Aerosol delivery of synthetic DNA containing CpG motifs in broiler chicks at hatch under field conditions using a commercial-scale prototype nebulizer provided protection against lethal Escherichia coli septicemia.

Synthetic DNA containing CpG motifs (CpG-ODN) are potent innate immune stimulators in neonatal and adult broiler chickens against bacterial septicemia. We have recently demonstrated that intrapulmonary (IPL) delivery of CpG-ODN as microdroplets under laboratory conditions can protect neonatal chickens against lethal Escherichia coli septicemia. The objectives of this study were to develop a commercial-scale poultry nebulizer (CSPN) that can deliver CpG-ODN as microdroplets in neonatal broiler chicks in the hatcheries and study the efficacy of CSPN in inducing immune-protective effects under different environmental conditions in 2 geographical locations in Canada. Three field experiments were conducted in commercial poultry hatcheries during different seasons of the year in Saskatchewan and British Columbia, Canada. Neonatal broiler chicks (n = 8,000/experiment) received CpG-ODN by the IPL route in the CSPN chamber for 30 min, and control chicks received distilled water (DW) for 30 min. Broiler chicks (CpG-ODN-240 chicks/experiment and DW-40 chicks/experiment) were randomly sampled from all locations of the CSPN after nebulization and challenged with a lethal dose of E. coli to examine the CpG-ODN nebulization induced protection. We found a significant level (P < 0.05) of protection in broiler chicks against E. coli challenge, suggesting that the newly built CSPN successfully delivered CpG-ODN via the IPL route. We found that when the CSPN was maintained at humidex 28°C or below and relative humidity (RH) between 40 and 60%, neonatal birds were significantly (P < 0.05) protected against E. coli septicemia after IPL delivery of CpG-ODN. By contrast, protection in chicks was adversely affected when the CSPN was maintained at the humidex of 29°C or higher and RH of 70%. Overall, the present study successfully built a CSPN for CpG-ODN delivery in chicks at the hatchery and revealed that the temperature, humidity, and humidex were critical parameters in CSPN for efficient delivery of CpG-ODN.

Indoor mold levels and current asthma among school-aged children in Saskatchewan, Canada.

Current knowledge regarding the association between indoor mold exposures and asthma is still limited. The objective of this case-control study was to investigate the relationship between objectively measured indoor mold levels and current asthma among school-aged children. Parents completed a questionnaire survey of health history and home environmental conditions. Asthma cases had a history of doctor-diagnosed asthma or current wheeze without a cold in the past 12 months. Controls were age- and sex-matched to cases. Vacuumed dust samples were collected from the child's indoor play area and mattress. Samples were assessed for mold levels and quantified in colony-forming units (CFU). Sensitization to mold allergens was also determined by skin testing. Being a case was associated with family history of asthma, pet ownership, and mold allergy. Mold levels (CFU/m2 ) in the dust samples of children's mattress and play area floors were moderately correlated (r = 0.56; P < 0.05). High mold levels (≥30 000 CFU/m2 ) in dust samples from play [adjusted odds ratio (aOR) = 2.6; 95% CI: 1.03-6.43] and mattress (aOR) = 3.0; 95% CI: 1.11-8.00) areas were significantly associated with current asthma. In this study high levels of mold are a risk factor for asthma in children.

Bladder injuries in emergency/expedited laparoscopic surgery in the absence of previous surgery: a case series.

INTRODUCTION: The use of laparoscopy as a diagnostic and therapeutic tool is being used increasingly in the emergency setting with many of these procedures being performed by trainees. While the incidence of iatrogenic injuries is reported to be low, we present six emergency or expedited cases in which the bladder was perforated by the suprapubic trocar. CASES: Three cases were related to the management of appendicitis, two to negative diagnostic laparoscopies for lower abdominal pain and one to an ectopic pregnancy. Management of the bladder injuries varied from a urinary catheter alone to laparotomy with debridement of the abdominal wall due to sepsis and later reconstruction. Four of the six cases were performed by registrars. CONCLUSIONS: Although the incidence of bladder injury is low, its importance is highlighted by the large number of laparoscopies being performed. In addition to catheterisation of the patient, care must be taken with the insertion of low suprapubic ports and consideration should be made regarding alternative sites. Adequate laparoscopic supervision and training in port site planning is required for surgical trainees.

Electrosurgery ignition of a pneumoperitoneum secondary to prior spontaneous perforation of the small bowel: a cautionary tale.

