Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing.

BACKGROUND: Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. METHODS: Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. RESULTS: In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. CONCLUSION: The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

Comparative qualitative and quantitative analysis of lactic acid bacteria by molecular methods in different Greek cheeses.

In the present research communication, we report on identification and quantification of four main lactic acid bacteria (LAB) genera (Lactococcus, Lactobacillus, Streptococcus and Leuconostoc), most common in Greek cheeses, by a novel culture-independent method. More specifically, new primers were designed to be used in both multiplex PCR for simultaneous identification and in real-time PCR for quantification of the LAB. The method was validated by applying it in parallel to culture-dependent method in a variety of cheeses from different Greek geographical locations, of different animal milk origins and of different production methods. While the standard plate culture method showed absence of Leuconostoc sp. in all cheeses, the culture-independent methods detected all four LAB genera studied. Furthermore, the relative presence of the four genera detected by the culture-independent method showed a pattern present in almost all cheese samples tested, indicating Lactococcus genus as the dominant one.

Identification of Brucella spp. isolates and discrimination from the vaccine strain Rev.1 by MALDI-TOF mass spectrometry.

Brucellosis' surveillance and control programs require robust laboratory techniques that can reliably identify and biotype Brucella strains and discriminate between vaccine and field infection. In the recent years, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the routine identification of several microorganisms in clinical microbiology laboratories. Nevertheless, its application on Brucella spp. identification is limited since there are no reference spectra in the commercial databases, due to the microorganism's potential bioterrorist use. In this study, a custom MALDI-TOF MS reference library was constructed and its performance on identification at species level was evaluated using 75 Brucella spp. isolates. Furthermore, distinct peak biomarkers were detected for biovar assignment and discrimination from vaccine strain Rev.1. Analysis of mass peak profiles allowed Brucella accurate identification at genus and species level (100%) with no misidentifications. Despite the high intrageneric similarity, MALDI-TOF MS database succeeded in classifying at biovar level, 47 out of 62 B. melitensis bv. 3 isolates (75.81%), whereas all B. melitensis strains, except for one, were correctly discriminated from vaccine strain Rev.1. MALDI-TOF MS appeared to be a rapid, cost-effective and reliable method for the routine identification of brucellae which reduces time consumption in pathogen identification and could replace in the near future the current conventional and molecular techniques. Its ability to differentiate vaccine from field infection could facilitate brucellosis' monitoring systems contributing in the effective control of the disease.

Application of triplex-PCR with an innovative combination of 3 pairs of primers for the detection of milk's animal origin in cheese and yoghurt.

In this research communication we describe an innovative protocol that combines three pairs of primers, two from the literature and one designed in our laboratory, for application in triplex-PCR on somatic cell DNA to enable identification of the species origin (cow, sheep, goat) of cheeses and yogurts with a detection limit of 0.1%. Mislabeling was detected in 15 out of 40 cheeses and in 18 out of 40 yogurts tested. The suggested procedure is a quick and reliable tool for identifying the animal origin of cheeses and yogurts and it can be used to certify product reliability on the domestic and international market. Additionally, in combination with a serological test it can offer a reliable tool for detecting the presence of cow's whey.

Prevalence of Staphylococcus aureus and of methicillin-resistant S. aureus (MRSA) along the production chain of dairy products in north-western Greece.

The objective of this study was to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the production chain of dairy products. Of 367 tested samples (36 bulk tank milk (BTM), 19 dairy products, 72 human, 185 animal, 55 equipment), 212 (57.8%) were found positive for S. aureus. Almost all isolates (99.6%) were resistant to at least one antimicrobial and 13.3% were multi-drug resistant (MDR), exhibiting resistance to three or more antibiotic classes. Eleven samples (3%) were found contaminated by MRSA carrying the mecA gene. None of the MRSA isolates carried the mecC or the Pandon-Valentine leucocidin (PVL) genes. Four spa types were identified among the MRSA isolates: t127, t3586, t1773, t4038, with t127 being the most prevalent (7 out of 11). Two of them, t3586 and t1773, were isolated for the first time in Greece. Furthermore, Pulse-Field Gel Electrophoresis (PFGE) analysis indicated clonal circulation through the dairy production chain. The presence of MDR S. aureus, and especially MRSA, in animals and dairy products represents a potential threat for the spread of this pathogen in the community. The results indicated that human, animal and environmental sources could be involved in the contamination of dairy products along their production chain and therefore further investigation of contamination sources is needed to control the dispersion of MRSA in the community.

