The horizontal transfer of Salmonella between the lesser mealworm (Alphitobius diaperinus) and poultry manure.

There is need to determine the nature of enduring reservoirs of Salmonella contributing to perpetual contamination within poultry flocks. The dispersal of Salmonella between birds, litter and the lesser mealworm has been established, but the extent that these act as critical components in the epidemiology of Salmonella infection during broiler grow-out and flock rotation has not been delineated; in particular, the level of participation by the lesser mealworm beetles (LMB) as agents of retention and dispersal. This study defines this route of transmission and provides empirical data on bacterial loads that facilitate Salmonella transfer. Results showed differential Salmonella transfer dependent on bacterial concentration. At 103  cfu/ml, only a small, but not significant, amount of Salmonella was transferred, from the LMB to the manure and back to uninfected LMB; while from 105 to 107  cfu/ml, a significant acquisition and transfer occurred both internally and externally to the LMB over 4 and 24 hr exposures. These data will be used in correlation with facility management practices to develop intervention strategies to mitigate the establishment and spreading of reservoir Salmonella populations contributing to pre-harvest contamination of poultry flocks.

Characterization of antibiotic and disinfectant susceptibility profiles among Pseudomonas aeruginosa veterinary isolates recovered during 1994-2003.

AIMS: To evaluate susceptibility of Pseudomonas aeruginosa veterinary isolates to antibiotics and disinfectants. METHODS AND RESULTS: Pseudomonas aeruginosa isolates collected from dogs (n = 155) and other animals (n = 20) from sixteen states during 1994-2003 were tested for susceptibility. Most isolates were resistant to twenty-one antimicrobials tested, and the highest prevalence of resistance was to ß-lactams (93.8%) and sulphonamides (93.5%). Fluoroquinolone resistance did not increase from 1994 to 2003. Ciprofloxacin and enrofloxacin had a 5 and 16% prevalence of resistance, respectively, while sarafloxacin and nalidixic acid had a prevalence of resistance of 97 and 98%, respectively. Strains were pan-resistant to triclosan and chlorhexidine, were highly resistant to benzalkonium chloride and demonstrated high susceptibility to other disinfectants. Didecyldimethylammonium chloride was the most active ammonium chloride. Inducible resistance was observed to cetyl ammonium halides, chlorhexidine and benzyl ammonium chlorides, which formulate disinfectants used in veterinary clinics and dairies. Organic acid inhibition was associated with the dissociated acid species. CONCLUSIONS: Dissociated organic acids appear able to inhibit Ps. aeruginosa, and rates of fluoroquinolone resistance merit sustained companion animal isolate surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of Ps. aeruginosa susceptibility to 24 disinfectants and illustrates the high resistance of Ps. aeruginosa to both antibiotics and disinfectants.

Transient gut retention and persistence of Salmonella through metamorphosis in the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

AIMS: This study was undertaken to determine the retention of Salmonella through Alphitobius diaperinus metamorphosis and its contribution, through defecation, to external contamination. METHODS AND RESULTS: Insects were exposed to a tagged Salmonella enterica and evaluated for external elimination. (i) Each day for 3 weeks, a filter collected frass from a restrained insect for analysis. (ii) Exposed larvae in a closed container were followed through pupation, and newly emerged adults were examined for their retention of marker bacteria. CONCLUSIONS: Exposed adults and larvae produced Salmonella-positive frass for an average of 8 days, ranging from 6 to 11 days and 6 to 12 days, respectively. Nineteen per cent of the larvae carried Salmonella through metamorphosis and eclosion, with 5% of the pupal exuviae being positive as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Many sources of foodborne pathogens within the poultry production facilities, including reservoir populations, currently go unrecognized. This diminishes the ability of producers to mitigate the transfer of pathogens between animals, humans and the environment. Poultry management standards accept the reutilization of litter. Alphitobius diaperinus survive between flock rotations on the reutilized litter, and it was demonstrated in this study that the Salmonella they carry can survive with them.

Planktonic and biofilm communities from 7-day-old chicken cecal microflora cultures: characterization and resistance to Salmonella colonization.

