Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.     

Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence

Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD₅₀ experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.     

Use of radiographic measures and three-dimensional computed tomographic imaging in surgical correction of an antebrachial deformity in a dog

CASE DESCRIPTION: A 1-year-old 7.4-kg (16.3-lb) castrated male mixed-breed dog was evaluated because of intermittent lameness and an antebrachial angular limb deformity. CLINICAL FINDINGS: The left forelimb had gross antebrachial external rotation (approx 90 degrees ) and marked procurvatum. Radiography revealed a severe partially compensated biapical antebrachial angular limb deformity. Measurements of medial proximal radial angle (MPRA) and lateral distal radial angle (LDRA) were obtained from orthogonal radiographs of the proximal and distal segments of the radius, respectively. Elbow joint-to-carpus translation was quantified. Deformities were localized and quantified by the center of rotation of angulation (CORA) method. Computed tomographic 3-dimensional image reconstructions of the antebrachium and carpus were completed to create 3 life-size stereolithographic models. TREATMENT AND OUTCOME: 2 closing wedge radial osteotomies were performed at the level of the CORAs and stabilized with bone plates and screws. RESULTS: Frontal and sagittal plane alignments were corrected to 8 degrees and 15 degrees , respectively (reference limits, 0 degrees to 8 degrees and 8 degrees to 35 degrees , respectively). The MPRA was corrected from 55 degrees to 68 degrees , and LDRA was corrected from 32 degrees to 76 degrees (values considered normal are approx 85 degrees and 87 degrees , respectively). Elbow joint-to-carpus translation was improved by 42.5%. After 8 weeks, radiography revealed bone union. Owners considered the outcome acceptable, on the basis of limb appearance and lack of lameness at 1 year after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: A segmental radiographic planning technique combined with the CORA method, computed tomography, and stereolithography may be useful in the characterization of and planning corrective surgery for forelimb deformities in dogs.     

A real-time polymerase chain reaction assay for the detection of the myxozoan parasite Henneguya ictaluri in channel catfish

Proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya ictaluri, is the most prevalent parasitic infection affecting commercial channel catfish (Ictalurus punctatus) aquaculture. There are currently no effective chemotherapeutic or biological control measures for PGD, which often peaks during the spring and fall when water temperatures are between 16-25 degrees C. The current diagnostic techniques of gross examination of gill clip wet mounts and histopathology are subject to false-negatives during the early stages of infection, and the quantifiable nature of end-point polymerase chain reaction (PCR) is subjective. Consequently, a rapid and more sensitive quantitative real-time PCR assay was developed for the detection of H. ictaluri during the early stages of infection in channel catfish. A 23 base-pair TaqMan probe was designed based on previously published H. ictaluri PCR protocols. The sensitivity of the assay was the equivalent of a single H. ictaluri actinospore, and in a pond challenge study, quantitative real-time PCR proved to be more sensitive than gross examination, microscopic examination of gill clip wet mounts, and histopathologic examination of gill tissue sections. Future applications of this assay will focus on developing methodologies to be used in conjunction with current pond-monitoring protocols to evaluate potential treatments and better manage this significant seasonal disease.