Quercetin Protects Blood-Brain Barrier Integrity via the PI3K/Akt/Erk Signaling Pathway in a Mouse Model of Meningitis Induced by Glaesserella parasuis.

Glaesserella parasuis (G. parasuis) causes serious inflammation and meningitis in piglets. Quercetin has anti-inflammatory and anti-bacterial activities; however, whether quercetin can alleviate brain inflammation and provide protective effects during G. parasuis infection has not been studied. Here, we established a mouse model of G. parasuis infection in vivo and in vitro to investigate transcriptome changes in the mouse cerebrum and determine the protective effects of quercetin on brain inflammation and blood-brain barrier (BBB) integrity during G. parasuis infection. The results showed that G. parasuis induced brain inflammation, destroyed BBB integrity, and suppressed PI3K/Akt/Erk signaling-pathway activation in mice. Quercetin decreased the expression of inflammatory cytokines (Il-18, Il-6, Il-8, and Tnf-α) and BBB-permeability marker genes (Mmp9, Vegf, Ang-2, and Et-1), increased the expression of angiogenetic genes (Sema4D and PlexinB1), reduced G. parasuis-induced tight junction disruption, and reactivated G. parasuis-induced suppression of the PI3K/Akt/Erk signaling pathway in vitro. Thus, we concluded that quercetin may protect BBB integrity via the PI3K/Akt/Erk signaling pathway during G. parasuis infection. This was the first attempt to explore the protective effects of quercetin on brain inflammation and BBB integrity in a G. parasuis-infected mouse model. Our findings indicated that quercetin is a promising natural agent for the prevention and treatment of G. parasuis infection.

Inhibition of Haemophilus parasuis by berberine and proteomic studies of its mechanism of action.

Haemophilus parasuis is the main agent of Glässer's disease, which causes substantial losses in pig production. However, the pathogenic mechanism and virulence factors of H. parasuis have not been fully determined. In this study, berberine is shown to have a good therapeutic effect in vivo against H. parasuis; the minimal inhibitory concentration (MIC) in vitro was 2 µg/mL. Berberine inhibited H. parasuis adhesion to and invasion of PK-15 pig kidney cells. Proteomics studies of H. parasuis after berberine treatment identified a total of 97 differentially-expressed proteins; 35 upregulated and 62 downregulated. Bioinformatics analysis showed that berberine may inhibit the growth of H. parasuis by affecting outer membrane proteins, transferrins, and energy metabolism. This study provides a basis for the development of new antibacterial agents.

Gamithromycin in swine: Pharmacokinetics and clinical evaluation against swine respiratory disease.

The pharmacokinetics of gamithromycin were evaluated in 26 male castrated and female crossbred swine administered gamithromycin 15% w/v (Zactran®, Boehringer Ingelheim) intravenously at 6 mg/kg bodyweight or intramuscularly at 3, 6 or 12 mg/kg bodyweight. Blood samples were collected up to Day 10 to establish the plasma profile of gamithromycin, bioavailability and dose proportionality. When administered by intramuscular injection at 6 mg/kg BWT, pharmacokinetic parameters were as follows: area under the curve until last quantifiable plasma concentration, 5.13 ± 0.957 µg*hours/ml; maximum plasma concentration, 960 ± 153 ng/ml at 5 to 15 min; terminal half-life of 94.1 ± 20.4 hr. Absolute bioavailability was 92.2%. Increase in systemic exposure was proportional to the gamithromycin dose level over the range 3-12 mg/kg BWT. No gender-related statistically significant difference in exposure was observed. For clinical evaluation of Zactran® against swine respiratory disease, 305 pigs from six commercial farms in three countries in Europe with signs associated with Actinobacillus pleuropneumoniae and/or Haemophilus parasuis and/or Pasteurella multocida and/or Bordetella bronchiseptica were used. At each site, animals were treated once in a 1:1 ratio with a single intramuscular dose of Zactran® (6 mg gamithromycin/kg bodyweight) or Zuprevo® (4% w/v tildipirosin at 4 mg/kg bodyweight; MSD Animal Health) at the recommended dose respectively. Animals were observed and scored daily for 10 consecutive days for signs of swine respiratory disease (depression, respiration and rectal temperature), and animals presenting signs of clinical swine respiratory disease (Depression Score 3 and/or Respiratory Score 3 associated with Rectal Temperature > 40.0°C) were removed from the study and considered as treatment failure. Animals which remained in the study were individually assessed for 'treatment success' or 'treatment failure' (Depression Score ≥ 1 and Rectal Temperature > 40.0°C or Respiratory Score ≥ 1 and Rectal Temperature > 40.0°C). Using a non-inferiority hypothesis test (non-inferiority margin = 0.10), the proportion of treatment successes in the Zactran® group (97%) was equivalent to or better than that in the Zuprevo® group (93%).

