Antimicrobial resistance of Pasteurella multocida isolates recovered from swine pneumonia in Spain throughout 2017 and 2018.

A total of 32 Pasteurella multocida isolates were obtained from 60 cases of swine pneumonic lungs collected in "Castilla y León" (northwestern Spain) between November 2017 and April 2018. Capsular type A isolates were isolated from 96.9% cases and capsular type D from the remaining 3.1%. All isolates were characterized for their susceptibilities to eight antimicrobial agents and the presence of eight resistance genes. The frequency of susceptibility was lower than 60% in four of the drugs, 84.4% of the isolates showed resistance to at least two compounds, and 46.9% to a combination of three drugs. The resistance patterns suggested that enrofloxacin, chloramphenicol, tetracycline and cefotaxime were the compounds most likely active to P. multocida. The usage of PCR revealed that ermC, bla ROB1, tetB and msrE genes occurred in more than 37.0% isolates, that suggested its putative accountability in the resistance of the strains harbor them. However, most were detected in susceptible strains and only a genetic explanation for the resistance could be linked to erythromycin. Therefore, the resistances to clyndamicin, cotrimoxazol, ß-lactams and tetracyclin observed by phenotypic testing remains genetically unexplained and further investigations are required.

Molecular study of an outer fragment of Haemophilus parasuis neuraminidase and utility with diagnostic and immunogen purposes.

Haemophilus parasuis is a swine pathogenic organism, being the causative agent of Glässer's disease. It has got some virulence factors, some of which act as potential candidates for the vaccine developing. Among them there is the neuraminidase enzyme, which is located inside the outer membrane and contains a ß-barrel domain with seven external loops. By using the polymerase chain reaction technique, the ß-barrel fragment was amplified, sequenced and analysed for the 15 H. parasuis reference serotypes. The results showed a small diversity for them, except for serotype 2, which has a deletion that covers the loops with potential to be used as vaccine antigen. However, some of the other serotypes showed the same nucleotidic sequence between them, such those 6 and 7 or those 12 and 13. This fact was also confirmed by means of phylogenetic analysis. For these reasons, the tested fragment might result in a putative candidate for the development of subunit vaccines against all the serotypes causing Glässer's disease outbreaks, with the exception of serotype 2, alone or in combination with other proven immunogenicity molecules. Anyway, further studies should be carried out in pigs in order to confirm this hypothesis. Finally, this outer fragment of H. parasuis neuraminidase could be used as a suitable diagnostic tool at a species level, for instance, by PCR.

Altered indirect hemagglutination method for easy serotyping of Haemophilus parasuis / Método de Hemaglutinação Indireta alterado para facilitar a sorotipificação de Haemophilus parasuis

Glässer's disease is an emergent bacterial disease that affects swine husbandries worldwide causing important economic losses. The aetiological agent, Haemophilus parasuis, is currently divided in fifteen serovars but an increasing number of non-typeable serovars have been reported. Indirect hemagglutination (IHA) is indicated as a serotyping method for H. parasuis. In the present study, we describe an additional step that aims to work around a possible obstacle in the original protocol that may compromise the outcome of this assay. We observed that the choice of anticoagulant for blood collection influences and/or impairs spontaneous adsorption of H. parasuis antigens on sheep red blood cells (SRBCs). However, regardless of the anticoagulant used, chemical treatment of SRBCs with tannic acid induces a stable antigen adsorption (sensitization step). The addition of 1% BSA to SRBCs washing buffer and to antisera dilution augments IHA specificity. Tannic acid treated SRBCs combined with thermo-resistant H. parasuis antigens increases the assay resolution. Thus, our results demonstrate an improvement in the technique of H. parasuis serotyping that will prove valuable to understand Glässer's disease epidemiology and to better characterize serovars involved in outbreaks.(AU)

