Antimicrobial Resistance Genes in Porcine Pasteurella multocida Are Not Associated with Its Antimicrobial Susceptibility Pattern.

Forty-eight Pasteurella multocida isolates were recovered from porcine pneumonic lungs collected from farms in "Castilla y León" (north-western Spain) in 2017-2019. These isolates were characterized for their minimal inhibition concentrations to twelve antimicrobial agents and for the appearance of eight resistance genes: tetA, tetB, blaROB1, blaTEM, ermA, ermC, mphE and msrE. Relevant resistance percentages were shown against tetracyclines (52.1% for doxycycline, 68.7% for oxytetracycline), sulphamethoxazole/trimethoprim (43.7%) and tiamulin (25.0%), thus suggesting that P. multocida isolates were mostly susceptible to amoxicillin, ceftiofur, enrofloxacin, florfenicol, marbofloxacin and macrolides. Overall, 29.2% of isolates were resistant to more than two antimicrobials. The tetracycline resistance genes (tetA and tetB) were detected in 22.9% of the isolates, but none were positive to both simultaneously; blaROB1 and blaTEM genes were found in one third of isolates but both genes were detected simultaneously in only one isolate. The ermC gene was observed in 41.7% of isolates, a percentage that decreased to 22.9% for msrE; finally, ermA was harbored by 16.7% and mphE was not found in any of them. Six clusters were established based on hierarchical clustering analysis on antimicrobial susceptibility for the twelve antimicrobials. Generally, it was unable to foresee the antimicrobial susceptibility pattern for each family and the association of each particular isolate inside the clusters established from the presence or absence of the resistance genes analyzed.

Transcriptomics of Haemophilus (Glässerella) parasuis serovar 5 subjected to culture conditions partially mimetic to natural infection for the search of new vaccine antigens.

BACKGROUND: Haemophilus (Glässerella) parasuis is the etiological agent of Glässer's disease in pigs. Control of this disorder has been traditionally based on bacterins. The search for alternative vaccines has focused mainly on the study of outer membrane proteins. This study investigates the transcriptome of H. (G.) parasuis serovar 5 subjected to in vitro conditions mimicking to those existing during an infection (high temperature and iron-restriction), with the aim of detecting the overexpression of genes coding proteins exposed on bacterial surface, which could represent good targets as vaccine candidates. RESULTS: The transcriptomic approach identified 13 upregulated genes coding surface proteins: TbpA, TbpB, HxuA, HxuB, HxuC, FhuA, FimD, TolC, an autotransporter, a protein with immunoglobulin folding domains, another large protein with a tetratricopeptide repeat and two small proteins that did not contain any known domains. Of these, the first six genes coded proteins being related to iron extraction. CONCLUSION: Six of the proteins have already been tested as vaccine antigens in murine and/or porcine infection models and showed protection against H. (G.) parasuis. However, the remaining seven have not yet been tested and, consequently, they could become useful as putative antigens in the prevention of Glässer's disease. Anyway, the expression of this seven novel vaccine candidates should be shown in other serovars different from serovar 5.

Correction to: Mixed-method tutoring support improves learning outcomes of veterinary students in basic subjects.

The original article [1] contains an error whereby Fig. 2a and b are mistakenly swapped with each other, and thus do not correspond to their correct respective sub-headings in the caption.

Mixed-method tutoring support improves learning outcomes of veterinary students in basic subjects.

BACKGROUND: Tutoring is a useful tool in the university teaching-learning binomial, although its development is impaired in large classes. Recent improvements in information and communication technologies have made tutoring possible via the Internet. The aim of this study was to evaluate the efficacy of mixed-method academic tutoring in two basic subjects in Veterinary Science studies at the University of León (Spain) to optimize the usefulness of tutoring support in the college environment. This quasi-experimental study was firstly carried out as a pilot study in a small group of tutored students of "Cytology and Histology" (CH) (47/186; 25.3%) and "Veterinary Pharmacology" (VP) (33/141; 23.4%) subjects, and was implemented in a large class of CH the next academic year (150 students) while comparing the results with those obtained in a previous tutorless course (162 students). Tutored students were given access to online questionnaires with electronic feedback on each subject. In addition to traditional tutoring carried out in both tutored and tutorless students, the pilot study included three sessions of face-to-face tutoring in order to monitor the progress of students. Its efficacy was assessed by monitoring students' examination scores and attendance as well as a satisfaction survey. RESULTS: Although the examination attendance rate in the pilot study was not significantly different between tutored and tutorless groups in both subjects, an increase for numerical scores in tutored groups was observed, with a significant higher final score in VP (p = 0.001) and in the CH practice exams (first term, p = 0.009; final, p = 0.023). Good and merit scores were also better in tutored students with significant differences in VP (p = 0.005). Students felt comfortable with the tutoring service (100% in CH; 91.7% in VP). Implementation of this additional support in CH also resulted in a significant increase of attendance at the final exam in tutored courses (87.3% versus 77.2%; p = 0.026), scaled (p = 0.001) and numerical scores (final score, p = 0.001). CONCLUSIONS: Online tutoring support, together with conventional teaching methods, may be a useful method to incorporate student-centered learning in basic subjects in Veterinary Science.

