Genetic architecture of gene transcription in two Atlantic salmon (Salmo salar) populations.

Gene expression regulation has an important role in short-term acclimation and long-term adaptation to changing environments. However, the genetic architecture of gene expression has received much less attention than that of traditional phenotypic traits. In this study, we used a 5 × 5 full-factorial breeding design within each of two Atlantic salmon (Salmo salar) populations to characterize the genetic architecture of gene transcription. The two populations (LaHave and Sebago) are being used for reintroduction efforts into Lake Ontario, Canada. We used high-throughput quantitative real-time PCR to measure gene transcription levels for 22 genes in muscle tissue of Atlantic salmon fry. We tested for population differences in gene transcription and partitioned the transcription variance into additive genetic, non-additive genetic and maternal effects within each population. Interestingly, average additive genetic effects for gene transcription were smaller than those reported for traditional phenotypic traits in salmonids, suggesting that the evolutionary potential of gene transcription is lower than that of traditional traits. Contrary to expectations for early life stage traits, maternal effects were small. In general, the LaHave population had higher additive genetic effects for gene transcription than the Sebago population had, indicating that the LaHave fish have a higher adaptive potential to respond to the novel selection pressures associated with reintroduction into a novel environment. This study highlights not only the profound variation in gene transcription possible among salmonid populations but also the among-population variation in the underlying genetic architecture of such traits.

Effects of competition on fitness-related traits.

While interspecific competition is prevalent in natural systems, we do not yet understand how it can influence an individual's phenotype within its lifetime and how this might affect performance. Morphology and swimming performance are two important fitness-related traits in fishes. Both traits are essential in acquiring and defending resources as well as avoiding predation. Here, we examined if interspecific competition could induce changes in morphology and affect the swimming performance of two strains of juvenile Atlantic salmon (Salmo salar). We imposed competitive scenarios on the fish using artificial streams containing different combinations of four interspecific competitors. Exposure to interspecific competitors induced morphological changes over time, through the development of deeper bodies, whereas controls free of interspecific competitors developed more fusiform body shapes. Furthermore, swimming performance was correlated to fusiform morphologies and was weaker for Atlantic salmon in competitive scenarios vs. CONTROLS: This implies that interspecific competition has direct effects on these fitness-related traits in Atlantic salmon. To the best of our knowledge, this is the first time that morphology, an important fitness-related trait linked to swimming performance, has been shown to be negatively impacted through interactions with an interspecific competitor.

Genetic architecture and maternal contributions of early-life survival in lake trout Salvelinus namaycush.

The influences of additive, non-additive and maternal effects on early survival (uneyed embryo survival, eyed embryo survival, alevin survival and overall survival to first feeding) were quantified in lake trout Salvelinus namaycush using a 7 × 7 full-factorial breeding design. Maternal effects followed by non-additive genetic effects explained around one third of the phenotypic variance of the survival traits. Although the amount of additive genetic effects were low (<1%), suggesting a limited potential of the traits to respond to new selection pressures, how maternal and non-additive genetic effects may respond to selection under certain circumstances are discussed.

Predictability of multispecies competitive interactions in three populations of Atlantic salmon Salmo salar.

Juvenile Atlantic salmon Salmo salar from three allopatric populations (LaHave, Sebago and Saint-Jean) were placed into artificial streams with combinations of four non-native salmonids: brown trout Salmo trutta, rainbow trout Oncorhynchus mykiss, Chinook salmon Oncorhynchus tshawytscha and coho salmon Oncorhynchus kisutch. Non-additive effects, as evidenced by lower performance than predicted from weighted summed two-species competition trials, were detected for S. salar fork length (LF ) and mass, but not for survival, condition factor or riffle use. These data support emerging theory on niche overlap and species richness as factors that can lead to non-additive competition effects.

Genetic architecture of survival and fitness-related traits in two populations of Atlantic salmon.

The additive genetic effects of traits can be used to predict evolutionary trajectories, such as responses to selection. Non-additive genetic and maternal environmental effects can also change evolutionary trajectories and influence phenotypes, but these effects have received less attention by researchers. We partitioned the phenotypic variance of survival and fitness-related traits into additive genetic, non-additive genetic and maternal environmental effects using a full-factorial breeding design within two allopatric populations of Atlantic salmon (Salmo salar). Maternal environmental effects were large at early life stages, but decreased during development, with non-additive genetic effects being most significant at later juvenile stages (alevin and fry). Non-additive genetic effects were also, on average, larger than additive genetic effects. The populations, generally, did not differ in the trait values or inferred genetic architecture of the traits. Any differences between the populations for trait values could be explained by maternal environmental effects. We discuss whether the similarities in architectures of these populations is the result of natural selection across a common juvenile environment.

