FISHBIRD 2 For Serum

Spring viremia of carp (SVC) is an important disease affecting cyprinids, mainly common carp Cyprinus carpio. The disease is widespread in European carp culture, where it causes significant morbidity and mortality. Designated a notifiable disease by the Office International des Epizooties, SVC is caused by a rhabdovirus, spring viremia of carp virus (SVCV). Affected fish show destruction of tissues in the kidney, spleen and liver, leading to hemorrhage, loss of water-salt balance and impairment of immune response. High mortality occurs at water temperatures of 10 to 17 degrees C, typically in spring. At higher temperatures, infected carp develop humoral antibodies that can neutralize the spread of virus and such carp are protected against re-infection by solid immunity. The virus is shed mostly with the feces and urine of clinically infected fish and by carriers. Waterborne transmission is believed to be the primary route of infection, but bloodsucking parasites like leeches and the carp louse may serve as mechanical vectors of SVCV. The genome of SVCV is composed of a single molecule of linear, negative-sense, single-stranded RNA containing 5 genes in the order 3'-NPMGL-5' coding for the viral nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase, respectively. Polyacrylamide gel electrophoresis of the viral proteins, and sequence homologies between the genes and gene junctions of SVCV and vesicular stomatitis viruses, have led to the placement of the virus as a tentative member of the genus Vesiculovirus in the family Rhabdoviridae. These methods also revealed that SVCV is not related to fish rhabdoviruses of the genus Novirhabdovirus. In vitro replication of SVCV takes place in the cytoplasm of cultured cells of fish, bird and mammalian origin at temperatures of 4 to 31 degrees C, with an optimum of about 20 degrees C. Spring viremia of carp can be diagnosed by clinical signs, isolation of virus in cell culture and molecular methods. Antibodies directed against SVCV react with the homologous virus in serum neutralization, immunofluorescence, immunoperoxidase, or enzyme-linked immunosorbent assays, but they cross-react to various degrees with the pike fry rhabdovirus (PFR), suggesting the 2 viruses are closely related. However, SVCV and PFR can be distinguished by certain serological tests and molecular methods such as the ribonuclease protection assay.

FISHBIRD 2 for Serum

Carnitine represents an essential cofactor in fatty acid metabolism in mammals, as it transports fatty acids under the form of acylcarnitines into the mitochondria of muscle cells for cellular energy production by lipid β-oxidation [58]. Carnitine may be synthetized in the body from the essential amino acids lysine and methionine. Most of the carnitine in omnivores comes from dietary sources, particularly beef and lamb [20]. Carnitine and acetylcarnitine were higher in the urine [59] and serum [60] of subjects consuming high-meat diets compared to vegetarians, and their urinary values were representative of habitual intake of red and processed meat in free-living subjects [61, 62]. Three acylcarnitines (acetylcarnitine, propionylcarnitine and 2-methylbutylcarnitine) were significantly higher in the urine of subjects from the EPIC cohort after the intake of meat and fish compared to control subjects with no meat intake, with no distinction among the different animal protein sources [38]. Propionylcarnitine and acetylcarnitine were also observed in plasma after a dietary intervention with meat and fish from the same research group [38]. Acetylcarnitine had slower kinetics than propionylcarnitine, reaching the highest value after 24 h when propionylcarnitine had already returned to the baseline level. In the EPIC-Oxford cohort, a series of acylcarnitines were also characteristic of the dietary pattern associated with high meat intake [63]. In particular, the concentrations of carnitine and acylcarnitines C-4 and C-5 were highest in meat eaters, followed by fish eaters, vegetarians, and vegans. Furthermore, C-3 and C-16 were higher in meat eaters and lower in vegans. Carnitine and acylcarnitines may reflect the intake of highly accessible amino acids and fatty acids contained in meat and fish and may be considered generic markers of intake of foods of animal origin. However, given that physiological conditions such as age, gender, and health status, as well as the intake of other foods with highly accessible amino acids or fatty acids may affect plasma acylcarnitine levels and their excretion into urine [64, 65], these metabolites may not be suitable markers per se for specific and quantitative assessment of meat intake and are therefore not considered for further validation as single markers.

