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X.D. was funded by a grant from the National Institute for Health Research (NIHR) Health Protection Research Unit in Genomics and Enabling Data (no. NIHR200892). M.A.H. was supported by grants from the Medical Research Council (nos. G1001787/1, MR/N002660/1 and MR/P007201/1) and the Economic and Social Research Council (no. ES/S000186/1). E.M.H. was supported by a UK Research and Innovation (UKRI) Fellowship (no. MR/S00291X/1). The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus—a notorious human pathogen—appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two β-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development. Publisher PDF

In the Dampier Archipelago of Western Australia's Pilbara Region, several locally endemic, morphologically distinctive species of Rhagada land snails occur, contrasting with the morphologically conservative species with wider distributions on the adjacent mainland. To test alternative origins of this unusual local diversity in a continental archipelago, we examined sequences of the cytochrome oxidase subunit 1 and 16S mitochondrial genes in 22 described species and eight undescribed forms, including all known morphospecies from the Pilbara Region's Dampier Archipelago and adjacent mainland. Phylogenetic analyses consistently resolved four, deep clades within the Pilbara Region, with a mean sequence divergence of 15–18%. All but one of the species from the Dampier Archipelago formed one of the major clades, indicating that the morphological radiation in the archipelago evolved locally, rather than through multiple, relictual mainland lineages. Morphological divergence spanning almost that of the entire genus was within a subclade with sequence divergence

A study of polymorphisms of mitochondrial DNA (mtDNA) of West African N'Dama (Bos taurus) and East African Zebu (B. indicus) cattle was carried out to obtain information on maternal phylogenetic relationships between these breeds. A relatively large sample size was made possible by using polymerase chain reaction (PCR) amplification of DNA prepared from small blood samples to generate fragments of two known polymorphic mtDNA regions, one within the gene encoding subunit 5 of NADH dehydrogenase and one encompassing the entire D-loop. This approach allowed us to achieve a higher resolution restriction analysis on mtDNA from more animals than would have been possible by conventional methods. PCR-amplified mtDNA of 58 animals from five populations was examined at 26 restriction sites by 16 enzymes. In this way 154 nucleotides of mtDNA were scanned for polymorphism. Six polymorphic sites were located by this means, five of which were within the D-loop and one of which was within the NADH dehydrogenase 5 gene. None of the polymorphisms observed could be considered typical of breed or type.

Summary. It has hitherto proved difficult to phenotype cattle for class II histocompatibility antigens using standard serological techniques because of problems of reagent specificity and antigen expression on peripheral blood mononuclear cells (PBMs). We recently described the production of class II-specific alloreactive bovine T cell clones characterized by the BoT4+ phenotype. In this report we describe studies of the application of four such clones, derived from a single mixed leucocyte culture (MLC), for class II phenotyping in proliferation and cytotoxicity assay systems. Proliferation assays used irradiated PBM as stimulator cells and cytotoxicity assays used Theileria parva-infected lymphoblastoid cells as targets. Proliferation assays revealed three distinct specificities among the four clones indicating that they detected three different class II determinants. Furthermore, in a family study, the genes encoding the determinants recognized by the clones were found to be linked to the gene encoding the w10 class I A locus product on one of the w10-bearing haplotypes in our study population. Two of the clones were studied in cytolysis assays. Lack of cytolysis of one of the targets, which was derived from the PBM of an animal carrying a class II determinant detected in proliferation assay, was explained by the total lack of expression of class II antigens on the target cell line in question, as determined with 4 class II-specific monoclonal antibodies (mAb). We conclude that BoT4+ alloreactive clones provide a potentially useful and particularly discriminating way of detecting polymorphic class II antigens of cattle, especially when applied in assays of proliferative response to PBM.

Summary By amplification of pools of DNA representative of different bovine populations with single short oligonucleotide primers of random sequence, we were able rapidly to identify markers which distinguish the two major subspecies of domestic cattle, Bos taurus and B. indicus. One of the marker polymorphisms was found to be in a novel, dispersed DNA sequence which occurs in several ruminant species. The marker will assist in the detection of crossbreeding between Zebu and B. taurus types where this threatens a potentially valuable trypanosomiasis-resistant B. taurus genetic resource in West Africa. In addition, the marker will be useful for exploration of the evolutionary relationships of the major subspecies of domestic cattle. The general approach used to identify population-specific DNA polymorphisms has potentially broad application in definition of species, breeds and populations and will be of generic value in studies of genome evolution.

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in polymerase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61% of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.

