Fatal Meningitis in Swine after Intrathecal Administration of Adeno-associated Virus Expressing Syngeneic Interleukin-10.

Interleukin-10 (IL-10) delivered by intrathecal (i.t.) gene vectors is a candidate investigational new drug (IND) for several chronic neurological disorders such as neuropathic pain. We performed a preclinical safety study of IL-10. A syngeneic large animal model was used delivering porcine IL-10 (pIL-10) to the i.t. space in swine by adeno-associated virus serotype 8 (AAV8), a gene vector that was previously found to be nontoxic in the i.t. space. Unexpectedly, animals became ill, developing ataxia, seizures, and an inability to feed and drink, and required euthanasia. Necropsy demonstrated lymphocytic meningitis without evidence of infection in the presence of normal laboratory findings for body fluids and normal histopathology of peripheral organs. Results were replicated in a second animal cohort by a team of independent experimenters. An extensive infectious disease and neuropathology workup consisting of comprehensive testing of tissues and body fluids in a specialized research veterinary pathology environment did not identify a pathogen. These observations raise the concern that i.t. IL-10 therapy may not be benign, that previously used xenogeneic models testing the human homolog of IL-10 may not have been sensitive enough to detect toxicity, and that additional preclinical studies may be needed before clinical testing of IL-10 can be considered.

Comprehensive Colony Health Management and Emerging Pathogens of the Annual Killifish Species Nothobranchius furzeri.

The Leibniz Institute on Aging has maintained killifish colonies for over 15 y. Our veterinarians, scientists, and animal technicians developed a fish health scoring system and routine colony health surveillance program for our colonies. Over a 4-y period, health data from the African turquoise killifish Nothobranchius furzeri colony were systematically collected and analyzed. The fish health assessment system facilitated categorization of clinical signs and differentiation of fish with mild clinical signs from fish that required euthanasia. This report provides new information on clinical signs and conditions that may occur in young and aged N. furzeri. To be comprehensive, a colony health surveillance program incorporates animal health at both the individual and the population levels. The quarterly routine health monitoring program identified Mycobacterium spp. as the most common agent in our facility and identified the killifish pathogen (Loma acerinae) for the first time. Taken together, these findings demonstrate the importance of a comprehensive colony health management system in a fish research facility. By improving the health and welfare of fish used for research, the scientific community will benefit from less variable and more reliably reproducible research results.

Serial evaluation of segmental esophageal reconstruction using a polyurethane scaffold in a pig model.

Background: Many esophageal pathologies are clinically treated by resection and reconstruction of the esophagus. Surgical esophagectomy remains a morbid procedure and despite minimally invasive advances, has changed little in decades. Novel approaches to esophageal segmental resection and reconstruction are an unmet need. Methods: Circumferential thoracic esophageal transection was performed in both male and female pigs and the defects reconstructed using 5 or 10 cm polyurethane (PU) tubular grafts and stented. A subset were treated with stent only. Animals were survived to 14, 30, 60, and 399 days. Tissues were evaluated histologically, and via non-invasive serial endoscopy and contrast swallowing studies in long-term animals. Results: Luminal patency was achieved in all animals with no clinical evidence of leak. In short-term animals, there was healing noted in all cases with a variably sized region of ulceration remaining at the most central part of the repaired tube (between the proximal and distal anastomosis). In four long-term animals following stent removal, two resumed normal diet and thrived, while two animals were euthanized prior to the proposed endpoint because of stricture formation and inability to tolerate a normal diet. Re-epithelialization was observed in all groups, and more complete over time. Conclusions: The PU scaffold provides a matrix across which formation of new tissue can occur. The mechanisms through which this happens remain unclear, but likely a combination of fibrosis and tissue contraction, in conjunction with new tissue formation.

Evaluation of plasma antithrombin activity and D-dimer concentration in populations of healthy cats, clinically ill cats, and cats with cardiomyopathy.