Antibacterial effect of Blumea balsamifera DC. essential oil against Haemophilus parasuis.

Haemophilus parasuis (H. parasuis), the cause of the Glasser's disease, is a potentially pathogenic gram-negative organism that colonizes the upper respiratory tract of pigs. The extraction of Blumea balsamifera DC., as a traditional Chinese herb, has shown great bacteriostatic effect against several common bacteria. To study the antibacterial effect on H. parasuis in vitro, this study evaluated the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Blumea balsamifera DC. essential oil (BBO) as well as morphological changes in H. parasuis treated with it. Furthermore, changes in expression of total protein and key virulence factors were also assessed. Results showed that the MIC and MBC were 0.625 and 1.25 µg/mL, respectively. As the concentration of BBO increased, the growth curve inhibition became stronger. H. parasuis cells were damaged severely after treatment with BBO for 4 h, demonstrating plasmolysis and enlarged vacuoles, along with broken cell walls and membranes. Total protein and virulence factor expression in H. parasuis was significantly downregulated by BBO. Taken together, these results indicated a substantial antibacterial effect of BBO on H. parasuis.

Epidemiological and PK/PD cutoff values determination and PK/PD-based dose assessment of gamithromycin against Haemophilus parasuis in piglets.

BACKGROUND: Gamithromycin is a macrolide approved for the treatment of bovine and swine respiratory diseases. Our study aims to establish the clinical breakpoint and optimum dose regimen for gamithromycin against Haemophilus parasuis in piglets. RESULTS: Gamithromycin was well absorbed and fully bioavailable (87.2-101%) after intramuscular and subcutaneous administrations. The MICs of gamithromycin for 192 clinical H. parasuis isolates ranged from 0.008 to 128 mg/L and the epidemiological cutoff (ECOFF) was calculated as 1.0 mg/L. A large potentiation effect of serum on in vitro susceptibility of gamithromycin was observed for H. parasuis, with broth/serum ratios of 8.93 for MICs and 4.46 for MBCs, respectively. The postantibiotic effects were 1.5 h (1 × MIC) and 2.4 h (4 × MIC), and the postantibiotic sub-MIC effects ranged from 2.7 to 4.3 h. Gamithromycin had rapid and concentration-dependent killing against H. parasuis, and the AUC24h/MIC ratio correlated well with ex vivo efficacy (R2 = 0.97). The AUC24h/MIC targets in serum associated with bacteriostatic, bactericidal and eradication activities were 15.8, 30.3 and 41.2, respectively. The PK/PD-based population dose prediction indicated a probability of target attainment (PTA) for the current marketed dose (6 mg/kg) of 88.9% against H. parasuis. The calculated gamithromycin dose for a PTA ≥ 90% was 6.55 mg/kg. Based on Monte Carlo simulations, the PK/PD cutoff (COPD) was determined to be 0.25 mg/L. CONCLUSION: The determined cutoffs and PK/PD-based dose prediction will be of great importance in gamithromycin resistance surveillance and serve as an important step in the establishment of optimum dose regimen and clinical breakpoints.

