Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle.

Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens.

Cross protection of a Mannheimia haemolytica A1 Lkt-/Pasteurella multocida ΔhyaE bovine respiratory disease vaccine against experimental challenge with Mannheimia haemolytica A6 in calves.

Bovine respiratory disease causes significant economic losses in both beef and dairy calf industries. Although multi-factorial in nature, the disease is characterized by an acute fibrinous lobar pneumonia typically associated with the isolation of Mannheimia haemolytica. M. haemolytica A1 and A6 are the two most commonly isolated serotypes from cattle, however, the majority of vaccines have not demonstrated cross-serotype protection. In the current study, the efficacy of a novel, attenuated live vaccine, containing both M. haemolytica serotype A1 and Pasteurella multocida, was evaluated in calves challenged with M. haemolytica serotype A6. Although the challenge was more severe than expected, vaccinated calves had reduced clinical scores, lower mortality, and significantly lower lung lesion scores compared to the placebo-vaccinated control group. The results demonstrate that vaccination with an attenuated live vaccine containing M. haemolytica serotype A1 can protect calves against clinical disease following challenge with M. haemolytica serotype A6.

Safety and efficacy of a maternal vaccine for the passive protection of broiler chicks against necrotic enteritis.

Necrotic enteritis is a potentially fatal multifactorial disease of chickens, which under commercial conditions is often associated with increased levels of mortality and reduced bird performance. The safety and efficacy of a Clostridium perfringens type A alpha-toxoid (Netvax™) formulated as an oil emulsion was investigated, following maternal immunization of broiler breeder hens, housed under commercial conditions, by the intramuscular route. A total of 11,234 hens were vaccinated across two integrated poultry sites. The vaccine was safe with no systemic reactions or adverse effects on bird performance detected. Vaccination resulted in a significant increase in anti-alpha toxin antibody in the hen that was maintained throughout the study, and subsequently transferred to their progeny throughout the laying period via egg yolk. Chicks hatched from eggs produced from vaccinated hens were shown to have reduced mortality specifically related to progeny flocks where gross gut lesions associated with necrotic enteritis were observed in control chicks. Further, whilst C. perfringens was isolated from control chicks with necrotic enteritis lesions, no such isolations were made at these time points from chicks from vaccinated hens. These results indicate that, under commercial conditions, maternal vaccination with Netvax™ can help to control losses related to necrotic enteritis.

Ilizarov treatment of infected nonunions of the distal humerus after failure of internal fixation: an outcomes study.

OBJECTIVE: To report the functional outcomes of Ilizarov treatment of infected nonunion of the distal humerus. DESIGN: Prospective case series. SETTING: Tertiary referral center. PATIENTS: Between July 1998 and August 2003, 6 consecutive patients (age 33 to 73 years) were referred to us with an infected nonunion of the distal humerus following failure of open reduction and internal fixation. The average time from initial injury to presentation with the nonunion was 27 months (range, 6 to 99 months). The average number of prior surgeries was 2.8 (range, 1 to 4). INTERVENTION: Hardware removal, ulnar nerve neurolysis, 1 stage debridement, autogenous bone grafting, and application of an Ilizarov external fixator with acute compression in the operating room followed by slow gradual compression (0.25-0.50 mm per day) for several weeks postoperatively. MEASUREMENTS: Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire; SF-12 Physical Component Scale (PCS); Brief Pain Inventory; quality-adjusted life years. RESULTS: All patients attained bony union. One patient refractured 3 weeks after removal of the external fixator following a fall and ultimately underwent total elbow arthroplasty. At an average follow-up of 4.1 years (range, 2 to 7 years), none of the remaining 5 patients had undergone any additional surgery on their arm and all were free of infection. For these 5 patients, significant improvements were seen in standardized DASH scores (42% initially to 78% at follow-up, P = 0.017), worst pain intensity ratings (5.4 initially to 0.8 at follow-up, P = 0.007), and SF-12 PCS scores (37 initially to 44 at follow-up, P = 0.041). On average, the pretreatment to posttreatment improvement was equivalent to 3.8 quality-adjusted life years. CONCLUSIONS: Ilizarov treatment of infected distal humeral nonunions that have failed internal fixation restores function, decreases pain, and improves quality of life. The Ilizarov method should be considered a primary treatment option for this disabling and difficult clinical problem.

