Evaluation of two devices for point-of-care testing of haemoglobin in neonatal pigs

In veterinary medicine, point-of-care testing (POCT) techniques have become popular, since they provide immediate results and only small amounts of blood are needed. However, their accuracy is controversial. Pigs are often used for research purposes and accurate measurement of haemoglobin (Hb) is important during invasive procedures. The aim of this study was to evaluate two different Hb POCT devices in neonatal pigs. A prospective study with 57 pigs of 3-6 weeks of age, weighing 4.1-6.2 kg (median 5.1 kg) was performed. Fifty-seven blood samples were analysed for Hb using a conductivity-based and a photometrical POCT device and compared with a photometrical reference method. Statistical analysis was performed with Bland-Altman analysis, Spearman correlation and Passing-Bablok regression analysis. Hb values ranged from 32 to 108 g/L (median 80 g/L) using the reference method. The bias of the photometrical method (HemoCue(?)) to the reference method was -1 g/L, with limits of agreement (LOA) of -7 to 6 g/L. The conductivity-based method (i-STAT(?)) had a bias of -15 g/L with LOA from -24 to -6 g/L. There was a significant association between protein values and the bias of i-STAT versus CellDyn (r(2) = 0.27, P < 0.05) but not with the bias of HemoCue versus CellDyn (r(2) = 0.001, P = 0.79). The lower the protein values were, the lower the Hb values were measured by the i-STAT. The conductivity-based measurement of Hb constantly underestimated Hb values, whereas the photometrical method demonstrated a better accuracy and is therefore more reliable for on-site measurement of Hb in pigs.     

Mutations in MITF and PAX3 cause "splashed white" and other white spotting phenotypes in horses

During fetal development neural-crest-derived melanoblasts migrate across the entire body surface and differentiate into melanocytes, the pigment-producing cells. Alterations in this precisely regulated process can lead to white spotting patterns. White spotting patterns in horses are a complex trait with a large phenotypic variance ranging from minimal white markings up to completely white horses. The "splashed white" pattern is primarily characterized by an extremely large blaze, often accompanied by extended white markings at the distal limbs and blue eyes. Some, but not all, splashed white horses are deaf. We analyzed a Quarter Horse family segregating for the splashed white coat color. Genome-wide linkage analysis in 31 horses gave a positive LOD score of 1.6 in a region on chromosome 6 containing the PAX3 gene. However, the linkage data were not in agreement with a monogenic inheritance of a single fully penetrant mutation. We sequenced the PAX3 gene and identified a missense mutation in some, but not all, splashed white Quarter Horses. Genome-wide association analysis indicated a potential second signal near MITF. We therefore sequenced the MITF gene and found a 10 bp insertion in the melanocyte-specific promoter. The MITF promoter variant was present in some splashed white Quarter Horses from the studied family, but also in splashed white horses from other horse breeds. Finally, we identified two additional non-synonymous mutations in the MITF gene in unrelated horses with white spotting phenotypes. Thus, several independent mutations in MITF and PAX3 together with known variants in the EDNRB and KIT genes explain a large proportion of horses with the more extreme white spotting phenotypes.     

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.     

Probing Mixed-Genotype Infections I: Extraction and Cloning of Infections from Hosts of the Trypanosomatid Crithidia bombi

We here present an efficient, precise and reliable method to isolate and cultivate healthy and viable single Crithidia bombi cells from bumblebee faeces using flow cytometry. We report a precision of >99% in obtaining single trypanosomatid cells for further culture and analysis (“cloning”). In the study, we have investigated the use of different liquid media to cultivate C. bombi and present an optimal medium for obtaining viable clones from all tested, infected host donors. We show that this method can be applied to genotype a collection of clones from natural infections. Furthermore, we show how to cryo-preserve C. bombi cells to be revived to become infective clones after at least 4 years of storage. Considering the high prevalence of infections in natural populations, our method provides a powerful tool in studying the level and diversity of these infections, and thus enriches the current methodology for the studies of complex host-parasite interactions.     

A Mutation in the SUV39H2 Gene in Labrador Retrievers with Hereditary Nasal Parakeratosis (HNPK) Provides Insights into the Epigenetics of Keratinocyte Differentiation

Hereditary nasal parakeratosis (HNPK), an inherited monogenic autosomal recessive skin disorder, leads to crusts and fissures on the nasal planum of Labrador Retrievers. We performed a genome-wide association study (GWAS) using 13 HNPK cases and 23 controls. We obtained a single strong association signal on chromosome 2 (p_raw = 4.4×10⁻¹⁴). The analysis of shared haplotypes among the 13 cases defined a critical interval of 1.6 Mb with 25 predicted genes. We re-sequenced the genome of one case at 38× coverage and detected 3 non-synonymous variants in the critical interval with respect to the reference genome assembly. We genotyped these variants in larger cohorts of dogs and only one was perfectly associated with the HNPK phenotype in a cohort of more than 500 dogs. This candidate causative variant is a missense variant in the SUV39H2 gene encoding a histone 3 lysine 9 (H3K9) methyltransferase, which mediates chromatin silencing. The variant c.972T>G is predicted to change an evolutionary conserved asparagine into a lysine in the catalytically active domain of the enzyme (p.N324K). We further studied the histopathological alterations in the epidermis in vivo. Our data suggest that the HNPK phenotype is not caused by hyperproliferation, but rather delayed terminal differentiation of keratinocytes. Thus, our data provide evidence that SUV39H2 is involved in the epigenetic regulation of keratinocyte differentiation ensuring proper stratification and tight sealing of the mammalian epidermis.     