A Doença de Glässer é uma doença bacteriana emergente que afeta a produção de suínos em todo o mundo e causa importantes perdas econômicas. O agente etiológico, Haemophilus parasuis, é atualmente dividido em quinze sorovares; no entanto, um número crescente de cepas não tipificáveis tem sido relatado. O teste de hemaglutinação indireta (IHA) tem sido utilizado para a sorotipificação de H. parasuis. Neste estudo, descrevemos uma alteração no protocolo original de IHA e que supera uma limitação específica que pode comprometer o uso geral deste ensaio. Descobrimos que o tipo de anticoagulante utilizado para coletar os eritrócitos ovinos (SRBCs) pode comprometer a adsorção espontânea dos antígenos do H. parasuis. Por outro lado, o tratamento químico dos SRBCs com ácido tânico promove uma adsorção antigênica estável (passo de sensibilização) e independente do anticoagulante utilizado. O uso de 1% de BSA durante as lavagens dos SRBCs e na diluição dos antissoros incrementa a especificidade da IHA e, a combinação dos SRBCs tratados quimicamente com antígenos de H. parasuis termo-resistentes aumentam a resolução da IHA. Nossos resultados destacam uma melhoria na principal técnica de sorotipificação de H. parasuis, que auxiliará diretamente no entendimento da epidemiologia da Doença de Glässer e na caracterização dos sorovares envolvidos em surtos da doença.(AU)

Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis.

The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs.

Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin prevent the expression of proinflammatory chemokines and cytokines in pigs.

The expression of chemokines (CCL-2 and CXCL-8) and cytokines (IL-1 α , IL-1 ß , IL-6, TNF- α , and IL-10) was evaluated by RT-qPCR in colostrum-deprived pigs vaccinated and challenged with Haemophilus parasuis serovar 5. Two vaccines containing native proteins with affinity to porcine transferrin (NPAPTim and NPAPTit) were tested, along with two control groups: one inoculated with PBS instead of antigen (challenge group (CHG)), and another one nonimmunized and noninfected (blank group). The use of NPAPTim and NPAPTit resulted in complete protection against H. parasuis (no clinical signs and/or lesions), and both vaccines were capable of avoiding the expression of the proinflammatory molecules to levels similar to physiological values in blank group. However, overexpression of all proinflammatory molecules was observed in CHG group, mainly in the target infection tissues (brain, lungs, and spleen). High expression of CCL-2, CXCL-8, IL-1 α , IL-1 ß , and IL-6 can be considered one of the characteristics of H. parasuis infection by serovar 5.

New insights in cellular immune response in colostrum-deprived pigs after immunization with subunit and commercial vaccines against Glässer's disease.

Four groups of colostrum-deprived pigs were immunized with Porcilis Glässer® (PG) or with subunit vaccines developed by us (rTbpA, NPAPT(M) or NPAPT(Cp)) against Glässer's disease, and they were challenged with 3×10(8)CFU of Haemophilus parasuis. A strong reduction in CD3(+)γδTCR(+) cells was seen in non-immunized control and scarcely protected (rTbpA) groups, suggesting that these cells could represent a target of H. parasuis infection. A significant increase in CD172α(+)CD163(+) cells was detected in all groups but PG, while a reduction in SLAIIDR(+) molecules expression was observed after challenge in control animals. Significant increases in CD3ε(+)CD8α(+)CD8ß(+) and B cells were detected respectively in control and NPAPT groups, and in scarcely (rTbpA) and well-protected (NPAPT(M) and NPAPT(Cp)) groups. Finally, a greater response in CD4(+)CD8α(-) cells was observed in NPAPT(Cp) compared to NPAPT(M) and PG groups. These results state the potential of NPAPT antigen for developing effective vaccines against Glässer's disease.

Comparison of real-time PCR and culture isolation in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis.

AIMS: A real-time PCR (RT-PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol 18, 50-58.), evaluating different subunit or commercial vaccines. METHODS AND RESULTS: Samples from different tissues of 24 experimentally infected and challenged colostrum-deprived piglets were tested. The RT-PCR gave globally a 23·3% more of positive results than culture, and all samples being positive by culture were positive by RT-PCR also. H. parasuis could not be cultured from any of the samples of the piglets included in the three vaccinated groups resulting in a strong protection, but it could be detected by RT-PCR in six samples in the group immunized with the commercial vaccine, in three in that vaccinated with native proteins with affinity to porcine transferrin (NPAPT) administered intramuscularly and in only two in that immunized with NPAPT intratracheally. CONCLUSIONS: The RT-PCR was more sensitive than culture for H. parasuis detection in the organs compared. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-PCR evidenced that NPAPT vaccines were those yielding the best protection results in terms of H. parasuis clearance.

Complementary tools for the control and eradication of caprine and ovine brucellosis in the European Union.