PK/PD modeling of Ceftiofur Sodium against Haemophilus parasuis infection in pigs.

BACKGROUND: Ceftiofur Sodium is widely used in China. Our aim was to determine Ceftiofur Sodium activity and optimize dosing regimens against the pathogen Haemophilus parasuis using an in vitro and ex vivo pharmacokinetics/pharmacodynamics modeling approach. By adopting these strategies, we wanted to extend the effective life of Ceftiofur Sodium in reduce drug-resistance in pigs. RESULTS: We established an H. parasuis infection model in pigs, and assessed the pharmacokinetics of Ceftiofur Sodium in both healthy and infected animals. After Ceftiofur Sodium (10 mg/kg, i.m.) administration to healthy and H. parasuis-infected pigs, plasma based desfuroylceftiofur (a metabolite of Ceftiofur Sodium) was measured by High Performance Liquid Chromatography. The pharmacokinetics of Ceftiofur Sodium (desfuroylceftiofur) was consistent with a two-compartment open model, with first-order absorption. We observed no significant differences (P > 0.05) in pharmacokinetic parameters between healthy and infected pigs. Pharmacodynamics data showed that Ceftiofur Sodium was highly inhibitory against H. parasuis, with MIC, MBC, and MPC values of 0.003125, 0.0125 and 0.032 µg/mL, respectively. Desfuroylceftiofur in plasma also had strong bactericidal activity. Almost all H. parasuis cultured in plasma medium of Ceftiofur Sodium-inoculated healthy pigs, at each time point, were killed within 24 h. A weaker antibacterial activity was measured in infected-pig plasma medium at 18, 24, 36, and 48 h, after Ceftiofur Sodium inoculation. Pharmacokinetic parameters were combined with ex vivo pharmacodynamic parameters, and the bacteriostatic effect (36.006 h), bactericidal effect (71.637 h) and clearance (90.619 h) within 24 h, were determined using the Hill equation. Dose-calculation equations revealed the optimal dose of Ceftiofur Sodium to be 0.599-1.507 mg/kg. CONCLUSIONS: There were no significant differences in Ceftiofur Sodium pharmacokinetic parameters between healthy and infected pigs, although pharmacokinetics/pharmacodynamics fitting curves showed obviously differences. The optimal dose of Ceftiofur Sodium was lower than recommended (3 mg/kg), which may provide improved treatments for Glässers disease, with lower drug-resistance possibility.

Investigation of Haemophilus parasuis from healthy pigs in China.

Haemophilus parasuis is a common colonizer of the upper respiratory tract of swine and frequently causes disease, especially in weaner pigs. To date, limited epidemiological data was available for H. parasuis from healthy pigs, which might be carriers of potential pathogenic strains. In this study, from September 2016 to October 2017, we investigated the prevalence and characteristics of H. parasuis from healthy pigs in China. Totally, we obtained 244 isolates from 1675 nasal samples from 6 provinces. H. parasuis isolation was more successful in weaner pigs (22.6%, 192/849), followed by finisher pigs (9.3%, 43/463), and sows (2.5%, 9/363). The most prevalent serovars were 7 (20.1%, 49/244), followed by 3 (14.8%, 36/244), 2 (14.3%, 35/244), 11 (12.7%, 31/244), 5/12 (5.7%, 14/244) and 4 (2.5%, 6/244). Bimodal or multimodal distributions of MICs were observed for most of the tested drugs, which suggested the presence of non-wild type populations. It was noted that the MIC90 values of tilmicosin (64 µg/ml) was relatively higher than that reported in previous studies. Our results suggest that: 1) potentially pathogenic serovars of H. parasuis are identified in healthy pigs, and 2) elevated MICs and presence of mechanisms of resistance not yet described for clinically important antimicrobial agents would increase the burden of disease caused by H. parasuis.