Comparison of different RT-qPCR assays for the detection of human and bovine group A rotaviruses and characterization by sequences analysis of genes encoding VP4 and VP7 capsid proteins.

AIMS: The aim of this study was to compare the performance of four RT-qPCR assays for the detection of human and bovine group A rotaviruses and to characterize the positive samples by sequence analysis of VP4 and VP7 genes. METHODS AND RESULTS: RNA extracted from eight human rotavirus strains, and a panel of 33 human and 25 bovine faecal samples was subjected to different RT-qPCR detection systems. Among these assays, only RT-qPCR primers and probe systems B and C were able to detect all human rotavirus strains from cell culture solutions and faecal samples. However, the results showed that the system C was generally more sensitive by one or two logs than the other RT-qPCR assays tested. With the bovine faecal samples, the most efficient RT-qPCR systems were B and A with the detection in 100 and 92% of samples tested, respectively. Human group A rotavirus G1P[8] and bovine G6P[11] were the most frequently used strains identified in this study. A G3P[9] strain, closely related to a feline rotavirus isolated in the USA, was also discovered in a human rotavirus infection. CONCLUSION: The RT-qPCR system B was the only TaqMan assay evaluated in this study able to detect rotavirus RNA in all positive human and bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Utilization of only one RT-qPCR for the detection of human and bovine group A rotaviruses and the possibility of human infection by a feline rotavirus strain.

A systematic review/meta-analysis of primary research investigating swine, pork or pork products as a source of zoonotic hepatitis E virus.

The objectives of our study were to identify and categorize primary research investigating swine/pork as a source of zoonotic hepatitis E virus (HEV) using the relatively new technique of scoping study, and to investigate the potential association between human exposure to swine/pork and HEV infection quantitatively using systematic review/meta-analysis methodology. From 1890 initially identified abstracts, 327 were considered for the review. Five study design types (cross-sectional, prevalence, genotyping, case-report and experimental transmission studies) were identified. A significant association between occupational exposure to swine and human HEV IgG seropositivity was reported in 10/13 cross-sectional studies. The association reported between pork consumption and HEV IgG seropositivity was inconsistent. The quantification of viral load in swine and retail pork, viral load required for infection in primates, cohort and case-control studies in humans, and formal risk assessment are recommended before specific public-health policy actions are taken.

The use of bovine serum albumin to improve the RT-qPCR detection of foodborne viruses rinsed from vegetable surfaces.

AIMS: To demonstrate that produce rinsates used for RT-qPCR detection of foodborne viruses may cause significant PCR inhibition and propose a means to reduce its impact on sensitivity. METHODS AND RESULTS: Here, it is shown that rinsing and concentration from spinach and precut lettuce have the potential to generate RNA extracts that are inhibitory to RT-qPCRs assembled from commercial kits for the detection of norovirus GII (NoV GII), hepatitis A virus (HAV), hepatitis E virus (HEV), rotavirus (RV) and feline calicivirus (FCV) as sample process control. It is further shown that the addition of bovine serum albumin (BSA) to those reactions restored a positive signal in all cases. The effect of BSA was dependent upon the primer/probe combination. Moreover, two of the detection systems (FCV and HAV) strongly benefited from the addition of BSA even in the absence of PCR inhibitors. CONCLUSIONS: BSA was shown to restore positive signals in five different RT-qPCR systems that were otherwise completely inhibited by produce rinsate extracts. It is therefore suggested to consider the addition of BSA to RT-qPCRs for the detection of foodborne viruses when inhibition is observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study clearly demonstrates the potency of PCR inhibitors generated during routine virus concentration from produce and that it can be alleviated by the addition of BSA to the RT-qPCRs. Although used elsewhere, the addition of BSA to PCRs is not a common practice in this growing field of research.

Development of a real-time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus.

AIMS: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. METHODS AND RESULTS: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real-time PCR assay specifically detected bovine and porcine TTV DNA without cross-amplification of other common pathogens. The assay was compared with conventional PCR and nested-PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. CONCLUSIONS: The real-time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri-food continuum.

Correlated Evolution of Female Mating Preferences and Male Color Patterns in the Guppy Poecilia reticulata.

