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A collection of 177 Francisella tularensis subsp. holarctica clinical isolates (29 from humans and 148 from animals, mainly hares and voles) was gathered from diverse tularemia outbreaks in the Castilla y León region (northwestern Spain) that occurred from the end of the 20th century to the 2020s. Along with four F. tularensis subsp. holarctica reference strains, all of these clinical isolates were tested using a broth microdilution method to determine their susceptibility to 22 antimicrobial agents, including β-lactams, aminoglycosides and one member each of the tetracycline, glycylcycline, quinolone and sulphonamide classes. Many multi-resistance profiles were found among the tested isolates, but especially among those of human origin (all but two isolates showed resistance to at least 13 of 18 antimicrobial agents). Even so, all human isolates were susceptible to gentamicin and tobramycin, while more than 96% of animal isolates were susceptible to these two aminoglycosides. Ciprofloxacin showed activity against more than 92% of animal and human isolates. However, almost 21% of human isolates were resistant to tetracycline, and more than 65% were resistant to tigecycline. Finally, a quite similar activity to other F. tularensis subsp. holarctica isolates collected 20 years earlier in Spain was observed.

In this study, tularemia outbreaks associated with humans and several domestic and wild animals (Iberian hares, wild rabbits, voles, mice, grey shrews, sheep, dogs, foxes, wolves, ticks, and river crayfish) are reported in Spain from 2007 to 2020. Special attention was paid to the outbreaks in humans in 2007-2009 and 2014-2015, when the most important waves occurred. Moreover, positive rates of tularemia in lagomorphs were detected in 2007-2010, followed by negative results in 2011-2013, before again returning to positive rates in 2014 and in 2017 and in 2019-2020. Lagomorphs role in spreading Francisella tularensis in the epidemiological chain could not be discarded. F. tularensis is described for the first time infecting the shrew Crocidura russula worldwide, and it is also reported for the first time infecting wild rabbits ( Oryctolagus cuniculus ) in Spain. Serological positives higher than 0.4% were seen for sheep only from 2007-2009 and again in 2019, while serological rates greater than 1% were revealed in dogs in 2007-2008 and in wild canids in 2016. F. tularensis were detected in ticks in 2009, 2014-2015, 2017, and 2019. Lastly, negative results were achieved for river crayfish and also in environmental water samples from 2007 to 2020.

TLRs, Siglecs and CD163 are cell surface receptors that play an important role in immune response and sepsis. The objective of this study was to assess changes in the expression levels of several of these receptors (TLR2, TLR4, CD163, Siglec-1, Siglec-3, Siglec-5 and Siglec-10) on the surface of peripheral blood mononuclear cells from pigs with sepsis caused by Haemophilus parasuis. Flow cytometry was employed to analyze samples from an experimental infection and from cell cultures. A significant increase in CD163, TLR2 and Siglec-3 expression during infection was seen. However, in vitro exposure of peripheral blood monocytes to bacteria or sera from infected pigs did not increase the expression of these receptors. These changes may be due to recruitment of monocytes into the blood compartment in response to H. parasuis-induced sepsis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 erm C, bla ROB1 , tet B and msr E 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, β-lactams and tetracyclin observed by phenotypic testing remains genetically unexplained and further investigations are required., (© 2018 The Authors.)

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.

