Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior.

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

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.

Recommended guidelines for the conduct and evaluation of prognostic studies in veterinary oncology.

There is an increasing need for more accurate prognostic and predictive markers in veterinary oncology because of an increasing number of treatment options, the increased financial costs associated with treatment, and the emotional stress experienced by owners in association with the disease and its treatment. Numerous studies have evaluated potential prognostic and predictive markers for veterinary neoplastic diseases, but there are no established guidelines or standards for the conduct and reporting of prognostic studies in veterinary medicine. This lack of standardization has made the evaluation and comparison of studies difficult. Most important, translating these results to clinical applications is problematic. To address this issue, the American College of Veterinary Pathologists' Oncology Committee organized an initiative to establish guidelines for the conduct and reporting of prognostic studies in veterinary oncology. The goal of this initiative is to increase the quality and standardization of veterinary prognostic studies to facilitate independent evaluation, validation, comparison, and implementation of study results. This article represents a consensus statement on the conduct and reporting of prognostic studies in veterinary oncology from veterinary pathologists and oncologists from around the world. These guidelines should be considered a recommendation based on the current state of knowledge in the field, and they will need to be continually reevaluated and revised as the field of veterinary oncology continues to progress. As mentioned, these guidelines were developed through an initiative of the American College of Veterinary Pathologists' Oncology Committee, and they have been reviewed and endorsed by the World Small Animal Veterinary Association.

Vascular lesions in pigs experimentally infected with porcine circovirus type 2 serogroup B.

Vasculitis is a hallmark lesion of the severe form of systemic porcine circovirus-associated disease (PCVAD). In 2 experimental studies with porcine circovirus type 2 serogroup b (PCV2b), 2 pigs developed fatal PCVAD with acute vasculitis, and 5 related pigs developed chronic lymphohistiocytic and plasmacytic peri- and endarteritis. Five of these pigs (1 with acute vasculitis and 4 with chronic vasculitis) had also been inoculated with bovine viral diarrhea virus type 1 (BVDV1) or BVDV1-like virus. Vascular lesions were similar, independent of whether pigs had been inoculated singly with PCV2b or dually with PCV2b and BVDV1 or BVDV1-like virus. The acute vasculitis was accompanied by marked pulmonary and mesenteric edema and pleural effusion. In situ hybridization demonstrated abundant intracytoplasmic porcine circovirus type 2 (PCV2) nucleic acid in endothelial, smooth muscle-like, and inflammatory cells within and around affected arteries. The pigs with lymphohistiocytic and plasmacytic vasculitis had lesions of systemic PCVAD, including multisystemic lymphoplasmacytic and histiocytic or granulomatous inflammation. PCV2 nucleic acid was detected in renal tubule epithelial cells, mononuclear inflammatory cells, and rare endothelial cells in noninflamed vessels in multiple tissues of these animals. The 2 pigs with acute vasculitis had no PCV2-specific antibodies (or a low titer of), whereas the pigs with lymphohistiocytic and plasmacytic vasculitis developed high antibody titers against this virus. These observations suggest that (1) acute vasculitis observed in the current studies is directly caused by PCV2b, (2) chronic vasculitis may in part be mediated by the subsequent immune response, and (3) host factors and viral strain may both contribute to vasculitis in animals infected with PCV2b.

BioGlue surgical adhesive for thoracic aortic repair during coagulopathy: efficacy and histopathology.

