Occurrence, antimicrobial susceptibility, and pathogenic factors of Pseudomonas aeruginosa in canine clinical samples

Background and Aim: Pseudomonas aeruginosa is a relevant opportunistic and difficult to treat pathogen due to its widespread environmental diffusion, intrinsic resistance to many classes of antimicrobials, high ability to acquire additional resistance mechanisms, and wide range of pathogenic factors. The present study aimed to investigate the prevalence of P. aeruginosa in canine clinical samples, the antimicrobial susceptibility against antipseudomonal antibiotics, and the presence of extracellular pathogenic factors of the isolates, as well as their ability to produce biofilm. Materials and Methods: Overall, 300 clinical specimens from dogs with pyoderma or abscesses (n=58), otitis (n=59), and suspected bladder infection (n=183) were analyzed by standard bacteriological methods. P. aeruginosa isolates were tested for their antimicrobial susceptibility by disk and gradient diffusion methods to determine the minimum inhibitory concentrations. The ability of the isolates to produce biofilm was investigated by a microtiter plate assay, while virulence genes coding for elastase (lasB), exotoxin A (toxA), alkaline protease (aprA), hemolytic phospholipase C (plcH), and exoenzyme S (ExoS) were detected by polymerase chain reaction method. Results: A total of 24 isolates of P. aeruginosa were found in clinical specimens (urine n=3, skin/soft tissue n=6, and ear canal n=15). No resistance was found to ceftazidime, gentamicin, aztreonam, and imipenem (IMI), while low levels of resistance were found to enrofloxacin (ENR) (4.2%) and piperacillin-tazobactam (8.3%). However, 41.7% and 29.2% of the isolates showed intermediate susceptibility to ENR and IMI, respectively. Disk and gradient diffusion methods showed high concordance. The majority of the isolates revealed a weak (33.3%) or intermediate (45.8%) ability to form biofilm, while the strong biofilm producers (20.8%) derived exclusively from the ear canal samples. All isolates (100%) were positive for lasB, aprA, and plcH genes, while exoS and toxA were amplified in 21 (87.5%) and 22 (91.7%) isolates, respectively. Conclusion: In the present study, P. aeruginosa isolates from canine clinical samples were characterized by low levels of antimicrobial resistance against antipseudomonal drugs. However, the high presence of isolates with intermediate susceptibility for some categories of antibiotics, including carbapenems which are not authorized for veterinary use, could represent an early warning signal. Moreover, the presence of isolates with strong ability to produce biofilm represents a challenge for the interpretation of the antimicrobial susceptibility profile. In addition, the high prevalence of the extracellular pathogenic factors was indicative of the potential virulence of the isolates

Immunity in Dicentrarchus labrax culture: in vivo effects of arginate immunostimulation.

Parameters of cellular and humoral immunity wereanalysed in Dicentrarchus labrax to evaluate themodulatory activity of Ergosan®, an algal extractcontaining 1% alginic acid. Four cycles using normal fishfeed formulation supplemented with 0.5% Ergosan wereperformed at 60-days intervals (15-days treatment + 45-days suspension). Serum complement (C), lysozyme andtotal proteins and tissue HSP were measured 15, 30 and45 days after the first 15-days feeding cycle (short-term)and 45 days after each feeding cycle over a 35-weeksperiod (long-term). The percentages of B and Tlymphocytes in PBL and gut were measured over longtermtrial using mAbs against homologous Ig and T cells.Short-term alginic acid treatment raised serum C activity(p<0.05) at 15 days and serum C and lysozyme, gill andliver HSP (p<0.05) at 30 days. No significant differenceswith control group were found at 45 days. Over the longtermperiod, innate and specific immune parameters,survival, growth performances and conversion index didnot differ in treated and control fish.An experimental group received an oral Vibrioanguillarum vaccine (AVL, UK) according to the followingadministration protocol: 5-days vaccine and 0.5%Ergosan diet, 5-days normal diet, 5-days vaccine andErgosan diet (vaccination cycle, total dose: 0.2 ml/fish).Controls received the same dose of vaccine and were fedwith normal diet throughout the cycle. Total and specificserum Ig, C activity and percentages of lymphocytes inPBL and gut were measured 30 days after the start of thevaccination cycle.Oral vaccine raised significantly PBL Ig+ cells (p<0.05)and gut T-cells (p<0.01) in the Ergosan group. The titre ofspecific serum anti-Vibrio Ig was higher (p<0.05) inErgosan group compared with controls, while total Ig wereunmodified. These effects were accompanied by a rise ofPBL (T-cells +12%, B-cells +27%), intestine lymphocytes(T-cells +17%) and serum C (+19%) compared withvaccinated controls.The present studies in the sea bass indicate veryinteresting immunostimulatory properties of alginate.This work was granted by MIPAF Projects 5C68 and5C116.[...

