Descriptive epidemiology of spotty liver disease in Australian cage-free brown egg layer chicken flocks with a scratch area.

Spotty Liver Disease (SLD), caused by Campylobacter hepaticus or C. bilis infection in adult female chickens continues to emerge as a major disease problem in cage-free production systems. Free range production has become the predominant system in Australian egg production and SLD is widespread in these farms. Previous studies have identified having a scratch area as a key determinant for SLD occurrence. An Australia-wide survey of egg production flocks with scratch areas was conducted regarding SLD including 48 individual flocks. Descriptive information on the facilities and flock management practices was reported. The incidence of SLD, age of first outbreak, initial mortality rate, duration of elevated mortality, and magnitude and duration of any associated egg production decline are described. Recurrence of SLD in the same flock was also reported and discussed. Therapies applied were recorded and assessed across SLD severity and duration. SLD occurred in 66.7% of layer flocks whose facility included a scratch area. Recurrent SLD outbreaks occurred in 31% of flocks experiencing SLD. Antibiotic medication reduced duration of mortality and egg production decline. Antibiotic therapy was associated with reduced duration of mortality and a less severe and shorter duration of egg production drops compared to untreated flocks. PCR detection of C. hepaticus in cloacal swabs and house dust samples and a serological ELISA test were compared and evaluated as diagnostic aids or as possible predictors of SLD outbreaks. The ELISA showed substantial agreement with detection of C. hepaticus in cloacal swabs by PCR. Examining composite house dust samples by PCR for C. hepaticus DNA appeared to be the most convenient and cost-effective aid to diagnosis and as a putative predictor for SLD outbreaks.

Feeling the Stress: Salivary Cortisol Responses of Softball Umpires during National Championships.

Stress research in sports tends to focus on athletes, with sports officials typically being overlooked. In the current study, baseline, pre-game, and post-game cortisol levels among a sample of softball umpires were measured to assess the pattern of stress responses and determine if umpire performance (pass/fail) and position on the diamond (plate/field) could be predicted from cortisol levels. Nine male and four female participants aged 25-68 years (N = 13, M = 47.06 ± 15.65 years) each provided saliva samples on multiple occasions prior to and after officiating games at two Australian National Softball Championships. Data from 65 games were analysed. Performance was assessed using Softball Australia's official umpire assessment tool. Cortisol levels increased significantly from baseline to pre-game (p < 0.001, d = -0.69) and declined significantly from pre-game to post-game (p < 0.001, d = 0.47). Umpiring performances were correctly classified as pass or fail from baseline and pre-game cortisol levels in 61.5% of cases and umpire position on the diamond from pre-game cortisol in 63.1% of cases. Findings suggest that stress management strategies should be recommended to softball umpires for performance enhancement and to safeguard their mental health.

Developing a selective culturing approach for Campylobacter hepaticus.

Campylobacter hepaticus, the causative agent of Spotty Liver Disease (SLD) is an important disease in cage-free egg producing chickens causing mortality and production drops. C. hepaticus is a slow growing Campylobacter easily overgrown by fecal bacteria. It is currently only reliably isolatable from bile samples. A selective media for isolation from feces or environment would assist diagnosis and impact assessment. Growth of five Australian C. hepaticus isolates was studied using Horse blood agar (HBA), sheep blood agar (SBA), Bolton, Preston and Brain Heart Infusion (BHI) base media. Blood and/or bile were added to Bolton, Preston and BHI medias. C. jejuni was used as a positive control. Plates were incubated in duplicate under microaerophilic conditions at 42°C for 10 days and examined at days 3-5 and 7-10 of incubation. Each isolate was examined for sensitivity to 14 antimicrobials using HBA sensitivity plates. Growth was inhibited by BHI and by added bile, while blood improved growth. Further replicates using SBA, HBA, Bolton and Preston media showed best growth on Bolton agar with blood. All five C. hepaticus isolates were resistant to trimethoprim and vancomycin, while four were also resistant to rifampicin and bacitracin. Media based upon Bolton plus blood supplemented with vancomycin and trimethoprim might be used as the most appropriate media for selective growth of C. hepaticus. The addition of bile to media for C. hepaticus isolation and growth will inhibit growth and is not advised.