Summary. An account is given of the serologically defined class I specificities encoded by the bovine MHC (expressed as the BoLA system) in two populations of African cattle and in European breeds. The BoLA typing was performed using alloantisera raised against tissue antigens of both European and African breeds of cattle. All of the specificities agreed in the first two international BoLA workshops were found in the African cattle, although there were significant differences in the frequency of some specificities between the African and European animals. Many of the European antisera, which are operationally monospecific in Bos taurus cattle, were multispecific in the African animals. Subgroups of two specificities (w8 and w10) were demonstrated. Five new BoLA-A locus alleles were detected by means of antisera raised against alloantigens of African cattle. Two of these occurred at an extremely high frequency in the African populations; one being unique to these cattle. Monoclonal antibodies proved to be useful typing reagents, particularly in the elucidation of subgroups.

Summary. For a long time, the bovine major histocompatibility complex (MHC) (BOLA) class I region was characterized, rather uniquely among mammalian species, as having one expressed locus. Recent reports have suggested otherwise. Selective immunoprecipitation and molecular characteriztion of products enable a decisive answer to the question of whether there is indeed more than one locus expressed. Therefore, we characterized serologically defined w10 encoding haplotypes in European and African cattle by immunoprecipitation of [35S]-methioninelabelled peripheral blood mononuclear cells (PBMC), followed by one- and two-dimensional isoelectric focusing (1D/2D-IEF) of cell lysates. Monoclonal antibodies (mAb) used were directed against either human class I monomorphic determinants (W6/32 and B1.1G6) or bovine polymorphic determinants expressed on products encoded by serologically defined w10 encoding haplotypes of Boran and Friesian cattle. Sequential immunoprecipitations with W6/32 and B1.1G6 using lysates of PBMC of British Friesian cattle, revealed that from this haplotype W6/32 precipitated one product, whereas B1.1G6 precipitated two products. The product precipitated in addition appeared to be the one that was selectively precipitated by the mAb directed against polymorphic determinants on a product of w10 encoding haplotypes. Additionally, peptide maps of protease V8-digested precipitates showed that this particular ‘w10’ associated product was distinctly different from the product recognized by W6/32. Thus, we suggest that the two products are distinct gene products and that the product with higher PI is associated with the serologically defined A-locus product, whereas the product with lower PI is the putative second locus product. In the African Boran breed, variants of the serologically defined w10 specificity were found on the basis of IEF typing. These variants appeared to be associated with different second locus products. Therefore, we conclude that serologically defined w10 encoding haplotypes encode at least two independent class I locus products, expressed on normal bovine PBMC. In IEF analysis the additional use of mAb recognizing polymorphic determinants on serologically defined A-locus products highly facilitated the detection and typing of second locus products.

Summary. Blood samples from 54 animals were exchanged between 15 laboratories in nine countries to improve and expand BoLA class I and class II typing. A total of 27 out of 33 (82%) of previously accepted BoLA-w specificities were represented within the cell panel. Seventeen new serum-defined BoLA specificities were accepted by the workshop participants, thus expanding the number of internationally recognized BoLA specificities to 50. The large number of new specificities detected resulted from the number of serological reagents used (n= 1139) and the genetic diversity of the cell panel. Confidence derived from the high percentage of agreement between the laboratories on antigen detection (97.3%; r= 0.84) permitted the removal of the workshop (w) notation from 23 BoLA-w specificities and their acceptance as full status BoLA-A antigens. Two new non-BoLA antigens were also detected, one completely included within the red blood cell factor S' (BoLy-S'), whereas a second (BoLy-w1) did not show any association with tested red blood cell factors. A comparison between serological, isoelectric focusing (IEF) and DNA typing for BoLA class II polymorphism was conducted with a subset of workshop cells. Correlation between the three methods was significant for three combinations of alleles. Three other serologically defined class II specificities were correlated with DR and/or DQ restriction fragment length polymorphism (RFLP) types, whereas six additional IEF types were correlated with DR and/or DQ RFLP types (r≥ 0.50). Several new IEF, DRB, DQA and DQB RFLP patterns were identifed. In 46 animals that were typed for BoLA-DR and DQ genes by RFLP analysis, 46 different BoLA haplotypes were tentatively defined. These 46 haplotypes were distinguished by 31 serologically-defined BoLA-A alleles (and 2'blanks'), 15 DRB RFLP types (plus up to 10 new DRB RFLP patterns) and 23 DQA-DQB haplotypes.

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.

Local childhood obesity monitoring data can be a valuable resource for health communities and provide useful feedback for schools. However care must be taken in how the data are used. What role can a school nurse play to help improve school children’s health?