Information implicating bacterial biofilms as contributory factors in the development of environmental bacterial resistance has been increasing. There is a lack of information regarding the role of biofilms within the microbial ecology of the gastrointestinal tract of food animals. This work used a continuous-flow chemostat model derived from the ceca of 7-day-old chicks to characterize these communities and their ability to neutralize invasion by Salmonella enterica serovar Typhimurium. We characterized and compared the biofilm and planktonic communities within these microcosms using automated ribotyping and the Analytical Profile Index biotyping system. Eleven species from eight different genera were identified from six culture systems. Klebsiella pneumoniae was isolated from all planktonic communities and four of the biofilm communities. Three of the communities resisted colonization by Salmonella enterica serovar Typhimurium, two communities suppressed growth, and one community succumbed to colonization. In cultures that resisted colonization, no Salmonella could be isolated from the biofilm; in cultures that succumbed to colonization, Salmonella was consistently found within the biofilms. This study was one of a series that provided a molecular-based characterization of both the biofilm and planktonic communities from continuous-flow culture systems derived from the cecal microflora of chicks, ranging in age from day-of-hatch to 14 days old. The one common factor relating to successful colonization of the culture was the presence of Salmonella within the biofilm. The capacity to sequester the introduced Salmonella into the biofilm appears to be a contributing factor to the inability of these cultures to withstand colonization by the Salmonella.

Persistence of a vancomycin-resistant Enterococcus faecium in an anaerobic continuous-flow culture of porcine microflora in the presence of subtherapeutic concentrations of vancomycin.

Recombined porcine continuous-flow culture (RPCF) maintained in a continuous-flow fermentation system is effective in protecting neonatal and weaned pigs against infection by enteropathogens. In the current study, we demonstrate the effect of RPCF on vancomycin-resistant enterococci (VRE) in the presence and absence of subtherapeutic levels of vancomycin. Also examined was the ability of VRE to transfer vancomycin resistance to endogenous Enterococcus faecalis 137.1. When RPCF was challenged with VRE, the rate of VRE clearance was dependent on the method of challenge. In the control experiment, RPCF was challenged with 7.0 log10/CFU/ml VRE. Clearance of VRE from the culture was observed within 7 days at a rate of 1.44 log10/day. RPCF containing 0.001 microg/ml vancomycin cleared VRE at a slightly lower rate of 0.94 log10/day. RPCF containing 0.01 microg/ml or 0.1 microg/ml vancomycin reduced the level of VRE from 7.0 log10/CFU/ml to 2.0 log10/CFU/ml within 9 days, but failed to clear the VRE after 24 days. During the period of decline, the VRE clearance rate for the 0.01 microg/ml and 0.1 microg/ml vancomycin-treated cultures was 0.52 log10/day, and 0.53 log10/day, respectively. E. faecalis 137.1 endogenous to RPCF did not acquire the vancomycin resistance genes throughout the experiment as evidenced by direct selection, ribotyping, and pulsed-field gel electrophoresis.

Effect of oral sodium chlorate administration on Escherichia coli O157:H7 in the gut of experimentally infected pigs.

Strategies are sought to reduce pathogenic Escherichia coli concentrations in food animals. Because E. coli possess respiratory nitrate reductase activity, which also reduces chlorate to cytotoxic chlorite, we tested and found that oral sodium chlorate administration reduced gut concentrations of E. coli O157:H7 in experimentally infected pigs and wildtype E. coli concentrations in nonchallenged pigs. Mean +/- S.E. concentrations (log10 CFU/g) of E. coli O157:H7 in ileal, cecal, colonic and rectal contents from placebo-treated pigs were 4.03 +/- 0.66, 3.82 +/- 0.24, 4.42 +/- 0.25 and 4.03 +/- 0.16, respectively. In contrast, E. coli O157:H7 concentrations were reduced (P < 0.05) in ileal (1.56 +/- 0.22) cecal (2.65 +/- 0.38), colonic (3.05 +/- 0.38) and rectal (3.00 +/- 0.29) contents from pigs orally administered three successive (8 h apart) 10-ml doses of 100 mM chlorate. Wildtype E. coli concentrations in gut contents of non-E. coli O157:H7-challenged pigs likewise treated with chlorate were reduced by 1.1 to 4.5 log10 units compared to concentrations in placebo-treated pigs, which exceeded 6.0 log10 CFU/g. As before, the reductions were greater in anterior regions of the gut than regions more caudal. Similar treatment of E. coli O157:H7-challenged pigs with 200 mM chlorate caused reductions in gut concentrations of E. coli O157:H7; however, the reductions were not necessarily greater than those achieved with the 100 mM chlorate treatment.