The AI-2/luxS Quorum Sensing System Affects the Growth Characteristics, Biofilm Formation, and Virulence of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis) is a kind of opportunistic pathogen of the upper respiratory tract of piglets. Under certain circumstances, virulent strains can breach the mucosal barrier and enter the bloodstream, causing severe Glässer's disease. Many virulence factors are found to be related to the pathogenicity of H. parasuis strain, but the pathogenic mechanism remains unclear. LuxS/AI-2, as a kind of very important quorum sensing system, affects the growth characteristics, biofilm formation, antibiotic production, virulence, and metabolism of different strains. In order to investigate the effect of luxS/AI-2 quorum sensing system on the virulence of H. parasuis, a deletion mutant strain (ΔluxS) and complemented strain (C-luxS) were constructed and characterized. The results showed that the luxS gene participated in regulating and controlling stress resistance, biofilm formation and virulence. Compared with wild-type strain, ΔluxS strain decreased the production of AI-2 molecules and the tolerance toward oxidative stress and heat shock, and it reduced the abilities of autoagglutination, hemagglutination, and adherence, whereas it increased the abilities to form biofilm in vitro. In vivo experiments showed that ΔluxS strain attenuated its virulence about 10-folds and significantly decreased its tissue burden of bacteria in mice, compared with the wild-type strain. Taken together, the luxS/AI-2 quorum sensing system in H. parasuis not only plays an important role in growth and biofilm formation, but also affects the pathogenicity of H. parasuis.

Effects of Baicalin on piglet monocytes involving PKC-MAPK signaling pathways induced by Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (HPS) is the causative agent of Glässer's disease, characterized by arthritis, fibrinous polyserositis and meningitis, and resulting in worldwide economic losses in the swine industry. Baicalin (BA), a commonly used traditional Chinese medication, has been shown to possess a series of activities, such as anti-bacterial, anti-viral, anti-tumor, anti-oxidant and anti-inflammatory activities. However, whether BA has anti-apoptotic effects following HPS infection is unclear. Here, we investigated the anti-apoptotic effects and mechanisms of BA in HPS-induced apoptosis via the protein kinase C (PKC)-mitogen-activated protein kinase (MAPK) pathway in piglet's mononuclear phagocytes (PMNP). RESULTS: Our data demonstrated that HPS could induce reactive oxygen species (ROS) production, arrest the cell cycle and promote apoptosis via the PKC-MAPK signaling pathway in PMNP. Moreover, when BA was administered, we observed a reduction in ROS production, suppression of cleavage of caspase-3 in inducing apoptosis, and inhibition of activation of the PKC-MAPK signaling pathway for down-regulating p-JNK, p-p38, p-ERK, p-PKC-α and PKC-δ in PMNP triggered by HPS. CONCLUSIONS: Our data strongly suggest that BA can reverse the apoptosis initiated by HPS through regulating the PKC-MAPK signaling pathway, which represents a promising therapeutic agent in the treatment of HPS infection.

Tilmicosin enteric granules and premix to pigs: Antimicrobial susceptibility testing and comparative pharmacokinetics.