We describe explosive combustion of a gas filled peritoneum from a handheld electrosurgery electrode used to enter the abdomen. The pneumoperitoneum was due to small bowel perforation and peritonitis had been established for at least two days. No injury was caused to either the patient or medical staff. This rare occurrence has only been described once before. Surgeons should be aware of the possible combustion of bowel gas, whether on opening bowel or the peritoneum after bowel perforation.

Passage of pathogenic microorganisms through breathing system filters used in anaesthesia and intensive care.

SUMMARY: Invasive ventilation poses a risk of respiratory infection that can be drug-resistant. One means of reducing transmission of infection is the use of a breathing system filter. Filters are intended to be used with dry gas. Current international standards do not require that filters prevent bacterial transfer when wet. It is not known whether microorganisms pass through wet filters, but theory predicts that this might occur. We tested six filters from three different manufacturers. We passed a suspension of microorganisms through the filters using the least pressure necessary, and incubated a sample of the filtrate on blood agar. All the filters tested allowed free passage of both Candida albicans and coagulase-negative staphylococci. The median (IQR [range]) pressure required for fluid to flow across the filter varied greatly between different filter types (20 (0-48 [0-138]) cmH(2)O). We conclude that even large microorganisms pass across moist breathing system filters in conditions that are found in clinical practice.

In vitro production of tumor necrosis factor-alpha by human monocytes stimulated with lipopolysaccharide is positively correlated with increased blood monocytes after exposure to a swine barn.

Recently there has been interest in the air quality in and around intensive livestock production facilities, such as modern swine production barns, where agricultural workers and surrounding residents may be exposed to elevated levels of organic dusts. The health effects of these exposures are not completely understood. The study that is reported here is a component of a larger investigation of the relationships among the acute effects of high-concentration endotoxin exposure (swine barn dust), polymorphisms in the TLR4 gene, and respiratory outcomes following exposure to swine confinement buildings. The relationships among a mediator of acute lung inflammation, tumor necrosis factor alpha (TNF-alpha), and clinical responses to acute swine barn exposure were characterized. Analysis of the results showed that in vitro stimulation of human monocytes with as little as 1 ng/ml of lipopolysaccharide (LPS) produced a significant increase in the monocytes that produced TNF-alpha. Although the proportion of TNF-alpha-positive monocytes after in vitro stimulation with 1 ng/ml of LPS was not associated with gender or TLR4 genotype, it was positively associated with the concentration of monocytes in blood after barn exposure. Thus, these two responses to different forms of LPS exposure are significantly correlated, and more responsive monocytes in vitro indicate a forthcoming relative monocytosis, post barn exposure, which may initiate a cascade of chronic inflammation.

Assessment of endotoxin levels in the home and current asthma and wheeze in school-age children.

UNLABELLED: The relationship between household endotoxin and asthma in children is not clear. To further investigate the relationship between sources of endotoxin and childhood asthma, we conducted a case-control study of children with and without asthma and examined their more frequent household exposures in the home. Children ages 6-13 years with current asthma (n = 70) or wheeze only (n = 19) were sex and age matched (+/-1 year) to 107 controls. Play area and mattress dust were collected for endotoxin analysis. Atopic status was determined by skin prick testing for allergies. A family size of >4 per household was associated with higher endotoxin levels (EU/mg) in the bed dust (P < 0.05). Passive smoking (P < 0.05) and the presence of a cat were associated with higher levels of endotoxin in mattress dust. Endotoxin levels in either the play dust or the bed dust did not differ between cases and controls. Within atopic cases, those with higher endotoxin loads (EU/m2) in bed or play areas were more likely to miss school for chest illness (P < 0.05). In this study, household endotoxin is not a risk factor for current asthma overall but may be associated with increased severity in children with atopic asthma. PRACTICAL IMPLICATIONS: This study did not find that household sources of endotoxin were associated with asthma. However, within atopic asthmatics, asthma severity (as measured by a history of being kept home from school because of a chest illness in the past year) was associated with higher levels of endotoxin in dust from the child's bed. There is a need to further investigate the nature of the relationship between household endotoxin and asthma severity in children which could lead to better management of childhood asthma.

Correlation between in vitro secretion of virulence-associated proteins of Campylobacter jejuni and colonization of chickens.

The mechanisms used by Campylobacter jejuni to colonize the (chicken) intestinal tract have not been defined. In this study, we obtained evidence that in the presence of chicken serum and mucus, C. jejuni secreted proteins that may play a role in the colonization of chicken gut (Campylobacter invasion antigen = Cia). C. jejuni strains NCTC11168V1 and 81-176, as well as an NCTC11168V1 flaA mutant, were found to colonize intestinal tract and secrete proteins in the presence of chicken mucus, chicken serum, or fetal bovine serum in cell culture-conditioned medium. C. jejuni strain NCTC11168V26, which was observed to be a poor colonizer compared with the other C. jejuni isolates, did not secrete Cia proteins. Secreted proteins were also recognized by Western immunoblot using sera from birds that had been colonized by C. jejuni. These data suggest that C. jejuni secretes Cia proteins during colonization of chicken gut and that these Cia proteins play an important role in colonization.