Chimeric hepatitis B surface antigen virus-like particles expressing the strep A epitope p*17 elicit a humoral immune response in mice.

To optimize immunogenicity, bacterial epitopes in putative vaccine constructs can be presented to immune cells as multiple repeated structures on a defined nanoparticle. Virus-like particles (VLPs) are viral capsid proteins that self-assemble to form compact and highly ordered nanoparticles that are within the optimal size range for uptake by dendritic cells. VLPs mimic the live virus in size and form but contain no viral genetic material, are therefore noninfective and are the basis of safe and effective vaccines against hepatitis B virus (HBV) and human papillomavirus (HPV). Due to their particulate nature, molecular stability, and expression of high density and repetitive antigen displays, recombinant cell culture-derived VLPs are ideal platforms for the delivery of small molecules, including bacterial epitopes. We developed a putative vaccine by expressing a minimal epitope from the bacterium Streptococcus pyogenes (Strep A) on the surface of a recombinant VLP comprising multiple copies of HBV small envelope protein (HBsAg-S). Strep A is responsible for a wide spectrum of human infections and postinfectious diseases that disproportionately affect children and young adults living in resource-poor communities. No vaccine is currently available to offer sufficiently broad protection from the numerous and diverse strains of Strep A endemic in these at-risk populations. The Strep A antigen targeted by our vaccine construct is p*17, a cryptic epitope from a highly conserved region of the Strep A M-protein with demonstrated enhanced immunogenicity and broad protective potential against Strep A. To ensure surface expression and optimal immunogenicity, we expressed p*17 within the immunodominant "a" determinant of HBsAg-S. The recombinant VLPs (VLP-p*17) expressed in HEK293T cells spontaneously formed 22 nm particles and induced the production of high titers of p*17-specific IgG in BALB/c mice immunized with three 0.5 µg doses of VLP-p*17 formulated with adjuvant.

Strike 3 Out! Investigating Pre-Game Moods, Performance, and Mental Health of Softball Umpires.

Mood research in sports typically focuses on athletes, with sports officials being largely overlooked. In the current study, mood profiling was used to determine if softball umpires reported an identifiable and consistent mood profile and if mood was predictive of umpiring performance and/or reflective of positive mental health. Eleven male and five female participants aged 25-68 years (M = 48.5 ± 15.5 years) each completed the Brunel Mood Scale on multiple occasions prior to officiating games at the 2020 U18 National Softball Championships. A total of 185 mood profiles were analysed. Performance was assessed using Softball Australia's official umpire assessment tool. Overall, participants reported an iceberg mood profile, which tends to be associated with positive mental health and good performance. Umpiring performances (pass/fail) were correctly classified in 75.0% of cases from tension, depression, and confusion scores (p = 0.003). Participant sex explained 25.7% of the variance in mood scores (p < 0.001); age, 25.8% of the variance (p < 0.001); position on the diamond, 10.5% of the variance (p = 0.003); and accreditation level, 14.3% of the variance (p < 0.001). Australian softball umpires typically reported mood profiles associated with positive mental health, and none reported profiles associated with risk of mental ill-health.

Identification of epidemiological risk factors for spotty liver disease in cage-free layer flocks in houses with fully slatted flooring in Australia.

Spotty liver disease (SLD) is recognized to be caused by infection with Campylobacter hepaticus in adult layer hens farmed in cage-free environments. SLD is an emerging disease as cage-free egg production increases in popularity in response to desires for improved welfare of poultry. Outbreaks of SLD are frequently experienced around peak egg production in flocks, commonly between 25 and 40 wk of age. The disease becomes manifest with increased exposure and access of the birds to the feces of the flock. This study follows from a previous epidemiological survey of free-range and barn flocks in Australia which identified the presence of a scratch area within the laying house as a major risk factor for the occurrence of SLD. However, that survey also observed SLD occurrence in 45% of houses with a fully slatted floor (no scratch area). The present study describes a further analytical survey aimed at identification of risk factors for SLD in houses with fully slatted flooring. A comprehensive questionnaire was completed for 49 cage-free flocks from point of lay until 40 wk of age across Australia, retrieving information on house design, bird breed, flock size, stocking densities, bird growth, and performance and the occurrence of SLD. Multiple logistic regression model building was used to separate factors and identify important management factors that may be amenable to modify the occurrence of SLD in egg layers. Key determinants of SLD identified from the analyses were that houses with mechanical ventilation (such as tunnel ventilation) have some protection from SLD and an increase of an extra 1 bird/m2 of nest space increased odds of occurrence of SLD by 1.172 times. A recommendation to not exceed 112 brown egg layer hens/m2 of nest space in naturally ventilated houses with a full slat floor was suggested. A delay in birds reaching 60% hen day production (HD) by 1 wk is suggested as a possible predictor for a subsequent outbreak of SLD.

