Discovering the pharmacodynamics of conolidine and cannabidiol using a cultured neuronal network based workflow.

Determining the mechanism of action (MOA) of novel or naturally occurring compounds mostly relies on assays tailored for individual target proteins. Here we explore an alternative approach based on pattern matching response profiles obtained using cultured neuronal networks. Conolidine and cannabidiol are plant-derivatives with known antinociceptive activity but unknown MOA. Application of conolidine/cannabidiol to cultured neuronal networks altered network firing in a highly reproducible manner and created similar impact on network properties suggesting engagement with a common biological target. We used principal component analysis (PCA) and multi-dimensional scaling (MDS) to compare network activity profiles of conolidine/cannabidiol to a series of well-studied compounds with known MOA. Network activity profiles evoked by conolidine and cannabidiol closely matched that of ω-conotoxin CVIE, a potent and selective Cav2.2 calcium channel blocker with proposed antinociceptive action suggesting that they too would block this channel. To verify this, Cav2.2 channels were heterologously expressed, recorded with whole-cell patch clamp and conolidine/cannabidiol was applied. Remarkably, conolidine and cannabidiol both inhibited Cav2.2, providing a glimpse into the MOA that could underlie their antinociceptive action. These data highlight the utility of cultured neuronal network-based workflows to efficiently identify MOA of drugs in a highly scalable assay.

Revision of the Australian leaf beetle genus Cheiloxena Baly, 1860 (Coleoptera: Chrysomelidae: Spilopyrinae).

The endemic Australian leaf beetle genus Cheiloxena Baly, 1860 is revised, with eight valid species, three new: C. aitori sp. nov.; C. blackburni Reid, 1992; C. conani sp. nov.; C. frenchae Blackburn, 1893; C. insignis Blackburn, 1896; C. monga sp. nov.; C. tuberosa Reid, 1992; C. westwoodii Baly, 1860. A key is provided for their identification and all species are described. Cheiloxena species occur from southern Victoria to central Queensland. Hosts are Araliaceae (Astrotricha), Proteaceae (Lomatia) and possibly Myrtaceae (Eucalyptus).

Neurons Specifically Activated by Fear Learning in Lateral Amygdala Display Increased Synaptic Strength.

The lateral amygdala (LA) plays a critical role in the formation of fear-conditioned associative memories. Previous studies have used c-fos regulated expression to identify a spatially restricted population of neurons within the LA that is specifically activated by fear learning. These neurons are likely to be a part of a memory engram, but, to date, functional evidence for this has been lacking. We show that neurons within a spatially restricted region of the LA had an increase in both the frequency and amplitude of spontaneous postsynaptic currents (sPSC) when compared to neurons recorded from home cage control mice. We then more specifically addressed if this increased synaptic activity was limited to learning-activated neurons. Using a fos-tau-LacZ (FTL) transgenic mouse line, we developed a fluorescence-based method of identifying and recording from neurons activated by fear learning (FTL+ ) in acute brain slices. An increase in frequency and amplitude of sPSCs was observed in FTL+ neurons when compared to nonactivated FTL- neurons in fear-conditioned mice. No learning-induced changes were observed in the action potential (AP) input-output relationships. These findings support the idea that a discrete LA neuron population forms part of a memory engram through changes in synaptic connectivity.

miRNA-mediated post-transcriptional silencing of transgenes leads to increased adeno-associated viral vector yield and targeting specificity.

The production of high-titer recombinant adeno-associated virus (rAAV) vector is essential for treatment of genetic diseases affecting the retina and choroid, where anatomical constraints may limit injectable volumes. Problematically, cytotoxicity arising from overexpression of the transgene during vector production frequently leads to a reduction in vector yield. Herein, we evaluate the use of microRNA (miRNA)-mediated silencing to limit overexpression of cytotoxic transgenes during packaging as a method of increasing vector yield. We examined if post-transcriptional regulation of transgenes during packaging via miRNA technology would lead to increased rAAV yields. Our results demonstrate that silencing of cytotoxic transgenes during production resulted in up to a 22-fold increase in vector yield. The inclusion of organ-specific miRNA sequences improved biosafety by limiting off-target expression following systemic rAAV administration. The small size (22-23 bp) of the target site allows for the inclusion of multiple copies into the vector with minimal impact on coding capacity. Taken together, our results suggest that inclusion of miRNA target sites into the 3'-untranslated region of the AAV cassette allow for silencing of cytotoxic transgenes during vector production leading to improved vector yield, in addition to increasing targeting specificity without reliance on cell-specific promoters.

