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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.

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.

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.

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.

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.

Simple Summary Scrapie is a transmissible spongiform encephalopathy affecting sheep and goats. Due to similarities with mad cow disease (BSE) which is transmissible to humans, scrapie is considered of significance for its theoretical zoonotic potential. A variety of control strategies have been proposed and applied over the years. While herd depopulation, culling of affected susceptible animals, and disinfection measures are poorly successful, selective breeding schemes focused on amino acid substitutions (polymorphisms) in prion protein (PrP) would be the best way for eradication. In sheep, the association between PrP gene polymorphism argininine on codon 171 is highly effective for scrapie resistance breeding. Although in goats this polymorphism is absent, three other candidate PrP amino acid polymorphisms have been recognized for resistance breeding. For Greece with the largest goat population in Europe, the frequencies of these polymorphisms has yet to be sufficiently documented. This study performed a large-scale, cross-sectional survey for a large part of the country, regarding PrP polymorphic codons 110, 146, 211, and 222, and estimated effects of these polymorphisms on (i) the likelihood to be infected by scrapie, and (ii) scrapie status at the herd level, revealing possible candidate PrP amino acid polymorphisms to be used in breeding for scrapie-resistance programs. Abstract 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.

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.

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.

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.

Polymorphisms at codon 171 of the sheep prion protein gene (PRNP) are known to be genetically associated with scrapie ( Hunter et al., 1993 , Hunter et al., 1997 ). Glutamine (Q) and histidine (H) at codon 171 contribute to scrapie susceptibility, while arginine (R) is associated with resistance. In some breeds, lysine (K) occurs at codon 171 (171 K), but its effect on scrapie resistance has not been fully determined yet. To investigate the role of 171 K in dairy sheep in Greece, we initially analyzed blood samples from 2368 sheep with no clinical signs of the disease, from thirteen scrapie-affected flocks that were then monitored for 5 years for scrapie clinical signs. During this period 177 sheep resulted positive in classical scrapie, after obex testing. The 177 scrapie-affected brain samples PRNP polymorphisms were analyzed using a Real Time-PCR protocol while the 2368 sheep blood samples were analyzed by a micro-array method based on primer extension (Homogenous Mass Extend Assay – SEQUENOM, Genaissance Pharmaceuticals) in MALDI-TOF-MS. Single Nucleotide Polymorphisms (SNPs) present at PRNP codons 136A/V (Alanine/Valine), 154R/H and 171Q/R/H/K were detected. The ARQ haplotype as well as the ARQ/ARQ genotype predominated. In the studied population, ARK haplotype was found at the highest frequency reported so far (6.98%) whereas, ARK/-genotypes had a total frequency of 12.81%. Relative risk (RR) and the percent relative effect were calculated to assess the association between ARK/-genotypes and scrapie affection. The results showed that carriers of K allele at codon 171 compared to Q and H alleles had ca. 0.25 and 0.40 times the risk of scrapie affection, respectively, indicating a possible association of K allele at codon 171 with scrapie resistance.

Despite the intensive implementation of control programmes goat, sheep and human brucellosis remains endemic in Greece. As the discrimination between field endemic strains and vaccine strain Rev.1 is not feasible, it is essential to develop new diagnostic tools for brucellosis diagnosis. Moreover, effective disease control requires enhanced epidemiological surveillance in both humans and animals including robust laboratory support. Two new multiplex (duplex) polymerase chain reactions (PCRs) were developed and the results were compared with those obtained by real‐time PCR and bacteriological biotyping. A total of 71 Brucella spp. Greek endemic strains were identified at species and biovar level, using both molecular and conventional techniques. Their discrimination from the vaccine strain Rev.1 was achieved, using polymerase chain reaction‐restriction fragment length polymorphism assay (PCR‐RFLP). All 71 strains were identified as Brucella melitensis by multiplex PCR as well as by real‐time PCR and conventional biotyping. Sixty‐two (87.3%) out of 71 strains were identified as B. melitensis biovar 3, eight (11,3%) strains as biovar 1 and only one (1,4%) as biovar 2. Digestion with PstI restriction enzyme revealed that all strains were field endemic strains, as they gave different patterns from the vaccine strain Rev.1. Brucella melitensis biovar 3 appears to be the predominant type in Greece. The novel multiplex PCR produced results concordant to ones obtained by real‐time PCR and conventional biotyping. This technique could support and facilitate the surveillance of Brucellosis in Greece contributing in the control of the disease.

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.

This Real-Time PCR genotyping protocol can be used for the detection of polymorphisms at codons 146 (N/S/D), 211 (R/Q) and 222 (Q/K) in the caprine PRNP gene. We developed four seperate Real-Time PCR reactions with four different CustomTaqMan®SNP Genotyping Assays (Applied Biosystems, Foster City, California, USA). Each SNP Genotyping Assay consisted of a mix of sequence-specific forward and reverse primers to amplify the polymorphic sequence of interest and twoTaqMan®MGB probes, FAM and VIC dye-labeled to detect the amplified product.