The purpose of this study was to compare the pharmacokinetics and relative bioavailability of tilmicosin enteric granules and premix after oral administration at a dose of 40 mg/kg in pigs. Three kinds of different respiratory pathogens were selected for determination of minimal inhibitory concentration (MIC) to tilmicosin. Eight healthy pigs were assigned to a two-period, randomized crossover design. A modified rapid, sensitive HPLC method was used for determining the concentrations of tilmicosin in plasma. Pharmacokinetic parameters were calculated by using WinNonlin 5.2 software. The MIC90 of tilmicosin against Haemophilus parasuis, Actinbacillus pleuropneumoniae, and Pasteurella multocida were all 8 µg/ml. These results indicated that these common pig respiratory bacteria are sensitive to tilmicosin. The main parameters of time to reach maximum plasma concentration (Tmax ), elimination half-life (t1/2ß ), mean residence time (MRT), and apparent volume of distribution (VF ) were 2.03 ± 0.37 hr, 29.31 ± 5.56 hr, 25.22 ± 2.57 hr, 4.06 ± 1.04 L/kg, and 3.05 ± 0.08 hr, 17.06 ± 1.77 hr, 15.55 ± 1.37 hr, 2.95 ± 0.62 L/kg after the orally administrated tilmicosin enteric granules and premix. The relative bioavailability of tilmicosin enteric granules to premix was 114.97 ± 7.19%, according to the AUC0-t values. These results demonstrated that tilmicosin enteric granules produced faster tilmicosin absorption, slower elimination, larger tissue distribution, and higher bioavailability compared to the tilmicosin premix. The present study results manifest that tilmicosin enteric granules can be used as a therapeutic alternative to premix in clinical treatment.

Antimicrobial susceptibility of Haemophilus parasuis isolates from Germany by use of a proposed standard method for harmonized testing.

Haemophilus parasuis-related infections, especially among weaners are responsible for major economic losses on pig farms. A method for broth microdilution susceptibility testing of this fastidious organism has recently been developed, but the suitability of this method needs to be validated in a large collection of current field isolates. Using the proposed method, this study tested 123 H. parasuis isolates from different geographic regions in Germany (including five isolates from the Netherlands and Belgium) against a panel of 24 antimicrobial agents and antimicrobial combinations. The isolates were collected between 2013 and 2016. As there are no H. parasuis specific breakpoints available, the tested isolates could not be classified as susceptible, intermediate or resistant. Bi- or multi-modal distributions of minimum inhibitory concentration (MIC) values were observed for some antimicrobial agents (e.g. aminoglycosides, ß-lactams, fluoroquinolones and tetracyclines), indicative of non-wild type populations of H. parasuis. Susceptibility testing revealed broad distributions of MIC values for various antimicrobials (e.g. neomycin, streptomycin, tetracycline, tiamulin, tilmicosin, trimethoprim/sulfamethoxazole and tulathromycin). The lowest MIC90 (i.e. the concentration at which 90% of isolates were inhibited) was obtained for cefotaxime (≤0.015 µg/ml), and the highest MIC90 (512 µg/ml) was obtained for streptomycin. This study tested a large set of current field isolates and included the most common serovars (serovars 4 and 5). The results point to the suitability of the broth microdilution susceptibility testing method proposed previously for determining H. parasuis MIC values. In addition, the study provides a reliable overview of the susceptibility status of H. parasuis at present in Germany.

Characterisation of a novel plasmid containing a florfenicol resistance gene in Haemophilus parasuis.

Thirty clinical isolates of H. parasuis from pig farms in eastern China were screened for antimicrobial susceptibility. A novel plasmid, designated pHPSGC, was extracted from one isolate with evidence of resistance (elevated minimum inhibitory concentration) to florfenicol. DNA sequencing demonstrated that pHPSGC (5297 base pairs) contains three open reading frames (ORFs), corresponding to the genes rep, floR and lysR. The rep gene of pHPSGC shared 99% sequence identity with the rep gene of pHPS1019. In addition, the region containing floR and lysR in pHPSGC shared 99% similarity with the corresponding region of pCCK381. pHPSGC may be derived from a recombination event between pHPS1019 and pCCK381. A florfenicol resistance gene in H. parasuis may have been transferred via recombination between different plasmids.

Integration of pharmacokinetic-pharmacodynamic for dose optimization of doxycycline against Haemophilus parasuis in pigs.