The Peroxisome Proliferator-Activated Receptors of Ray-Finned Fish: Unique Structures, Elusive Functions.

The Actinopterygian and specifically the Teleostean peroxisome proliferator-activated receptors (PPARs) present an impressive variability and complexity in their structures, both at the gene and protein levels. These structural differences may also reflect functional divergence from their mammalian homologs, or even between fish species. This review, taking advantage of the data generated from the whole-genome sequencing of several fish species, highlights the differences in the primary structure of the receptors, while discussing results from the literature pertaining to the functions of fish PPARs and their activation by natural and synthetic compounds.

Indigenous Lactic Acid Bacteria Isolated from Raw Graviera Cheese and Evaluation of Their Most Important Technological Properties.

The aim of the present study was to characterize LAB isolates from raw-milk cheeses, to evaluate some of their technological properties and to select a few 'wild' LAB strains that could potentially be used as starter cultures. LAB strains were isolated and identified from raw milk, curd, and cheese at 30, 60, and 90 days of ripening. A total of 100 strains were isolated, 20 from each phase of ripening. All isolates were tested for acidification ability, curd formation, and aroma production at 32 °C and 42 °C after 24 and 48 h. Following the acidification test, 42 strains were selected for identification and characterization of their technological properties. A high proportion of lactic acid bacteria and Gram + cocci were found throughout the cheese-making process. Enterococci reached their maximum proportion on the 7th day of ripening while Lactobacilli increased significantly during the first month of ripening. Forty-two strains were identified by phenotypic, biochemical, and molecular techniques. Lactococci were predominant in raw milk and curd while Lactobacilli in the ripening of the cheese. Four LAB strains including one Leuconostoc pseudomenteroides, two Lacticaseibacillus paracasei subsp. paracasei and one Enterococcus hirae, were proposed for their potential use as starters or secondary cultures.

Circulation of Pestiviruses in Small Ruminants from Greece: First Molecular Identification of Border Disease Virus.

The incidence of small ruminant pestivirus infections in Greece remains unknown as they have not been diagnosed in the country since 1974 when the most recent Border Disease Virus (BDV) outbreak was reported. The objective of our study was to explore the possible occurrence of pestiviral infections among sheep and goat farms in Greece and to further determine the variants of major concern. Thus, serum samples were collected from 470 randomly selected animals belonging to 28 different flocks/herds. ELISA on p80 antibody revealed the existence of seropositive animals in four out of the 24 studied sheep flocks, whereas all the goats in the four studied herds were seronegative. Viral RNA and antigens were detected in two sheep out of the four seropositive flocks by RT-PCR and ELISA, respectively. Sequencing and phylogenetic analysis showed that the newly identified Greek variants were closely related to the strains of the BDV-4 genotype. One of the BDV-positive sheep demonstrated the diagnostic profile of a persistently infected (PI) animal, providing additional information regarding the source of the infection. This is the first molecular identification of BDV isolates in Greece. Our findings indicate that BDV infections are likely to remain undiagnosed, highlighting the need for further epidemiological studies and active surveillance programs to determine the prevalence and impact of BDV infections on a countrywide level.

Spatio-Temporal Distribution of Brucellosis in European Terrestrial and Marine Wildlife Species and Its Regional Implications.