Revision of the stag beetle genus Ryssonotus MacLeay (Coleoptera: Lucanidae), with descriptions of a new genus and three new species.

The Australian lucanid genus Ryssonotus MacLeay, 1819 is redefined and reduced to one species: R. nebulosus (Kirby, 1819). The supposed senior synonym of this species, Lucanus foveolatus Thunberg, 1806 is a junior synonym of the North American species Lucanus capreolus (Linnaeus, 1763) (new synonym). Safrina new genus, is described for the remaining species formerly in Ryssonotus and three new species: S. dekeyzeri new species, S. grandis (Lea, 1915) new combination, S. jaedoni new species, S. jugularis (Westwood, 1863) new combination, S. laticeps (Macleay, 1885) new combination, S. moorei new species, S. parallela (Deyrolle, 1881) new combination, and S. polita (Carter, 1921) new combination. Safrina grandis is a senior synonym of S. costata (Carter, 1929) new synonym. The type species of Safrina is Rhyssonotus laticeps Macleay, 1885. All species of Safrina are described and a key is provided to the adults of Ryssonotus and Safrina. The species are confined to the ranges of eastern mainland Australia. Diagnostic descriptions of the larvae of Ryssonotus and Safrina are provided. Safrina and Ryssonotus are placed in the tribe Chiasognathini and their systematic position is discussed.

Use of local field potentials of dissociated cultures grown on multi-electrode arrays for pharmacological assays.

In vitro Multi-Electrode Arrays (MEA) are an extracellular recording technology that enables the analysis of networks of neurons in vitro. Neurons in culture exhibit a range of behavioral dynamics, which can be measured in terms of individual action potentials, network-wide synchronized firing and a host of other features that characterize network activity. MEA data analysis was historically focused on high frequency spike data forgoing the low frequency content of the signal. In this study, we use local field potentials, which are low frequency components of MEA signals, to differentiate between two types of antiepileptic drugs (p<;0.0001) with different mechanisms of action.

Disentangling a taxonomic nightmare: a revision of the Australian, Indomalayan and Pacific species of Altica Geoffroy, 1762 (Coleoptera: Chrysomelidae: Galerucinae).

The genus Altica Geoffroy, 1762, is revised for Australia, the west Pacific region and the Indomalayan Archipelago, with 6 valid species: A. aenea (Olivier, 1808); A. birmanensis (Jacoby, 1896); A. caerulea (Olivier, 1791); A. corrusca (Erichson, 1842); A. cyanea Weber, 1801; A. gravida (Blackburn, 1896). The following new synonymy is recognised, in original combinations, senior synonym first: Galeruca aenea Olivier = Haltica ignea Blackburn, 1889, syn. nov., = Haltica bicolora Jacoby, 1904, syn. nov., = Altica jussiaeae Gressitt, 1955, syn. nov.; Galeruca caerulea Olivier = Haltica elongata Jacoby, 1884, syn. nov., = Altica brevicosta Weise, 1922; Haltica corrusca Erichson = Haltica pagana Blackburn, 1896, syn. nov.; Haltica birmanensis Jacoby = Haltica indica Shukla, 1960, syn. nov. Altica brevicosta and A. birmanensis are removed from synonymy with A. cyanea and A. indica is removed from synonymy with A. caerulea. The Altica caerulea of Maulik and subsequent authors (not Olivier) is a misidentification of two species, correctly named A. cyanea and A. birmanensis. The Altica cyanea of Maulik and subsequent authors (not Weber) is a misidentification, correctly named A. aenea. Altica bicosta Shukla, 1960, is removed from synonymy with A. brevicosta and regarded as a valid species. Altica splendida Olivier, 1808, and Haltica ferruginis Blackburn, 1889, are transferred to Sutrea Baly, 1876, as S. splendida (comb. nov.) and S. ferruginis (comb. nov.). The type species of Sutrea is designated as S. elegans Baly, 1876. Altica albicornis Medvedev, 2004, is transferred to Phygasia Dejean, 1836, as P. albicornis (comb. nov.). Lectotypes are designated for A. australis, A. birmanensis, A. caerulea, A. cyanea, A. elongata, A. ignea and A. pagana. A neotype is designated for A. aenea. Altica caerulea is newly recorded from Australia and A. cyanea is removed from the Australian fauna. Altica corrusca and A. gravida are endemic to Australia; all published records of these species from outside Australia refer to the widespread Asian-Pacific species A. aenea. The single record of the European Altica oleracea (L., 1758) from New Caledonia is regarded as a label error and this species removed from the Pacific fauna. A key, based primarily on genitalic structures, is provided for the six regional species and all are redescribed. Host plant records are reviewed: A. corrusca is a minor agricultural pest; A. aenea, A. caerulea and A. cyanea may be useful for biocontrol of weeds.

