Andrea Fuchs-Baumgartinger

Detection of Encephalitozoon cuniculi in the feline cataractous lens

PURPOSE Identification of Encephalitozoon cuniculi (E. cuniculi) as a possible causative agent for cataracts and uveitis in cats. METHODS Within a 12-month study period, cats that were presented with focal anterior cortical or mature cataract and secondary uveitis underwent a complete ophthalmic examination, complete blood count, serum biochemistry, serologic tests for E. cuniculi and tests for feline immunodeficiency virus (FIV), feline infectious peritonitis (FIP), feline leukemia virus (FeLV) and Toxoplasma gondii (T. gondii). PCR for DNA detection of E. cuniculi and T. gondii as well as cytologic examination of aqueous humor after paracentesis and phacoemulsified lens material were also performed. In addition histopathologic examination of the resected anterior lens capsule and attached lens epithelial cells was performed. Serologic testing for antibodies against E. cuniculi was also performed in 100 ophthalmologically healthy cats. RESULTS Eleven (19 eyes) European shorthair cats with a median age of 3.5 years were included. Nine of 11 cats had bilateral cataracts, with 12/19 eyes having focal anterior cortical cataracts and 7/19 eyes having mature cataracts. In 14/19 eyes anterior uveitis was present. All cats had a positive antibody titer (1:80-1:10,000) for E. cuniculi. Encephalitozoon cuniculi DNA was detected by PCR and sequencing in 18/19 lenses and in 10/19 aqueous samples. Five tentative positive results were detected by cytologic examination. Spores were detected in 15/19 samples of lens material with histopathologic staining. Only 2/100 ophthalmologically healthy cats showed a positive antibody titer for E. cuniculi. CONCLUSION Encephalitozoon cuniculi is a cause of focal anterior cortical cataract and anterior uveitis in cats.

Investigations on the conjunctival goblet cells and on the characteristics of glands associated with the eye in the guinea pig.

OBJECTIVE To investigate the distribution and density of conjunctival goblet cells (GC) and to study the anatomy and microscopic characteristics of glands associated with the eye in the guinea pig. PROCEDURES Twenty-five guinea pigs were used. Meibomian gland openings were counted using biomicroscopy. Conjunctiva, eyelids and glands were embedded in glycol methacrylate and paraffin. Sections were stained with hematoxylin and eosin (H&E), periodic acid Schiffs reaction (PAS) and Alcian blue (AB). RESULTS Highest GC densities were found in the bulbar and palpebral region of the nasal conjunctiva (GC index: 13.7-16.4%). Lowest GC densities (GC index: 0.0-1.0%) were found in 3/4 limbal regions (nasal and temporal upper eyelid, temporal lower eyelid). Guinea pigs have 27.1±3.0 (mean±SD) meibomian gland openings in the upper lid and 25.7±2.3 in the lower lid. Difference between upper and lower lid was significant (P=0.037). Two subconjunctival sebaceous glands occur temporal to each eye. The Harderian gland is very large. In the lacrimal gland three different cell types were distinguished both according to the cell structure and histochemical staining. CONCLUSIONS Goblet cell densities are lower in guinea pigs than in dogs and horses. Positive staining with PAS and AB could be an indication that mucins are produced in the lacrimal gland. If so, they may contribute to the mucin layer of the tear film. Both the extraordinarily large Harderian gland and the subconjunctival sebaceous glands produce lipids and may contribute to the lipid layer of the tear film.

Angiogenic markers in canine lymphoma tissues do not predict survival times in chemotherapy treated dogs

Angiogenesis, which is essential for malignancies to progress, depends on various signalling proteins including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2). Microvessel density (MVD) is frequently used to evaluate angiogenesis. This study assessed the relationship between expression of VEGF, VEGFR-1 and VEGFR-2, MVD and the survival time in dogs with lymphoma. VEGF, VEGFR-1 and VEGFR-2 expression was evaluated immunohistochemically and microvessel profiles were counted in 34 lymphoma samples. Seventy-nine percent of the samples showed high VEGF expression and 62% were highly positive for VEGFR-1; VEGFR-2 immunoreactivity was mostly negative. Dogs treated with chemotherapy had a median survival time of 266days, but no significant relationships were found between overall survival time, MVD and expression of VEGF, VEGFR-1 or VEGFR-2. In this study, VEGF its receptors and the MVD were no prognostic factors in dogs with lymphoma.

Authentication of Primordial Characteristics of the CLBL-1 Cell Line Prove the Integrity of a Canine B-Cell Lymphoma in a Murine In Vivo Model

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkins Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2−/−γc −/− mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45+, MHCII+, CD11a+ and CD79αcy+. PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.

Thymomas in Rabbits: Clinical Evaluation, Diagnosis, and Treatment

Thymomas are rarely recorded in rabbits, and the literature includes comparatively few cases. Medical records were reviewed to identify all pet rabbits in which a mediastinal mass was diagnosed between Feb 2007 and Jan 2010. Signalment, history, clinical signs, diagnostic work-up (including laboratory data, diagnostic imaging, and ultrasound-guided fine-needle aspiration of the mediastinal mass), treatment modalities, survival time, and histologic findings were evaluated. Cytologic and/or histopathologic examinations revealed thymomas in all rabbits with mediastinal masses (n=13). Rabbits with thymomas showed clinical signs of dyspnea (76.9%), exercise intolerance (53.9%), and bilateral exophthalmos (46.2%). In seven rabbits the thymoma was removed surgically. Two rabbits were treated conservatively, and four rabbits were euthanized because of their poor clinical condition. The two rabbits that underwent surgery were euthanized 6 mo and 34 mo later. Mediastinal masses in rabbits appear to be more common than previously believed and consist primarily of thymomas rather than thymic lymphomas. Cytology of samples collected by ultrasound-guided fine-needle aspiration is an accurate diagnostic tool for the identification of thymomas in rabbits. Due to a high rate of perioperative mortality, intensive perioperative care and the provision of a low-stress environment are recommended for a successful thoracotomy.

Uterine Disorders in 50 Pet Rabbits

Although the incidence of uterine disorders in pet rabbits is high there are only a few retrospective studies and case reports on genital tract disease in female rabbits. Uterine disorders were assessed in 50 pet rabbits. In 31 pet rabbits with suspected clinical uterine disease, medical records were further reviewed regarding clinical signs, diagnostic workup, treatment as well as the outcome itself. Uterine adenocarcinoma (54%) was most frequently diagnosed, followed by endometrial hyperplasia (26%). Serosanguineous vaginal discharge was the predominant clinical sign observed by the rabbit owners. In approximately 50% of the rabbits with suspected uterine disorders, abdominal palpation revealed enlarged and/or irregular masses in the caudoventral abdomen indicating uterine lesions. Out of 23 rabbits undergoing ovariohysterectomy, four were either euthanized or died shortly after surgery because they were clinically unstable. Overall, 80% of the ovariohysterectomized animals were still alive 6 mo after surgery. In female pet rabbits that are not breeding, either ovariohysterectomy should be performed at an early age or routine checks including ultrasonography of the abdomen are recommended on a regular basis.

Sudden death in a dog with lymphoplasmacytic hypophysitis.

A 10-year-old dog with a history of progressive anorexia and weight loss died suddenly despite treatment. Histopathological examination revealed severe follicular lymphoplasmacytic adenohypophysitis and atrophy of the zona fasciculata and zona reticularis of the adrenal cortex. It is likely that lack of production of adrenocorticotropic hormone and cortisol was the cause of death of this dog.

Investigations on the conjunctival goblet cells and the characteristics of the glands associated with the eye in chinchillas (Chinchilla Laniger)

OBJECTIVE To investigate the density and distribution of conjunctival goblet cells (GC) and study the anatomy and microscopic characteristics of glands associated with the eye in chinchillas (Chinchilla Laniger). PROCEDURE 12 chinchillas were included in the study. Conjunctiva (divided into four regions), eyelids, and glands were embedded in paraffin wax, sectioned, stained, and analyzed. RESULTS Highest GC densities were found in the palpebral region of the nasal and temporal conjunctiva of both eyelids (GC index: 25.1-18.2%), and lowest densities, in the bulbar and marginal region of the nasal and temporal conjunctiva of both eyelids (GC index: 1.5-0.0%). Meibomian glands extend along the entire length of both eyelids, and the whole glandular complex broadens toward the temporal canthus. This is macroscopically visible through the conjunctiva. The openings of the Meibomian glands are macroscopically not discernible. The light pink, smooth, and crescent-shaped lacrimal gland lies next to the aforementioned broadened part of the Meibomian glands in the temporal canthus. The whitish, 0.9-cm-long, smooth Harderian gland is firmly attached to the posterior part of the globe and extends nasally from the optic nerve to the equator. Furthermore, chinchillas possess two lacrimal puncta, situated on the inner conjunctival surface of both eyelids near the medial canthus. A pigmented lacrimal canaliculus originates from each punctum. The vestigial nictitating membrane is supported by a hyaline cartilage and is pigmented at its free margin. CONCLUSIONS Chinchillas possess a Harderian gland, a lacrimal gland, and Meibomian glands. The GC density in the nasal and temporal palpebral conjunctiva is higher than in guinea pigs.

Characterization of the T-cell receptor gamma chain gene rearrangements as an adjunct tool in the diagnosis of T-cell lymphomas in the gastrointestinal tract of cats.

Feline alimentary lymphoma is the most common hematopoietic neoplasia in cats. It affects mainly the small intestines and is most frequently of T-cell origin. Evaluation of a fine needle aspirate is often the first step in the diagnostic work-up. Differentiation between a resident mature lymphocyte population as encountered in inflammatory bowel disease and small cell lymphoma cannot be achieved by cytology alone. Even full thickness biopsies evaluated by histopathology can be inconclusive. These cases warrant the application of complementary tools like PCR-based T-cell receptor (TCR) clonality testing for confirmation. The aim of this study was to optimize the DNA extraction protocol for formalin fixed and paraffin embedded tissues (FFPE) and to establish a heteroduplex analysis to enhance resolution of the PCR fragments of the T-cell receptor gamma (TCRG) V-J gene. The new protocols resulted in improved quantity and quality of the extracted DNA. Heteroduplex analysis of the samples improved the resolution of the electrophoresis results so that rules for interpretation of the different patterns could be established. Application of this improved setup detected clonal rearrangements in at least one TCRG primer reaction in 31 of 36 of our feline intestinal lymphoma samples after DNA quality testing.

Characterization of a PCR-based lymphocyte clonality assay as a complementary tool for the diagnosis of feline lymphoma.

Differentiation between resident mature lymphocyte populations and small cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. In this prospective study, diagnostic sensitivity and specificity of different primer sets for routine diagnosis of feline TCR gamma (TCRG) and complete IG heavy chain (IGH) gene rearrangements were assessed. Fine needle aspirates from 20 feline lymphoma cases and lymph node material from 10 cats without hematopoietic neoplasia were subjected to clonality testing. Feline lymphoma cell lines and previously confirmed patient material served as positive control. Detection limits for clonal populations within a polyclonal background was 90% for B-cells and 50% for T-cells. Diagnostic sensitivity and specificity of the clonality assay were 70% and 90%. Overall diagnostic accuracy was 77%, positive predictive value 93% and negative predictive value 60%.