In the course of an international research project on hazard analysis of antimicrobial resis- tance in SE Asian aquaculture environments, 2 European Union and 3 SE Asian laboratories attempted to harmonize a procedure for antimicrobial agent susceptibility testing based on disk diffusion (DD). For this purpose, a selected panel of 10 bacterial control strains of relevance for monitoring warm-water aquacul- ture environments was sent by the central laboratory to the other participating laboratories. In each lab- oratory, 10 independently replicated DD determinations of each control strain to 6 antibiotics were per- formed using Iso-Sensitest Agar (ISA) according to a standard operating procedure (SOP); in total, this study thus yielded 300 data sets for all 5 laboratories. At the end of the study, strain authenticity of sub- cultures of the control strains used by the respective participating laboratories was verified by the central laboratory. Based on the arithmetic mean of 10 inhibition-zone diameter measurements and standard deviation (SD), intralaboratory SD variations ranged from 0 to 2 mm when 79% of the recorded data sets were considered. In 8% of the data sets, the SD value exceeded 4 mm, which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distrib- uted in one of the laboratories. At the interlaboratory level, 81% of the SD values based on global aver- aging of 50 data points per strain-disk combination were situated in the 0 to 5 mm range. Comparison with a minimal data set from literature of DD testing performed with Mueller-Hinton (MH) medium indi- cated that the use of either ISA or MH medium in DD testing has a limited impact on the method's preci- sion among different laboratories. In conclusion, the current study has provided a validated SOP to pro- mote the coordination and harmonization of DD-susceptibility methodologies for aquaculture-associated organisms at an international level. As one of the main action items for the future, new interpretive break- points should be specifically designed and validated for aquaculture drugs and organisms.

Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b(5) domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an omega-3 delta5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from alpha-linolenic acid (18:3n-3). The desaturase showed only low levels of delta6 activity toward C(18) polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C(18)C(20)C(22). Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.

Fish are an important dietary source of the long-chain C 20 and C 22 highly unsaturated fatty acids (HUFA), arachidonate (20:4 n −6), eicosapentaenoate (20:5 n −3) and docosahexaenoate (22:6 n −3), which are crucial to the health of higher vertebrates and that can be beneficial in human diets. Δ5 and Δ6 fatty acid desaturases and fatty acid elongases are critical enzymes in the biosynthetic pathways of HUFA from shorter chain C 18 polyunsaturated fatty acids (PUFA) such as linoleic (18:2 n −6) and α-linolenic (18:3 n −3) acids. Recently, full-length cDNAs for fatty acid desaturase and elongase enzymes have been cloned from Atlantic salmon. Functional characterisation of the desaturase revealed n −3 Δ5 desaturase activity, whereas the elongase had broad substrate specificity for PUFA with a range of chain lengths from C 18 to C 22 . The study described here was primarily focused on the nutritional regulation of genes involved in the HUFA biosynthetic pathway in Atlantic salmon. A feeding trial was performed whereby salmon smolts in seawater pens were fed for 40 weeks on five different diets. The diets consisted of a control diet containing fish oil (FO) and four diets in which the FO was replaced in a graded manner by linseed oil (LO). Specifically, in terms of added oils, the five diets were 100% FO (FO), 100% LO (LO100) and FO/LO in ratios of 3:1 (LO25), 1:1 (LO50) and 1:3 (LO75). Fish were sampled at 20 and 40 weeks, and samples of liver were collected for lipid analyses and total RNA extraction. Hepatocytes were also prepared and the activity of the HUFA biosynthetic pathway determined. Expression of fatty acid desaturase and elongase genes was determined by quantitative real time polymerase chain reaction (PCR) and the ratio of the copy number of the targeted gene against that of β-actin was calculated. The results showed that after 20 weeks of feeding, desaturase and elongase gene expression in liver was increased in a graded manner by increasing dietary LO. Expression of both genes was positively and negatively correlated with dietary 18:3 n −3 and n −3 HUFA, respectively. By 40 weeks of feeding, expression of neither gene showed the same degree of correlation with dietary fatty acid composition. In contrast, activity of the HUFA biosynthetic pathway, which showed some association with diet at 20 weeks, was positively and significantly correlated with dietary LO after 40 weeks of feeding. Elongation activity reflected the overall activity of the HUFA biosynthetic pathway to a greater degree than Δ5 desaturation activity. The possible mechanisms underlying the observed results and the regulation of the HUFA biosynthetic pathway are discussed.

A study was undertaken to compare first-feeding mortality among 10 brown trout families fed high (100%) and low (25%) rations in replicate mixed family tanks. Microsatellite DNA profiling was used to assign individual brown trout to family of origin. At the end of the 35 day experimental period, highly significant differences in overall mortality were observed between the two treatments, and within the treatments there were highly significant differences in family mortality. Both replicates displayed similar patterns of family mortality. Notably, the distribution of mortality among families differed significantly between the two ration treatments, although this was more distinct for some families than others. No correlation between mean family egg diameter and family mortality were observed within either feeding treatment, although a significant positive correlation between mean family egg diameter and timing of family mortality was observed. It is suggested that these data indicate the existence of a genotype x environment interaction relating to feeding level during the critical start-feeding period.