BACKGROUND: Current coagulation tests lack sensitivity and detect disseminated intravascular coagulation (DIC) only when it is severe. Measurement of antithrombin (AT) activity and D-dimer concentration permits early diagnosis and more precise classification of coagulopathies in some species. OBJECTIVES: The objectives of this study were to validate and determine the diagnostic utility of a chromogenic AT assay and an immunoturbidimetric D-dimer assay for the diagnosis of DIC in cats. METHODS: Citrated plasma samples were collected from 30 healthy cats, 30 ill cats, and 13 cats with cardiomyopathy. Partial thromboplastin time, prothrombin time, fibrin(ogen) degradation products, platelet concentration, and erythrocyte morphology were determined on all samples to document the presence or the absence of DIC. AT activity and D-dimer concentration were then measured. RESULTS: The chromogenic AT assay was linear and precise. Mean AT activity was higher in ill cats and cats with cardiomyopathy compared with healthy cats, but the difference was only significant in ill cats (P = .003). Seven cats met the criteria for DIC. Of the cats with DIC, 2 had decreased AT activity, 1 had increased AT activity, and 4 had AT activities within normal limits. The immunoturbidimetric D-dimer assay did not appear to accurately measure feline D-dimer. CONCLUSIONS: The chromogenic AT assay appeared to measure AT in cats but was not useful for the diagnosis of DIC. AT may be an acute phase reactant in cats. The immunoturbidimetric D-dimer assay was not useful for the diagnosis of DIC in cats.

Detection of activated platelets in dogs with primary immune-mediated hemolytic anemia.

Thromboembolism is a major cause of morbidity and mortality in dogs with immune-mediated hemolytic anemia (IMHA). To the authors' knowledge, the role of platelets in thromboembolic events associated with IMHA has not been extensively investigated. In the study reported here, we evaluated cell membrane expression of P-selectin with flow cytometry to determine whether platelets circulate in an activated state in association with primary IMHA. Median P-selectin expression for 20 dogs with primary IMHA was 8.1-fold greater, compared with values for 20 healthy dogs. Fifteen of 20 dogs (75%) with IMHA had P-selectin median fluorescence intensity (MFI) values that exceeded the reference interval for healthy dogs. Additionally, P-selectin MFI after activation of platelets with phorbol myristate acetate was 2.1-fold greater for dogs with IMHA than for healthy control dogs. Despite treatment of all dogs with immunosuppressive therapy and 18 dogs with subcutaneously administered low-dose unfractionated heparin, 7 dogs developed clinical signs consistent with thromboembolism. These data provide support for the hypothesis that platelets circulate in an activated state in many dogs with IMHA.

A retrospective study of aplastic pancytopenia in the dog: 9 cases (1996-2003).

BACKGROUND: Aplastic pancytopenia is defined by the presence of pancytopenia in blood and a hypocellular bone marrow with the hematopoietic space replaced by adipose tissue. Several causes of acquired aplastic pancytopenia are known; however, in some cases, an underlying cause is never determined. OBJECTIVE: The objective of this retrospective study was to identify the incidence, potential causes, and outcome of aplastic pancytopenia in dogs. METHODS: Bone marrow cytologic and core biopsy reports were reviewed to identify dogs diagnosed with aplastic pancytopenia between July 1, 1996 and June 30, 2003. Four-hundred eighty-six bone marrow reports that included aspirate and core biopsy evaluations were reviewed. Signalment, treatment given, previous and current disease conditions, clinical signs of disease, clinical laboratory data, therapy, response to therapy, and survival time were recorded. RESULTS: Nine dogs (1.85% of bone marrow samples reviewed) met the criteria for inclusion. Two dogs (22%) had associated diseases that included monocytic ehrlichiosis and Sertoli cell tumor. In 7 dogs (78%), the cause of aplastic pancytopenia could not be definitively determined, although an idiosyncratic drug reaction to griseofulvin was suspected in 1 of the dogs. The median age of dogs diagnosed with aplastic pancytopenia was 3.2 years, and apparent breed or sex predilection was not identified. Median HCT, total WBC count, and platelet count on the day of presentation were 21.8%, 1.0 x 10(3)/microL, and 2.0 x 10(3)/microL, respectively. Six of 9 dogs diagnosed with aplastic pancytopenia died or were euthanized within 21 days. Two dogs had complete hematologic recovery. One dog was living 3 years after diagnosis, but hematologic recovery was never documented. CONCLUSIONS: Aplastic pancytopenia is diagnosed infrequently and idiopathic aplastic pancytopenia may account for up to 67% or more of canine cases. Although the prognosis is guarded, some dogs with aplastic pancytopenia recover.

