Dodd G. Sledge

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.

Immunohistochemical Diagnosis of Canine Oral Amelanotic Melanocytic Neoplasms

Definitive diagnosis of canine oral melanocytic neoplasms is often difficult because of variability in pigmentation and cellular pleomorphism. These neoplasms can resemble carcinomas, sarcomas, and round cell neoplasms, which differ in prognosis and treatment. A variety of immunohistochemical antibodies have been used for diagnosis of melanocytic neoplasms in humans and dogs; however, sensitivity and specificity of many markers have not been determined in amelanotic melanocytic neoplasms in dogs. The authors investigated a comprehensive panel of immunohistochemical markers in 49 canine oral amelanotic melanocytic neoplasms—namely, Melan-A, PNL2, HMB-45, microphthalmia transcription factor (MiTF), S-100, tyrosine hydroxylase, tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2), and CD34. Ten well-differentiated cutaneous soft tissue spindle cell sarcomas were negative controls. Melan-A, PNL2, TRP-1, and TRP-2 were highly sensitive and 100% specific for the diagnosis of canine oral amelanotic melanocytic neoplasms. S-100 and MiTF showed high sensitivity but were less specific; that is, they also labeled a proportion of the soft tissue spindle cell sarcomas. HMB-45, tyrosinase, and tyrosine hydroxylase were 100% specific but had low sensitivities. CD34 did not label any of the melanocytic neoplasms but did label 80% of the soft tissue spindle cell sarcomas. A cost-effective and efficient immunodiagnostic cocktail containing antibodies against PNL2, Melan-A, TRP-1, and TRP-2 was created that had 100% specificity and 93.9% sensitivity in identifying canine oral amelanotic melanocytic neoplasms. The spindloid variant was the variant with the lowest sensitivity to the cocktail. The likelihood of correctly diagnosing canine oral amelanotic melanocytic neoplasms was dramatically higher when biopsy samples contained ample overlying and adjacent epithelium.

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.

Outbreak of Neonatal Diarrhea in Farmed Mink Kits (Mustella vison) Associated With Enterotoxigenic Staphylococcus delphini

An outbreak of diarrhea on a large commercial mink farm affected 5,000 of 36,000 neonatal mink kits, with 2,000 dying within a 2-week period. Affected kits were severely dehydrated, and their furcoats and paws were covered with yellow- to green-tinged mucoid feces. On necropsy, the small intestines of examined animals were markedly distended by serous to mucoid fluid. Microscopically, there was prominent colonization of the intestinal villar epithelium by gram-positive bacterial cocci in the absence of inflammation and morphologic changes in villous enterocytes. The colonizing bacteria were phenotypically identified as belonging to the Staphylococcus intermedius group of bacteria. This was confirmed by nucleic acid sequence analysis of the 16S ribosomal RNA gene. Further nucleic acid sequencing of polymerase chain reaction (PCR) amplicons from the superoxide dismutase gene and the heat shock protein 60 gene differentiated the isolate as Staphylococcus delphini. Production of staphylococcal enterotoxins A and E was demonstrated with a commercial ELISA-based immunoassay. Sequencing of PCR amplicons confirmed the presence of the enterotoxin E gene, but PCR amplification of the enterotoxin A, B, C, or D genes was not successful. Although direct causation was not confirmed in this study, the authors postulate that the observed hypersecretory diarrhea in these mink kits was the result of colonization of the small intestine by S delphini and subsequent production of enterotoxin.

Surgical Amputation of a Digit and Vacuum-Assisted-Closure (V.A.C.) Management in a Case of Osteomyelitis and Wound Care in an Eastern Black Rhinoceros (Diceros bicornis michaeli)

Abstract:  A 14-yr-old female eastern black rhinoceros (Diceros bicornis michaeli) presented with progressive suppurative osteomyelitis in her left hind lateral toe. β-Hemolytic Streptococcus sp. was isolated. The animal was treated with multiple systemic antibiotics, and topical wound cleansing. Repeated debridements and nail trimmings were performed for 5 mo prior to electing amputation. The toe was surgically amputated under general anesthesia between the first and second phalanges. Analgesia was diffused into the wound topically via a catheter and elastomeric pump. The open amputation site was covered with adherent drapes and a negative-pressure wound therapy device provided vacuum-assisted closure (V.A.C.®) for 72 hr. Three months later this animal developed a deep dermal ulcer on the lateral aspect of the right hind limb, at the level of the stifle. Methicillin-resistant Staphylococcus aureus was isolated. The wound was managed by initial daily lavage, followed by 1 mo of V.A.C. therapy, with 72 hr between dressing changes. Clinically, this therapy expedited the formation of healthy granulation tissue and overall healing was accelerated. The animal tolerated the machine and bandage changes well via operant conditioning. The use of negative-pressure wound therapy appeared to shorten time to resolution of slow-healing wounds in black rhinoceros.

Coinfection of a cow with Bovine leukemia virus and Mycobacterium bovis.

Bovine leukosis associated with infection with the delta retrovirus Bovine leukemia virus (BLV) is endemic in many cattle herds in the United States. Infection has been associated with immunosuppression and decreased productivity. Cases of tuberculosis in cows due to infection with Mycobacterium bovis reemerged in Michigan in 1998, and despite intensive eradication attempts, new cases of bovine tuberculosis are sporadically identified. The present report details a coinfection with BLV and M. bovis in a Holstein cow from Michigan that presented as part of a bovine tuberculosis screening program. Peripheral and visceral lymph nodes of this animal were markedly enlarged, homogeneously pale white, and bulged on the cut surface. The submandibular, mesenteric, and caudal mediastinal lymph nodes contained multifocal to coalescing caseogranulomas that ranged from 1 to 5 cm in diameter. Histologically, dense sheets of monomorphic populations of neoplastic lymphocytes obliterated the normal architecture of all lymph nodes. Caseogranulomas were characterized by central pools of amorphous degenerate eosinophilic and occasionally mineralized granular debris surrounded by thick rims of epithelioid macrophages, occasional Langhans type giant cells, and fibrosis. Polymerase chain reaction assay was positive for BLV. Cultures of affected lymph nodes yielded growth of M. bovis.

Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection.

In view of the varied biologic behavior and the costs of treatment for canine cutaneous mast cell tumors, development of appropriate treatment plans for individual affected dogs can be difficult, but decisions regarding treatment should be made using a systematic, evidence-based approach. This manuscript reviews the current state of diagnostics and prognostication of canine cutaneous mast cell tumors, and suggests a combined approach based on clinical and pathologic assessment for decision making regarding treatment choices. The current state of histologic grading, evaluation of proliferation indices, evaluation of mutations in the c-kit gene and KIT expression, evaluation of excision and clinical staging are examined. On the basis of the current understanding of prognostication and treatment response, algorithms for selection of local and systemic therapy are presented.

Outbreaks of severe enteric disease associated with Eimeria furonis infection in ferrets (Mustela putorius furo) of 3 densely populated groups

CASE DESCRIPTION 3 unrelated, densely populated, dynamic ferret populations with severe outbreaks of enteric coccidiosis were evaluated. CLINICAL FINDINGS In each outbreak, morbidity rate was high, there were an appreciable number of deaths, and ferrets of all ages were affected. Affected individuals had acute onset of diarrhea, and feces often contained frank or digested blood. Other clinical signs included dehydration, weakness, lethargy, and weight loss. Fecal examinations of affected ferrets revealed sporadic and inconsistent shedding of coccidial oocysts. Necropsy findings included moderate to marked atrophic enteritis associated with numerous intraepithelial and fewer extracellular coccidial life stages. Sporulated oocysts isolated from feces were consistent with Eimeria furonis. A PCR assay was performed on formalin-fixed, paraffin-embedded sections of intestine for the gene encoding the small subunit of rRNA yielded products with sequences identical to those described for E furonis. TREATMENT AND OUTCOME Supportive care and treatment with sulfadimethoxine over the course of these outbreaks was palliative, but long-term treatment was required and failed to completely eradicate infection as identified by the subsequent finding of oocysts in fecal samples. CLINICAL RELEVANCE Enteric coccidiosis due to infection with E furonis has typically been reported to be subclinical rather than to cause severe gastrointestinal disease in ferrets. This report indicated that infection with E furonis may have contributed to severe enteric disease with high morbidity and mortality rates in 3 densely populated, dynamic groups of ferrets. Furthermore, long-term treatment with anti-coccidials may be required in outbreak situations, but may be ineffectual in completely eradicating infection.

Differences in expression of uroplakin III, cytokeratin 7, and cyclooxygenase-2 in canine proliferative urothelial lesions of the urinary bladder.

The expression of immunohistochemical markers that have been used in diagnosis and/or prognostication of urothelial tumors in humans (uroplakin III [UPIII], cytokeratin 7 [CK7], cyclooxygenase-2 [COX-2], and activated caspase 3) was evaluated in a series of 99 canine proliferative urothelial lesions of the urinary bladder and compared to the lesion classification and grade as defined by the World Health Organization / International Society of Urologic Pathology consensus system. There were significant associations between tumor classification and overall UPIII pattern (P = 1.49 × 10–18), loss of UPIII (P = 1.27 × 10–4), overall CK7 pattern (P = 4.34 × 10–18), and COX-2 pattern (P = 8.12 × 10–25). In addition, there were significant associations between depth of neoplastic cell infiltration into the urinary bladder wall and overall UPIII pattern (P = 1.54 × 10–14), loss of UPIII (P = 2.07 × 10–4), overall CK7 pattern (P = 1.17 × 10–13), loss of CK7 expression (P = .0485), and COX-2 pattern (P = 8.23 × 10–21). There were no significant associations between tumor classification or infiltration and caspase 3 expression pattern.

Regulatory T cells and immune profiling in johne’s disease lesions

Johnes disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease of ruminants. Hallmark symptoms of clinical Johnes disease include diarrhea, progressive weight loss, and premature death; symptoms due largely to chronic inflammation in the small intestine. MAP colonizes resident macrophages within the ileum of the small intestine, subsequently establishing a persistent infection in the host. It has been proposed that regulatory T cells may play a role in the progression of Johnes disease, either through promotion of tolerance to MAP or via a loss in homeostasis that subsequently allows widespread inflammation. In this report, we evaluated the presence of Tregs, as well as other immune parameters, in the ileum and draining lymph nodes of MAP associated lesions. A lesion classification scheme was developed to categorize severity of MAP-induced lesions within infected tissues and subsequently regulatory T cell presence and overall immune activity were assessed corresponding to lesions of varying severity, in comparison to tissues from healthy control animals. Our results revealed a relationship between animal health and overall lesion severity within the infected tissues, as well as a relationship between bacterial burden and severity of pathology. Regulatory T cell abundance was shown to decrease with increasing lesion severity. Within the ileum, the expression of many Th1, Th2, and Treg-associated genes increased in mild lesions and decreased in severe lesions, whereas in the lymph nodes the expression of these genes tended to increase with increasing lesion severity. Based on our results, we conclude that a local loss of T cell (including Treg) activity occurs within severe ileal lesions associated with MAP, resulting in a loss of homeostasis that ultimately leads to the progression of clinical Johnes disease.