Titanium Dioxide Nano-Formulation: Characterization, Antimicrobial Activity, and Wound Healing in Animals.

The use of metal oxide nanoparticles as an alternative antimicrobial agent has gained attention due to the increasing problem of antimicrobial resistance. Understanding its properties and potential benefits can contribute to the development of more effective and sustainable treatments in veterinary medicine. The aim of this study was to characterize TiO2-NP formulations and evaluate their antibacterial and wound healing abilities. The diameters and zeta potentials were determined using the Zetasizer in conjunction with dynamic light scattering. The agar-well diffusion method, time-kill kinetic assay and crystal violet assay were used to evaluate their antimicrobial activities. Wound healing assays were conducted both in vitro and in vivo. The study demonstrated that TiO2-NP formulations exhibit significant antimicrobial properties against various bacterial strains such as S. aureus and E. coli. No measurable E. coli growth was observed within a 15-min period following exposure to TiO2-NP formulations. The TiO2-NP formation can improve wound healing by enhancing cell migration and collagen formation in both in vitro and in vivo conditions. In summary, our study suggests that TiO2-NP has the potential for use as an antimicrobial agent for animal wound treatment due to its ability to suppress bacterial growth and biofilm formation, as well as to enhance wound healing.

Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii.

Background and Aim: Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and although there are several clinical diagnoses and molecular methods, these are complicated and time-consuming for veterinarians. This study aimed to develop a visual diagnostic assay that is less time-consuming and can be used by veterinarians to screen for sporotrichosis. Materials and Methods: To develop a loop-mediated isothermal amplification (LAMP) assay for sporotrichosis, primers specific for fragments of the 18S rRNA gene of S. schenckii were designed. Then, the time and temperature were optimized to successfully achieve LAMP. Ten-fold serial dilutions of DNA were used to determine the detection limit using both LAMP and nested polymerase chain reaction (nPCR) assays. Results: The optimal LAMP conditions were incubation at 73°C for 30 min. Agarose gel electrophoresis revealed a ladder-like pattern of the LAMP product, and a sky-blue color indicated a positive result. A comparison of the LAMP assay with nPCR revealed that it was 10 times more sensitive than nPCR, with a detection limit of 10 pg. The use of a heat box compared with a thermocycler gave the same results. Conclusion: Loop-mediated isothermal amplification gives good results and may represent a future alternative diagnostic tool for screening fungal pathogens before the results of conventional fungal cultures are received. However, this method should be further studied to clarify its use with clinical samples.

Clinical field trial of parenteral amoxicillin for the treatment of clinical and subclinical mastitis in smallholder dairy farms in the upper region of Northern Thailand.

Background and Aim: Mastitis, primarily caused by intramammary bacterial infection, is the most expensive disease in the global dairy industry due to its negative impact on milk composition and manufacturing properties. This study aimed to evaluate the efficacy of parenteral amoxicillin in the treatment of clinical and subclinical mastitis in smallholder dairy farms in Northern Thailand. Materials and Methods: A total of 51 cows with clinical and subclinical mastitis from dairy cooperatives in Lamphun and Chiang Mai provinces, Northern Thailand, were enrolled in this study. Conventional bacteriological procedures were applied to identify the causative bacteria in milk samples from these cows before and 7 days after treatment, and antibiotic susceptibility tests were conducted using the disk diffusion method for all bacteria isolated before treatment. All cows with mastitis were administered 15 mg/kg of amoxicillin (LONGAMOX®, Syva Laboratories SA, Spain) intramuscularly every other day for 3 days. Results: Environmental streptococcal bacteria (Streptococcus uberis and Streptococcus spp.) were commonly isolated from infected quarters and were highly susceptible to amoxicillin (100%). The clinical efficacy of amoxicillin treatment for clinical mastitis cases was 80.43%, and the bacteriological efficacy was 47.82%, with opportunistic staphylococcal bacteria (coagulase-negative staphylococci) and contagious streptococcal bacteria (Streptococcus agalactiae) being the most sensitive microorganisms (100%). In subclinical mastitis cases, the bacteriological efficacy of parenteral amoxicillin was 70.45%, with environmental streptococcal bacteria (S. uberis) being the most (100%) sensitive microorganisms. Conclusion: Amoxicillin is highly efficacious and can be used to treat clinical and subclinical mastitis in dairy cows, particularly mastitis caused by environmental Streptococcus spp. These findings could be used to guide treatment regimens in veterinary practice in smallholder dairy farms in Thailand.

In Vitro Bacterial Competition of Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli against Coagulase-Negative Staphylococci from Bovine Mastitis Milk.

