Quantification of Clostridioides (Clostridium) difficile in feces of calves of different age and determination of predominant Clostridioides difficile ribotype 033 relatedness and transmission between family dairy farms using multilocus variable-number tandem-repeat analysis.

BACKGROUND: Community acquired Clostridioides (Clostridium) difficile infection (CA-CDI) is a significant health problem in human and veterinary medicine. Animals are often considered as potential reservoirs for CA-CDI. In Europe, family farming is the most predominant farming operation, with a complex interaction between animals and the community. Therefore, it is pertinent to evaluate transmission patterns of C. difficile on such prominent European farming model. Fecal samples from calves (n = 2442) were collected biweekly over a period of one year on 20 mid-size family dairy farms. Environmental samples (n = 475) were collected in a three month interval. Clostridioides difficile was detected using qPCR in 243 fecal samples (243/2442); positive samples were then quantified. Association between prevalence/load of C. difficile and age of the calves was estimated with logistic regression model. Most common C. difficile isolate from calves (n = 76) and the environment (n = 14) was C. difficile ribotype 033, which was further analyzed using multilocus variable-number tandem-repeat analysis (MLVA) to assess intra- and between-farm relatedness. RESULTS: Clostridioides difficile was detected in feces of calves less than 24 h old. Results showed a non-linear statistically significant decrease in shedding load of C. difficile with age (P < 0.0001). A nonlinear relationship was also established between the number of calves and the farm C. difficile prevalence, whereas the prevalence of C. difficile ribotype 033 increased linearly with the number of calves. MLVA revealed close intra-farm relatedness among C. difficile ribotypes 033. It also revealed that the between-farms close relatedness of C. difficile ribotypes 033 can be a direct result of farm to farm trade of calves. CONCLUSIONS: Implementation of better hygiene and management measures on farms may help decrease the risk of spreading CA-CDI between animals and the community. Trading calves older than 3 weeks would decrease the possibility C. difficile dissemination in the community because of lower prevalence and lower load of C. difficile in feces.

Computational Modeling and Characterization of Peptides Derived from Nanobody Complementary-Determining Region 2 (CDR2) Targeting Active-State Conformation of the ß2-Adrenergic Receptor (ß2AR).

This study assessed the suitability of the complementarity-determining region 2 (CDR2) of the nanobody (Nb) as a template for the derivation of nanobody-derived peptides (NDPs) targeting active-state ß2-adrenergic receptor (ß2AR) conformation. Sequences of conformationally selective Nbs favoring the agonist-occupied ß2AR were initially analyzed by the informational spectrum method (ISM). The derived NDPs in complex with ß2AR were subjected to protein-peptide docking, molecular dynamics (MD) simulations, and metadynamics-based free-energy binding calculations. Computational analyses identified a 25-amino-acid-long CDR2-NDP of Nb71, designated P4, which exhibited the following binding free-energy for the formation of the ß2AR:P4 complex (ΔG = -6.8 ± 0.8 kcal/mol or a Ki = 16.5 µM at 310 K) and mapped the ß2AR:P4 amino acid interaction network. In vitro characterization showed that P4 (i) can cross the plasma membrane, (ii) reduces the maximum isoproterenol-induced cAMP level by approximately 40% and the isoproterenol potency by up to 20-fold at micromolar concentration, (iii) has a very low affinity to interact with unstimulated ß2AR in the cAMP assay, and (iv) cannot reduce the efficacy and potency of the isoproterenol-mediated ß2AR/ß-arrestin-2 interaction in the BRET2-based recruitment assay. In summary, the CDR2-NDP, P4, binds preferentially to agonist-activated ß2AR and disrupts Gαs-mediated signaling.

Effects of season and sex on the concentrations of fecal glucocorticoid metabolites in captive and free-ranging endangered mountain gazelles (Gazella gazella).

