Droplet digital PCR applied to environmental DNA, a promising method to estimate fish population abundance from humic-rich aquatic ecosystems

Measures of environmental DNA (eDNA) concentrations in water samples have the potential to be both a cost-efficient and a nondestructive method to estimate fish population abundance. However, the inherent temporal and spatial variability in abiotic and biotic conditions in aquatic systems have been suggested to be a major obstacle to determine relationships between fish eDNA concentrations and fish population abundance. Moreover, once water samples are collected, methodological biases are common, which introduces additional sources of variation to potential relationships between eDNA concentrations and fish population abundance. Here, we evaluate the performance of applying the droplet digital PCR (ddPCR) method to estimate fish population abundance in experimental enclosures. Using large-scale enclosure ecosystems that contain populations of nine-spined stickleback (Pungitius pungitius), we compared the concentrations of fish eDNA (COI mitochondrial region, 134 bp) obtained with the ddPCR method with high precision estimates of fish population abundance (i.e., number of individuals) and biomass. To evaluate the effects of contrasted concentrations of humic substances (potential PCR inhibitors) on the performance of ddPCR assays, we manipulated natural dissolved organic carbon (DOC) concentrations (range 4–11 mg/L) in the enclosures. Additionally, water temperature (+2°C) was manipulated in half of the enclosures. Results showed positive relationships between eDNA concentration and fish abundance and biomass estimates although unexplained variation remained. Still and importantly, fish eDNA estimates from high DOC enclosures were not lowered by potential inhibitory effects with our procedure. Finally, water temperature (although only 2°C difference) was neither detected as a significant factor influencing fish eDNA estimates. Altogether, our work highlights that ddPCR-based eDNA is a promising method for future quantification of fish population abundance in natural systems

15-Ketodihydro-PGF2α, Progesterone and Cortisol Profiles in Heifers after Induction of Parturition by Injection of Dexamethasone

In order to study rapid changes in 15-ketodihydro-PGF2α, cortisol and progesterone in the period preceding parturition in cattle, pre-term parturition was induced in 4 late pregnant heifers. Parturitions were induced by 2 intramuscular injections of 20 mg dexamethasone with a 24-h interval. The first injection was made on days 254, 258, 264 and 265 in gestation, respectively. Twenty-four h before the first injection an intravenous polyurethane cannula was inserted. Blood samples were collected at least every hour until 12 h after parturition and during the second stage of labour at least 6 times per hour. Plasma was analysed for 15-ketodihydro-PGF2α and progesterone by radioimmunoassays, and for cortisol by an ELISA. The average time from injection to parturition was 7.7 (6.6–8.9) days (mean (range)). Two of the heifers had retained foetal membranes (RFM). At the start of the experiment the levels of PGF2α metabolite were low (< 300 pmol/L) and increased slowly to levels between 1000 and 2000 pmol/L at one day before parturition. During the last day, however, the levels increased rapidly and the highest levels (>10000 pmol/L) were reached at the time of delivery. No pulsatile release was seen. Immediately after foetal expulsion the PG-metabolite levels decreased rapidly in all animals. In the 2 animals with RFM, however, this decline ceased within a few h. The PG-metabolite levels in these animals then started to increase and reached levels as high as during parturition. Luteolysis occurred between 1.6 and 0.4 days before parturition in all animals. The cortisol profile showed a distinct peak at the time of parturition in the RFM heifers. This peak was absent in the non-RFM heifers. This study shows that the PGF2α release at prepartal luteolysis and parturition is not pulsatile in cattle and that cortisol profiles in heifers with retained foetal membranes might differ from the profiles in non-RFM heifers at the time of parturition

Pharmacokinetics and Pharmacodynamic Effects of Flunixin after Intravenous, Intramuscular and Oral Administration to Dairy Goats