The aims of this study were to establish optimal doses of doxycycline (dox) against Haemophilus parasuis on the basis of pharmacokinetic-pharmacodynamic (PK-PD) integration modeling. The infected model was established by intranasal inoculation of organism in pigs and confirmed by clinical signs, blood biochemistry, and microscopic examinations. The recommended dose (20 mg/kg b.w.) was administered in pigs through intramuscular routes for PK studies. The area under the concentration 0- to 24-hr curve (AUC0-24 ), elimination half-life (T½ke ), and mean residence time (MRT) of dox in healthy and H. parasuis-infected pigs were 55.51 ± 5.72 versus 57.10 ± 4.89 µg·hr/ml, 8.28 ± 0.91 versus 9.80 ± 2.38 hr, and 8.43 ± 0.27 versus 8.79 ± 0.18 hr, respectively. The minimal inhibitory concentration (MIC) of dox against 40 H. parasuis isolates was conducted through broth microdilution method, the corresponding MIC50 and MIC90 were 0.25 and 1 µg/ml, respectively. The Ex vivo growth inhibition data suggested that dox exhibited a concentration-dependent killing mechanism. Based on the observed AUC24 hr /MIC values by modeling PK-PD data in H. parasuis-infected pigs, the doses predicted to obtain bacteriostatic, bactericidal, and elimination effects for H. parasuis over 24 hr were 5.25, 8.55, and 10.37 mg/kg for the 50% target attainment rate (TAR), and 7.26, 13.82, and 18.17 mg/kg for 90% TAR, respectively. This study provided a more optimized alternative for clinical use and demonstrated that the dosage 20 mg/kg of dox by intramuscular administration could have an effective bactericidal activity against H. parasuis.

Pharmacokinetic/pharmacodynamic (PK/PD) evaluation of tulathromycin against Haemophilus parasuis in an experimental neutropenic guinea pig model.

The objective of the study was to develop an ex-vivo PK/PD model of intramuscular (IM) administration of tulathromycin and to test its efficacy against Haemophilus parasuis (H. parasuis) infection in intraperitoneal-inoculated neutropenic guinea pigs. The pharmacokinetics (PKs) of tulathromycin at doses of 1 and 10 mg/kg in H. parasuis-infected neutropenic guinea pig were studied by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). In vitro minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), mutant prevention concentration (MPC), post-antibiotic effect (PAE) and dynamic time-kill curve experiments were carried out using H. parasuis strain 13R. Tulathromycin exhibited concentration-dependent activity and PAE persisted long after administration of the antibiotic. The ratio of the 24-h area under the concentration-time curve (AUC) to MIC in serum (AUC24h/MICserum) was recognized as an important PK/PD parameter that positively correlated with the in vitro antibacterial effectiveness of tulathromycin (R2 = 0.9961 or R2 = 1). For the 1 and 10 mg/kg treatments with tulathromycin, the values of AUC24h/MIC for H. parasuis bacteriostatic action, bactericidal action and virtual bacterial eradication were respectively 22.73, 34.5 and 88.03 h for the 1 mg/kg treatment and respectively 24.94, 30.94 and 49.92 h for the 10 mg/kg treatment. In addition, we demonstrated that doses of 7.2-8.0 mg/kg of tulathromycin resulted in high eradication rates (99.99%). Using a previously published conversion factor of 0.296, we were able to estimate an approximate dose, 2.1-2.4 mg/kg, that should also obtain high eradication rates in the target animal, pigs. This study can help optimize tulathromycin efficacy against H. parasuis infections in swine farming.

Haemophilus parasuis CpxRA two-component system confers bacterial tolerance to environmental stresses and macrolide resistance.

