The role of fimbriae and flagella in the colonization, invasion and persistence of Escherichia coli O78 : K80 in the day-old-chick model

To understand the role of flagella and fimbriae of Escherichia coli O78:K80 in avian colibacillosis, day-old chicks were dosed orally with defined afimbriate and or aflagellate mutants and colonization, invasion and persistence compared with that of the wild-type. In an invasion model, chicks were dosed with 1 x 10(5) c.f.u. of a single strain and mutants defective for type 1 fimbriae, curli fimbriae or flagella colonized livers by 24 h although the numbers of bacteria present were significantly less than the wild-type, Mutants colonized between 50 and 75 % of spleens whereas the wild-type colonized 100 % of spleens. Additionally, the numbers of mutant bacteria in colonized spleens were significantly less than the wild-type. Surprisingly, mutants defective for the elaboration of more than one appendage were no more attenuated than single mutants. In a persistence model, chicks were dosed with 1 x 10(2) c.f.u. of a single strain and mutants defective for type 1 or curli or flagella or any combination thereof persisted as assessed by cloacal swabbing for 5 weeks of the experiment less well than the wild-type. In an additional persistence model, chicks were dosed with 5 x 10(2) c.f.u. of each of wild-type and one mutant together. All mutants were significantly less persistent than the wild-type (P < 0.001) and one mutant which lacked type 1, curli and flagella, was eliminated within 2 weeks. Analysis of the trends of elimination indicated that flagella contributed to persistence more than curli, which contributed more than type 1 fimbriae. Here was evidence for a major role in colonization, invasion and persistence played by type 1, curli and flagella.

Bacillus subtilis spores competitively exclude Escherichia coli O78:K80 in poultry

Newly hatched specific pathogen-free chicks were dosed with a suspension of Bacillus subtilis spores prior to challenge with Escherichia coli O78:K80, a known virulent strain associated with avian colibacillosis. 24 h later. A single oral inoculum of 2.5 x 10(8) spores was sufficient to suppress all aspects of E. coli O78:K80 infection. Colonisation of deep organs was reduced by a factor of over 2 log(10) whilst colonisation of the intestine, as measured by direct caecal count, was reduced over 3 log(10). Shedding of E. coli O78:K80 was measured by semi-quantitative cloacal swabbing and was reduced significantly for the: duration of the experiment, 35 days. B, subtilis persisted in the intestine although with decreasing numbers over the same period. Challenge with the same dose 5 days after pre-dosing with spores overcame any suppressive effect of the spores. Crown Copyright (C) 2001 Published by Elsevier Science B.V. All rights reserved.

The efficacy of Salenvac, a Salmonella enterica subsp. Enterica serotype Enteritidis iron-restricted bacterin vaccine, in laying chickens

The protective effect of two vaccination regimes using Salenvac, a commercially available iron-restricted Salmonella enterica subsp. Enterica serotype Enteritidis PT4 bacterin vaccine, was verified in laying birds. Immunization was intramuscular at 1 day old and again at 4 weeks of age (V2), or at 1 day and 4 weeks with a third dose at 18 weeks of age (V3). Challenge S. Enteritidis (5 to 7.5) x 10(7) colony forming units) was given intravenously at 8, 17, 23, 30 and 59 weeks of age. For all age groups, both vaccination regimes reduced significantly the number of tissues and faecal samples that were culture positive for the challenge strain. For laying birds, fewer eggs (P < 0.001) were culture positive for S. Enteritidis after challenge from vaccinated laying birds ( 56/439 batches of eggs) than unvaccinated birds (99/252 batches). The data give compelling evidence that the vaccine is efficacious and may contribute to the reduction of layer infection and egg contamination.

Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens

Cost effective control of avian diseases and food borne pathogens remains a high priority for all sectors of the poultry industry with cleansing and disinfection, vaccination and competitive exclusion approaches being used widely. Previous studies showed that Bacillus subtilis PY79(hr) was an effective competitive exclusion agent for use in poultry to control avian pathogenic Escherichia coli serotype O78:K80. Here we report experiments that were undertaken to test the efficacy of B. subtilis PY79(hr) in the control of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. To do this, 1-day-old and 20-day-old specific pathogen free (SPF) chicks were dosed with a suspension of B. subtilis spores prior to challenge with S. Enteritidis (S1400) and C. perfringens, respectively. For both challenge models, a single oral inoculum of 1 x 10(9) spores given 24 h prior to challenge was sufficient to suppress colonisation and persistence of both S. Enteritidis and C perfringens. In particular, the faecal shedding of S. Enteritidis, as measured by a semi-quantitative cloacal swabbing technique, was reduced significantly for the 36 days duration of the experiment. B. subtilis persisted in the intestine although with decreasing numbers over the same period. These data add further evidence that B. subtilis spores may be effective agents in the control of avian diseases and food borne pathogens.