Scratch area as an epidemiological risk factor for Spotty Liver Disease in cage-free layers in Australia.

Spotty Liver Disease (SLD) is a serious problem in laying hens farmed in cage-free systems. The causative organism, Campylobacter hepaticus, is regarded as having a fecal-oral method of transmission and hence may build up and spread readily in housing systems which allow ease of direct contact of hens with the flock's fecal material. The epidemiology of SLD has not been thoroughly investigated. An initial cross-sectional analytical epidemiological survey of SLD in free range and barn layer systems was conducted in Australia over 2019 to 2021.The survey involved rearing flocks (n = 32) which were then followed through into laying flocks (n = 24) up to 40 wk of age. Cloacal swabs were collected during rearing and lay for C. hepaticus detection by PCR. Flocks were classified as "Cases" (n = 18) where clinical SLD according to the case definition was observed or "Controls" (n = 6) which were clinically unaffected. No C. hepaticus was detected in cloacal swabs from rearing houses whereas the organism was detected in 18 Case flocks in lay and from 2 Control flocks in lay. All layer houses that incorporated a scratch area (n = 13) were categorized as Cases. Thus, having a scratch area is a key determinant for SLD and no analyses of further contributory factors from these flocks were able to be made. Of the remaining 11 flocks which had floors fully covered by slats, 5 were Cases (45%). Further risk factor analysis was compromised by this small sample size and identification of other significant associations was not possible. A larger survey investigating flocks laying in houses with fully slatted floors was undertaken to further the understanding of SLD epidemiology and is reported in a companion paper.

Detection, characterization, and persistence of Campylobacter hepaticus, the cause of spotty liver disease in layer hens.

A Campylobacter species was first described as the etiological agent of Spotty Liver Disease (SLD) in 2015 and subsequently named as Campylobacter hepaticus in 2016. The bacterium predominantly affects barn and/or free-range hens at peak lay, is fastidious and difficult to isolate, which has impeded elucidation of its sources, means of persistence and transmission. Ten farms from South-Eastern Australia, of which 7 were free range entities participated in the study. A total of 1,404 specimens from layers and 201 from environmental sources, were examined for the presence of C. hepaticus. In this study, our principal findings included the continuing detection of C. hepaticus infection in a flock following an outbreak, indicating a possible transition of infected hens to asymptomatic carriers, that was also characterized by no further occurrence of SLD in the flock. We also report that the first outbreaks of SLD on newly commissioned free-range farms affected layers ranging from 23 to 74 wk of age, while subsequent outbreaks in replacement flocks on these farms occurred during the more conventional peak lay period (23-32 wk of age). Finally, we report that in the on-farm environment, C. hepaticus DNA was detected in layer feces, inert elements such as stormwater, mud, soil, as well as in fauna such as flies, red mites, Darkling beetles, and rats. While in off-farm locations, the bacterium was detected in feces from a variety of wild birds and a canine.

Rheumatic heart disease: A review of the current status of global research activity.

Rheumatic heart disease (RHD) is a serious and long-term consequence of acute rheumatic fever (ARF), an autoimmune sequela of a mucosal infection by Streptococcus pyogenes (Group A Streptococcus, Strep A). The pathogenesis of ARF and RHD is complex and not fully understood but involves host and bacterial factors, molecular mimicry, and aberrant host innate and adaptive immune responses that result in loss of self-tolerance and subsequent cross-reactivity with host tissues. RHD is entirely preventable yet claims an estimated 320 000 lives annually. The major burden of disease is carried by developing nations and Indigenous populations within developed nations, including Australia. This review will focus on the epidemiology, pathogenesis and treatment of ARF and RHD in Australia, where: streptococcal pyoderma, rather than streptococcal pharyngitis, and Group C and Group G Streptococcus, have been implicated as antecedents to ARF; the rates of RHD in remote Indigenous communities are persistently among the highest in the world; government register-based programs coordinate disease screening and delivery of prophylaxis with variable success; and researchers are making significant progress in the development of a broad-spectrum vaccine against Strep A.