Laser therapy: a randomized, controlled trial of the effects of low intensity Nd:YAG laser irradiation on lateral epicondylitis.

OBJECTIVE: To assess the effectiveness of low intensity laser therapy in the treatment of lateral epicondylitis. DESIGN: A double-masked, placebo-controlled, randomized clinical trial. SETTING: A physical medicine and rehabilitation clinic. PARTICIPANTS: Fifty-two ambulatory men and women (age range, 18-70 yr) with symptomatic lateral epicondylitis of more than 30 days in duration and a normal neurologic examination. INTERVENTION: Subjects were bloc randomized into 2 groups with a computer-generated schedule. All underwent irradiation for 60 seconds at 7 points along the symptomatic forearm 3 times weekly for 4 weeks by a masked therapist. The sole difference between the groups was that the probe of a 1.06-microm continuous wave laser emitted 204 mW/cm2 (12.24 J/cm2) for the treated subjects and was inactive for the control subjects. Subjects were assessed at the beginning, midpoint (session 6), and end (session 12) of treatment, as well as at follow-up 28 to 35 days after their last treatment. MAIN OUTCOME MEASURES: Pain in last 24 hours, tenderness to palpation, and patient's perception of change (benefit). RESULTS: The treated and untreated groups were well matched demographically. Masking was maintained for subjects and therapists; however, the groups did not vary to a statistically significant extent in terms of the main outcome measures either during treatment or at follow-up. Secondary outcome variables, such as grasp and pinch strength, medication use, and pain with grasp and pinch, also failed to statistically differ significantly between the groups. No significant treatment side effects were noted. CONCLUSION: Treatment with low intensity 1.06-microm laser irradiation within the parameters of this study was a safe but ineffective treatment of lateral epicondylitis. Further research seems warranted in this controversial area.

Outcome of de novo hepatitis C virus infection in heart transplant recipients.

The outcome of de novo hepatitis C virus (HCV) infection in heart transplant recipients of HCV-antibody positive organs is not known. The aim of the study was to determine the short-term outcome of de novo HCV infection in recipients of HCV-positive donor organs. HCV-antibody negative recipients of HCV-antibody positive hearts were identified from January 1, 1991 to February 28, 1998. Control patients matched for year of transplantation were also identified. Twenty-eight patients (22 males, mean age of 56 +/- 11 SD) received hearts from HCV-antibody-positive donors. The control group was similar to the patients in all clinical and demographic aspects. Twenty-three patients had detectable viremia by reverse-transcription polymerase chain reaction (RT-PCR). Of these 23 patients with de novo HCV infection, 7 (30%) developed HCV-related liver disease. Three patients (13%) had chronic hepatitis and 4 patients (17%) developed severe acute cholestatic hepatitis (ACH). Mycophenolate mofetil (MMF) use (P =.04) and high viral load at onset of acute liver disease (P =.02) were associated with ACH. Overall survival was similar between patients with de novo HCV infection and controls (P =.20). Development of ACH (P =.02) and MMF use (P =.0009) were associated with decreased survival in patients with de novo HCV infection. The present study showed that survival of patients with de novo HCV infection was similar to a matched control group. HCV-related severe ACH is associated with a poor short-term outcome in patients with de novo HCV infection. MMF use may be associated with a higher incidence of HCV-related severe ACH and a poor short-term outcome.

Laser therapy: a randomized, controlled trial of the effects of low-intensity Nd:YAG laser irradiation on musculoskeletal back pain.