Creatine is present in animal muscles, mainly in the form of phosphocreatine, and can be synthesized endogenously from arginine, glycine, and methionine. The main dietary source is meat, including red meat, fish, and poultry [66]. Several studies reported an increase in creatine levels after meat intake in urine [59, 62, 67, 68], erythrocytes [60], plasma, and serum [60, 69, 70]. Even a single meal containing meat has been shown to increase creatine in urine [71]. Associations between urinary creatine and habitual intake of shellfish [62] and oily fish such as salmon [42] have also been reported. Pallister et al. [69] proposed circulating levels of creatine as a possible marker of habitual intake of red meat and poultry, as observed in the UK Twin cohort study (n = 3559 subjects). However, experimental studies supporting this observation are lacking. Creatine in urine and plasma/serum are therefore candidate markers of total meat intake.

Intake of white meat, i.e., poultry, but also some species of fish, may be assessed by the urinary excretion of 3-MH or anserine, or by 3-MH in plasma. Anserine is present in hen and turkey muscle and to a lesser extent in meat from several species of fish, including cod, salmon and tuna; excretion in humans may therefore mainly reflect poultry intake, while more studies are needed to assess the influence of fish on this marker. Human excretion of anserine has been shown to be strongly associated only with chicken intake and not with fish at the group level (high vs. non-consumers) in a small European sub-study [38]. Anserine excretion after dietary intake has acceptable analytical performance in several analytical systems, stability after longer-term storage, and robustness; but experimental evidence for dose-response and time-response is still missing, including for experimental studies with fish intake. On the other hand, the validation of 3-MH in urine shows reproducible dose-, and time-response in experimental studies, although it is strongly associated with the intake of both bird and fish meat in observational studies. Increased excretion is also observed after red meat intake, albeit less strongly [276]; even so, experimental studies showing time- and dose-response of 3-MH excretion have been observed after intake of beef [43]. Inter-individual variation seems limited and background excretion rates with lacto-ovo-vegetarian diets are low. Stability is excellent and analytical performance is good in systems able to discriminate 1- and 3-MH [277, 278], but more data are needed regarding relative excretion levels after different sources of meat. Postprandial plasma 3-MH has been shown to respond stronger to chicken than to fish (haddock) with clear dose-response but with only marginal increases after high intakes of beef compared with other sources [38]. Plasma or serum 3-MH has not been extensively used as a marker in observational studies and most aspects of validation are therefore still missing, needing further research. From the scoring pattern, 3-MH in urine can be considered a valid compliance marker of poultry intake but may reflect general meat intake more broadly in samples from observational studies. Further validation studies are needed to study the relationship of 3-MH excretion with different kinds of aquatic meats.

A range of biomarkers have potential to estimate meat and fish intake. The δ15N and/or δ13C ratios in hair are good determinants of long-term animal protein. Blood levels of creatine, hydroxyproline, and 3-MH are promising markers of total meat while carnosine works for terrestrial meats. However, more studies are needed to validate these markers, both alone and in combinations. The best validated markers are DHA and EPA in blood, serum, and blood lipids, clearly recognized as markers of habitual oily fish consumption. CMPF in blood and urine and TMAO in urine may also be useful markers to measure fish intake but depend on dietary habits. Overall, fish markers need validation in different populations. 3-MH seems to be a good biomarker of poultry intake showing dose-response, time-response, and good analytical performance but its robustness may be questioned because intake of some fish and possibly other meats are likely to interfere. Anserine is particularly good at characterizing poultry in both intervention and observational settings, but studies on time-response and dose-response are lacking. Guanidinoacetate excretion in urine is a promising new biomarker of chicken intake showing dose-response and robustness. No robust markers have been recognized so far to specifically assess red meat intake. BFIs able to discriminate between red and processed meats are also lacking. For fried and grilled meats, MeIQx in hydrolyzed urine and PhIP in hair are candidate biomarkers but need further work for their full validation. Additional studies on BFI discovery and validation are therefore needed before we can reliably assess or quantify intakes of all meat and most meat subgroups, except for aquatic n-3 LCPUFA.