Comparison of intradermal and serum testing for allergen-specific IgE using a Fcepsilon RIalpha-based assay in atopic dogs in the UK

Atopic dermatitis in dogs is a common allergic skin disease that affects substantial numbers of dogs in the UK. The purpose of this study was to compare the results of an intradermal test (IDT) and an in vitro test in a large cohort of dogs. Dogs were intradermal tested with Greer allergens (Greer Labs Inc, Lenoir, NC, USA) using standard techniques. At the same time blood samples were drawn and submitted for evaluation by ELISA using the ALLERCEPT Definitive Allergen Panels for allergen-specific IgE, a commercial assay that uses a biotinylated recombinant extracellular domain of the high affinity Fc-epsilon receptor alpha chain protein (Fcepsilon RIalpha). The allergens used in the two tests included grass, tree and weed pollens, moulds, flea saliva/whole flea extract and house dust mite species. The optical density readings from the ELISA for each allergen were compared with the results of the IDT for 265 dogs. The prevalence of positive reactions in the ELISA was equal to or greater than the results of the IDT in the case of almost all of the allergens, but two notable exceptions were the house dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus. These two allergens were the most common positive reactions by IDT (prevalence D. farinae 78.9%, D. pteronyssinus 66.4%). The results of the two tests were significantly different (McNemar's test, P<0.05) for 16 of the 22 allergens. The sensitivities of the ELISA compared to the IDT (where there were more than 3 dogs with positive reactions in both tests) varied between 19.3 and 77.1% (D. pteronyssinus 19.3% and D. farinae 67.9%) and the specificities varied between 64.2 and 96.6% (D. pteronyssinus 96.6% and D. farinae 89.3%).     

Morphological relationship between intercalated duct and pancreatic islet in streptozotocin and/or camostat mesilate administrations in the chicken

Present electron microscopical and immunocytochemistrical studies elucidated some morphological relationship between intercalated duct (ICD) and pancreatic islet cells in the chicken in streptozotocin (STZ) and/or camostat mesilate (CM) administrations. Twenty-one chickens were set into four experimental groups: (1) control group, (2) STZ administration group, (3) CM administration group, and (4) STZ + CM administration group. Cytoplasms of ICD cells stained more strongly with eosin in STZ administration group than other groups, and electron-dense materials and intercalated processes between ICD and islet cells were also increasing in time dependence in STZ administration. Number of pancreatic islet in STZ + CM co-administration was about 3.1 times larger than other groups. Many small sized cells were detected at surrounding area of ICD and they incorporated 5-bromo-2'-deoxyuridine better than other experimental groups. Present morphological data suggested that ICD cells might support some tolerances of pancreatic endocrine cells against toxic substances and also involve in regeneration of new pancreatic islet cells in STZ + CM co-administration.     

Characterisation of Hungarian porcine circovirus 2 genomes associated with PMWS and PDNS cases

The authors report the data of the first survey on the incidence of post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) in Hungary. A PCR method specific for the detection of porcine circovirus 2 (PCV-2) was developed, which proved to be suitable for diagnostic purposes. PCR screening of organ samples from pigs suspected to be affected with PMWS or PDNS revealed the presence of PCV-2 in 80% of the cases. Six PCV-2 genomes from Hungarian isolates were completely sequenced. Phylogenetic comparison with all the available PCV-2 sequences showed that porcine circoviruses circulating in Hungary are more variable than in several other European countries. Two Hungarian strains clustered together with the Spanish strains forming a distinct group; two others fell in a common group with the French, UK, and Dutch strains, whereas another two strains showed the closest relationship to two of the three known German PCV-2 sequences.     

Recombinant Iss as a potential vaccine for avian colibacillosis

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 microg of GST-Iss, and 1/12 chickens immunized with 50 microg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.     