The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis

The ability of an isogenic set of mutants of Salmonella enterica serovar Typhimurium L354 (SL1344) with defined deletions in genes encoding components of tripartite efflux pumps, including acrB, acrD, acrF and tolC, to colonize chickens was determined in competition with L354. In addition, the ability of L354 and each mutant to adhere to, and invade, human embryonic intestine cells and mouse monocyte macrophages was determined in vitro. The tolC and acrB knockout mutants were hyper-susceptible to a range of antibiotics, dyes and detergents; the tolC mutant was also more susceptible to acid pH and bile and grew more slowly than L354. Complementation of either gene ablated the phenotype. The tolC mutant poorly adhered to both cell types in vitro and was unable to invade macrophages. The acrB mutant adhered, but did not invade macrophages. In vivo, both the acrB mutant and the tolC mutant colonized poorly and did not persist in the avian gut, whereas the acrD and acrF mutant colonized and persisted as well as L354. These data indicate that the AcrAB-TolC system is important for the colonization of chickens by S. Typhimurium and that this system has a role in mediating adherence and uptake into target host cells.

How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles

Powered by advances in electron tomography, recent studies have extended our understanding of how viruses construct "replication factories" inside infected cells. Their function, however, remains an area of speculation with important implications for human health. It is clear from these studies that whatever their purpose, organelle structure is dynamic (M. Ulasli, M. H. Verheije, C. A. de Haan, and F. Reggiori, Cell. Microbiol. 12:844-861, 2010) and intricate (K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, et al., PLOS Biol. 6:e226, 2008). But by concentrating on medically important viruses, these studies have failed to take advantage of the genetic variation inherent in a family of viruses that is as diverse as the archaea, bacteria, and eukaryotes combined (C. Lauber, J. J. Goeman, M. del Carmen Parquet, P. T. Nga, E. J. Snijder, et al., PLOS Pathog. 9:e1003500, 2013). In this climate, Maier et al. (H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, et al., mBio 4:e00801-13, 2013) explored the replicative structures formed by an avian coronavirus that appears to have diverged at an early point in coronavirus evolution and shed light on controversial aspects of viral biology.

Evidence for systemic spread of the potentially zoonotic intestinal spirochaete Brachyspira pilosicoli in experimentally challenged laying chickens

Brachyspira pilosicoli is a potentially zoonotic anaerobic intestinal spirochaete that is one of several species causing avian intestinal spirochaetosis. The aim of this study was to develop a reproducible model of infection in point-of-lay chickens and compare the virulence of two strains of B. pilosicoli in a model using experimentally challenged laying chickens. Seventeen-week-old commercial laying chickens were experimentally challenged by oral gavage with either B. pilosicoli strain B2904 or CPSp1, following an oral dose of 10 % sodium bicarbonate to neutralize acidity in the crop. Approximately 80 % of the chickens became colonized and exhibited increased faecal moisture content, reduced weight gain and delayed onset of lay. Tissues sampled at post-mortem examination were analysed to produce a quantitative output on the number of spirochaetes present and hence, the extent of colonization. The liver and spleen were colonized, and novel histopathology was observed in these tissues. The infection model we report here has potential use in studies to improve our understanding of the mechanisms by which Brachyspira elicit disease in poultry and in testing novel intervention strategies.

Oral treatment of chickens with Lactobacillus reuteri LM1 reduces Brachyspira pilosicoli-induced pathology

Avian intestinal spirochaetosis (AIS) results from the colonization of the caeca and colon of poultry by pathogenic Brachyspira, notably Brachyspira pilosicoli. Following the ban on the use of antibiotic growth promoters in the European Union in 2006, the number of cases of AIS has increased, which, alongside emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Lactobacillus-based probiotics have been shown to protect against infection with common enteric pathogens in livestock. Our previous studies have shown that Lactobacillus reuteri LM1 antagonizes aspects of the pathobiology of Brachyspira in vitro. Here, we showed that L. reuteri LM1 mitigates the clinical symptoms of AIS in chickens experimentally challenged with B. pilosicoli. Two groups of 15 commercial laying hens were challenged experimentally by oral gavage with B. pilosicoli B2904 at 18 weeks of age; one group received unsupplemented drinking water and the other received L. reuteri LM1 in drinking water from 1 week prior to challenge with Brachyspira and thereafter for the duration of the study. This treatment regime was protective. Specifically, B. pilosicoli was detected by culture in fewer birds, bird weights were higher, faecal moisture contents were significantly lower (P<0.05) and egg production as assessed by egg weight and faecal staining score was improved (P<0.05). Also, at post-mortem examination, significantly fewer B. pilosicoli were recovered from treated birds (P<0.05), with only mild–moderate histopathological changes observed. These data suggest that L. reuteri LM1 may be a useful tool in the control of AIS.

