Characterization of <Emphasis Type="Italic">pax3a</Emphasis> and <Emphasis Type="Italic">pax3b</Emphasis> genes in artificially induced polyploid and gynogenetic olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) during embryogenesis

Although chromosome set manipulation techniques including polyploidy induction and gynogentic induction in flatfish are becoming increasingly mature, there exists a poor understanding of their effects on embryonic development. PAX3 plays crucial roles during embryonic myogenesis and neurogenesis. In olive flounder (Paralichthys olivaceus), there are two duplicated pax3 genes (pax3a, pax3b), and both of them are expressed in the brain and muscle regions with some subtle regional differences. We utilized pax3a and pax3b as indicators to preliminarily investigate whether chromosome set manipulation affects embryonic neurogenesis and myogenesis using whole-mount in situ hybridization. In the polyploid induction groups, 94 % of embryos in the triploid induction group had normal pax3a/3b expression patterns; however, 45 % of embryos in the tetraploid induction group showed abnormal pax3a/3b expression patterns from the tailbud formation stage to the hatching stage. Therefore, the artificial induction of triploidy and tetraploidy had a small or a moderate effect on flounder embryonic myogenesis and neurogenesis, respectively. In the gynogenetic induction groups, 87 % of embryos in the meiogynogenetic diploid induction group showed normal pax3a/3b expression patterns. However, almost 100 % of embryos in the gynogenetic haploid induction group and 63 % of embryos in the mitogynogenetic diploid induction group showed abnormal pax3a/3b expression patterns. Therefore, the induction of gynogenetic haploidy and mitogynogenetic diploidy had large effects on flounder embryonic myogenesis and neurogenesis. In conclusion, the differential expression of pax3a and pax3b may provide new insights for consideration of fish chromosome set manipulation.     

CRISPR-Cas Technology in Plant Science

CRISPR-Cas technology has raised considerable interest among plant scientists, both in basic science and in plant breeding. Presently, the generation of random mutations at a predetermined site of the genome is well mastered, just like the targeted insertion of transgenes, although both remain restricted to species or genotypes amenable for plant transformation. On the other hand, true genome editing, i.e. the deliberate replacement of one or several nucleotides of the genome in a predetermined fashion, is limited to some rather particular examples that generally concern genes allowing positive selection, for example tolerance to herbicides. Therefore, further technological developments are necessary to fully exploit the potential of genome editing in enlarging the gene pool beyond the natural variability available in a given species. In principle, the technology can be applied to any quality related, agronomical or ecological trait, under the condition of upstream knowledge on the genes to be targeted and the precise modifications necessary to improve alleles. Published proof of concepts concern a wide range of agronomical traits, the most frequent being disease resistance, herbicide tolerance and the biochemical composition of harvested products. The regulatory status of the plants obtained by CRISPR-Cas technology raises numerous questions, in particular with regard to the plants that carry in their genomes the punctual modifications caused by the presence of the Cas9 nuclease but not the nuclease itself. Without clarification by the competent authorities, CRISPR-Cas technology would continue to be a powerful tool in functional genomics, but its potential in plant breeding would remain untapped.     

Effects of Dietary Supplementation of Barodon on Growth Performance,Innate Immunity and Disease Resistance of Juvenile Olive Flounder,Paralichthys olivaceus,Against Streptococcus iniae

This study was conducted to evaluate the effects of dietary supplementation of Barodon, an anionic alkali mineral complex, on growth, feed utilization, humoral innate immunity and disease resistance of olive flounder. A basal experimental diet was used as a control and supplemented with 0.1, 0.2, 0.3, 0.4, or 0.5% Barodon. Triplicate groups of fish (26.4 ± 0.2 g) were fed one of the diets to apparent satiation twice daily for 10 wk. The growth performance was enhanced (P < 0.05) linearly and quadratically in fish fed diets containing Barodon compared with that in fish fed the control. Feed utilization was significantly improved by Barodon supplementation. Serum lysozyme and antiprotease activities were increased quadratically in Barodon fed groups. Also, significantly higher superoxide dismutase activity was found in Barodon‐fed fish. Dietary supplementation of 0.1–0.3% Barodon resulted in significant enhancement of fish disease resistance against Streptococcus iniae. The findings in this study indicate that dietary supplementation of Barodon can enhance growth, feed utilization, innate immunity, and disease resistance of olive flounder and that the optimum level seems to be 0.1% in diets.     