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)

Nonbinding site-directed mutants of transferrin binding protein B exhibit enhanced immunogenicity and protective capabilities.

Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines.

Immunoproteomic analysis of the protective response obtained with subunit and commercial vaccines against Glässer's disease in pigs.

An immunoproteomic analysis of the protective response of subunit and commercial vaccines in colostrum-deprived pigs against Glässer's disease was carried out. A mixture of proteins with affinity to porcine transferrin (PAPT) from Haemophilus parasuis Nagasaki strain (serovar 5) was inoculated intramuscularly (PAPT(M)) and intratracheally (PAPT(Cp)), along with a commercial bacterin. PAPT were separated using 2 dimensional electrophoresis (2DE) gels and with them, 2DE Western blots were carried out. A total of 17 spots were identified as positive with sera of pigs from any of the three vaccinated groups, the highest number of immunoreactive proteins being detected in those having received PAPT(Cp). Among them, six proteins (FKBP-type peptidyl-prolyl cis-trans isomerase, neuraminidase exo-α-sialidase, xanthine-guanine phosphoribosyl transferase, CMP-N-acetylneuraminic acid synthetase, phenylalanyl-tRNA synthetase and glyceraldehyde 3-phosphate dehydrogenase) were found to be novel immunogens in H. parasuis. These proteins showed a high potential as candidates in future subunit vaccines against Glässer's disease. The three experimental groups developed specific systemic total IgG (IgGt), IgG1, IgG2 and IgM antibodies after immunizations. In addition, those receiving PAPT(Cp) yielded a serum IgA response.

Haemophilus parasuis serovar 5 Nagasaki strain adheres and invades PK-15 cells.

Haemophilus parasuis is the agent responsible for causing Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. The purpose of this study was to investigate the in vitro ability of two H. parasuis serovars of different virulence (serovar 5, Nagasaki strain, highly virulent, belonging to serovar 5, and SW114 strain, nonvirulent, belonging to serovar 3) to adhere to and invade porcine kidney epithelial cells (PK-15 line). Nagasaki strain was able to attach at high levels from 60 to 180 min of incubation irrespective of the concentrations compared (10(7)-10(10)CFU), and a substantial increase of surface projections could be seen in PK-15 cells by scanning electron microscopy. This virulent strain was also able to invade effectively these epithelial cells, and the highest invasion capacity was reached at 180 min of infection. On the contrary, nonvirulent SW114 strain hardly adhered to PK-15 cells, and it did not invade these cells, thus suggesting that adherence and invasion of porcine kidney epithelial cells could be a virulence mechanism involved in the lesions caused by H. parasuis Nagasaki strain in this organ.

Acute phase protein concentrations in colostrum-deprived pigs immunized with subunit and commercial vaccines against Glässer's disease.

Haemophilus parasuis is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. This study was focused on the characterization of the acute-phase response after immunization and infection of colostrum-deprived pigs with H. parasuis serovar 5, by measuring serum concentrations of three positive acute-phase proteins (APPs) (pig major acute-phase protein pig, MAP; haptoglobin, HPG; C-reactive protein, CRP) and one negative APP (apolipoprotein A-I, ApoA-I). Six experimental groups were established: a non-immunized but infected control group (CTL); two groups immunized with either a recombinant transferrin-binding protein (Tbp) A or TbpB fragment from H. parasuis Nagasaki strain (rTbpA and rTbpB, respectively); two groups immunized with native outer membrane proteins with affinity to porcine transferrin (NPAPT), one of them inoculated intramuscularly (NPAPTim) and the other intratracheally (NPAPTit), and the last group receiving a commercially available bacterin (PG). The greatest concentrations of the three positive APPs and the lowest concentration of the negative APP were detected in CTL group, as well as in those animals belonging to rTbpA or rTbpB groups that died in response to challenge. Significant differences (P<0.005) were found in these groups when comparing challenge with the following days after it. However, no significant differences were seen for the remaining vaccinated groups (NPAPTim, NPAPTit and PG), which were effectively protected against Glässer's disease. Therefore, APPs could be used as useful biomarkers for both evaluating disease progression and determining vaccination effectiveness.