Intramammary infection (IMI) from the environment and infected quarters can cause co-infection. The objective of this study was to determine the ability of coagulase-negative staphylococci (CNS) to survive in the same environment as Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli as major pathogens. In total, 15 and 242 CNS strains were used in Experiment I and Experiment II, respectively. Both experiments were separated into three conditions: culture with CNS 24 h before (PRIOR), after (AFTER), and at the same time (EQUAL). The lack of a clear zone, regardless of size, was determined to be the key to the survival of both. The CNS species' percentages of survival against major pathogens were tested using Fisher's exact test. Differences in the percentages of survival were evident among the CNS species in all conditions. For the PRIOR condition, all CNS mostly survived when living with major strains; however, S. chromogenes could degrade S. agalactiae. Although most CNS strains were degraded in the AFTER and EQUAL conditions, some strains of S. hominis and S. simulans could resist S. aureus and S. agalactiae. In conclusion, some specific strains of CNS are able to survive in an environment with major pathogens. Research into the survival strains may indicate that the concept of novel bacteria with bacteriolytic capabilities might be possible as a novel mastitis treatment.

Extended-spectrum beta-lactamase-producing Escherichia coli from pork in Muang district, Chiang Mai Province, Thailand.

Background and Aim: Contaminated pork is one of the transmission routes for pathogens. Extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli is one of the critical threats to global public health. This study aimed to examine pork from different types of markets in Muang district, Chiangmai Province, Thailand, for a proportion of ESBL-producing E. coli, antibiotic resistance of ESBL-producing E. coli and ESBL-producing E. coli genotypes. Materials and Methods: Samples were collected from different market types; fresh markets, pork stores, and supermarkets, enriched and inoculated on selective media. Extended-spectrum beta-lactamases-producing E. coli was identified using double-disk diffusion method according to Clinical and Laboratory Standards Institute 2016. Antibiotic susceptibility test was performed through VITEK® System and ESBL-encoding genes were detected using a multiplex polymerase chain reaction. Results: About 69% of the samples were positive to ESBL-producing E. coli and showed high rates of resistance for ampicillin (100%), piperacillin (100%), cefalexin (100%), cefpodoxime (100%), cefovecin (100%) and ceftiofur (100%), gentamycin (89.86%), and tetracycline (TE) (84.06%). All isolates were multiple drug resistant; resistance patterns of beta-lactams, aminoglycosides, TEs, nitrobenzene derivatives, and sulfonamide groups were observed. The ESBL-producing E. coli-positive isolates carried blaCTX-M groups (100%), blaTEM (98.55%), and blaSHV (1.45%). None of blaOXA was found in this study. Conclusion: Extended-spectrum beta-lactamases-producing E. coli was found in various types of markets; all isolates were detected as multidrug-resistant. The dissemination of such strains can conceivably cause concerning public health, implying that supervised antimicrobial use in pork production and sanitary food preparation is recommended.

Effects of supplemental Bacillus subtilis, injectable vitamin E plus selenium, or both on health parameters during the transition period in dairy cows in a tropical environment.

The objective of this study was to determine the effects of supplemental Bacillus subtilis (BS, 0.5 × 1011 CFU/day), injectable vitamin E and selenium (ES, 1000 mg α-tocopherol acetate and 10 mg sodium selenite), or both during the transition period on health parameters and the incidence of retained fetal membranes (RFM) of dairy cows under tropical conditions (average temperature humidity index = 77.0). Thirty-two crossbred Holstein-Friesian cows were used in a randomized design trial with a 2 × 2 factorial arrangement of treatments. Cows were randomly assigned to one of four treatments, including no supplementation (CON), single intramuscular injection of ES on day - 21 before the expected calving date (ES), daily oral supplementation of BS between day - 21 and day 21 relative to calving, or both ES and BS. Body condition score (BCS) and blood samples were collected on days - 28, - 14, 0, 14, and 28 relative to calving. Mean concentrations of corpuscular hemoglobin were higher (33.12 vs 34.03 g/dL, p = 0.06) and platelets were lower (380.97 vs 302.32 × 103/µL, p = 0.10) with ES than without ES. Cows fed supplemental BS had lower concentrations of creatinine and albumin and tended to have lower AST and ß-hydroxybutyrate (BHBA) levels. However, concentrations of glucose were higher for cows fed BS than for those without BS. No differences in the incidence of RFM were observed. In summary, supplemental B. subtilis could reduce indicators of negative energy balance by increasing glucose and lowering BHBA and improve health parameters by keeping WBCs and monocytes in a healthy range during the transition period.

First Evidence of Carp Edema Virus Infection of Koi Cyprinus carpio in Chiang Mai Province, Thailand.