Effect of hydrogen peroxide on the oxidative burst of neutrophils in pigs and ruminants

Background and Aim: Neutrophils represent between 20% and 75% of white blood cells in animals and play a key role in an effective immune response. The generation of reactive oxygen species (ROS) is commonly referred to as an oxidative burst and is crucial under healthy and disease conditions. Interestingly, ROS are emerging as regulators of several neutrophil functions, including their oxidative burst. The present study aimed to investigate the effect of hydrogen peroxide on the oxidative burst of neutrophils, collected from domestic animal species (namely, pig, cattle, and sheep), and exposed to different stimuli. Materials and Methods: A total of 65 slaughtered animals were included in the present study: Twenty-two pigs, 21 cattle, and 22 sheep. Blood samples were collected at bleeding and neutrophils were then purified using ad hoc developed and species-specific protocols. Neutrophils were treated with hydrogen peroxide at micromolar-to-millimolar concentrations, alone, or combined with other stimuli (i.e., opsonized yeasts, and phorbol 12-myristate 13-acetate). The generation of ROS was evaluated using a luminol-derived chemiluminescence (CL) assay. For each animal species, data were aggregated and reported as mean area under curve±standard deviation. Finally, data were statistically analyzed by one-way ANOVA, followed by Tukey's post hoc test. Results: Exposure of bovine and ovine neutrophils to hydrogen peroxide alone resulted in a dose-dependent enhancement of the CL response, which was significantly stronger at its highest concentration and proved particularly prominent in sheep. Opsonized yeasts and phorbol 12-myristate 13-acetate both proved capable of stimulating the generation of ROS in all animal species under study. Hydrogen peroxide negatively modulated the oxidative burst of neutrophils after exposure to those stimuli, observed response patterns varying between pigs and ruminants. Porcine neutrophils, pre-exposed to micromolar concentrations of hydrogen peroxide, showed a decreased CL response only to opsonized yeasts. Conversely, pre-exposure to hydrogen peroxide reduced the CL response of ruminant neutrophils both to yeasts and phorbol 12-myristate 13-acetate, the effect being most prominent at 1 mM concentration. Conclusion: These results indicate that hydrogen peroxide is capable of modulating the oxidative bursts of neutrophils in a species-specific and dose-dependent manner, substantial differences existing between pigs and ruminants. Further investigation is required to fully comprehend such modulation, which is crucial for the proper management of the generation of ROS under healthy and disease conditions

Hemocyte morphological parameters and Heat Shock Protein 70 kDa (HSP 70) mantle expression as stress parameters in Mytilus galloprovincialis

The present study aim of investigating the role of the hemocyte morphological parameters (diameter and circularity) and the mantle heat shock protein 70 kDa (HSP 70) as stress parameters in the mussel Mytilus galloprovincialis. The mussel specimens were exposed to the heat stress (40° C) for different times and then placed to the control temperature (18° C); after the stress and during the recovery, the hemocyte parameters were measured using a cell viability analyzer and the results showed the significant increase of the diameter and circularity, demonstrating also the relation between the treatment duration and the mussels ability to recover their homeostasis. Moreover, hemocyte stimulation with Vibrio alginolyticus was conduced in vitro and a significant decrease of the diameter and circularity was observed, whereas the stressed hemocytes maintained their roundish shape, so confirming the inhibitory effect of the stress on the cellular immune system. A third mussel group was also maintained out of the water at cooling temperature (4° C for 24 hours), showing the significant increase of the parameters investigated in a time-dependent manner. The HSP 70 synthesis increase was observed in the heat stressed mussels, particularly after 24 hours of recovery, whereas the cooling temperature exposure induced a time-dependent decrease of the protein expression. In conclusion, the two parameters investigated could represent important stress markers both in field and in experimental study, so suggesting their use in the environmental monitoring, shellfish culture or natural beds management and seafood quality assessment.[...

Presenza di Bonamia ostreae in ostriche piatte (Ostrea edulis)del Nord Adriatico.

Nella presente ricerca si espongono i risultati conclusivi di un monitoraggio, finalizzato all’accertamento della presenza di patogeni dell’ostrica piatta da evidenziarsi mediante esame istopatologico, condotto nel periodo compreso tra il 1997 ed il 2000 su ostriche piatte (Ostrea edulis) presenti in una stazione di ingrasso e mantenimento posta al largo della laguna di Chioggia-Venezia.Le ostriche esaminate, provenienti in origine da banchi naturali del Nord Adriatico, sono state in tutto 600 suddivise in quattro campionamenti costituiti ciascuno da 150 soggetti prelevati durante il 1997 (due campionamenti), 1998 e 2000.Mentre nei due campionamenti del 1998 e 2000 non si sono rilevati segni evidenti di patologie, in 8 soggetti, relativi ai due campionamenti del 1997, è stata riscontrata la presenza di elementi rotondi, nucleati, sia intraemocitari che liberi negli spazi intracellulari, dalle dimensioni di 2-3 m.L’esame ultrastrutturale confermava la presenza, sia liberi che all’interno del citoplasma degli emociti, di cellule parassitarie: tali cellule, nella maggior parte con citoplasma denso ed in taluni casi con citoplasma più chiaro, mostravano aplosporosomi, e grandi mitocondri.Era inoltre presente una forma binucleata.Le caratteristiche succitate fanno riferire gli elementi parassitari a Bonamia ostreae, protozoo diffuso in ambito europeo in grado di causare elevate mortalità in Ostrea edulis: il suo riscontro nel Nord Adriatico, zona ad elevata vocazione conchiglicola, rende necessarie ulteriori ricerche finalizzate a definirne la diffusione e pericolosità per le ostriche piatte mediterranee. [...