OBJECTIVE: To assess the effectiveness of low-intensity laser therapy in the treatment of musculoskeletal low back pain. DESIGN: A double-masked, placebo-controlled, randomized clinical trial. SETTING: A physical medicine and rehabilitation clinic. PARTICIPANTS: Sixty-three ambulatory men and women between the ages of 18 and 70yrs with symptomatic nonradiating low back pain of more than 30 days' duration and normal neurologic examination results. INTERVENTION: Subjects were bloc randomized into two groups with a computer-generated schedule. All underwent irradiation for 90 seconds at eight symmetric points along the lumbosacral spine three times a week for 4 weeks by a masked therapist. The sole difference between the groups was that the probes of a 1.06 microm neodymium:yttrium-aluminum-garnet laser emitted 542mW/cm2 for the treated subjects and were inactive for the control subjects. MAIN OUTCOME MEASURES: Subject's perception of benefit, level of function as assessed by the Oswestry Disability Questionnaire, and lumbar mobility. RESULTS: The treated group had a time-dependent improvement in two of the three outcome measures: perception of benefit and level of function. These results were most marked at the midpoint evaluation (p < .005, p < .01) and end of treatment (p < .017, p < .001) but tended to lessen at the 1-month follow-up (p < .10, p < .004). Lumbar mobility did not differ between the groups at any time. All tests were two-sample t tests with unequal variances. CONCLUSIONS: Treatment with low-intensity 1.06 microm laser irradiation produced a moderate reduction in pain and improvement in function in patients with musculoskeletal low back pain. Benefits, however, were limited and decreased with time. Further research is warranted.

Centrally and locally mediated thermoregulatory responses alter subcutaneous oxygen tension.

Mild perianesthetic hypothermia decreases resistance to infections. Decreased resistance likely results in part from direct immune inhibition. However, decreased tissue oxygen partial pressure also decreases resistance to infection by impairing oxidative killing by neutrophils and collagen deposition. Thermoregulatory vasoconstriction decreases skin blood flow and may also decrease subcutaneous tissue oxygen tension. Accordingly, we determined the influence of centrally and locally mediated thermoregulatory vasomotion on subcutaneous oxygen tension. We also compared subcutaneous oxygen tension to other potential markers of tissue perfusion: laser Doppler flowmetry and transcutaneous oxygen tension. Arterial oxygen tension was maintained near 325 mm Hg in five volunteers. Control subcutaneous oxygen tension values were recorded after 1 hour of euthermia (no sweating or vasoconstriction). Volunteers were then cooled with a circulating-water mattress positioned under the trunk and legs. After 1.5 hours of cooling sufficient to produce shivering, the right upper arm was covered for 1 hour with a small circulating water blanket set to 40 degrees C while systemic cooling continued. The volunteers were then systematically warmed to produce sweating, and the right arm was locally cooled. There was no correlation among laser Doppler flowmetry, transcutaneous oxygen tension, and subcutaneous oxygen tension. Systemic cooling significantly decreased subcutaneous oxygen tension, but subcutaneous oxygen tension in the right arm returned to control values during local heating. Systemic warming significantly increased subcutaneous oxygen tension, and 1 hour of local cooling failed to fully reverse the increase. These data indicate that thermoregulatory vasoconstriction significantly decreases tissue oxygen availability. Decreased subcutaneous oxygen tension may be one mechanism by which mild perianesthetic hypothermia facilitates development of surgical wound infections.

Production and characterization of a monoclonal antibody against bovine haptoglobin and its use in an ELISA.

Haptoglobin (Hp) is the major acute phase reactant found in cattle. As such, it is an excellent indicator of early disease processes and could be used as a marker for pre-clinical illness in cattle. The production of monoclonal antibodies (mAbs) directed against bovine Hp and bovine hemoglobin is described. The anti-haptoglobin mAbs (Hap1, Hap2, Hap3) and the anti-bovine hemoglobin (Hb) mAb (BoHem1) were characterized and tested for cross-reactivity by means of enzyme-linked immunosorbent assay (ELISA) and immunoblotting analyses. Additionally, the development of an ELISA based on an anti-haptoglobin mAb is discussed.

Prolonged cardiac xenograft survival is induced by intrathymic splenocyte injection.

