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.     

The localization and connections of suboviductal lymphatic vessels in the bovine uterine broad ligament

The distribution of oviductal lymphatic vessels in the bovine mesosalpinx and bursa ovarica, and their communications with other lymphatics in the uterine broad ligament were examined after exposition of all lymphatic pathways with varicoloured microfil and/or ink-gelatin mixture. However, filling of the lumen of the oviductal infundibulum lymphatics was difficult or impossible because of its slender walls. Lymphatics of the isthmus and ampulla with short precollectors formed branches 1.5-2.0 cm long on both sides: dorsal and ventral of the uterine broad ligament. Collectors with slender and long lymphangions, visible after filling their lumen, encircled the alymphatic area in the parainfundibullar mesosalpinx. The greatest number of lymphatic branches, which originated from the paraisthmal part of the oviductal ampulla were observed in the subovarian area. The characteristic feature was their immediate proximity with uterine and oviductal arterial vessels. Subsequent studies have provided convincing evidence that there are direct connections of lymphatics leaving the oviduct and ovarian sac with lymphatics emerging from the uterus and ovary (after bilateral filling of lymphatics pathways in the whole broad liagment of the uterus). The performed investigations showed particularly characteristic feature in the cow--mixing lymph leaving various reproductive organs in the area of the right and left ligament--before two long collector branches reach the nearest lymphatic node/-s.     

Effect of Endobronchial Challenge with Actinobacillus pleuropneumoniae Serotype 10 of Pigs Vaccinated with Bacterins Consisting of A. pleuropneumoniae Serotype 10 Grown under NAD‐Rich and NAD‐Restricted Conditions

The efficacy of two bacterins containing an Actinobacillus pleuropneumoniae serotype 10 strain was evaluated. The bacterial cells constituting bacterin 1 and 2 were grown under nicotinamide adenine dinucleotide (NAD)‐rich (low‐adherence capacity to alveolar epithelial cell cultures) and NAD‐restricted (high‐adherence capacity to alveolar epithelial cell cultures) conditions, respectively. Ten pigs were vaccinated twice with the bacterin 1 and nine pigs with the bacterin 2. Ten control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 106.5 colony‐forming units (CFU) of an A. pleuropneumoniae serotype 10 strain. In the bacterin 1 and 2 group, three and two pigs died after inoculation, respectively. Only two pigs of the control group survived challenge. Surviving pigs were killed at 7 days after challenge. The percentage of pigs with severe lung lesions (>10% of the lung affected) was 100% in the control group, 70% in the bacterin 1 group and 22% in the bacterin 2 group. Actinobacillus pleuropneumoniae was isolated from the lungs of all animals. The mean bacterial titres of the caudal lung lobes were 7.0 × 10⁶ CFU/g in the control group, 6.3 × 10⁵ CFU/g in the bacterin 1 group and 1.3 × 10⁶ CFU/g in the bacterin 2 group. It was concluded that both bacterins induced partial protection against severe challenge. Furthermore, there are indications that the bacterin 2, containing A. pleuropneumoniae bacteria grown under conditions resulting in high in vitro adhesin, induced better protection than the bacterin 1.     

Azotemia and mortality among Babesia microti-like infected dogs

Babesia microti-like piroplasms are a recently recognized cause of illness in dogs in northwest Spain. Our objective was to describe the clinical characteristics and investigate the risk factors for azotemia and death among 58 B microti-like infected dogs. Twenty-one of the 58 (36%) dogs were azotemic at the time that the infection was diagnosed. The case fatality rate during the following week was 22%. Dogs with azotemia at the time of diagnosis were 10 times (95% CI, 3.26-28.8) more likely to die during the following week. Azotemia was the main cause of death for B microti-like infected dogs (attributable fraction = 90%). Severe anemia was present in 45 of the 58 (78%) dogs. Azotemic dogs also presented with hyperphosphatemia, hypoalbuminemia, hypercholesterolemia, proteinuria, and high urine protein: creatinine ratios, suggesting a glomerular component to the disease. Age was the only factor significantly associated with the risk of azotemia (P = .042): on average, a 4-year age increase doubled the risk of an infected dog being azotemic. The only factor significantly associated with mortality was azotemia (P = .001). We concluded that B microti-like infection is associated with a high risk of azotemia and mortality.     

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.     