Evaluation of efficacy of several disinfectants against Campylobacter jejuni strains by a suspension test.

The objective of this study was to determine the efficacy of 16 active compounds and 11 commercial disinfectants against Campylobacter jejuni. Two reference strains (one of avian origin and the other isolated from bovine) and two avian field strains were tested in suspension test in the presence and absence of serum. Chloramine-T, povidone-iodine (1% available iodine), cetylpiridinium chloride, ethanol, isopropanol, chlorhexidine digluconate, formaldehyde, phenol, and 10 of the 11 commercial formulations (eight of them based on quaternary ammonium compounds) showed an excellent disinfectant capability, resulting in the highest level of reduction (>6-log(10)) in colony-forming units of the four C. jejuni strains compared regardless of the presence or absence of organic material. These compounds might be helpful in the adoption of environmental control measures against C. jejuni.

Development and characterization of protective Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin and comparison with other subunit and commercial vaccines.

Haemophilus parasuis is the agent responsible for causing Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuis serovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was potentiated with a bacterial neuraminidase from Clostridium perfringens. The potential protective effect conferred by these two vaccines was compared to that afforded by two other vaccines, consisting of recombinant transferrin-binding protein (rTbp) A or B fragments from H. parasuis, Nagasaki strain, and by a commercially available inactivated vaccine. Five groups of colostrum-deprived piglets immunized with the vaccines described above, one group per each vaccine, and a group of nonvaccinated control animals were challenged intratracheally with a lethal dose (3 × 108 CFU) of H. parasuis, Nagasaki strain. The two vaccines containing rTbps yielded similar results with minimal protection against death, clinical signs, gross and microscopic lesions, and H. parasuis invasion. In contrast, the two vaccines composed of NPAPT antigen and commercial bacterin resulted in a strong protection against challenge (without deaths and clinical signs), mild histopathological changes, and no recovery of H. parasuis, thus suggesting their effectiveness in preventing Glässer's disease outbreaks caused by serovar 5.

Characterization of a recombinant transferrin-binding protein A (TbpA) fragment from Haemophilus parasuis serovar 5.

Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, possesses iron acquisition pathways mediated by a surface receptor that specifically bind porcine transferrin. This receptor is composed of transferrin-binding protein A (TbpA) and TbpB. As it has been reported for other gram-negative organisms, H. parasuis TbpA could be useful as a candidate target for H. parasuis vaccination. In this study, a 600-bp tbpA fragment of the gene encoding TbpA from H. parasuis serovar 5, the Nagasaki strain, was amplified by PCR and cloned into a pBAD/Thio-TOPO expression vector, generating the pBAD-Thio-TbpA-V5-His (TbpA-His) construction. Escherichia coli LMG194-competent cells were transformed with this construction, followed by the induction of protein expression with arabinose. A band (38.5 kDa) corresponding to a 200-amino acid recombinant TbpA (rTbpA) fragment was seen on the sodium dodecyl sulfate polyacrylamide gel electrophoresis and confirmed by immunoblotting. Polyclonal antibodies raised against this fragment were specific for H. parasuis and Actinobacillus pleuropneumoniae, reacted at the cell surface with H. parasuis, and a significant bactericidal activity was also detected. Therefore, this rTbpA fragment induces an immunological response and might be useful as an antigen for vaccination against Glässer's disease.

Nucleotide sequence and transfer properties of two novel types of Actinobacillus pleuropneumoniae plasmids carrying the tetracycline resistance gene tet(H).

OBJECTIVES: To analyse the sequence and transfer properties of two tetracycline resistance plasmids found in clinical isolates of Actinobacillus pleuropneumoniae in order to assess their role in the spread of tetracycline resistance. METHODS: The plasmids designated p9956 and p12494 were purified from A. pleuropneumoniae and completely sequenced. The transfer properties of both plasmids were evaluated by electroporation and/or conjugation into Pasteurella multocida and Escherichia coli. RESULTS: Both plasmids showed a modular structure consisting of three regions involved in mobilization, tetracycline resistance or replication. The mobilization regions included the mobA gene, encoding a relaxase, a protein involved in plasmid transfer. The tetracycline resistance regions were closely related and consisted of the tet(H) gene and its repressor gene tetR. The tetracycline resistance phenotype was transferred successfully to P. multocida and in the case of p9956 also to E. coli by electroporation of the plasmids. Moreover, plasmid p9956 could be mobilized in E. coli with the assistance of RP4 conjugal transfer functions. CONCLUSIONS: For the first time, the complete sequences of two tet(H)-carrying plasmids from A. pleuropneumoniae were determined. These two plasmids differed from one another and from known tet(H)-carrying plasmids from Pasteurella or Mannheimia spp. Structural analysis confirmed that these plasmids consisted of segments that have been previously detected in members of the families Pasteurellaceae and Enterobacteriaceae.

