Canine cutaneous histiocytomas are clonal lesions as defined by X-linked clonality testing.

Canine cutaneous histiocytoma (CCH) is the most common skin tumour of young dogs. CCH is assumed to be a tumour, but as these lesions often undergo complete regression, they have also been proposed to represent hamartomas or unregulated hyperplasia of polyclonal histiocytic cells. To demonstrate a monoclonal origin of CCH, and thereby a probable neoplastic nature, a clonality assay was established based on the heterozygosity of a polymorphic microsatellite within the canine androgen receptor located on the X chromosome. Analysis of 11 histiocytomas identified a non-random X chromosome inactivation of one androgen receptor allele. CCH may, therefore, be a clonal lesion and of neoplastic nature.

Weigners fixative-an alternative to formalin fixation for histology with improved preservation of nucleic acids.

Formalin fixation and paraffin embedding (FFPE) is the standard method for tissue storage in histopathology. However, FFPE has disadvantages in terms of user health, environment, and nucleic acid integrity. Weigners fixative has been suggested as an alternative for embalming cadavers in human and veterinary anatomy. The present study tested the applicability of Weigners for histology and immunohistochemistry and the preservation of nucleic acids. To this end, a set of organs was fixed for 2 days and up to 6 months in Weigners (WFPE) or formalin. WFPE tissues from the skin, brain, lymphatic tissues, liver, and muscle had good morphologic preservation, comparable to formalin fixation. The quality of kidney and lung samples was inferior to FFPE material due to less accentuated nuclear staining and retention of proteinaceous interstitial fluids. Azan, Turnbull blue, toluidin, and immunohistochemical stainings for CD79a, cytokeratin, vimentin, and von Willebrand factor led to comparable results with both fixates. Of note, immunohistochemical detection of CD3 was possible after 6 months in WFPE but not in FFPE tissues. mRNA, miRNA, and DNA from WFPE tissues had superior quality and allowed for amplification of miRNA, 400-bp-long mRNA, and 1000-bp-long DNA fragments after 6 months of fixation in WFPE. In summary, Weigners fixative is a nonhazardous alternative to formalin, which provides a good morphologic preservation of most organs, a similar sensitivity for protein detection, and a superior preservation of nucleic acids. Weigners may therefore be a promising alternative to cryopreservation and may be embraced by people affected by formalin allergies.

Iron storage disease in red deer (Cervus elaphus elaphus) is not associated with mutations in the HFE gene.

Iron storage diseases are rare conditions of dysregulated iron metabolism in man and animals. A genetic basis has been confirmed only for human haemochromatosis. Iron storage disease was diagnosed in six related, 2-year-old male red deer of the same herd. These animals presented with weight loss and rough hair coats. Haematological examination was unremarkable. At necropsy examination, gross lesions were restricted to cachexia. Microscopical examination revealed severe, diffuse hepatocellular necrosis and iron accumulation in hepatocytes, Kupffer cells, cardiac myocytes and renal tubular cells in all affected animals. Four animals also had moderate bridging fibrosis in the liver. Hepatic iron concentrations were increased (1108-2275 mg/kg wet weight; reference range 100-200 mg/kg). Drinking water in rusty iron tubs in the deer park contained eight times more iron than the accepted level for human drinking water. To test for a possible genetic basis of increased iron uptake and storage in red deer, the cervid haemochromatosis gene (HFE) was identified. Sequence comparisons between the six diseased animals and three healthy free-ranging unrelated animals failed to identify differences in the HFE sequences. Furthermore, the disease was not associated with common amino acid substitutions reported in human patients with haemochromatosis, including C282Y and H63D. Polymorphisms in other non-HFE genes involved in iron metabolism may have led to a higher sensitivity to iron and this, together with the high iron content of the drinking water, may have been the cause of the observed iron storage in these red deer.

T-cell receptor γ chain variable and joining region genes of subgroup 1 are clonally rearranged in feline B- and T-cell lymphoma.

