Derivation of Canine Induced Pluripotent Stem Cells

Dogs and humans have many inherited genetic diseases in common and conditions that are increasingly prevalent in humans also occur naturally in dogs. The use of dogs for the experimental and clinical testing of stem cell and regenerative medicine products would benefit canine health and welfare and provide relevant animal models for the translation of therapies to the human field. Induced pluripotent stem cells (iPSCs) have the capacity to turn into all cells of the body and therefore have the potential to provide cells for therapeutic use and for disease modelling. The objective of this study was to derive and characterize iPSCs from karyotypically abnormal adult canine cells. Aneuploid adipose‐derived mesenchymal stromal cells (AdMSCs) from an adult female Weimeraner were re‐programmed into iPSCs via overexpression of four human pluripotency factors (Oct 4, Sox2, Klf4 and c‐myc) using retroviral vectors. The iPSCs showed similarity to human ESCs with regard to morphology, pluripotency marker expression and the ability to differentiate into derivatives of all three germ layers in vitro (endoderm, ectoderm and mesoderm). The iPSCs also demonstrated silencing of the viral transgenes and re‐activation of the silent X chromosome, suggesting full reprogramming had occurred. The levels of aneuploidy observed in the AdMSCs were maintained in the iPSCs. This finding demonstrates the potential for generating canine induced pluripotent stem cells for use as disease models in addition to regenerative medicine and pharmaceutical testing.     

Expression of genes associated with the antigen presentation and processing pathway are consistently regulated in early Mycobacterium avium subsp. paratuberculosis infection

The primary objective of this study was to evaluate early gene expression profiles associated with paratuberculosis in cattle exposed to known infectious doses of Mycobacterium avium sub-species paratuberculosis (MAP). A Johne's disease experimental infection field trial was conducted on a mixed population of Holstein and Holstein Red cattle. Blood samples from four MAP exposed and four unexposed cattle, selected based on IFNγ expressions were taken at 9, 13 and 21 weeks and RNA processed to Affymetrix GeneChip? Bovine Genome arrays. Ontological analysis revealed consistent differences in gene expression between MAP exposed and control animals. A stark variation was observed in expression of a number of genes along antigen presentation pathways, suggesting that MAP exposure potentially results in the host immune response switching to a CD8(+) biased antigen presentation profile. This requires further in-depth analysis since it exposes a hitherto unconfirmed association between MAP exposure and in vivo MHC gene modulation.     

Ataxia and paralysis in cats in Australia associated with exposure to an imported gamma-irradiated commercial dry pet food

Between June 2008 and March 2009, 87 cats in Australia developed symmetrical hindlimb ataxia, paraparesis, tetraparesis, paraplegia or tetraplegia in association with eating an imported, irradiated dry pet food. This communication reports the clinical signs and outcomes of those cats.     

The effects of antigenic competition on the efficacy of multivalent footrot vaccines

SUMMARY A multivalent footrot vaccine has been developed, containing pilus antigens produced in recombinant Pseudomonas aeruginosa and representing all nine serogroups of Dichelobacter (Bacteroides) nodosus commonly recognised in the field. The responses of sheep to the multivalent vaccine have been compared with those to monovalent vaccines representing only a single serogroup. Antigenie competition between serogroups occurred in sheep immunised with the multivalent formulation, but high levels of protection were still achieved. The study showed that in multivalent footrot vaccines, antigenic competition is predominantly due to the presence of a family of immunologically-related pilus antigens rather than to interference by extraneous proteins.     

Conversion of Cortisone to Cortisol and Prostaglandin F2α Production by the Reproductive Tract of Cows at the Late Luteal Stage In Vivo

Previous in vitro studies demonstrated that bovine endometrium has the capacity to convert inactive cortisone to biologically active cortisol (Cr) and that Cr inhibits cytokine‐stimulated prostaglandin F_2α (PGF) production. This study was carried out to test the hypothesis that bovine reproductive tract has the capacity to convert cortisone to Cr in vivo and to evaluate the effects of intravaginal application of exogenous cortisone on uterine PGF secretion during the late luteal stage. The temporal relationships between PGF and Cr levels in uterine plasma were also determined. Catheters were inserted into jugular vein (JV), uterine vein (UV), vena cava caudalis (VCC) and aorta abdominalis (AA) of six cows on Day 15 of the oestrous cycle (ovulation = Day 0) for frequent blood collection. On Day 16, the cows were divided randomly into two groups and infused intravaginally with vaseline gel (10 ml; control; n = 3) or cortisone dissolved in vaseline gel (100 mg; n = 3). Blood samples were collected at ?2, ?1, ?0.5, 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h after treatments (0 h). Intravaginal application of cortisone increased plasma concentrations of Cr between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in JV and at 1.5 h in AA. The plasma concentrations of PGF in UV and of PGF metabolite in JV were greater at 0.5 and 1 h in the cortisone‐treated animals than in control animals. The levels of PGF in UV blood plasma decreased after Cr reached its highest levels. The overall findings suggest that the female reproductive tract has the capacity to convert cortisone to Cr in vivo. Based on the temporal changes of PGF and Cr levels in the uterine plasma, a biphasic response in PGF secretion was found to be associated to the Cr increase induced by the cortisone treatment at the late luteal stage in non‐pregnant cows.     

