Functional and evolutionary analysis of host Synaptogyrin-2 in porcine circovirus type 2 susceptibility.

Mammalian evolution has been influenced by viruses for millions of years, leaving signatures of adaptive evolution within genes encoding for viral interacting proteins. Synaptogyrin-2 (SYNGR2) is a transmembrane protein implicated in promoting bacterial and viral infections. A genome-wide association study of pigs experimentally infected with porcine circovirus type 2b (PCV2b) uncovered a missense mutation (SYNGR2 p.Arg63Cys) associated with viral load. In this study, CRISPR/Cas9-mediated gene editing of the porcine kidney 15 (PK15, wtSYNGR2+p.63Arg) cell line generated clones homozygous for the favorable SYNGR2 p.63Cys allele (emSYNGR2+p.63Cys). Infection of edited clones resulted in decreased PCV2 replication compared to wildtype PK15 (P<0.05), with consistent effects across genetically distinct PCV2b and PCV2d isolates. Sequence analyses of wild and domestic pigs (n>700) revealed the favorable SYNGR2 p.63Cys allele is unique to domestic pigs and more predominant in European than Asian breeds. A haplotype defined by the SYNGR2 p.63Cys allele was likely derived from an ancestral haplotype nearly fixed within European (0.977) but absent from Asian wild boar. We hypothesize that the SYNGR2 p.63Cys allele arose post-domestication in ancestral European swine. Decreased genetic diversity in homozygotes for the SYNGR2 p.63Cys allele compared to SYNGR2 p.63Arg, corroborates a rapid increase in frequency of SYGNR2 p.63Cys via positive selection. Signatures of adaptive evolution across mammalian species were also identified within SYNGR2 intraluminal loop domains, coinciding with the location of SYNGR2 p.Arg63Cys. Therefore, SYNGR2 may reflect a novel component of the host-virus evolutionary arms race across mammals with SYNGR2 p.Arg63Cys representing a species-specific example of putative adaptive evolution.

Safe and just Earth system boundaries.

The stability and resilience of the Earth system and human well-being are inseparably linked1-3, yet their interdependencies are generally under-recognized; consequently, they are often treated independently4,5. Here, we use modelling and literature assessment to quantify safe and just Earth system boundaries (ESBs) for climate, the biosphere, water and nutrient cycles, and aerosols at global and subglobal scales. We propose ESBs for maintaining the resilience and stability of the Earth system (safe ESBs) and minimizing exposure to significant harm to humans from Earth system change (a necessary but not sufficient condition for justice)4. The stricter of the safe or just boundaries sets the integrated safe and just ESB. Our findings show that justice considerations constrain the integrated ESBs more than safety considerations for climate and atmospheric aerosol loading. Seven of eight globally quantified safe and just ESBs and at least two regional safe and just ESBs in over half of global land area are already exceeded. We propose that our assessment provides a quantitative foundation for safeguarding the global commons for all people now and into the future.

Host-genetic-based outcome of co-infection by PCV2b and PRRSV in pigs.

Replication of porcine circovirus type 2 (PCV2), an important worldwide swine pathogen, has been demonstrated to be influenced by host genotype. Specifically, a missense DNA polymorphism (SYNGR2 p.Arg63Cys) within the SYNGR2 gene was demonstrated to contribute to variation in PCV2b viral load and subsequent immune response following infection. PCV2 is known to induce immunosuppression leading to an increase in susceptibility to subsequent infections with other viral pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). In order to assess the role of SYNGR2 p.Arg63Cys in co-infections, pigs homozygous for the favorable SYNGR2 p.63Cys (N = 30) and unfavorable SYNGR2 p.63Arg (N = 29) alleles were infected with PCV2b followed a week later by a challenge with PRRSV. A lower PCV2b viremia (P < 0.001) and PCV2-specific IgM antibodies (P < 0.005) were observed in SYNGR2 p.63Cys compared to SYNGR2 p.63Arg genotypes. No significant differences in PRRSV viremia and specific IgG antibodies were observed between SYNGR2 genotypes. Lung histology score, an indicator of disease severity, was lower in the pigs with SYNGR2 p.63Cys genotypes (P < 0.05). Variation in the lung histology scores within SYNGR2 genotypes suggests that additional factors, environmental and/or genetic, could be involved in disease severity.