Tumour necrosis factor stimulated gene 6 intrinsically regulates PD-L1 expressions in breast cancer cells,leading to modulation of tumour microenvironment

Recent studies have shown that tumour cells express tumour necrosis factor-inducible gene 6 (TSG-6) and its protein, which is known to play a key role in regulating excessive immune responses and proliferation and growth of mesenchymal stem cells (MSCs). It has not been confirmed whether the inhibition of TSG-6 for tumour cells can suppress tumour cell growth and regulate the activation of immune cells in the tumour microenvironment (TME). TSG-6-specific small interfering RNA was transfected into canine and human breast cancer cells (CIPp, CIPm and BT-20). TSG-6-down-regulated (siTSG-6) cells showed decreased cell proliferation, migration, and invasion abilities. Decreased mRNA expressions of NF-κB, STAT3 and Sox2, confirming that TSG-6 is an upper factor governing tumour growth and metastasis. Notably, siTSG-6 cells showed significantly decreased expression levels of CD44 and PD-L1. Direct and indirect co-culture of canine peripheral blood mononuclear cells (cPBMCs) and the siTSG-6 cells showed significant activation in M1 type macrophages and cytotoxic T cells. They also showed a tendency to decrease in the expression of CTLA-4 and increase in the expression of PD-1. In conclusion, this study suggests that the down-regulation of TSG-6 in breast cancer cells could not only suppress tumour growth and metastasis, and but also regulate TME. Since modulation of immune checkpoint proteins occurs in both tumour cells and immune cells, inhibiting TSG-6 and its protein within the TME could be novel therapeutic target for anticancer treatment.     

Abnormal changes in both mandibular salivary glands in a dog: Non-mineral radiopaque sialoliths

A 10-year-old Maltese dog was presented with a firm mass on the left side of his neck. Physical examination confirmed a firm mass in the left and a submandibular swelling in the right cervical region. Sialolithiasis and associated sialocele in both mandibular salivary glands were suspected and bilateral sialoadenectomy was performed. The stones were identified as non-mineral sialoliths.     

Perianesthetic development of diaphragmatic hernia in a horse with equine pituitary pars intermedia dysfunction (PPID)

A 21-year-old Thoroughbred gelding with a history of equine pituitary pars intermedia dysfunction (PPID) presented with priapism of 2 days’ duration. The horse received a caudal morphine epidural and then underwent corpus cavernosum lavage and phallectomy under general anesthesia. The patient’s recovery featured multiple unsuccessful attempts to stand and his respiratory distress persisted for several hours until he acutely developed severe colic and was euthanized. Necropsy findings revealed a pituitary adenoma of the pars intermedia, bilateral adrenal cortical hyperplasia, and diaphragmatic herniation. This report suggests that horses with PPID may present a greater risk for diaphragmatic hernia under general anesthesia or during procedures placing stress on the diaphragm, including anesthetic recovery.     

Use of serology and real time PCR to control an outbreak of bovine brucellosis at a dairy cattle farm in the Nile Delta region,Egypt

Background

Bovine brucellosis remains one of the most prevalent zoonotic infections affecting dairy cattle in developing countries where the applied control programs often fail. We analyzed the epidemiologic pattern of bovine brucellosis in a dairy cattle herd that showed several cases of abortions after regular vaccination with RB51 (B. abortus vaccine). In 2013 thirty dairy cows, from a Holstein-Friesian dairy herd with a population of 600 cattle, aborted five months post vaccination by a regular RB51 vaccine. Blood samples were drawn from milking cows and growing heifers, as well as heifers and cows pregnant up to 6 months. These samples were collected in June 2013 (n?=?257) and May 2014 (n?=?263) and were tested by real time (rt)-PCR as well as serological tests, in particular Rose Bengal Test (RBT), Enzyme-Linked Immunosorbent Assays (ELISA) and Fluorescence Polarization Assay. Tissue specimens were also collected from an aborted fetus and cultured. Isolates were subjected to bacteriological typing tests at the genus and species levels.

Results

Five months post vaccination with RB51 vaccine, Brucella (B.) DNA was detected in blood samples of cows by rt-PCR. The serological tests also revealed the spread of Brucella field strains within the herd in 2013. Four Brucella isolates were recovered from specimens collected from the aborted fetus. These isolates were typed as follows: one B. abortus RB51 vaccine strain and three isolates of B. abortus field strain. The seropositive cows with positive rt-PCR might indicate an infection by the Brucella field strain; while the positive rt-PCR results from seronegative animals may either be due to circulating RB51 vaccine DNA in vaccinated animals or to circulating field strain in infected animals before seroconversion.

Conclusion

The results herein suggest that PCR can be a good supplementary tool in an outbreak situation, if an assay is available that can differentiate vaccine and field strains with a high analytical sensitivity. We recommend using RBT and ELISA in parallel in outbreak situations, to identify as many infected animals as possible during the initial screenings. This test procedure should be repeated for at least three successive negative tests, with one month interval.