Correlation between Fatty Acid Profile and Anti-Inflammatory Activity in Common Australian Seafood by-Products.

Marine organisms are a rich source of biologically active lipids with anti-inflammatory activities. These lipids may be enriched in visceral organs that are waste products from common seafood. Gas chromatography-mass spectrometry and fatty acid methyl ester (FAME) analyses were performed to compare the fatty acid compositions of lipid extracts from some common seafood organisms, including octopus (Octopus tetricus), squid (Sepioteuthis australis), Australian sardine (Sardinops sagax), salmon (Salmo salar) and school prawns (Penaeus plebejus). The lipid extracts were tested for anti-inflammatory activity by assessing their inhibition of nitric oxide (NO) and tumor necrosis factor alpha (TNFα) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse cells. The lipid extract from both the flesh and waste tissue all contained high amounts of polyunsaturated fatty acids (PUFAs) and significantly inhibited NO and TNFα production. Lipid extracts from the cephalopod mollusks S. australis and O. tetricus demonstrated the highest total PUFA content, the highest level of omega 3 (ω-3) PUFAs, and the highest anti-inflammatory activity. However, multivariate analysis indicates the complex mixture of saturated, monounsaturated, and polyunsaturated fatty acids may all influence the anti-inflammatory activity of marine lipid extracts. This study confirms that discarded parts of commonly consumed seafood species provide promising sources for the development of new potential anti-inflammatory nutraceuticals.

Spotty Liver Disease: A review of an ongoing challenge in commercial free-range egg production.

Spotty Liver Disease is an acute infectious disease of layer chickens that was likely first described in the USA and Canada in the 1950s and 1960's. The disease occurs almost exclusively in barn and free-range production systems. Outbreaks usually, but not exclusively occur in young layers (≅25 weeks) at peak of lay. Indicators of SLD include an acute drop in egg production of up to 35%, together with increased mortality of up to 15%. A presumptive diagnosis at post mortem is made with the detection of characteristic small yellow-white necrotic hepatic lesions, together with a fibrinous peri-hepatitis, excess pericardial and peritoneal fluid, and usually enteritis with diarrhoea. Histopathology reveals a multifocal acute hepatocellular necrosis with fibrin and occasional haemorrhage. Control measures trialled include use of antibiotics, improved biosecurity and hygiene, as well as management practices directed at reducing stress in flocks. However, none other than treatment with antibiotics has been consistently effective which suggested a bacterial aetiology. In 2015, a novel fastidious thermophilic, microaerobic campylobacter was isolated from symptomatic SLD flocks in the UK. Subsequently, an Australian group isolated and further characterised a genetically similar bacterium and named it Campylobacter hepaticus. The bacterium can be cultured from the liver and bile of infected birds, although recovery from non-sterile organs such as the caecum and duodenum remains elusive. Consequently, the route of transmission remains unconfirmed, although molecular detection by PCR of C. hepaticus DNA in the gastrointestinal tract and faeces of SLD infected birds is highly suggestive of a faecal-oral route.

Review of anti-inflammatory, immune-modulatory and wound healing properties of molluscs.