Porcine circovirus type 2 (PCV2) is an important virus involved in the onset of a group of severe disease symptoms commonly known as porcine circovirus associated diseases (PCVAD). Vaccination options exist for PCV2, though the severity of PCVAD can be influenced by the presence of additional co-infecting pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), for which vaccination is still a challenge. Host genetic resistance is a potential avenue for solving this problem. Previously, a genetic polymorphism in the SYNGR2 gene was found to be associated with PCV2b viremia and immune response. The aim of this study was to determine the impact of this polymorphism in pigs experimentally co-infected with PCV2b and PRRSV. Pigs were weighed, and blood was collected at various days following infection to measure viremia and antibodies. Histological analysis was performed at the experiment completion to assess disease severity in lungs and lymph nodes. The results showed that variation within the SYNGR2 gene is involved in PCV2b disease progression including lung histology scores, but no evidence was seen in response to PRRSV infection.

DNA methylation profile in beef cattle is influenced by additive genetics and age.

DNA methylation (DNAm) has been considered a promising indicator of biological age in mammals and could be useful to increase the accuracy of phenotypic prediction in livestock. The objectives of this study were to estimate the heritability and age effects of site-specific DNAm (DNAm level) and cumulative DNAm across all sites (DNAm load) in beef cattle. Blood samples were collected from cows ranging from 217 to 3,192 days (0.6 to 8.7 years) of age (n = 136). All animals were genotyped, and DNAm was obtained using the Infinium array HorvathMammalMethylChip40. Genetic parameters for DNAm were obtained from an animal model based on the genomic relationship matrix, including the fixed effects of age and breed composition. Heritability estimates of DNAm levels ranged from 0.18 to 0.72, with a similar average across all regions and chromosomes. Heritability estimate of DNAm load was 0.45. The average age effect on DNAm level varied among genomic regions. The DNAm level across the genome increased with age in the promoter and 5' UTR and decreased in the exonic, intronic, 3' UTR, and intergenic regions. In addition, DNAm level increased with age in regions enriched in CpG and decreased in regions deficient in CpG. Results suggest DNAm profiles are influenced by both genetics and the environmental effect of age in beef cattle.

Genetic diversity and detection of atypical porcine pestivirus infections.

Atypical porcine pestivirus (APPV), an RNA virus member of the Flaviviridae family, has been associated with congenital tremor in newborn piglets. Previously reported quantitative polymerase chain reaction (qPCR)-based assays were unable to detect APPV in novel cases of congenital tremor originated from multiple farms from U.S. Midwest (MW). These assays targeted the viral polyprotein coding genes, which were shown to display substantial variation, with sequence identity ranging from 58.2% to 70.7% among 15 global APPV strains. In contrast, the 5'-untranslated region (5' UTR) was found to have a much higher degree of sequence conservation. In order to obtain the complete 5' UTR of the APPV strains originated from MW, the 5' end of the viral cDNA was obtained by using template switching approach followed by amplification and dideoxy sequencing. Eighty one percent of the 5' UTR was identical across 14 global and 5 MW strains with complete or relatively complete 5' UTR. Notably, some of the most highly conserved 5' UTR segments overlapped with potentially important regions of an internal ribosome entry site (IRES), suggesting their functional role in viral protein translation. A newly designed single qPCR assay, targeting 100% conserved 5' UTR regions across 19 strains, was able to detect APPV in samples of well documented cases of congenital tremor which originated from five MW farm sites (1-18 samples/site). As these fully conserved 5' UTR sequences may have functional importance, we expect that assays targeting this region would broadly detect APPV strains that are diverse in space and time.

Effects of restricting energy during the gilt development period on growth and reproduction of lines differing in lean growth rate: responses in reproductive performance and longevity.

