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Antimicrobial resistance and molecular epidemiological analysis of Escherichia fergusonii harboring the mcr gene in pigs and broiler chickens in Okinawa, Japan
Get PDFjournal articleThe dissemination of mcr-harboring Enterobacteriaceae, e.g., Escherichia fergusonii, with resistance to colistin via animal products is a public health concern. In our previous study, E. fergusonii harboring the mcr gene were isolated from 11 pigs and 43 chickens. To understand the spread of mcr-harboring E. fergusonii in Okinawa, Japan, and to gain further insights into how they can be controlled, an antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), a conjugation test for the transferability of mcr-harboring plasmids, and PCR-based replicon typing (PBRT) were performed using the 54 strains. According to the disk-diffusion and broth microdilution methods, 9 of the 11 strains from pigs and 9 of the 43 strains from chickens had multidrug resistance (MDR). The broth microdilution method showed that all strains were resistant to colistin, and the minimum inhibitory concentration of colistin was 4–16 μg/mL. PFGE suggested identical PFGE types were being transmitted within one pig farm, within one chicken farm, and among several chicken farms. These findings showed that some mcr-harboring E. fergusonii in Okinawa exhibited MDR, and these had spread within farms and between farms. In the mcr gene conjugation test and PBRT, a type IncI2 plasmid replicon was detected in all mcr-1-harboring transconjugants. Therefore, evidence suggests that the IncI2 plasmid is probably involved in the transmission of the mcr-1 gene. It is important to monitor the antimicrobial resistance profile and dissemination of the IncI2 plasmid in mcr-harboring E. fergusonii.journal articl
A Comparative Study on Fungal Diversity in Organic and Conventionally Cultivated Lemons During Accelerated Storage
Get PDFjournal articleThere has been a growing interest in organic farming as a countermeasure to the environmental burden caused by chemical pesticides. We analyzed and compared the fungal diversity of lemon fruits from organic and conventional cultivation by automated rRNA intergenic spacer analysis (ARISA), accompanied by isolation of cultured colonies and metagenomic analysis. Lemon peels were cut out and subjected to the analyses at purchase and after accelerated storage at 28 °C. The organic lemons did not decay even after 14 weeks, while most of the conventional lemons did decay. The fungal colony counts were not significantly different, although the number of fungal species together with the Shannon index, considering the abundance of each species, clearly showed more diversity in organic lemons than in conventional lemons (p = 0.011). Fusarium sp. (putative F. solani) accounted for as much as 90% of the relative abundance in the decayed conventional lemons. Metagenomic analysis also supported the lack of fungal diversity in conventional lemons. These results may suggest that organic cultivation maintains the diversity of native fungal flora in lemon fruit and could contribute to preventing decay during ambient storage.journal articl
Changes in CCN activity of ship exhaust particles induced by fuel sulfur content reduction and wet scrubbing
Get PDFjournal articleMaritime transport remains a large source of airborne pollutants, including exhaust particles that can act as cloud condensation nuclei (CCN). While primary diesel engine exhaust particles are generally considered hydrophobic, international regulations targeting a reduction of particulate emissions from ships may have secondary effects, and therefore influence how exhaust interacts within the atmosphere. The effect of international fuel sulfur content (FSC) regulations on the cloud forming abilities of exhaust particles was investigated using a marine test engine operating on compliant low FSC fuels, non-compliant high FSC distillate fuels and in conjunction with a marine wet scrubber (fresh- and seawater). Particle sizing and liquid droplet activation measurements reveal that compliance measures can have opposing effects on the CCN activity of exhaust particles. For a non-compliant, high FSC fuel, wet scrubbing leads to an increase in CCN activity but not to significant increases in CCN emission factors. However, switching to low FSC fuels resulted in emissions of highly hydrophobic particles, causing a significant reduction in CCN activity resulting in smaller CCN emission factors by at least one order of magnitude. Our observations are supported by chemical analysis of exhaust particles using scanning transmission X-ray microscopy and near edge X-ray absorption fine structure (STXM/NEXAFS) spectra. Potential implications of effects on ship exhaust particles for cloud and climate interactions due to different compliance measures are discussed.journal articl
Economic losses associated with mastitis due to bovine leukemia virus infection
