RNA‐transfected CD40‐activated B cells as a vaccine strategy in canine malignancies

Introduction: Cell‐based vaccine strategies using dendritic cells as cellular adjuvant have entered phase III trials in humans and have been found to be safe, feasible, and potentially efficacious. Canine patients are generally smaller than adult human patients, which makes production of canine dendritic cell (DC) vaccines problematic, given patient size and the small number of available DC precursors. Here we describe feasibility studies of a novel cell‐based vaccine strategy which uses CD40‐activated B‐cells (CD40‐B) loaded with RNA. This strategy is based on our observations that RNA‐transfected human CD40‐B can drive anti‐tumor T cell responses. One advantage of using CD40‐B cells is the ability to expand this cell population ex vivo, allowing for the numbers of cells required for therapeutic vaccines. Methods: Twenty milliliters of blood were drawn from 6 normal dogs and 5 canine lymphoma patients. Peripheral blood mononuclear cells were separated by Ficoll centrifugation. Culture conditions for B cell activation were optimized using CD40‐ligand, canine IL‐4, and Toll‐like receptor stimulus with CpGoligodinucleotides (ODN). Cyclosporine was added to eliminate peripheral T lymphocytes. Proliferation and activation of CD40‐B cells were demonstrated by CFSE dilution of B cells quantified by flow cytometry. Gene transfer was achieved by mRNA electroporation. Results: Marked in vitro stimulation and proliferation of canine peripheral B cells were achieved with soluble trimeric CD40L, canine IL‐4, and ODN. CD40‐B cells showed dramatic upregulation of MHC class II molecules and CD21 (B‐cell activation marker). After two weeks in culture, cells were negative for CD3 and CD4. Canine CD40‐B cells were efficiently transfected with mRNA, with >60% of CD40‐B expressing green fluorescent protein after GFP mRNA electroporation. Conclusion: RNA‐transfected CD40‐B cells can be efficiently generated from normal and tumor‐bearing dogs. These results provide rationale to test tumor RNA‐transfected CD40‐B as a novel therapeutic approach to treating canine malignancies. Clinical trials in canine lymphoma have been proposed.     

Genotypic prevalence of the adhesin involved in diffuse adherence in Escherichia coli isolates in pre-weaned pigs with diarrhoea in Korea

A total of 1002 Escherichia coli strains isolated from pre-weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat-stable (STa, STb) and heat-labile (LT) enterotoxin, enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty-three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre-weaned pigs with diarrhoea.     

Balloon dilation of oesophageal strictures in two horses

Oesophageal strictures in 2 horses with chronic recurrent oesophageal obstruction or choke was diagnosed by endoscopy and contrast radiography. Balloon dilation of the strictures was performed under endoscopic and fluoroscopic guidance. Follow‐up endoscopic and radiological examinations performed 6 weeks after the procedure showed clear dilation of the strictures. Both horses did not have any recurrence during follow‐up periods of 10 and 12 months.     

Clinical evaluation of clonidine added to lidocaine solution for subarachnoid analgesia in sheep

Clonidine (CL) is a alpha2-adrenergic agonist that produces analgesia in animals and humans by a non-opiate alpha2-adrenergic action in the spinal cord dorsal horn. The objective of this prospective randomized study was to investigate the clinical effects of CL/lidocaine (LD) combination administered by the subarachnoid route in sheep. Each sheep received each of three treatments, at no shorter than weekly intervals. Treatments consisted of 0.003 mg/kg CL, 1.2 mg/kg LD and a combination of CL (0.003 mg/kg) and LD (1.2 mg/kg) (CLLD). Subarachnoid injections were given in all animals between the last lumbar and first sacral vertebra. Heart rate (HR), arterial pressures, respiratory rate, rectal temperature, analgesia, sedation, and motor blockade were determined before drug administration (basal) and 5, 10, 15 and 30 min after drug administration, and at 30-min intervals until loss of analgesia occurred. The duration of analgesia after subarachnoid CLLD administration was 187 +/- 24 min (mean +/- SD), i.e. more than twice of that obtained with CL (99 +/- 19 min) or LD (55 +/- 4.4 min) alone. In all sheep, CL, administered either alone or with LD, induced moderate sedation. After subarachnoid administration of three treatments, all sheep had ataxia and subsequent sternal recumbency. The CL treatment causes decreases in blood pressure (diastolic arterial pressure and mean arterial pressure) and HR. Data suggest that the CLLD combination could be used subarachnoidally in sheep requiring prolonged surgery.     