The shortage of suitable human organs currently limits widespread utilization of clinical transplantation. Successful xenotransplantation could potentially eliminate the shortage of suitable organs for transplantation. The present study determines the effect of intrathymic donor spleen cell injection on the survival of cardiac xenografts performed without post-transplant immunosuppression. Wistar-Furth rats served as recipients of Lewis rat cardiac allografts or Golden Syrian hamster cardiac xenografts either with or without intrathymic donor spleen cell injection. Some xenograft cohorts were pretreated with four doses of cyclophosphamide prior to cardiac transplantation. Graft function was assessed by daily graft palpation and rejection was defined as cessation of graft function. Cardiac allograft median survival time (MST) was 8 days in untreated recipients. Pretreatment with intrathymic donor spleen cell injection led to essentially indefinite allograft survival (MST > 100 days). Control xenograft survival was 3 days with no significant prolongation seen when intrathymic donor spleen cell injection was performed (MST = 2.5 days). The addition of pulse-dose cyclophosphamide resulted in markedly prolonged xenograft survival (MST = 30 days) compared to control (P < 0.01) and to cyclophosphamide alone (MST = 9.5 days, P < 0.01). Cardiac xenografts undergo a vigorous rapid rejection. The time course of rejection was not altered by the intrathymic presence of donor cells under conditions which lead to indefinite survival of cardiac allografts. A brief period of pretransplant cyclophosphamide treatment markedly improved the success of the thymic tolerance protocol in xenografts. This finding suggests that temporary inhibition of humoral immunity may be permissive for the development of thymic tolerance to cell-mediated immunity in xenotransplants.

Mild hypothermia during isoflurane anesthesia decreases resistance to E. coli dermal infection in guinea pigs.

Small changes in core temperature profoundly alter cutaneous blood flow, a major factor influencing resistance to wound infection. Furthermore, when measured in vitro, various immune functions are temperature dependent in the physiological range. Accordingly, we tested the hypothesis that mild hypothermia impairs and mild hyperthermia improves resistance to dermal infections. Thirty-two guinea pigs were anesthetized for 6 h using 1.5% (1.25 MAC) inspired isoflurane. Their core temperatures were maintained at either 39 degrees C (normal for guinea pigs, n = 11), 36 degrees C (n = 12), or 41 degrees C (n = 9). One h after induction of anesthesia, 2 x 10(8) E. coli were injected intradermally with a 26-g needle at eight sites on each animal's back. Core temperatures were not controlled after recovery from anesthesia, and animals in each group were maintained in the same environment. Twenty-four h after injection, the area of induration surrounding each injection site was measured. This is a standard test of resistance to wound infection. Values were compared using one-way ANOVA and Scheffé's S tests. Results are presented as means +/- standard deviations; differences were considered significant when P < 0.05. Areas of inflammation on the hypothermic animals were significantly larger (48 +/- 10 mm2) than those on normothermic (36 +/- 10 mm2) or hyperthermic (37 +/- 6 mm2) animals. These data suggest that mild hypothermia during anesthesia significantly impairs resistance to dermal infection. In contrast, mild hyperthermia does not appear to be protective.

Mild hypothermia during halothane-induced anesthesia decreases resistance to Staphylococcus aureus dermal infection in guinea pigs.

Because various immune functions are impaired at temperatures only 1 degrees to 3 degrees C less than normal, we tested the hypothesis that mild hypothermia during anesthesia impairs resistance to dermal infections. Guinea pigs were anesthetized for 6 hours with 1% inspired halothane. Their core temperatures were maintained at either 39 degrees C (normal for guinea pigs, n = 12) or 36 degrees C (n = 12). Two hours after induction of anesthesia, three doses each of Staphylococcus aureus (10(8), 10(7), and 10(6) organisms) were injected intradermally at nine sites on each animal's back. Core temperatures were not controlled after recovery from the anesthetic, and animals in each group were maintained in the same environment. Four days after anesthesia, each injection site was excised to obtain a count of viable bacteria. Subcutaneous oxygen partial pressure values, averaged over time, were 53 +/- 3 mm Hg (mean +/- SEM) in the hypothermic group and 62 +/- 4 mm Hg in the normothermic group (p = 0.06). Capillary perfusion, as assessed by laser Doppler flowmetry, was comparable in the two groups. One day after injection of 10(8) bacteria, the area of induration was 89 +/- 11 mm(2) in the hypothermic group but only 61 +/- 6 mm(2) in the normothermic group (p < 0.05). On postanesthetic day 4, the area of induration was 72 +/- 6 and 59 +/- 6 mm(2) in the hypothermic and normothermic groups, respectively (p > 0.05). After inoculation with 10(8) bacteria, the fraction recovered was 1.0 +/- 0.2 in the hypothermic groups and 0.6 +/- 0.2 in the normothermic group (p < 0.05). After inoculation with 10(7) and 10(6) bacteria, the fraction recovered was less than 0.2, and no difference was found between the hypothermic and normothermic animals. Thus mild hypothermia during halothane-induced anesthesia slightly impairs resistance to dermal infection.

Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis.

Hapten-carrier protein conjugates were made using five different small haptens (MW < 1000), two carrier proteins and two methods of conjugation. Nondenaturing agarose gel electrophoresis was used to demonstrate that when as few as two molecules of a small hapten are attached to the carrier, the conjugate band migrates differently from that of the carrier alone or of the coupling reagent-treated carrier. Furthermore, the direction of the change in migration of each conjugate correlates with the change in charge which occurs upon attachment of the hapten to the carrier.

Composite kidney-islet transplantation prevents recurrent autoimmune beta-cell destruction.

BACKGROUND: Clinically successful islet transplantation has been rare despite adequate isolation techniques. Reenactment of the original autoimmune beta-cell destruction may contribute to the poor results. Distinguishing autoimmune effects from rejection can be accomplished with isogeneic transplants exchanged between diabetes-prone (BB-DP) and diabetes-resistant (BB-DR) rats. These experiments determine the relative sensitivity of islet, whole pancreas, and composite kidney-islet transplants to recurrent autoimmunity. METHODS: Acutely diabetic (BB-Ac) BB rats served as recipients of vascularized pancreas, intraportal (IPo) or renal capsular (KC) islet transplants, or vascularized composite kidney-islet grafts from BB-DR or BB-DP donors. Graft function was assessed by daily blood glucose level, and the outcome was confirmed on histologic examination. Cyclosporine 5 mg/kg/day intramuscularly was administered to assess its effect on recurrent beta-cell injury. RESULTS: BB-DP pancreases developed recurrent autoimmunity in 55% of cases; cyclosporine afforded complete protection if maintained. Diabetes resistance was transplanted with 23 of 23 BB-DR pancreas grafts; however, islet isolation led to a loss of diabetes resistance for islet grafts to the KC and IPo. Cyclosporine protected KC but not IPo islets. Composite BB-DR kidney-islet transplants functioned indefinitely in all cases. CONCLUSIONS: Transplanted islets initially survive by passive diffusion but are ultimately revascularized by capillary ingrowth. The finding that composite kidney-islet transplants function indefinitely suggests that the revascularizing endothelium may play a role in resistance or susceptibility to autoimmune beta-cell destruction.

Extracardiac to coronary anastomoses support regional left ventricular function in dogs.

Intramyocardial implantation of a systemic artery [the internal mammary artery (IMA)] causes angiogenesis, with formation of systemic to coronary anastomoses. In dogs, we assessed the magnitude of IMA-derived nutritive flow and determined its influence on regional contraction. We also sought to determine whether acidic fibroblast growth factor (FGF), an angiogenic peptide, could enhance myocardial neovascularization. Ameroid constrictors and hydraulic balloon occluders were placed on the left anterior descending coronary artery (LAD) of 23 dogs, and the left IMA was implanted in the LAD territory. Dogs were randomized to receive continuous infusions of acidic FGF with heparin, heparin alone, or placebo directly into the IMA for 8 wk. Regional myocardial blood flow was assessed in the conscious state 3 days and 8 wk after operation. Left ventricular function was determined in the anesthetized state at the 8-wk conclusion of treatment. In all dogs, IMA occlusion reduced mean maximal LAD zone perfusion by 28% (P < 0.001), without influencing regional contraction. When IMA occlusion was superimposed on left circumflex coronary artery (LCX) occlusion, LAD zone perfusion declined by 34% (relative to LCX occlusion alone), significantly impairing regional contraction. Treatment with either acidic FGF plus heparin or heparin alone improved IMA-derived collateral flow; however, addition of acidic FGF to heparin afforded no additional advantage over heparin by itself. We conclude that acidic FGF did not enhance myocardial angiogenesis in this model. IMA-derived collateral flow has significant functional importance; however, it is evident in the dog only when other sources of collateral flow are compromised.