Brucellosis is an important bacterial zoonosis of domestic and wildlife species. This disease has a significant public health concern and is characterized by reproductive failure resulting in economic losses in the livestock industry. Among thirteen known species, B. abortus, B. melitensis, B. suis, and B. canis are human pathogens. Brucellosis has been extensively investigated in humans and domestic animals. However, the situation in wildlife is still not completely reported and studied. Therefore, a systematic literature search and screening were done to clarify the situation of brucellosis in wildlife in Europe. Sixty-five articles from a total of 13,424 reports published between 1991 and 2021 were selected, applying defined inclusion criteria. Wild boars and brown hares were the most often studied terrestrial wildlife species, whereas seals and porpoises were the most often investigated marine wildlife. Poland, Croatia, and Belgium showed the highest seroprevalences of wild boars caused by B. suis biovar 2. In marine wildlife, brucellosis was mainly caused by B. ceti and B. pinnipedialis. Most samples were from carcasses. Thus, sera could not be collected. It is worrisome that B.abortus and B. melitensis were reported from both terrestrial and marine wild animals, posing a zoonotic threat to people exposed to wild animals. Currently, there is no approved vaccine available for wild animals. The main challenges are the development of specific diagnostics and their validation for use in wildlife.

Polymorphisms of Codons 110, 146, 211 and 222 at the Goat PRNP Locus and Their Association with Scrapie in Greece.

Scrapie is considered an endemic disease in both sheep and goats in Greece. However, contrary to sheep, in goats more than one prion protein (PrP) polymorphism has been recognized as a candidate for resistance breeding against the disease. For an impression, candidates which are circulating, (i) brain samples (n = 525) from scrapie-affected (n = 282) and non-affected (n = 243) animals within the national surveillance program, and (ii) individual blood samples (n = 1708) from affected (n = 241) and non-affected (n = 1467) herds, in a large part of mainland Greece and its islands, were collected and assayed. A dedicated Taqman method was used to test for amino acid polymorphisms 110T/P, 146N/S/D, 211R/Q, and 222Q/K. Highly prevalent genotypes were 110TT, 146NN, 211RR, and 222QQ. The frequencies of polymorphisms in blood and negative brain samples for codons 110P, 211Q, and 222K were 4.0%, 3.0%, and 1.9%, respectively, while 146D (0.7%) was present only on Karpathos island. Codon 110P was exclusively found in scrapie-negative brains, and homozygous 110P/P in two scrapie-negative goats. It is concluded that breeding programs in Karpathos could focus on codon 146D, while in other regions carriers of the 110P and 222K allele should be sought. Case-control and challenge studies are now necessary to elucidate the most efficient breeding strategies.

MLVA-16 Genotyping of Brucella abortus and Brucella melitensis Isolates from Different Animal Species in Egypt: Geographical Relatedness and the Mediterranean Lineage.

Brucellosis is a common zoonotic disease in Egypt. However, there are limited data available on the genetic diversity of brucellae circulating in Egypt and other Mediterranean areas. One hundred and nine Brucella (B.) strains were isolated from different animal species in thirteen Egyptian governorates. Multi-locus variable number tandem repeats (VNTRs) analysis (MLVA-16) was employed to determine the geographical relatedness and the genetic diversity of a panel of selected Egyptian strains (n = 69), with strains originating from Italy (n = 49), Portugal (n = 52), Greece (n = 63), and Tunisia (n = 4). Egyptian B. melitensis strains clustered into two main clusters containing 21 genotypes. Egyptian B. abortus strains clustered into three main clusters containing nine genotypes. The genotypes were irregularly distributed over time and space in the study area. Egyptian strains of B. melitensis showed MLVA-16 patterns closer to that of Italian strains. Egyptian B. abortus strains isolated from cattle share the same genotype with strains from Portugal and similar to strains from Italy with low genetic diversity. Strains with similar MLVA patterns isolated from different governorates highlight the movement of the pathogen among governorates. Hence, it may also reflect the long endemicity of brucellosis in Egypt with earlier dispersal of types and great local genetic diversity. Open markets may contribute to cross-species transmission and dissemination of the new types nationwide. The presence of West Mediterranean lineages of B. melitensis and relatedness of B. abortus strains from the studied countries is a result of the socio-historical connections among the Mediterranean countries. Transnational eradication of brucellosis in the Mediterranean basin is highly demanded.

Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats.

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds.

Molecular characterization of a cDNA from the gilthead sea bream (Sparus aurata) encoding a fish prion protein.

