Immunomodulatory Effects of Herbal Compounds Quercetin and Curcumin on Cellular and Molecular Functions of Bovine-Milk-Isolated Neutrophils toward Streptococcus agalactiae Infection.

Herbal phytochemicals featuring active ingredients including quercetin and curcumin have shown potential in treating human and animal diseases. The current study investigated their potential function in vitro for host immunomodulation associated with Streptococcus agalactiae subclinical bovine mastitis via milk-isolated neutrophils. Our results showed a positive influence on cellular migration, reactive oxygen species (ROS) generation, phagocytosis, and bacterial killing as well as neutrophil extracellular traps (NETs) release. This study also highlighted several important molecular aspects of quercetin and curcumin in milk-isolated neutrophils. Gene expression analyses by RT-PCR revealed significant changes in the expression of proinflammatory cytokines (IL1B, IL6, and TNF), ROS (CYBA), phagocytosis (LAMP1), and migration (RAC). The expression levels of apoptotic genes or proteins in either pro-apoptosis (CASP3 and FAS) or anti-apoptosis (BCL2, BCL2L1, and CFLAR) were significantly manipulated by the effects of either quercetin or curcumin. A principal component analysis (PCA) identified the superior benefit of quercetin supplementation for increasing both cellular and molecular functions in combating bacterial mastitis. Altogether, this study showed the existing and potential benefits of these test compounds; however, they should be explored further via in vivo studies.

Antimicrobial drug resistance profile of isolated bacteria in dogs and cats with urologic problems at Chiang Mai University Veterinary Teaching Hospital, Thailand (2012-2016).

The present study aimed to estimate the proportion of bacterial urinary tract infection (UTI) in dogs and cats, assess risks associated with bacterial UTI, and to determine bacterial isolates' antimicrobial susceptibility and resistance pattern from the urinary tract of dogs and cats with urologic problems. The medical records from animals visiting Chiang Mai University Small Animal Veterinary Teaching Hospital between January 2012 and December 2016 were reviewed. In total, 203 dogs and 49 cats with urinary tract diseases that had samples submitted for bacterial culture were identified;198 and 24 bacterial isolates were recovered from dogs' and cats' submitted samples, respectively. At least one episode of bacterial UTI was detected in 75.4% (95% CI: 69.4-81.3) of dogs and in 40.8% (95% CI: 26.6-55.1) of cats with UTI and submitted urine cultures. Of 242 submitted urinary samples from dogs and 60 urinary samples from cats, bacteria were identified in 74.0% (95% CI: 68.4-79.5) and 38.3% (95% CI: 26.0-50.6), respectively. The most common pathogen of bacteria positive cultured from dogs was Staphylococcus spp. (30.3%), followed by Escherichia coli (16.7%), and Proteus spp. (13.6%). For cats, the most common pathogen was Pseudomonas spp. (25.0%), followed by E. coli (20.8%) and Proteus spp. (16.7%). Staphylococcus spp. isolates from dogs and Proteus spp. isolates from cats were highly susceptible to Amoxicillin/clavulanic acid (AMC) at 88% and 75%, respectively. Of all isolated bacteria, 67.1% of the bacteria from dogs and 83.3% from cats were multidrug-resistant (MDR). The proportion of MDR-bacterial urinary tract infections in dogs and cats with urologic problems in this study was high. This observation raises concerns regarding the potential of zoonotic transmission of MDR-bacteria from these companion animals. The results suggested that AMC remains a good empirical drug for treating UTIs in dogs in this region.

Bactericidal Effect of Clove Oil against Multidrug-Resistant Streptococcus suis Isolated from Human Patients and Slaughtered Pigs.

Streptococcus suis is a zoonotic pathogen that is currently considered an emerging multidrug-resistant (MDR). Increasing antibiotic resistance can lead to the unsuccessful treatment of S. suis infection. Recently, many investigations of medicinal plants were conducted for the treatment of infection as a result of the increase of antibiotic-resistant bacteria. The aims of this study were to determine the chemical composition of essential oil from Syzygium aromaticum (L.) Merr. & L.M. Perry and the antibacterial activities of clove oil on MDR S. suis. Using gas chromatography coupled to a mass spectrometer, eugenol (97.76%) was found to be the major active ingredient of clove oil. In vitro antibacterial activities of clove oil against MDR S. suis were evaluated. Using the agar disc diffusion test, the clove oil showed a maximum zone of inhibition at 15% (v/v) oil concentration. In a broth microdilution method, the minimum bactericidal concentration of clove oil against all MDR S. suis isolates was 0.1% (v/v). A time-kill analysis was performed, and the killing kinetics of clove oil showed that MDR S. suis was completely reduced after 15 min of exposure to clove oil. In addition, clove oil exhibited a strong antibacterial activity at all pH values applied following incubation of MDR S. suis in pH-adjusted media with clove oil. Moreover, scanning electron microscopy revealed the nonviable S. suis isolates clearly showed atypical form and cell membrane lysis after incubation with clove oil. This study confirms the efficacy of clove oil as a natural antimicrobial against MDR S. suis and suggests the possibility of employing it as a promising alternative product for control of infectious diseases caused by S. suis in animal and human patients.