Chrysomelid males with enlarged mandibles: three new species and a review of occurrence in the family (Coleoptera: Chrysomelidae).

Three new species of Chrysomelidae with extraordinary extensions of the male mandibles are described: Scaphodius drehu sp. nov. and S. ferox sp. nov. (Cryptocephalinae), from New Caledonia, and Chaloenus gajah sp. nov. (Galerucinae), from Borneo. Designation of the type species of Chaloenus Westwood, 1861, is clarified. Synonymy of Scaphodius Chapuis, 1874, with Nyetra Baly, 1877, is supported. Four species of Ditropidus Erichson, 1842, described from New Caledonia, but hitherto regarded as nomina nuda, are shown to be available and are placed in Scaphodius: S. aeneus (Fauvel, 1907), comb. nov., S. nitidus (Fauvel, 1907) comb. nov., S. striolatus (Fauvel, 1907) comb. nov., S. sulcatus (Fauvel, 1907) comb. nov. Ditropidus opacicollis Fauvel, 1907, is also transferred to Scaphodius, as S. opacicollis (Fauvel) comb. nov. The genus Ditropidus does not occur on New Caledonia. Male mandible enlargment in the Chrysomelidae is reviewed: it is common in Cryptocephalinae, but otherwise restricted to a few species of Chrysomelinae, Eumolpinae and Galerucinae. Possible reasons for its distribution in the Chrysomelidae are discussed.

Stargazin and AMPA receptor membrane expression is increased in the somatosensory cortex of Genetic Absence Epilepsy Rats from Strasbourg.

Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.

Differential expression of exon 5 splice variants of sodium channel alpha subunit mRNAs in the developing mouse brain.

Sodium channel alpha subunit genes expressed in the human brain, SCN1A, SCN2A, SCN3A and SCN8A, are subject to alternative splicing of coding exons 5N and 5A. In this study we examined expression of alpha subunit mRNA and exon 5 splicing in the developing mouse brain. Expression levels of Scn1a, Scn2a and Scn8a mRNAs increase postnatally, whereas Scn3a mRNA expression levels decrease. Scn1a mRNA contains only exon 5A, due to the absence of exon 5N in the mouse Scn1a gene. At birth, Scn2a is the only sodium channel alpha subunit mRNA that contains higher or equal amounts of the 5N isoform compared to the 5A isoform in most brain regions. In contrast, the predominant isoform of Scn3a and Scn8a mRNAs in the newborn mouse brain is 5A. 5N/5A ratios for each of the three mRNAs vary across brain regions, with cortex >or= hippocampus>thalamus>cerebellum. In all brain regions and for all three alpha subunits, 5N/5A ratios gradually decrease with age, levelling at a value between 0.1 and 0.2. These findings suggest potential involvement of common factors in the alternative splicing of exon 5 for all three transcripts, and that expression of these factors varies between brain regions and changes during development. Differences in the strength of exon 5N and/or exon 5A splice sites in Scn2a pre-mRNA as compared to Scn1a and Scn8a may underlie the observed differences in 5N/5A ratios in the three alpha subunit mRNAs.