Distribution of tetracycline resistance genes in Actinobacillus pleuropneumoniae isolates from Spain.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia. Tetracycline is used for therapy of this disease, and A. pleuropneumoniae carrying the tet(B) gene, coding for an efflux protein that reduces the intercellular tetracycline level, has been described previously. Of the 46 tetracycline-resistant (Tc(r)) Spanish A. pleuropneumoniae isolates used in this study, 32 (70%) carried the tet(B) gene, and 30 of these genes were associated with plasmids. Eight (17%) isolates carried the tet(O) gene, two (4%) isolates carried either the tet(H) or the tet(L) gene, and all these genes were associated with plasmids. This is the first description of these tet genes in A. pleuropneumoniae. The last two Tc(r) isolates carried none of the tet genes examined. Except for tet(O)-containing plasmids, the other 34 Tc(r) plasmids were transformable into an Escherichia coli recipient. Two plasmids were completely sequenced. Plasmid p11745, carrying the tet(B) gene, was 5,486 bp and included a rep gene, encoding a replication-related protein, and two open reading frames (ORFs) with homology to mobilization genes of Neisseria gonorrhoeae plasmid pSJ7.4. Plasmid p9555, carrying the tet(L) gene, was 5,672 bp and, based on its G+C content, consisted of two regions, one of putative gram-positive origin containing the tet(L) gene and the other comprising four ORFs organized in an operon-like structure with homology to mobilization genes in other plasmids of gram-negative bacteria.

Changes in antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolated from pigs in Spain during the last decade.

A total of 229 Spanish Actinobacillus pleuropneumoniae isolates recovered from diseased pigs with pleuropneumonia from 1997 to 2004 was tested for their susceptibility to 11 antimicrobials in a broth microdilution method. All the isolates were susceptible to florfenicol and most of them to cephalothin; however, a high rate of resistance was observed to tetracycline. A bimodal or multimodal distribution of isolates over the MIC range were observed for penicillins, tetracycline, trimethoprim, sulfisoxazole and nalidixic acid, suggesting the development of acquired resistance. Eight resistance patterns were established, and 21.1% of the isolates were resistant to at least two antimicrobials. In addition, a considerable increase in the resistance to tetracyclines was observed during the last decade in Spain, when compared with other A. pleuropneumoniae strains isolated during 1987-1988 (Gutiérrez, C.B., Píriz, S., Vadillo, S., Rodríguez Ferri, E.F., 1993. In vitro susceptibility of Actinobacillus pleuropneumoniae strains to 42 antimicrobial agents. Am. J. Vet. Res. 54, 546-550); this finding was also observed for gentamicin in minor percentage.

Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis.

Haemophilus parasuis is the causative agent of Glässer's disease, which is responsible for considerable economic losses in the pig-rearing industry. The aim of the study reported here was the identification, sequencing and molecular characterization of the TonB region that includes tonB, exbBD, and tbpBA genes in H. parasuis. In addition, two fusion proteins were generated. One of them (pGEX-6P-1-GST-TbpB) contained the first 501 amino acids of H. parasuis TbpB protein, while the second (pBAD-Thio-TbpB-V5-His) included the first 102 amino acids of H. parasuis TbpB N-terminus domain. A panel of 14 hybridomas secreting monoclonal antibodies was raised against the two recombinant TbpB fusion proteins. Furthermore, to assess whether the expression of the H. parasuis ExbB, TbpB, and TbpA proteins was upregulated under conditions of restricted availability of iron, a rabbit polyclonal antibody against H. parasuis TbpB-His fusion protein was produced. A rabbit polyclonal antibody against serotype 7 of Actinobacillus pleuropneumoniae ExbB and TbpA proteins was also used for the detection of the homologous proteins in H. parasuis. Overall, the data indicate that H. parasuis, like other members of the Pasteurellaceae family, possesses the genetic elements of the TonB region for iron acquisition and the transferrin-binding proteins encoded under this region are upregulated under restricted iron availability.