Chromosomal DNA probes for the identification of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity of cats.

A dot-blot hybridisation assay using isolated high molecular weight DNA as whole chromosomal probes of the cat pigmented asaccharolytic Bacteroides/Porphyromonas species was used against both purified high molecular weight DNA and DNA released on membranes from whole cells for the identification of B. salivosus and for its differentiation from the other anaerobic species isolated from normal and diseased mouths of cats and horses. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes (Boehringer-Mannheim). The whole chromosomal probes were specific--differentiating B. salivosus from a variety of species (including members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. Likewise, asaccharolytic black pigmented Group 2 strains were distinguishable from all strains tested. However, cat strains of P. gingivalis which show 68-76% DNA-DNA homology with human strain P. gingivalis ATCC 33277T, were not distinguishable from each other using either 32P-labelled or DIG-labelled probes. The minimum amount of pure Bacteroides DNA which could be detected by the 32P-labelled probe was 100-300 pg, while the amount of pure DNA detected by the DIG system was 1-3 mg after room temperature colour development for 1 h and 100-300 pg after 6 h colour development.     

Recovery of Campylobacter jejuni in feces and semen of caged broiler breeder roosters following three routes of inoculation

We previously reported the recovery of Campylobacter (naturally colonized) from the ductus deferens of 5 of 101 broiler breeder roosters, and four of those five positive roosters had previously produced Campylobacter-positive semen samples. Those results prompted further evaluation to determine if inoculation route influenced the prevalence or level of Campylobacter contamination of semen, the digestive tract, or reproductive organs. Individually caged roosters, confirmed to be feces and semen negative for Campylobacter, were challenged with a marker strain of Campylobacter jejuni either orally using 1.0 ml of a diluted cell suspension (log(10)4.3 to 6.0 cells), by dropping 0.1 ml of suspension (log(10)5.3 to 7.0 cells) on the everted phallus immediately after semen collection or by dip coating an ultrasound probe in the diluted cell suspension (log(10)4.3 to 6.0 cells) and then inserting the probe through the vent into the colon. Six days postinoculation, individual feces and semen samples were again collected and cultured for Campylobacter. Seven days postinoculation, roosters were killed, the abdomen aseptically opened to expose the viscera, and one cecum, one testis, and both ductus deferens were collected. The samples were then suspended 1:3 (weight/volume) in Bolton enrichment broth for the culture of Campylobacter. Samples were also directly plated onto Cefex agar to enumerate Campylobacter. Campylobacter was recovered 6 days after challenge from feces in 82% of samples (log(10)4.1 colony-forming units [CFU]/g sample), 85% of semen samples (log(10)2.9 CFU/ml), and on the seventh day postchallenge from 88% of cecal samples (log(10)5.8 CFU/g sample). Campylobacter was not directly isolated from any testis sample but was detected following enrichment from 9% (3/33) of ductus deferens samples. Roosters challenged with Campylobacter orally, on the phallus, or by insertion of a Campylobacter dip-coated ultrasound probe were all readily colonized in the ceca and produced Campylobacter-positive semen and feces on day 6 after challenge. The low prevalence of recovery of Campylobacter from the ductus deferens samples and failure to recover from any testis sample suggests that semen may become Campylobacter positive while traversing the cloaca upon the everted phallus. The production of Campylobacter-positive semen could provide a route in addition to fecal-oral for the horizontal transmission of Campylobacter from the rooster to the reproductive tract of the hen.     

Endotoxin-induced digital vasoconstriction in horses: associated changes in plasma concentrations of vasoconstrictor mediators

REASONS FOR PERFORMING STUDY: Lipopolysaccharide (LPS) infusion reduces digital perfusion, but the mediators responsible remain undetermined. OBJECTIVES: To identify vasoconstrictor mediators released following LPS infusion and relate their appearance in plasma to digital blood flow alterations. METHODS: Blood flow in the lateral digital vessels of 6 Thoroughbred horses, following a 30 min infusion of LPS (E. coli 055:B5; 30 ng/kg), was measured using Doppler ultrasonography. Concomitant measurements of hoof wall and coronary band surface temperatures (HWST and CBST) were made. Serial blood samples were collected and plasma LPS, tumour necrosis factor alpha (TNFalpha), 5-HT, thromboxane B2 (TxB2) and endothelin measured. RESULTS: Plasma LPS concentrations reached a maximum of 13.2 pg/ml during the infusion, followed by an increase in plasma TNFalpha concentration. Digital arterial and venous blood flow decreased by 43 and 63%, respectively; HWST and CBST similarly decreased. Systemic blood pressure remained unaltered. Plasma concentrations of TxB2 and 5-HT increased, coinciding with the onset of digital hypoperfusion. Plasma endothelin concentrations remained unchanged. CONCLUSIONS: The temporal relationship between the onset of digital hypoperfusion and increases in plasma 5-HT and TxB2 concentrations is consistent with these platelet-derived mediators being associated with LPS-induced laminitis. POTENTIAL RELEVANCE: These experimental data support the use of anti-platelet therapy in the prevention of laminitis associated with endotoxaemic conditions.     

