Experimental paratuberculosis in calves following inoculation with a rabbit isolate of Mycobacterium avium subsp. paratuberculosis.

The role of wildlife species in the epidemiology of paratuberculosis has been the subject of increased research efforts following the discovery of natural paratuberculosis in free-living rabbits from farms in east Scotland. This paper describes the experimental inoculation of young calves with an isolate of Mycobacterium avium subsp. paratuberculosis recovered from a free-living rabbit. After a 6-month incubation period, all eight calves inoculated with the rabbit isolate had developed histopathological and/or microbiological evidence of M. avium subsp. paratuberculosis infection. Similar results were obtained from a group of calves infected with a bovine isolate of M. avium subsp. paratuberculosis. The virulence of the rabbit isolate for calves demonstrated in this study suggests that rabbits are capable of passing paratuberculosis to domestic ruminants and that wildlife reservoirs of M. avium subsp. paratuberculosis should therefore be considered when formulating control plans for the disease.

Extra-anatomic redo of MIDCAB and OPCAB: an early experience.

BACKGROUND: Eighteen patients with unstable angina underwent repeat myocardial revascularization without cardiopulmonary bypass using saphenous vein grafts from either the left (13) or right (2) axillary arteries or the descending thoracic aorta (3). Patients' ages ranged from 53 to 85 years. Left ventricular ejection fractions ranged from 15% to 60%. METHODS: In 14 patients, the heart was exposed through an anterior thoracotomy, a minimally invasive direct coronary artery bypass (MIDCAB) technique. In 3 patients a left posterolateral thoractomy (lateral MIDCAB) was performed. One patient underwent repeat sternotomy (off-pump coronary artery bypass: OPCAB). In MIDCAB and lateral MIDCAB patients, the "target" vessel was a coronary artery in 8 patients and a previously placed vein graft in the remaining 9 patients. One patient underwent repeat sternotomy, and 3 coronary arteries were bypassed with a complex vein graft attached to the left axillary artery. Two patients died of mesenteric ischemia on the 2nd and 7th postoperative day. The remainder of patients were discharged from the hospital free of angina. Early graft patency was demonstrated by noninvasive vascular laboratory testing and/or angiography in the 13 survivors in whom the axillary artery had been the site of the proximal anastomosis. RESULTS: Follow-up ranged from 1 to 25 months. No other patients have died, and none have undergone additional surgical or catheter-based procedures. Three patients have developed recurrent angina, and in 4 patients the extra-anatomic bypass grafts have apparently become occluded. CONCLUSION: Extra-anatomic, off-pump bypass from the axillary artery or descending thoracic aorta to one or more coronary arteries can be performed safely in seriously ill patients requiring a repeat bypass procedure. The early results, regarding relief of angina, are encouraging.

Modulation of gammadelta T cells and CD1 in Mycobacterium avium subsp. paratuberculosis infection.

M.a. paratuberculosis is the causal agent of paratuberculosis (Johne's disease). Recent work has suggested that gammadelta T cells may play an important role in the early immunological response to mycobacterial diseases, and that CD1 may act as a non-classical MHC molecule in antigen presentation to these gammadelta T cells. Experimental infection of neonatal lambs with M.a. paratuberculosis was used to investigate the changes in gammadelta T cells and CD1 molecules in the gut associated lymphoid tissue 4 weeks after inoculation. Immunohistochemistry was used to label the gammadelta lymphocytes and CD1 molecules. An increase in the number of gammadelta T cells was noted in both the jejunal and ileal Peyer's patches in the gut of infected lambs, but no statistically significant change was found in the mesenteric lymph nodes. There were no obvious changes in the CD1 molecules in any tissue. This work suggests that gammadelta T cells may play a role in the initial immunological events of paratuberculosis infection.

Bovine IgG repertoire is dominated by a single diversified VH gene family.

Using methods based upon the PCR, we have analyzed sequences that encode the V domain of bovine Ig heavy chain. Pairwise comparison indicates that the heavy chain repertoire in cattle is dominated by a single VH gene family that is homologous to human VHII. The third complementarity-determining region (CDR3) is particularly variable and 60% of the sequences encode a CDR3 in excess of 21 amino acids. Southern blots, single-stranded conformation polymorphism analysis, and sequencing of segments from the germline indicate that the dominant VHII gene family is of small size, comprising as few as 10 unique members, and carries a very low level of diversity in CDRs 1 and 2. Comparison of germline VH sequences with those gathered from adult bovine splenic cDNA demonstrates a pattern of nucleotide substitution that is consistent with diversification through somatic hypermutation.

Splenic infarction caused by a large thoracic aortic thrombus.

We report on a patient with left upper quadrant pain as a result of splenic infarction; the patient was subsequently found to have a thoracoabdominal aortic thrombus extending through the celiac axis. The patient was successfully treated with an aortic thrombectomy guided by intraoperative transesophageal echocardiography.