Population fluctuations of light-attracted chrysomelid beetles in relation to supra-annual environmental changes in a Bornean rainforest.

In Southeast Asian tropical rainforests, two events, severe droughts associated with the El Niño-Southern Oscillation and general flowering, a type of community-wide mass flowering, occur at irregular, supra-annual intervals. The relationship between these two supra-annual events and patterns of insect population fluctuations has yet to be clearly elucidated. Leaf beetles (Chrysomelidae) are major herbivores and flower-visitors of canopy trees, affecting their growth and reproduction and, in turn, affected by tree phenology; but their population fluctuations in the Southeast Asian tropics have not been extensively investigated. We examined population fluctuation patterns of the 34 most dominant chrysomelid species in relation to the two supra-annual events by conducting monthly light-trapping over seven years in a lowland dipterocarp forest in Borneo. Our results showed large community variation in population fluctuation patterns and a supra-annual (between-year) variation in abundance for most of the dominant chrysomelids that was significantly larger than the annual (within-year) variation. Specifically, in response to a severe drought in 1998, chrysomelid species exhibited different population responses. These results show that population fluctuations of individual species, rather than the entire assemblage, must be analyzed to determine the effects of changes in environmental conditions on the structure of insect assemblages in the tropics, especially in regions where supra-annual environmental changes are relatively more important than seasonal changes.

Genetic absence epilepsy rats from Strasbourg have increased corticothalamic expression of stargazin.

Stargazin is membrane bound protein involved in trafficking, synapse anchoring and biophysical modulation of AMPA receptors. A quantitative trait locus in chromosome 7 containing the stargazin gene has been identified as controlling the frequency and duration of absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Furthermore, mutations in this gene result in the Stargazer mouse that displays an absence epilepsy phenotype. GAERS stargazin mRNA expression is increased 1.8 fold in the somatosensory cortex and by 1.3 fold in the thalamus. The changes were present before and after the onset of absence seizures indicating that increases are not a secondary consequence of the seizures. Stargazin protein expression was also significantly increased in the somatosensory cortex after the onset of spontaneous seizures. The results are of significant importance beyond the GAERS model, as they are the first to show that an increase in stargazin expression may be pro-epileptic.

Oro-nasal fistula development and velopharyngeal insufficiency following primary cleft palate surgery--an audit of 148 children born between 1985 and 1997.

We present an audit of primary cleft palate surgery in our unit including rates of two important post-operative complications. Multidisciplinary audit clinics ran from March 1998 to April 2002 to follow up all local patients with a cleft lip or palate who had undergone primary palatal surgery in our unit. One hundred and forty eight patients were studied. Patient ages at follow-up ranged from 3 years and 10 months to 17 years and 4 months. Two surgeons performed the primary surgery. One hundred and twenty eight Wardill-Kilner and 20 Von Langenbeck repairs were performed. We found a 4.7% rate of oro-nasal fistula development requiring surgical closure, and a 26.4% rate of velopharyngeal insufficiency (VPI) requiring subsequent pharyngoplasty. We noted that the type of cleft involved affected the rate of VPI, 16% of patients with unilateral cleft lip and palate versus 29.2% of patients with a solitary cleft palate requiring secondary surgery. Outcome of surgery was determined by a 'Cleft Audit Protocol for Speech' (CAPS) speech therapy assessment at follow-up clinics. Only 14.9% of all patients assessed demonstrated any degree of hypernasality. Our results compare favourably with other recent studies including the Clinical Standards Advisory Group (CSAG) report into treatment of children with cleft lip and palate.

Antiretroviral therapy in sub-Saharan Africa: adherence lessons from tuberculosis and leprosy.

Declining drug costs and increases in international donor interest are leading to greater availability of antiretroviral treatment programmes for persons living with the human immunodeficiency virus in parts of sub-Saharan Africa. Ensuring adequate adherence to antiretroviral drug therapy is one of the principal challenges facing successful implementation in Africa, where 70% of the world's infected persons live. Tuberculosis and leprosy are two diseases of global importance whose control programmes can provide important lessons for developing antiretroviral drug adherence strategies. This paper examines various approaches used in tuberculosis and leprosy control which could help enhance adherence to antiretroviral therapy in resource-limited settings.

