Identification of Susceptibility Genes Underlying Bovine Respiratory Disease in Xinjiang Brown Cattle Based on DNA Methylation.

DNA methylation is a form of epigenetic regulation, having pivotal parts in controlling cellular expansion and expression levels within genes. Although blood DNA methylation has been studied in humans and other species, its prominence in cattle is largely unknown. This study aimed to methodically probe the genomic methylation map of Xinjiang brown (XJB) cattle suffering from bovine respiratory disease (BRD), consequently widening cattle blood methylome ranges. Genome-wide DNA methylation profiling of the XJB blood was investigated through whole-genome bisulfite sequencing (WGBS). Many differentially methylated regions (DMRs) obtained by comparing the cases and controls groups were found within the CG, CHG, and CHH (where H is A, T, or C) sequences (16,765, 7502, and 2656, respectively), encompassing 4334 differentially methylated genes (DMGs). Furthermore, GO/KEGG analyses showed that some DMGs were involved within immune response pathways. Combining WGBS-Seq data and existing RNA-Seq data, we identified 71 significantly differentially methylated (DMGs) and expressed (DEGs) genes (p < 0.05). Next, complementary analyses identified nine DMGs (LTA, STAT3, IKBKG, IRAK1, NOD2, TLR2, TNFRSF1A, and IKBKB) that might be involved in the immune response of XJB cattle infected with respiratory diseases. Although further investigations are needed to confirm their exact implication in the involved immune processes, these genes could potentially be used for a marker-assisted selection of animals resistant to BRD. This study also provides new knowledge regarding epigenetic control for the bovine respiratory immune process.

A timely, user-friendly, and flexible marker-assisted speed congenics method.

Mice are the most widely used mammalian animal model worldwide. Their use presents many advantages, including our ability to manipulate their genome. Unfortunately, transgenic mice often need to be introgressed to transfer the transgene of interest in a specific mouse line. This time-consuming process can be shortened using the speed congenics technique. However, the need for a panel of informative markers to evaluate the proportion of donor and receiver genomes in different individuals produced at each generation hinders the utilisation of speed congenics. In this study, we present 255 microsatellites and 10 RFLPs which can be used in 18 marker panels, allowing the easy and fast introgression of genes of interest from three mouse lines commonly used for transgenesis (C57BL/6, 129/Sv and FVB) to six mouse lines relevant for biomedical research (BALB/c, C3H, DBA/1, DBA/2, SJL and SWR/J). In addition, our markers analysis confirmed a recently described lack of isogeny in well-established inbred mouse lines available from commercial breeders.

Salinity significantly affects intestinal microbiota and gene expression in striped catfish juveniles.

In the present study, juvenile striped catfish (Pangasianodon hypophthalmus), a freshwater fish species, have been chronically exposed to a salinity gradient from freshwater to 20 psu (practical salinity unit) and were sampled at the beginning (D20) and the end (D34) of exposure. The results revealed that the intestinal microbial profile of striped catfish reared in freshwater conditions were dominated by the phyla Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Alpha diversity measures (observed OTUs (operational taxonomic units), Shannon and Faith's PD (phylogenetic diversity)) showed a decreasing pattern as the salinities increased, except for the phylogenetic diversity at D34, which was showing an opposite trend. Furthermore, the beta diversity between groups was significantly different. Vibrio and Akkermansia genera were affected differentially with increasing salinity, the former being increased while the latter was decreased. The genus Sulfurospirillium was found predominantly in fish submitted to salinity treatments. Regarding the host response, the fish intestine likely contributed to osmoregulation by modifying the expression of osmoregulatory genes such as nka1a, nka1b, slc12a1, slc12a2, cftr, and aqp1, especially in fish exposed to 15 and 20 psu. The expression of heat shock proteins (hsp) hsp60, hsp70, and hsp90 was significantly increased in fish reared in 15 and 20 psu. On the other hand, the expression of pattern recognition receptors (PRRs) were inhibited in fish exposed to 20 psu at D20. In conclusion, the fish intestinal microbiota was significantly disrupted in salinities higher than 10 psu and these effects were proportional to the exposure time. In addition, the modifications of intestinal gene expression related to ion exchange and stressful responses may help the fish to adapt hyperosmotic environment. KEY POINTS: • It is the first study to provide detailed information on the gut microbiota of fish using the amplicon sequencing method. • Salinity environment significantly modified the intestinal microbiota of striped catfish. • Intestinal responses may help the fish adapt to hyperosmotic environment.