The cervical vertebral column of horses--a clinico-radiological study

For various anamnestic reasons the cervical vertebral column of 78 horses was examined radiologically. Statistical comparisons showed that male "warmbloods" were more frequently affected than was expected. Our horses were older than comparable animals in the international literature. Independently of their history the radiologic diagnosis of "cervical spondylarthrosis" was established in 56 horses. Arthrosis and other conditions were clinically manifested by spinal ataxia (44 horses) or mechanical impairment of the neck's mobility (21 horses). 10 of the arthrotic patients had synovial cysts causing compression of the spinal cord. 79 percent of all dorsal intervertebral arthrotic joints were found in the caudal column (C6/7 to C7/Th1). With 38 percent, C6/7 was the most frequently affected joint. Clinical and radiological findings could be compared with patho-anatomic results in 29 horses. The correlation was satisfactory.     

The oxygen uptake capacity of swine

Comprehensive studies into domestic pigs and wild boars together with literature data provided a basis for an assessment of aerobic metabolic capacity (VO2max) of swine. The values quoted, from 20 to 94 ml/min-1/kg, had been due to several factors of methodological approach, growth, training, and nutrition as well as to pathophysiological aspects. For full capacity utilisation of VO2max, untrained pigs can be challenged at belt velocities between 0.8 and 1.8 m/s-1 and trained animals at 5 m/s-1.     

Technetium-99m-HDP uptake characteristics in equine fractures: a retrospective study

Bone scintigraphy is a very sensitive diagnostic tool to detect elevated bone metabolism. In cases of fractures and fissure fractures, the radiopharmaceutical uptake in the bone is said to be increased within a few hours after the injury. In this retrospective study, the scintigraphic uptake characteristics at the fracture site of 36 horses with radiographically confirmed fractures or fissure fractures were evaluated. Uptake ratios between the fracture region and adjacent normal bone or soft tissue activity respectively were calculated and compared to different anamnestic and radiographic data. The overall sensitivity of bone scintigraphy was 94.4% (34 positive cases out of 36). In the 36 horses, no correlation between the age of the fracture and the radiopharmaceutical uptake was found. However, there seems to be a lack of sensitivity in early detection of equine pelvic fractures when a standing bone scintigraphy examination protocol is used.     

Studies of cardiac pathomorphology in swine which died during transport

The following findings were obtained from pathomorphological examination of the myocardium of 100 pigs which had died on transport: Degenerative or necrobiotic changes of certain myocardial fibres were recorded from 73 per cent of all cases, usually in the form of granular degeneration (60 per cent). They were "scattered" in 33 per cent of all cases, "multiple" in 22 per cent, and "frequent" in five per cent. In 26 per cent, the myocardial fibres had undergone coarse granular or hyalin plaque-type decomposition, among them with "scattered" manifestation in 15 per cent, "multiple" in four per cent, "frequent" in five per cent, and "massive" in two per cent. Moderate focal resorptive myositis was recorded from eight per cent, while earlier myocardial scars were exhibited by four per cent. The figure for reactive inflammatory processes was twelve per cent. Prestatic hyperaemia of different degrees had taken place in 75 per cent of all animals, and it had been accompanied by varied congestion. The following results were obtained from comparative checks of 100 hearts of clinically intact pigs after slaughter under normal conditions: Granular degeneration was recorded only from 18 per cent of all cases, with actual manifestations "scattered" in 16 per cent and "multiple" in two per cent. Their myocardial fibres had undergone coarse granular or hyalin plaque-type decomposition of moderate intensity in three per cent only, all of them having been "scattered". Reactive inflammatory changes were not recorded at all and signs of prestatic hyperaemia only in 46 per cent. The degenerative-necrobiotic alterations of myocardial fibres which had been observed in the context of death on transport are interpreted as a result of hypoxaemic or hypoxic conditions accompanying acute shock-type circulatory failure in pigs.     

Plasma pharmacokinetics and faecal excretion of ivermectin (Eqvalan paste) and doramectin (Dectomax, 1%) following oral administration in donkeys

Ivermectin (IVM- Eqvalan paste, 1.87%) and doramectin (DRM-Dectomax 1%) were each administered orally to donkeys at 200 microgkg(-1) bodyweight. Blood and faecal samples were collected at predetermined times over 30 days and plasma pharmacokinetics and faecal excretion determined. Maximum plasma concentrations (C(max)) of IVM (23.6 ngml(-1)) and DRM (33.9 ngml(-1)) were obtained at (t(max)) 19.2 and 24h, respectively. The area under the concentration curve (AUC) of DRM (228.9 ngdayml(-1)) was significantly larger than that of IVM (119.3 ngdayml(-1)) and mean residence time (MRT) was 6.5 days for IVM and 9.1days for DRM. The highest (dry weight) faecal concentrations (9.33 microgg(-1) - IVM, 12.12 microgg(-1) - DRM) were detected at 55.9 and 48.0 h, respectively and each compound was detected (0.05 microgg(-1)) in faeces between 11h and 9 days following oral administration in donkeys.