ETHNOPHARMACOLOGICAL RELEVANCE: This review focuses on traditional and contemporary anti-inflammatory uses of mollusc-derived products summarising all the in vitro, in vivo and human clinical trials that have tested the anti-inflammatory activity of molluscan natural products. Inflammatory conditions, burns and wounds have been an ongoing concern for human health since the early era of civilisation. Many texts from ancient medicine have recorded the symptoms, signs and treatments for these conditions. Natural treatments are well-documented in traditional European medicine, Traditional Chinese Medicine (TCM), Siddha and ancient Mediterranean and African traditional medicine and include a surprisingly large number of molluscan species. MATERIALS AND METHODS: An extensive review of the Materia Medica and scientific literature was undertaken using key word searches for "mollusc" and "anti-inflammatory" or "immunomodulatory" or "wound healing". RESULTS: Molluscs have been used in ethnomedicine by many traditional cultures to treat different aspects of inflammatory conditions. We found 104 different anti-inflammatory preparations from a variety of molluscan species, of which 70 were from the well-documented Traditional Chinese Medicine (TCM). This traditional use of molluscs has driven the testing for inflammatory activity in extracts from some species in the phylum Mollusca, with 20 in vitro studies, 40 in vivo animal studies and 14 human clinical trials performed to substantiate the anti-inflammatory and wound healing activity of molluscs. Some of these studies have led to the approval of mollusc-derived products to be used as over-the-counter (OTC) nutraceuticals, like Lyprinol® and Biolane™ from the New Zealand green lipped mussel Perna canaliculus. CONCLUSION: Natural products provide important leads for the development of pharmaceuticals, including anti-inflammatory agents. Only a small proportion of the molluscan traditional medicines have been tested to confirm their anti-inflammatory activity and most screening studies have tested crude extracts from molluscs without any chemical characterisation. This highlights the need for further research to strategically identify the anti-inflammatory compounds in molluscan medicines to provide leads for novel anti-inflammatory drugs in the future.

Brominated indoles from a marine mollusc inhibit inflammation in a murine model of acute lung injury.

New drug leads for the treatment of inflammation are urgently needed. Marine molluscs are widely used as traditional medicines for the treatment of inflammation. Here we report the positive effects of a hypobranchial gland (HBG) extract and the dominant bioactive compound 6-bromoisatin from the Muricidae mollusc Dicathais orbita, for reducing lipopolysaccharide (LPS) induced acute lung inflammation in a mouse model. Both 6-bromoisatin and the HBG extract suppressed the inflammatory response in mice that were pre-treated by oral gavage at 48, 24 and 1 h prior to LPS infusion. The inflammatory antagonists were tested at concentrations of 0.5 mg/g and 0.1 mg/g HBG extract and 0.1 mg/g and 0.05 mg/g 6-bromoisatin in carrier oil and all treatments reduced inflammation as indicated by a significant suppression of inflammatory markers present in bronchoalveolar lavage fluid (BALF), in comparison to LPS induced positive control mice administered the carrier oil alone (p < 0.0001). Tumour necrosis factor-alpha (TNFα) and interleukin-1 beta (IL-1ß) levels, in addition to total protein concentration were all significantly reduced in BALF from mice treated with the extract or 6-bromoisatin. Furthermore, all treatment groups showed significant reductions in neutrophil sequestration and preservation of the lung tissue architecture compared to the positive control (p < 0.0001). The combined results from this study and our previous in vitro studies indicate that 6-bromoisatin in the HGB extracts inhibit the activation of inflammatory signalling pathway. The results from this study further confirm that the HBG extract from Muricidae molluscs and 6-bromoisatin are bioavailable and effective in vivo, thus have potential for development as natural therapeutic agents for inflammation.

Anti-Inflammatory Activity and Structure-Activity Relationships of Brominated Indoles from a Marine Mollusc.

Marine molluscs are rich in biologically active natural products that provide new potential sources of anti-inflammatory agents. Here we used bioassay guided fractionation of extracts from the muricid Dicathais orbita to identify brominated indoles with anti-inflammatory activity, based on the inhibition of nitric oxide (NO) and tumour necrosis factor α (TNFα) in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages and prostaglandin E2 (PGE2) in calcium ionophore-stimulated 3T3 ccl-92 fibroblasts. Muricid brominated indoles were then compared to a range of synthetic indoles to determine structure-activity relationships. Both hypobranchial gland and egg extracts inhibited the production of NO significantly with IC50 of 30.8 and 40 µg/mL, respectively. The hypobranchial gland extract also inhibited the production of TNFα and PGE2 with IC50 of 43.03 µg/mL and 34.24 µg/mL, respectively. The purified mono-brominated indole and isatin compounds showed significant inhibitory activity against NO, TNFα, and PGE2, and were more active than dimer indoles and non-brominated isatin. The position of the bromine atom on the isatin benzene ring significantly affected the activity, with 5Br > 6Br > 7Br. The mode of action for the active hypobranchial gland extract, 6-bromoindole, and 6-bromoisatin was further tested by the assessment of the translocation of nuclear factor kappa B (NFκB) in LPS-stimulated RAW264.7 mouse macrophage. The extract (40 µg/mL) significantly inhibited the translocation of NFκB in the LPS-stimulated RAW264.7 macrophages by 48.2%, whereas 40 µg/mL of 6-bromoindole and 6-bromoistain caused a 60.7% and 63.7% reduction in NFκB, respectively. These results identify simple brominated indoles as useful anti-inflammatory drug leads and support the development of extracts from the Australian muricid D. orbita, as a new potential natural remedy for the treatment of inflammation.