Haemophilus parasuis is an opportunistic pathogen localized in the upper respiratory tracts of pigs, its infection begins from bacterial survival under complex conditions, like hyperosmosis, oxidative stress, phagocytosis, and sometimes antibiotics as well. The two-component signal transduction (TCST) system serves as a common stimulus-response mechanism that allows microbes to sense and respond to diverse environmental conditions via a series of phosphorylation reactions. In this study, we investigated the role of TCST system CpxRA in H. parasuis in response to different environmental stimuli by constructing the ΔcpxA and ΔcpxR single deletion mutants as well as the ΔcpxRA double deletion mutant from H. parasuis serotype 4 isolate JS0135. We demonstrated that H. parasuis TCST system CpxRA confers bacterial tolerance to stresses and bactericidal antibiotics. The CpxR was found to play essential roles in mediating oxidative stress, osmotic stresses and alkaline pH stress tolerance, as well as macrolide resistance (i.e. erythromycin), but the CpxA deletion did not decrease bacterial resistance to abovementioned stresses. Moreover, we found via RT-qPCR approach that HAPS_RS00160 and HAPS_RS09425, both encoding multidrug efflux pumps, were significantly decreased in erythromycin challenged ΔcpxR and ΔcpxRA mutants compared with wild-type strain JS0135. These findings characterize the role of the TCST system CpxRA in H. parasuis conferring stress response tolerance and bactericidal resistance, which will deepen our understanding of the pathogenic mechanism in H. parasuis.

Tilmicosin- and florfenicol-loaded hydrogenated castor oil-solid lipid nanoparticles to pigs: Combined antibacterial activities and pharmacokinetics.

The combined antibacterial effects of tilmicosin (TIL) and florfenicol (FF) against Actinobacillus pleuropneumoniae (APP) (n = 2), Streptococcus suis (S. suis) (n = 2), and Haemophilus parasuis (HPS) (n = 2) were evaluated by chekerboard test and time-kill assays. The pharmacokinetics (PKs) of TIL- and FF-loaded hydrogenated castor oil (HCO)-solid lipid nanoparticles (SLN) were performed in healthy pigs. The results indicated that TIL and FF showed synergistic or additive antibacterial activities against APP, S. suis and HPS with the fractional inhibitory concentration (FIC) ranging from 0.375 to 0.75. The time-kill assays showed that 1/2 minimum inhibitory concentration (MIC) TIL combined with 1/2 MIC FF had a stronger ability to inhibit the growth of APP, S. suis, and HPS than 1 MIC TIL or 1 MIC FF, respectively. After oral administration, plasma TIL and FF concentrations could maintain about 0.1 µg/ml for 192 and 176 hr. The SLN prolonged the last time point with detectable concentrations (Tlast ), area under the concentration-time curve (AUC0-t ), elimination half-life (T½ke ), and mean residence time (MRT) by 3.1, 5.6, 12.7, 3.4-fold of the active pharmaceutical ingredient (API) of TIL and 11.8, 16.5, 18.1, 12.1-fold of the API of FF, respectively. This study suggests that the TIL-FF-SLN could be a useful oral formulation for the treatment of APP, S. suis, and HPS infection in pigs.

Antimicrobial susceptibility patterns of Brazilian Haemophilus parasuis field isolates / Perfil de susceptibilidade antimicrobiana de isolados clínicos brasileiros de Haemophilus parasuis

Haemophilus parasuis is the etiological agent of Glässer's disease (GD), an ubiquitous infection of swine characterized by systemic fibrinous polyserositis, polyarthritis and meningitis. Intensive use of antimicrobial agents in swine husbandries during the last years triggered the development of antibiotic resistances in bacterial pathogens. Thus, regular susceptibility testing is crucial to ensure efficacy of different antimicrobial agents to this porcine pathogen. In this study, 50 clinical isolates from South Brazilian pig herds were characterized and analyzed for their susceptibility to commonly used antibiotic. The identification and typing of clinical isolates was carried out by a modified indirect hemagglutination assay combined with a multiplex PCR. The susceptibility of each isolate was analyzed by broth microdilution method against a panel of 21 antimicrobial compounds. We found that field isolates are highly resistance to gentamycin, bacitracin, lincomycin and tiamulin, but sensitive to ampicillin, clindamycin, neomycin, penicillin, danofloxacin and enrofloxacin. Furthermore, an individual susceptibility analysis indicated that enrofloxacin is effective to treat clinical isolates with the exception of those classified as serovar 1. The results presented here firstly demonstrate the susceptibility of Brazilian clinical isolates of H. parasuis to antimicrobials widely used by swine veterinary practitioners and strengthen the need to perform susceptibility test prior to antibiotic therapy during GD outbreaks. In addition, because only six antimicrobial drugs (28.6%) were found effective against field isolates, a continuous surveillance of the susceptibility profile should be of major concern to the swine industry.(AU)