Trypanosomosis, or sleeping sickness, is a major disease constraint on livestock productivity in sub-Saharan Africa. To identify quantitative trait loci (QTL) controlling resistance to trypanosomosis in cattle, an experimental cross was made between trypanotolerant African N'Dama ( Bos taurus ) and trypanosusceptible improved Kenya Boran ( Bos indicus ) cattle. Sixteen phenotypic traits were defined describing anemia, body weight, and parasitemia. One hundred seventy-seven F 2 animals and their parents and grandparents were genotyped at 477 molecular marker loci covering all 29 cattle autosomes. Total genome coverage was 82%. Putative QTL were mapped to 18 autosomes at a genomewise false discovery rate of 2 cross between N'Dama and Boran cattle could produce a synthetic breed with higher trypanotolerance levels than currently exist in the parental breeds.

Quantitative trait loci (QTL) associated with resistance to an intestinal worm in a well-defined murine model are described. These have been identified in an F2 population derived from resistant (SWR) and susceptible (CBA) parental mouse strains infected with the gastro-intestinal nematode parasite Heligmosomoides polygyrus. Seven QTL located on six chromosomes are described, associated with components of the complex host response and the differential regulation of parasite survival and reproduction. The combined additive effects of the five significant QTL associated with worm survival (total worm count at necropsy) account for about 60% of the difference in worm count between the parental lines. The dominance effect for these five QTL are all in the direction of resistance, supporting the heterosis for resistance established from the mean worm count for the F2 line relative to the parental lines. It is now possible to identify the comparative chromosomal regions of these QTL in livestock and humans and to consider the possibility of future improved control strategies. These may include breeding of resistant or tolerant livestock, development of vaccines, or identification of new anthelmintic drugs.

Three Norwegian sea trout (Salmo trutta L.) stocks and a farmed Atlantic salmon (Salmo salar L.) stock were challenged with salmon lice (Lepeophtheirus salmonis (Krøyer, 1837)), in a “common garden” experiment. Sea trout from the River Guddal exhibited a significantly lower level of infection, as measured by louse abundance and louse density, than other stocks. In addition, salmon lice developed significantly more slowly on the Guddal stock than on the other stocks. Salmon louse abundance and density were similar for the Rivers Fortun and Sima stocks of sea trout, and abundance of lice, though not density, was highest for farmed Atlantic salmon. Within stocks, there were no differences in infection levels of salmon louse between mature and immature fish, between sexes, or between anal-fin-clipped and non-clipped salmon. Differences in infection level among the sea trout stocks may, it is suggested, reflect genetic differences.

Theileria parva is a protozoan parasite that infects lymphocytes of cattle and African buffalo. As is the case with certain viruses, the parasite causes antigenic changes on the cell surface against which the host mounts cytotoxic T-cell. Precise definition of the cells participating in these response and their specificity has been facilitated by the recent identification of markers for bovine T-cell subpopulations and functional analyses of bovine lymphocytes at the clonal level. In this paper Ivan Morrison and his colleagues discuss current information on the parasite specificity and MHC restriction of anti-Theileria cytotoxic T cell, in relation to their role in protective immunity.

Two experimental groups comprising mixed Norwegian sea run and freshwater resident brown trout Salmo trutta were infected with sea lice Lepeophtheirus salmonis in replicate tanks. Comparison of mean abundance and louse development between the sea run and resident groups revealed highly significant differences in lice abundance. The resident trout had an average abundance ± S.E. of 6.3 ± 0.37 and 6.6 ± 0.43 lice whilst the sea trout had an average abundance of 3.5 ± 0.25 and 3.3 ± 0.28 lice 29 days post infection at 9° C. No differences in development of lice, of either sex, were detected between the groups. As host groups were naive to sea lice at the start of the experiment, this suggests that there was a significant difference in susceptibility to sea lice infection between them, which may be genetically determined.

Δ5 and Δ6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids arachidonic, eicosapentaenoic, and docosahexaenoic acids. They are encoded by distinct genes in mammals and Caenorhabditis elegans . This paper describes a cDNA isolated from zebrafish ( Danio rerio ) with high similarity to mammalian Δ6 desaturase genes. The 1,590-bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b 5 domain and three histidine boxes, believed to be involved in catalysis. When the zebrafish cDNA was expressed in Saccharomyces cerevisiae it conferred the ability to convert linoleic acid (18:2 n -6) and α-linolenic acid (18:3 n -3) to their corresponding Δ6 desaturated products, 18:3 n -6 and 18:4 n -3. However, in addition it conferred on the yeast the ability to convert di-homo-γ-linoleic acid (20:3 n -6) and eicosatetraenoic acid (20:4 n -3) to arachidonic acid (20:4 n -6) and eicosapentaenoic acid (20:5 n -3), respectively, indicating that the zebrafish gene encodes an enzyme having both Δ5 and Δ6 desaturase activity. The zebrafish Δ5/Δ6 desaturase may represent a component of a prototypic vertebrate polyunsaturated fatty acids biosynthesis pathway.