Interleukin 17 (IL-17) manipulates mouse bone marrow- derived neutrophils in response to acute lung inflammation.

Interleukin 17 (IL-17) mediates neutrophil migration to the lungs during acute inflammation, potentially leading to lung tissue damage. In the present study, we evaluated whether IL-17 could facilitate certain neutrophil functions in a mouse model. Mice were divided into four groups and intranasally challenged with PBS (1 = Control), Influenza A (H1N1) and Klebsiella pneumoniae (2 = Mix), Influenza A alone (3 = Flu), or K. pneumoniae (4 = KP) alone. Bone marrow, BAL cells, and lung specimens were collected seven days post-challenge for analysis. Mice in the Flu group showed the highest mortality rate. Neutrophils were the prominent cell type in BAL from Mix and KP, whereas lymphocytes were most numerous in Flu. Lesions in the lungs revealed considerably damage in the Mix, Flu, and KP groups. Isolated bone marrow-derived neutrophils were in vitro primed with mouse recombinant IL-17A protein (rIL-17A) followed by various functional assays. The reactive oxygen species (ROS) levels in rIL-17A primed cells showed significant elevations in all groups. Phagocytosis and bacterial destruction showed no significant difference between (+) or (-) rIL-17A groups. The formation of neutrophil extracellular traps (NETs) in rIL-17A-primed neutrophils showed elevated NET production. We next monitored expressions of genes in neutrophils. IL-17A mRNA expression was significantly increased in Mix and Flu; IL-1ß mRNA only significantly increased in Flu, and IL-17RA showed constitutive expressions in all groups. In summary, neutrophils may cause tissue damage during lung inflammation through specific functions influenced by IL-17.

Preferential pattern of mouse neutrophil cell death in response to various stimulants.

Neutrophils undergo cell death processes once their physiological function has been fulfilled. Apoptosis, necrosis, pyroptosis, or NETosis, a unique form of cell death, could occur, depending on the type of stimulant or inhibitory intervention. We investigated whether phorbol myristate acetate (PMA) and Klebsiella pneumoniae (KP), serving as stimulants, or whether an inhibitor (cytochalasin B, CytB) could alter the morphology and gene expression pattern associated with mouse neutrophil cell death. Fluorescence microscopy, flow cytometry, and real-time PCR approaches were used to identify morphological changes, percentages of cell death, and gene expression patterns, respectively. The results showed an increase in the percentage of cell death in PMA and KP groups, whereas CytB exerted inhibitory effects. Fluorescently stained cell nuclei revealed significantly different percentages of cell death among treatments. Moreover, there was a stepwise increase in cell death from 90 to 150 min after stimulation. Quantification of the release of neutrophil extracellular traps (NETs) demonstrated clearly elevated amounts in cells stimulated with KP, while the amount of NETs was slightly increased or unchanged with PMA or CytB treatments. Analysis of the genes involved in cell death revealed that mRNA expression of CASP1, IL1ß, IL18, MCL1, NLRP3, and PYCARD was down-regulated in the PMA group, with the exception of AIM2 and CASP3. The expression of AIM2, IL1ß, MCL1, and NLRP3 was up-regulated in KP-stimulated neutrophils, while CASP1, CASP3, IL18, and PYCARD genes were down-regulated. In summary, a spectrum of specific cell stimulants and exposure durations accounted for different outcomes in mouse neutrophil cell death.

Serotype- and virulence-associated gene profile of Streptococcus suis isolates from pig carcasses in Chiang Mai Province, Northern Thailand.

In this present study, the serotype of 40 Streptococcus suis isolates from submaxillary glands of pig carcasses sold in wet markets in Chiang Mai Province, northern Thailand, was investigated. Eleven serotypes, including types 2, 3, 4, 5, 7, 8, 9, 17, 21, 22 and 31, were found in the isolates by a Multiplex PCR combined with serum agglutination. Of the eleven serotypes present, type 3 was the most prevalent, while types 2, 4, 5 and 21 were of primary interest due to their human isolate serotype. The mrp+/epf - /sly - genotype was found to be the most prevalent genotype. This study indicates the importance of effective control of human S. suis infection due to raw pork or pig carcass handling in northern Thailand.