Salinity affects growth performance, physiology, immune responses and temperature resistance in striped catfish (Pangasianodon hypophthalmus) during its early life stages.

In this study, striped catfish larvae were gradually exposed to the increase of different salinities, and then they reached the levels of 0, 5, 10, 15, and 20 psu after 10 days, followed by heat shock at 39 °C to determine stress tolerance. After the 10-day experiment, the survival rate of fish exposed to the 20 psu treatment was only 28.6 ± 4%, significantly lower than that of the other treatments. The results showed that the osmolality of the whole-body (WB) homogenate was gradually and significantly increased with salinity elevation, except in fish exposed to freshwater and 5 psu treatments, while there were no significant changes in WB Na+/K+-ATPase activity. Digestive enzymatic activities, i.e., pepsin, α-amylase, alkaline phosphatase, and leucine alanine peptidase (leu-ala) generally increased with salinity, but not aminopeptidase and trypsin. Lysozyme and peroxidase activities increased in fish larvae exposed to 15 and 20 psu. These increases proportionally improved growth performance, with the lowest and the highest final weights observed in fish reared at 0 psu (0.08 ± 0.03 g/larvae) and 20 psu (0.11 ± 0.02 g/larvae), respectively, although the average growth recorded at 20 psu could be biased by the high mortality in this group. Occurrence of skeleton deformities, such as in caudal vertebrae and branchiostegal rays, was significantly higher in fish exposed to the higher osmotic conditions (15.0 ± 1.2% and 10.3 ± 2.1% respectively at 0 psu vs. 31.0 ± 2.9% and 49.0 ± 5.6%, respectively at 15 psu). After the 12.5-h heat shock, survival rates significantly differed between treatments with the highest survival observed in fish submitted to 5 psu (68.9%), followed by those exposed to 0 (27%) and 10 (20%) while all fish died at 15 psu. These findings suggest that the striped catfish larvae could be reared in salinity up to 5 to 10 psu with a higher survival and tolerance to thermal stress when compared to fish maintained in freshwater.

Two-dimensional speckle tracking echocardiography in goats: repeatability, variability, and validation of the technique using an exercise test and an experimentally induced acute ischemic cardiomyopathy.

BACKGROUND: Two-dimensional speckle tracking (2DST) technique has been validated in numerous animal species, but neither studies of repeatability nor measurements after exercise or in animals with cardiac disease have been reported in goats. Goats are an attractive candidate for animal models in human cardiology because they are easy to handle and have a body and heart size comparable to that of humans. Therefore, the aim of this study was to validate this technique in goats for further clinical and experimental applications in this species. RESULTS: This study was divided into several steps. First, a standardized echocardiographic protocol was performed and 5 cineloops of a right parasternal short-axis view at papillary muscles level were recorded three times at one-day intervals in ten healthy adult unsedated Saanen goats to test repeatability and variability of 2DST measurements. Then, the same measurements were performed immediately before and after a standardized exercise on treadmill in seven of the goats, and at 24 h after induction of an experimental ischemic cardiomyopathy in five of the goats, to test the reliability of the technique to assess physiological and pathological changes. Average and regional measurements of radial and circumferential strain and strain rate, radial displacement, rotation and rotation rate were obtained. Comparisons were performed using two-way ANOVA (p < 0.05). Caprine 2DST average measurements have demonstrated a good repeatability with a low to moderate variability for all measurements except for the diastolic peaks of the circumferential strain rate, radial strain rate and rotation rate. Segmental 2DST measurements were less repeatable than average measurements. Time effect of two-way ANOVA was significant for anteroseptal segment diastolic peaks measurements, rotation and rotation rate measurements. Overall variability of segmental measurements was moderate or high. Segmental and average peak values obtained after exercise and after myocardial ischemia were significantly different than curves obtained at baseline. CONCLUSIONS: The results of this study are consistent with those previously described in other animal species and humans. 2DST echocardiography is a valid technique to evaluate physiological and pathological changes in myocardial function in goats, despite the technical limitations observed in this species.