Intra-abdominal mass aspirate from an alopecic dog.

An 11-year-old female Yorkshire terrier was presented to the University of Minnesota Veterinary Medical Center for evaluation of a palpable intra-abdominal mass and alopecia. Abdominal ultrasonography revealed a large, complex, cavitary mass in the left caudal region of the abdomen. A fine needle aspirate of the mass was collected. A population of markedly pleomorphic, large, round to polygonal cells were found singly and in small noncohesive aggregates. The cells contained scant, clear to blue-gray cytoplasm, large, round to oval nuclei, and distinctly stippled to reticular chromatin. Cytologic findings were consistent with a tumor of ovarian origin, with a primary differential diagnosis of germ cell tumor. Hormonal analysis of serum revealed a marked increase in 14-OH-progesterone concentration (2.71 ng/mL, reference interval 0.05-0.69 ng/mL). Ovariohysterectomy was performed, and the mass was found to be in the area of the left ovary. Histologic evaluation of the reproductive tract confirmed a diagnosis of left ovarian dysgerminoma. Based on immunohistochemical stains, the tumor was negative for c-kit (CD117c) and single cells were positive for neuron-specific enolase. A right ovarian cyst and squamous metaplasia of the right uterine horn also were diagnosed. The cyst was presumed to be the source of 14-OH-progesterone, which likely resulted in the squamous metaplasia and dermatopathy. Three months after surgery, the progesterone concentration had returned to normal and the alopecia had nearly resolved. Dysgerminomas in dogs are reported rarely, but have a distinctive, recognizable, cytologic appearance and should be included in the differential diagnosis of an intra-abdominal mass in a reproductively intact female dog.

Bold fusion: transtracheal wash from a Jersey calf.

A 10-week-old, female, Jersey calf was referred to the University of Minnesota Veterinary Medical Center for evaluation of difficult breathing and inappetence of 18-hours duration. Cytologic examination of a transtracheal wash specimen revealed pyogranulomatous inflammation, with increased numbers of multinucleated cells. Differential diagnoses included established bacterial infection, viral infection, fungal infection, and foreign body reaction. Immunocytochemical staining was done on a direct smear of the transtracheal wash using a cocktail of 4 mouse monoclonal antibodies against 3 human respiratory syncytial virus proteins. Strong intracytoplasmic staining within mononuclear cells was observed and a diagnosis of bovine respiratory syncytial virus infection was made. Immunocytochemistry may be used as a rapid, inexpensive, adjunctive diagnostic tool for the antemortem diagnosis of bovine respiratory syncytial virus on transtracheal wash specimens.

Platelet dysfunction after association with Anaplasma phagocytophilum in vitro.

Granulocytic anaplasmosis is a tick-borne, zoonotic disease caused by an obligate intracellular granulocytotropic bacterium, Anaplasma phagocytophilum. A. phagocytophilum and other Anaplasma and Ehrlichia spp. have been variably implicated with an associated hematologic alteration, platelet dysfunction, that can result in potentially serious clinical sequelae including hemorrhage. The objective of this work was to define an in vitro model to assess alterations in platelet function associated with A. phagocytophilum.