Supplementary feeding of wild birds indirectly affects ground beetle populations in suburban gardens

Supplementary feeding of wild birds by domestic garden-holders is a globally widespread and popular form of human–wildlife interaction, particularly in urban areas. Vast amounts of energy are thus being added to garden ecosystems. However, the potential indirect effects of this activity on non-avian species have been little studied to date, with the only two previous studies taking place under experimentally manipulated conditions. Here we present the first evidence of a localised depletive effect of wild bird feeding on ground beetles (Coleoptera: Carabidae) in suburban gardens under the usual feeding patterns of the garden-holders. We trapped significantly fewer ground beetles directly under bird-feeding stations than in matched areas of habitat away from feeders. Video analysis also revealed significantly higher activity by ground-foraging birds under the feeding stations than in the control areas. Small mammal trapping revealed no evidence that these species differ in abundance between gardens with and without bird feeders. We therefore suggest that local increases in ground-foraging activity by bird species whose diets encompass arthropods as well as seed material are responsible for the reduction in ground beetle numbers. Our work therefore illustrates that providing food for wild birds can have indirect negative effects on palatable prey species under typical conditions.

Efficacy of a live attenuated Escherichia coli O78:K80 vaccine in chickens and turkeys

A candidate live vaccine for avian pathogenic Escherichia coli (APEC) was constructed from a virulent field APEC O78 strain by mutation of the aroA gene. The mutant was highly similar to the parent wild-type strain in respect of colony morphology, motility, growth in suspension, hemagglutination, Congo Red binding, HEp-2 cell adhesion, and the elaboration of surface antigens type 1 fimbriae and flagella, although production of curli fimbriae was reduced marginally. The mutant proved avirulent when inoculated into 1-day-old chicks by spray application and when presented again in the drinking water at 7 days of age. Chickens and turkeys vaccinated with an O78 aroA mutant were protected against a challenge at 6 wk of age by virulent APEC strains.

Mercury and methylmercury bioaccumulation by polychaete worms is governed by both feeding ecology and mercury bioavailability in coastal mudflats

Polychaete worms are abundant in many mudflats but their importance to coastal food web Hg biomagnification is not known. We sampled sediments and polychaete worms from mudflats in the Bay of Fundy to investigate the bioaccumulation of mercury (Hg) and methylmercury (MeHg) in the coastal invertebrate food web. Hg concentrations in the sediments were low (<20 μg kg−1). Labile Hg (methanol/KOH sediment extraction) in surface sediments (0–1 cm) was positively correlated with Hg bioaccumulation by surface sediment-ingesting polychaetes but, surprisingly, there was a negative correlation between δ15N (i.e. trophic level) and THg bioaccumulation factors in polychaete worms. Worms feeding on deeper sediments contained the greatest MeHg concentrations (69.6 μg kg−1). Polychaetes are an important vector for Hg biomagnification to the coastal avian food web. This research demonstrates that feeding depth and method of feeding are more important than trophic position or sediment Hg concentrations for predicting Hg bioaccumulation.

Cadaver decomposition in terrestrial ecosystems

A dead mammal (i.e. cadaver) is a high quality resource (narrow carbon:nitrogen ratio, high water content) that releases an intense, localised pulse of carbon and nutrients into the soil upon decomposition. Despite the fact that as much as 5,000 kg of cadaver can be introduced to a square kilometre of terrestrial ecosystem each year, cadaver decomposition remains a neglected microsere. Here we review the processes associated with the introduction of cadaver-derived carbon and nutrients into soil from forensic and ecological settings to show that cadaver decomposition can have a greater, albeit localised, effect on belowground ecology than plant and faecal resources. Cadaveric materials are rapidly introduced to belowground floral and faunal communities, which results in the formation of a highly concentrated island of fertility, or cadaver decomposition island (CDI). CDIs are associated with increased soil microbial biomass, microbial activity (C mineralisation) and nematode abundance. Each CDI is an ephemeral natural disturbance that, in addition to releasing energy and nutrients to the wider ecosystem, acts as a hub by receiving these materials in the form of dead insects, exuvia and puparia, faecal matter (from scavengers, grazers and predators) and feathers (from avian scavengers and predators). As such, CDIs contribute to landscape heterogeneity. Furthermore, CDIs are a specialised habitat for a number of flies, beetles and pioneer vegetation, which enhances biodiversity in terrestrial ecosystems.

