Expression of interleukin-1beta in the digital laminae of horses in the prodromal stage of experimentally induced laminitis

OBJECTIVE: To study expression of interleukin-1beta (IL-1beta) in the digital laminae of horses in the prodromal stage of experimentally induced laminitis. ANIMALS: 8 healthy adult horses with no signs of laminitis. PROCEDURES: Black walnut extract was administered via nasogastric tube to 4 horses, and water was administered to the remaining 4 (controls). Complete blood counts and physical examinations were performed every 30 minutes after administration of black walnut extract or water. General anesthesia was induced when total WBC count decreased by 30% in horses given the black walnut extract and 3 hours after water administration in control horses. The left forefoot was perfusion fixed with neutral-buffered 10% formalin, and paraffin-embedded sections of the digit were used for in situ hybridization with an equine-specific IL-1beta probe. RESULTS: IL-1beta mRNA expression was observed in perivascular cells of the small laminar venules and capillaries in all 4 horses given black walnut extract and in interstitial cells remote from the microvasculature in 1 of the 4. Other cellular components of the laminar tissue and cellular components of the digital arterioles and veins did not exhibit IL-1beta mRNA expression. Expression of IL-1beta mRNA was not detected in laminae from control horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that IL-1beta mRNA is expressed by perivascular cells in the laminar tissues of horses in the prodromal stage of experimentally induced laminitis. This provides evidence of an inflammatory process during the prodromal stage of laminitis, indicating that local digital proinflammatory cytokine expression may be an initiating factor in laminitis.     

Experimental infection of normal and immunosuppressed pigs with Pseudomonas pseudomallei

A single dose of 5 x 10(8) bacilli of Pseudomonas pseudomallei by intratracheal injection resulted in acute (21 cases) or chronic (19 cases) melioidosis in 40 of 48 pigs. Fifteen (10 acute and 5 chronic) had been immunosuppressed by cyclophosphamide before inoculation. The major clinical signs were initial fever, marked neutrophilia and, in the acute cases, respiratory distress. There were no signs of the nasal and ocular discharge, paresis or diarrhoea seen in acute cases in south-east Asia. The cyclophosphamide treatment caused a significant decrease in the neutrophil count by 7 d after inoculation in all 15 immunosuppressed pigs, and all were culture positive at necropsy. Eight of the 33 non-treated pigs were culture negative at necropsy. Pigs overcoming the initial phase of infection had more abscess-like nodules that were bacteriologically sterile at necropsy than the pigs with acute cases of melioidosis. P. pseudomallei was isolated predominantly from the spleen, lungs and the injection site. Although only one strain was used in this study, it is likely that Australian strains of P. pseudomallei are not as virulent as the south-east Asian isolates.     

Inhibitory effects of plant phenols on the activity of selected enzymes

Selected enzymes (alpha-amylase, trypsin, and lysozyme) were allowed to react with some simple phenolic and related compounds (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, m-, o-, and p-dihydroxybenzenes, quinic acid, and p-benzoquinone). The derivatized enzymes obtained were characterized in terms of their activity. In vitro experiments showed that the enzymatic activity of the derivatives was adversely affected. This enzyme inhibition depended on the reactivity of the phenolic and related substances tested as well as on the kind of substrate applied. The decrease in the activity was accompanied by a reduction in the amount of free amino and thiol groups, as well as tryptophan residues, which resulted from the covalent attachment of the phenolic and related compounds to these reactive nucleophilic sites in the enzymes. The enzyme inhibition correlates well with the blocking of the mentioned amino acid side chains.     

Root surface-inhabiting bacteria and the expression of clubroot of radish in controlled environments

The effect of bacteria on the expression of clubroot of radish caused by the obligate parasite, Plasmodiophora brassicae, was studied. Inoculations with the bacterial strains isolated from roots of radish plants increased slightly, decreased slightly or did not change the number of root hairs colonized by P. brassicae in the root hair, i.e. primary phase of colonization. Five of six bacterial strains decreased the incidence of clubroot which was associated with a decrease in the extent of P. brassicae -colonization of the stele, i.e. secondary phase colonization. The role of root surface-inhabiting bacteria in clubroot disease development in radish under controlled environmental conditions is discussed.     

Antioxidant activity of protein-bound quercetin

Bovine serum albumin (BSA) was derivatized by covalent attachment of different amounts of quercetin [ratios of BSA to quercetin were 20:1, 10:1, 7:1, 5:1, and 2:1 (w/w)]. The antioxidant activity of the protein-phenol derivatives was investigated using a modified TEAC assay. The results show that the covalent attachment of quercetin to BSA decreases the total antioxidant activity in comparison to an equivalent amount of free quercetin depending on the degree of derivatization. The derivative with the highest amount of covalently bound quercetin (the 2:1 derivative) showed an antioxidant activity of only 79% compared to an equivalent amount of free quercetin. After the enzymatic proteolysis of the BSA-quercetin derivatives with trypsin, the total antioxidant activity of the degradation products increases in comparison to the respective undigested derivatives but does not reach the activity of an equivalent amount of free quercetin. Even after 240 min of tryptic degradation, there is still a lack in antioxidant activity (for the 7:1 derivative nearly 33%) as compared to free quercetin.     