Aggregation of experts: an application in the field of "interactomics" (detection of interactions on the basis of genomic data).

BACKGROUND: Despite the successful mapping of genes involved in the determinism of numerous traits, a large part of the genetic variation remains unexplained. A possible explanation is that the simple models used in many studies might not properly fit the actual underlying situations. Consequently, various methods have attempted to deal with the simultaneous mapping of genomic regions, assuming that these regions might interact, leading to a complex determinism for various traits. Despite some successes, no gold standard methodology has emerged. Actually, combining several interaction mapping methods might be a better strategy, leading to positive results over a larger set of situations. Our work is a step in that direction. RESULTS: We first have demonstrated why aggregating results from several distinct methods might increase the statistical power while controlling the type I error. We have illustrated the approach using 6 existing methods (namely: MDR, Boost, BHIT, KNN-MDR, MegaSNPHunter and AntEpiSeeker) on simulated and real data sets. We have used a very simple aggregation strategy: a majority vote across the best loci combinations identified by the individual methods. In order to assess the performances of our aggregation approach in problems where most individual methods tend to fail, we have simulated difficult situations where no marginal effects of individual genes exist and where genetic heterogeneity is present. we have also demonstrated the use of the strategy on real data, using a WTCCC dataset on rheumatoid arthritis. Since we have been using simplistic assumptions to infer the expected power of the aggregation method, the actual power we estimated from our simulations has turned out to be a bit smaller than theoretically expected. Results nevertheless have shown that grouping the results of several methods is advantageous in terms of power, accuracy and type I error control. Furthermore, as more methods should become available in the future, using a grouping strategy will become more advantageous since adding more methods seems to improve the performances of the aggregated method. CONCLUSIONS: The aggregation of methods as a tool to detect genetic interactions is a potentially useful addition to the arsenal used in complex traits analyses.

The Major Envelope Glycoprotein of Murid Herpesvirus 4 Promotes Sexual Transmission.

Gammaherpesviruses are important human and animal pathogens. Infection control has proven difficult because the key process of transmission is ill understood. Murid herpesvirus 4 (MuHV-4), a gammaherpesvirus of mice, is transmitted sexually. We show that this depends on the major virion envelope glycoprotein gp150. gp150 is redundant for host entry, and in vitro, it regulates rather than promotes cell binding. We show that gp150-deficient MuHV-4 reaches and replicates normally in the female genital tract after nasal infection but is poorly released from vaginal epithelial cells and fails to pass from the female to the male genital tract during sexual contact. Thus, we show that the regulation of virion binding is a key component of spontaneous gammaherpesvirus transmission.IMPORTANCE Gammaherpesviruses are responsible for many important diseases in both animals and humans. Some important aspects of their life cycle are still poorly understood. Key among these is viral transmission. Here we show that the major envelope glycoprotein of murid herpesvirus 4 functions not in entry or dissemination but in virion release to allow sexual transmission to new hosts.

Altered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy.