We have identified and characterized a cDNA from the brain tissue of the highly commercial marine fish species, the gilthead sea bream (Sparus aurata), which encodes a 496 amino acid residue protein sharing the organizational and structural features of the mammalian prion proteins. Tissue mRNA expression analyses revealed the presence of this transcript in various tissues of the gilthead sea bream including the brain, the spleen, and the heart. Sequence comparison and phylogenetic analysis showed the gilthead sea bream protein to be the homologue of one of the long form prion proteins identified from the model fish species Takifugu rubripes and Danio rerio. Unique to this fish prion protein is an extended Gly-Tyr-Pro-rich region, a structural feature that apparently resulted from multiple duplications of a core motif. The presence of this feature in other seemingly unrelated proteins suggests the involvement of common mechanism(s) in its formation and infers possible evolutionary trends related to its function.

Piscine UDP-glucuronosyltransferase 1B.

Glucuronidation is an important detoxification pathway for organic pollutants in fish. We report here the isolation and characterisation of UDP-glucuronosyltransferases (UGT) genes from the closely related marine flatfish, plaice (Pleuronectes platessa) and flounder (Platichthys flesus). The deduced amino acid sequences share greater similarity with mammalian UGT1 family genes than UGT2 genes (44-47% and 39-40% amino acid identity, respectively) and have been designated UGT1B. Both plaice and flounder UGT1B mRNAs are expressed in all tissues and are most highly expressed in liver, with high levels in intestine, gill, kidney and adipose tissue and much lower levels in muscle, heart and brain. Plaice UGT1B mRNA is undetectable in gametes or fertilised eggs and there is a large increase in expression between gastrulation and myotome formation after which levels decline some 5-10-fold. Flounder UGT1B mRNA was increased in liver after intraperitoneal injection of Arochlor 1254 or lindane (gamma-hexachlorocyclohexane), but not after perflourooctanoic acid or 3-methylcholanthrene treatment. In isolated flounder hepatocytes UGT1B mRNA was increased after exposure to benzo(a)pyrene but not by 17alpha-ethynylestradiol. Expression of a cDNA for plaice UGT1B in cos7 cells resulted in higher 1-naphthol conjugation in cell homogenates compared to steroid conjugation, whilst bilirubin and bile acid conjugation were undetectable. This indicates that the plaice gene codes for the phenol-conjugating UGT previously purified in our laboratory from this species and that it is likely to play a major role in the detoxification of polyaromatic hydrocarbons in flatfish. Its role in development is unknown. UGT1B genes are also present in pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio) genomes, but they differ in their genic organisation. Pufferfish possess multiple (repeated) complete UGT1 genes and Southern blots indicate that the homologous plaice UGT1B gene may also be organised in this way. In contrast, zebrafish appear to have two UGT1 loci whose sequences and intron/exon structures are closely related to that of plaice, however, the organisation of these genes is similar to the mammalian UGT1 family since each has multiple repeated exon 1's which are alternatively spliced to a common set of exons encoding the aglycone binding domain. Taken together with evidence from phylogenetic comparison of fish sequences with UGT1 and UGT2 families in mammals, we suggest these homologous fish UGTs should all be included within the vertebrate UGT1 family and designated as UGT1B.

Three peroxisome proliferator-activated receptor isotypes from each of two species of marine fish.

The cloning and characterization of cDNAs and genes encoding three peroxisome proliferator-activated receptor (PPAR) isotypes from two species of marine fish, the plaice (Pleuronectes platessa) and the gilthead sea bream (Sparus aurata), are reported for the first time. Although differences in the genomic organization of the fish PPAR genes compared with their mammalian counterparts are evident, sequence alignments and phylogenetic comparisons show the fish genes to be homologs of mammalian PPARalpha, PPARbeta/delta, and PPARgamma. Like their mammalian homologs, fish PPARs bind to a variety of natural PPAR response elements (PPREs) present in the promoters of mammalian or piscine genes. In contrast, the mRNA expression pattern of PPARs in the two fish species differs from that observed in other vertebrates. Thus, PPARgamma is expressed more widely in fish tissues than in mammals, whereas PPARalpha and beta are expressed similarly in profile to mammals. Furthermore, nutritional status strongly influences the expression of all three PPAR isotypes in liver, whereas it has no effect on PPAR expression in intestinal and adipose tissues. Fish PPARalpha and beta exhibit an activation profile similar to that of the mammalian PPAR in response to a variety of activators/ligands, whereas PPARgamma is not activated by mammalian PPARgamma-specific ligands. Amino acid residues shown to be critical for ligand binding in mammalian PPARs are not conserved in fish PPARgamma and therefore, together with the distinct tissue expression profile of this receptor, suggest potential differences in the function of PPARgamma in fish compared with mammals.