SUMMARY The TNF-a gene on mouse chromosome MMU17 is among the candidates for the trypanosomosis resistance QTL Tir1. Tir1 has the largest effect of those loci so far detected which influence degree of resistance to murine trypanosomosis caused by Trypanosoma congolense infection. We therefore studied the survival to 180 days after challenge with T. congolense of mice that were homozygous and hemizygous with respect to a disruption of the TNF-a gene on a . 99% C57BL/6 (resistant) background. We also examined the responses of TNF-a hemizygous mice produced by crossing the deletion line with mice of the C57BL/6J strain, and with mice of the susceptible A/J strain. Mice lacking a functional TNF-a gene were shown to be highly susceptible to challenge with T. congolense with a median survival time of 37 days. This was comparable to 71 days for control wild-type mice, and 61 and 111 days for mice of the susceptible A/J and resistant C57BL/6J strains, respectively. In mice of the deletion line, the C57BL/6 TNF-a allele tended to be dominant to the TNF knockout in terms of resistance. We conclude that TNF-a plays an important role in resistance to the effects of T. congolense infection in mice.

Groups of eight full-sibling wild sea trout Salmo trutta L. families, communally reared, exhibited highly significant differences in family representation in the largest 25 percentile within each tank at the end of the observation period (referred to as family performance). Despite these large differences in family performance within tanks, there were no significant differences in performances of families relative to each other between high and low feeding levels. These results are discussed in the context of domestication selection.

The desaturation and elongation of [1-14C]18:3n-3 was investigated in hepatocytes of the tropical warm freshwater species, zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus). The hepatocyte fatty acid desaturation/elongation pathway was assayed before and after the fish were fed two experimental diets, a control diet containing fish oil (FO) and a diet containing vegetable oil (VO; a blend of olive, linseed and high oleic acid sunflower oils) for 10 weeks. The VO diet was formulated to provide 1% each of 18:2n-6 and 18:3n-3, and so satisfy the possible EFA requirements of zebrafish and tilapia. At the end of the dietary trial, the lipid and fatty acid composition was determined in whole zebrafish, and liver, white muscle and brain of tilapia. Both zebrafish and tilapia expressed a hepatocyte fatty acid desaturation/elongation pattern consistent with them being freshwater and planktonivorous fish. The data also showed that hepatic fatty acid desaturation/elongation was nutritionally regulated with the activities being higher in fish fed the VO diet compared to fish fed the FO diet. In zebrafish, the main effect of the VO diet was increased fatty acid Δ6 desaturase activity resulting in the production of significantly more 18:4n-3 compared to fish fed the FO diet. In tilapia, all activities in the pathway were greater in fish fed the VO diet resulting in increased amounts of all fatty acids in the pathway, but primarily eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). However, the fatty acid compositional data indicated that despite increased activity, desaturation of 18:3n-3 was insufficient to maintain tissue proportions of EPA and DHA in fish fed the VO diet at the same level as in fish fed the FO diet. Practically, these results indicate that manipulation of tilapia diets in commercial culture in response to the declining global fish oil market would have important consequences for fish fatty acid composition and the health of consumers. Scientifically, zebrafish and tilapia, both the subject of active genome mapping projects, could be useful models for studies of lipid and fatty acid metabolism at a molecular biological and genetic level.

A polymorphism was detected in the 3' untranslated region of the bovine gamma-S-crystallin gene by direct sequencing of polymerase chain reaction (PCR) products from genomic DNA of an N'Dama bull and a Boran cow. A set of three PCR primers was designed to detect this difference and thus give allele-specific amplification. The two allele-specific primers differ in length by 20 nucleotides so that the allelic products may be distinguished by simple agarose gel electrophoresis following a single PCR reaction. This provides a simple and rapid assay for this polymorphism.

The applicability of bovine autosomal microsatellite markers for population genetic studies on African buffalo was investigated. A total of 168 microsatellite markers were tested for PCR amplification on a test panel of seven African buffalo. Amplification was observed for 139 markers (83%), and 101 markers were studied further with 91 (90%) being polymorphic. The mean number of alleles per marker was 5.0 (SE = 0.2) and the mean heterozygosity per marker was 0.61 (SE = 0.03). Considering the overall high level of polymorphism, it was concluded that most bovine microsatellite markers are applicable in African buffalo.

Under most circumstances, certain breeds of domestic ruminants show a remarkable resistance to the effects of African trypanosomiasis: they can tolerate the presence of parasites while apparently controlling levels of parasitaemia and, crucially, not showing the severe anaemia and production loss that are characteristic of infection in susceptible hosts. As discussed here by Stephen Kemp and Alan Teale, the genetic control of this phenomenon might finally be yielding to gene mapping studies. Genetic regions determining susceptibility to trypanosomiasis in mice have been identified and parallel studies are well advanced in cattle. There is growing evidence that only modest numbers of genes are involved in determining the difference between a susceptible and a resistant animal. These observations raise a new series of important questions concerning the possible exploitation of major trypanotolerance genes and the way that they might function in different genetic and physical environments.