Τo exploit the solid residue of olive mill wastewaters (OMWS) towards the production of an olive paste-type product, natural fermentation of OMWS in NaCl (6% w/v) was implemented for 14 days at 20 °C. The microbiological and physicochemical characteristics of OMWS were examined during fermentation. Fermentation of OMWS was shown to be relied on yeasts while absence of enterobacteria, pseudomonads and staphylococci as well as LAB was observed. Molecular identification of yeast included RFLP analysis of the 5.8S-ITS region. For some selected isolates, sequence analysis of the 5.8S-ITS region was performed. The results were compared to sequences held in public databases (NCBI) and data of papers published previously. These techniques allowed to identify the isolates as Saccharomyces cerevisiae (99% homology) and Pichia membranefasciens (100% homology). Beta-glucosidase was the most important biochemical activity observed, mainly responsible for removing the bitter taste. Paste preparations of fermented OMWS did not differ in sensory characteristics than the controls.

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.

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.

To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.

The correlation between the natural levels of GST and the tolerance to the insecticide decamethrin (dMT), as well as the interaction between the molecules of affinity purified enzyme and the insecticide were investigated in order to collect further information on the obscure role of the Glutathione S-transferase system (GST) as a mechanism of defence against pyrethroids. The studies were carried out, comparatively, on the larvae and pupae developmental stages of the coleopteran Tenebrio molitor, which exhibit varying natural levels of GST activity. No stage dependent susceptibility of the insect against pyrethroid insecticides was found during the first 24 h, however 48 h after treatment, the KD50 dose increased significantly due to the recovery of some individuals from the larvae stage. Simultaneous injection of decamethrin with compounds which inhibit GST activity in vitro, resulted in an increased tolerance, which was more pronounced in the pupae stage. Inhibition studies combined with competitive fluorescence spectroscopy and high pressure liquid chromatography (HPLC) showed that the insecticide binds probably to the active site of the enzyme inhibiting its activity towards CDNB in a competitive manner, but is not conjugated with GSH. According to this, GST offers a passive protection towards pyrethroid insecticides by binding to their molecule in a sequestering mechanism.

The correlation between the natural levels of glutathione S-transferase (GST) and the tolerance to the organophosphorus insecticides parathion-methyl and paraoxon-methyl, as well as the interaction of affinity-purified enzyme and the insecticides were investigated in order to collect further information on the role of the glutathione S-transferase system as a mechanism of defence against insecticides in insects. The studies were carried out on the larvae and pupae of the coleopteran Tenebrio molitor L, which exhibit varying natural levels of GST activity. Stage-dependent susceptibility of the insect against insecticides was observed during the first 24 h. However, 48 h after treatment, the KD50 value increased significantly due to the recovery of some individuals. Simultaneous injection of insecticide with compounds which inhibit GST activity in vitro caused an alteration in susceptibility of insects 24 or 48 h post-treatment, depending on stage and insecticide used. Inhibition studies combined with competitive fluorescence spectroscopy revealed that the insecticides probably bind to the active site of the enzyme, thus inhibiting its activity towards 1-chloro-2,4-dinitrobenzene in a competitive manner. High-performance liquid chromatography and gas chromatography revealed that T molitor GST catalyses the conjugation of the insecticides studied to a reduced form of glutathione (GSH). From the above experimental results, it is considered that GST offers a protection against the organophosphorus insecticides studied by active site binding and subsequent conjugation with GSH.

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. ispartof: Journal of Agricultural and Food Chemistry vol:60 issue:32 pages:7941-7948 ispartof: location:United States status: published

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 12 h after last feeding, but returns to initial, non-fasted levels after 72 h. In contrast, in liver non-fasted expression is low, but strongly increases at 24 and 72 h after last feeding. In white muscle and liver, CPT1B mRNA expression is highly correlated with the expression of peroxisomal proliferator-activated receptor β (PPARβ). 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.

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.

To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.

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.

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.

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 PPARα, PPARβ/δ, and PPARγ. 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, PPARγ is expressed more widely in fish tissues than in mammals, whereas PPARα and β 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 PPARα and β exhibit an activation profile similar to that of the mammalian PPAR in response to a variety of activators/ligands, whereas PPARγ is not activated by mammalian PPARγ-specific ligands. Amino acid residues shown to be critical for ligand binding in mammalian PPARs are not conserved in fish PPARγ and therefore, together with the distinct tissue expression profile of this receptor, suggest potential differences in the function of PPARγ in fish compared with mammals.