Evidence of cell cycle re-entry in post-mitotic, terminally differentiated feline neurons.

Parvovirus infections in dogs and cats are restricted to highly mitotically active tissues, predominantly to the epithelium of the gastrointestinal tract and, in cases of prenatal infections in cats, also to Purkinje cell neuroblasts. The evidence of parvovirus-infected mature feline neurons gave rise to reconsider the dogma of post-mitotically fixed and terminally differentiated neurons in the adult central nervous system. To elucidate the postulated capability of certain terminally differentiated feline neurons to re-enter the cell cycle, immunohistochemical double labeling using the transcription factor Sox2 and the tumor suppressor and cell cycle regulator retinoblastoma protein in its phosphorylated state (pRb) was performed. Formalin-fixed and paraffin-embedded brain tissue negative for parvovirus-antigen from 14 cats was compared to brain tissue from 13 cats with immunohistochemically confirmed cerebral parvovirus infection; the 27 cats were aged between 50 days of gestation (E50) and 5 years. Both groups revealed nuclear Sox2 and pRb immunosignals in numerous neurons, suggesting a more active state than mature neurons should have. Accordingly, parvovirus is not exclusively involved in the reactivation of the cell cycle machinery in those post-mitotic, terminally differentiated feline neurons.

Immunohistochemical staining of immunoglobulin G in healthy equine, canine, and feline corneas.

OBJECTIVE: Establishing an immunohistochemical approach for semi-quantitative assessment of the presence of immunoglobulin G (IgG) in equine, canine, and feline corneas. PROCEDURES: Healthy corneas of horses, dogs, and cats, euthanized because of a fatal disease or an unrecoverable trauma unrelated to and without a history of ophthalmic disease were formalin-fixed, paraffin-embedded, and determined to be pathomorphologically healthy by light microscopy. Automated immunohistochemistry was performed using primary antibodies against IgG, biotin-conjugated secondary antibodies, and streptavidin-peroxidase, as well as diaminobenzidine for visualization. After counterstaining with hematoxylin, epithelium, stroma, Descemet´s membrane (DM), and endothelium were semi-quantitatively scored for the presence of IgG on a 4-grade scale (0 = no, 1 = faint, 2 = medium, 3 = strong staining) by light microscopy. RESULTS: Corneal specimens of 20 horses (40 eyes) with a median age of 15.5 years (range 2-31 years), 12 dogs (21 eyes) with a median age of 10.0 years (range 4-16), and 13 cats (24 eyes) with a median age of 10.0 years (range 2-18) were included in the study. Different sexes and breeds were represented. In all corneas (100%), significant medium signal intensity in the stroma was observed. Variable immunosignal was obtained in epithelium, DM, and endothelium. CONCLUSION: This method reproducibly allows for the detection of IgG in healthy equine, canine, and feline corneas, particularly stroma. Semi-quantitative results evidence medium presence of IgG in the corneal stroma. Further research is needed to evaluate IgG presence in diseased corneas.

The first RT-qPCR confirmed case of tick-borne encephalitis in a dog in Scandinavia.

BACKGROUND: Tick-borne encephalitis (TBE) is a zoonotic neurological disease caused by tick-borne encephalitis virus (TBEV), a flavivirus endemic in parts of Europe and Asia. Seroconversion without signs of clinical disease is common in dogs and most of the cases previously described have been tentatively diagnosed by combining neurologic signs with serum antibody titres. Here, the first Scandinavian RT-qPCR-confirmed clinical case of TBE in a dog is reported. CASE PRESENTATION: A 4-year old castrated male Pointer Labrador cross was presented with acute-onset ataxia. During hospitalisation, the dog developed seizures. Despite aggressive treatment with steroids, antimicrobials and sedation/anaesthesia, there was continued deterioration during the following 24 h after admission and the dog was euthanised and submitted for necropsy. Histopathological changes in the brain were consistent with lymphoplasmacytic and histiocytic meningoencephalomyelitis. RT-qPCR examination of the brain was positive for TBEV, confirming infection. CONCLUSIONS: Meningoencephalomyelitis caused by TBEV should be a diagnostic consideration in dogs presenting with clinical signs of central nervous system disease such as acute-onset ataxia and seizures in areas where TBEV-positive ticks are endemic. Clinical TBE may be underdiagnosed in dogs due to lack of specific testing.

In vitro study to assess the efficacy of CDK4/6 inhibitor Palbociclib (PD-0332991) for treating canine mammary tumours.