Total dust and endotoxin in poultry operations: comparison between cage and floor housing and respiratory effects in workers.

OBJECTIVE: The objective of this study was to assess respiratory outcomes and environmental exposure levels of workers in cage-housed and floor-housed poultry operations. METHODS: Poultry operations were evaluated for total dust, endotoxin, and ammonia, and respiratory symptoms and lung function tests of workers were conducted. RESULTS: Workers in floor-housed poultry operations had significantly greater exposures to total dust and ammonia, whereas workers from cage-housed poultry operations reported greater frequency of current and chronic symptoms overall and significantly greater current and chronic phlegm (39% vs 18% and 40% vs 11%, respectively). Endotoxin concentration (EU/mg) was a significant predictor (P = 0.05) of chronic phlegm for all poultry workers. CONCLUSIONS: Greater endotoxin concentration in the presence of significantly lower total dust, in conjunction with greater respiratory symptoms in workers from cage-housed poultry operations, as compared with workers from floor-housed poultry operations, appears to indicate that differences in environmental exposures may impact respiratory outcomes of workers.

Intranasal immunization using biphasic lipid vesicles as delivery systems for OmlA bacterial protein antigen and CpG oligonucleotides adjuvant in a mouse model.

The nasal mucosa is an important arm of the mucosal system since it is often the first point of contact for inhaled antigens. The ineffectiveness of the simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies in appropriate delivery systems and adjuvants. We have evaluated biphasic lipid vesicles as a novel intranasal (i.n.) delivery system (designated as vaccine targeting adjuvant, VTA) containing bacterial antigens and CpG oligodeoxynucleotides (ODNs). Results show that administration of antigen and CpG ODNs in biphasic lipid vesicles resulted in greater induction of IgA levels in serum (P< 0.05) and mucosal antibody responses such as IgA in nasal secretions and lung (P< 0.01) after immunization with a combined subcutaneous (s.c.)/i.n. as compared to s.c./s.c. approach. Based on antibody responses, VTA formulations were found to be suitable as delivery systems for antigens and CpG ODNs by the intranasal route, resulting in a Th2-type of immune response, characterized by IgG1 and IL-4 production at the systemic level.

Early laparoscopy as a routine procedure in the management of acute abdominal pain: a review of 1,320 patients.

BACKGROUND: Acute abdominal pain is a common cause for presentation to the emergency room and hospital admission. Many of these patients will undergo exploration for suspected appendicitis, but in 20-35% of cases a normal appendix is found. Because of the limited access provided by the gridiron incision, a definitive diagnosis may not be found. Other patients may be treated conservatively and discharged, only to return with recurrent pain or more definitive symptoms of pathology. In patients with acute abdominal pain, early laparoscopy is an accurate means of both making a definitive diagnosis and avoiding a delay in the diagnosis. METHODS: We performed a retrospective analysis of 1,320 consecutive patients with acute abdominal pain over a 62-month period. All patients underwent diagnostic laparoscopy within 48 h of admission. We evaluated the initial clinical diagnosis, the laparoscopic diagnosis, and the subsequent outcome in this group of patients. Individuals with abdominal trauma were excluded from the study, and all patients were >12 years of age. RESULTS: A definitive diagnosis was made in 90% of patients after diagnostic laparoscopy. Laparoscopy changed the clinical diagnosis in 30% of cases. (83%) of patients underwent a laparoscopic operation for management of their condition at the time of diagnosis. In 92 patients (7%), conversion to laparotomy was required to manage their condition. Peritonitis was present in 180 patients; of 110 of them had appendicitis. Twelve patients developed complications related to the diagnostic laparoscopy or the laparoscopic operation, and there was one postoperative death due to a perforated gastric malignancy. Mean operating time was 30 min (range, 17-90). CONCLUSION: Early diagnostic laparoscopy and treatment results in the accurate, prompt, and efficient management of acute abdominal pain. This technique reduces the rate of unnecessary laparotomy and right iliac fossa gridiron incisions and increases the diagnostic accuracy in these patients. This treatment method is feasible where facilities are available to accommodate the workload and there are practitioners with the requisite expertise.

