Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef-pef region, Ranked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI-orf9 region although polymorphism was evident. Crown Copyright (C) 2001 Published by Elsevier Science B.V. All rights reserved.

The O-antigen of Salmonella enterica serotype Enteritidis PT4: a significant factor in gastrointestinal colonisation of young but not newly hatched chicks

The lipopolysaccharide of Salmonella and other Gram negative pathogenic species has been implicated as a major virulence determinant and in this study we report the role of LPS of S. Enteritidis in the colonisation and persistent gastrointestinal infection of young poultry. The gene encoding the unique O-antigen ligase, waaL, was mutated by insertional inactivation in a well characterised S. Enteritidis strain, S1400/94. The waaL mutant, designated PCP, produced rough colonies on agar medium, did not agglutinate O9 antiserum, did not produce an LPS ladder on silver stained gels and was serum sensitive. PCP and a nalidixic acid marked derivative of S1400/94 (S1400/94 Nal(r)) were used to orally challenge young chicks, separately and together in competitive index experiments. At post-mortem examination of 1-day-old chicks challenged S1400/94 Nal(r) and PCP separately there were no significant differences in the numbers of S1400/94 Nal(r) and PCP bacteria in tissues sampled on days 1, 2. and 5. By day 42 after challenge S1400/94 Nal(r) bacteria were recovered in significantly higher numbers than PCP from the caecal contents (P < 0.001). In competitive index studies in the 1-day-old chick PCP colonised, invaded and persisted in lower numbers than S1400/94 Nal(r). In 4-week-old chicks challenged separately, PCP bacteria were recovered from all tissues examined in significantly lower numbers than S1400/94 Nal(r). In competitive index experiments in 4-week-old chicks, PCP was not detected at any site and at any time point. Therefore, the O-antigen of S. Enteritidis plays art important role in poultry infections although this role is less important in the newly hatched chick. Crown Copyright (C) 2004 Published by Elsevier B.V. All rights reserved.

Investigation on Cacao swollen shoot virus (CSSV) pollen transmission through cross-pollination

DNA- and RNA-based polymerase chain reaction (PCR) systems were used with Cacao swollen shoot virus (CSSV) primers designed from conserved regions of the six published genomic sequences of CSSV to investigate whether the virus is transmissible from infected trees through cross-pollination to seeds and seedlings. Pollen was harvested from CSSV infected cocoa trees and used to cross-pollinate flowers of healthy cocoa trees (recipient parents) to generate enough cocoa seeds for the PCR screening. Adequate precautions were taken to avoid cross-contamination during duplicated DNA extractions and only PCR results accompanied by effective positive and negative controls were scored. Results from the PCR analyses showed that samples of cocoa pod husk, mesocarp and seed tissues (testa, cotyledon and embryo) from the cross-pollinations were PCR negative for CSSV DNA. Sequential DNA samples from new leaves of seedlings resulting from the cross-pollinated trees were consistently PCR negative for presence of portions of CSSV DNA for over 36 months after germination. A reverse transcription-PCR analysis performed on the seedlings showed negative results, indicating absence of functional CSSV RNA transcripts in the seedlings. None of the seedlings exhibited symptoms characteristic of the CSSV disease, and all infectivity tests on the seedlings were also negative. Following these results, the study concluded that although CSSV DNA was detected in pollen from CSSV infected trees, there was no evidence of pollen transmission of the virus through cross-pollination from infected cocoa parents to healthy cocoa trees.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

An improved methodology for the recovery of Zea mays and other large crop pollen, with implications for environmental archaeology in the Neotropics

