants secrete the pro-enzymes matrix metalloproteinse-2 (MMP-2, 72 kDa) and MMP-2 (92 kDa). Untreated explants remained intact tested on a calibrated force transducer, but when treated with an NIMP activator, developed in-vitro laminitis, separating at the dermal-epidermal junction. Explants treated with the bacterial protease thermolysin separated dose-dependently; this was accompanied by activation of both MMP-2 and -9. Thermolysin-mediated NIP activation did not occur in a cell-free system and was not inhibited by the addition of the MMP inhibitor and batimastat. These findings suggest that thermolysin-mediated gelatinase activation is not dependent on membrane-bound matrix metalloproteinase (MT-MMP) activation, providing further evidence that bacteria can produce potent MMP activators that probably facilitate host invasion. (C) 2002 Harcourt Publishers Ltd.

Matrix metalloproteinase-2 and-9 involvement in canine tumors

Matrix metalloproteinases (MMPs) are a family of enzymes implicated in the degradation and remodeling of extracellular matrix and in vascularization. They are also involved in pathologic processes such as tumor invasion and metastasis in experimental cancer models and in human malignancies. We used gelatin zymography and inummohistochemistry to determine whether MMP-2 and MMP-9 are present in canine tumors and normal tissues and whether MMP production correlates with clinicopathologic parameters of prognostic importance. High levels of pro-MMP-9, pro-MMP-2, and active MMP-2 were detected in most canine tumors. Significantly higher MMP levels were measured in canine tumors than in nontumors, malignancies had higher MMP levels than benign tumors, and sarcomas had higher active MMP-2 than carcinomas. Cartilaginous tumors produced higher MMP levels than did nonsarcomatous malignancies, benign tumors, and normal tissues, and significantly greater MMP-2 than osteosarcomas and fibrosarcomas. Pro-MMP-9 production correlated with the histologic grade of osteosarcomas. The 62-kd form of active MMP-2 was detected only in high-grade, p53-positive, metastatic malignancies. Zymography proved to be a sensitive and quantitative technique for the assessment of MMP presence but has the limitation of requiring fresh tissue; inummohistochemistry is qualitative and comparatively insensitive but could be of value in archival studies. MMP presence was shown in a range of canine tumors, and their link to tumor type and grade was demonstrated for the first time. This study will allow a substantially improved evaluation of veterinary cancer patients and provides baseline information necessary for the design of clinical trials targeting MMPs.

Immunohistochemical Characterization of Canine Prostatic Intraepithelial Neoplasia

The development of prostate cancer is believed to be a multistep process, progressing sequentially from normal epithelium, to prostatic intraepithelial neoplasia (PIN) and, finally, to invasive neoplasia. Malignant stem cells within the basal cell layer of the prostatic epithelium are believed to play an important role in the failure of androgen-ablation therapy that occurs in the most advanced form of prostate cancer. The aim of the present study was to immunohistochemically characterize the lesions of canine PIN. Prostatic tissue from five dogs with PIN was compared with normal prostate tissue from nine further dogs. There was an increase in the number of basal epithelial cells in lesions consistent with PIN as defined by expression of the nuclear protein p63. These lesions had elevated expression of proliferating cell nuclear antigen (PCNA) and heterogeneous labelling for the nuclear androgen receptor (AR). These findings suggest that the basal cells present in PIN may play a role in canine prostate carcinogenesis and that the proliferation of these cells occurs despite the heterogeneous expression of the AR. (C) 2009 Elsevier Ltd. All rights reserved.