Polysaccharide storage myopathy (PSSM) is a widely described cause of exertional rhabdomyolysis in horses. Mitochondria play a central role in cellular energetics and are involved in human glycogen storage diseases but their role has been overlooked in equine PSSM. We hypothesized that the mitochondrial function is impaired in the myofibers of PSSM-affected horses. Nine horses with a history of recurrent exercise-associated rhabdomyolysis were tested for the glycogen synthase 1 gene (GYS1) mutation: 5 were tested positive (PSSM group) and 4 were tested negative (horses suffering from rhabdomyolysis of unknown origin, RUO group). Microbiopsies were collected from the gluteus medius (gm) and triceps brachii (tb) muscles of PSSM, RUO and healthy controls (HC) horses and used for histological analysis and for assessment of oxidative phosphorylation (OXPHOS) using high-resolution respirometry. The modification of mitochondrial respiration between HC, PSSM and RUO horses varied according to the muscle and to substrates feeding OXPHOS. In particular, compared to HC horses, the gm muscle of PSSM horses showed decreased OXPHOS- and electron transfer (ET)-capacities in presence of glutamate&malate&succinate. RUO horses showed a higher OXPHOS-capacity (with glutamate&malate) and ET-capacity (with glutamate&malate&succinate) in both muscles in comparison to the PSSM group. When expressed as ratios, our results highlighted a higher contribution of the NADH pathway (feeding electrons into Complex I) to maximal OXPHOS or ET-capacity in both rhabdomyolysis groups compared to the HC. Specific modifications in mitochondrial function might contribute to the pathogenesis of PSSM and of other types of exertional rhabdomyolyses.

Influence of a single dose of buprenorphine on rabbit (Oryctolagus cuniculus) gastrointestinal motility.

OBJECTIVE: To establish a noninvasive imaging protocol for rabbit gastrointestinal transit evaluation. To assess the effect of a single injection of buprenorphine on the digestive transit of rabbits via this new technique. STUDY DESIGN: Prospective, parallel study. ANIMALS: Fifteen specific pathogen-free male New Zealand White rabbits weighing 2.68 ± 0.28 kg. METHODS: A 10 mL kg-1 barium meal was administered and the rabbits were subjected to serial radiographic and ultrasound examinations without treatment and 1 week later following a single intramuscular dose of 100 µg kg-1 of buprenorphine. Radiographic data from the stomach and caecum were collected and assigned a retention score ranging from 0 (no barium) to 3 (large amount of barium). The resulting scores and pyloric and duodenal contraction counts were analysed using a mixed linear model and are expressed as least square mean (lsm) ± standard error. Transit was estimated based on the apparition time of faeces in the pelvic area and analysed using a Wilcoxon test. A p < 0.05 was considered significant. RESULTS: Buprenorphine treatment induced a higher lsm number of pyloric (1.73 ± 0.19 versus 0.78 ± 0.19, p < 0.01) and lsm duodenal contractions (17.35 ± 1.04 versus 13.44 ± 1.04, p < 0.01). Buprenorphine administration decreased the lsm barium retention score in the stomach (2.44 ± 0.05 versus 2.64 ± 0.05, p < 0.01), but had no effect on the lsm barium retention score in the caecum. The time to apparition of faeces in the pelvic area was not influenced by buprenorphine administration (p = 0.66). CONCLUSIONS AND CLINICAL RELEVANCE: A single high dose of buprenorphine appears to have no adverse effect on gastrointestinal motility in healthy rabbits.

KNN-MDR: a learning approach for improving interactions mapping performances in genome wide association studies.

BACKGROUND: Finding epistatic interactions in large association studies like genome-wide association studies (GWAS) with the nowadays-available large volume of genomic data is a challenging and largely unsolved issue. Few previous studies could handle genome-wide data due to the intractable difficulties met in searching a combinatorial explosive search space and statistically evaluating epistatic interactions given a limited number of samples. Our work is a contribution to this field. We propose a novel approach combining K-Nearest Neighbors (KNN) and Multi Dimensional Reduction (MDR) methods for detecting gene-gene interactions as a possible alternative to existing algorithms, e especially in situations where the number of involved determinants is high. After describing the approach, a comparison of our method (KNN-MDR) to a set of the other most performing methods (i.e., MDR, BOOST, BHIT, MegaSNPHunter and AntEpiSeeker) is carried on to detect interactions using simulated data as well as real genome-wide data. RESULTS: Experimental results on both simulated data and real genome-wide data show that KNN-MDR has interesting properties in terms of accuracy and power, and that, in many cases, it significantly outperforms its recent competitors. CONCLUSIONS: The presented methodology (KNN-MDR) is valuable in the context of loci and interactions mapping and can be seen as an interesting addition to the arsenal used in complex traits analyses.

Moku Virus in Invasive Asian Hornets, Belgium, 2016.