PCR strategies for isolation of the 5' end of an immunoglobulin-encoding bovine cDNA.

We have employed two in vitro amplification strategies in our attempts to characterise the variable region of bovine immunoglobulin heavy chains (VH). Products derived by 5' RACE (rapid amplification of cDNA ends) spanned the coding sequence, but diversity in the complementarity determining regions was highly restricted, indicating that a limited subset of the heavy-chain repertoire had been recovered. In contrast, unique, full-length VH determinants were obtained with ease by an inverse application of the polymerase chain reaction. The strength of this approach is considerable: it is straightforward to perform, requiring none of the tailing procedures inherent to RACE strategies, yet it enables the rapid isolation of uncharacterized regions of genes for which limited sequence data are available. Our findings suggest strongly that the heavy-chain repertoire of cattle is highly dependent upon a very limited number of germline VH and JH (joining region) gene families.

Molecular characterization of bovine V lambda regions.

Using an inverse PCR technique with primers directed against constant region sequences, 18 V lambda regions have been isolated at random from bovine splenic cDNA for detailed analysis. On the basis of protein sequences in FR1 and their homologies to the human lambda chains NEWM and MCG, the bovine determinants can be separated into two families that have been designated V lambda 1 (94% of all clones isolated) and V lambda 2 (6% of all clones isolated). Scrutiny of the nucleotide sequences has allowed subdivision of the dominant family into V lambda 1a, V lambda 1b and V lambda 1c groupings, which represent 29, 65, and 6% of all V lambda 1 clones respectively. It appears that the size of CDR1 is V lambda group-specific--the region comprises 14 amino acids in V lambda 1a determinants and 13 residues in V lambda 1b and V lambda 1c,--whereas with a single exception, seven amino acids make up CDR2. The size of CDR3 is variable, ranging from 10 to 15 amino acids in length. Conservation of those nucleotide sequences that encode the signal peptide and FR3 has enabled us to amplify and characterize 14 V lambda 1 genes from kidney genomic DNA. Interestingly, half of the germline sequences were not represented in the data gathered from splenic cDNA, while the remaining genes could be identified as members of the V lambda 1a and V lambda 1b groups. The findings indicate that the V lambda 1 family dominates the expressed bovine light chain repertoire but that a large proportion of the germline segments from which this repertoire is derived may be nonfunctional.

Vectors to facilitate the creation of translational fusions to the maltose-binding protein of Escherichia coli.

A set of vectors has been constructed to facilitate the fusion of heterologous sequences to the C terminus of the maltose-binding protein (MBP) of Escherichia coli. The plasmids carry a cloning region comprising two blunt cloning sites, a BamHI site and multiple stop codons, and this has been placed in each reading frame so that translational fusions to MBP can be generated and manipulated with ease. To demonstrate the utility of this system, recombinant proteins have been engineered in which staphylococcal enterotoxin A has been fused to MBP.

Characterization of a herpes simplex virus type 1 deletion variant (1703) which under-produces Vmw63 during immediate early conditions of infection.

The herpes simplex virus type 1 deletion variant 1703 apparently fails to synthesize the essential IE2 gene product Vmw63 despite the deletion leaving the gene intact. Sequence analysis revealed that the deletion removes a region to the right of IE2 comprising the 3' end of IE1, UL56 and the 3' part of UL55, stopping 555 bp downstream of the IE2 polyadenylation signal. Further DNA sequencing has shown that there is no secondary mutation in the IE2 gene. Western blot analysis demonstrated that Vmw63 is made at reduced levels compared to that produced by the wild-type virus during immediate early conditions of infection. S1 nuclease protection mapping has revealed that this reduction is also apparent at the level of mRNA synthesis. A direct link between the deletion and the change in mRNA synthesis was provided by the insertion of a deletion-spanning fragment from 1703 into a 17+ genome, which resulted in the recombinant having a 1703-like phenotype. Evidence that down-regulation of IE2 mRNA during immediate early conditions of infection could be due to antisense RNA initiating from the IE1 promoter was obtained by the insertion of a novel transcriptional termination signal between IE1 and IE2 in the variant and the subsequent detection of wild-type levels of IE2 mRNA and protein.

Penetrating abdominal injuries in children and adolescents.

A total of 107 children and adolescents underwent laparotomies for penetrating trauma. There were 64 patients with stab wounds and 43 with gunshot wounds. Only 8 per cent of the patients were under 12 years of age. Three patients died--two from gunshot wounds of major vessels and one from a gunshot wound of the colon. Most of the injuries were related to crimes of violence or were self-inflicted. Multiple organ injuries and postoperative complications were significantly more common among patients with gunshot wounds than in those with stab wounds. Associated extra-abdominal injuries appeared to be more common among patients with gunshot wounds but the difference was not statistically significant. Unnecessary laparotomies were twice as frequent in victims of stab wounds.