Expression of Connexins in Normal and Neoplastic Canine Bone Tissue

Previous studies showed that intercellular communication by gap junctions has a role in bone formation. The main connexin involved in the development, differentiation, and regulation of bone tissue is connexin (Cx) 43. In addition, Cx46 is also expressed, mostly localized within the trans-Golgi region. Alterations in the expression pattern and aberrant location of these connexins are associated with oncogenesis, demonstrating a deficient gap junctional intercellular communication (GJIC) capacity in neoplastic tissues. In this study, we evaluated normal and neoplastic bone tissues regarding the expression of Cx43 and Cx46 by immunofluorescence, gene expression of these connexins by real-time PCR, and their correlation with cell proliferation index and deposition of collagen. Fourteen neoplastic bone lesions, including 13 osteosarcomas and I multilobular tumor of bone, were studied. The mRNA levels of Cx43 were similar between normal and neoplastic bone tissue. In normal bone tissue, the Cx43 protein was found mainly in the intercellular membranes. However, in all bone tumors studied here, the Cx43 was present in both cell membranes and also aberrantly in the cytoplasm. Regarding only tumor samples, we determined a possible inverse correlation between Cx43 expression and cellular proliferation, although a positive correlation between Cx43 expression and collagen deposition was also noted. In contrast, Cx46 had lower levels of expression in neoplastic bone tissues when compared with normal bone and was found retained in the perinuclear region. Even though there are differences between these two connexins regarding expression in neoplastic versus normal tissues, we concluded that there are differences regarding the subcellular location of these connexins in normal and neoplastic dog bone tissues and suggest a possible correlation between these findings and some aspects of cellular proliferation and possibly differentiation.

Effects of physical and chemical immobilization on hematologic and biochemical variables in captive brown brocket deer (Mazama gouazoubira)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

Background: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. Objective: the objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. Methods: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. Results: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. Conclusion: the microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations

Leukocyte and thrombocyte reference values for channel catfish (Ictalurus punctatus Raf), with an assessment of morphologic, cytochemical, and ultrastructural features

Background: Hematology tests are useful to evaluate physiologic disturbances in fish and can provide important information for the diagnosis and prognosis of disease. Objectives: the primary purpose of this study was to define reference intervals for thrombocytes and leukocytes in healthy channel catfish (Ictalurus punctactus). In addition, the morphologic, cytochernical, and ultrastructural features of blood cells were assessed. Methods: Blood samples (0.5 mL were collected into EDTA from 40 clinically healthy catfish on a commercial fish farm in Jaboticabal, Brazil. Thrombocyte, total WBC, and differential WBC counts were determined and reference intervals were calculated as the 25-95th percentiles of data. Thrombocyte and leukocyte morphology was assessed in blood smears stained with May Griinwald-Giemsa-Wright and ultrastructurally by transmission electron microscopy. Cytochemical staining patterns were described using periodic acid-Schiff (PAS), peroxidase, nonspecific esterase, alkaline phosphatase, and toluidine blue. Results: Reference intervals were as follows: thrombocytes 58,802-99,569/mu L; total WBCs 27,460-41,523/mu L; lymphocytes 5380-11,581/mu L; monocytes 2949-7459/mu L; neutrophils 12,529-22,748/mu L, and basophils 736-2003/mu L. Neutrophils were positive for peroxidase and PAS; monocytes were positive for nonspecific esterase; and basophils were positive with toluidine blue. Conclusion: the morphologic and staining features of neutrophils and monocytes of channel catfish are similar to those of mammals, and the presence of basophils in this species was verified. These reference intervals and morphologic findings provide a foundation for future investigations on the functions and alterations of blood cells in channel catfish.

Characterization and quantification of blood cells from the umbilical cord of dogs

Background: Models for the study of hematopoietic stem cells in dogs provide important information for bone marrow transplantation in humans. Recent studies have reported the importance of human umbilical cord blood (UCB) as an alternative to allogenic bone marrow for hematopoietic reconstitution. However, there are no studies on the UCB cells of dogs. Objective: the aim of this experiment was to characterize and quantify the blood cells of the umbilical cord of dogs. Methods: the blood of the umbilical cord of 20 neonatal dogs, delivered at term, with a median gestation time of 58 days, was collected with a 5-mL syringe containing EDTA. Total RBC, WBC, and platelet counts, HCT, hemoglobin (Hgb) concentration, and RBC indices were determined using an automatic cell counter. The differential leukocyte count was determined manually in blood smears stained with May-Grunwald-Giemsa. Reticulocyte percentages were determined on blood smears stained with brilliant cresyl blue and counterstained with May-Grunwald Giemsa. Results: the MCHC and numbers of RBCs, WBCs, neutrophils, and eosinophils in UCB were lower as compared with reference values for the peripheral blood of healthy neonatal and adult dogs; whereas, the MCV and reticulocyte percentages were higher. Conclusion: Erythrocyte macrocytosis and hypochromasia in UCB were consistent with marked reticulocytosis and indicative of high erythropoietic activity. The results of this study are an important first step in the characterization of UCB from neonatal dogs.