Sensitivity of segregation analysis for data structure and data transformation was studied using data from two trials in which mice were challenged at three months of age with a cloned isolate of Trypanosoma congolense and survival time was recorded. Data included records from three inbred strains (C57BL/6 (tolerant), A/J, and BALB/c (both susceptible)) and their crosses. Data were standardized and normalized using a modified power transformation. Segregation analysis was applied to both untransformed and transformed data to determine the genetic inheritance of trypanotolerance in these mice. Data from the two trials were analysed separately and combined. Four genetic models were compared; a one locus model, a polygenic model, a mixed model with common variance, and a mixed model with different variances for each major genotype. Even though the separate data sets and the combined data set all supported the hypothesis of a major gene (or a tightly linked cluster of genes) with different variances within each genotype, parameter estimates were highly sensitive to data transformation and several sets of parameter estimates gave similar likelihood values because of high dependency between parameters. Based on the results segregation analysis can be very sensitive to data structure in a crossbreeding design and to data transformation. Interpretation of the results can be misleading if the entire parameter space is not studied carefully.

To map the genetic sources of trypanotolerance in mice, a linkage analysis of survival following trypanosome challenge was performed by selective genotyping in a large F2 population produced by crossing the resistant C57BL/6 and susceptible BALB/c inbred mouse lines. We report evidence of a chromosomal region of large effect, possibly comprising more than one resistance locus, on Chromosome 17; and of further loci on Chromosomes 1 and 5. Together, these genes can account for all of the difference between the mean parental phenotypes.

The parasite strain specificity of CTL responses to Theileria parva varies among cattle immunized with the same parasite stock. We have investigated the influence of class I MHC on the strain specificity of CTL responses to T. parva in 19 cattle of defined class I phenotype immunized with either of two T. parva populations, in which protection to subsequent reciprocal challenge correlated with CTL strain specificity. In the majority of animals the response was restricted by the products of one MHC haplotype and there was a consistent bias to some haplotypes in preference to others. In 10 of 13 cattle expressing the molecularly defined MHC specificities A10 and KN104 on one haplotype, the CTL response was restricted entirely by this haplotype, thus allowing a precise analysis of the MHC restriction specificities. The MHC restriction specificity and the parasite population used for immunization both influenced the strain specificity of the response. By examining responses in identical twins immunized with different parasites or in animals before and after challenge with heterologous parasites, animals that mounted a strain-specific response to primary infection were shown to be capable of responding to Ags shared by the two parasite populations. These findings indicate that the strain specificity of CTL responses to T. parva is not determined primarily by immune response genes that define the inherent capacity to respond, but rather is a consequence of the response in individual animals being biased toward a limited number of immunodominant peptide-MHC determinants.

The World Organisation for Animal Health (OIE) TerrestrialAnimal Health Code considers the prudent use of antimicrobial agents in veterinary medicine to comprise a series of practical measures and recommendations which confer benefits to animal and public health while preserving and maintaining the therapeutic efficacy of antimicrobials. This paper reviews some of the main veterinary prudent use guidelines which have been published in English and the responsibilities of those involved at all levels in the administration of antimicrobials to animals, including national regulatory authorities. The OIE guidelines are considered comprehensive and cover all of those levels, from regulatory authorities to veterinarians and food producers. Guidelines produced by national authorities, professional veterinary associations or farming associations and which are targeted at particular individuals, for example veterinarians or food animal producers, will, obviously, restrict their coverage to those aspects considered relevant for their target audience.

A cattle genetic linkage map was constructed which marks about 90% of the expected length of the cattle genome. Over 200 DNA polymorphisms were genotyped in cattle families which comprise 295 individuals in full sibling pedigrees. One hundred and seventy-one loci were found linked to one other locus. Twenty nine of the 30 chromosome pairs are represented by at least one of the 36 linkage groups. Less than a 50 cM difference was found in the male and female genetic maps. The conserved loci on this map show as many differences in gene order compared to humans as is found between humans and mice. The conservation is consistent with the patterns of karyotypic evolution found in the rodents, primates and artiodactyls. This map will be important for localizing quantitative trait loci and provides a basis for further mapping.