We present a simple sieving methodology to aid the recovery of large cultigen pollen grains, such as maize (Zea mays L.), manioc (Manihot esculenta Crantz), and sweet potato (Ipomoea batatas L.), among others, for the detection of food production using fossil pollen analysis of lake sediments in the tropical Americas. The new methodology was tested on three large study lakes located next to known and/or excavated pre-Columbian archaeological sites in South and Central America. Five paired samples, one treated by sieving, the other prepared using standard methodology, were compared for each of the three sites. Using the new methodology, chemically digested sediment samples were passed through a 53 µm sieve, and the residue was retained, mounted in silicone oil, and counted for large cultigen pollen grains. The filtrate was mounted and analysed for pollen according to standard palynological procedures. Zea mays (L.) was recovered from the sediments of all three study lakes using the sieving technique, where no cultigen pollen had been previously recorded using the standard methodology. Confidence intervals demonstrate there is no significant difference in pollen assemblages between the sieved versus unsieved samples. Equal numbers of exotic Lycopodium spores added to both the filtrate and residue of the sieved samples allow for direct comparison of cultigen pollen abundance with the standard terrestrial pollen count. Our technique enables the isolation and rapid scanning for maize and other cultigen pollen in lake sediments, which, in conjunction with charcoal and pollen records, is key to determining land-use patterns and the environmental impact of pre-Columbian societies.

Characterisation of Bolivian savanna ecosystems by their modern pollen rain and implications for fossil pollen records

The majority of vegetation reconstructions from the Neotropics are derived from fossil pollen records extracted from lake sediments. However, the interpretation of these records is restricted by limited knowledge of the contemporary relationships between the vegetation and pollen rain of Neotropical ecosystems, especially for more open vegetation such as savannas. This research aims to improve the interpretation of these records by investigating the vegetation and modern pollen rain of different savanna ecosystems in Bolivia using vegetation inventories, artificial pollen traps and surface lake sediments. Two types of savanna were studied, upland savannas (cerrado), occurring on well drained soils, and seasonally-inundated savannas occurring on seasonally water-logged soils. Quantitative vegetation data are used to identify taxa that are floristically important in the different savanna types and to allow modern pollen/vegetation ratios to be calculated. Artificial pollen traps from the upland savanna site are dominated by Moraceae (35%), Poaceae (30%), Alchornea (6%) and Cecropia (4%). The two seasonally-inundated savanna sites are dominated by Moraceae (37%), Poaceae (20%), Alchornea (8%) and Cecropia (7%), and Moraceae (25%), Cyperaceae (22%), Poaceae (19%) and Cecropia (9%), respectively. The modern pollen rain of seasonally-inundated savannas from surface lake sediments is dominated by Cyperaceae (35%), Poaceae (33%), Moraceae (9%) and Asteraceae (5%). Upland and seasonally-flooded savannas were found to be only subtly distinct from each other palynologically. All sites have a high proportion of Moraceae pollen due to effective wind dispersal of this pollen type from areas of evergreen forest close to the study sites. Modern pollen/vegetation ratios show that many key woody plant taxa are absent/under-represented in the modern pollen rain (e.g., Caryocar and Tabebuia). The lower-than-expected percentages of Poaceae pollen, and the scarcity of savanna indicators, in the modern pollen rain of these ecosystems mean that savannas could potentially be overlooked in fossil pollen records without consideration of the full pollen spectrum available.

Pollen-based differentiation of Amazonian rainforest communities and implications for lowland palaeoecology in tropical South America

An ongoing controversy in Amazonian palaeoecology is the manner in which Amazonian rainforest communities have responded to environmental change over the last glacial–interglacial cycle. Much of this controversy results from an inability to identify the floristic heterogeneity exhibited by rainforest communities within fossil pollen records. We apply multivariate (Principal Components Analysis) and classification (Unweighted Pair Group with Arithmetic Mean Agglomerative Classification) techniques to floral-biometric, modern pollen trap and lake sediment pollen data situated within different rainforest communities in the tropical lowlands of Amazonian Bolivia. Modern pollen rain analyses from artificial pollen traps show that evergreen terra firme (well-drained), evergreen terra firme liana, evergreen seasonally inundated, and evergreen riparian rainforests may be readily differentiated, floristically and palynologically. Analogue matching techniques, based on Euclidean distance measures, are employed to compare these pollen signatures with surface sediment pollen assemblages from five lakes: Laguna Bella Vista, Laguna Chaplin, and Laguna Huachi situated within the Madeira-Tapajós moist forest ecoregion, and Laguna Isirere and Laguna Loma Suarez, which are situated within forest patches in the Beni savanna ecoregion. The same numerical techniques are used to compare rainforest pollen trap signatures with the fossil pollen record of Laguna Chaplin.