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC)

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of S?o Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.     

Heat stroke in a great Pyrenees dog

A 2-year-old, male Great Pyrenees presented with a history and clinical signs suggesting heat exhaustion. Treatment with intravenous fluids, antibiotics, and surface cooling was unsuccessful, and euthanasia was elected. Histological evaluation of the dog's tissues revealed lesions consistent with severe hyperthermia and shock.     

Performance and digestibility characteristics of finishing diets containing distillers grains, composites of corn processing coproducts, or supplemental corn oil

Three experiments evaluated the lipids in distillers grains plus solubles compared with corn or other sources of lipid in finishing diets. Experiment 1 utilized 60 individually fed yearling heifers (349 +/- 34 kg of BW) fed treatments consisting of 0, 20, or 40% (DM basis) wet distillers grains plus solubles (WDGS), or 0, 2.5, or 5.0% (DM basis) corn oil in a finishing diet based on high-moisture corn (HMC) and dry-rolled corn. Cattle fed 20 and 40% WDGS had greater (P < 0.10) G:F than cattle fed 0% WDGS. Cattle fed the 5.0% corn oil had less overall performance than cattle fed the other diets. Results from Exp. 1 indicated that adding fat from WDGS improves performance, whereas supplementing 5.0% corn oil depressed G:F, suggesting that the fat within WDGS is different than corn oil. Experiment 2 used 234 yearling steers (352 +/- 16 kg of BW) fed 1 of 5 treatments consisting of 20 or 40% (DM basis) dry distillers grains plus solubles, 1.3 or 2.6% (DM basis) tallow, or HMC. All diets contained 20% (DM basis) wet corn gluten feed as a method of controlling acidosis. No differences between treatments for any performance variables were observed in Exp. 2. The dry distillers grains plus solubles may be similar to tallow and HMC in finishing diets containing 20% wet corn gluten feed. Experiment 3 used 5 Holstein steers equipped with ruminal and duodenal cannulas in a 5 x 5 Latin square design. Treatments were a 40% WDGS diet, 2 composites, one consisting of corn bran and corn gluten meal; and one consisting of corn bran, corn gluten meal, and corn oil; and 2 dry-rolled corn-based diets supplemented with corn oil or not. Cattle fed the WDGS diet had numerically less rumen pH compared with cattle fed other treatments. Cattle fed WDGS had greater (P < 0.10) molar proportions of propionate, decreased (P < 0.10) acetate:propionate ratios, greater (P < 0.10) total tract fat digestion, and a greater (P < 0.10) proportion of unsaturated fatty acids reaching the duodenum than cattle fed other treatments. Therefore, the greater energy value of WDGS compared with corn may be due to more propionate production, greater fat digestibility, and more unsaturated fatty acids reaching the duodenum.     

Pharmacokinetics and intramuscular bioavailability of difloxacin in dromedary camels (Camelus dromedarius)

Single-dose disposition kinetics of difloxacin (5mg/kg bodyweight) were determined in clinically normal male dromedary camels (n=6) following intravenous (IV) and intramuscular (IM) administration. Difloxacin concentrations were determined by high performance liquid chromatography with fluorescence detection. The concentration-time data were analysed by compartmental and non-compartmental kinetic methods. Following a single IV injection, the plasma difloxacin concentration-time curve was best described by a two-compartment open model, with a distribution half-life (t(1/2alpha)) of 0.22+/-0.02h and an elimination half-life (t(1/2beta)) of 2.97+/-0.31h. Steady-state volume of distribution (V(dss)) and total body clearance (Cl(tot)) were 1.02+/-0.21L/kg and 0.24+/-0.07L/kg/h, respectively. Following IM administration, the absorption half-life (t(1)(/)(2ab)) and the mean absorption time (MAT) were 0.44+/-0.03h and 1.53+/-0.22h, respectively. The peak plasma concentration (C(max)) of 2.84+/-0.34microg/mL was achieved at 1.42+/-0.21h. The elimination half-life (t(1/2el)) and the mean residence time (MRT) was 3.46+/-0.42h and 5.61+/-0.23h, respectively. The in vitro plasma protein binding of difloxacin ranged from 28-43% and the absolute bioavailability following IM administration was 93.51+/-11.63%. Difloxacin could be useful for the treatment of bacterial infections in camels that are sensitive to this drug.