A new multidrug-resistant enterotoxigenic Escherichia coli pulsed-field gel electrophoresis cluster associated with enrofloxacin non-susceptibility in diseased pigs.

AIMS: To describe the temporal trends in Escherichia coli pathotypes and antimicrobial resistance detected in isolates from diseased-pig cases submitted to the EcL from 2008 to 2016, in Quebec, Canada, and to investigate the presence of spatiotemporal and phylogenetic clusters. METHODS AND RESULTS: Detection of 12 genes coding for virulence factors in pathogenic E. coli in pigs by PCR and antimicrobial resistance standard disc diffusion assay were performed. Demographic and clinical data were entered in the Animal Pathogenic and Zoonotic E. coli (APZEC) database. ETEC:F4 was the most prevalent pathovirotype among the 3773 cases submitted. The LT:STb:F4 virotype was predominant until 2014, then was overtaken by the LT:STb:STa:F4 virotype. More than 90% of the ETEC:F4 isolates were multidrug resistant. A spatiotemporal cluster of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin was detected between 4/2015 and 9/2016. Pulsed-field gel electrophoresis analysis of 137 ETEC:F4 isolates revealed the presence of a cluster composed mainly of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin. CONCLUSIONS: The APZEC database was useful to highlight temporal trends in E. coli pathotypes. A high-risk ETEC:F4 clone might disseminate in the pig population in Quebec since 2015. SIGNIFICANCE AND IMPACT OF THE STUDY: Surveillance is crucial to identify new clones and develop control strategies.

Developmental and epigenetic anomalies in cloned cattle.

Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the 'large offspring syndrome' (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.

Aberrant expression of E-cadherin and ß-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer.

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and ß-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or ß-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total ß-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-ß-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/ß-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and ß-catenin proteins, along with defective ß-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Endocrine profiles of somatic nuclear transfer-derived pregnancies in dairy cattle.

In cattle, several hormones and proteins are necessary for maintenance of a normal pregnancy that will result in a viable calf. Deviation from the normal cascade or expected profile of reproductive hormones and proteins may be associated with impairment of somatic nuclear transfer-derived pregnancies and the high rate of fetal loss. The objectives of this study were to characterize maternal plasma concentrations of pregnancy-specific protein B (PSPB), progesterone (P4), estrone sulphate (E(1)S), and estradiol (E2) during the last two-thirds of pregnancy (cloned calves), and to determine associations with gestational abnormalities. Cows with cloned fetuses, produced by either commercial (N = 16) or zona-free (N = 4) cloning techniques, were compared with pregnant animals derived from traditional embryo transfer (N = 6) or AI (N = 6), at various stages of gestation (Days 80, 120, 150, 180, 210, and 240; Day 0 = estrus). Fetal well-being was monitored with ultrasonography throughout gestation. At Day 80, progesterone concentration was lower (P < 0.0001) in nuclear transfer (NT) recipients than in control groups. Mean estrone sulphate concentrations did not vary significantly between NT and control groups. At Day 150, pregnancy-specific protein B concentrations were elevated (P < 0.002) in NT cows. Estradiol concentration was higher in NT recipients than control cows throughout the study period.

Ultrasonographic and histological characterization of the placenta of somatic nuclear transfer-derived pregnancies in dairy cattle.

The high incidence of pregnancy loss and prenatal morbidity and mortality in cloned animals may be due to placental insufficiency, thereby compromising fetal survival. Our objective was to characterize morphological changes in fetal membranes of cloned bovine pregnancies. Two groups of cows with cloned fetuses, produced by two cloning techniques, a commercial group (n=16) and a hand-made group (n=4), and control fetuses derived from traditional embryo transfer (n=6) or AI (n=6), were compared at various stages of gestation (Days 80, 120, 150, 180, 210, and 240; Day 0=estrus). Thickness and shape of the amniotic membrane, placentome shape and length, umbilical cord shape and diameter, and fetal fluid echodensities were assessed by ultrasonography, and the placenta was evaluated histologically. Only eight (40%) of cloned pregnancies reached term and seven calves (35%) were alive at birth. Both placentome length and umbilical cord diameter were larger (P<0.05) in clones than in normal fetuses at all stages of gestation. Amniotic membrane abnormalities (Day 120) including focal edema and the presence of a series of nodules were detected in 38% of the clones and were always accompanied by hyper-echodense spikes or irregularities (detected ultrasonographically) around the umbilical cord. Histopathology revealed degenerate inflammatory cells, edematous chorioallantoic membranes, and decreased epithelial thickness. We inferred that these morphological anomalies of placentomes compromised fetal development, and we concluded that ultrasonographic monitoring of pregnancies enabled characterization of changes in the placentae and may be useful to assess fetal well-being.