BACKGROUND: We hypothesized that induction of coagulopathy in sheep would model clinical needle hole and surgical bleeding from synthetic graft anastomoses, and that a new tissue bioadhesive (BioGlue) would control postoperative blood loss during surgical repair of the thoracic aorta. METHODS: Sheep were anticoagulated with aspirin and heparin. A bypass was made using end-to-side anastomoses of a graft to a partially occluded descending thoracic aorta. Experimental anastomoses (EXP, n = 9) were treated with BioGlue, and control anastomoses (CON, n = 5) were treated with Surgicel to gain intraoperative hemostasis. RESULTS: EXP animals exhibited significantly reduced postsurgical bleeding (CON median 955 mL versus EXP median 470 mL, p < 0.003), a reduced rate of blood loss over the first 2 postoperative hours (CON median 210 mL/hr versus EXP median 92.5 mL/hr, p < 0.006), and over the entire recovery period (CON median 158 mL/hr versus EXP median 86 mL/hr, p < 0.05), and reduced total blood loss (CON mean 1,497 +/- 691 mL versus EXP mean 668 +/- 285 mL, p < 0.008). On histologic examination of tissues explanted after 3 months, BioGlue was relatively inert and demonstrated a minimal inflammatory response. CONCLUSIONS: The use of BioGlue significantly reduced the volume and rate of postsurgical bleeding in a coagulopathic sheep model for thoracic aortic operations. Histopathologically, BioGlue generated only a minimal inflammatory response. This new surgical tissue bioadhesive should prove extremely beneficial for coagulopathic patients undergoing thoracic aortic or vascular procedures.

Ventral surgical approach to the caudal brain stem in dogs.

OBJECTIVE: To develop a safe neurosurgical procedure that accessed the ventral pons and medulla of the dog primarily for the removal of brain stem neoplasms. STUDY DESIGN: In vivo study. METHODS: A cadaver study was performed on mesocephalic dog heads to develop a neurosurgical approach to the ventral brain stem. Based on this study, an approach to the ventral brain stem was developed by basioccipital craniectomy. This procedure was performed on 4 young neurologically normal Beagle dogs to assess morbidity and mortality associated with the procedure. Morbidity was evaluated by subjective criteria, daily complete neurologic examinations, comparison of preoperative and postoperative brain stem auditory evoked response (BAER) tests, and postmortem examinations. RESULTS: Three dogs developed a transient cough but were neurologically normal after surgery. One dog was euthanatized within 12 hours of surgery because of severe postoperative morbidity associated with basilar artery disruption due to improper development of the craniectomy. Prolongations of postoperative BAER latencies were observed in 2 dogs but did not appear to be associated with clinical deficits or histopathologic changes in the brain stem. Minimal histopathologic changes were observed except in the dog with basilar artery disruption. Results of this study indicate that, although technically challenging, this procedure can be performed with minimal morbidity. CLINICAL IMPLICATIONS: The main indication for this procedure is surgical reduction or biopsy of ventrally located brain stem neoplasms in dogs. The major disadvantage is anatomic restrictions that prevent access to laterally oriented ventral brain stem masses.

Inoculation of Sarcocystis neurona merozoites into the central nervous system of horses.

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. A horse model of EPM is needed to test the efficacy of chemotherapeutic agents and potential vaccines. Five horses that were negative for antibodies to S. neurona in their serum and cerebrospinal fluid (CSF) were injected in the subarachnoid space with living merozoites of the SN2 isolate of S. neurona. None of the horses developed clinical disease or died over a 132-day observation period. All five horses developed antibodies to S. neurona in their CSF and serum 3-4 weeks after injection. Two of the horses were examined at necropsy and no parasite induced lesions were observed in their tissues and no parasites were recovered from portions of their spinal cords inoculated on to cell cultures. Results of this study demonstrate that merozoites of the SN2 isolate of S. neurona will induce seroconversion but not clinical disease when inoculated directly into the CSF of nonimmune horses.

Prevalence of antibodies to Neospora sp. in horses from Alabama and characterisation of an isolate recovered from a naturally infected horse [corrected].

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.

Characterization of temperature-sensitive strains of Neospora caninum in mice.

Temperature-sensitive (ts) strains of the Neospora caninum tachyzoites were selected by chemical mutagenesis and selection for growth at 32 C. Three ts strains and the parental, N. caninum wild-type strain, NC-1, were examined in the present study for their ability to cause disease in inbred BALB/c mice, outbred ICR mice, and chemically immunosuppressed ICR mice. In BALB/c mice, all 3 strains failed to induce clinical disease, whereas infection with the NC-1 strain caused central nervous system disease and death in some mice. No disease was observed in ICR mice inoculated with the 3 ts strains or the NC-1 strain. All immunosuppressed ICR mice inoculated with the NC-1 strain died, whereas no immunosuppressed mice inoculated with the NCts-4 strain and only 1 of 5 mice inoculated with the NCts-8 and NCts-12 strains died. The NCts-4 and NCts-12 strains reverted to a wild-type phenotype when grown at 37 C. Vaccination of BALB/c mice with live, but not frozen NCts-8 strain tachyzoites induced significant (P < 0.05) protection following NC-1 strain challenge.

