L. Gómez

Capacitative and non-capacitative signaling complexes in human platelets.

Discharge of the intracellular Ca(2+) stores activates Ca(2+) entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca(2+) signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca(2+) stores with thapsigargin, independently of changes in cytosolic Ca(2+) concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca(2+) entry. These results indicate that Ca(2+) store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca(2+) influx in human platelets.

Comparative pathology of the natural infections by Mycobacterium bovis and by Mycobacterium caprae in wild boar (Sus scrofa).

The potential role of wild animals in the maintenance and spread of tuberculosis (TB) infection in domestic livestock is of particular importance in countries where eradication programs have substantially reduced the incidence of bovine tuberculosis but sporadic outbreaks still occur. Mycobacterium bovis is the agent mainly isolated in wildlife in Spain, but recently, infections by Mycobacterium caprae have increased substantially. In this study, we have analysed 43 mandibular lymph nodes samples containing TB-like lesions from 43 hunted wild boar from Madrid and Extremadura (central and south-western regions of Spain). After isolation, identification and typing of Mycobacterium tuberculosis complex isolates, we found that 23 mandibular lymph nodes involved M. caprae infections and 20 M. bovis. The lesions were compared for histopathology (different granuloma stage and number of multinucleated giant cells (MNGCs)), and acid-fast bacilli (AFBs) were quantified in the Ziehl-Neelsen-stained slides. Granulomas produced by M. caprae showed more stage IV granulomas, more MNGCs and higher AFBs counts than those induced by M. bovis. In conclusion, lesions caused by M. caprae would be more prone to the excretion of bacilli, and infected animals result as a high-risk source of infection for other animals.

Molecular modulators of store-operated calcium entry.

Three decades ago, store-operated Ca(2+) entry (SOCE) was identified as a unique mechanism for Ca(2+) entry through plasma membrane (PM) Ca(2+)-permeable channels modulated by the intracellular Ca(2+) stores, mainly the endoplasmic reticulum (ER). Extensive analysis of the communication between the ER and the PM leads to the identification of the protein STIM1 as the ER-Ca(2+) sensor that gates the Ca(2+) channels in the PM. Further analysis on the biophysical, electrophysiological and biochemical properties of STIM1-dependent Ca(2+) channels has revealed the presence of a highly Ca(2+)-selective channel termed Ca(2+) release-activated Ca(2+) channel (CRAC), consisting of Orai1 subunits, and non-selective cation channels named store-operated channels (SOC), including both Orai1 and TRPC channel subunits. Since the identification of the key elements of CRAC and SOC channels a number of intracellular modulators have been reported to play essential roles in the stabilization of STIM-Orai interactions, collaboration with STIM1 conformational changes or mediating slow Ca(2+)-dependent inactivation. Here, we review our current understanding of some of the key modulators of STIM1-Orai1 interaction, including the proteins CRACR2A, STIMATE, SARAF, septins, golli and ORMDL3.

Severity of bovine tuberculosis is associated with co-infection with common pathogens in wild boar

Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa), a recognized reservoir of bovine tuberculosis (bTB) in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes), or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs), was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2), Aujeszkys disease virus (ADV), swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under controlled conditions. Furthermore, more research including other important pathogens, such as gastro-intestinal nematodes, will be necessary to complete this picture.

Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain.

Research on management of bovine tuberculosis (bTB) in wildlife reservoir hosts is crucial for the implementation of effective disease control measures and the generation of practical bTB management recommendations. Among the management methods carried out on wild species to reduce bTB prevalence, the control of population density has been frequently used, with hunting pressure a practical strategy to reduce bTB prevalence. However, despite the number of articles about population density control in different bTB wildlife reservoirs, there is little information regarding the application of such measures on the Eurasian wild boar (Sus scrofa), which is considered the main bTB wildlife reservoir within Mediterranean ecosystems. This study shows the effects of a management measure leading to a radical decrease in wild boar population density at a large hunting estate in Central Spain, in order to assess the evolution of bTB prevalence in both the wild boar population and the sympatric fallow deer population. The evolution of bTB prevalence was monitored in populations of the two wild ungulate species over a 5-year study period (2007-2012). The results showed that bTB prevalence decreased in fallow deer, corresponding to an important reduction in the wild boar population. However, this decrease was not homogeneous: in the last season of study there was an increase in bTB-infected male animals. Moreover, bTB prevalence remained high in the remnant wild boar population.