Spread of marker bacteria from the hides of cattle in a simulated livestock market and at an abattoir.

The spread of microbial contamination on the hides of beef was investigated at two stages in the meat chain: (i) in a simulated livestock market ("the market") using 33 animals, and (ii) in the unloading-to-skinning area of a commercial abattoir using 18 animals. At both stages, harmless bacterial markers (nalidixic acid-resistant Escherichia coli K-12; rifampicin- and nalidixic acid-resistant Pseudomonas fluorescens; and a tetracycline-resistant E. coli) were inoculated on the hides of a small number of selected animals, and their transfer to other animals and the environment was examined. At the market, the initial prevalence of animals positive for the hide markers (9.1% in each phase) introduced in the presale pen, sale ring, and postsale pen changed to 39.4, 15.1, and 54.5%, respectively, by the end of the market process. In addition, widespread contamination of the market environment with the hide markers was observed. At the abattoir, the initial prevalence of animals positive for the hide marker (11.1%) inoculated at unloading increased to 100% (hide before skinning) and 88.8% (skinned carcass). In addition, another marker inoculated on environmental surfaces in lairage pens, races, and stunning box was detected on 83.3% (hide before skinning) and 88.8% (skinned carcass). These results, although obtained with a relatively small number of animals, demonstrate that both the livestock market process and the unloading-to-skinning process at abattoirs can facilitate the extensive spread of microbial contamination on hides not just within, but also between, batches of animals.

Development of low-affinity, membrane-targeted Ca2+ sensors suitable for measuring presynaptic Ca2+.

1. Our aim is to measure near-membrane Ca(2+) flux within the presynaptic terminals of central neurons by modifying new genetically encoded Ca(2+) sensors to develop tools capable of measuring localized Ca(2+) signals. 2. We used standard recombinant DNA technologies to generate the DNA coding for a fusion construct of a modified fluorescent 'pericam' Ca(2+) biosensor with a presynaptic P2X7 receptor (P2X7R). The Ca(2+) sensitivity of the biosensor was modified by rational site-directed mutagenesis of the calmodulin portion of the pericam. 3. Biosensor-receptor fusions were transfected into expression systems for evaluation. Expression studies in HEK-293 cells showed that biosensor-receptor fusion construct-delivered protein was localized exclusively to the plasma membrane, confirming that fusion did not affect the ability of the receptor to undergo normal protein synthesis and trafficking. 4. The Ca(2+)-dependent fluorescence of the pericam portion of the fusion protein was also retained. Site-direct mutagenesis within the calmodulin moiety of the pericam significantly reduced the Ca(2+) affinity of the complex. The dynamic range of the sensor following this modification is better matched to the higher Ca(2+) levels expected within presynaptic Ca(2+) microdomains.

Conditions in lairages at abattoirs for ruminants in southwest England and in vitro survival of Escherichia coli O157, Salmonella Kedougou, and Campylobacter jejuni on lairage-related substrates.

Information on lairages (regarding design, construction materials, and use of bedding and cleaning regimes) was collected for 21 commercial cattle and/or sheep abattoirs in southwest England. Overall, roughened or grooved concrete was the most common lairage flooring material. Straw bedding was used in the majority of lairages and was changed between animal batches, daily, weekly, and monthly in roughly 5, 60, 15, and 10%, respectively, of the surveyed lairages. Lairages were commonly washed with cold water with no detergents and/or disinfectants, and only about half the lairages were washed daily. Also, a three-pathogen cocktail inoculum comprising Escherichia coli O157 (NCTC 12900), Salmonella Kedougou (VLA S488/01), and Campylobacter jejuni (VLA C4) (at 8, 8, and 7 log CFU/ml or 8, 8, and 7 log CFU/g, respectively) was suspended in either broth (for nonfecal contamination) or bovine feces (for fecal contamination). Samples of the four most common substrates present in lairages (concrete, straw, metal, and hide) were contaminated in vitro with either fecal or nonfecal inocula and subsequently held in the laboratory at 10 or 25 degrees C for 1 week. Bacterial counts for these samples were monitored daily and used to assess the number of days required for a 90% reduction of each pathogen population. In most cases, pathogens survived for >1 week, with survival rates being higher for straw or hide than for concrete or metal and higher for fecal contamination than for nonfecal contamination. Overall, if survival rates for the three pathogens under practical lairage conditions were similar to the in vitro survival rates found in this study, contamination of lairages with pathogens could be carried over from one batch of animals to another and/or from one day to the next.

Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease.

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease XbaI, while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously. The 207 E. coli O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E. coli O157 isolates in the study (Simpson's index of diversity [D] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E. coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E. coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.

Pulsed-field gel electrophoresis characterization of Shiga toxin-producing Escherichia coli O157 from hides of cattle at slaughter.

Contamination of the brisket areas of the hides of healthy adult cattle with Shiga toxin-producing Escherichia coli O157 at slaughter in England was studied. In total, 73 cattle consignments comprising 584 animals delivered to one abattoir over 3 days during 1 week in July 2001 were studied: 26 cattle consignments arriving on Monday, 32 consignments arriving on Wednesday, and 15 consignments arriving on Friday. Consignment sizes ranged from 1 to 23 animals, with a mean consignment size of 8. The hide of the first animal to be slaughtered in each consignment was sampled by using a sponge swab moistened with 0.85% saline to rub an unmeasured brisket (ventral) area (ca. 30 by 30 cm). The process of isolating E. coli O157 from the swabs consisted of enrichment, screening with immunoprecipitation assay kits, and immunomagnetic separation. E. coli O157 was found on 24 of 73 (32.9%) cattle hides examined, and 21 of these 24 isolates produced Shiga toxins. The 24 E. coli O157 isolates produced six different pulsed-field gel electrophoresis profiles, and 18 (75%) of the isolates were of one prevalent clone. The high prevalence of one E. coli O157 clone on the hides of cattle at slaughter could be due to a high prevalence of that clone on the 18 farms involved (not investigated in the current study), in the postfarm transport or lairage environments, or both. Since the lairage environment, but not the farm of origin or the postfarm transport vehicle, was a factor common to all 18 cattle consignments, it could have played an important role in spreading the prevalent E. coli O157 clone to the cattle hides. Lairage pen floors and the stunning box floor were identified as the most probable sites along the unloading-to-slaughter route at which the brisket areas of cattle hides could become contaminated.

Potential for the spread of Escherichia coli O157, Salmonella, and Campylobacter in the lairage environment at abattoirs.

Prevalences of Escherichia coli O157, Salmonella spp., and Campylobacter spp. were examined in 270 swabs taken from selected sites along the unloading-to-slaughter routes of animal movement in lairages of six commercial abattoirs, three for cattle and three for sheep. The overall prevalences of the pathogens in the respective lairage environments were compared with those for 270 swabs from the pelts of 90 lambs examined in the present study and 270 swabs from the hides of 90 cattle examined in a previous study that were slaughtered at the same abattoirs on the same days. Also, the results obtained were analyzed with the aim of identifying critical points at which animal-environment-animal transfer of the pathogens in lairages occurs. The results showed that (i) the overall prevalences of E. coli O157, Salmonella spp., and Campylobacter spp. were 27.2, 6.1, and 1.1%, respectively, in cattle lairages and 2.2, 1.1, and 5.6%, respectively, in sheep lairages; (ii) the overall prevalences of the three pathogens on cow hides (28.8, 17.7, and 0%, respectively) and sheep pelts (5.5, 7.8, and 0%, respectively) were higher than the overall prevalences in the respective lairage environments; (iii) the most frequently contaminated sites in cattle lairages were holding pen floors (50% of swabs positive for one or more pathogens), entrance gates of stun boxes (27.8% of swabs positive for one or more pathogens), and stun box floors (22.2% of swabs positive for one or more pathogens); (iv) the most frequently contaminated sites in sheep lairages were unloading ramp floors, holding pen floors, and water troughs (33.3, 22.2, and 22.2%, respectively); and (v) overall, cattle lairages and cow hides were more frequently contaminated with the pathogens than were lamb lairages and lamb pelts. Further research is needed to develop strategies for the incorporation of pathogen control in lairages into integrated microbial meat safety systems.