The use of a systemic prime/mucosal boost strategy with an equine influenza ISCOM vaccine to induce protective immunity in horses.

In horses, natural infection confers long lasting protective immunity characterised by mucosal IgA and humoral IgGa and IgGb responses. In order to investigate the potential of locally administered vaccine to induce a protective IgA response, responses generated by vaccination with an immunostimulating complex (ISCOM)-based vaccine for equine influenza (EQUIP F) containing A/eq/Newmarket/77 (H7N7), A/eq/Borlänge/91 (H3N8) and A/eq/Kentucky/98 (H3N8) using a systemic prime/mucosal boost strategy were studied. Seven ponies in the vaccine group received EQUIP F vaccine intranasally 6 weeks after an initial intramuscular immunisation. Following intranasal boosting a transient increase in virus-specific IgA was detected in nasal wash secretions. Aerosol challenge with the A/eq/Newmarket/1/93 reference strain 4 weeks after the intranasal booster resulted in clinical signs of infection and viral shedding in seven of seven influenza-naive control animals whereas the seven vaccinated ponies had statistically significantly reduced clinical signs and duration of virus excretion. Furthermore, following this challenge, significantly enhanced levels of virus-specific IgA were detected in the nasal washes from vaccinated ponies compared with the unvaccinated control animals. These data indicate that the intranasal administration of EQUIP F vaccine primes the mucosal system for an enhanced IgA response following exposure to live influenza virus.

Immune responses and protective efficacy in ponies immunised with an equine influenza ISCOM vaccine containing an 'American lineage' H3N8 virus.

Protective responses generated by vaccination with an immuno-stimulating complex (ISCOM)-based vaccine for equine influenza (EQUIP F), containing a new 'American lineage' H3N8 virus, were studied. Seven ponies in the vaccine group received two intramuscular injections of EQUIP F given 6 weeks apart. Aerosol challenge with an A/eq/Newmarket/1/93 reference strain 4 weeks after booster vaccination resulted in clinical signs of infection and viral shedding in 7 influenza-naive control animals whereas the vaccinated ponies were significantly protected from both clinical signs and virus excretion. Influenza virus-specific IgG responses in serum following immunisation with the ISCOM vaccine were predominantly of the IgGa and IgGb sub-isotypes, a pattern similar to that generated by equine influenza virus infection. However, in contrast to the response following infection, virus-specific antibody responses in nasal washes following immunisation were characterised by the presence of IgG but not IgA.These results demonstrated that an ISCOM-based vaccine containing A/eq/Kentucky/98 provides strong protective immunity against challenge with an 'American lineage' H3N8 reference virus.

Utilization of orthopaedic services for hand and wrist conditions in a capitated population.

BACKGROUND: The utilization of orthopaedic services (office visits and surgery) to treat hand and wrist conditions is not well known. In this study, we report the utilization rates for patients referred for orthopaedic treatment of hand and wrist conditions in a large population of individuals enrolled in a capitated insurance plan. METHODS: The study population consisted of individuals enrolled, between January 1998 and December 2001, in a capitated insurance plan that had an annual average membership of 135,188 during that period. This plan was serviced by an independent physician association of sixty-two orthopaedic surgeons who were responsible for all orthopaedic care. Data were collected prospectively in a centralized database as patients with various hand or wrist conditions were referred for orthopaedic services. Odds ratios were used to compare gender-specific and age-specific utilization rates. RESULTS: Overall utilization rates were 18.06 office visits and 6.47 surgical procedures per 1000 members per year. The most frequent hand or wrist conditions were fractures, carpal tunnel syndrome, tendinitis or tenosynovitis, and ganglion or synovial cysts. These four diagnoses accounted for 70% of all office visits and 71% of all surgical cases. Across all age groups, males had a significantly higher rate of utilization of office visits (p < 0.001). Between the ages of thirty-five and fifty-five years, utilization of office visits and surgery increased approximately linearly with age. CONCLUSIONS: A comparison of these data with those of previous reports indicates that approximately one of every ten patients who are referred for orthopaedic services has a hand or wrist condition, and nearly half will require surgery.