Introduction: The aim of our study was to measure fecal glucocorticoid metabolite (FGM) concentrations in captive and free-ranging male and female mountain gazelles (Gazella gazella) during their circannual cycle. In addition, FGM concentrations were used to track the intensity of the adrenocortical response in mountain gazelles during the same period. Methods: Fecal samples were collected from the ground in the Hatay Mountain Gazelle Wildlife Development Area in the Hatay Province of Türkiye (36°32' N, 36°32' E) in each season of the year (December, April, July, September). The sex of the animals was determined by detecting the SRY gene of the Y chromosome in DNA isolated from the fecal samples. FGM was extracted from dried fecal samples with methanol, and its concentration was measured using a previously partially validated ELISA. Results and discussion: The results indicate that season is the most important factor explaining the variability in FGM concentrations in mountain gazelles. In animals of both sexes, the highest concentrations of FGM were observed in September. The values were significantly higher in the captive population, perhaps due to unpredictable stress. In July, FGM concentrations were low in both populations. As a result of the overall analysis across seasons, the comparison of FGM concentrations between captive and free-ranging animals revealed higher concentrations in captive animals only in September but not in other seasons, although higher concentrations have been previously reported for several wild captive species. Due to predation risk, the presence of offspring can be considered a critical point in the biological cycle for the welfare of free-ranging mountain gazelles, as suggested by the higher FGM concentrations in the free-ranging population in July. The high number of visitors could be a challenge for mountain gazelles in captivity, as indicated by higher FGM concentrations during September. Sex had no effect on the FGM concentrations of either population.

Genome-Wide Homozygosity Patterns and Evidence for Selection in a Set of European and Near Eastern Horse Breeds.

Intensive artificial and natural selection have shaped substantial variation among European horse breeds. Whereas most equine selection signature studies employ divergent genetic population structures in order to derive specific inter-breed targets of selection, we screened a total of 1476 horses originating from 12 breeds for the loss of genetic diversity by runs of homozygosity (ROH) utilizing a 670,000 single nucleotide polymorphism (SNP) genotyping array. Overlapping homozygous regions (ROH islands) indicating signatures of selection were identified by breed and similarities/dissimilarities between populations were evaluated. In the entire dataset, 180 ROH islands were identified, whilst 100 islands were breed specific, all other overlapped in 36 genomic regions with at least one ROH island of another breed. Furthermore, two ROH hot spots were determined at horse chromosome 3 (ECA3) and ECA11. Besides the confirmation of previously documented target genes involved in selection for coat color (MC1R, STX17, ASIP), body size (LCORL/NCAPG, ZFAT, LASP1, HMGA2), racing ability (PPARGC1A), behavioral traits (GRIN2B, NTM/OPCML) and gait patterns (DMRT3), several putative target genes related to embryonic morphogenesis (HOXB), energy metabolism (IGFBP-1, IGFBP-3), hair follicle morphogenesis (KRT25, KRT27, INTU) and autophagy (RALB) were highlighted. Furthermore, genes were pinpointed which might be involved in environmental adaptation of specific habitats (UVSSA, STXBP4, COX11, HLF, MMD).

Polycystic kidney disease: a review and occurrence in Slovenia with comparison between ultrasound and genetic testing.

Polycystic kidney disease (PKD) is an inherited autosomal kidney disease which is most commonly identified in Persian and Persian related cats. Positive cats have multiple cysts of various sizes that occur in the renal cortex and medulla and occasionally in other abdominal organs. PKD often leads to renal failure which occurs from mid to late in life. Renal cysts can be diagnosed ultrasonographically after 7 weeks of age by an experienced ultrasonographer and a high resolution machine. However, ultrasonography is now being replaced by genetic screening. A total of 340 cats of variable breeds aged from 5 months to 18 years were ultrasonographically examined in the past 7 years at the University Veterinary Small Animal Clinic. Of these, 13.8% were PKD positive with very high prevalence in Persian cats (36%). There was no sex predilection identified. The C>A transversion at position 3284 on exon 29 of PKD1 gene, resulting in a stop mutation has been identified in the heterozygous state in eight affected cats examined (Persian breed). All heterozygous cats were also ultrasonographically positive.

A decade of using small-to-medium throughput allele discrimination assay to determine prion protein gene ( Prnp) genotypes in sheep in Slovenia.

Sheep with valine (V) at codon 136 and glutamine (Q) at codon 171 of the prion protein gene ( Prnp) are highly susceptible to classical scrapie, whereas phenylalanine (F) at codon 141 and histidine (H) at codon 154 play a major role in the susceptibility to atypical scrapie. A TaqMan real-time PCR assay was developed to determine Prnp alleles at codons 136, 141, 154, and 171 and used in classical scrapie eradication and breeding programs adopted in Slovenia. The frequency of the most resistant genotypes ARR/ARR and ARR/ARQ increased significantly in tested animals ( n = 35,138) from 6.7 and 27.1% of the tested sheep in 2006 to 12.1 and 32.4%, respectively, in 2015. Frequencies of more susceptible genotypes ARQ/ARQ and ARQ/VRQ decreased significantly from 36.4 and 3.5% in 2006 to 31.1 and 1.8%, respectively, in 2015. The most susceptible genotype VRQ/VRQ was detected in <0.5% of tested sheep. Frequencies of alleles AFRQ and AHQ affecting the susceptibility to atypical scrapie did not change significantly. The developed assay was suitable for genotyping on a small-to-medium throughput scale and was successfully used in classical scrapie eradication, as well as for the selection of classical scrapie-resistant sheep within breeding programs in Slovenia.