Sexual selection may explain why secondary sexual traits of males are so strongly developed in some species that they seem maladaptive. Female mate choice appears to favor the evolution of conspicuous color patterns in male guppies (Poecilia reticulata) from Trinidad, but color patterns vary strikingly among populations. According to most theory, correlated evolution of female mating preferences and preferred male traits within populations could promote this kind of divergence between populations. But mating preferences could also constrain the evolution of male traits. In some guppy populations, females discriminate among males based on variation in the extent of orange pigment in male color patterns, and populations differ significantly in the degree offemale preferences for orange area. In a comparison ofseven populations, the degree offemale preference based on orange is correlated with the population average orange area. Thus male traits and female preferences appear to be evolving in parallel.

Comparative analysis of different TaqMan real-time RT-PCR assays for the detection of swine Hepatitis E virus and integration of Feline calicivirus as internal control.

AIMS: The aim of this study was to compare the performance of four TaqMan RT-PCR assays with a commonly used nested RT-PCR and to include the Feline calicivirus (FCV) as an internal control. METHODS AND RESULTS: RNA extracted from 87 swine faecal samples and 103 swine blood samples was subjected to different detection systems. Faecal samples naturally contaminated with Hepatitis E virus (HEV) and negative samples were artificially inoculated with 3.2 x 10(3) PFU of FCV. Detection results obtained on faecal and plasma samples were 35.6% and 4.9% with the nested RT-PCR assay, 8.0% and 0%, 0% and 0%, 13.8% and 0% and 36.8% and 3.9% with TaqMan systems A, B, C and D respectively. The Ct means obtained with the multiplex TaqMan assay were 30.11 and 30.43 for the detection of FCV with HEV contaminated samples and negative samples. CONCLUSIONS: The TaqMan system D was more suitable for the detection of swine HEV strains than the three others and FCV was integrated successfully as an internal control. SIGNIFICANCE AND IMPACT OF THE STUDY: FCV was demonstrated as an efficient control to monitor the RNA extraction process and HEV amplification procedure in a multiplex HEV/FCV TaqMan assay. This control would be helpful in limiting false negative results.

Development of a TaqMan real-time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of botrytis leaf blight of onion.

The use of a DNA-based method for quantifying airborne inoculum of Botrytis squamosa, a damaging pathogen of onion, was investigated. A method for purifying DNA from conidia collected using rotating-arm samplers and quantifying it using a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay is described. The sensitivity of the qPCR assay was high, with a detection limit of 2 conidia/rod. A linear relationship between numbers of conidia counted with a compound microscope and those determined with the qPCR assay was obtained. Receiver operating characteristic curve analysis was used to evaluate the reliability of the two methods of conidia quantification (microscope examination and qPCR assay) to predict the risk of disease being below or above a damage threshold (D(th)). In total, 142 field samples from commercial onion fields were analyzed. At damage thresholds of 5 or 10 lesions/leaf, conidia quantification with the qPCR assay was more reliable at predicting disease risk than conidia quantification based on microscope counts. The proportion of decisions where the disease was present and predicted was higher for the qPCR assay than for the microscope counts, with values of 0.95 and 0.89 compared with 0.79 and 0.81 for D(th) of 5 and 10 lesions/leaf, respectively. The proportion of decisions where the disease was present but not predicted was lower for the qPCR assay than for microscope counts, with values of 0.05 and 0.11 compared with 0.20 and 0.19 for D(th) of 5 and 10 lesions/leaf, respectively. The results demonstrated that this new qPCR assay was reliable for quantifying B. squamosa airborne inoculum in commercial onion fields and that molecular conidia quantification could be used as a component of a risk management system for Botrytis leaf blight.

Molecular detection of bovine and porcine Torque teno virus in plasma and feces.

Torque teno virus (TTV) is frequently detected in humans, livestock and some companion animals. Very little is known about presence of TTV in Canadian livestock and the goal of this study was to evaluate the presence of TTV in swine and cattle using molecular tools. TTV DNA was detected and confirmed by sequencing in the plasma of 90.5% and in the feces of 60.3% of the animals tested in a single swine herd as well as 80.9% and 1.1% in the plasma of individuals from general Quebec swine and cattle populations, respectively. The impact of the TTV presence in livestock population for the agri-food chain should be further investigated.

Characterization of swine adiponectin and adiponectin receptor polymorphisms and their association with reproductive traits.

