Understanding the molecular epidemiology of the footrot pathogen Dichelobacter nodosus to support control and eradication programs.

The Gram-negative anaerobe Dichelobacter nodosus is the primary etiologic agent of ovine footrot. Few studies of the genetic diversity and epidemiology of D. nodosus have been done, despite the economic cost and welfare implications of the disease. This study examined a large collection of Australian isolates; 735 isolates from footrot-infected sheep from 247 farms in Western Australia (WA) were tested by pulsed-field gel electrophoresis (PFGE), and a subset of 616 isolates was tested by infrequent restriction site PCR (IRS-PCR). The genetic diversity of WA isolates was compared to that of 61 isolates from three other Australian states. WA isolates were genetically diverse, with 181 molecular types resolved by PFGE, resulting in a simple diversity ratio (SDR) of 1:4 and a Simpson's index of discrimination value (D) of 0.98. IRS-PCR resolved 77 molecular types (SDR = 1:8 and D = 0.95). The isolates were grouped into 67 clonal groups by PFGE (SDR = 1:11, D = 0.90) and 36 clonal groups by IRS-PCR (SDR = 1:17, D = 0.87). Despite the high genetic diversity, three common clonal groups predominated in WA and were found in other Australian states. On some farms, molecular type was stable over a number of years, whereas on other farms genetically diverse isolates occurred within a flock of sheep or within a hoof. This study provides a large database from which to appropriately interpret molecular types found in epidemiological investigations and to identify common and unknown types that may compromise footrot eradication or control programs.

Eradication of ovine footrot by repeated daily footbathing in a solution of zinc sulphate with surfactant.

OBJECTIVE: To investigate the effect on ovine footrot of repeated daily footbathing in a solution of zinc sulphate with surfactant. DESIGN: Merino sheep were allocated to control and treatment groups of 119 sheep each at week 0. The sheep had a history of S1, U1, T and/or U6 types of Dichelobacter nodosus in interdigital and underrunning footrot lesions. Feet were not pared prior to treatment. PROCEDURE: Treatment sheep were footbathed in a 15 to 18% (w/v) solution of zinc sulphate with surfactant for 10 min on five consecutive days during week 1. At week 2, and fortnightly to week 52, all feet were inspected, lesion scores were recorded and samples were taken for laboratory tests. At week 53, all feet with no lesions at week 52, but with underrunning lesions prior to week 1, were pared and samples were taken. RESULTS: After footbathing, there were no lesions in any treatment sheep at any inspection to week 52. The percentage of feet of control sheep with lesions increased from 9% (391 of 4,284) between weeks 20 and 36, to 14% (593 of 4,284) between weeks 36 and 52. Ninety-five of 96 control sheep with no lesions at week 20 were still asymptomatic at week 52. D nodosus was not isolated from samples taken from 99 and 87 pared feet of treatment and control sheep, respectively. CONCLUSION: Repeated daily footbathing combined with prolonged exposure to a dry environment eradicated footrot in sheep with both interdigital and underrunning lesions in feet that were not pared prior to treatment.

Clinical practice and the UK Human Rights Act 1998: protecting individual rights in the interest of the wider community.

Health professionals have expressed concern that the UK Human Rights Act 1988, which came into force in 2000, may threaten their autonomy in clinical practice/decision-making and resource allocation by health authorities. Although healthcare-related cases have so far been slow to emerge under the Act, it seems clear that in clinical practice the transition from duty to legal obligation will involve a degree of change for health professionals, in attitude if not in behaviour. With regard to resource allocation, it appears that the UK courts are likely to consider challenges to health authorities' decisions in a way that takes into account the need to set priorities, so long as these decisions do not discriminate unfairly and can be shown to have been made in the best interest of the wider community.

Occurrence of different strains of Dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections.