The pharmacokinetics and the prostaglandin (PG) synthesis inhibiting effect of flunixin were determined in 6 Norwegian dairy goats. The dose was 2.2 mg/kg body weight administered by intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) routes using a cross-over design. Plasma flunixin content was analysed by use of liquid chromatography and the PG synthesis was evaluated by measuring plasma 15-ketodihydro-PGF(2α )by a radioimmuno-assay. Results are presented as median (range). The elimination half-lives (t(1/2·λ)) were 3.6 (2.0–5.0), 3.4 (2.6–6.8) and 4.3 (3.4–6.1) h for i.v., i.m. and p.o. administration, respectively. Volume of distribution at steady state (Vd(ss)) was 0.35 (0.23–0.41) L/kg and clearance (CL), 110 (60–160) mL/h/kg. The plasma concentrations after oral administration showed a double-peak phenomenon with the two peaks occurring at 0.37 (0.25–1) and 3.5 (2.5–5.0) h, respectively. Both peaks were in the same order of magnitude. Bioavailability was 79 (53–112) and 58 (35%–120)% for i.m. and p.o. administration, respectively. 15-Ketodihydro-PGF(2α )plasma concentrations decreased after flunixin administration independent of the route of administration

Droplet digital PCR assays for the quantification of brown trout (Salmo trutta) and Arctic char (Salvelinus alpinus) from environmental DNA collected in the water of mountain lakes

Classical methods for estimating the abundance of fish populations are often both expensive, time-consuming and destructive. Analyses of the environmental DNA (eDNA) present in water samples could alleviate such constraints. Here, we developed protocols to detect and quantify brown trout (Salmo trutta) and Arctic char (Salvelinus alpinus) populations by applying the droplet digital PCR (ddPCR) method to eDNA molecules extracted from water samples collected in 28 Swedish mountain lakes. Overall, contemporary fish CPUE (catch per unit effort) estimates from standardized survey gill nettings were not correlated to eDNA concentrations for either of the species. In addition, the measured environmental variables (e.g. dissolved organic carbon concentrations, temperature, and pH) appear to not influence water eDNA concentrations of the studied fish species. Detection probabilities via eDNA analysis showed moderate success (less than 70% for both species) while the presence of eDNA from Arctic char (in six lakes) and brown trout (in one lake) was also indicated in lakes where the species were not detected with the gillnetting method. Such findings highlight the limits of one or both methods to reliably detect fish species presence in natural systems. Additional analysis showed that the filtration of water samples through 1.2 mu m glass fiber filters and 0.45 mu m mixed cellulose ester filters was more efficient in recovering DNA than using 0.22 mu m enclosed polyethersulfone filters, probably due to differential efficiencies of DNA extraction. Altogether, this work showed the potentials and limits of the approach for the detection and the quantification of fish abundance in natural systems while providing new insights in the application of the ddPCR method applied to environmental DNA

Gene diversity and demographic turnover in central and peripheral populations of the perennial herb Gypsophila fastigiata

Within-population gene diversity (H-S) was estimated (using allozyme markers) for 16 populations of the perennial, outcrossing plant, Gypsophila fastigiata, on the Baltic island of Oland. The populations were characterized by data on extent, density, life-stages, and habitat diversity. Populations were classed as central or peripheral in relation to the distribution of ''alvar" (habitats with shallow, calcareous soils on limestone bedrock) on southern Oland. Three minimal adequate models were used to explain H-S and the proportions of juveniles and dead adults. In the first model, H-S was significantly lower in peripheral populations and there were no significant additional effects of other explanatory variables. The lower diversity in peripheral populations can be explained by a combination of genetic drift (in populations that vary in size in response to habitat fragmentation) and lower levels of interpopulation gene flow than in central populations. In the two life-stage models, peripheral populations had significantly larger proportions of both juveniles and dead adults indicating a greater demographic turnover than in the central populations. There were also significant effects of H-S and species diversity on the proportion of juveniles. The central or peripheral position of populations is the strop est predictor of both within-population gene diversity and life-stage dynamics in Oland G. fastigiata

Addition of meloxicam to the treatment of clinical mastitis improves subsequent reproductive performance