In this study, polymorphisms in genes encoding porcine adiponectin (ADIPOQ) and its receptors (ADIPOR1 and ADIPOR2) were evaluated for associations with reproductive traits in a Landrace sow population. Sixteen SNPs were identified, and among these, associations were found between reproductive traits and five SNPs. Heterozygous multiparous females for SNP ADIPOQEF601160:c.178G>A had fewer stillborn piglets (P < 0.05) and shorter weaning-to-oestrus intervals (P < 0.05). Multiparous females bearing the mutant allele for SNP ADIPOQEF601160:c.*1094_1095insC gave birth to fewer stillborn piglets (P < 0.05). In addition, selection for the ADIPOQ [A;C] haplotype is expected to result in multiparous sows having the lowest number of stillborn piglets and shorter weaning-to-oestrus intervals. In second-parity sows, the polymorphism in ADIPOR1 (AY856513:c.*129A>C) showed significant associations with live-born (P < 0.01) and stillborn (P < 0.05) piglets. In multiparous sows, a significant association was observed for an ADIPOR2 polymorphism (AY856514:c.*112G>A), with the c.*112GA genotype associated with shorter weaning-to-oestrus intervals (P < 0.01). Haplotype analyses of ADIPOR2 SNPs revealed that selection in favour of the [A;C] haplotype and against the [G;G] haplotype may result in sows having an increased number of live-born piglets and shorter weaning-to-oestrus intervals. We have therefore described specific SNPs and haplotypes that are associated with large litter size, fewer stillborn and mummified piglets and shorter weaning-to-oestrus intervals. Selection for these SNPs and haplotypes is a strategy to improve reproductive success in pigs.

Risk Profile of Hepatitis E Virus from Pigs or Pork in Canada.

The role and importance of pigs and pork as sources of zoonotic hepatitis E virus (HEV) has been debated in Canada and abroad for over 20 years. To further investigate this question, we compiled data to populate a risk profile for HEV in pigs or pork in Canada. We organized the risk profile (RP) using the headings prescribed for a foodborne microbial risk assessment and used research synthesis methods and inputs wherever possible in populating the fields of this RP. A scoping review of potential public health risks of HEV, and two Canadian field surveys sampling finisher pigs, and retail pork chops and pork livers, provided inputs to inform this RP. We calculated summary estimates of prevalence using the Comprehensive Meta-analysis 3 software, employing the method of moments. Overall, we found the incidence of sporadic locally acquired hepatitis E in Canada, compiled from peer-reviewed literature or from diagnosis at the National Microbiology Laboratory to be low relative to other non-endemic countries. In contrast, we found the prevalence of detection of HEV RNA in pigs and retail pork livers, to be comparable to that reported in the USA and Europe. We drafted risk categories (high/medium/low) for acquiring clinical hepatitis E from exposure to pigs or pork in Canada and hypothesize that the proportion of the Canadian population at high risk from either exposure is relatively small.

Factors Affecting Detection of Hepatitis E Virus on Canadian Retail Pork Chops and Pork Livers Assayed Using Real-Time RT-PCR.

We collected 599 Canadian retail pork chops and 283 pork livers routinely (usually weekly) from April 2011 to March 2012 using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) retail sampling platform. Samples were assayed using validated real-time (q) reverse transcriptase polymerase chain reaction (RT-PCR) and nested classical RT-PCR for the detection of hepatitis E virus (HEV), porcine enteric calicivirus (PEC) and rotavirus (RV). The presence of Escherichia coli, Salmonella spp. and Campylobacter spp. was measured on a subset of our samples. Exact logistic regression models were fitted for predictors for HEV detection, for each assay. For both assays, sample type (pork chop versus liver) was a significant predictor for HEV RNA detection. For nested classical RT-PCR but not qRT-PCR, region of sample collection was a significant predictor (P = 0.008) of HEV detection. Odds of HEV detection were greatest in spring relative to other seasons. E. coli was a significant predictor for HEV RNA detection using the qRT-PCR (P = 0.03). Overall, the prevalence of E. coli, Salmonella spp. and Campylobacter spp. was significantly greater than HEV, PEC or RV on our retail pork samples. Our sparse data set for the detection of PEC and RV precluded modelling of risk factors for the detection of these viruses.

Cloning, developmental expression, and immunohistochemistry of cyclooxygenase 2 in the endometrium during embryo implantation and gestation in the mink (Mustela vison).