Therapy of canine mammary tumours (CMTs) with classical antitumour drugs is problematic, so better therapeutic options are needed. Palbociclib (PD-0332991) is an innovative and effective anticancer drug for the treatment of breast cancer in women. Palbociclib is an inhibitor of cyclin-dependent kinase 4 (CDK4) and CDK6, which are key regulators of the cell cycle machinery and thus cell proliferation. In the present in vitro study, we investigated whether Palbociclib also represents a candidate drug to combat CMT. For this purpose, the effect of Palbociclib was analysed in P114 and CF41 cells, two CMT cell lines with an endogenous CDK4/6 co-expression. Incubation of P114 and CF41 cells with Palbociclib resulted in a dose- and time-dependent loss of phosphorylated retinoblastoma protein (pRb), a classical CDK4/6 substrate within the cell cycle machinery. Moreover, treatment of CMT cells with Palbociclib-induced cell cycle arrest affected cell viability, prevented colony formation and impaired cell migration activity. Palbociclib also inhibited the growth of P114 and CF41 cell spheroids. Immunohistochemical analysis of canine patient samples revealed a consistent expression of CDK6 in different canine mammary carcinoma types, but an individual and tumour-specific expression pattern of phosphorylated pRb independent of the tumour grade. Together, our findings let us suggest that Palbociclib has antitumour effects on CMT cells and that canine patients may represent potential candidates for treatment with this CDK4/6 inhibitor.

Meningoencephalitis in cats in Austria: a study of infectious causes, including Encephalitozoon cuniculi.

Objectives Despite comprehensive diagnostics, the aetiology of meningoencephalitis (ME) in cats often remains undetermined. As a result of recently published surveys, Encephalitozoon cuniculi has gained growing importance in cats not only with ocular disorders, but also with central nervous system disease. Therefore, it was hypothesised that E cuniculi may be an underestimated pathogen in the development of feline non-suppurative and/or granulomatous ME. Methods As a first step, histopathological sections of the brain of cats with encephalopathy were retrospectively reviewed to identify cases of granulomatous ME. In a second step, an immunohistochemical screening for detection of E cuniculi was performed in cases with ME of unknown origin. Results In 59/89 (66.3%) cats with ME, an aetiologically relevant pathogen was detected. Forty-three of 89 (48.3%) cats had a diagnosis of feline infectious peritonitis. In 14/89 (15.7%) cats, protozoan cysts were identified and infection with Toxoplasma gondii was confirmed by immunohistochemistry (IHC) in all cases. In 2/89 (2.3%) cats with granulomatous ME, fungal organisms were identified. Thirty of 89 (33.7%) cats with ME of unknown origin that underwent IHC for the detection of E cuniculi remained negative. Conclusions and relevance The results of this study suggest that E cuniculi is unlikely to be directly associated with (non-suppurative and/or granulomatous) ME in cats in Austria.

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV).

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch's postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.

NEPHROPATHIES IN THE EUROPEAN CAPTIVE CHEETAH (ACINONYX JUBATUS) POPULATION.

According to previous studies in captive cheetah ( Acinonyx jubatus ) populations, one of the most threatening diseases besides amyloidosis, myelopathy, veno occlusive disease, and gastritis, is renal failure. Contrary to captive cheetahs in North America and South Africa, morphological data concerning renal lesions in the cheetah European Endangered Species Program (EEP) are lacking. This study details the histological characterization as well as immunohistochemical and morphometrical analysis of nephropathies in 35 captive cheetahs from the EEP, which were necropsied between 1985 and 2003. Examination of paraffin- and glycolmethacrylate-methylmethacrylate (GMA-MMA) embedded kidney samples by light microscopy revealed glomerulonephritis in 91%, with a high prevalence for glomerulosclerosis and glomerulonephritis with the histologic pattern of membranous glomerulonephritis (77%). Besides these predominating glomerulopathies, a wide range of other renal lesions, like acute tubular necrosis, interstitial nephritis, calcinosis, and amyloidosis, were present. Pathological expression of collagen type IV, complement C3, fibronectin, and IgG was demonstrated in the glomeruli of the cheetah kidneys with the use of the avidin-biotin complex method. Morphometrical analysis was performed on GMA-MMA embedded kidney samples to obtain glomerulosclerosis index and glomerulosclerosis incidence.

Clinical and biochemical signs in Fleckvieh cattle with genetically confirmed Fanconi-Bickel syndrome (cattle homozygous for Fleckvieh haplotype 2).