Protective efficacy of a live attenuated anti-coccidial vaccine administered to 1-day-old chickens.

The efficacy of a live attenuated anti-coccidial vaccine, Paracox-5, administered to 1-day-old chicks was investigated by assessing protection against changes in weight gain following virulent challenge. Vaccinated birds were challenged independently 28 days later with each of the component species (Eimeria acervulina, Eimeria maxima, Eimeria mitis or Eimeria tenella), and protection was demonstrated against associated reduction in weight gain and lesion formation. In addition, an improvement in bird performance, in terms of feed conversion ratio, was also observed following vaccination. Furthermore, under conditions designed to more closely mimic those in the field and using hatchery spray administration, protection against a mixed virulent challenge introduced by 'seeder birds' was demonstrated evenly across a flock of broiler birds within 21 days after vaccination. These data demonstrate that Paracox-5 vaccine will protect broiler chickens against the adverse effects on performance induced by Eimeria spp.

Serological, colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and Escherichia coli F5 (K99).

Twenty-five Ayrshire/Friesian cows were vaccinated once with a new combined vaccine against rotavirus, coronavirus and Escherichia coli F5 (K99) or given a saline placebo 31 days before the first expected calving date. Blood samples were taken from the cows at intervals from vaccination until seven days after calving and from their calves up to 28 days after birth, and colostrum and milk samples were collected from the cows at intervals for 28 days after calving. There was a significant increase in the mean specific antibody titre against all three antigens in the serum of the vaccinated animals (even in the presence of pre-existing antibody) which was accompanied by increased levels of protective antibodies to rotavirus, coronavirus and E coli F5 (K99) in their colostrum and milk for at least 28 days.

Comparison of length of hospital stay for patients with known or suspected methicillin-resistant Staphylococcus species infections treated with linezolid or vancomycin: a randomized, multicenter trial.

STUDY OBJECTIVE: To compare hospital length of stay (LOS), weekly discharges, and days of antibiotic treatment with linezolid (intravenous with oral follow-up) and vancomycin (intravenous only). DESIGN: Multinational, randomized, phase III trial. SETTINGS: Hospitals in North America, Latin America, and Europe. PATIENTS: Four hundred sixty hospitalized patients with infections of known or suspected methicillin-resistant Staphylococcus species. INTERVENTION: Administration of linezolid or vancomycin. MEASUREMENTS AND MAIN RESULTS: For linezolid recipients, median LOS was 5 and 8 days shorter (p=0.05 and 0.003) in the complicated skin and soft tissue infection intent-to-treat (230 patients) and clinically evaluable (144) samples, and slightly but not significantly shorter in the overall intent-to-treat (460) and clinically evaluable (254) samples. In all samples, linezolid recipients had more discharges in the first week of treatment and fewer days of intravenous therapy than vancomycin recipients. CONCLUSION: Our results support linezolid's ability to reduce medical resource use.

Lactogenic immunity following vaccination of cattle with bovine coronavirus.

In order to investigate the ability of an oil adjuvanted vaccine containing bovine coronavirus antigen to enhance lactogenic immunity in the calf, pregnant cows and heifers were vaccinated and specific virus neutralising antibody levels determined in serum, colostrum and milk. Pre-existing antibody titres (as a result of natural infection) in the serum of these animals were found to be significantly increased as a result of a single shot vaccination carried out between 2 and 12 weeks before calving. This was reflected in a similar increase in the titre and duration of specific antibody in milk and colostrum that was passed on to the calves. The overall response observed was highly dependent on an adequate antigen payload being incorporated within the single dose vaccine. No abnormal local or systemic reactions were observed as a result of vaccination. It is hoped that this approach will lead to the production of a superior commercial vaccine for the protection of neonatal calves against enteric coronavirus infection.

Motivation for change and psychological distress in homeless substance abusers.