Three recombinant outer membrane proteins (rOmps) from the Haemophilus parasuis Nagasaki strain (serovar 5 reference strain), rOmpP2, rOmpP5 and rOmpD15, which have previously shown protection against H. parasuis infection in mice, were cloned, expressed and evaluated as vaccine antigens in colostrum-deprived pigs. When these animals were immunized with these rOmps and were later challenged intratracheally with 10 8 CFUs of the Nagasaki strain, no protection was seen in terms of survival, clinical signs, pathological results and recovery of H. parasuis. We hypothesized that a possible explanation for this lack of protection could be the low number of epitopes accessible to the immune system as a consequence of their poor exposure on the bacterial surface so that the immune response would not be able to protect against experimental infection by H. parasuis when a fully susceptible animal model, such as pigs, was used., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 β-barrel domain with seven external loops. By using the polymerase chain reaction technique, the β-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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vaccines have become fundamental in the control and elimination of Glässer Disease, a systemic disease of pigs caused by Haemophilus parasuis. The classic vaccines available for prevention of this infection were developed without a robust knowledge about host immunological mechanisms. In this study, we demonstrated the presence of cross-reactive epitopes on both the N-lobe and C-lobe of variants of transferrin binding protein B (TbpBs) expressed on the surface of 6 virulent serovars of H. parasuis. Antibodies against TbpB-derived antigens were capable of increasing the phagocytic capacity of neutrophils and were also capable of blocking porcine transferrin from binding to TbpB. Surprisingly, none of the pig or mice antisera from animals immunized with TbpB-derived antigens mixed with Montanide IMS 2215 VG PR adjuvant were able to activate the classical complement pathway (CCP). In contrast, antisera from mice immunized with TbpB-derived antigens adjuvanted with Freund's adjuvants or Montanide Gel 01 were able to activate the CCP and kill H. parasuis. Our results demonstrate that the type of adjuvant can modulate the functional response induced by TbpB-derived antigens. Based on these results, we propose that a properly formulated TbpB-based vaccine may elicit a functional protective antibody response with broad cross-reactivity against heterologous strains of H. parasuis.

This study aimed to characterize the type of immune response induced by an experimental vaccine based on a mutant Haemophilus parasuis transferrin binding protein (Tbp) B (Y167A) defective in its ability to bind porcine transferrin. Clinical and pathological signs, bacterial clearance, antibody response and the cytokine profile in alveolar macrophages and spleen after the vaccination and challenge of twenty-two colostrum-deprived pigs with 10(8) CFU of H. parasuis were analysed. Pigs vaccinated with Y167A were compared to those vaccinated with native TbpB (nTbpB), those treated with a commercial bacterin (CB) against Glässer's disease, those unvaccinated challenged (CH) and those unvaccinated unchallenged (UNCH) pigs. The rectal temperatures of Y167A pigs resembled those of UNCH pigs and were significantly lower than those of the nTbpB, CB and CH animals. A major reduction in pathological changes of the challenged pigs was observed in the Y167A group. H. parasuis was cleared from 88.9% of the samples from Y167A pigs versus 60.0% and 55.6% from those of the CB and nTbpB groups, respectively. The antibody response elicited by Y167A by ELISA was notably higher than that observed for nTbpB and CB pigs and was capable of preventing the expression and secretion of IL-8. The expression of IL-4 and IL-5, which were associated with the specific antibody levels, suggests that the main mechanism of protection conferred by Y167A vaccine is based on a strong T-helper 2 response., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Tularemia outbreaks occurred in northwestern Spain in 1997-1998 and 2007-2008 and affected >1,000 persons. We assessed isolates involved in these outbreaks by using pulsed-field gel electrophoresis with 2 restriction enzymes and multilocus variable number tandem repeat analysis of 16 genomic loci of Francisella tularensis, the cause of this disease. Isolates were divided into 3 pulsotypes by pulsed-field gel electrophoresis and 8 allelic profiles by multilocus variable number tandem repeat analysis. Isolates obtained from the second tularemia outbreak had the same genotypes as isolates obtained from the first outbreak. Both outbreaks were caused by genotypes of genetic subclade B.Br:FTNF002-00, which is widely distributed in countries in central and western Europe. Thus, reemergence of tularemia in Spain was not caused by the reintroduction of exotic strains, but probably by persistence of local reservoirs of infection.

Background: The pathogenesis of Haemophilus parasuis depends on the bacterium's ability to interact with endothelial cells and invade adjacent tissues. In this study, we investigated the abilities of eight H. parasuis reference strains belonging to serovars 1, 2, 4, 5, 7, 9, 10 and 13 to adhere to and invade porcine aortic endothelial cells (AOC-45 cell line)., Results: The strains belonging to serovars 1, 2 and 5 were able to attach at high rates between 60 and 240 min of incubation, and serovars 4, 7 and 13 had moderate attachment rates; however, the strains belonging to serovars 9 and 10 had low adherence at all time points. Strong adherence was observed by scanning electron microscopy for the strains of serovars 5 and 4, which had high and moderate numbers, respectively, of H. parasuis cells attached to AOC-45 cells after 240 min of incubation. The highest invasiveness was reached at 180 min by the serovar 4 strain, followed by the serovar 5 strain at 240 min. The invasion results differed substantially depending on the strain., Conclusion: The reference strains of H. parasuis serovars 1, 2, 4 and 5 exhibited high adhesion and invasion levels to AOC-45 porcine aorta endothelial cells, and these findings could aid to better explain the pathogenesis of the disease caused by these serovars.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The expression of chemokines (CCL-2 and CXCL-8) and cytokines (IL-1 α , IL-1 β , 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 β , and IL-6 can be considered one of the characteristics of H. parasuis infection by serovar 5.