Putative mannose-specific phosphotransferase system component IID represses expression of suilysin in serotype 2 Streptococcus suis.

In this study, we generated a genomic mutant library from a North American strain of serotype 2 Streptococcus suis using the pGh9:ISS1 transposition vector. Suilysin is the hemolysin made by S. suis. A hyper-hemolytic mutant was identified by screening for hemolytic phenotype using media with human blood. The hyper-hemolytic phenotype was characterised by a quantitative hemolysis microplate method. The use of green fluorescent protein (GFP) as a reporter also showed that suilysin gene expression was greater in the mutant. DNA sequence analysis of 3.8 kb surrounding the ISS1 insertion site revealed four open reading frames (ORFs) with three consecutive ORFs that belong to a putative mannose-specific phosphotransferase system (PTS). The S. suis gene homologous to mannose permease IID, manN, was interrupted by the transposon. A complementation test showed that manN repressed the expression of suilysin and the absence of manN was responsible for the hyper-hemolytic phenotype. However, both wild type and isogenic hyper-hemolytic mutant S. suis fermented mannose, glucose and lactose. Thus, despite its potential roles in carbohydrate transport, phosphorylation and metabolism, the manN homologue in the putative mannose-specific PTS regulates gene expression in S. suis.

Expression of green fluorescent protein and its application in pathogenesis studies of serotype 2 Streptococcus suis.

We investigated the interaction between type 2 Streptococcus suis and swine phagocytes during infection of the natural host, by using green fluorescent protein (GFP) as a specific marker to observe the challenge organism. We compared the strength of the S. suis sly promoter (SP332) and the synthetic promoter (CP25) in driving GFP expression. Two GFP alleles, gfpP11 and gfpmut3*, were also compared. The two promoters and two alleles were efficiently compared using three different promoter-GFP gene combinations on a shuttle vector, which were transformed into S. suis strains SX332, SX932 or M2. Plasmid pSL6.81 has SP332 with gfpP11, pSL5.24 has SP332 with gfpmut3*, and pSL5.28 has CP25 with gfpmut3*. The transformants were fluorescent with green light when viewed with an epifluorescence microscope or during flow cytometry. The signal was also detected using a laser scanning confocal microscope. The GFP expression level varied and CP25 with gfpmut3* led to greatest expression. For optimizing GFP detection, fluorescence-based cell sorting was applied to SX332(pSL5.28) and the mean fluorescence intensity increased 25.9% after optimization. Fluorescence activated cell sorting (FACS)-based phagocytosis assay showed that, without opsonization, phagocytosis rates of SX332, SX932 and M2 by both neutrophils and monocytes were similar and low. After opsonization, the phagocytosis of M2 increased 10-fold while phagocytosis of SX332 and SX932 did not change. GFP-labeled S. suis was identified in fresh pig tonsil tissue 18 h after infection. The results of this study indicated that GFP was expressed in type 2 S. suis and GFP labeled S. suis could be used in phagocytosis and pathogenesis studies.

Induction of protective immunity in pigs after immunisation with CpG oligodeoxynucleotides formulated in a lipid-based delivery system (Biphasix).

A large number of studies demonstrated the immunostimulatory effects of CpG oligonucleotides (ODN), particularly in mice. In the present study, we evaluated the ability of lipid-based delivery systems to enhance the adjuvant effect of CpG-ODN and protect against infection in a porcine pleuropneumonia model. Increased levels of OmlA-specific antibody were detected in animals immunised with OmlA and CpG-ODN formulated in the delivery system Biphasix-vaccine targeting adjuvant (VTA), compared to pigs immunised with VTA without CpG-ODN or CpG-ODN alone. In addition, the responses induced by VTA/CpG formulation were similar to those induced by the commercial adjuvant VSA; however, VTA formulations caused significantly less tissue damage than VSA.

Role of suilysin in pathogenesis of Streptococcus suis capsular serotype 2.