Haemophilus parasuis é o agente etiológico da doença de Glässer (GD), um processo infeccioso que acomete suínos e que se caracteriza por poliserosites fibrinosas sistêmicas, poliartrites e meningites. O uso intensivo de agentes antimicrobianos na produção de suínos, durante os últimos anos, tem disparado a seleção de cepas bacterianas resistentes a antibióticos. Desta maneira, a avaliação rotineira de susceptibilidade torna-se crucial para assegurar a correta seleção de um antimicrobiano eficaz contra este patógeno. Neste estudo, analisou-se a susceptibilidade antimicrobiana de 50 isolados clínicos de H. parasuis procedentes de granjas localizadas na região sul do Brasil. A identificação e tipificação dos isolados clínicos foi realizada através de uma PCR multiplex combinada com o teste de hemaglutinação indireta modificada. A susceptibilidade de cada isolado foi analisada pelo método de microdiluição em caldo utilizando-se um painel composto por 21 agentes antimicrobianos. Os resultados deste estudo indicam que as cepas clínicas de H. parasuis apresentam alta resistência à gentamicina, bacitracina, lincomicina e tiamulina, no entanto, são susceptíveis a ampicilina, clindamicina, neomicina, penicilina, enrofloxacina e danofloxacina. A análise de susceptibilidade realizada dentro de cada grupo de cepas de um mesmo sorovar indicou que a enrofloxacina é o antibiótico mais efetivo para tratar todos isolados clínicos com exceção daqueles pertencentes ao sorovar 1. Em termos gerais, neste trabalho, demonstra-se o perfil de susceptibilidade de isolados clínicos de H. parasuis aos antimicrobianos comumente utilizados pelos médicos veterinários especialistas em suínos, e reforça-se a necessidade da realização de testes de susceptibilidade antes do início da terapia com antibióticos durante surtos de DG. Além disso, como somente seis antimicrobianos (28.6%) foram efetivos contra os isolados clínicos, uma vigilância contínua do perfil de susceptibilidade aos antimicrobianos deve ser de grande preocupação para a indústria de suínos.(AU)

Comparative transcriptional profiling of tildipirosin-resistant and sensitive Haemophilus parasuis.

Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current study, transcriptional profiling was applied to analyse the variation in gene expression of JS0135 and tildipirosin-resistant JS32. The growth curves showed that JS32 had a higher growth rate but fewer bacteria than JS0135. The cell membranes of JS32 and a resistant clinical isolate (HB32) were observed to be smoother than those of JS0135. From the comparative gene expression profile 349 up- and 113 downregulated genes were observed, covering 37 GO and 63 KEGG pathways which are involved in biological processes (11), cellular components (17), molecular function (9), cellular processes (1), environmental information processing (4), genetic information processing (9) and metabolism (49) affected in JS32. In addition, the relative overexpression of genes of the metabolism pathway (HAPS_RS09315, HAPS_RS09320), ribosomes (HAPS_RS07815) and ABC transporters (HAPS_RS10945) was detected, particularly the metabolism pathway, and verified with RT-qPCR. Collectively, the gene expression profile in connection with tildipirosin resistance factors revealed unique and highly resistant determinants of H. parasuis to macrolides that warrant further attention due to the significant threat of bacterial resistance.