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.

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(+)CD8β(+) 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2008 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

Bacteria have evolved a set of highly specialized proteins to capture iron in iron-depleted environments. The acquisition and uptake of iron present in the extracellular milieu of eukaryotic organisms is indispensable for the growth and survival of microbial pathogens in the course of infection. Haemophilus parasuis is the causative agent of Glässer disease, which is responsible for considerable financial losses in pig-rearing worldwide. To gain insight into the mechanisms involved in siderophore-mediated iron uptake in H. parasuis, genes in the H. parasuis ferric hydroxamate uptake (Fhu) region were amplified in the work being reported here. As has been described in A. pleuropneumoniae, an Fhu genomic region was also present in H. parasuis, being composed of four potential consecutive open reading frames (ORF) designated as fhuC, fhuD, fhuB, and fhuA, respectively. By immunoblotting, using a cross-reactive polyclonal antibody raised against Actinobacillus pleuropneumoniae FhuA protein, it was demonstrated that this protein was constitutively expressed in H. parasuis and its level of expression was not modified under conditions of restricted iron availability. This is the first report describing the presence of the fhu genes in H. parasuis. Our results indicate that FhuA protein expression is not affected under iron-restricted conditions, however, it is one of the targets of the humoral immune response.

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.

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.

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 authors analyse the organisational structure of the OIE (World organisation for animal health), highlighting the roles of the Central Bureau, the Specialist Commissions, Regional Commissions, working groups and ad hoc groups, Regional Representations, Reference Laboratories and Collaborating Centres. The paper also includes some suggestions as to how the OIE could work more closely with its 'customers', that is, the Member Countries. These suggestions are based on current theories of organisational flexibility, and take into account not only the current organisational structure of the OIE, but also the Strategic Plan and the Working Plan, which were adopted at the 69th General Session of the OIE International Committee in 2001.

Objective: To determine duration and rates of recovery of Actinobacillus pleuropneumoniae and Haemophilus parasuis from 4 liquid media and 2 swab specimen transport systems and compare findings with those of Escherichia coli., Sample Population: One strain each of A pleuropneumoniae (biovar 1, serotype 1), H parasuis (serovar 5), and E coli (serotype O149:K91:H19)., Procedure: Strains were incubated in brain heart infusion broth supplemented with horse serum and other nutrients or in horse serum alone, with and without nicotinamide-adenine dinucleotide in both instances, for 150 days at 4 degrees C or room temperature (21 degrees C). Similarly, strains were tested in Stuart and Amies transport systems after storage at room temperature for 8 days., Results: Colony counts greater than those of the initial inoculum were observed after incubation in horse serum for A pleuropneumoniae but not for H parasuis. Overall, incubation at 4 degrees C in the 4 liquid media resulted in longer recovery duration and higher rates than at room temperature. Culture of H parasuis resulted in lower recovery rates and shorter durations of recovery than culture of A pleuropneumoniae, except for culture in horse serum. Haemophilus parasuis survived longer than A pleuropneumoniae in the transport systems, and all organisms survived longer in the Amies system., Conclusions and Clinical Relevance: Survival of A pleuropneumoniae and H parasuis indicated that horse serum prolongs survivability, which may result in exposure of more animals during a prolonged period. The Amies system might be a good choice for collection of clinical samples from animals, especially for recovery of H parasuis.

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.

An indirect hemagglutination test (IHA) and a coagglutination test (CA) were evaluated using saline, boiled, and autoclaved extracts for serotyping Haemophilus parasuis. CA showed several cross-reactions, whereas IHA gave rise to specific reactions, with minor exceptions. IHA was further compared with the immunodiffusion test (the "gold standard") for the serotyping of 67 field isolates. As a conclusion, IHA is recommended as a useful method for sensitive and specific serotyping of H. parasuis.