Differentiation between Neotropical rainforest, dry forest and savannah ecosystems by their modern pollen spectra and implications for the fossil pollen record

Accurate differentiation between tropical forest and savannah ecosystems in the fossil pollen record is hampered by the combination of: i) poor taxonomic resolution in pollen identification, and ii) the high species diversity of many lowland tropical families, i.e. with many different growth forms living in numerous environmental settings. These barriers to interpreting the fossil record hinder our understanding of the past distributions of different Neotropical ecosystems and consequently cloud our knowledge of past climatic, biodiversity and carbon storage patterns. Modern pollen studies facilitate an improved understanding of how ecosystems are represented by the pollen their plants produce and therefore aid interpretation of fossil pollen records. To understand how to differentiate ecosystems palynologically, it is essential that a consistent sampling method is used across ecosystems. However, to date, modern pollen studies from tropical South America have employed a variety of methodologies (e.g. pollen traps, moss polsters, soil samples). In this paper, we present the first modern pollen study from the Neotropics to examine the modern pollen rain from moist evergreen tropical forest (METF), semi-deciduous dry tropical forest (SDTF) and wooded savannah (cerradão) using a consistent sampling methodology (pollen traps). Pollen rain was sampled annually in September for the years 1999–2001 from within permanent vegetation study plots in, or near, the Noel Kempff Mercado National Park (NKMNP), Bolivia. Comparison of the modern pollen rain within these plots with detailed floristic inventories allowed estimates of the relative pollen productivity and dispersal for individual taxa to be made (% pollen/% vegetation or ‘p/v’). The applicability of these data to interpreting fossil records from lake sediments was then explored by comparison with pollen assemblages obtained from five lake surface samples.

Pollen-based biome reconstructions for Latin America at 0, 6000 and 18000 radiocarbon years

The biomisation method is used to reconstruct Latin American vegetation at 6000±500 and 18 000±1000 radiocarbon years before present (14C yr BP) from pollen data. Tests using modern pollen data from 381 samples derived from 287 locations broadly reproduce potential natural vegetation. The strong temperature gradient associated with the Andes is recorded by a transition from high altitude cool grass/shrubland and cool mixed forest to mid-altitude cool temperate rain forest, to tropical dry, seasonal and rain forest at low altitudes. Reconstructed biomes from a number of sites do not match the potential vegetation due to local factors such as human impact, methodological artefacts and mechanisms of pollen representivity of the parent vegetation.

Pollen, plant macrofossil and charcoal records for palaeovegetation reconstruction in the Mediterranean-Black Sea Corridor since the Last Glacial Maximum

New reconstructions of changing vegetation patterns in the Mediterranean-Black Sea Corridor since the Last Glacial Maximum are being produced by an improved biomisation scheme that uses both pollen and plant macrofossil data, in conjunction. Changes in fire regimes over the same interval will also be reconstructed using both microscopic and macroscopic charcoal remains. These reconstructions will allow a diagnosis of the interactions between climate, fire and vegetation on millennial timescales, and will also help to clarify the role of coastline and other geomorphic changes, salinity and impacts of human activities in this region. These new data sets are being produced as a result of collaboration between the Palynology Working Group (WG-2) within the IGCP-521 project and the international Palaeovegetation Mapping Project (BIOME 6000). The main objective of this paper is to present the goals of this cooperation, methodology, including limitations and planned improvements, and to show the initial results of some applications.