Drinking water application of Denargard Tiamulin for control of Brachyspira pilosicoli infection of laying poultry

Avian intestinal spirochaetosis (AIS) caused by Brachyspira spp., and notably Brachyspira pilosicoli, is common in layer flocks and reportedly of increasing incidence in broilers and broiler breeders. Disease manifests as diar- rhoea, increased feed consumption, reduced growth rates and occasional mortality in broilers and these signs are shown in layers also associated with a delayed onset of lay, reduced egg weights, faecal staining of eggshells and non-productive ovaries. Treatment with Denagard® Tiamulin has been used to protect against B. pilosicoli colonisation, persistence and clinical presentation of AIS in commercial layers, but to date there has been no de- finitive study validating efficacy. Here, we used a poultry model of B. pilosicoli infection of layers to compare the impact of three doses of Denagard® Tiamulin. Four groups of thirty 17 week old commercial pre-lay birds were all challenged with B. pilosicoli strain B2904 with three oral doses two days apart. All birds were colonised within 2 days after the final oral challenge and mild onset of clinical signs were observed thereafter. A fifth group that was unchallenged and untreated was also included for comparison as healthy birds. Five days after the final oral Brachypira challenge three groups were given Denagard® Tiamulin in drinking water made up following the manufacturer's recommendations with doses verified as 58.7 ppm, 113 ppm and 225 ppm. Weight gain body condition and the level of diarrhoea of birds infected with B. pilosicoli were improved and shedding of the organism reduced significantly (p = 0.001) following treatment with Denagard® Tiamulin irrespective of dose given. The level and duration of colonisation of organs of birds infected with B. pilosicoli was also reduced. Confirming previous findings we showed that the ileum, caeca, colon, and both liver and spleen were colonised and here we demonstrated that treatment with Denagard® Tiamulin resulted in significant reduction in the numbers of Brachyspira found in each of these sites and dramatic reduction in faecal shedding (p b 0.001) to ap- proaching zero as assessed by culture of cloacal swabs. Although the number of eggs produced per bird and the level of eggshell staining appeared unaffected, egg weights of treated birds were greater than those of untreated birds for a period of approximately two weeks following treatment. These data conclusively demonstrate the ef- fectiveness of Denagard® Tiamulin in reducing B. pilosicoli infection in laying hens.

Drinking water application of Denagard® Tiamulin for control of Brachyspira pilosicoli infection of laying poultry

Avian intestinal spirochaetosis (AIS) caused by Brachyspira spp., and notably Brachyspira pilosicoli, is common in layer flocks and reportedly of increasing incidence in broilers and broiler breeders. Disease manifests as diarrhoea,increased feed consumption, reduced growth rates and occasional mortality in broilers and these signs are shown in layers also associated with a delayed onset of lay, reduced egg weights, faecal staining of eggshells and non-productive ovaries. Treatment with Denagard® Tiamulin has been used to protect against B. pilosicoli colonisation, persistence and clinical presentation of AIS in commercial layers, but to date there has been no definitive study validating efficacy. Here, we used a poultry model of B. pilosicoli infection of layers to compare the impact of three doses of Denagard® Tiamulin. Four groups of thirty 17 week old commercial pre-lay birds were all challengedwith B. pilosicoli strain B2904with three oral doses two days apart. All birdswere colonised within 2 days after the final oral challenge and mild onset of clinical signs were observed thereafter. A fifth group that was unchallenged and untreated was also included for comparison as healthy birds. Five days after the final oral Brachypira challenge three groups were given Denagard® Tiamulin in drinking water made up following the manufacturer's recommendations with doses verified as 58.7 ppm, 113 ppm and 225 ppm. Weight gain body condition and the level of diarrhoea of birds infected with B. pilosicoli were improved and shedding of the organism reduced significantly (p = 0.001) following treatment with Denagard® Tiamulin irrespective of dose given. The level and duration of colonisation of organs of birds infected with B. pilosicoli was also reduced. Confirming previous findings we showed that the ileum, caeca, colon, and both liver and spleen were colonised and here we demonstrated that treatment with Denagard® Tiamulin resulted in significant reduction in the numbers of Brachyspira found in each of these sites and dramatic reduction in faecal shedding (p b 0.001) to approaching zero as assessed by culture of cloacal swabs. Although the number of eggs produced per bird and the level of eggshell staining appeared unaffected, egg weights of treated birds were greater than those of untreated birds for a period of approximately two weeks following treatment. These data conclusively demonstrate the effectiveness of Denagard® Tiamulin in reducing B. pilosicoli infection in laying hens.