Cyclooxygenase (COX) is the first rate-limiting enzyme in the biosynthesis of PGs. There are two isoforms, COX-1, a constitutive enzyme and COX-2, the induced form, products of two different genes. In this study, we report COX-2 complementary DNA cloning, uterine expression, and immunohistochemical localization in the mink uterus during postimplantation gestation. The open reading frame of mink COX-2 contains 1812 nucleotides encoding 604 amino acids. The homologies are 86%, 83%, 83%, 83%, 85%, and 84% in nucleotides and 86%, 87%, 87%, 85%, 86%, and 88% in amino acids with human, mouse, rat, guinea pig, sheep, and rabbit, respectively. All domains associated with biological activity are conserved in the mink. Northern analysis revealed a transcript of 4.2 kb for COX-2 in mink uterus and adrenal. Semiquantitative RT-PCR showed that COX-2 messenger RNA is not present during diapause. The abundance of COX-2 messenger RNA reached its maxima (P < 0.05) on days 3-5 of postimplantation, gradually decreased through day 9, and was not present thereafter. By immunohistochemistry, COX-2 was present in uterine epithelium, stroma, and necks of endometrial glands at sites of implantation. COX-2 expression appears to be induced in the endometrium by the embryo and may play a role in implantation and placentation in the mink.

Adenovirus proteinases: comparison of amino acid sequences and expression of the cloned cDNA in Escherichia coli.

Adenoviruses (Ad) synthesize serine-center endoproteinases (AdEPs) responsible for maturation cleavages within the virus particle. Many questions regarding these enzymes remain unanswered because previous studies utilized crude cells or viral lysates as the enzyme source. Here, we report on the comparison of the amino acid (aa) sequences of several AdEPs and on the expression of the cDNA of the Ad2Ep in Escherichia coli. The AdEPs consist of about 200 aa and their size is around 23 kDa. Among the seven sequences known, 60% of aa were strictly conserved. The usual serine proteinase active site sequence, GDSGG, is absent. The recombinant Ad2EP, produced by an inducible vector as a protein-A fusion product is capable of autocatalytic cleavage, and of cleaving its natural viral substrates as well as foreign proteins. Therefore, other viral proteins or mammalian specific post-translational modifications are not required for enzyme activity.

Sequence of the protease of human subgroup E adenovirus type 4.

A fragment of DNA containing the protease gene and 3' and 5' flanking regions of human adenovirus type 4 (Ad4) has been cloned and sequenced. The gene is located between 59 and 62 map units and codes for a protein of 201 amino acids with a calculated Mr of 22,758. The Ad4 protease has a 72% amino acid homology with the Ad2 protease, the divergence being concentrated in the middle of the molecule. Comparison with other mammalian and bacterial proteases failed to reveal any significant homology and in particular a putative active site. The adenoviral proteases may therefore represent a novel class of enzymes.

Bovine thyrotropin receptor cDNA is characterized by full-length and truncated transcripts.

The complete coding sequence for the bovine thyrotropin (TSH) receptor was derived using a modified PCR cloning strategy. The bovine thyrotropin receptor conforms to the pattern of receptor interacting with membrane-bound G-protein already established in other species for TSH and gonadotropins receptors. The cDNA for the bovine TSH receptor consists of an open reading frame 2289 nucleotides in length, corresponding to a protein of 763 amino acids (estimated molecular mass of 86.4 kDa) which includes a 20 amino acid putative leading signal peptide. The receptor consists of a large NH2-terminal extracellular membrane domain of 417 amino acids with 5 potential N-linked glycosylation sites, a transmembrane domain (265 amino acids) consisting of 7 putative membrane alpha-helix spanning segments, and an intracytoplasmic COOH-terminal domain (82 amino acids). The bovine TSH receptor is one amino acid less than the corresponding sequence in dog, human, rat and mouse. Cysteine residues (n = 22) were conserved when compared with other TSH receptors. Three potential phosphorylation sites were found in the transmembrane domain and the COOH-terminal domain. As with other members of this receptor family, alternative splicing was observed. A transcribed but truncated TSH receptor of 1769 nucleotides was demonstrated, lacking half of the V segment of the transmembrane domain up to the COOH-terminal domain of the full length TSH receptor. Additionally, alternative transcriptional start sites were observed. Northern blot analysis using a probe (1170 bp) spanning part of the extracellular domain up to the first loop of the transmembrane domain showed specific expression in the bovine thyroid gland with major transcripts of 9.3 and 4.3 kb, and a minor transcript of 3.8 kb being detected.