Relationships between plant traits and climate in the Mediterranean region: an analysis based on pollen data

Question: What are the correlations between the degree of drought stress and temperature, and the adoption of specific adaptive strategies by plants in the Mediterranean region? Location: 602 sites across the Mediterranean region. Method: We considered 12 plant morphological and phenological traits, and measured their abundance at the sites as trait scores obtained from pollen percentages. We conducted stepwise regression analyses of trait scores as a function of plant available moisture (α) and winter temperature (MTCO). Results: Patterns in the abundance for the plant traits we considered are clearly determined by α, MTCO or a combination of both. In addition, trends in leaf size, texture, thickness, pubescence and aromatic leaves and other plant level traits such as thorniness and aphylly, vary according to the life form (tree, shrub, forb), the leaf type (broad, needle) and phenology (evergreen, summer-green). Conclusions: Despite conducting this study based on pollen data we have identified ecologically plausible trends in the abundance of traits along climatic gradients. Plant traits other than the usual life form, leaf type and leaf phenology carry strong climatic signals. Generally, combinations of plant traits are more climatically diagnostic than individual traits. The qualitative and quantitative relationships between plant traits and climate parameters established here will help to provide an improved basis for modelling the impact of climate changes on vegetation and form a starting point for a global analysis of pollen-climate relationships

Pollen-based reconstructions of biome distributions for Australia, Southeast Asia and the Pacific (SEAPAC region) at 0,6000 and 18,000 14C yr B.P.

Aim  This paper documents reconstructions of the vegetation patterns in Australia, Southeast Asia and the Pacific (SEAPAC region) in the mid-Holocene and at the last glacial maximum (LGM). Methods  Vegetation patterns were reconstructed from pollen data using an objective biomization scheme based on plant functional types. The biomization scheme was first tested using 535 modern pollen samples from 377 sites, and then applied unchanged to fossil pollen samples dating to 6000 ± 500 or 18,000 ± 1000 14C yr bp. Results  1. Tests using surface pollen sample sites showed that the biomization scheme is capable of reproducing the modern broad-scale patterns of vegetation distribution. The north–south gradient in temperature, reflected in transitions from cool evergreen needleleaf forest in the extreme south through temperate rain forest or wet sclerophyll forest (WSFW) and into tropical forests, is well reconstructed. The transitions from xerophytic through sclerophyll woodlands and open forests to closed-canopy forests, which reflect the gradient in plant available moisture from the continental interior towards the coast, are reconstructed with less geographical precision but nevertheless the broad-scale pattern emerges. 2. Differences between the modern and mid-Holocene vegetation patterns in mainland Australia are comparatively small and reflect changes in moisture availability rather than temperature. In south-eastern Australia some sites show a shift towards more moisture-stressed vegetation in the mid-Holocene with xerophytic woods/scrub and temperate sclerophyll woodland and shrubland at sites characterized today by WSFW or warm-temperate rain forest (WTRF). However, sites in the Snowy Mountains, on the Southern Tablelands and east of the Great Dividing Range have more moisture-demanding vegetation in the mid-Holocene than today. South-western Australia was slightly drier than today. The single site in north-western Australia also shows conditions drier than today in the mid-Holocene. Changes in the tropics are also comparatively small, but the presence of WTRF and tropical deciduous broadleaf forest and woodland in the mid-Holocene, in sites occupied today by cool-temperate rain forest, indicate warmer conditions. 3. Expansion of xerophytic vegetation in the south and tropical deciduous broadleaf forest and woodland in the north indicate drier conditions across mainland Australia at the LGM. None of these changes are informative about the degree of cooling. However the evidence from the tropics, showing lowering of the treeline and forest belts, indicates that conditions were between 1 and 9 °C (depending on elevation) colder. The encroachment of tropical deciduous broadleaf forest and woodland into lowland evergreen broadleaf forest implies greater aridity. Main conclusions  This study provides the first continental-scale reconstruction of mid-Holocene and LGM vegetation patterns from Australia, Southeast Asia and the Pacific (SEAPAC region) using an objective biomization scheme. These data will provide a benchmark for evaluation of palaeoclimate simulations within the framework of the Palaeoclimate Modelling Intercomparison Project.