Longevity and reproductive performance are economically important traits in the swine industry that are largely influenced by nutrition and other environmental factors. Reproductive performance and longevity through 4 parities was assessed in gilts of 2 genetic lines developed on ad libitum access to feed or restricted to 75% of ad libitum intake. A total of 661 gilts were used in a 2 × 2 factorial with half of the gilts allocated to an ad libitum diet (AL; n = 330), while the other half were energy restricted by 25% (R; n = 331) from 123 to 235 d of age. All gilts were sired by an industry maternal line. Dams of the gilts were from either a Large White (W) by Landrace (L) industry maternal line or Nebraska Selection Line 45X, producing gilts designated as W × L (n = 355) and L45X (n = 306), respectively. Daily estrus detection began at 140 d of age to obtain age at puberty (AP). Gilts (n = 510) were mated on their second or later estrus, beginning at 240 d of age. Sow weight and backfat were recorded at 110 d of gestation and weaning of each parity. Number of live-born, stillborn, and mummified pigs per litter and piglet birth and weaning weights were recorded through 4 parities. More L45X than W × L and more AL than R gilts reached puberty by 230 d of age (P < 0.01). Dietary treatment did not affect probability to produce parities 1 to 4 or any litter trait analyzed. The L45X females tended to be more likely to produce parities 1 (P < 0.08) and 3 (P < 0.06), while W × L had heavier litters at birth (P < 0.01) and weaning (P = 0.01). Treatment by parity interactions (P < 0.01) existed for weight and backfat prior to farrowing and backfat at weaning, and weight at weaning exhibited a line by treatment by parity interaction (P = 0.04) as R sows had lower weights and backfats in earlier parities, but caught up to AL sows in later parities. A treatment by parity interaction (P < 0.01) was also present for backfat loss from farrowing to weaning as R gilts lost less backfat than AL in parities 1 and 2, but more in parities 3 and 4. No significant differences were detected between lines or treatments for lifetime production traits. The populations of pigs and data presented here provide a framework for a diverse array of further studies. Alternative approaches to restrict energy have been assessed in addition to methods of marker-assisted and genomic selection for improvement of litter size and sow longevity.

Fine mapping genetic variants associated with age at puberty and sow fertility using SowPro90 genotyping array.

Sow fertility traits, such as litter size and the number of lifetime parities produced (reproductive longevity), are economically important. Selection for these traits is difficult because they are lowly heritable and expressed late in life. Age at puberty (AP) is an early indicator of reproductive longevity. Here, we utilized a custom Affymetrix single-nucleotide polymorphisms (SNPs) array (SowPro90) enriched with positional candidate genetic variants for AP and a haplotype-based genome-wide association study to fine map the genetic sources associated with AP and other fertility traits in research (University of Nebraska-Lincoln [UNL]) and commercial sow populations. Five major quantitative trait loci (QTL) located on four Sus scrofa chromosomes (SSC2, SSC7, SSC14, and SSC18) were discovered for AP in the UNL population. Negative correlations (r = -0.96 to -0.10; P < 0.0001) were observed at each QTL between genomic estimated breeding values for AP and reproductive longevity measured as lifetime number of parities (LTNP). Some of the SNPs discovered in the major QTL regions for AP were located in candidate genes with fertility-associated gene ontologies (e.g., P2RX3, NR2F2, OAS1, and PTPN11). These SNPs showed significant (P < 0.05) or suggestive (P < 0.15) associations with AP, reproductive longevity, and litter size traits in the UNL population and litter size traits in the commercial sows. For example, in the UNL population, when the number of favorable alleles of an SNP located in the 3' untranslated region of PTPN11 (SSC14) increased, AP decreased (P < 0.0001), while LTNP increased (P < 0.10). Additionally, a suggestive difference in the observed NR2F2 isoforms usage was hypothesized to be the source of the QTL for puberty onset mapped on SSC7. It will be beneficial to further characterize these candidate SNPs and genes to understand their impact on protein sequence and function, gene expression, splicing process, and how these changes affect the phenotypic variation of fertility traits.

Diet modulates cecum bacterial diversity and physiological phenotypes across the BXD mouse genetic reference population.

The BXD family has become one of the preeminent genetic reference populations to understand the genetic and environmental control of phenotypic variation. Here we evaluate the responses to different levels of fat in the diet using both chow diet (CD, 13-18% fat) and a high-fat diet (HFD, 45-60% fat). We studied cohorts of BXD strains, both inbred parents C57BL/6J and DBA/2J (commonly known as B6 and D2, respectively), as well as B6D2 and D2B6 reciprocal F1 hybrids. The comparative impact of genetic and dietary factors was analyzed by profiling a range of phenotypes, most prominently their cecum bacterial composition. The parents of the BXDs and F1 hybrids express limited differences in terms of weight and body fat gain on CD. In contrast, the strain differences on HFD are substantial for percent body fat, with DBA/2J accumulating 12.5% more fat than C57BL/6J (P < 0.0001). The F1 hybrids born to DBA/2J dams (D2B6F1) have 10.6% more body fat (P < 0.001) than those born to C57BL/6J dams. Sequence analysis of the cecum microbiota reveals important differences in bacterial composition among BXD family members with a substantial shift in composition caused by HFD. Relative to CD, the HFD induces a decline in diversity at the phylum level with a substantial increase in Firmicutes (+13.8%) and a reduction in Actinobacteria (-7.9%). In the majority of BXD strains, the HFD also increases cecal sIgA (P < 0.0001)-an important component of the adaptive immunity response against microbial pathogens. Host genetics modulates variation in cecum bacterial composition at the genus level in CD, with significant quantitative trait loci (QTLs) for Oscillibacter mapped to Chr 3 (18.7-19.2 Mb, LRS = 21.4) and for Bifidobacterium mapped to Chr 6 (89.21-89.37 Mb, LRS = 19.4). Introduction of HFD served as an environmental suppressor of these QTLs due to a reduction in the contribution of both genera (P < 0.001). Relations among liver metabolites and cecum bacterial composition were predominant in CD cohort, but these correlations do not persist following the shift to HFD. Overall, these findings demonstrate the important impact of environmental/dietary manipulation on the relationships between host genetics, gastrointestinal bacterial composition, immunological parameters, and metabolites-knowledge that will help in the understanding of the causal sources of metabolic disorders.

Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality1.

Recently, piglets from a high-health status farm began exhibiting congenital tremors, high preweaning mortality and incidence of splayed legs. Postmortem histological examination identified a small number of scattered white matter vacuoles in the cerebellum and underlying brainstem of affected piglets. Presence of potential viral sources associated with this neurologic condition was initially infirmed using quantitative PCR for atypical porcine pestivirus (APPV), porcine teschovirus, and porcine sapelovirus. Using metagenomic analysis, APPV was identified as the main microbial species in serum obtained from piglets affected by congenital tremor. These piglets had higher preweaning mortality rates (46.4% vs. 15.3%) and incidence of splayed legs (33.0% vs. 0.8 %) compared to unaffected piglets. Piglets affected by congenital tremor had higher viral titer (P < 0.15) and larger birth weights (P < 0.05) compared to normal litter mates. Whole-genome sequencing and genome assembly of the novel APPV strain (MK728876) was carried out using Oxford Nanopore and related bioinformatics pipelines. Phylogenic analysis demonstrated that this strain along with other completely sequenced APPV strains were grouped into 2 clades, both including strains-inducing congenital tremor. Strains appear to cluster based on region but there were still significant differences within regions. Future research needs to address potential underdiagnosis due to genetic diversity but also to understand mode of transmission, variation in virulence, and the role of host genetics in APPV susceptibility.

Genome-wide association analysis for porcine reproductive and respiratory syndrome virus susceptibility traits in two genetic populations of pigs1.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen that continues to threaten swine industry sustainability. The complexity and high genetic diversity of PRRSV has prevented vaccines from conferring adequate protection against disease outbreaks. Genome-wide association analyses of PRRSV experimentally infected pigs representing two genetic lines (n = 174 to 176) revealed two major genomic regions accounting for ~1.2% of the genetic variation in PRRSV-specific antibody level in serum or lung. The major region for serum antibody was mapped to SSC7 near the SLAII complex, which has also been implicated in susceptibility to other swine viral pathogens. Haplotype substitution analysis uncovered potential DQB1 haplotypes associated with divergent effects. A novel major region for lung antibody was mapped to the proximal end of SSC17 with the top SNP overlapping two genes, PRAG1 and LONRF1. Sequencing LONRF1 uncovered polymorphisms within the coding region that may play a role in regulating PRRSV-specific antibody production in lung tissue following PRRSV infection. These data implicate novel host genomic regions (SSC17) that influence PRRSV-specific immune response as well as a common region (SSC7) potentially involved in susceptibility to multiple viral pathogens.

Evaluation of genotype quality parameters for SowPro90, a new genotyping array for swine1.