Agreement between direct, oscillometric and Doppler ultrasound blood pressures using three different cuff positions in anesthetized dogs

ObjectiveTo evaluate the agreement between invasive blood pressure (IBP) and Doppler ultrasound blood pressure (DUBP) using three cuff positions and oscillometric blood pressure (OBP) in anesthetized dogs.Study designProspective study.AnimalsNine adult dogs weighing 14.5–29.5 kg.MethodsThe cuff was placed above and below the tarsus, and above the carpus with the DUBP and above the carpus with the OBP monitor. Based on IBP recorded via a dorsal pedal artery catheter, conditions of low, normal, and high systolic arterial pressures [SAP (mmHg) <90, between 90 and 140, and >140, respectively] were induced by changes in isoflurane concentrations and/or dopamine administration. Mean biases ± 2 SD (limits of agreement) were determined.ResultsAt high blood pressures, regardless of cuff position, SAP determinations with the DUBP underestimated invasive SAP values by more than 20 mmHg in most instances. With the DUBP, cuff placement above the tarsus yielded better agreement with invasive SAP during low blood pressures (0.2 ± 16 mmHg). The OBP underestimated SAP during high blood pressures (?42 ± 42 mmHg) and yielded better agreement with IBP for mean (MAP) and diastolic (DAP) arterial pressure measurements [overall bias: 2 ± 15 mmHg (MAP) and 0.2 ± 16 mmHg (DAP)].ConclusionsAgreement of SAP determinations with the DUBP is poor at SAP > 140 mmHg, regardless of cuff placement. Measurement error of the DUBP with the cuff placed above the tarsus is clinically acceptable during low blood pressures. Agreement of MAP and DAP measurements with this OBP monitor compared with IBP was clinically acceptable over a wide pressure range.Clinical relevanceWith the DUBP device, placing the cuff above the tarsus allows reasonable agreement with IBP obtained via dorsal pedal artery catheterization. Only MAP and DAP provide reasonable estimates of direct blood pressure with the OBP monitor evaluated.     

Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells

The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4(+)CD25(+) cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar kinetics to those of the three classical SLA class I genes. The expression profiles detected by flow cytometry of the SLA molecules on the cell surface of PBMCs were maintained at a consistently high level during cell stimulation with either TSST-1 or IFN-γ, which was distinct from the kinetics of mRNA expression. These results showed that miniature swine SLA class I mRNA expression was effectively and equally up-regulated among the three loci and coordinately with IRF-1 gene expression after stimulation of T cell activation by sAG or IFN-γ.     

First detection of 'Candidatus Mycoplasma haemolamae' infection in alpacas in England

This is the first report of detection of Candidatus Mycoplasma haemolamae in alpacas in England. The primary case occurred in a three year-old male alpaca in the south-east of England which presented with a history of progressive weight loss, lethargy, swelling of the scrotum and pale mucous membranes. Blood smear examination revealed a moderate, regenerative anaemia, with numerous small basophilic coccoid structures consistent with Candidatus M haemolamae. To confirm the presence of Candidatus M haemolamae, a portion of the 16S rDNA gene was amplified and analysed by denaturing gradient gel electrophoresis (DGGE). 16S rDNA gene sequencing showed a 99.8 per cent homology with Candidatus M haemolamae sequences deposited in GenBank. Subsequently, a cross-sectional study was carried out to investigate the presence of Candidatus M haemolamae infection in the alpaca herd from which the primary case was detected (n=131). Blood smear examinations and PCR with DGGE were used and compared with a species-specific PCR. The prevalence of infection when PCR positive results were combined was 29 per cent. A substantial agreement between the PCR/DGGE and the species-specific PCR was found (κ=0.86). A significant association was also found between age and infection (P=0.04) while no significant association was found with sex or origin.     

Nuclear Replacement of In Vitro‐Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

The objective of the present study was to establish a method for nuclear replacement in metaphase‐II (M‐II) stage porcine oocytes. Karyoplasts containing M‐II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro‐matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona‐free M‐II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2–77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2–32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0–15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri‐Fusion) is an effective method for producing M‐II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.