OBJECTIVE: To investigate the occurrence of S1, U1 and T strains of Dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections. DESIGN: Seventy-seven donor sheep were grazed with 84 recipients for 33 weeks. The donor sheep were Merinos with a history of clinically virulent footrot associated with protease type S1, U1 and T strains of D nodosus that hybridised with gene sequences pJIR314B, pJIR318 and/or pB645-335. The recipient sheep were Merinos with no history of footrot. PROCEDURE: Each fortnight, all feet were examined, their lesion scores were recorded and samples of lesion material were taken for laboratory tests. RESULTS: Eighty-nine percent (299 of 336) of feet of recipient sheep developed new clinical lesions. S1, U1 and T strains of D nodosus were recovered from 58%, 22% and 18%, respectively, of these lesions at a ratio that remained constant during two apparent peaks in footrot transmission. Gene sequences homologous to pJIR314B and pB645-335 were detected in 56% (93 of 166) and 29% (48 of 166), respectively, of S1 strains of D nodosus at a ratio that was not constant during the experiment. CONCLUSIONS: S1 was the dominant protease type of D nodosus in new clinical lesions. The occurrence of S1 strains did not increase relative to U1 and T strains of D nodosus during the experiment. S1, U1 and T strains of D nodosus remained in equilibrium despite changes in environment, genetic types in the population of S1 strains, and host resistance to footrot.

Severity and persistence of footrot in Merino sheep experimentally infected with a protease thermostable strain of Dichelobacter nodosus at five sites.

OBJECTIVE: To test the hypothesis that ovine footrot associated with a thermostable protease strain of Dichelobacter nodosus undergoes self cure or is sustained as an annually recurring disease, depending on the environment. DESIGN AND PROCEDURE: Forty Merino sheep from a single blood line were infected with a protease thermostable strain of D nodosus at each of five sites in Western Australia. Footrot lesions and microscopic evidence of D nodosus were recorded every fortnight for 2.5 years, supplemented by laboratory culture. Rainfall, soil and air temperature, pasture quantity and composition and soil types were also recorded. Flocks that apparently self cured were relocated to a more favourable site for footrot in the final spring season. RESULTS: The maximum prevalence of feet with clinical footrot lesions was 80.6, 1.3, 14.4, 3.8 and 88.1% at the five sites. Severe footrot occurred for three consecutive spring seasons at one site that had clay loam soil and at least 3500 kg/ha total pasture dry matter annually. However, the infection was asymptomatic for up to 10 weeks between outbreaks. D nodosus was isolated from flocks for 2.5 years at only two sites, although there was microscopic evidence of the organism at other sites in the final year. A thermolabile variant (strain U6) of D nodosus was isolated from the two sites where footrot persisted. CONCLUSION: Depending on time and location, ovine footrot induced by a protease thermostable strain of D nodosus either self cured or persisted as annual outbreaks interspersed with periods of asymptomatic infection.

Comparison of gene probe and conventional methods for the differentiation of ovine footrot isolates of Dichelobacter nodosus.

In a collaborative study that involved four Australian veterinary diagnostic laboratories a gene probe test based on the recombinant plasmids pJIR318, pJIR314B, and pJIR313, which contain genomic vap or vrl regions, was compared with conventional tests used for the differential diagnosis of ovine footrot. A total of 771 clinical dichelobacter nodosus isolates were tested and designated as belonging to one of several gene probe categories. The results showed that 87% of the virulent isolates belonged to gene probe category 1, compared to only 6% of the benign isolates. It was concluded that there was good correlation between the gene probe test and the virulence designation of these isolates as well as the results of elastase, gelatin-gel and protease isoenzyme tests. Furthermore, the gene probe test was converted to a polymerase chain reaction (PCR)-based test. It is suggested that diagnostic laboratories consider carrying out both this PCR test and tests based on the extracellular proteases of D. nodosus.

Effects of exposure to stressors of varying predictability on adrenal function in rats.

For 5 days, rats were exposed to shocks that were signalled by a light 0, 33, 66, or 100% of the time. Basal hormone levels and responses to a light-shock pair were measured daily. Greater predictability was associated with higher basal plasma corticosterone and norepinephrine levels indicative of chronic stress. Habituation of the corticosterone response was also less in the groups with greater predictability. However, predictability did not affect plasma prolactin or epinephrine responses. Because the endocrine systems responded differently, it is unlikely that the changes were due to a unitary process. Greater predictability appeared to be more stressful in this paradigm. Both associative and nonassociative factors have major roles in determining the hormonal responses to repeated presentation of stressors.

The laboratory culture of Dichelobacter nodosus in a footrot eradication program.