Diversity of Virulence Factors Associated with West Australian Methicillin-Sensitive Staphylococcus aureus Isolates of Human Origin.

An extensive array of virulence factors associated with S. aureus has contributed significantly to its success as a major nosocomial pathogen in hospitals and community causing variety of infections in affected patients. Virulence factors include immune evading capsular polysaccharides, poly-N-acetyl glucosamine, and teichoic acid in addition to damaging toxins including hemolytic toxins, enterotoxins, cytotoxins, exfoliative toxin, and microbial surface components recognizing adhesive matrix molecules (MSCRAMM). In this investigation, 31 West Australian S. aureus isolates of human origin and 6 controls were analyzed for relative distribution of virulence-associated genes using PCR and/or an immunoassay kit and MSCRAMM by PCR-based typing. Genes encoding MSCRAMM, namely, Spa, ClfA, ClfB, SdrE, SdrD, IsdA, and IsdB, were detected in >90% of isolates. Gene encoding α-toxin was detected in >90% of isolates whereas genes encoding ß-toxin and SEG were detectable in 50-60% of isolates. Genes encoding toxin proteins, namely, SEA, SEB, SEC, SED, SEE, SEH, SEI, SEJ, TSST, PVL, ETA, and ETB, were detectable in >50% of isolates. Use of RAPD-PCR for determining the virulence factor-based genetic relatedness among the isolates revealed five cluster groups confirming genetic diversity among the MSSA isolates, with the greatest majority of the clinical S. aureus (84%) isolates clustering in group IIIa.

The diversity and antimicrobial activity of Preussia sp. endophytes isolated from Australian dry rainforests.

Limited knowledge currently exists regarding species diversity and antimicrobial activity of endophytic isolates of Preussia within Australia. This report describes endophytic Preussia species that were identified through molecular analysis of the internal transcribed spacer region. Screening for antimicrobial secondary metabolites was determined by testing crude ethyl acetate (EtOAc) extracts derived from fungal mycelia against a panel of ATCC type strains which included Bacillus cereus, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Serratia marcescens, methicillin-resistant Staphylococcus aureus (MRSA) and the opportunist yeast pathogen Candida albicans. Subsequently, high-performance liquid chromatography generated fractions of bioactive EtOAc extracts which were subject to confirmatory testing using the Clinical and Laboratory Standards Institute reference microdilution antimicrobial activity assay. A total of 18 Preussia were isolated from nine host plants with 6/18 having a <97 % sequence similarity to other known species in Genbank, suggesting that they are new species. In preliminary screening, 13/18 Preussia isolates revealed antimicrobial activity against at least one of the microbes tested, whilst 6/18 isolates, including 4/6 putative new species showed specific antimicrobial activity against MRSA and C. albicans. These results highlight the antimicrobial potential of Australian Preussia spp. and also the importance of Australian dry rainforests as an untapped repository of potentially significant bioactive compounds.

Anti-inflammatory activity of hyperimmune plasma in a lipopolysaccharide-mediated rat air pouch model of inflammation.