Gastrointestinal pathogenicity of adenoviruses and reoviruses isolated from broiler chickens in Alabama.

Adenoviruses and reoviruses isolated from commercial broiler chickens were evaluated for gastrointestinal pathogenicity in specific-pathogen-free Leghorn chickens. The viruses were originally isolated from either the proventriculus or a gastrointestinal pool of tissues of broiler chickens with proventriculitis or enteritis. Isolates were cloned by terminal dilution. Day-old chickens were inoculated by oral and ocular routes with undiluted tissue culture fluids (titers of 10[2]-10[4] TCID50/ml) and then examined at necropsy on days 5, 10, and 15 postinoculation. Chickens in all virus groups (but not the control group) developed wet, unformed fecal droppings that persisted for the duration of the study. Mild lesions occurred in reovirus-inoculated chickens and included hyperplasia of lymphocyte aggregates in various organs and mild gizzard erosions. Chickens inoculated with adenovirus isolates developed marked gizzard erosions and necrotizing pancreatitis as well as mild proventriculitis. Intranuclear viral inclusion bodies occurred in gizzard epithelium and pancreatic acinar cells at the sites of lesions. Lymphocytic atrophy occurred in the bursa of Fabricius. Respective viruses were reisolated from proventriculus and duodenum collected from chickens of each group; no viruses were isolated from controls. Under the conditions of this study, adenovirus isolates were more pathogenic than the reovirus isolates in the digestive system.

Vaccination of mice with Neospora caninum: response to oral challenge with Toxoplasma gondii oocysts.

Neospora caninum is a protozoan parasite that can cause severe disease in mammals. Two experiments were conducted to examine the effects of subcutaneous (s.c.) vaccination with Hank's balanced salt solution (HBSS), 1 x 10(5) N. caninum NC-1 strain tachyzoites or 1 x 10(5) Toxoplasma gondii TS-4 strain tachyzoites on challenge oral infections in mice with sporulated VEG strain T. gondii oocysts (1 X 10(3) oocysts exp. 1 and 5 x 10(3) oocysts exp. 2). An additional study, experiment 3, evaluated s.c. challenge with 2.5 X 10(3) tachyzoites of the highly virulent RH strain of T. gondii after vaccination with HBSS, NC-1 tachyzoites, or TS-4 tachyzoites. Mice vaccinated with NC-1 strain tachyzoites survived significantly (P < 0.05) longer than mice given HBSS in experiment 1, but not in experiments 2 and 3. Mice vaccinated with TS-4 strain tachyzoites survived significantly longer than HBSS-vaccinated mice in experiments 1, 2, and 3 and significantly longer than mice vaccinated with the NC-1 strain in experiments 2 and 3. Toxoplasma gondii tissue cyst numbers were significantly lower for mice vaccinated with TS-4 strain tachyzoites than mice vaccinated with HBSS or the NC-1 strain tachyzoites in experiment 1. No difference was observed in tissue cyst numbers in mice vaccinated with HBSS or NC-1 strain tachyzoites in experiment 1. No HBSS-vaccinated mice survived experiment 2, and the numbers of T. gondii tissue cysts were significantly lower for mice vaccinated with the TS-4 strain tachyzoites compared to NC-1 strain tachyzoites. No HBSS- or NC-1-vaccinated mice survived RH strain challenge in experiment 3. Results of these experiments indicate that infection with N. caninum provides some protection against fatal oral infection with T gondii oocysts of a moderately pathogenic strain but not tachyzoites of a highly pathogenic strain. The protection provided by N. caninum is much less than that provided by previous exposure to T. gondii, and the numbers of tissue cysts in the brains of mice are not significantly (P > 00.5) lowered.

A new Hepatozoon species from dogs: description of the causative agent of canine hepatozoonosis in North America.