We report the detection of Moku virus in invasive Asian hornets (Vespa velutina nigrithorax) in Belgium. This constitutes an unexpected report of this iflavirus outside Hawaii, USA, where it was recently described in social wasps. Although virulence of Moku virus is unknown, its potential spread raises concern for European honeybee populations.

Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging.

Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.

Age-based partitioning of individual genomic inbreeding levels in Belgian Blue cattle.

BACKGROUND: Inbreeding coefficients can be estimated either from pedigree data or from genomic data, and with genomic data, they are either global or local (when the linkage map is used). Recently, we developed a new hidden Markov model (HMM) that estimates probabilities of homozygosity-by-descent (HBD) at each marker position and automatically partitions autozygosity in multiple age-related classes (based on the length of HBD segments). Our objectives were to: (1) characterize inbreeding with our model in an intensively selected population such as the Belgian Blue Beef (BBB) cattle breed; (2) compare the properties of the model at different marker densities; and (3) compare our model with other methods. RESULTS: When using 600 K single nucleotide polymorphisms (SNPs), the inbreeding coefficient (probability of sampling an HBD locus in an individual) was on average 0.303 (ranging from 0.258 to 0.375). HBD-classes associated to historical ancestors (with small segments ≤ 200 kb) accounted for 21.6% of the genome length (71.4% of the total length of the genome in HBD segments), whereas classes associated to more recent ancestors accounted for only 22.6% of the total length of the genome in HBD segments. However, these recent classes presented more individual variation than more ancient classes. Although inbreeding coefficients obtained with low SNP densities (7 and 32 K) were much lower (0.060 and 0.093), they were highly correlated with those obtained at higher density (r = 0.934 and 0.975, respectively), indicating that they captured most of the individual variation. At higher SNP density, smaller HBD segments are identified and, thus, more past generations can be explored. We observed very high correlations between our estimates and those based on homozygosity (r = 0.95) or on runs-of-homozygosity (r = 0.95). As expected, pedigree-based estimates were mainly correlated with recent HBD-classes (r = 0.56). CONCLUSIONS: Although we observed high levels of autozygosity associated with small HBD segments in BBB cattle, recent inbreeding accounted for most of the individual variation. Recent autozygosity can be captured efficiently with low-density SNP arrays and relatively simple models (e.g., two HBD classes). The HMM framework provides local HBD probabilities that are still useful at lower SNP densities.

COMPARISON BETWEEN SEDATION AND GENERAL ANESTHESIA FOR HIGH RESOLUTION COMPUTED TOMOGRAPHIC CHARACTERIZATION OF CANINE IDIOPATHIC PULMONARY FIBROSIS IN WEST HIGHLAND WHITE TERRIERS.

Canine idiopathic pulmonary fibrosis is a progressive interstitial lung disease mainly affecting West Highland white terriers. Thoracic high-resolution computed tomographic (T-HRCT) findings for Canine idiopathic pulmonary fibrosis acquired under general anesthesia have been described previously. However, the use of general anesthesia may be contraindicated for some affected dogs. Sedation may allow improved speed and safety, but it is unknown whether sedation would yield similar results in identification and grading of Canine idiopathic pulmonary fibrosis lesions. The aim of this prospective, observational, method-comparison, case-control study was to compare findings from T-HRCT images acquired under sedation versus general anesthesia for West Highland white terriers affected with Canine idiopathic pulmonary fibrosis (n = 11) and age-matched controls (n = 9), using the glossary of terms of the Fleischner Society and a scoring system. Ground-glass opacity was identified in all affected West Highland white terriers for both sedation and general anesthesia acquisitions, although the Ground-glass opacity extent varied significantly between the two acquisitions (P < 0.001). Ground-glass opacity was the sole lesion observed in control dogs (n = 6), but was less extensive compared with affected West Highland white terriers. Identification and grading of a mosaic attenuation pattern differed significantly between acquisitions (P < 0.001). Identification of lesions such as consolidations, nodules, parenchymal and subpleural bands, bronchial wall thickening, and bronchiectasis did not differ between acquisitions. The present study demonstrated that T-HRCT obtained under sedation may provide different information than T-HRCT obtained under general anesthesia for identification and grading of some Canine idiopathic pulmonary fibrosis lesions, but not all of them. These differences should be taken into consideration when general anesthesia is contraindicated and sedation is necessary for evaluating West Highland white terriers with Canine idiopathic pulmonary fibrosis.