Three suilysin (SLY) knockout mutant strains of Streptococcus suis serotype 2 were generated by allelic replacement from one North American and two European wild type strains. The mutants were characterized by Southern blot, Western blot and phenotyping. In vitro bactericidal testing showed that both wild type and SLY mutants were resistant to bactericidal factors in whole pig blood. To demonstrate the role of SLY during S. suis infection, four animal trials were carried out using young pigs. Either high dose (4 x 10(6)CFU/ml/pig) or low dose (0.5 x 10(6)CFU/ml/pig) live cell aerosol was applied to the pharynx. In one trial, a low challenge dose of North American strain SX332 and its isogenic sly(-) mutant strain (SX932) resulted in acute disease in 3/5 of pigs exposed to the wild type strain, while 5/5 of pigs exposed to the mutant strain survived the trial. In the repeat trial, 1/8 of pigs in wild type group and 6/8 of pigs in mutant group developed disease. The high dose trial with 332/932 pair showed that 4/8 pigs challenged with wild type and 5/8 of pigs challenged with mutant strain developed disease respectively. The third low dose trial, using European strain 31533 and its isogenic sly(-) mutant strain SX911, showed that 1/8 of pigs challenged with the wild type strain and 4/8 of pigs challenged with the corresponding mutant strain developed disease. All the diseased pigs showed fever, clinical signs and developed septicemia. S. suis was isolated from tissue samples such as brain, submandibular lymph node, lung, spleen, liver, heart or joint. Serum antibody titer against cell surface proteins changed little while the antibody titer against SLY increased only in the wild type group after challenge. sly gene was cloned and expressed in E. coli. The recombinant SLY (rSLY) protein showed 800 hemolysin units per microg protein. In vitro study showed that rSLY triggered TNFalpha production by human monocytes and IL-6 production by pig pulmonary alveolar macrophages and monocytes. Thus, the results of this study suggest that SLY does not seem to be a critical virulence factor for S. suis serotype 2 respiratory infection, but by stimulating cytokine release it may play a role in innate immunity.

CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D.

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.

Changes in the leucocyte subpopulations of the palatine tonsillar crypt epithelium of pigs in response to Streptococcus suis type 2 infection.

The tonsils are portal of entry and a site of multiplication and persistence for a variety of pathogens, including Streptococcus suis (S. suis), which is a common cause of meningitis, septicemia and arthritis in pigs. Understanding the early changes that occur in the first barrier of the tonsil, i.e. the crypt epithelium, in response to S. suis infection is critical in clarifying the pathogenesis of this disease and for the future development of efficient methods of mucosal vaccination. In this study, we investigated the early changes, from 18 to 72 h, that occur in leucocyte subpopulations of the crypt epithelium of the palatine tonsils of 3-week-old pigs in response to S. suis type 2 infection. Monoclonal antibodies against leucocyte markers CD3, CD4, CD8, gammadelta T cell receptor, lambda-immunoglobulin light-chain, myeloid cells, and major histocompatibility complex class II molecule (MHC-II) were used in an avidin-biotin immunoperoxidase technique. An increase in the number of lambda-immunoglobulin light-chain positive cells (B cell subset) was noticed in crypts of S. suis-infected animals from 18 h after infection onwards. This increase was significant at 18 and 48 h after infection. The number of CD4 and CD8 cells was greater from 18 h onwards, with a significant increase at 24 and 72 h post-infection. No significant difference in numbers of CD3, gammadelta T cell receptor and MHC-II positive cells was detected in the crypts of infected animals compared to controls. Macrophages, neutrophils and crypt epithelial cells stained positively with the myeloid marker, and the area of crypt epithelium positive for this marker was increased in the crypts of infected animals, with a significant difference detected at 24 and 72 h after infection. These results suggest that there is participation of the innate immunity in the early phase of S. suis infection, represented by neutrophils, macrophages and likely epithelial cells, and that there is a potential for the initiation of both humoral and cellular responses against S. suis within the crypt epithelium of the palatine tonsil.

Suppository-mediated DNA immunization induces mucosal immunity against bovine herpesvirus-1 in cattle.

Mucosal surfaces are the primary sites for the transmission of infectious agents including viruses, so effective vaccines generally should induce mucosal immunity. Furthermore, noninvasive delivery is desirable because of the ease of application, the high degree of patient compliance, and the improved safety for patients and clinicians due to the elimination of needles. Unfortunately, most of the conventional vaccines are parenterally administered and result in systemic rather than mucosal immunity. Here we present the first report of mucosal immunity by noninvasive DNA immunization in a target species. As an approach to induce mucosal immunity against bovine herpesvirus-1, cows were immunized intravaginally with suppositories containing plasmid coding for glycoprotein D. Serum IgG, as well as IgA both in the serum and in the nasal fluids, were detected, which supports the contention of a common mucosal immune system. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge, which demonstrates the efficacy of suppository-based administration of DNA vaccines to target species. As this is a very practical method of delivery, it has great potential to be applied as vaccine or therapy in a variety of species.

Prevalence of hepatitis E virus antibodies in Canadian swine herds and identification of a novel variant of swine hepatitis E virus.

Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation. In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds. A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in Escherichia coli. These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada. From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody. The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%). By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs. The nucleotide sequence identity between the U.S. swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America. Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein. Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.