A new species of Adeleina, Hepatozoon americanum, is described from the skeletal muscle, cardiac muscle, visceral organs, and blood of dogs (Canis familiaris) in the Southern United States. The organism was previously identified as Hepatozoon canis (James, 1905) Wenyon, 1926; however, differences in clinical signs, histopathological and serological findings, gamont size, and ultrastructure define the new species of Hepatozoon. Attempts to transmit the protozoan from infected dogs to nymphal Rhipicephalus sanguineus ticks, the definitive host of H. canis, were not successful.

Drinking water contaminants (arsenic, cadmium, lead, benzene, and trichloroethylene). 1. Interaction of contaminants with nutritional status on general performance and immune function in broiler chickens.

The objective of this study was to examine possible interactions between drinking water contaminants and suboptimal nutritional status for performance and immune function in male broiler chickens. Experimental drinking water contained a mixture of arsenic, benzene, cadmium, lead, and trichloroethylene (TCE) at low concentrations (0.80, 1.3, 5.0, 6.7, and 0.65 ppm) and high concentrations (8.6, 13, 50, 67, and 6.5 ppm). These chemicals were selected because they are among the most common contaminants found in ground water near hazardous waste sites. The experimental diets included feed containing 50% added vitamins and minerals (V&M) and feed without added V&M. Increasing levels of drinking water contaminants and decreasing levels of V&M in diet resulted in significantly (P < or = 0.05) decreased water and feed intake, decreased weight gain, and suppression of natural, humoral, and cell-mediated immune response. In a paired-water study, feed consumption, body weight, and immune function were decreased in chickens provided low and high concentrations of the chemical mixture in drinking water compared with chickens given control drinking water equal to the volumes consumed by the chickens given the low and high concentration of mixture, respectively. A deficiency of dietary V&M caused increased sensitivity to adverse effects of drinking water contaminants.

Drinking water contaminants (arsenic, cadmium, lead, benzene, and trichloroethylene). 2. Effects on reproductive performance, egg quality, and embryo toxicity in broiler breeders.

Broiler breeder hens were used to determine the effect of drinking water containing a low concentration of a chemical mixture (arsenic, 0.8 ppm; benzene, 1.3 ppm; cadmium, 5.1 ppm; lead, 6.7 ppm; and trichloroethylene, 0.65 ppm) and a high (10 times greater than the low concentration of the chemical mixture) levels of the chemical mixture. These chemicals are present in ground water near hazardous waste sites. Water consumption significantly decreased in chickens provided the high concentration of the chemical mixture, whereas feed consumption was not affected in any treatment. There was a linear relationship between increasing concentration of the chemical mixture in drinking water and decreasing body weight of hens. The low concentration of the chemical mixture significantly decreased egg production and egg weight, and increased percentage embryonic mortality. These results suggest that reproductive function in hens is sensitive to adverse effects of contaminated drinking water.

Effects of fusarium moniliforme isolates on tissue and serum sphingolipid concentrations in horses.

Disruption in sphingolipid (SL) metabolism is a biomarker of exposure to fumonisins. The role of altered SL metabolism in the pathogenesis of fumonisin toxicoses is not understood. A 27-d feeding trial in horses compared the toxic effects of 3 strains of Fusarium moniliforme: RRC 415, cultured from corn in MS; AU 2/3, cultured from feed associated with clinical signs of duodenitis-proximal jejunitis (DPJ) in horses in AL; and MRC 826, cultured from corn in South Africa and shown to cause equine leukoencephalomalacia (ELEM). These were cultured on corn and diluted with clean corn and grain mixed with molasses (sweet feed) to final concentrations of < 1, 65-130 and 200 mg fumonisin B1 (FB1)/kg, respectively. None of the horses developed DPJ, but horses fed MRC 826 had intestinal lesions consistent with DPJ and horses fed AU 2/3 and MRC 826 developed ELEM. Serum SL concentrations were analyzed in horses fed control and F moniliforme culture material, in brain and/or intestinal tissues in healthy horses (euthanized in AL and IL), and in horses fed AU 2/3 and MRC 826. Serum sphinganine (Sa): sphingosine (Szero) ratios were significantly elevated in horses fed AU 2/3 and MRC 826. Sphinganine concentrations were significantly elevated in the hypothalamus-dentate gyrus and brain stem in horses fed AU 2/3, compared to healthy horses. Sphingosine concentrations were significantly elevated in the proximal duodenum and ileum of MRC 826 horses compared to healthy horses. The brain and intestinal tissues in horses with and without pathological lesions did not have changed Sa:S(zero).