Understanding early predictors of sow fertility has the potential to improve genomic predictions. A custom SNP array (SowPro90 produced by Affymetrix) was developed to include genetic variants overlapping quantitative trait loci for age at puberty, one of the earliest indicators of sow fertility, as well as variants related to innate and adaptive immunity. The polymorphisms included in the custom genotyping array were identified using multiple genomic approaches including deep genomic and transcriptomic sequencing and genome-wide associations. Animals from research and commercial populations (n = 2,586) were genotyped for 103,476 SNPs included in SowPro90. To assess the quality of data generated, genotype concordance was evaluated between the SowPro90 and Porcine SNP60 BeadArray using a subset of common SNP (n = 44,708) and animals (n = 277). The mean genotype concordance rate per SNP was 98.4%. Differences in distribution of data quality were observed between the platforms indicating the need for platform specific thresholds for quality parameters. The optimal thresholds for SowPro90 (≥97% SNP and ≥93% sample call rate) were obtained by analyzing the data quality distribution and genotype concordance per SNP across platforms. At ≥97% SNP call rate, there were 42,151 SNPs (94.3%) retained with a mean genotype concordance of 98.6% across platforms. Similarly, ≥94% SNPs and ≥85% sample call rates were established as thresholds for Porcine SNP60 BeadArray. At ≥94% SNPs call rate, there were 41,043 SNPs (91.8%) retained with a mean genotype concordance of 98.6% across platforms. Final evaluation of SowPro90 array content (n = 103,476) at ≥97% SNPs and ≥93% sample call rates allowed retention of 89,040 SNPs (86%) for downstream analysis. The findings and strategy for quality control could be helpful in identifying consistent, high-quality genotypes for genomic evaluations, especially when integrating genotype data from different platforms.

Immunohistochemical evaluation of tumor budding in colorectal cancer: an important parameter with prognostic value.

We analyzed 82 patients with colorectal cancer (CRC) [75 patients with mucinous adenocarcinoma (ADK) and seven patients with "signet ring cell" ADK] using multi-cytokeratin (CK) AE1∕AE3 immunohistochemical assay. In order to determine the mucinous nature of some of the lymph node metastases of the mucinous colorectal ADKs studied, Periodic Acid Schiff-Alcian Blue (PAS-AB) histochemical staining was used. The counting results were systematized in the following ranges: 0 budding areas; between 1-4 budding areas; between 5-9 budding areas; and =10 tumor budding (TB) areas. The statistical analysis was performed using the Student's t-test. More than half of the cases of mucinous ADK revealed an increased intensity of TB, whereas in the case of "signet ring cell" ADK, an average intensity of this phenomenon. Mucinous ADKs, which were pT3 staged, showed an increased intensity of TB, and those in pT2 stage demonstrated, in the vast majority of cases, the absence of TB. There was a predominance of TB intensity in the absence of vascular-lymphatic invasion. Our study shows the existence of a concordance between tumor progression, the histological type of CRC, vascular-lymphatic invasion and the phenomenon of TB.

Synaptogyrin-2 influences replication of Porcine circovirus 2.

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (>24 hpi) and supernatant (>48hpi)(P < 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load.

A 16.7 kb deletion in Sipa1l3 is associated with juvenile cataract in mice.

Congenital or juvenile cataract is a disease condition in which opacification of the lenses is present at birth or manifests early in life. It has been attributed to different monogenic factors with a high degree of heterogeneity and is often studied using mouse models. A spontaneous mutation was identified in a mouse line selected for heat loss that influenced lens formation and resulted in juvenile cataracts in mice homozygous for the recessive allele. Genetic dissection of this selection line by combining high-density genotypes and homozygosity mapping uncovered a 906 kb fragment on MMU7 encompassing 21 SNPs split into two groups of consecutive, homozygous segments specific to the cataract phenotype. Haplotype analysis revealed a 197.5 kb segment unique to cataract-affected mice that included a single known transcript consisting of the first 14 exons of Sipa1l3. In this region, we discovered a deletion of 1114 bp at the mRNA level, spanning four coding exons, predicted to produce a truncated Sipa1l3 protein lacking a portion of a Rap-GAP domain and two other potentially vital domains. At the genome level, the deletion consisted of 16,733 bp. Genotyping across different samples confirmed that only affected mice were homozygous for the deletion and normal mice were either heterozygous or homozygous for the wild-type allele. Further studies will be required to determine the impact of the truncated Sipa1l3 domains on eye development.

Tardigrades from Nahuel Huapi National Park (Argentina, South America) with descriptions of two new Macrobiotidae species.

In 31 samples of mosses and lichens collected in the Argentinean province of Río Negro, 657 tardigrades, 53 exuviae and 219 free-laid eggs were found. In total, 20 species were identified: Diphascon chilenense, Dip. mitrense, Echiniscus bigranulatus, Ech. corrugicaudatus, Ech. merokensis merokensis, Ech. testudo, Hebesuncus mollispinus, Hypsibius convergens, Macrobiotus cf. anderssoni, Mac. andinus, Mac. kazmierskii, Mac. patagonicus, Mesobiotus szeptyckii, Mes. pseudoblocki sp. nov., Milnesium argentinum, Mil. beatae, Mil. brachyungue, Mil. granulatum, Mopsechiniscus granulosus, Minibiotus pseudostellarus sp. nov. Of the two new species, Mesobiotus pseudoblocki sp. nov. is most similar to Mes. blocki, but it differs mainly by the lack of dentate lunules, smaller eggs and presence of reticular design on egg processes. Minibiotus pseudostellarus sp. nov. is most similar to Min. constellatus, Min. eichhorni, Min. sidereus or Min. vinciguerrae, but it differs from them by the presence of 'pseudo-star'-shaped pores in the dorsal cuticle instead of fully developed 'stars' and by other morphometric characters.