Molecular characterization of three peroxisome proliferator-activated receptors from the sea bass (Dicentrarchus labrax).

Peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that control the expression of genes involved in lipid homeostasis in mammals. We searched for PPAR in sea bass, a marine fish of particular interest to aquaculture, after hypothesizing that the physiological and molecular processes that regulate lipid metabolism in fish are similar to those in mammals. Here, we report the identification of complementary DNA and corresponding genomic sequences that encode three distinct PPAR from sea bass. The sea bass PPAR are the structural homologs of the mammalian PPAR alpha, beta/delta, and gamma isotypes. As revealed by RNase protection, the tissue expression profile of the fish PPAR appears to be very similar to that of the mammalian PPAR homologs. Thus, PPAR alpha is mainly expressed in the liver, PPAR gamma in adipose tissue, and PPAR beta in all tissues tested, with its highest levels in the liver, where it is also the dominant isotype expressed. Like mammalian PPAR, the sea bass isotypes recognize and bind to PPAR response elements of both mammalian and piscine origin, as heterodimers with the 9-cis retinoic acid receptor. Through the coactivator-dependent receptor ligand assay, we also demonstrated that natural FA and synthetic hypolipidemic compounds can act as ligands of the sea bass PPAR alpha and beta isotypes. This suggests that the sea bass PPAR act through similar mechanisms and perform the same critical lipid metabolism functions as mammalian PPAR.

Restriction fragment length analysis of the cytochrome b gene and muscle fatty acid composition differentiate the cryptic flatfish species Solea solea and Solea aegyptiaca.

Overlapping external morphometric characters easily confound the flatfishes Solea aegyptiaca and Solea solea (Soleidae) in areas of the Mediterranean Sea where both species live in sympatry. This leads to uncertainties in the fisheries and marketing of the species, in addition to misinterpretations in biogeography and conservation studies. This paper describes a simple restriction fragment length-based diagnostic test that differentiates S. solea from S. aegyptiaca, as well as from other species of the Soleidae family. Furthermore, the two species living in sympatry in the Gulf of Kavala (North Aegean Sea, Greece) present significant qualitative differences in muscle fatty acid composition, a property that can also be used to distinguish the two cryptic species.

Molecular characterization of a gilthead sea bream (Sparus aurata) muscle tissue cDNA for carnitine palmitoyltransferase 1B (CPT1B).

Understanding the control of piscine fatty acid metabolism is important for determining the nutritional requirements of fish, and hence for the production of optimal aquaculture diets. The regulation and expression of carnitine palmitoyltransferase 1 (CPT1; EC No 2.3.1.21) are critical processes in the control of fatty acid metabolism, and here we report a cDNA from gilthead sea bream (Sparus aurata) which encodes a protein with high identity to vertebrate CPT1. This sea bream CPT1 mRNA is predominantly expressed in skeletal and cardiac muscle, with little expression in other tissues. Phylogenetic analysis of other vertebrate CPT1 sequences show that fish genomes contain a single gene related to mammalian CPT1B, and a further two multi-gene families related to mammalian CPT1A. Genes related to mammalian CPT1C are absent in fish. Therefore, based on both functional and evolutionary orthology to mammalian CPT1B, the sea bream CPT1 reported here is a CPT1B isoform. Sea bream CPT1B mRNA expression progressively decreases in heart and muscle up to 12h after last feeding, but returns to initial, non-fasted levels after 72h. In contrast, in liver non-fasted expression is low, but strongly increases at 24 and 72h after last feeding. In white muscle and liver, CPT1B mRNA expression is highly correlated with the expression of peroxisomal proliferator-activated receptor beta (PPARbeta). Thus fatty acid metabolism by CPT1B and its control by PPARs are similar in fish and mammals, but multiple genes for CPT1A-like proteins in fish also suggest different and more complex pathways of lipid utilisation than in mammals.