Fanconi-Bickel Syndrome (FBS) is an autosomal recessive disorder of the carbohydrate metabolism, which has been reported in human and some animals (OMIA 000366-9913). In Fleckvieh cattle it is caused by mutations in SLC2A2, a gene encoding for glucose transporter protein 2 (GLUT2), which is primarily expressed in liver, kidney, pancreas and intestines. The causal mutation resides in a previously reported Fleckvieh Haplotype 2 (FH-2). FH-2 homozygous individuals are rare, but due to widespread use of heterozygous bulls in artificial insemination, heterozygous animals are likely to be present in a larger number in the cattle population. Two clinical cases of Fleckvieh cattle with a syndrome resembling the phenotypic appearance of FBS are presented in the present study describing the association between the clinical manifestations of FBS and the postulated frameshift mutation in bovine SLC2A2. Clinical examination showed poor growth, retarded development, polyuria, and polydipsia. Laboratory analyses showed an increased plasma glucose but normal insulin concentration and increased renal glucose excretion. Histopathological examination of kidney and liver samples revealed massively increased liver glycogen storage and nephrosis. Sires of both cases were tested positive for being heterozygous carriers for the same frameshift mutation in SLC2A2 as was originally reported in Fleckvieh cattle. DNA of both cases described was analyzed and Sanger sequencing confirmed homozygosity for the frameshift mutation in SLC2A2.

Evaluation of a gene-directed enzyme-product therapy (GDEPT) in human pancreatic tumor cells and their use as in vivo models for pancreatic cancer.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) is a two-step treatment protocol for solid tumors that involves the transfer of a gene encoding a prodrug-activating enzyme followed by administration of the inactive prodrug that is subsequently activated by the enzyme to its tumor toxic form. However, the establishment of such novel treatment regimes to combat pancreatic cancer requires defined and robust animal model systems. METHODS: Here, we comprehensively compared six human pancreatic cancer cell lines (PaCa-44, PANC-1, MIA PaCa-2, Hs-766T, Capan-2, and BxPc-3) in subcutaneous and orthotopical mouse models as well as in their susceptibility to different GDEPTs. RESULTS: Tumor uptake was 83% to 100% in the subcutaneous model and 60% to 100% in the orthotopical mouse model, except for Hs-766T cells, which did not grow orthotopically. Pathohistological analyses of the orthotopical models revealed an infiltrative growth of almost all tumors into the pancreas; however, the different cell lines gave rise to tumors with different morphological characteristics. All of the resultant tumors were positive for MUC-1 staining indicating their origin from glandular or ductal epithelium, but revealed scattered pan-cytokeratin staining. Transfer of the cytochrome P450 and cytosine deaminase suicide gene, respectively, into the pancreatic cancer cell lines using retroviral vector technology revealed high level infectibility of these cell lines and allowed the analysis of the sensitivity of these cells to the chemotherapeutic drugs ifosfamide and 5-fluorocytosine, respectively. CONCLUSION: These data qualify the cell lines as part of valuable in vitro and in vivo models for the use in defined preclinical studies for pancreas tumor therapy.

Beta-escin has potent anti-allergic efficacy and reduces allergic airway inflammation.

BACKGROUND: Type I hypersensitivity is characterized by the overreaction of the immune system against otherwise innocuous substances. It manifests as allergic rhinitis, allergic conjunctivitis, allergic asthma or atopic dermatitis if mast cells are activated in the respective organs. In case of systemic mast cell activation, life-threatening anaphylaxis may occur. Currently, type I hypersensitivities are treated either with glucocorticoids, anti-histamines, or mast cell stabilizers. Although these drugs exert a strong anti-allergic effect, their long-term use may be problematic due to their side-effects. RESULTS: In the course of a routine in vitro screening process, we identified beta-escin as a potentially anti-allergic compound. Here we tested beta-escin in two mouse models to confirm this anti-allergic effect in vivo. In a model of the early phase of allergic reactions, the murine passive cutaneous anaphylaxis model, beta-escin inhibited the effects of mast cell activation and degranulation in the skin and dose-dependently prevented the extravasation of fluids into the tissue. Beta-escin also significantly inhibited the late response after antigen challenge in a lung allergy model with ovalbumin-sensitized mice. Allergic airway inflammation was suppressed, which was exemplified by the reduction of leucocytes, eosinophils, IL-5 and IL-13 in the bronchoalveolar lavage fluid. Histopathological examinations further confirmed the reduced inflammation of the lung tissue. In both models, the inhibitory effect of beta-escin was comparable to the benchmark dexamethasone. CONCLUSIONS: We demonstrated in two independent murine models of type I hypersensitivity that beta-escin has potent anti-allergic properties. These results and the excellent safety profile of beta-escin suggest a therapeutic potential of this compound for a novel treatment of allergic diseases.

Evidence of canine distemper and suggestion of preceding parvovirus-myocarditis in a Eurasian badger (Meles meles).

