A survey of antimicrobial resistance in Escherichia coli isolated from wild sika deer (Cervus nippon) in Japan

We examined the antimicrobial susceptibility of 848 Escherichia coli isolates from 237 feces samples of wild sika deer (Cervus nippon) captured between 2016 and 2019 in 39 of the 47 prefectures of Japan. Five of the 237 wild sika deer (2.1%) carried E. coli with resistance to at least one antimicrobial, and all the resistant isolates showed resistance to tetracycline. The resistant isolates contained antimicrobial resistance genes that were similar to those in E. coli derived from humans and farm animals. Although wild sika deer are not currently likely to be a source for the transmission of antimicrobial resistance in Japan, they can potentially mediate antimicrobial resistance spread by coming into contact with humans, animals, and their surroundings.     

Expression and functional analysis of chemokine receptor 7 in canine lymphoma cell lines

C-C chemokine receptor 7 (CCR7) contributes to cell homing to lymph nodes (LNs). Recent studies reported that CCR7 is also expressed in tumor cells, which correlates with LN metastasis in various cancers. However, the expression of CCR7 in tumor cells is unknown in dogs due to the lack of appropriate antibodies. In the present study, a fusion protein of C-C chemokine ligand 19 (CCL19) was employed as an alternative method to CCR7 antibodies. The fusion CCL19 protein specifically detected CCR7 expressed in canine lymphoma cell lines, which showed active chemotaxis to both canine and mouse ligands. The present study will help further research on the involvement of canine CCR7 in LN metastasis.     

Comparison of extracapillary and endocapillary blood flow oxygenators for open heart surgery in dogs: efficiency of gas exchange and platelet conservation

The goal of the current study was to compare the efficiency of gas exchange and platelet conservation of a new extracapillary blood flow oxygenator versus an endocapillary blood flow oxygenator during open heart surgery with extracorporeal circulation in dogs. Dilation and remodeling of the right ventricular outflow tract of dogs was performed using a patch graft technique to simulate pulmonary stenosis. Sequential pre- and post-operative blood analysis revealed that gas exchange efficiency and platelet conservation was significantly greater with the extracapillary blood flow oxygenator than with the endocapillary blood flow oxygenator. However, the priming volume of the extracapillary blood flow oxygenator was significantly greater, leading to hemodilution. We conclude that while the extracapillary blood flow oxygenator provided benefits in terms of gas exchange and platelet conservation, development of a smaller extracapillary blood flow type oxygenator to reduce hemodilution effects would be beneficial.     

LC-MS/MS measurement of ampicillin residue in swine tissues at 5 days after in-feed administration

We assessed ampicillin (ABPC) concentrations of kidney, muscle and intestine after a 5–day withdrawal period in 2 male and a female young Large White pigs fed the diet containing ABPC (ABPC medicated feed, 24 mg/kg/day) for a week. The ABPC residues were measured with liquid chromatography–tandem mass spectrometry, and the mean recoveries and quantitation limits ranged from 91.8 to 97.2% and from 0.1 to 0.12 ng/g, respectively. The residual ABPC concentrations were ≤1.18 ng/g for the muscle, ≤0.53 ng/g for the kidney and ≤1.93 ng/g for the intestine, suggesting below the Japanese provisional maximum residue limits. These results reveal that the analytical method is developed for residual ABPC and that the withdrawal period is appropriate.     

The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.     

Hypoxia inducible factor 1α expression and effects of its inhibitors in canine lymphoma

Hypoxic conditions in various cancers are believed to relate with their malignancy, and hypoxia inducible factor-1α (HIF-1α) has been shown to be a major regulator of the response to low oxygen. In this study, we examined HIF-1α expression in canine lymphoma using cell lines and clinical samples and found that these cells expressed HIF-1α. Moreover, the HIF-1α inhibitors, echinomycin, YC-1 and 2-methoxyestradiol, suppressed the proliferation of canine lymphoma cell lines. In a xenograft model using NOD/scid mice, echinomycin treatment resulted in a dose-dependent regression of the tumor. Our results suggest that HIF-1α contributes to the proliferation and/or survival of canine lymphoma cells. Therefore, HIF-1α inhibitors may be potential agents to treat canine lymphoma.     

In vitro development of bovine nuclear transfer embryos reconstructed with mammary gland epithelial cells at different passages

In the present study, the effect of passage of nuclear donor cells on the in vitro development of nuclear transfer (NT) embryos was investigated using colostrum‐derived mammary gland epithelial (MGE) cells at different passages (3–30 passages) to find reliable passages for the efficiency of cloning. Development of NT embryos to the blastocyst stage was affected by the number of passages of MGE cells (P < 0.05). Nuclear transfer embryos reconstructed with MGE cells at 3–7 passages showed a significantly higher blastocyst development (31.3–48.5%) than those with the cells at 10–30 passages (2.5–12.5%, P < 0.05). No difference in the proportion of the MGE cells with normal diploid was observed among passage of 3, 15 and 30 (P > 0.05). The use of MGE cells at early passages for nuclear donor cells may be advantageous for the production of NT embryos.     

Fixation to the canine bone of artificial implant with new surface structure

Screw and laser (SL) column by making screw threads and forming small holes using laser irradiation on the base metal and conventional beads coating (BC) columns were embedded into the shaft of canine femurs, and compared the implant fixation to the host bone. The interfacial strength in SL columns was almost equivalent as BC columns, and bone-column contact rate was higher than BC columns significantly at twelve weeks after implantation. The newly devised SL surface had almost equivalent bone fixation strength comparable to the conventional BC surface. Also, this surface should provide a useful porous surface for use in artificial joints since there is no risk of surface structure detachment.     

Development of a monoclonal antibody for the detection of anti-canine CD20 chimeric antigen receptor expression on canine CD20 chimeric antigen receptor-transduced T cells

Chimeric antigen receptor (CAR) CAR-T cell therapy targeting CD20 can be a novel adoptive cell therapy for canine patients with B-cell malignancy. After injection of the CAR-T cells in vivo, monitoring circulating CAR-T cells is essential to prove in vivo persistence of CAR-T cells. In this study, we developed a novel monoclonal antibody against canine CD20 CAR, whose single-chain variable fragment was derived from the our previously reported anti-canine CD20 therapeutic antibody. Furthermore, we proved that this monoclonal antibody can detect therapeutic anti-canine CD20 chimeric antibody in the serum from healthy beagle dogs injected with the therapeutic antibody for safety study. This monoclonal antibody is a useful tool for monitoring both canine CD20-CAR-T cells and anti-canine CD20 therapeutic antibody for canine lymphoma.