Tumour necrosis factor-α (TNFα) and polymorphonuclear neutrophils play key and interrelated roles in the inflammatory response against infectious agents. However, these entities can mediate significant tissue damage if their biological activity becomes deregulated. We have previously shown that canine hyperimmune frozen plasma (HFP) contains anti-TNFα activity that is attributable to elevated levels of soluble TNFα receptor 1 (sTNFR1). The aim of this study was to determine the effect of HFP on TNFα levels and neutrophil infiltration in a lipopolysaccharide (LPS)-mediated rat air pouch model of inflammation. Rats were administered either HFP, HFP which had been pre-incubated with anti-sTNFR1 antibody (5 ng/ml), fresh frozen plasma (FFP), physiological saline (PS) at 2 ml/day or Carprofen at 5 mg/kg for 3 days prior to LPS challenge. Pouch fluid was withdrawn at 1, 6, 12, 24 and 48 h post-LPS challenge and assayed for TNFα by ELISA, and for total leukocytes and neutrophils by microscopic examination. At 6 h post-LPS challenge, both TNFα levels and neutrophil counts were significantly lower in HFP-treated rats than was found in FFP, PS or Carprofen treated animals (p<0.05). In a sTNFR1 blocking experiment, incubation of HFP with anti-sTNFR1 antibody resulted in significant increases in neutrophil numbers and TNFα levels, which suggests that the anti-TNFα activity observed in HFP may be due to elevated levels of sTNFR1. The data also revealed a significant inverse correlation between total leukocyte counts and sTNFR1 levels present in pouch fluid (r= -0.73, p<0.0001). Our observations suggest that HFP warrants further investigation as a possible means for modulating acute inflammatory processes where TNFα is a key mediator.

Immunological response to parenteral vaccination with recombinant hepatitis B virus surface antigen virus-like particles expressing Helicobacter pylori KatA epitopes in a murine H. pylori challenge model.

Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S-KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 10(3)) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 10(2)). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 10(4) and 2.6 × 10(4), respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens.

Caelestines A-D, brominated quinolinecarboxylic acids from the Australian ascidian Aplidium caelestis.

Four new brominated natural products, caelestines A-D (1-4), have been isolated from the Australian ascidian Aplidium caelestis. The structures of 1-4 were determined by analysis of their NMR and MS data. This is the first report of brominated quinolinecarboxylic acids from nature. Compound 1 has been previously synthesized but not spectroscopically characterized. Compounds 1-4 were tested against three mammalian cell lines (MCF-7, NFF, and MM96L) and a panel of microbial strains and showed only minor cytotoxicity.

Detection of anti-TNFalpha activity in canine hyperimmune serum using a TNFalpha inhibition assay.

BACKGROUND: Increased serum tumor necrosis factor-alpha (TNFalpha) activity has been associated with onset of serious inflammatory diseases in dogs. Development of treatment with TNFalpha-antagonists has been limited by the unavailability of suitable reagents and potency assays for TNFalpha. OBJECTIVES: The objectives of this study were to optimize a cell-based assay to measure anti-TNFalpha activity in serum and plasma from hyperimmune (vaccinated with an Escherichia coli J5 bacterin) and unvaccinated canine donors; to use the assay to determine whether hyperimmune serum inhibits TNFalpha activity in vivo; and to determine whether soluble TNF receptor-1 (sTNFR1, a naturally occurring TNFalpha antagonist) contributes to anti-TNFalpha activity. METHODS: Commercial plasma and serum from hyperimmune-frozen plasma (HFP) donors and unvaccinated fresh-frozen plasma (FFP) donors were used in the study. An L929-cell TNFalpha-inhibition assay (LTIA) was optimized to measure anti-TNFalpha activity. Using a rat subcutaneous pouch model of inflammation, the effects of HFP, FFP, a synthetic TNFalpha antagonist (Etanercept), and carprofen on TNFalpha activity were compared in vivo. Immunofluorescence was used to measure soluble sTNFR1 concentration. RESULTS: Using the optimized LTIA, HFP serum but not FFP serum decreased canine TNFalpha activity (P<.01). HFP plasma and Etanercept (but not FFP plasma or carprofen) significantly decreased TNFalpha activity in pouch exudates (P<.05). A significantly higher concentration of sTNFR1 was found in HFP than FFP serum. CONCLUSIONS: Using the LTIA, anti-TNFalpha activity is readily measured in canine serum and inflammatory exudates. sTNFR1 appears to contribute to anti-TNFalpha activity in HFP serum. These results suggest HFP should be investigated further as a potential immunotherapeutic agent for controlling canine diseases in which TNFalpha is implicated.