Uptake of 5-hydroxytryptamine by equine digital vein endothelial cells: inhibition by amines found in the equine caecum

REASONS FOR PERFORMING STUDY: 5-hydroxytryptamine (5-HT; serotonin) is a potent vasoconstrictor of equine digital blood vessels and has been implicated in the pathogenesis of acute laminitis. OBJECTIVES: The aims of this study were firstly to examine whether cells of the digital blood vessel wall exhibited an active uptake mechanism for 5-HT and to characterise its efficiency; and secondly, to study the potential inhibitory effect on this process of other amines, produced in the equine caecum. METHODS: Confluent monolayers of equine digital vein endothelial cells (EDVEC) and equine digital vein smooth muscle cells (EDVSMC) were incubated with [3H]5-HT (0.1-250 micromol/l) and the total and active uptake calculated. Equine pulmonary vein endothelial cells (EPVEC) were used as a positive control. RESULTS: Both EDVEC and EDVSMC showed uptake of [3H]5-HT by nonfaci litated diffusion; however, only EDVEC showed evidence of saturable facilitated uptake mechanism, with a Km of 41.6 +/- 9.3 micromol/l, which was significantly higher than that of EPVEC (9.9 +/- 2.1 micromol/l). All 6 caecally-derived amines examined (tyramine, spermine, isoamylamine, tryptamine, phenylethylamine and isobutylamine) inhibited the total uptake of [3H]5-HT in a concentration-dependent manner, tyramine having the lowest IC50 (3.7 x 10(-6) mol/l). CONCLUSIONS: These data suggest that facilitated uptake into the endothelium could play a role in moderating the vasoconstrictor response to 5-HT in the equine digital circulation. POTENTIAL CLINICAL RELEVANCE: The vasoconstrictor action of 5-HT could be potentiated by gut-derived amines, providing a feasible link between GI disturbances and the pathophysiology of laminits.     

Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells

The mechanisms by which Mycobacterium avium subspecies paratuberculosis (M. a. ptb) survives within macrophages are not well characterized. One strategy for intracellular survival developed by Mycobacterium tuberculosis is inhibition of phagosomal maturation. In this study it was hypothesized that M. a. ptb is capable of survival within macrophages by residing within a phagosomal compartment that does not mature into a functional phagolysosome. To test this hypothesis the following objectives were determined. Phagosomal maturation was evaluated by comparison of stage specific markers on the membranes of phagosomes containing live M. a. ptb with those containing killed M. a. ptb, Mycobacterium smegmatis, and zymosan A using immunofluorescent labeling and confocal microscopy. Intracellular survival of live M. a. ptb within J774 macrophages was compared to that of M. smegmatis by direct determination of bacterial viability by differential live/dead staining. The results of this study show that the phagosomes containing live M. a. ptb had increased levels of an early marker (transferrin receptor [TFR]) and decreased levels of a late maturation marker (lysosome associated membrane protein one [Lamp-1]), relative to those containing killed M. a. ptb, M. smegmatis, and zymosan A. Additionally, compared to M. smegmatis, M. a. ptb has enhanced ability to survive within cultured macrophages. These data indicate that M. a. ptb resists intracellular killing by residing within a phagosomal compartment that retains the characteristics of early phagosomes and resists maturation into functional phagolysosome.     

Incidence and tracking of Clostridium perfringens through an integrated broiler chicken operation

Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations. In a previous study, ribotypes of certain strains of C. perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining trays used to transport commercial chicks from the hatchery to the grow-out facility on the farm. These results suggest that C. perfringens contaminating the processed product could originate from facilities in the integrated poultry operation prior to grow out. In this study, samples were collected from the breeder farm, hatchery, previous grow-out flock, during grow out and after processing. In the first trial, C. perfringens was recovered from the breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, from processed carcasses, and from the breeder farm after processing in 4%, 30%, 4%, 0%, 2% and 16%, and 4% of the samples, respectively. In the second trial, the incidence of C. perfringens in samples collected from breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, and fromprocessed carcasses was 38%, 30%, 32%, 8%, 4%, and 8%, respectively. The genetic relatedness of the isolated strains as determined by ribotyping suggests that C. perfringens may be transmitted between facilities within the integrated broiler chicken operation.     

Alterations in the organisation, ultrastructure and biochemistry of the myocardial collagen matrix in doberman pinschers with dilated cardiomyopathy

Remodelling of the collagen matrix of the myocardium has been implicated in the pathogenesis of dilated cardiomyopathy, a major cause of heart failure in Doberman pinschers. The aim of this study was to characterise the myocardial collagen matrix of Dobermans. In clinically normal Dobermans there was evidence of focal fibrosis. Collagen cross-links were altered in both diseased and clinically normal Doberman myocardium as compared with myocardium from control dogs. Extensive remodelling, in the form of a loss of collagen tethers, increased collagen synthesis and alterations in the collagen cross-links, occurs in diseased Doberman myocardium. Changes in the collagenous matrix are also present in apparently normal Dobermans. These changes are likely to be involved in the progression of the disease and may explain the predisposition of this breed to dilated cardiomyopathy.