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.

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.

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.

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.

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.

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.

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.

Preliminary evidence that different domains are involved in cytolytic activity and receptor (cholesterol) binding in listeriolysin O, the Listeria monocytogenes thiol-activated toxin.

The inactive truncated 52 Kilodaltons (kDa) Listeriolysin O (LLO) produced by a transposon Tn1545-induced Listeria monocytogenes non-hemolytic/avirulent mutant previously described (Gaillard et al. (1986) Infect. Immun. 52, 50-55), that lacks a 48 aminoacid fragment at the C-terminal end including the single cysteine residue essential for activity (Mengaud et al. (1988) Infect. Immun. 56, 766-772), bound to the SH-cytolysin membrane receptor cholesterol, as did the active 60 kDa toxin. These results indicate that the missing fragment is a functionally important region needed in the 60 kDa LLO to cause membrane-disruption but not to bind to cholesterol, which strongly suggests that in LLO (and presumably in the other SH-cytolysins, in accordance with their structural and functional homologies) different domains are involved in cytolytic activity and cholesterol binding. The cysteine residue contained in the missing fragment, therefore, would not be essential for cholesterol binding, as is currently thought, rather it seems to be essential specifically for cell lysis.

Actinobacillus pleuropneumoniae does not require urease activity to produce acute swine pleuropneumonia.

The role in virulence of Actinobacillus pleuropneumoniae urease activity was investigated. A urease-negative mutant was isolated following transposon mutagenesis with a mini-Tn10 derivative. Both the parent strain and the urease-negative mutant exhibited identical LD50 values in a murine infection model. Pig challenge confirmed that the urease-negative mutant was fully virulent, since experimental inoculation with 5 x 10(7) colony forming units resulted in an acute disease indistinguishable from that produced by the wild-type strain at the same dose. Our results demonstrate that urease activity is not required for the development of acute pleuropneumonia.

Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide of Actinobacillus pleuropneumoniae serotype 2 and their use in the classification of field isolates.

Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.

Characterization of monoclonal antibodies that recognize common epitopes located on O antigen of lipopolysaccharide of serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae.

Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae (reference strain Shope 4074) were produced and characterized. All hybridomas secreting mAbs were reactive with whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5D6 that failed to recognized serotype 9. They did not react with other taxonomically related Gram-negative organisms tested. The predominant isotype was immunoglobulin (Ig) M, although IgG2a, IgG2b, and IgG3 were also obtained. The epitopes identified by these mAbs were resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however, they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection assay showed that mAbs could be divided in two groups according to the patterns of immunoreaction observed. Group 1 (mAbs 3E10, 4B7, 9H5 and 11C3) recognized a ladder-like banding profile consistent with the O antigen of lipopolysaccharide (LPS) from smooth strains. Group II (mAbs 3B10 and 9H1) recognized a long smear of high molecular weight which ranged from 60 to 200 kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis, immunodiffusion and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae which were located on the O antigen of LPS.

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.

The insertion element IS 6110 is a useful tool for DNA fingerprinting of Mycobacterium bovis isolates from cattle and goats in Spain.

A total of 129 Mycobacterium bovis strains from 5 different Spanish locations were fingerprinted using the IS6110 repetitive element. We demonstrated the presence of multiple copies (from 2 to 13) of IS6110 in a large proportion (47.4%) of the M.bovis strains isolated from cattle and we showed that these strains can be successfully differentiated by means of the RFLP with IS6110. All of the M. bovis strains isolated from goats had multiple copies of IS6110 and 4 bands of 2, 1.7, 1.4 and 1.3 kb were common in all the caprine RFLP patterns. The caprine strains formed a clearly separate cluster from the bovine strains.

Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain.

A field comparison of the interferon-gamma (IFN-gamma) assay and the single intradermal cervical tuberculin (SICT) test for the diagnosis of bovine tuberculosis was conducted. A total of 1136 cattle belonging to 85 herds placed in 'Castilla y León' (northwestern Spain) were chosen, and 21 of these herds were subjected to the diagnostic assays two or three times at intervals of at least 4 months. All the animals positive to any of the tests were slaughtered and tuberculosis was confirmed by culture isolation method (CIM) and further identification by means of PCR. Only 10.6% of cattle reacted with the bovine PPD in the SICT test, a percentage that increased to 12.8% in the IFN-gamma assay. The sensitivity of the IFN-gamma assay compared to CIM was shown to be higher (84.9%) than that of the SICT test (80.2%), but the combination of both tests offered the highest sensitivity (92.9%). The number of false positive reactors (those animals in which CIM was negative) was considerably higher for the IFN-gamma assay than for the SICT test and, conversely, the number of false negative animals (M. bovis isolation but negative immunological result) was higher for the skin test than for the interferon assay. In the herds tested twice, tuberculosis was eradicated after the second cycle of testing in 50%, and in 75% after the third cycle in herds tested three times. The combination of these two techniques instead of separately seems, therefore, to be useful in eradication programmes against bovine tuberculosis.

Typing of Haemophilus parasuis strains by PCR-RFLP analysis of the tbpA gene.

On the basis of a species-specific PCR assay, a RFLP analysis for typing of Haemophilus parasuis strains was developed and evaluated. Amplification was based on the gene tbpA, encoding a transferrin-binding protein. RFLP analysis of the 1.9-kb tbpA-amplicon using TaqI, AvaI and RsaI endonucleases produced 12 different patterns for the reference strains of the 15 known H. parasuis serovars, and showed a high heterogeneity (33 RFLP groups) for 101 H. parasuis clinical isolates tested. The sensitivity, typeability (100% versus 65% for immunodiffusion), high degree of discrimination (0.93 versus 0.84 for immunodiffusion), simplicity and low cost per test make this PCR-RFLP assay a useful method for typing H. parasuis and, therefore, for studying the epidemiology of outbreaks of Glässer's disease.

Characterization of V factor-dependent organisms of the family Pasteurellaceae isolated from porcine pneumonic lungs in Spain.

116 V factor (NAD)-dependent strains belonging to the family Pasterurellaceae isolated from porcine pneumonic lungs were collected in Spain over a period of 1 yr and studied using 52 biochemical characters. In addition to Actinobacillus pleuropneumoniae (72 strains), Haemophilus taxon minor group (37 strains) and Taxon D (four strains), other taxon (three strains) were observed. This taxon, provisionally designated as Haemophilus sp. sorbitol+, is closed to A. pleuropneumoniae but differed by some biochemical characteristics. Among A. pleuropneumoniae strains, nine different serotypes were detected, the most frequent being serotypes 4 and 2.