Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera

Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of antibodies to Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA). However, none are commercially available in the UK. It was therefore necessary to develop a new, economic ELISA for use in a research project studying the epidemiology of CLA in UK sheep. The ELISA with its diagnostic qualities is presented. The ELISA was developed using sonicated C. pseudotuberculosis and optimised to detect total antibody or IgG class antibody in serum. Receiver operating characteristic (ROC) curves were obtained and the area under the ROC curve was used to compare the sensitivity and specificity of the two ELISAs. Both versions of the ELISA were evaluated on a panel of 150 positive reference sera and 103 negative reference sera. Using the test at 100% specificity, the sensitivity of detection of total antibody was 71% (95% confidence interval 63-78%), and the sensitivity of detection of IgG antibody to C. pseudotuberculosis was 83% (76-89%), which compares favourably with other reported ELISA tests for CLA in sheep. The sensitivity of the IgG antibody assay may be higher because of the greater affinity of IgG class antibodies compared with the IgM antibodies also detected by the total antibody ELISA. The results of ROC analysis indicated that the IgG isotype ELISA was more accurate than the total antibody ELISA. The efficiency of the test was greatest when serum samples were run in a dilution series than when any single serum dilution was used. The ELISA is considered to be suitable for application in field studies of CLA in UK sheep.     

Ontogeny of systemic cellular immunity in the neonatal pig: correlation with the development of post-weaning multisystemic wasting syndrome

The aetiology of porcine post-weaning multisystemic wasting syndrome (PMWS) is poorly understood. Porcine circovirus type 2 (PCV-2) is an essential component of the experimental disease model for PMWS: however, evidence from experimental and field studies indicates that additional factors play a critical role in the aetiopathogenesis of PMWS. Current candidates include (1) immune stimulation (for example, via co-infection or vaccination), and (2) a novel infectious agent. A prospective, longitudinal case-control study was designed to investigate molecular triggers in leucocytes of neonatal piglets that may predispose to the development of PMWS. Blood samples were collected weekly from pigs (n=125) within five farms, from 1 week to 8 weeks of age: that is, before the appearance of clinical signs. Four colour flow cytometry was used to investigate changes in subsets of peripheral blood mononuclear cells, using monoclonal antibodies against the following cell associated markers; sIgG, CD3, MHCII dR, CD14, CD4a, CD8a, CD45RC, CD25, SWC3a, SWC8, CD163 and CD45. Sampling and laboratory analysis was supported by monitoring of clinical signs from 1 week to 20 weeks of age, or until disease supervened. At the conclusion of the study, 68 pigs (54%) were classified in Group 1 (no signs of clinical disease), 34 pigs (27%) in Group 2 (signs of clinical disease but not characteristic of PMWS), 17 pigs (14%) in Group 3 (suspect PMWS case) and 5 pigs (4%) in Group 4 (PMWS case). A single case of Porcine Dermatitis and Nephropathy syndrome (PDNS) was also diagnosed. Significant changes with age were demonstrated in clinically normal, neonatal pigs (Group 1), including an increase in B-cells and T-cells, and an increase in the proportion of total T-cells expressing MHCII. Within the T-cell subset, the proportion of CD8(+high) CD4(-) T-cells increased, in addition to the proportion of CD4(+) T-cells co-expressing CD8. Of the factors recorded, farm was found to have a highly significant effect on immune system development in the neonate. Comparison of Groups 1 and 4 cases identified significant differences between pigs which remained normal and those which subsequently developed PMWS. Pigs which went on to develop PMWS had a greater proportion of T-cells expressing MHCII in early life, higher mean intensity of expression of MHCII on T-cells, higher mean intensity of expression of MHCII on B cells and higher expression of CD25 on CD45RC(-) T-cells. These findings suggest that lymphocyte activation may be a key early event in the aetiology of PMWS.     

Characteristics of Granular Cold‐Water Gelling Starches of Cereal Grains and Legumes Prepared Using Liquid Ammonia and Ethanol

Starches of wheat, corn, smooth and wrinkled peas, and chickpeas were modified to a free‐flowing powder of granular cold‐water gelling (GCWG) starch using liquid ammonia and ethanol at 23°C and atmospheric pressure. Amylose content of starches was 26.3% in wheat, 27.1% in corn, 35.4% in chickpeas, 43.2% in smooth peas, and 79.9% in wrinkled peas. The modified starches remained in granular form with an increased number of grooves and fissures on the surface of the granules compared with native starch, while the crystallinity was mostly lost, as shown by X‐ray diffractograms and DSC endothermic enthalpies. Pasting viscosity of modified starches at 23°C was 171 BU and 305 BU in wheat and corn, respectively, and much higher in legume starches, ranging from 545 BU to 814 BU. Viscosities of modified legume starches at 23°C were at least twice as high as those of native starches determined at 92.5°C. Swelling power of modified starches at 23°C ranged from 8.7 g/g to 15.3 g/g, while swelling power of native starches heated to 92.5°C ranged from 4.8 g/g to 16.0 g/g. GCWG starches exhibited higher dextrose equivalent (DE) values of enzymatic hydrolysis, ranging from 25.2 to 27.0 compared with native starches (1.5–2.9). Modified starches from wheat, corn, smooth peas, and chickpeas formed weak gels without heat treatment and experienced no changes in gel hardness during storage, while native starch gels formed by heat treatment showed an increase in hardness by 1.1–7.5 N during 96 hr of storage at 4°C.     

Post‐disturbance haulout behaviour of harbour seals

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Estimate of the annual economic impact of treatment of cranial cruciate ligament injury in dogs in the United States

OBJECTIVE: To estimate the economic impact to veterinary clients for the medical and surgical treatment of rupture of the cranial cruciate ligament (RCCL) in dogs for the year 2003. DESIGN: Economic impact survey. SAMPLE POPULATION: 501 diplomates of the American College of Veterinary Surgeons (ACVS) indicating that their area of surgical emphasis was small animal orthopedic surgery or small animal general and orthopedic surgery and 4,000 veterinarians indicating to the AVMA that their professional area was small animal practice exclusive or mixed animal practice (at least 80% small animal). PROCEDURE: Veterinarians were surveyed concerning the cost for medical and surgical treatment of RCCL for 2003. The economic impact was calculated by multiplying the number of RCCL surgeries performed by the mean cost of surgery. This was added to the number of RCCL cases managed medically multiplied by the mean cost of medical management. This estimate for survey responders was extrapolated to the total number of veterinarians in the study population for the ACVS or AVMA. RESULTS: Estimates for the total cost of surgery were $171,730,134.72 and $1,020,167,907 for veterinarians in the ACVS and AVMA populations, respectively. The cost of medical management was $2,885,687.86 and $126,558,155.16 for veterinarians in the ACVS and AVMA populations, respectively. After combining the ACVS and AVMA populations, we estimated that owners spent $1.32 billion for the treatment of RCCL in the United States in 2003. CONCLUSIONS AND CLINICAL RELEVANCE: RCCL is a prevalent, costly injury. Results may motivate veterinary and consumer agencies to prioritize funding for a better understanding of the injury.     

Jug r 4, a legumin group food allergen from walnut (Juglans regia Cv. Chandler)

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.