Hepatic gene expression in flounder chronically exposed to multiply polluted estuarine sediment: Absence of classical exposure 'biomarker' signals and induction of inflammatory, innate immune and apoptotic pathways.

The effects of chronic long-term exposure to multiply polluted environments on fish are not well understood, but environmental surveys suggest that such exposure may cause a variety of pathologies, including cancers. Transcriptomic profiling has recently been used to assess gene expression in European flounder (Platichthys flesus) living in several polluted and clean estuaries. However, the gene expression changes detected were not unequivocally elicited by pollution, most likely due to the confounding effects of natural estuarine ecosystem variables. In this study flounder from an uncontaminated estuary were held on clean or polluted sediments in mesocosms, allowing control of variables such as salinity, temperature, and diet. After 7 months flounder were removed from each mesocosm and hepatocytes prepared from fish exposed to clean or polluted sediments. The hepatocytes were treated with benzo(a)pyrene (BAP), estradiol (E2), copper, a mixture of these three, or with the vehicle DMSO. A flounder cDNA microarray was then used to measure hepatocyte transcript abundance after each treatment. The results show that long-term chronic exposure to a multiply polluted sediment causes increases in the expression of mRNAs coding for proteins of the endogenous apoptotic programme, of innate immunity and inflammation. Contrary to expectation, the expression of mRNAs which are commonly used as biomarkers of environmental exposure to particular contaminants were not changed, or were changed contrary to expectation. However, acute treatment of hepatocytes from flounder from both clean and polluted sediments with BAP or E2 caused the expected changes in the expression of these biomarkers. Thus transcriptomic analysis of flounder exposed long-term to chronic pollution causes a different pattern of gene expression than in fish acutely treated with single chemicals, and reveals novel potential biomarkers of environmental contaminant exposure. These novel biomarkers include Diablo, a gene involved in apoptotic pathways and highly differentially regulated by both chronic and acute exposure to multiple pollutants.

Multiple peroxisome proliferator-activated receptor beta subtypes from Atlantic salmon (Salmo salar).

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily that functions as critical regulators of lipid and energy homeostasis. Although intensively studied in mammals, their basic biological functions are still poorly understood. The objective of this work was to characterize PPARbeta subtypes in a fish, the Atlantic salmon (Salmo salar), in order to address PPAR function and the regulation of lipid homeostasis in lower vertebrates. The screening of an Atlantic salmon genomic library revealed the presence of four genes for PPARbeta subtypes. Based on comparisons of exons and exon-flanking regions, these genes were assigned into two families, ssPPARbeta1 and ssPPARbeta2, each family containing two isotypes: ssPPARbeta1A and beta1B and ssPPARbeta2A and beta2B. Two full-length cDNAs for ssPPARbeta1A and ssPPPARbeta2A were isolated. Transcripts for ssPPARbeta1A and ssPPARbeta2A have distinct tissue expression profiles, with ssPPARbeta1A predominating in liver and ssPPARbeta2A predominating in gill. Expression levels of mRNA of either isotypes were up to tenfold lower in kidney, heart, spleen, muscle, and brain. In cellular transfection assays, ssPPARbeta1A is activated by monounsaturated fatty acids, 2-bromopalmitate, and mammalian PPARbeta-specific ligand GW501516. In contrast, PPARbeta2A was not activated by any of the compounds tested. Furthermore, ssPPARbeta2A repressed both the basal reporter gene activity and the GW501516-induced activity of ssPPARbeta1A. The results indicate unexpected levels of variety and complexity in PPAR subtype and mechanism of action in lower vertebrates.