Get PDFjournal articleBovine leukemia virus (BLV), which causes enzootic bovine leukosis and immunosuppression, is widely prevalent on Japanese dairy farms. However, in the absence of a national eradication scheme with compensation programs, it is important to estimate BLV-associated economic losses to raise farmers' awareness. Mastitis (includes both clinical and subclinical) is a common disease in the dairy industry and the most common reason for culling. We hypothesized that immunosuppression due to BLV predisposes subclinical mastitis. A retrospective cohort study was conducted to trace Holstein cows at 9 commercial dairy farms in the Nemuro and Kushiro regions of Hokkaido Prefecture, Japan, where monitoring of BLV proviral load is routine. Information regarding Dairy Herd Improvement data, parity number, and delivery day was collected at each farm. Cows with no confirmed infection with BLV during lactation were defined as non-infected. Low-proviral-load and high-proviral-load (H-PVL) cows were defined as those in which proviral load was below and over 2,465 copies/50 ng of DNA, respectively, or 56,765 copies/105 cells, respectively, throughout the lactation period. Survival analysis was performed using the frailty model to estimate the hazard ratio of subclinical mastitis for BLV infection status using data from 1,034 dairy cows after adjusting for parity number and delivery season as confounding factors. Kaplan–Meier survivor curves demonstrated that half of the H-PVL cows developed subclinical mastitis within 52 d after calving. The hazard ratio of subclinical mastitis for H-PVL cows was 2.61 times higher than that of non-infected cows. In 2017, there were 264,443 clinical mastitis cases in Hokkaido. Using field and published data, annual economic losses were estimated using Monte Carlo simulation. The economic loss due to mastitis associated with BLV infection per H-PVL cow was 6,097,225 (¥640,208,633). In summary, H-PVL cows were more susceptible to subclinical mastitis than non-infected and low-proviral-load cows, and mastitis due to BLV infection was projected to cause significant economic losses.journal articl
Antimicrobial susceptibility of bovine clinical mastitis pathogens in Japan and development of a simplified agar disk diffusion method for clinical practice
Get PDFjournal articleThis study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.journal articl
Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies
Get PDFjournal articleCoagulase-positive Staphylococci express protein A, which binds to host antibodies, to evade the immune system. Taking advantage of its specific binding to antibodies, protein A from Staphylococcus aureus, which is called SpA, is commonly used as an affinity chromatography ligand for human therapeutic antibodies. However, among four canine IgG subclasses (A, B, C, and D), only IgG-B binds to SpA strongly and establishing an efficient and robust purification scheme for canine therapeutic antibodies whose IgG subclass is A, C, or D remains difficult and depends on finding a suitable substitute to SpA. S. pseudintermedius, a major coagulase-positive Staphylococci found in dogs, expresses spsQ gene which is orthologous to S. aureus spa. We hypothesized that to serve S. pseudintermedius to better adapt to the dog immune system, SpsQ would bind to canine IgGs stronger than SpA, making it a better affinity chromatography ligand for canine therapeutic antibodies. To characterize SpsQ, we first determined the spsQ nucleotide sequence from S. pseudintermedius isolates. Based on the identified sequence, we prepared recombinant proteins containing the immunoglobulin-binding domains of SpA (r-SpA) and SpsQ (r-SpsQ) and determined their binding capacity for each canine IgG subclass. The binding capacity of r-SpsQ for IgG-B was almost as high as that of r-SpA. Interestingly, while both r-SpsQ and r-SpA showed no binding to IgG-C, the binding capacity of r-SpsQ for IgG-A and IgG-D was significantly higher than that of r-SpA. Finally, we performed affinity chromatography using r-SpsQ- or r-SpA-immobilized resin and revealed that the recovery rates of IgG-A and IgG-D using r-SpsQ were significantly higher than those using r-SpA. Our findings indicate that SpsQ has a strong potential to be used as an affinity chromatography ligand for canine therapeutic antibodies of subclass A, B, and D.journal articl
Pyruvate kinase deficiency mutant gene carriage in stray cats and rescued cats from animal hoarding in Hokkaido, Japan
Get PDFjournal articleThe mutant allele frequency of the Pyruvate kinase (PK) gene has been investigated mostly in pure breed cats. We investigated the PK mutant gene in stray and animal hoarding mongrel cats in Hokkaido, Japan. We also investigated the kinship of individuals carrying the mutant gene. Genotyping was conducted using the previously reported real-time PCR method. Fourteen microsatellite markers were used to identify the parents and offspring of cats carrying the PK mutant gene, and some kinship such as parent-offspring and siblings was observed. Some stray and animal hoarding cats carried the PK mutation gene and that consanguinity was confirmed among these cats indicated that the PK mutation gene was spread by unregulated interbreeding.journal articl