An approximately 1.5-yr-old free-ranging male Eurasian badger (Meles meles) from the eastern part of Austria had macroscopic and microscopic lesions consistent with canine distemper virus infection, including nonsuppurative meningoencephalitis, interstitial pneumonia with accumulation of macrophages in alveoli that contained intranuclear inclusion bodies, vesicular exanthema of the ventral abdomen, and atrophy of lymphoid tissues. Canine distemper virus-antigen was demonstrable in a variety of organs by using immunohistology. In addition, there were widespread areas of fibrosis in the myocardium that were rich in collagen and paucicellular. Because such changes are comparable with sequelae of the acute cardiac form of canine parvovirus (CPV) infection in dogs, it was speculated that this badger may have experienced CPV myocarditis as a cub but that the corresponding antigen or DNA was not detectable due to resolution of the disease.

A 470 bp WAP-promoter fragment confers lactation independent, progesterone regulated mammary-specific gene expression in transgenic mice.

The ability of a 470 bp sub-fragment of the murine whey acidic protein (WAP) promoter in the context of a retroviral expression plasmid to direct gene expression to mammary epithelial cells was analysed in a number of independent transgenic mouse lines. In contrast to previous findings with the genuine 2.5 kb promoter fragment, our studies revealed a highly mammary gland-specific expression detectable only in non-lactating animals. This suggested a mainly progesterone-regulated activity of the short fragment. Therefore, transgene expression was examined in the progesterone-determined estrous cycle and during pregnancy. In accordance with in vitro data from stably transfected cell lines, in both situations expression was upregulated at stages associated with high progesterone levels. Taken together these data provide deeper insight into WAP-promoter regulation and stress the usefulness of the shortened fragment for a lactation independent mammary-targeted expression.

Physical exercise retards the development of chronic nephropathy in the ageing rat as efficiently as food restriction does.

BACKGROUND: Obesity combined with decreasing physical fitness in the ageing Western populations promotes a number of degenerative diseases, including chronic kidney disease. It has further been shown in rodent models that prevention of obesity by food restriction mitigates development of kidney lesions. Whether lifelong physical activity also has a positive effect is not known. OBJECTIVE: To compare the effects of physical exercise and food restriction on the development of chronic kidney lesions in ageing rats. METHODS: Male Sprague-Dawley rats were divided into groups: voluntarily running in wheels (RW), food restriction to the degree necessary to attain pair weight to RW rats (PW), forced running in treadmills (TM) and sedentary controls housed individually (S1) or 4 in each cage (S4). The interventions began at the age of 5 months and kidneys were sampled and analysed histologically at the ages of 15, 19 and 23 months. RESULTS: Total score for kidney lesions (sum of the scores for glomerular changes, interstitial non-purulent inflammation, proteinaceous casts in tubules and increased amount of connective tissue, the possible maximum being 10.0) increased from 0.5 +/- 0.2 at 5 months of age to 1.6 +/- 0.3 for RW, 2.3 +/- 0.4 for PW, 4.5 +/- 0.4 for TM, 3.6 +/- 0.5 for S1 and 5.4 +/- 0.6 for S4 at the age of 23 months. The increase from 5 months of age was gradual for all groups through 15, 19 and 23 months. The patterns for the various lesions followed the same pattern with the exception of connective tissue, which did not increase. CONCLUSION: Voluntary running in wheels is as effective in mitigating kidney lesions as is food restriction, while forced running in a treadmill is not effective in this respect.

Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements.

Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.

Interferon resistance promotes oncolysis by influenza virus NS1-deletion mutants.

NS1 protein of influenza virus is a virulence factor that counteracts Type I interferon (IFN)-mediated antiviral response by the host. A recombinant influenza A virus that lacks the NS1 protein only replicates efficiently in systems that contain defective IFN pathways. We demonstrate that the conditional replication properties of NS1-modified influenza A virus mutants can be exploited for the virus-mediated oncolysis of IFN-resistant tumor cells. IFN resistance in analyzed tumor cell lines correlated with a reduced expression of STAT1. Addition of exogenous IFNalpha or supernatant of virus-infected endothelial cells inhibited viral oncolysis in IFN-sensitive but not in IFN-resistant cell lines. The oncolytic potential of NS1-modified influenza A virus mutants could be exploited in vivo in a SCID mouse model of a subcutaneously-implanted human IFN-resistant melanoma. The data indicate that IFN-resistant tumors are a suitable target for oncolysis induced by NS1-modified influenza virus mutants. STAT1 might serve as a marker to identify these IFN-resistant tumors.