Successful treatment of cryptococcal pneumonia in a pony mare

A 20-year-old Welsh Mountain Pony (212 kg) mare was initially presented for a chronic cough, fever, weight loss and low grade abdominal pain. She later developed dyspnoea, tachypnoea and exercise intolerance. The presence of multiple masses (up to 17 cm diameter) in the pulmonary parenchyma was established using lateral thoracic radiography and transthoracic ultrasonography. Encapsulated, budding yeasts were observed in smears made from transtracheal washings and needle aspirates of the pulmonary lesions. Cryptococcus gattii (synonym: Cryptococcus neoformans variety gattii; Cryptococcus bacillisporus) was cultured from the transtracheal washings and aspirates of the lung masses. The pony was successfully treated using daily intravenous infusions of amphotericin B (typically 0.5 mg/kg in 1 L 5% dextrose in water over 1 h, following premedication with 50 mg flunixin intravenously) over a 1 month period, until a cumulative dose of 3 g had been administered. Treatment was considered to be successful on the basis of progressive improvement in clinical signs, reduction in the size of pulmonary cryptococcomas, 48 kg weight gain and a reduction in the cryptococcal antigen titre from 4096 to 256, 1 year after cessation of treatment.     

Acquired arachnoid cyst in a cat

A 5-year-old Birman cat presented with paraparesis associated with a fracture of the third thoracic vertebra and was managed conservatively. Voluntary function was regained over the next 6 months, but the cat was referred 4 years after the trauma because of recurrence of hindlimb paresis and ataxia. Magnetic resonance imaging (MRI) showed a spinal arachnoid cyst at the level of the fourth thoracic vertebra, which was treated surgically by dorsal laminectomy and durectomy, and hindlimb function subsequently recovered. The cat re-presented 4 years later for recurrent hindlimb paresis. Myelography and computed tomography–myelography (CT-M) showed dorsolateral pooling of cerebrospinal fluid (CSF) at the previous laminectomy site. The neurological signs after the second surgery improved, but not as much as after the first surgery and the cat remained ataxic with moderate paresis. Seven months later repeat CT-M revealed an atrophic spinal cord, but negligible pooling of CSF at the previous site of the arachnoid cyst. The cat could walk, but was being treated for self-induced trauma of the left hindlimb that was thought to be related to paraesthesia. The neurological signs gradually deteriorated over 3 months and the cat was euthanased.     

Experimental animal infection models for Johne's disease, an infectious enteropathy caused by Mycobacterium avium subsp. paratuberculosis

A critical literature review of experimental infection models for Johne's disease in farm and laboratory animals was conducted. A total of 73 references were admitted. They were published between 1938 and 2006 and covered species as diverse as cattle, sheep, goats, deer, mice, pigs and others. The factors that appeared to influence the outcome of experimental infections with Mycobacterium avium subsp. paratuberculosis (Mptb) were the species, breed and age of subject used for the infection, the route of infection, and the strain, dose and number of doses of Mptb used to inoculate the subjects. Natural paratuberculosis infection passes through stages, generally over a period measured in years. However, the endpoints chosen by researchers using experimental infections have been determined by the need for immunological, microbiological, pathological or clinical outcomes, and these were the likely factors determining the duration of the trials. Studies have been lacking in the use of a defined type strain of Mptb in pure culture prepared from an archived seed stock of Mptb that can be used at the same passage level in a later trial. Replication of experimental groups has been very uncommon, temporal replication equally rare, as have sufficiently long time scales so as to be able to observe a full range of immunological and pathological changes at different stages of the disease process. While it may be difficult to develop a satisfactory experimental infection model, there is room for improvement in the way experiments have been designed and carried out to date. Choice of animal species/breed of host and strain of Mptb used in an experimental model should be based on the purpose of the study (for example, vaccine efficacy trial, diagnostic test evaluation, pathogenesis study) and local needs. The strain of Mptb used should be typed using IS900 RFLP analysis, IS1311 sequence analysis and other genotypic methods, and preferably be from an archived low passage pure culture with viable bacteria enumerated using a sensitive method rather than from an uncharacterised and unrepeatable tissue homogenate. It is generally agreed that the faecal-oral route is the most important natural route of exposure and the oral route is therefore the preferred route of experimental inoculation to achieve Johne's disease that closely resembles natural infection.