Caprine and ovine brucellosis is one of the most serious and complex animal health problems faced by Veterinary Services in countries where the disease is endemic. Various geographical factors and the nature of the disease itself influence its epidemiology, encouraging widespread distribution and, at the same time, impeding the ability of animal health programmes to prevent, control and eradicate it. Although strategies against brucellosis have traditionally been based on two specific tools (namely, vaccination of the at-risk population and testing and slaughter of animals which are suspected of or test positive for the disease), other complementary tools of a technical or administrative nature should also be considered. Experience in the European Union has shown that these tools are necessary to guarantee sustainable progress and success against this disease. However, these complementary tools have not always received sufficient attention during the strategic planning and subsequent implementation of animal health programmes, with consequent reductions in efficiency. The aim of this article is to review these complementary tools, in order to facilitate their adoption and use by official Veterinary Services, according to the resources available.

Comparative efficacy of several disinfectants in suspension and carrier tests against Haemophilus parasuis serovars 1 and 5.

The comparative efficacy of 16 active compounds (including the most commonly used chemical groups) and 10 commercial formulations against Haemophilus parasuis serovars 1 and 5 was studied. These organisms were tested in suspension and carrier tests in the presence and absence of serum as representative of organic matter. Chloramine-T and half of the formulations from commercial sources (most of them including quaternary ammonium compounds) were effective in both in vitro tests, regardless of the presence or absence of organic load. All 26 disinfectants except for an iodophor (0.1% available iodine) resulted in at least 3-log(10) reduction in colony-forming units in suspension test, and most of them resulted in the maximal level of detection (>6-log(10) reduction). On the other hand, disinfectants were not as effective in carrier test as in suspension test, and the presence of serum considerably reduced the activities of most of the compounds tested, especially in carrier test. These results suggest the importance of selecting suitable disinfection for routine use on surfaces contaminated with H. parasuis, particularly when organic matter is present. Chloramine-T and formulations 2 and 7-10 are recommended for a complete inactivation of H. parasuis in swine herds.

Acute-phase protein response in pigs experimentally infected with Haemophilus parasuis.

The acute-phase protein (APP) response to an infection caused by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized measuring serum concentrations of pig major acute-phase protein (pig MAP), haptoglobin (HPT), C-reactive protein (CRP) and apolipoprotein A-I (ApoA-I) in colostrum-deprived pigs. They were divided into six experimental groups: non-immunized control group (I); immunized with a non-commercial bacterin (II); with an OMP-vaccine (III); with a sublethal dose (IV); and with two commercial bacterins (V and VI). All groups were challenged intratracheally with 5 × 10(9)CFU of H. parasuis 37 days after immunisation. The highest levels of the positive APPs (pig MAP, HPT and CRP) and the lowest levels of the negative APPs (ApoA-I) were observed in the animals that died as a consequence of the infection, both those in the non-immunized and in the immunized groups. However, the surviving animals (all of them in groups II, V and VI, two pigs in group III, and three in group IV) showed a minor variation in APP response, mainly on day 1 post-challenge (p.c.), and then tended to recover the initial values. APP response was still less pronounced in the groups of pigs previously immunized with bacterins. In conclusion, APP response can reflect Glässer-disease ongoing, showing a correlation between the severity and duration of the clinical signs and lesions and the magnitude of changes in the APP levels.

Effect of different vaccine formulations on the development of Glässer's disease induced in pigs by experimental Haemophilus parasuis infection.

Four groups of pigs immunized with different vaccines and a group of non-vaccinated controls were challenged intratracheally with a lethal dose (5 x 10(9) colony-forming units) of Haemophilus parasuis, the aetiological agent of Glässer's disease. A vaccine containing inactivated whole organisms gave strong protection against clinical signs, death, pathological changes and persistence of organisms in vivo. However, all non-immunized pigs, all pigs given a vaccine consisting of the recombinant transferring-binding protein (Tbp) B, some pigs given an outer membrane protein (OMP) formulation enriched with TbpB and some pigs immunized with a sub-lethal dose of live organisms died at various times after challenge, yielding positive cultures from most organs post mortem and having shown hyperthermia and other clinical signs before death. Animals that died showed fibrinosuppurative polyserositis, exudative pneumonia, and lesions compatible with acute septicaemia, e.g., disseminated intravascular coagulation with multiple fibrinous thrombi in arterioles and capillaries, depletion of splenic white pulp, and acute lymphadenitis. The results suggested that, in addition to the protection given by inactivated whole organisms, partial protection was given by the OMP formulation and by a sub-lethal dose of living organisms; however, the recombinant TbpB preparation gave no protection.