Joint mouse-human phenome-wide association to test gene function and disease risk.

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ∼5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ∼4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets--by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). We tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.

Two new tardigrade species from Romania (Eutardigrada: Milnesiidae, Macrobiotidae), with some remarks on secondary sex characters in Milnesium dornensis sp. nov.

In two moss and lichen samples collected in Romania, two new eutardigrade species were found. Milnesium dornensis sp. nov. belongs to the granulatum group and differs from most of other species in this group mainly by having a different claw configuration ([3-3]-[3-3]) and by some morphometric characters. Minibiotus diversus sp. nov. is very similar to M. gumersindoi Guil & Guidetti, 2005 and M. weglarskae Michalczyk et al., 2005, but differs from these and other congeners by the cuticular pore arrangement and morphometric characters of both adults and eggs. Males and females of the newly described Milnesium species differ not only by the shape and structure of claws I, but also by other morphometric characters. Males of Milnesium dornensis sp. nov. are smaller, more slender, have shorter papillae and relatively longer claws on legs III-IV.

The first record of Pyxidium tardigradum Van der Land, 1964 (Ciliophora) in Romania.

In three lichen samples collected from eastern part of Romania, three populations of Ramazzottius cf. oberhaeuseri (Doyère, 1840) infested by Pyxidium tardigradum Van der Land 1964 were found. In this short correspondence we present a first record of P. tardigradum in Romania and infestation rates in studied populations according to the different life stages.

Variation in time and magnitude of immune response and viremia in experimental challenges with Porcine circovirus 2b.

BACKGROUND: Porcine circovirus 2 is the primary agent responsible for inducing a group of associated diseases known as Porcine Circovirus Associated Diseases (PCVAD), which can have detrimental effects on production efficiency as well as causing significant mortality. The objective of this study was to evaluate variation in viral replication, immune response and growth across pigs (n = 974) from different crossbred lines. The approach used in this study was experimental infection with a PCV2b strain of pigs at an average of 43 days of age. RESULTS: The sequence of the PCV2b isolate used in the challenge was similar with a cluster of PCV2b isolates known to induce PCVAD and increased mortality rates. The swine leukocyte antigen class II (SLAII) profile of the population was diverse, with nine DQB1 haplotypes being present. Individual viremia and antibody profiles during challenge demonstrate variation in magnitude and time of viral surge and immune response. The correlations between PCV2 specific antibodies and average daily gain (ADG) were relatively low and varied between - 0.14 to 0.08 for IgM and -0.02 and 0.11 for IgG. In contrast, PCV2 viremia was an important driver of ADG decline following infection; a moderate negative correlation was observed between viral load and overall ADG (r = - 0.35, P < 0.001). The pigs with the lowest 10% level of viral load maintained a steady increase in weekly ADG (P < 0.0001) compared to the pigs that had the 10% greatest viral load (P < 0.55). In addition, the highly viremic group expressed higher IgM and IgG starting with d 14 and d 21 respectively, and higher tumor necrosis factor - alpha (TNF-α) at d 21 (P < 0.005), compared to low viremic group. CONCLUSIONS: Molecular sources of the observed differences in viremia and immune response could provide a better understanding of the host factors that influence the development of PCVAD and lead to improved knowledge of swine immunity.

Milnesium berladnicorum sp. n. (Eutardigrada, Apochela, Milnesiidae), a new species of water bear from Romania.

In a lichen sample collected from a tree in Bârlad town (Vaslui County, Romania), a new tardigrade species belonging to the genus Milnesium (granulatum group) was found. Milnesium berladnicorum sp. n. is most similar (in the type of dorsal sculpture) to Milnesium beasleyi Kaczmarek et al., 2012 but differs from it mainly by having a different claw configuration and some morphometric characters. Additionally, the new species differs from other congeners of the granulatum group by the different type of dorsal sculpture, claw configuration and some morphometric characters.