This study explores the treatment needs of homeless individuals participating in a large urban day shelter program. Alcohol and drug use, psychological distress, and stage of change were assessed in 100 participants presenting for services. The associations among substance use, risk perception, and readiness to change were examined for alcohol and drugs separately. Participants had high levels of psychological distress compared to "non-patient" samples. Eighty percent had used alcohol in the past 6 months, with 65% of those drinking at higher-risk levels; 60% had used drugs, with 82% in the higher-risk levels. While the majority felt that they drank and/or used drugs "too much", most were in precontemplation or contemplation stages of change. Intervention efforts for this population should focus on motivation, facilitation through the stages, and the associations between psychiatric symptoms and substance use.

Toxicologic and carcinogenic effects of the type IV phosphodiesterase inhibitor RP 73401 on the nasal olfactory tissue in rats.

RP 73401, a type IV phosphodiesterase inhibitor, caused toxic effects in the nasal olfactory region of Sprague-Dawley rats when administered by either oral or inhalation exposure. A single oral administration of RP 73401 (at a dose of > or = 50 mg/kg) or 5-day inhalation exposure (1 hr/day) at a dose of approximately 1.0 mg/kg per day caused degeneration and sloughing of the olfactory surface epithelium. Degeneration and loss of Bowman's glands were noted in the underlying lamina propria and submucosa. Electron microscopy of these lesions demonstrated that sustentacular cells and the epithelial cells lining Bowman's glands were the primary target cells in the olfactory mucosa. The earliest ultrastructural changes detected in these cells were dilatation and vesiculation of the endoplasmic reticulum, suggesting that metabolic activation is important for the toxic effects. In repeated-dose studies, 13 wk of oral dosing at 2.0 or 6.0 mg/kg per day resulted in subtle disorganization of the olfactory epithelium, whereas basal cell hyperplasia in the olfactory epithelium was identified in a 6-month inhalation study at a dose of 1.0 mg/kg per day. A 2-yr inhalation carcinogenicity study resulted in tumors of the nasal olfactory region in rats treated at 0.5 and 1.0 mg/kg per day. Most tumors were classified as olfactory neuroblastomas, and immunohistochemistry on selected tumors was consistent with their being of neuroectodermal origin. Of the species studied (rat, mouse, and dog), the olfactory toxicity of RP 73401 was confined to the rat, and the toxicity was likely related to metabolic activation by olfactory epithelial cells rather than the phosphodiesterase activity of the compound.

Septic arthritis caused by chronic osteomyelitis.

We have treated four cases of previously quiescent osteomyelitis which presented as septic arthritis in an adjacent joint. The osteomyelitic focus was in the bone proximal to the involved joints (zero to ten centimeters above the joint line). Based on the presenting history, physical findings, laboratory tests and cultures of joint fluids, the joint sepsis was low grade in all patients which led to delays in diagnosis and treatment. Aggressive surgical debridement of both bone and joint, followed by a prolonged course of antibiotics led to resolution in all patients. A high index of suspicion combined with adequate radiographs of the surrounding bones should lead to the appropriate diagnosis and treatment.

Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

AIMS: To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS: Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS: Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS: The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM.

Failure of trapeziometacarpal arthrodesis with use of the Herbert screw and limited immobilization.

Eighteen patients with trapeziometacarpal joint arthritis had arthrodesis with use of the 1.9 mm Herbert screw for internal fixation. Thumb spica cast immobilization was maintained for an average of 8 weeks, followed by thumb spica orthoplast splintage on a part time basis. Fourteen patients had radiographic follow-up an average of 12 months after operation. Seven had union and seven had a nonunion. There were three fixation-related complications and two nonfixation-related complications. Eleven patients had clinical follow-up an average of 12 months after operation. Four patients had no pain, five had mild pain, and two had moderate pain after operation. All had severe pain before operation. All were satisfied with the procedure. Although subgroups of patients had a higher rate of union with bone grafts or with immobilization over 8 weeks, the theory that arthrodesis with 1.9 mm Herbert screws does not require external splintage and allows early mobilization is not supported by this series.