Gamma delta T cells are early responders to Mycobacterium avium ssp. paratuberculosis in colostrum-replete Holstein calves.

Peripheral blood mononuclear cells (PBMC) and mesenteric node lymphocytes (MNL) were obtained from 30 calves that were assigned randomly at birth to 1 of 6 treatment groups with 5 calves per treatment in a 14-d study: (1) colostrum-deprived (CD), no vitamins; (2) colostrum-replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; (6) CR, vitamins A, D3, E. Calves were injected with appropriate vitamin supplements and fed pasteurized whole milk (CD calves) or fractionated colostrum replacer (CR calves) at birth. Thereafter, all calves were fed pasteurized whole milk fortified with vitamins according to treatment group. Calves were orally inoculated with 108 cfu of Mycobacterium avium ssp. paratuberculosis (MAP) on d 1 and 3. The PBMC and MNL harvested on d 13 were analyzed by flow cytometry as fresh cells, after 3-d culture with phytohemagglutinin (PHA), and after 6-d culture with a whole-cell sonicate of MAP (MPS). Peripheral γδ T cells were a predominant lymphocyte subset in neonatal calves, with a decreased percentage noted in CD calves compared with CR calves. As well, CD25 expression was higher in γδ T cells compared with other cell subsets, regardless of treatment group. Stimulation of PBMC with PHA resulted in increased CD4+ and CD8+ subsets, whereas MNL response was dominated by expansion of B-cell subpopulations. Stimulation with PHA and MPS decreased the relative abundance of PBMC γδ T cells, but MNL γδ T cells increased upon stimulation with MPS. These results identify γδ T cells as key early responders to intracellular infection in neonatal calves and suggest that colostrum may be an important mediator of this response.

Brachyspira (Serpulina) hyodysenteriae gyrB mutants and interstrain transfer of coumermycin A(1) resistance.

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a lambdaZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A(1)-resistant (Cn(r)) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 microg of coumermycin A(1)/ml. The coumermycin A(1) MICs were 25 to 100 microg/ml for the resistant strains and 0.1 to 0.25 microg/ml for strain B204. Four Cn(r) strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly(78) to Ser (two strains), Gly(78) to Cys, and Thr(166) to Ala. When Cn(r) strain 435A (Gly(78) to Ser) and Cm(r) Km(r) strain SH (DeltaflaA1::cat Deltanox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56 degrees C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A(1), and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn(r) Km(r) Cm(r) strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn(r) Km(r) Cm(r) cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.

Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae.

Serpulina hyodysenteriae B204 cells treated with mitomycin (20 microg of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsC1 density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (deltaflaA1 593-762::cat) were added to growing cells of strain A216 (deltanox 438-760::kan), transductants (Cmr Kmr) were obtained at a frequency of 1.5 x l0(-6) per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete.

Mitomycin C induction of bacteriophages from Serpulina hyodysenteriae and Serpulina innocens.

A prophage was induced from cells of the pathogenic spirochaete Serpulina hyodysenteriae using mitomycin C. Five to seven hours after mitomycin C was added (8 micrograms/ml, final concentration) to S. hyodysenteriae B204 cultures in BHIS broth (OD620 = 0.9) cell lysis was detected as a decrease in culture optical density. Bacteriophage particles attached to whole cells and to cell debris were detected by electron microscopic analysis of negatively stained (2% PTA, pH 7.0) bacteria harvested by centrifugation from mitomycin C treated cultures. The phage particles consisted of a head (45 nm diameter) and a tail (64 nm x 9 nm). Bacteria from untreated cultures lacked phages detectable by electron microscopy. The appearance of bacteriophage particles in mitomycin C treated cultures correlated with the appearance of extrachromosomal DNA, 7-8 kb in size as estimated by agarose gel electrophoresis, in DNA preparations from treated S. hyodysenteriae cells. When cultures of other S. hyodysenteriae strains (B78, B169, A-1, B8044, B6933, Ack300/8, R-1) and S. innocens 4/71 in BHIS were treated with mitomycin C (8-15 micrograms/ml, final concentration), phages similar in morphology and size to the S. hyodysenteriae B204 were induced.

Left ventricular hypertrophy in left bundle branch block.

The detection of left ventricular hypertrophy (LVH) in the presence of left bundle branch block (LBBB) remains a difficult clinical problem. Its prevalence and significance have not previously been studied in a group of living patients. M-mode echocardiography was utilized to determine the prevalence of anatomic LVH in 28 patients with LBBB. Various ECG and chest x-ray criteria as predictors of LVH were assessed. Anatomic LVH was present in 89% by echocardiography. A left atrial abnormality on ECG and a cardio-thoracic ratio greater than .50 were the best predictors of LVH. Hypertension and/or ischemic heart disease was present in 78.5% of the patients while only one patient was free of any evidence of cardiovascular disease.

Prehospital brady-asystolic cardiac arrest.

Of 133 persons with spontaneous cardiac arrest attended by paramedics within 10 minutes, 100 (75%) had ventricular fibrillation as the initial rhythm and 33 (25%) had extreme bradycardia or asystole. The latter group of arrhythmias was characterized by sinus arrest or severe sinus bradycardia (90%) and complete A-V block (10%). Junctional escape rhythm was also absent or markedly retarded. Despite cardiopulmonary resuscitation and the administration of epinephrine, atropine, isoproterenol, and sodium bicarbonate, recovery of the sinus and junctional tissues was infrequent. Ventricular fibrillation developed in 11 cases (33%). One patient lived 12 days, but all others were dead on arrival or died in the emergency room. Among the 13 coronary causes of death proved at autopsy, 10 (77%) were due to a fresh thrombus and seven (54%) to an occluded proximal right coronary artery, suggesting a causal relation to this type of arrest.

Prehospital cardiac arrest after arrival of the Paramedic Unit.

Rapid response time by paramedic units made it possible to study 26 cases of "primary" cardiac arrests occurring after arrival of the unit. Ventricular fibrillation developed in 14 cases with prodromal ectopy in only two (14%) and rapidly increasing tachycardia in seven (50%). Countershock was successful in 12 (86%) and six (43%) survived. Bradycardia and asystole following countershock forecasted a fatal outcome. Brady-asystolic arrests (BAA) developed rapidly without much warning in 12 cases and were due to sinus arrest or severe sinus bradycardia in 92% and to atrioventricular block in 8%. BAA was 100% fatal. Coronary artery disease was diagnosed as the cause of BAA in seven (58%). All of the three cases, proven to be due to coronary artery disease at autopsy, had an occlusion of the proximal right coronary artery. In the remaining five (42%) cases, BAA was secondary to ruptured aneurysm (2), acute pancreatitis (1), chronic lung disease (1), and mitral stenosis (1). These observations emphasize a need for a more aggressive approach to prehospital management of brady-asystolic cardiac arrests.