Genetic Diversity and Antibiotic Resistance Patterns in a Campylobacter Population Isolated from Poultry Farms in Switzerland

The diversity and genetic interrelation of Campylobacter jejuni and C. coli isolated from Swiss poultry were assessed by three independent typing methods. Samples were derived prior to slaughter from 100 randomly selected flocks (five birds per flock) raised on three different farm types. The observed flock prevalence was 54% in total, with 50% for conventional and 69% for free-range farms. Birds held on farms with a confined roaming area had the lowest prevalence of 37%. Campylobacter isolates were characterized by amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism of flaA PCR fragments (flaA-RFLP), and disk diffusion testing for eight antimicrobial agents that are commonly used in veterinary or human medicine in Switzerland. Analysis of the genotypic results indicates that the Campylobacter population in Swiss poultry is genetically highly diverse. Nevertheless, occasionally, isolates with identical or nearly identical characteristics were isolated from different farms or farm types in different locations. Genetic typing by AFLP and flaA-RFLP was found to be complementary. The majority of isolates (67%) were susceptible to all tested antibiotics; however, single, double, and triple resistances were observed in 7%, 23%, and 2% of the strains, respectively. There was no correlation between genotype and antibiotic resistance. Surprisingly, sulfonamide resistance was frequently found together with streptomycin resistance. Our findings illustrate the results of common genetic exchange in the studied bacterial population.     

Differential and reversible responses of common fen meadow species to abandonment

Abstract. We studied the effects of abandonment on two common fen plant species. In mown and a chronosequence of abandoned fen meadows spanning 35 yr, we measured fitness traits of the sedge Carex davalliana and the forb Succisa pratensis. Cessation of mowing had little effect on fitness traits and seed production of C. davalliana, but seedling density decreased more than threefold. Population density of S. pratensis decreased with increasing community biomass, but was not affected by the cessation of mowing. However, flowering frequency increased threefold and seed production was 20% higher in fallow meadows. Consequently, seedling density of S. pratensis increased nearly threefold after abandonment. However, these changes were not dependent on the age of the fallow. In a common garden and germination experiment, we found no differences in either species between plants from fallows and mown fen meadows, except for the height of the flowering stalk of S. pratensis. The combined results from the common garden experiment and the field studies indicate that changes in fitness traits observed in fallows were mostly phenotypic and likely to be reversible. If other species react in similar ways, there is a high potential for re‐establishing traditional fen meadow communities from fallows by mowing.     

Novel microsatellite DNA loci for Bombus terrestris (Linnaeus, 1758)

We present details and characteristics of 123 novel polymorphic microsatellite DNA loci for Bombus terrestris. Thirty-four of these loci have been tested in nine other Bombus species and 25 of them showed polymorphisms in at least one species. These microsatellite DNA loci together with the already established 60 loci will be useful for characterizing wild and managed populations of B. terrestris and other Bombus species as well as for detailed genetic studies in including mapping studies and genome annotations.     

Phylogeography of native ploidy levels and invasive tetraploids of Solidago gigantea

Ploidy level is an important aspect of the genetic makeup of a plant, and can strongly influence ecological characteristics such as invasiveness. We used a phylogeographical approach to elucidate the history of polyploidization and colonization success of diploid and tetraploid Solidago gigantea Aiton (Asteraceae) within its native range in North America. We were also able to identify the probable source material of the haplotype lineages invasive in Europe and Asia, where only tetraploid plants occur. To do this, we sequenced 1275 bp of chloroplast intergenic spacer DNA in 268 individuals from 57 populations. In addition, we performed a crossing experiment, which supported the hypothesis that chloroplast inheritance in this species is maternal. The phylogeographical analysis showed a complex pattern of 20 haplotypes of diploid and tetraploid plants. In North America, we found significant differentiation among regions, private haplotypes, and isolation by distance. Ploidy levels were more differentiated in the northern regions than in the South. The haplotype network was shallow and included one tetraploid-only, star-shaped cluster of haplotypes that were particularly successful colonizers. Post-glacial migration of diploid S. gigantea occurred mainly northwards east of the Appalachian Mountains, and to a lesser degree also southward. Our data suggest that tetraploids have formed several times in North America. Haplotype number and diversity were lower in European populations than in the native range, and we found evidence that four haplotypes were introduced to Europe from two source areas, New England and the Southern Appalachian Mountains.