Systemic antibody response in colostrum-deprived pigs experimentally infected with Haemophilus parasuis.

The serum antibody response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized by ELISA measuring IgM and IgGt levels against whole-cells and outer-membrane-proteins (OMPs) as antigens. Five groups of pigs were studied, four of those were previously immunized with different formulations, and the fifth was maintained as non-immunized control. All groups were challenged with 5x10(9) CFU of H. parasuis. The non-commercial bacterin induced a full protection against disease, the OMP-vaccine and the exposure to a sublethal dose of 10(5) CFU protected only partially, and the recombinant TbpB-vaccine conferred no protection. The humoral response in the pigs that died after infection (all controls, all those vaccinated with the recombinant TbpB, and two of both those inoculated with OMPs and those exposed to the sublethal dose) could be only measured before it, but it was irrelevant in all cases. However, a specific IgM and IgGt production was observed before challenge in all the surviving pigs, irrespective of the type of immunization received. This antibody response was even greater after H. parasuis infection, especially in those survivors receiving the sublethal dose. These results suggest a role of the antibodies developed after the different immunization protocols in preventing infection and death; therefore, the humoral immunity is protective against experimental Glässer's disease.

Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis.

The cellular immune response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized studying changes in peripheral blood mononuclear cells (PBMC) in colostrum-deprived pigs. Five groups were studied, four of those were previously immunized with different formulations and the fifth was maintained as non-immunized control. All groups were challenged with 5 x 10(9) CFU of H. parasuis serotype 5. The non-commercial bacterin conferred a complete protection, while the OMP-vaccine and the exposure to a subletal dose of 10(5) CFU of H. parasuis protected only partially, and the recombinant Tbp B-vaccine induced no protection. PBMC were analyzed using monoclonal antibodies against porcine CD45(+), CD3(+), CD4(+), CD8alpha(+), CD25(+), CD4(+) naïve, alphaIgM(+) and SWC3(+) cells in single-colour fluorescence, and CD4(+)/CD8alpha(+) and CD8alpha(+)/CD8beta(+) combinations in two-colour fluorescence. The different groups showed no significant changes in PBMC subsets following vaccination, and only minor changes were encountered after challenge, consisting mainly of significant increases (P<0.05) in the relative proportions of monocytes and granulocytes (SWC3(+)) and B cells (alphaIgM(+)), as well as a significant reduction in CD3(+) cells (P<0.05). These changes were similar for the five groups compared, except for the significant increase of CD25(+) cells, which was only observed for the bacterin-vaccinated group. These results suggest an increase of trafficking of inflammatory cells and the onset of the adaptive antibody response against H. parasuis infection; in addition, the blood cellular response developed by the different groups was not relevant to protection.

Evaluation of different API systems for identification of porcine Pasteurella multocida isolates.

An exhaustive biochemical characterisation of 60 porcine Pasteurella multocida clinical isolates recovered from lesions indicative of pneumonia, previously confirmed by PCR and all belonging to the capsular serogroup A, was performed by means of four commercial systems. The API 20NE correctly identified almost all isolates (95%), but only 60% could be ascribed to this species by the API 20E method. The high diversity exhibited by the API 50CHB/E system, with six different patterns, does not advise its use as additional system for a definitive identification at the species level, but this method could be a potential tool for characterising P. multocida isolates below this level. The more uniform reactions yielded by the API ZYM test make this system helpful in the confirmatory identification of this organism. The high variability (20 profiles) obtained when the four systems are taken together also suggests their usefulness for epidemiological purposes in order to sub-type P. multocida isolates.

aroA gene PCR-RFLP diversity patterns in Haemophilus parasuis and Actinobacillus species.