AbstractA blinded, negative controlled, randomized intervention study was undertaken to test the hypothesis that addition of meloxicam, a nonsteroidal anti-inflammatory drug, to antimicrobial treatment of mild to moderate clinical mastitis would improve fertility and reduce the risk of removal from the herd. Cows (n=509) from 61 herds in 8 regions (sites) in 6 European countries were enrolled. Following herd-owner diagnosis of mild to moderate clinical mastitis within the first 120d of lactation in a single gland, the rectal temperature, milk appearance, and California Mastitis Test score were assessed. Cows were randomly assigned within each site to be treated either with meloxicam or a placebo (control). All cows were additionally treated with 1 to 4 intramammary infusions of cephalexin and kanamycin at 24-h intervals. Prior to treatment and at 14 and 21d posttreatment, milk samples were collected for bacteriology and somatic cell count. Cows were bred by artificial insemination and pregnancy status was subsequently defined. General estimating equations were used to determine the effect of treatment (meloxicam versus control) on bacteriological cure, somatic cell count, the probability of being inseminated by 21d after the voluntary waiting period, the probability of conception to first artificial insemination, the number of artificial insemination/conception, the probability of pregnancy by 120 or 200d postcalving, and the risk of removal by 300d after treatment. Cox’s proportional hazards models were used to test the effect of treatment on the calving to first insemination and calving to conception intervals. Groups did not differ in terms of age, clot score, California Mastitis Test score, rectal temperature, number of antimicrobial treatments given or bacteria present at the time of enrollment, but cows treated with meloxicam had greater days in milk at enrollment. Cows treated with meloxicam had a higher bacteriological cure proportion than those treated with the placebo [0.66 (standard error=0.04) versus 0.50 (standard error=0.06), respectively], although the proportion of glands from which no bacteria were isolated posttreatment did not differ between groups. No difference was observed in the somatic cell count between groups pre- or posttreatment. The proportion of cows that underwent artificial insemination by 21d after the voluntary waiting period was unaffected by treatment. Treatment with meloxicam was associated with a higher proportion of cows conceiving to their first artificial insemination (0.31 versus 0.21), and a higher proportion of meloxicam-treated cows were pregnant by 120d after calving (0.40 versus 0.31). The number of artificial inseminations required to achieve conception was lower in the meloxicam compared with control cows (2.43 versus 2.92). No difference was observed between groups in the proportion of cows pregnant by 200d after calving or in the proportion of cows that were culled, died, or sold by 300d after calving (17% versus 21% for meloxicam versus control, respectively). It was concluded that use of meloxicam, in conjunction with antimicrobial therapy, for mild to moderate cases of clinical mastitis, resulted in a higher probability of bacteriological cure, an increased probability of conception to first artificial insemination, fewer artificial inseminations, and a greater proportion of cows pregnant by 120d in milk

Identification of nine sequence types of the 16S rRNA genes of Campylobacter jejuni subsp. jejuni isolated from broilers

<p>Abstract</p> <p>Background</p> <p>Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. <it>Campylobacter jejuni </it>is the <it>Campylobacter </it>sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of <it>C. jejuni </it>and <it>C. coli </it>and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of <it>Sma</it>I restricted genomic DNA of the strains.</p> <p>Methods</p> <p>The 16S rRNA genes of 45 strains of <it>C. jejuni </it>and two <it>C. coli </it>strains isolated from broilers were sequenced and compared with 16S rRNA sequences retrieved from the Ribosomal Database Project or GenBank. The strains were also genotyped by PFGE after digestion with <it>Sma</it>I.</p> <p>Results</p> <p>Sequence analyses of the 16S rRNA genes revealed nine sequence types of the <it>Campylobacter </it>strains and the similarities between the different sequence types were in the range 99.6–99.9%. The number of nucleotide substitutions varied between one and six among the nine 16S rRNA sequence types. One of the nine 16S rRNA sequence profiles was common to 12 of the strains from our study and two of these were identified as <it>Campylobacter coli </it>by PCR/REA. The other 10 strains were identified as <it>Campylobacter jejuni</it>. Five of the nine sequence types were also found among the <it>Campylobacter </it>sequences deposited in GenBank. The three 16S rRNA genes in the analysed strains were identical within each individual strain for all 47 strains.</p> <p>Conclusion</p> <p><it>C. jejuni </it>and <it>C. coli </it>seem to lack polymorphisms in their 16S rRNA gene, but phylogenetic analysis based on 16S rRNA sequences was not always sufficient for differentiation between <it>C. jejuni </it>and <it>C. coli</it>. The strains were grouped in two major clusters according to 16S rRNA, one cluster with only <it>C. jejuni </it>and the other with both <it>C. jejuni </it>and <it>C. coli</it>. Genotyping of the 47 strains by PFGE after digestion with <it>Sma</it>I resulted in 22 subtypes. A potential correlation was found between the <it>Sma</it>I profiles and the 16S rRNA sequences, as a certain <it>Sma</it>I type only appeared in one of the two major phylogenetic groups.</p