Following the introduction of a national abattoir-based monitoring programme for Salmonella in pigs, advisory visits were made to pig farms in England and Wales with high Salmonella seroprevalence assessed by muscle tissue fluid (meat juice) ELISA. Samples (n = 15 790), including pooled pen floor faeces (n = 12 136), were collected for Salmonella culture from 296 farms, between October 2003 and February 2008. Salmonella was isolated from 4489 (28%) of all samples collected, including 3301 (27%) of pooled pen floor faecal samples, from 270 (91%) of farms visited. Salmonella Typhimurium and S. Derby were the most prevalent serovars, representing 64% and 16% of isolates serotyped, respectively. The main phage types of S. Typhimurium identified were U288 and DT193. Antimicrobial resistance (AMR) was seen in 92% of isolates tested, with the highest frequencies of resistance occurring to tetracyclines (T), sulphonamide compounds (SU), ampicillin (AM), sulphamethoxazole/trimethoprim (SXT), streptomycin (S) and chloramphenicol (C). Fifty-nine AMR patterns were observed, the most frequent of these being T, AM, SXT, C, S, SU, seen in 35% of isolates tested. Multi-drug resistance was commonly found, with 67% of isolates submitted for AMR testing showing resistance to between four and nine antimicrobials.

The cost of reproductive effort is known to result in a trade-off between current and future reproduction. Similarly, trade-offs in energy allocation may occur between components of reproductive effort, mating and parental effort, within a single reproductive episode. We investigated the energy allocated to mating effort (calling to attract females) and parental effort (donation of spermatophore nutrients at mating) by male bushcrickets, Requena verticalis, under two dietary regimes. Males provided with a low quality diet reduced the daily energy allocated to calling activity while maintaining their investment in spermatophores. Males provided with a high quality diet did not allocate more resources per day to their spermatophores but stored excess energy for future reproduction. Thus, on a per day basis, males appear to hold constant their investment in the spermatophore at the cost of reduced mating effort when resources are limited. Males on both diets, however, increased the size of their spermatophore donations when the interval between female encounters was increased. One explanation for this pattern could be a frequency-dependent optimization of spermatophore size.

Phylogenetic diversity of Rhagada land snails is high on the Burrup Peninsula, Western Australia, with four distinct clades, representing three of the four major clades of the Pilbara region. Detailed sampling indicated little geographic overlap of the four clades, conforming to the general rarity of congeneric sympatry in Australian camaenids. The diversity on the Burrup Peninsula includes three previously unclassified morphotypes. One of these lies within the broad endemic clade of the adjacent Dampier Archipelago, and is provisionally assigned to the island species R. perprima, based on phylogenetic evidence. The two other undescribed morphotypes constitute an endemic clade that is the sister group of the broader Dampier Archipelago clade. All COI p-distances within clades are less than 6%, whereas nearly all distances between clades exceed 10%, the gap corresponding to differences among species of Rhagada generally. One morphotype in the Burrup Peninsula endemic clade has a low spire and a distinctive keel, and is restricted to a single rockpile. Detailed local sampling revealed gradation between this form and the more widely distributed globose morphotype. On the basis of genetic similarity and morphological continuity, we describe the morphologically variable endemic Burrup Peninsula clade as Rhagada ngurrana, sp. nov., which has a distribution spanning only 9 km.

Background Historically there has been a wide variation in the proportion of inadequate smears between general practices. Cervical screening in the UK is undergoing a fundamental change by moving from conventional to liquid based cytology (LBC). The main driver for this change has been a predicted reduction in the proportions of inadequate samples. This study investigates the effect of LBC on the variation in the proportion of inadequate samples between general practices using Shewhart's theory of variation and control charts. Methods Routinely collected cervical cytology data was obtained for all general practices in two localities in South Staffordshire for periods before and after the introduction of liquid based cytology. Control charts of the proportion of inadequate smears were plotted for the practices stratified by laboratory. A standardised measure of variation for all of the practices in each laboratory and each time period was also calculated. Results Following the introduction of liquid based cytology the overall proportion of inadequate samples in the two localities fell from 11.8 to 1.3% (p < 0.05). This fall was associated with a reduction in the average variation between the GP practices in the two localities from 1.6 to 1.0 standard deviations. There has also been a reduction in the number of practices showing special cause variation from eight to one following the introduction of liquid based cytology. Conclusion A reduction in the proportion of inadequate samples has been realised in these localities. The reduction in the overall proportion of inadequate samples has also been accompanied by a reduction in variation between GP practices.

This study reports the cloning, functional characterization, tissue expression, and nutritional regulation of a delta6 fatty acyl desaturase of Atlantic cod (Gadus morhua). PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from cod liver, with full-length cDNA obtained by rapid amplification of cDNA ends. The cDNA for the putative desaturase was shown to comprise 1980 bp, including a 261-bp 5'-UTR, a 375-bp 3'-UTR, and an ORF of 1344 bp that specified a protein of 447 amino acids. The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all characteristic of microsomal fatty acyl desaturases. The cDNA displayed delta6 desaturase activity in a yeast expression system. Quantitative real-time PCR assay of gene expression in cod showed that the delta6 desaturase gene was expressed highly in brain, to a slightly lesser extent in liver, kidney, intestine, red muscle, and gill, and at much lower levels in white muscle, spleen, and heart. The expression of the delta6 desaturase gene did not appear to be under significant nutritional regulation, with levels in liver and intestine being barely altered in fish fed a vegetable oil blend, in comparison with levels in fish fed fish oil. This was reflected in enzyme activity, as hepatocytes or enterocytes showed very little highly unsaturated FA biosynthesis activity irrespective of diet. Further studies are required to determine why the delta6 desaturase appears to be barely functional in cod under the conditions tested.