Lymphoma is one of the most common tumours of domestic cats. Microscopically, it can be challenging to distinguish lymphoma (monoclonal lymphoid proliferation) from reactive lymphoid hyperplasia (polyclonal lymphoid proliferation). Molecular methods that characterize cellular clonality can overcome this diagnostic challenge; however, it is essential to know which variable and joining region variants are utilized by neoplastic lymphocytes before a sensitive and specific assay can be developed. The present study describes a polymerase chain reaction assay that allows for complete sequencing of clonally recombined T-cell receptor (TCR) γ chain genes from formalin-fixed and paraffin wax-embedded samples of feline lymphoma. The variable (V) and joining (J) region variants of the TCR γ chain were characterized in 50 feline lymphomas. Amplification and sequencing with primers directed against conserved framework regions 1, 2 and 4 of the TCR γ chain identified clonal rearrangement in 68% of T-cell lymphomas and 22% of B-cell lymphomas. The distribution of TCR variants present in B- and T-cell lymphomas was similar and included V region variants 1 and 3, and J region variants 1.2, 1.3 and 1.5. V region variants 2 and 4 were not identified in either tumour type. Some feline B-cell lymphomas had a clonally rearranged TCR, a finding reported in human, but not canine, B-cell lymphoma.

Leukoencephalomalacia and laminar neuronal necrosis following smoke inhalation in a dog.

Acute respiratory and neurologic disease after smoke inhalation are well documented, but human patients may also develop delayed-onset neurologic symptoms associated with leukoencephalomalacia after exposure to smoke or carbon monoxide. In this case, a dog developed progressive neurologic signs 6 days after rescue from an apartment fire. At necropsy 9 days after smoke inhalation, leukoencephalomalacia of the central cerebral white matter was accompanied by laminar necrosis of cerebrocortical neurons. This is the first report of delayed posthypoxic leukoencephalopathy in a nonhuman animal.

Efficient and cost-effective extraction of genomic DNA from formalin-fixed and paraffin-embedded tissues.

Diagnostic and investigative molecular pathology frequently has to resort to extraction of DNA from formalin-fixed and paraffin-embedded tissue samples. Although many different protocols are reported for this type of material, extraction of sufficient amounts of intact DNA is still challenging. Here, the authors report a reproducible, simple, cost-effective, and efficient protocol that yields up to 140 µg of DNA from approximately 10 to 15 mg of formalin-fixed and paraffin-embedded tissue samples and compare it to available protocols. The protocol allows stable amplification of DNA fragments up to 600 bp in length in a wide variety of tissues.

Canine alveolar echinococcosis: morphology and inflammatory response.

Echinococcus multilocularis is a tapeworm with zoonotic potential that is common in the northern hemisphere. Its natural definitive hosts are foxes and other canids and its intermediate hosts are rodents. Other mammalian species, including man, can become infected and develop infiltrative, tumour-like lesions that particularly affect the liver. The present study characterizes the relatively rare lesions of alveolar echinococcosis in 10 dogs. Metacestodes in these dogs frequently contained no protoscolices and were thus sterile. Metacestodes were surrounded by fibrous tissue intermixed with inflammatory cells comprising variable numbers of macrophages, T and B lymphocytes, and plasma cells, as well as neutrophils and/or eosinophils. These lesions resemble those observed in man and are in contrast to those observed in the natural intermediate hosts, which have less fibrosis and more protoscolices.

Characterization of C-, J- and V-region-genes of the feline T-cell receptor gamma.

Lymphomas and leukemias are important neoplasias of domestic cats and human beings. In some cases it can be difficult to differentiate these tumors from reactive lymphatic hyperplasia. To overcome this problem, the diagnosis of lymphomas and leukemias in man is often supported by molecular techniques. To be able to establish such a technique in the cat we had to sequence the genes coding for the antigen receptors. As primary target in this study we choose the T-cell receptor gamma. Using 5'-and 3'-RACE techniques we were able to clone and sequence four different V-region genes, which can be clustered into two subgroups as well as six variants of the C-region gene. Additionally, we found eight J-region genes which can be classified into three subgroups. One of the V-region genes, six of the J-region genes and all C-region genes had not been described previously. All together we analysed 112 clones containing V- and J-region genes and 31 clones containing C-region genes. Sixty-six of these clones were full length containing the L-region as well as the 5'-UTR of the feline T-cell receptor gamma. The sequences of the V-region- and J-region-genes show sufficiently homologous areas that can be used to establish a small number of consensus-primers to be applied in molecular diagnosis of feline lymphomas and leukemias.