RESUMO
Intensive livestock farming has become indispensable to meet the rapidly increasing demand for animal-based nutrition in low- and middle-income countries (LMICs) where antimicrobials are frequently used for treatment and prophylactic or metaphylactic purposes. However, very little is known about the trends of antimicrobial use (AMU) in dairy animals in LMICs. The objective of this study was to quantify AMU in two large commercial dairy farms in Pakistan. A retrospective study was conducted at two large corporate commercial dairy farms located in Punjab province for the year 2018. AMU was calculated using three metrics: active ingredient (AI; kg) and milligrams per population unit (mg/PU; mg/kg), which quantifies the amount of AI used, and antimicrobial treatment incidence (ATI; DDDA/1,000 cow-days), which estimates the per-day number of treatments to 1,000 cows. Total on-farm AMU was found to be 138.34 kg, 65.88 mg/kg, and 47.71 DDDA/1,000 cow-days. Measured in ATI, aminoglycosides (11.05 DDDA/1,000 cow-days), penicillins (8.29 DDDA/1,000 cow-days), and tetracyclines (8.1 DDDA/1,000 cow-days) were the most frequently used antimicrobial classes. A total of 42.46% of all the antimicrobials used belonged to the critically important antimicrobials for human medicine as defined by the World Health Organization. Considerably high AMU was found compared to other farm-level studies across the world. This was the first study to quantify AMU in the dairy industry in Pakistan. Our results showed that corporate commercial dairy management practices are associated with increased antimicrobial consumption and highlight the need for antimicrobial stewardship programs to encourage prudent use of antimicrobials in commercial dairy.
RESUMO
Small ubiquitin-like modifier (SUMO) fusion technology is widely used in the production of heterologous proteins from prokaryotic system to aid in protein solubilization and refolding. Due to an extensive clinical application of human bone morphogenetic protein 2 (hBMP2) in bone augmentation, total RNA was isolated from human gingival tissue and mature gene was amplified through RT-PCR, cloned (pET21a), sequence analyzed, and submitted to GenBank (Accession no. KF250425). To obtain soluble expression, SUMO3 was tagged at the N-terminus of hBMP2 gene (pET21a/SUMO3-hBMP2), transferred in BL21 codon+, and ~ 40% soluble expression was obtained on induction with IPTG. The dimerized hBMP2 was confirmed with Western blot, native PAGE analysis, and purified by fast protein liquid chromatography with 0.5 M NaCl elution. The cleavage of SUMO3 tag from hBMP2 converted it to an insoluble form. Computational 3D structural analysis of the SUMO3-hBMP2 was performed and optimized by molecular dynamic simulation. Protein-protein interaction of SUMO3-hBMP2 with BMP2 receptor was carried out using HADDOCK and inferred stable interaction. The alkaline phosphatase assay of SUMO3-hBMP2 on C2C12 cells showed maximum 200-ng/ml dose-dependent activity. We conclude that SUMO3-tagged hBMP2 is more suited for generation of soluble form of the protein and addition of SUMO3 tag does not affect the functional activity of hBMP2.