Age-Dependent Expression of MyHC Isoforms and Lipid Metabolism-Related Genes in the Longissimus Dorsi Muscle of Wild and Domestic Pigs.

This study aimed to compare age-dependent changes in the relative expression of genes encoding myosin heavy chain (MyHC) isoforms and selected lipid metabolism-related genes in the longissimus dorsi muscle of wild pigs (WPs) and domestic pigs (DPs). Muscles sampled from postnatal day one as well as three-week-old and two-year-old animals were used in quantitative polymerase chain reaction (qPCR) assays, histological evaluations of succinate dehydrogenase (SDH) activity, and intra-myofiber lipid (IMFL) assessment. Expression of the MyHC isoforms displayed the most extensive age- and breed-dependent changes within the first three postnatal weeks. The MyHCembry level decreased significantly faster in the WPs than in the DPs. The relative MyHC-I and -IIa expression was significantly higher in the WPs, and MyHC-IIb was substantially higher in the DPs. The differences in MyHC expression corroborated the number of SDH-positive myofibers and IMFLs. Expression of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) genes displayed only age-related variations. In summary, the evidence is provided for accelerated postnatal myofiber transformation directed towards oxidative myofibers in WPs. The SDH activity/staining intensity largely reflected the expression of MyHCs, and not genes involved in lipid uptake and utilization.

Pre-cholesterol precursors in gametogenesis.

Meiosis activating sterols (MAS) are biologically active post-lanosterol intermediates of cholesterol biosynthesis that are synthetized primarily in the gonads, including the sperm. MAS reinitiate the meiosis of oocytes in vitro while in vivo they seem to contribute to the oocyte quality and the progression of meiosis. The mRNAs for the MAS-producing enzyme lanosterol 14alpha-demethylase (CYP51) arise by alternative poly (A) signal selection. Only signals with low cleavage activity are used in the testis. Translation of mammalian CYP51s starts at one of the tandem in-frame ATGs. CYP51 protein of the bull is shorter compared to the human due to the usage of a more downstream translation start site. CYP51 proteins are post-translationally modified by glycosylations in the Golgi and on acrosomal membranes of the sperm. Green fluorescence protein-based ex vivo system has been developed to aid studying the intracellular transport of the MAS-producing CYP51. The influence of the post-translational modifications on MAS-synthesizing capacity is under investigation.

Detection of Clostridium difficile in animals: comparison of real-time PCR assays with the culture method.

Clostridium difficile has emerged as a pathogen or commensal in food animals. There is overlap between isolates from animals, retail meats and humans, suggesting that animals may be a C. difficile reservoir. For direct detection of variant C. difficile strains in faecal samples of symptomatic and asymptomatic animals, we developed and validated a new TaqMan real-time PCR (TMrtPCR) assay targeting the tcdA, tcdB and cdtB genes. We compared it with the enrichment culture method and with two real-time PCR (rtPCR) assays, BrtPCR and PCRFast, targeting tcdB and tcdA/tcdB, respectively. All ten tested C. difficile toxinotypes, except one (XIa) with PCRFast and two (X, XIa) with BrtPCR, were detected with the test assays. A total of 340 (100 %) samples were cultured and amplified with TMrtPCR. Results correlated in 75.3 % samples. Forty (11.8 %) samples were culture positive/TMrtPCR negative, possibly because of the low numbers of bacteria in the samples or because of DNA extraction failure. Forty (11.8 %) samples were TMrtPCR positive/culture negative. Among 79 samples included in the rtPCR assays/culture comparison, 50.6 % were in complete concordance. The results showed that TMrtPCR performed better than BrtPCR and PCRFast, and 67 % of the culture-positive samples were TMrtPCR positive in comparison to 40 % of the samples positive in BrtPCR and 7 % of the samples positive in PCRFast, respectively. Another advantage of TMrtPCR over BrtPCR and PCRFast is its ability to detect a binary toxin gene. Therefore, the TMrtPCR results can provide the first information about the toxin type present in the sample. According to the results of our study, TMrtPCR could be a preferred screening method for the rapid detection of C. difficile in animal faecal samples, although an enrichment culture has to be performed for the specimens with negative or inconclusive rtPCR results.