Histological and immunohistochemical characterisation of Mycobacterium bovis induced granulomas in naturally infected fallow deer (Dama dama).

Mycobacterium bovis infections in fallow deer have been reported in different countries and play an important role in the epidemiology of bovine tuberculosis (bTB), together with other deer species. There is little knowledge of the pathogenesis of bTB in fallow deer. The aim of this study was to perform a histopathological characterisation of the granulomas induced by M. bovis in this species and the immunohistochemical distribution of different cell subsets (CD3+, CD79+, macrophages) and chemical mediators (iNOS, TNF-α, IFN-γ) in the different developmental stages of granulomas. Stage I/II granulomas showed a marked presence of macrophages (MAC387+) expressing high iNOS levels while stage III/IV granulomas showed a decrease in the number of these cells forming a rim surrounding the necrotic foci. This was correlated with the presence of IFN-γ expressing cell counts, much higher in stage I/II than in stage III/IV. The number of B cells increased alongside the developmental stage of the granuloma, and interestingly the expression of TNF-α was very low in all the stages. This characterisation of the lesions and the local immune response may be helpful as basic knowledge in the attempts to increase the vaccine efficacy as well as for disease severity evaluation and for the development of improved diagnostic tools. Immunohistochemical methods using several commercial antibodies in fallow deer tissues are described.

Influence of Porcine Circovirus Type 2 Infections on Bovine Tuberculosis in Wild Boar Populations

The wild boar is an important reservoir of bovine tuberculosis (bTB) in south-western Spain. Some risk factors such as wild boar density or age have been associated with the presence of high prevalences of bTB in wild boar. However, the influence of other risk factors such as co-infections with other pathogens has not yet been studied. This work aims to assess the influence of porcine circovirus type 2 (PCV-2) infection on bTB prevalence and bTB lesional patterns observed in wild boar. The presence of bTB-like lesions was evaluated in 551 hunted wild boar from 11 different game estates in south-western Spain, with a known history of bTB. Tuberculosis prevalences in each estate were calculated based on the percentage of animals found with bTB-like lesions. The percentage of animals with generalized bTB lesional patterns (bTB lesions in more than one organ) was also assessed. The prevalence of PCV-2 was studied in each estate using a specific PCR assay. The relationship between PCV-2 and bTB prevalences and between PCV-2 infections and the presence of generalized lesional patterns in wild boar were analysed. A statistical relationship between the prevalences of bTB and PCV-2 was found, with bTB prevalences being higher in estates where prevalences of PCV-2 were high. On the other hand, animals infected with PCV-2 were more likely to develop a generalized lesional pattern. Porcine circovirus type 2 prevalences seem to be associated with prevalences of bTB in wild boar. PCV-2 infection may aggravate the development and severity of bTB, favouring the presence of generalized lesional patterns and raising the risk of contagion in these estates. The implementation of sanitary measures that focus on the control of PCV-2 infection may be necessary as a preliminary measure in bTB control programmes for wild boar.

Outbreak of swine erysipelas in a semi-intensive wild boar farm in Spain.

Swine erysipelas (SE) is a disease caused by the bacterium Erysipelothrix rhusiopathiae and is one of the best-known and most serious diseases affecting domestic pigs. However, few studies exist concerning the susceptibility of wild boars to this disease and the role of this species as a reservoir. This study investigates and describes an outbreak of SE that occurred on a semi-intensive wild boar breeding farm housing 40 boars in Extremadura (SW Spain) on 11-18 February 2010. Seven animals died, of which four were examined post-mortem. Of these, three (two females and one male) were approximately 3 months old, and one was 1 year old (male). Lesions were consistent with acute septicaemia, consisting of cutaneous erythema/cyanosis and petechial haemorrhages in kidneys, urinary bladder, lungs and meninges. The 1-year-old male also had proliferative polyarthritis. Histopathology confirmed the presence of disseminated intravascular coagulation and vasculitis. Additionally, a bilateral acute panuveitis with concurrent necrotizing vasculitis and diffuse corneal oedema, neither of which have been described before in this disease, were found in the 3-month-old male boar. E. rhusiopathiae was isolated from all four animals in pure cultures from several tissues. Of these four animals, antibodies against E. rhusiopathiae, using an indirect ELISA test, were only detected in the 1-year-old male boar with polyarthritis. Posteriorly, of nine live adults tested for antibodies, four (including an adult male with polyarthritis) were positive.