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Pasteurella canis biotype 1, usually associated with the oral cavity of dogs and cats, or with human wound infections following dog bites, was isolated from newborn puppies with a fatal systemic infection. The identity of P. canis was confirmed by arbitrarily primed polymerase chain reaction and the organism was susceptible to all the penicillins, cephalosporins, tetracyclines and fluoroquinolones tested and to most of the aminoglycosides tested. This represents the first report of systemic pasteurellosis caused by P. canis in dogs., (Copyright 2000 Harcourt Publishers Ltd.)

In this study, we evaluated three PCR methods for epidemiological typing of Francisella tularensis: repetitive extragenic palindromic element PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and random amplified polymorphic DNA (RAPD) assay with both M13 and T3-T7 primers. The analysis was performed with 40 strains of F. tularensis isolated from hares, humans, ticks, and a vole. On the basis of the combination of REP, ERIC, and RAPD fingerprints, F. tularensis strains were divided into 17 genetic groups (designated A to Q), and one Francisella novicida strain was classified in group R. The F. novicida strain is of special concern, since previous genetic methods have been unable to clearly distinguish between F. tularensis and F. novicida. The F. tularensis isolates recovered from hares were included in groups A to J, M, and P; those recovered from humans were included in groups A, D, G, J, L, O, and N; those isolated from ticks were included in groups B and Q; and that recovered from a vole was in group K. The diversities calculated for the 40 F. tularensis isolates, according to Simpson's index, were 0.14 for REP-PCR, 0.52 for ERIC-PCR, 0.39 for RAPD assay with the M13 primer (RAPD/M13-PCR), and 0.65 for RAPD/T3-T7-PCR, and the diversity increased up to 0.90 when ERIC-PCR, RAPD/M13-PCR, and RAPD/T3-T7-PCR were combined. Our results suggest that although limited genetic heterogeneity among F. tularensis strains was observed, this small variation is enough to validate the PCR methods used in this study and their combinations, because they can provide safe, useful, and rapid tools for the typing of F. tularensis.

Actinobacillus seminis isolates were cultured from the semen (two isolates) and the left testis (one isolate) of two one-year-old rams in León, Spain. One animal showed lesions of chronic unilateral orchitis and epididymitis while the other appeared to suffer a subclinical infection and only a moderate number of pleomorphic rods and inflammatory cells were present in its semen. The isolates were biochemically similar to the A. seminis type strain NCTC 10851 and two other European A. seminis isolates, except that they produced acid from sorbitol; their identity was confirmed by arbitrarily primed polymerase chain reaction. The isolates were also tested against 30 antimicrobial agents, and only marbofloxacin was found active against all of them. As far as is known, this is the first report of A. seminis isolation from rams in southern Europe.

A total of 198 pigs with tachypnoea and temperature >/= 40 degrees C were selected on a Spanish finishing unit, and their sera were examined for antibodies to Actinobacillus pleuropneumoniae (App), porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky' disease virus (ADV), and swine influenza virus (SIV). Eighty-nine point nine per cent of the pigs were seropositive to App, 88.6 per cent to PRRS, 73.0 per cent to ADV, and 30.6 per cent to SIV. Thirty-one pigs (15.6 per cent) were seropositive for App, PRRSV, ADV and SIV, and only one (0.5 per cent) was seronegative for all. Statistical association was assessed for dual infections but it was not found in any case (P > 0.05). Other parameters (dyspnoea, nasal discharge and coughing) were also recorded, and no significant associations between them and the presence of antibodies against any of the four infections was found., (Copyright 2000 Harcourt Publishers LtdCopyright 2000 Harcourt Publishers Ltd.)

Non-motile, Gram-negative rods, isolated from the intestinal tract and kidney of several dead animals in a gerbil colony, were identified as Citrobacter rodentium (formerly included in C. freundii species) on the basis of 31 biochemical tests. The isolates were tested against 40 antimicrobial agents and were all susceptible to ticarcillin plus clavulanate, ceftazidime and most of the quinolones studied, but were all resistant to most of the penicillins and aminoglycosides tested, and to fosfomycin, metronidazole and tiamulin. This bacterial species has been primarily associated with transmissible murine colonic hyperplasia, and this appears to be the first report of an epidemic infection in a gerbil colony with a fatal outcome in most of the animals affected.

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.