The presence of carp edema virus (CEV) was confirmed in imported ornamental koi in Chiang Mai province, Thailand. The koi showed lethargy, loss of swimming activity, were lying at the bottom of the pond, and gasping at the water's surface. Some clinical signs such as skin hemorrhages and ulcers, swelling of the primary gill lamella, and necrosis of gill tissue, presented. Clinical examination showed co-infection by opportunistic pathogens including Dactylogyrus sp., Gyrodactylus sp. and Saprolegnia sp. on the skin and gills. Histopathologically, the gill of infected fish showed severe necrosis of epithelial cells and infiltrating of eosinophilic granular cells. Electron microscope examination detected few numbers of virions were present in the cytoplasm of gill tissue which showed an electron dense core with surface membranes worn by surface globular units. Molecular detection of CEV DNA from gill samples of fish was performed by polymerase chain reaction (PCR) and confirmed by nested-PCR. Phylogenetic analyses revealed that CEV isolate had 99.8% homology with the CEV isolated from South Korea (KY946715) and Germany (KY550420), and was assigned to genogroup IIa. In conclusion, this report confirmed the presence of CEV infection of koi Cyprinus carpio in Chiang Mai province, Thailand using pathological and molecular approaches.

Molecular characterization of canine parvovirus in Vientiane, Laos.

The global emergence of canine parvovirus type 2c (CPV-2c) has been well documented. In the present study, 139 rectal swab samples collected from diarrheic dogs living in Vientiane, Laos, in 2016 were tested for the presence of the canine parvovirus (CPV) VP2 gene by PCR. The results showed that 82.73% (115/139) of dogs were CPV positive by PCR. The partial VP2 gene was sequenced in 94 of the positive samples; 91 samples belonged to CPV-2c (426Glu) subtype, while 3 samples belonged to the CPV-2a (426Asn) subtype. Notably, phylogenetic analysis of amino acid sequences revealed a close relationship between Laotian isolates and novel Chinese CPV-2c isolates. In Laotian CPV isolates, aligned protein sequences indicated a high rate of residue substitutions at positions 305, 324, 345, 370, 375, and 426 in the GH loop. The mutation at residue 370 (Q370R), a single mutation, was characterized as a unique mutant residue specific to the Laotian CPV-2c variant.

Transferase Activity of Lactobacillal and Bifidobacterial ß-Galactosidases with Various Sugars as Galactosyl Acceptors.

The ß-galactosidases from Lactobacillus reuteri L103 (Lreußgal), Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (Lbulßgal), and Bifidobacterium breve DSM 20281 (Bbreßgal-I and Bbreßgal-II) were investigated in detail with respect to their propensity to transfer galactosyl moieties onto lactose, its hydrolysis products D-glucose and D-galactose, and certain sugar acceptors such as N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc), and L-fucose (Fuc) under defined, initial velocity conditions. The rate constants or partitioning ratios (kNu/kwater) determined for these different acceptors (termed nucleophiles, Nu) were used as a measure for the ability of a certain substance to act as a galactosyl acceptor of these ß-galactosidases. When using Lbulßgal or Bbreßgal-II, the galactosyl transfer to GlcNAc was 6 and 10 times higher than that to lactose, respectively. With lactose and GlcNAc used in equimolar substrate concentrations, Lbulßgal and Bbreßgal-II catalyzed the formation of N-acetyl-allolactosamine with the highest yields of 41 and 24%, respectively, as calculated from the initial GlcNAc concentration.

Biochemical and structural characterization of a thermostable ß-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis.

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial ß-glucosidases (BGL) with significant ß-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific ß-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal ß-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are ß-D-Galp-(1→6)-D-Lac (6GALA) and ß-D-Galp-(1→3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

Two ß-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

Two ß-galactosidases, ß-gal I and ß-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both ß-gal I and ß-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-ß-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for ß-gal I, and at pH 6.5 and 55°C for ß-gal II, respectively. The kcat/Km values for oNPG and lactose hydrolysis are 722 and 7.4 mM-1s-1 for ß-gal I, and 543 and 25 mM-1s-1 for ß-gal II. Both ß-gal I and ß-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with ß-gal I and ß-gal II, respectively. The predominant transgalactosylation products are ß-D-Galp-(1→6)-D-Glc (allolactose) and ß-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by ß-gal I and ß-gal II, respectively, indicating that both enzymes have a propensity to synthesize ß-(1→6) and ß-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.

Nature and biosynthesis of galacto-oligosaccharides related to oligosaccharides in human breast milk.

Human milk oligosaccharides (HMO) are prominent among the functional components of human breast milk. While HMO have potential applications in both infants and adults, this potential is limited by the difficulties in manufacturing these complex structures. Consequently, functional alternatives such as galacto-oligosaccharides are under investigation, and nowadays, infant formulae are supplemented with galacto-oligosaccharides to mimic the biological effects of HMO. Recently, approaches toward the production of defined human milk oligosaccharide structures using microbial, fermentative methods employing single, appropriately engineered microorganisms were introduced. Furthermore, galactose-containing hetero-oligosaccharides have attracted an increasing amount of attention because they are structurally more closely related to HMO. The synthesis of these novel oligosaccharides, which resemble the core of HMO, is of great interest for applications in the food industry.