Investigation of regional glutathione levels in a model of chemically-induced renal papillary necrosis.

The effect of diphenylamine on renal cortical, outer medullary and inner medullary glutathione (GSH) concentrations and the effect of GSH depletion on the nephrotoxicity of diphenylamine were investigated in male Syrian hamsters. A dose-dependent decrease in renal cortical GSH was observed within 1 hr of a single oral dose of diphenylamine (200, 400 or 600 mg/kg body weight), but statistically significant changes in outer medullary or papillary GSH were not observed. Reduction of renal papillary GSH to 29% of basal concentration [by prior treatment with L-buthionine sulfoxime (500 mg/kg body weight, ip)] did not increase the papillotoxicity of a non-toxic dose of diphenylamine (400 mg/kg) administered orally. The findings indicate that diphenylamine-induced renal papillary necrosis in the Syrian hamster is not associated with a decrease in renal papillary or outer medullary GSH nor mediated by oxidative cell injury.

Intramedullary cisplatin chemotherapy: experience in four dogs with osteosarcoma.

Single-agent intramedullary cisplatin chemotherapy provided effective localised control in two of four dogs with advanced stage osteosarcoma unable to withstand an amputation or limb-sparing surgery. Complete remission of the local neoplasm was observed in one of the four dogs, partial remission of the local neoplasm in one dog and progressive disease in the other two. Limb function was preserved in one dog for seven months and that dog was found to be tumour-free when euthanased due to unrelated causes. These preliminary results warrant further investigation into the use of intramedullary cisplatin chemotherapy in the localised management of canine appendicular osteosarcoma. It is possible that this mode of treatment may be combined with other treatments to maximise the survival (i.e., systemic control) in other dogs affected with appendicular osteosarcoma.

Early ultrastructural lesions of diphenylamine-induced renal papillary necrosis in Syrian hamsters.

The ultrastructural lesions of diphenylamine-induced renal papillary necrosis in Syrian hamsters were characterized by transmission electron microscopy. Twenty-four male Syrian hamsters were orally administered 600 mg diphenylamine/kg body weight as a single dose. At 30 minutes and at 1, 2, 4, 8, 16 and 24 hours after administration of diphenylamine, three hamsters were anesthetized with pentobarbital, perfused via the left ventricle with half-strength KARNOVSKY's fixative, and the renal papilla and outer medulla collected. Three hamsters administered 0.5 ml peanut oil/kg body weight (vehicle controls) were anesthetized at 24 hours, perfused, and the renal papilla and outer medulla collected. Initial ultrastructural lesions were observed in the endothelial cells of the ascending vasa recta in the proximal portion of the renal papilla at 1 hour after diphenylamine administration. The endothelial cell basal plasma membrane was elevated from the basal lamina, forming large subendothelial vacuoles. Alterations in inner medullary interstitial cells, endothelial cells of the descending vasa recta, and the epithelial cells of the thin limbs of Henle and the medullary collecting tubules were observed subsequent to the lesion in the ascending vasa recta. It was concluded that the endothelial cell of the ascending vasa recta is the target cell in diphenylamine-induced renal papillary necrosis in Syrian hamsters.

Paraneoplastic bullous stomatitis in a horse.

An adult horse with a 2-month history of anorexia, ataxia, and oral blisters had developed these clinical signs just prior to the appearance and growth of a cervical mass. Bullous stomatitis was characterized histologically as subepidermal clefting. Clinical signs were unresponsive to treatment with antibiotics or corticosteroids; however, surgical removal of the mass coincided with remission of all signs. Histologic findings of the mass were consistent with hemangiosarcoma. Results of indirect immunofluorescence and immunoprecipitation on frozen serum from the horse were characteristic of paraneoplastic pemphigus in human beings, a newly recognized mucocutaneous autoimmune disease associated with neoplasia.