The Haemophilus parasuis aroA gene encodes 5-enolpyruvylshikimate-3-phosphate synthase and participates in the aromatic amino acids and the folic acid universal metabolic pathway of bacteria. The application of aroA-based PCR-RFLP methodology yields a significant degree of diversity in H. parasuis and Actinobacillus species. PCR amplification of the aroA gene rendered a 1,067-bp fragment in all 15 H. parasuis serovars, and also in Actinobacillus pleuropneumoniae serotypes 1-12, Actinobacillus lignieresii, Actinobacillus equuli, Actinobacillus porcinus, Actinobacillus rossii, Actinobacillus suis, Actinobacillus ureae, Actinobacillus minor and Actinobacillus indolicus. Sau3AI and RsaI digestions of the aroA PCR products rendered seven different restriction fragment length polymorphism (RFLP) patterns: group I (H. parasuis serovars 1, 2, 4-6, and 8-15, A. porcinus and A. ureae), group II (H. parasuis serovars 3 and 7, and A. pleuropneumoniae serotypes 1, 4, 5, 9, 11 and 12), group III (A. lignieresii), group IV (A. pleuropneumoniae serotype 7), group V (A. pleuropneumoniae serotypes 2, 3, 6 and 8, A. equuli, A. rossii, A. minor and A. indolicus), group VI (A. suis) and group VII (A. pleuropneumoniae serotype 10). This is the first report describing the presence of aroA gene in H. parasuis, A. lignieresii, A. porcinus, A. rossii, A. suis, A. ureae, A. minor and A. indolicus and the data presented here demonstrates a significant degree of aroA genetic diversity in H. parasuis and species of the genus Actinobacillus.

Identification of sulI allele of dihydropteroate synthase by representational difference analysis in Haemophilus parasuis serovar 2.

AIMS: Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA). METHODS AND RESULTS: Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7). CONCLUSION: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique. SIGNIFICANCE AND IMPACT OF THE STUDY: The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.

Kinetics of infection and effects on the placenta of Chlamydophila abortus in experimentally infected pregnant ewes.

A Chlamydophila abortus-induced abortion model was carried out on the basis of the experimental infection of ewes at day 75 of gestation. The infection induced abortions and the birth of weak lambs during the last 3 weeks of pregnancy. To study the kinetics of the infection in the placenta and in other organs, infected ewes were killed at 105, 120, and 130 days of gestation and also several days after abortion or parturition. Infected ewes developed a systemic infection that caused a mild and transient pneumonia and focal hepatitis. Pathologic changes were observed in placentas at 120 day of gestation, although the lesions varied between animals and even between placentomes of the same placenta. The first placental area infected was the maternal stroma and epithelium next to the intercaruncular areas, where neutrophilic response seemed to control the infection. A substantial degree of multiplication of C. abortus was then observed in the trophoblast cells of the placentome, periplacentomal choriallantoic membranes, and hilius, with an inflammatory exudate composed mainly of neutrophils, some macrophages, and very scarce lymphocytes. After abortion, the lesions affected the intercotyledonary areas of the aborted placentas, whereas in the uterus significant lymphocyte infiltration was observed, together with a rapid decrease of the C. abortus antigen in the degenerated caruncular tissues.

Efficacy of different commercial and new inactivated vaccines against ovine enzootic abortion.

The protective efficacy of two inactivated commercial (A, B) and two new inactivated vaccines (M7, QS) against ovine enzootic abortion was determined in two separate experiments in sheep. Vaccine A contained chlamydiae propagated in chicken embryos, adjuvated with Marcol 82, and vaccine B contained chlamydiae cultured in cell monolayers, adjuvated with aluminium hydroxide. For the preparation of the experimental vaccines, Chlamydophila abortus AB7 strain was cultured in McCoy cells and adjuvated with QS-21 (QS) or Montanide ISA 773 (M7). The ewes were vaccinated twice subcutaneously and challenged at 90 days of gestation. Protection was evaluated by clinical, bacteriological and serological examinations, and compared to two control groups: one of infected but not vaccinated ewes, and another of vaccinated but not infected ewes. The experimental vaccines induced considerably better protection than the two commercial ones. The new vaccine M7 especially showed no abortions, a good antibody response, the highest newborn lamb weights and the lowest level of C. abortus shedding at lambing.

In vitro susceptibility of field isolates of Francisella tularensis subsp. holarctica recovered in Spain to several antimicrobial agents.

Forty-two recent (1997-1999) Spanish isolates of Francisella tularensis subsp.holarctica were tested in a broth microdilution method for their susceptibilities to 29 antimicrobial agents, including penicillins, cephalosporins, cephamicins, monobactams, penems, aminoglycosides, tetracyclines, macrolides, quinolones, chloramphenicol and fosfomycin. The isolates were resistant to beta-lactam antibiotics and susceptible to chloramphenicol, ciprofloxacin, levofloxacin and norfloxacin.