[Acute lead poisoning in cows due to feeding of lead contaminated ash residue]. / Akute Bleivergiftung bei Kühen durch Aufnahme eines bleihaltigen Verbrennungsrückstandes.

In a dairy herd of 21 cows which were on pasture during the day at the end of May 2002, four eight years old cows were suddenly inappetent and showed severe diarrhoea consisting of black discolorate feces. A few days after the onset of the disease, three affected cows exhibited neurological disorders. These cows were admitted to the IInd Medical Clinic of the University for Veterinary Medicine in Vienna. Following clinical signs were observed: circulatory weakness, anorexia, atony of the rumen, diarrhoea and in accordance with acute lead poisoning typical signs of the central nervous system. One cow died and the other two animals were euthanized. Results of blood testing were anaemia, basophil spotting of erythrocytes, increase of liver enzymes and CK, hypocalcaemia, decrease of potassium and phosphate. The cerebrospinal fluid of two cows showed increased CK-, LDH- and AST-values. The lead contents of whole blood samples were between 0.486 and 0.928 mg/kg, of liver samples 13.3 to 114.4 mg/kg, of kidney samples 172.2 to 448 mg/kg and of rumen content 59 mg/kg fresh matter. At necropsy, enteritis, liver fluke disease and severe interstitial and alveolar pulmonary emphysema were found. Pathohistologically typical ischaemic necrosis of neurons predominantly at the tips of the gyri, disseminated petechial hemorrhages and moderate diffuse neovascularisation, but no acid-fast intranucleolar inclusion bodies in the renal tubules were observed. As causative agent of the acute lead poisoning a residue on combustion, taken up by the cows on the pasture, was confirmed. The ash residue was formed by combustion of three tires which contained 450 g heavy weights of 96.5% lead for wheel balance. The lead content of the ash residue was between 2.9 and 28 g/kg dry matter.

Evidence of parvovirus replication in cerebral neurons of cats.

The correlation between parvovirus infections and lesions in the central nervous system other than cerebellar hypoplasia was studied in 100 cats. The animals were necropsied with a history of various diseases, one third showing typical clinical and pathomorphological signs of panleukopenia. In 18 cats polyclonal antiserum against canine parvovirus consistently labeled neurons mainly in diencephalic regions, whereas the cerebellar cortex remained negative in all cases. In situ hybridization with digoxigenin-labeled minus-sense RNA probes, hybridizing with monomer-replicative form DNA or mRNA, revealed positive signals in nuclei of several neurons of the brain, again excluding the cerebellum. PCR applied to formalin-fixed and paraffin-embedded brain tissue and intestinal tissues of the diseased cats and subsequent DNA sequence analysis yielded canine parvovirus type 2 (CPV-2)-like sequences in the central nervous system. Two aspects of these findings are intriguing: (i). parvoviruses appear to be capable of replicating in neurons, cells that are considered to be terminally differentiated and (ii). CPV-like viruses of the old antigenic type CPV-2 appear to be able to infect cats.

Idiopathic acute onset myelopathy in cheetah (Acinonyx jubatus) cubs.

Numerous cases of ataxia, hind limb paresis, and paralysis have occurred in cheetah (Acinonyx jubatus) cubs over the past 10 yr within the European Endangered Species Program population, including 12 in mainland Europe, two in the British Isles, one in Namibia, and one in Dubai. The condition is the most important medical factor limiting European cheetah population growth. Eight cubs at the Salzburg Zoo, Austria, were affected. They demonstrated upper motor neuron lesions when alive and bilateral, symmetrical myelin degeneration of the spinal cord on necropsy. Ballooning of myelin sheaths surrounded mostly preserved axons, and no spheroids, characteristic of acute axonal degeneration, were found. Myelin loss markedly exceeded axonal degeneration. The syndrome's etiology is unclear, although viral, bacterial, parasitic, genetic, nutritional-metabolic, toxic, and physical causes have been considered.

Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe.

During late summer 2001 in Austria, a series of deaths in several species of birds occurred, similar to the beginning of the West Nile virus (WNV) epidemic in the United States. We necropsied the dead birds and examined them by various methods; pathologic and immunohistologic investigations suggested a WNV infection. Subsequently, the virus was isolated, identified, partially sequenced, and subjected to phylogenetic analysis. The isolates exhibited 97% identity to Usutu virus (USUV), a mosquito-borne Flavivirus of the Japanese encephalitis virus group; USUV has never previously been observed outside Africa nor associated with fatal disease in animals or humans. If established in central Europe, this virus may have considerable effects on avian populations; whether USUV has the potential to cause severe human disease is unknown.