Ampicillin trihydrate in foals: serum concentrations and clearance after a single oral dose

Five foals from two to three days old were given a single oral dose of ampicillin trihydrate (20 mg/kg bodyweight [bwt]). Serum ampicillin concentrations were measured serially over a 24 h period. The study was repeated in the same foals at 16 to 21 days old. The mean peak serum ampicillin concentration at two to three days old was 5.0 micrograms/ml at 1 h after treatment; the mean peak serum concentration at 16 to 21 days old was 2.7 micrograms/ml at 2 h. The concentrations steadily declined and ampicillin was not detected in the serum from any of the foals by 24 h. Serum clearance averaged 17.7 ml/min/kg at two to three days and 35.8 ml/min/kg at 16 to 21 days.     

Proliferative enteropathy: a global enteric disease of pigs caused by Lawsonia intracellularis

Proliferative enteropathy (PE; ileitis) is a common intestinal disease affecting susceptible pigs raised under various management systems around the world. Major developments in the understanding of PE and its causative agent, Lawsonia intracellularis, have occurred that have led to advances in the detection of this disease and methods to control and prevent it. Diagnostic tools that have improved overall detection and early onset of PE in pigs include various serological and molecular-based assays. Histological tests such as immunohistochemistry continue to be the gold standard in confirming Lawsonia-specific lesions in pigs post mortem. Despite extreme difficulties in isolating L. intracellularis, innovations in the cultivation and the development of pure culture challenge models, have opened doors to better characterization of the pathogenesis of PE through in vivo and in vitro L. intracellularis-host interactions. Advancements in molecular research such as the genetic sequencing of the entire Lawsonia genome have provided ways to identify various immunogens, metabolic pathways and methods for understanding the epidemiology of this organism. The determinations of immunological responsiveness in pigs to virulent and attenuated isolates of L. intracellularis and identification of various immunogens have led to progress in vaccine development.     

Analysis of differential protein expression in Actinobacillus pleuropneumoniae by Surface Enhanced Laser Desorption Ionisation--ProteinChip (SELDI) technology

Actinobacillus pleuropneumoniae (APP) is the aetiological agent of porcine pleuropneumonia. An increased understanding of its molecular basis of pathogenicity and vaccine development will be facilitated by the availability of sequence data from a complete genome which, by analogy to other bacteria, is predicted to encode many proteins in the molecular mass range 3-20kDa. However, conventional techniques to study bacterial protein expression, such as SDS-PAGE and 2-dimensional electrophoresis, typically focus on the 15-200kDa range. In this study we have evaluated Surface Enhanced Laser Desorption Ionisation-ProteinChip (SELDI) technology for the analysis of protein expression, in particular those of <20kDa, of APP grown under different environmental conditions. Cytoplasmic/periplasmic and outer membrane protein fractions were obtained from the APP wildtype serotype 1 strain 4074 grown in Brain Heart Infusion (BHI) broth (+different concentrations of NAD), BHI containing pig serum or defined medium. Optimum conditions for SELDI profiles included a sample size of 1 microg and the use of sinapinic acid as the energy absorbing matrix. In the <20kDa range, the SELDI profiles obtained from wild-type bacteria grown in rich medium plus 33-66% pig serum were most similar to those grown in defined medium. The SELDI profiles of extracts of the wild-type and of an rpoE mutant were similar although there were clear differences. The results suggest that SELDI is a useful complementary approach to conventional proteomic analytical methods with APP, and presumably other bacterial pathogens, being particularly suited for analysis of proteins in the <20kDa mass range.     

Evaluation of protective immunity in pigs following oral administration of an avirulent live vaccine of Lawsonia intracellularis

OBJECTIVE: To evaluate the efficacy of an orally administered avirulent live vaccine to protect pigs against challenge exposure with virulent Lawsonia intracellularis. ANIMALS: 108 weaned 3-week-old pigs (35 in experiment 1 and 73 in experiment 2). PROCEDURE: 2 experiments were conducted. On day 0, vaccinates were orally administered vaccine via drench or in drinking water, whereas challenge-control pigs were administered cultured medium. On day 21, pigs were challenge exposed with a virulent heterologous isolate of L. intracellularis. Clinical observations, weights, seroconversion, and fecal excretion of L. intracellularis were measured until day 42. At study termination, pigs were euthanatized and examined for L. intracellularis-specific lesion development of the ileum and colon. RESULTS: Pigs receiving a single dose of vaccine were protected when challenge exposed with virulent L. intracellularis (at least 10(77) TCID50/dose). In experiment 1, vaccinates had significantly less fecal excretion (47% and 40% for days 35 and 42, respectively), compared with challenge-control pigs. In experiment 2, vaccinates had significantly less fecal excretion (50% and 58% for days 35 and 42, respectively), compared with challenge-control pigs. Significant reductions in lesion development were evident in the ileum of vaccinated pigs (70% and 56% at day 42 for experiments 1 and 2, respectively), compared with challenge-control pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration by drench or via drinking water of an avirulent live vaccine against L. intracellularis resulted in substantial protection against proliferative enteropathy among vaccinates and offers a better way to reduce stress of pigs during vaccine administration.