Normal bacterial conjunctival flora in the Huacaya alpaca (Vicugna pacos).

OBJECTIVE: To describe the bacterial flora of the normal conjunctiva of Huacaya alpacas (Vicugna pacos) and to determine the effect of age and gender on this flora. ANIMALS STUDIED: Fifty Huacaya alpacas. PROCEDURES: After a complete ophthalmic examination, conjunctival swabs were obtained from both eyes and cultured for aerobic and anaerobic bacteria. Logistic and Poisson regression analyses were used to evaluate the effect of age and gender on bacterial isolation. RESULTS: Four animals were excluded because of signs of external ocular disease. Of the remaining 46 alpacas, bacteria were recovered from 96.7% (89/92) of the eyes. A total of 190 bacterial isolates were cultured with a mean of 2.1 bacterial isolates per eye. The majority of isolates (70%) were Gram-positive. Staphylococcus xylosus (44/190: 23.2%) predominated, followed by viridans streptococci (32/190: 16.8%) and Pantoea agglomerans (24/190: 12.6%). Other frequently isolated bacteria included Rothia mucilaginosa (12/190: 6.3%), Staphylococcus equorum (12/190: 6.3%), Bacillus species (9/190: 4.7%), Moraxella ovis (9/190: 4.7%), and Moraxella catarrhalis (6/190: 3.2%). Statistical analysis showed that alpacas harboring viridans streptococci and Moraxella species were significantly younger. Gender did not significantly affect type of bacterial isolation. There appeared to be no significant effect of age or gender on number of bacteria isolated. CONCLUSIONS: Gram-positive aerobes were most commonly cultured, with S. xylosus and viridans streptococci predominating. To the best of the authors' knowledge, this is the first report describing the presence of Moraxella species in the healthy conjunctival sac of alpacas. Alpacas harboring viridans streptococci and Moraxella species were significantly younger.

Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri.

In Arabidopsis halleri, the HMA4 gene has an essential function in Zn/Cd hypertolerance and hyperaccumulation by mediating root-to-shoot translocation of metals. Constitutive high expression of AhHMA4 results from a tandem triplication and cis-activation of the promoter of all three copies. The three AhHMA4 copies possess divergent promoter sequences, but highly conserved coding sequences, and display identical expression profiles in the root and shoot vascular system. Here, an AhHMA4::GFP fusion was expressed under the control of each of the three A. halleri HMA4 promoters in a hma2hma4 double mutant of A. thaliana to individually examine the function of each AhHMA4 copy. The protein showed non-polar localization at the plasma membrane of the root pericycle cells of both A. thaliana and A. halleri. The expression of each AhHMA4::GFP copy complemented the severe Zn-deficiency phenotype of the hma2hma4 mutant by restoring root-to-shoot translocation of Zn. However, each copy had a different impact on metal homeostasis in the A. thaliana genetic background: AhHMA4 copies 2 and 3 were more highly expressed and provided higher Zn tolerance in roots and accumulation in shoots than copy 1, and AhHMA4 copy 3 also increased Cd tolerance in roots. These data suggest a certain extent of functional differentiation among the three A. halleri HMA4 copies, stemming from differences in expression levels rather than in expression profile. HMA4 is a key node of the Zn homeostasis network and small changes in expression level can have a major impact on Zn allocation to root or shoot tissues.

Training modifies innate immune responses in blood monocytes and in pulmonary alveolar macrophages.