Immunopathology of granulomas produced by Mycobacterium bovis in naturally infected wild boar

Limited information has been published on the wild boar immune response against bovine tuberculosis (bTB) and the immunopathogenesis of the pathological hallmark (granuloma) in this species. The main objectives of this study were, on the one hand, to characterize the histopathological features (number of acid-fast bacilli (AFB) and multinucleated giant cells (MNGCs) and the immunohistochemical distribution of different cell subsets (CD3+, CD79a+ and MAC387+) and chemical mediators (iNOS and IFN-γ) in the different developmental stages of granulomas produced by the natural infection of Mycobacterium bovis (M. bovis) in wild boar. On the other hand, the study also aimed to analyze the mechanisms underlying the marked differences in the typical lesional patterns observed in M. bovis infections of wild boar (contained, not generalized) and those previously described in fallow deer (poorly contained, generalized). The majority of granulomas analyzed (95.3%) did not show any AFB with the ZN stain and a low number of MNGCs were identified in the different granuloma stages. The immunohistochemical analysis showed that MAC387 was the only immune marker that produced decreasing positivity by granuloma stage, being statistically significantly lower in stages III and IV when compared to stage I and II. Immune markers for lymphocyte cells (CD3 and CD79a) showed a slight rise in the positivity (which was not statistically significant) in the advanced granuloma stages. In keeping with the presence of large numbers of T cells and macrophages, there was a consistently high level of expression of IFN-γ at all stages of granuloma development without a statistical significant decrease in advanced stages. Also related with the higher presence of macrophages in stage I and II, the expression of iNOS was higher in early stages and sustained until stage III, showing a non statistical significant decrease in stage IV. The macrophage and iNOS activity are more intense and sustained along the granuloma development than those described in fallow deer. Immunohistochemical protocols with a panel of markers for wild boar different cells subsets (CD3+, CD79a+ and MAC387+) and chemical mediators (iNOS and IFN-γ), and their use to further investigate the immune response in this species are provided.

Evaluation of endotoxaemia in the prognosis and treatment of scouring merino lambs.

Summary This study looked at measurement of endotoxaemia as a tool in determining prognosis and probable response to treatment in scouring lambs. One hundred eighty‐three lambs in the first 15–20 days of life, from eight Merino sheep farms located in the region of La Serena, south‐west Spain, were used in this experiment. Scouring and normal/control lambs were selected following a clinical examination, the scouring group was further divided into subgroups, specifically those that did or did not survive 72 h following treatment. At the time of the clinical examination, faecal and blood samples were taken. Faecal culture and commercial faecal antigen tests for detection of enteropathogens in faeces and serum endotoxin measurement using chromogenic lymulus amoebocyte lysate (LAL) were carried out. Scouring lambs received 0.07 mg/kg liveweight halofuginone once a day for 3 days, a single oral dose of 0.20 mg/kg liveweight of spectinomycin and oral rehydration fluid. The pathogens isolated were Cryptosporidium spp. and Escherichia coli. The case fatality rate was 51% in the scouring lambs. Postmortem findings were consistent with enterotoxigenic E. coli infection. The concentration of endotoxin was 0.18 ± 0.12 ng/ml in the control group, 0.35 ± 0.17 ng/ml in the surviving lambs and 0.46 ± 0.14 ng/ml in the non‐surviving lambs. Significant differences between groups were found. Case fatality rate of the scouring lambs with endotoxaemia below 0.30 ng/ml was 0%, while it was 100% above 0.50 ng/ml. These results may be utilized as a prognostic indicator in lambs affected by E. coli and Cryptosporidium that will help aid in decision‐making as to whether to treat a lamb or not based on its chances of survival.