As part of a program to eradicate virulent footrot from Western Australia, 2745 isolates of Dichelobacter nodosus were isolated from 5263 specimens from 1883 submissions. The virulence of each isolate was assessed using protease thermostability and isoenzyme zymogram. We describe changes to the materials and methods required to handle these specimens and to reduce the interval between submission and report to 8 days.

Ridge augmentation using a subepithelial connective tissue pedicle graft.

The deficient labial ridge can present aesthetic problems for the patient and technical problems for the restorative dentist. The learning objective of this report is a technique for obtaining connective tissue from an adjacent donor site in the form of a pedicle and positioning it beneath a partial thickness flap. The technique offers advantages over previous methods in terms of patient comfort, vascularization of the graft, and specific placement of the grafted connective tissue.

Role of the adrenal gland in the thermal response to morphine withdrawal in rats.

Administration of naloxone to morphine-dependent rats results in an elevation of tail skin temperature and a fall in core temperature. Previous studies have demonstrated a role of the adrenal gland in the thermal responses that accompany morphine withdrawal in the rat. In the present study, experiments were designed to determine if the duration of adrenalectomy significantly influenced the thermal response observed in morphine withdrawal. In addition we evaluated the influence of the adrenal medulla and glucocorticoid replacement in adrenalectomized rats in mediating the thermal responses of the morphine-dependent rat. Ovariectomized rats were addicted to morphine and subsequently withdrawn by administration of naloxone. This treatment results in a significant rise in tail skin temperature and subsequent fall in colonic temperature. These thermal responses were not observed in morphine-naive rats. Adrenalectomy resulted in a significant attenuation of the rise in tail skin temperature associated with withdrawal. This reduced tail skin temperature response was not different among animals adrenalectomized for 1, 7, 14, 21, or 28 days. Likewise, the moderate increase in core temperature associated with morphine treatment was not observed in the adrenalectomized rats. Serum corticosteroid determinations confirmed the loss of the adrenal steroids in the adrenalectomized rats. In a subsequent experiment it was determined that adrenal demedullation did not reduce the tail skin temperature response during morphine withdrawal, and corticosteroids restored the naloxone-induced surge in tail skin temperature in morphine-dependent, adrenalectomized rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Classical aversive conditioning of catecholamine and corticosterone responses.

Some earlier work, not rigorously controlled, suggested that conditional increases in sympathoadrenal stress hormones could occur. The purpose of this experiment was to test this idea further using an appropriately controlled design. To do this, we subjected rats living in a tether-type apparatus to differential fear conditioning. Chronic catheterization allowed us to sample blood before and after conditional stimulus probes without having to touch the rats. No evidence for conditional changes in norepinephrine and corticosterone was found. In contrast, differential conditioning of epinephrine responses was found. The conditional response, however, was not a simple one in that conditional increases in epinephrine following CS+ probes were not always seen. These data support the idea that learned changes in hormonal stress respondents can occur. But they leave open the question of why clear cut conditional changes in these visceral systems are so difficult to obtain.

Effects of repeated stress on T cell numbers and function in rats.

Although stress has been reported to affect various functions of the immune system, the mechanism that mediate these effects remain unclear. Thus we examined the effects that 1, 7, and 14 days of stress could have on various aspects of immune and endocrine function in rats. Rats subjected to repeated stress (7 and 14 days) showed significant decreases in the total number of mononuclear cells, particularly suppressor/cytotoxic (CD8) T cells, in the spleen and blood. The mitogenic responses of T cells to phytohemagglutinin (PHA) and concanavalin-A (Con A) were also significantly diminished at these times, as well as after acute (1 day) stress in the case of PHA stimulation. The mechanisms of this impaired T cell mitogenesis were explored by assessing the effects of stress on T cell interleukin 2 (IL-2) production and T cell responsiveness to IL-2. T cells from repeatedly stressed rat showed a decreased production of IL-2 in response to PHA, although their proliferative response to exogenous IL-2 was normal. Repeated stress also decreased body weight and spleen weight, increased adrenal weight, and decreased plasma levels of triiodothyronine and testosterone. These results suggest that lower levels of IL-2 production during stress could be one reason for the decreased mitogen responsiveness of T cells, often seen with stress. This is important because defective IL-2 production could also lead to significant impairment of immunoregulatory T cell generation and thus a predisposition to malignancy or autoimmune disease that some have associated with stress.