In humans, strenuous exercise causes increased susceptibility to respiratory infections associated with down-regulated expression of Toll-like receptors (TLRs) and costimulatory and antigen-presenting molecules. Lower airway diseases are also a common problem in sport and racing horses. Because innate immunity plays an essential role in lung defense mechanisms, we assessed the effect of acute exercise and training on innate immune responses in two different compartments. Blood monocytes and pulmonary alveolar macrophages (PAMs) were collected from horses in untrained, moderately trained, intensively trained, and deconditioned states before and after a strenuous exercise test. The cells were analyzed for TLR messenger ribonucleic acid (mRNA) expression by real-time PCR in vitro, and cytokine production after in vitro stimulation with TLR ligands was measured by ELISA. Our results showed that training, but not acute exercise, modified the innate immune responses in both compartments. The mRNA expression of TLR3 was down-regulated by training in both cell types, whereas the expression of TLR4 was up-regulated in monocytes. Monocytes treated with LPS and a synthetic diacylated lipoprotein showed increased cytokine secretion in trained and deconditioned subjects, indicating the activation of cells at the systemic level. The production of TNF-α and IFN-ß in nonstimulated and stimulated PAMs was decreased in trained and deconditioned horses and might therefore explain the increased susceptibility to respiratory infections. Our study reports a dissociation between the systemic and the lung response to training that is probably implicated in the systemic inflammation and in the pulmonary susceptibility to infection.

Sensitivity and permissivity of Cyprinus carpio to cyprinid herpesvirus 3 during the early stages of its development: importance of the epidermal mucus as an innate immune barrier.

Cyprinid herpesvirus 3 (CyHV-3) causes a lethal disease in common and koi carp (Cyprinus carpio). The present study investigated the ability of CyHV-3 to infect common carp during the early stages of its development (from embryos to fingerlings) after inoculation by immersion in water containing the virus. Fish were inoculated at different times after hatching with a pathogenic recombinant CyHV-3 strain expressing luciferase. The sensitivity and permissivity of carp to CyHV-3 were investigated using in vivo bioluminescence imaging. The susceptibility of carp to CyHV-3 disease was investigated by measuring the survival rate. Carp were sensitive and permissive to CyHV-3 infection and susceptible to CyHV-3 disease at all stages of development, but the sensitivity of the two early developmental stages (embryo and larval stages) was limited compared to later stages. The lower sensitivity observed for the early developmental stages was due to stronger inhibition of viral entry into the host by epidermal mucus. In addition, independent of the developmental stage at which inoculation was performed, the localization of light emission suggested that the skin is the portal of CyHV-3 entry. Taken together, the results of the present study demonstrate that carp are sensitive and permissive to CyHV-3 at all stages of development and confirm that the skin is the major portal of entry after inoculation by immersion in infectious water. The results also stress the role of epidermal mucus as an innate immune barrier against pathogens even and especially at the early stages of development.

Assessment of Toll-like receptor 2, 4 and 9 SNP genotypes in canine sino-nasal aspergillosis.

BACKGROUND: The exact aetiology of canine sino-nasal aspergillosis (SNA) is unknown. In man, dysfunction in innate immunity, particularly in the function of pattern recognition receptors, is implicated in the pathogenesis of inflammatory sino-nasal disease and in fungal diseases. Associations between single nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) and these diseases have been identified. Similarly, in dogs SNPs in genes encoding TLRs may be important in the pathogenesis of SNA. The aims of the present study were (1) to identify the presence of non-synonymous SNPs in the coding regions of the TLR2, 4 and 9 genes in dogs suffering from SNA, and (2) to investigate the SNP genotypes in dogs with SNA compared with a control population. RESULTS: Direct sequencing of nine dogs of various breeds with SNA revealed two non-synonymous SNPs in the coding region of TLR2, eight in TLR4 and four in TLR9. These non-synonymous SNPs were further evaluated in a case-control study of affected Golden Retrievers, Labrador Retrievers, Rottweilers and Beaucerons. Genotyping was performed using a combination of allele-specific primers and hydrolysis probe assays in 31 dogs with SNA and 31 controls. No significant difference in minor allele frequency was identified between these groups, for all studied SNPs, in any of the four breeds. CONCLUSIONS: These findings do not support a role for non-synonymous SNPs in the TLR 2, 4 and 9 coding regions in the pathogenesis of canine SNA, but do not exclude a role for innate immunity in the pathogenesis of the disease.