In the present study, samples of pond water, sediment and farmed species were collected at 12 fish and shrimp farms in Malaysia, Thailand and Vietnam to determine the biodiversity and environmental distribution of chloramphenicol-resistant (CmR) mesophilic heterotrophs in Southeast Asian aquaculture sites. Following isolation on Iso-Sensitest agar supplemented with 35mug ml(-1) Cm and dereplication using (GTG)(5)-PCR fingerprinting, 557 genotypically unique CmR strains were subjected to polyphasic identification. The 557 mesophilic heterotrophic CmR isolates represented 18 different genera largely dominated by the genera Escherichia (40.2%), Pseudomonas (11.7%), Acinetobacter (11.1%), Klebsiella (7.5%) and Bacillus (5.9%). A total of 439 CmR isolates were further assigned to 31 described species or species groups, mainly including organisms that have been associated with various human opportunistic infections such as Escherichia coli (n=219), Pseudomonas putida (n=47), Klebsiella pneumoniae (n=38) and Acinetobacter baumannii (n=23). Strains of Escherichia, and most notably, of E. coli, were the only common group of CmR heterotrophs irrespective of country, sample type or farm type. Together with other predominant but less widespread groups such as acinetobacters and pseudomonads, the results of this biodiversity study suggest that E. coli can be regarded as a potential indicator of Cm resistance in Southeast Asian aquaculture environments.

The sediment and water inter-phase layer in aquaculture ponds is a dynamic zone that plays a crucial role in maintaining the ecological balance of the pond system. An accurate sediment profile of the soft-watery layer that includes several important parameters can only be determined by using an adequate sampling technique. Because most of the commercially available grabs and soil corers currently used do not fulfill these requirements, a new, simple device for sediment sampling was designed in the framework of the European Union (EU)-funded ASIARESIST project. The sampler was used to collect samples from the upper 5 to 10 cm soil layer for bacteriological analysis from aquaculture ponds in Malaysia, Thailand and Vietnam. The sampler consisted of a three-piece PVC pipe that could be sterilized and had a total length of 230 cm. Each section was threaded at the end except for the top of the first and last section that tapered into a sharp point at an angle of 45°. When dismantled into sections, the sampler was convenient to transport and to reassemble at the site of sampling. The sediment sampler proved to be effective in obtaining intact profiles of bottom sediment, including soft sediments, was user-friendly and could be easily constructed at low cost.

Thymic-derived lymphocytes (T cells) recognize and respond to antigens through the mediation of the T-cell receptor complex. Here we report polymorphism at each of the five loci that encode the different components of the T-cell receptor complex in cattle. These genes were mapped on the bovine genome by genetic linkage analysis in the International Bovine Reference Panel (IBRP). These mapping data provide additional type I markers for linking the bovine genetic map with the human and mouse maps and also permit investigation of the effect of T-cell receptor polymorphism on immune responsiveness and disease susceptibility.

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids are key to the biosynthesis of the highly unsaturated fatty acids, arachidonic, eicosapentaenoic and docosahexaenoic acids from linoleic and alpha-linolenic acids. A Mortierella alpina cDNA polyunsaturated fatty acid elongase sequence identified mammalian, amphibian, zebrafish and insect expressed sequence tags (ESTs) in GenBank. Consensus primers were designed in conserved motifs and used to isolate full length cDNA from livers of several fish species by Rapid Amplification of cDNA Ends (RACE). The amplified cDNAs encoded putative open reading frames (ORFs) of 288-294 amino acids that were highly conserved among the fish species. Heterologous expression in yeast, Saccharomyces cerevisiae, demonstrated that all of the ORFs encoded elongases with the ability to lengthen polyunsaturated fatty acid substrates with chain lengths from C18 to C22 and also monounsaturated fatty acids, but not saturated fatty acids. There were differences in the functional competence of the elongases from different fish species. Most of the fish elongases showed a pattern of activity towards different fatty acid substrates in the rank order C18>C20>C22, although the tilapia and turbot elongases had similar activity towards 18:4n-3 and 20:5n-3. The fish elongases generally showed greater activity or similar activities with n-3 than with n-6 homologues, with the exception of the cod enzyme which was more active towards n-6 fatty acids.

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a delta5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterized previously. Here we report cloning and functional characterization of a delta6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon delta5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly delta6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the delta6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both delta6 and delta5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both delta6 and delta5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil.