Paratuberculosis en España: Importancia de los reservorios silvestres

[ES]: Esta tesis aborda el papel de los animales silvestres en relación con la paratuberculosis, en la península ibérica bajo la hipótesis de que debido a su alta densidad poblacional de ciervos, la paratuberculosis podría ser detectada en un elevado numero de ciervos ibéricos. Además, otras especies, como el jabalí, los conejos o los carnívoros, también podrían constituir indicadores de paratuberculosis. En concreto, se plantean los siguientes objetivos: 1. Actualizar todas las informaciones vigentes sobre paratuberculosis en fauna silvestre 2. Analizar especificidad y sensibilidad de los métodos de diagnóstico en la detención de MAP; 3. Ver si MAP sea un factor significativo en la dinámica de la población de algunas especies de la fauna silvestre española 4. Caracterizar la diferencia de expresión de genes relacionados con la inmunidad en ciervos y bóvidos infectados con M. bovis y Anaplasma marginale. La tesis se estructura en cinco capítulos. El primero de ellos es una revisión bibliográfica sobre la paratuberculosis en la fauna silvestre, que permite conocer el estado actual de conocimientos sobre su posible implicación en el mantenimiento de MAP, su efecto sobre sus poblaciones, y las consecuencias de paratuberculosis sobre la vigilancia sanitaria de otras micobacteriosis, particularmente la tuberculosis. Los siguientes tres capítulos describen el papel del conejo como posible reservorio local de paratuberculosis en la península ibérica; la ausencia de utilidad de los cánidos ibéricos (lobo y zorro) como posibles centinelas de la circulación de MAP; y el hecho de que, contrariamente a lo esperado, el ciervo no constituye un reservorio de paratuberculosis en el área de estudio. El quinto y último capítulo analiza la expresión de genes relacionados con la inmunidad en respuesta a la infección de rumiantes por patógenos de los géneros Anaplasma y Mycobacterium.[EN]: This thesis attempts to evaluate the role of wildlife in relation to pararatuberculosis in the Iberian Peninsula under the assumption that the contact between wildlife and domestic ruminants is increasing due to the demographic and geographic expansion of some species and to changes in management systems. In particular, we established the following objetives: 1. To collect and review the information of the scientific literature about paratuberculosis in domestic animals and wildlife. 2. To optimize some paratuberculosis diagnostic methods for application in wildlife. 3. To study the putative role of wildlife as a reservoir for paratuberculosis. 4. To identify new molecular mechanisms important in the Mycobacterium avium paratuberculosis (MAP) - host interaction. The thesis is divided into five chapters. The first one is a review on paratuberculosis in wildlife, which identifies the current state of knowledge about their possible involvement in the maintenance of MAP, its effect on populations, and the impact of paratuberculosis on health surveillance for other mycobacterial infection, particularly tuberculosis. The next three chapters describe the role of rabbits as a possible local paratuberculosis reservoir in the iberian peninsula, the putative role of the iberian canids (wolf and fox) as sentinels for MAP circulation, and the fact that, contrary to our expectations, deer are not a reservoir of paratuberculosis in the study area. The fifth and final chapter analyzes the expression of genes related to immunity in response to infection of ruminants with pathogens of the genera Anaplasma and Mycobacterium.Peer Reviewe

Serosurvey for selected pathogens in Iberian roe deer

<p>Abstract</p> <p>Background</p> <p>The roe deer is the most abundant and widespread wild Eurasian cervid. Its populations are expanding and increasingly in contact with livestock. This may affect the distribution of infectious diseases shared with other wild and domestic ungulates.</p> <p>Methods</p> <p>We investigated the antibody seroprevalence against Pestivirus, Herpesvirus, Bluetongue (BT) virus, <it>M. avium paratuberculosis </it>(MAP), and <it>Brucella </it>sp. in 519 roe deer from different regions in Spain, south-western Europe.</p> <p>Results</p> <p>No antibodies were detected against BT and <it>Brucella </it>sp. However, antibodies were detected against Pestivirus (1.5%), Herpesvirus (0.2%) and MAP (9.2%). MAP antibodies were detected in seven of the eight populations (range 5-16.4%).</p> <p>Conclusions</p> <p>The detection of MAP antibodies in samples from most roe deer populations suggests that contact with MAP is widespread in this wildlife species. The highest prevalence was detected in sites with abundant dairy cattle and frequent use of liquid manure on pastures. Considering the results obtained regarding exposure to different pathogens, we suggest that antibody prevalences in this non-gregarious browser are largely determined by environmental factors, potentially modulating vector populations or pathogen survival in the environment.</p

Serologic tests for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa)

Boadella et al.New tools to detect exposure of free-range Eurasian wild boar (Sus scrofa scrofa) to pathogenic mycobacteria would be valuable for improved disease surveillance and wildlife management. Two hundred sera from wild boar of known Mycobacterium bovis infection status were used to evaluate test suitability for the detection of antibodies against M. bovis and Mycobacterium avium subsp. paratuberculosis (or crossreacting members of the M. avium complex). Two traditional enzyme-linked immunosorbent assays were evaluated using M. bovis purified protein derivative (bPPD) and paratuberculosis protoplasmatic antigen 3 (PPA3) as antigens, respectively, and a new point-of-care test format for bovine tuberculosis (bTB) that uses the innovative dual-path platform (DPP TB) test. The effect of individual factors (sex, age, lesions) on the diagnostic performance of the serologic tests was also determined. Although the DPP had a sensitivity of 89.6% and a specificity of 90.4%, for bPPD, the sensitivity was 79.2% and the specificity 100%. Both tests had a kappa agreement of 0.80. Sixty-five of 68 (95.6%) wild boar sera with antibodies against the PPA3 antigen corresponded to known M. bovis-infected wild boar. Significant differences were not observed in the bPPD and DPP readings among lesion categories or between age classes. A slight sex-related difference in sensitivity toward males in the DPP was found, but it was not detected in the bPPD enzyme-linked immunosorbent assay. The results support the use of antibody-based diagnostic tests for both large-scale and individual bTB testing of Eurasian wild boar and suggest that wild boar cannot be used as sentinels for infections caused by M. avium complex members.The current study is a contribution to MCINN Plan Nacional I+D+i research grant AGL2008-03875 and FEDER and to FP7 EU grant TB-STEP (212414). Studies on diseases shared between domestic animals and wildlife are also supported by Grupo Santander Fundacion Marcelino Botin. Tania Carta acknowledges a grant from Regione Sardegna.Peer Reviewe

The atypical antipsychotic drug amisulpride. Secretory effects on rat salivary glands observed by HRSEM

The atypical antipsychotic drug clozapine, used in the treatment of schizophrenia, induces sialorrhea, that is reported to be abolished by amisulpride (1), a dopamine and serotonin antagonist. Preclinical studies show, however, amisulpride to be without inhibitory effect on the clozapine-evoked salivary flow in rats. Unexpectedly, amisulpride, without evoking any secretion per se did, instead, enhance the salivary response to both nerve stimulation and autonomic drugs by unknown mechanisms. The aim of this study is to observe, by high resolution scanning electron microscopy (HRSEM), the morphological effects of amisulpride on serous cells of submandibular and parotid glands in vivo and in vitro. Rats were intravenously given amisulpride (2), glandular tissue was removed, fixed for electron microscopy, and subjected to our variant of the OsO4 maceration method (3); some specimens exposed in vitro (4) to the drug, were also included. In order to observe the morphological changes on the plasmalemma involved in secretory processes, we shook slices during maceration time removing all cytoplasmic organelles. In HRSEM images, on the cytoplasmic side of intercellular canaliculi, we calculated the density of microvilli, microbuds, and protrusions per µm2 of luminal membrane (4). Our morphological images and morphometrical data showed differences between control and treated specimens: in particular, in the canaliculi of both types of glands the density of protrusions, related to docked-granules, was increased. We conclude that amisulpride per se initiates secretory activity in the glands. This activity may be the pre-requisite for the potentiating effect of amisulpride

Development of a Real-Time PCR for detection of <i>Mycoplasma agalactiae</i> in bulk tank milk samples and epidemiology of infection in Sardinia

In this work the Mycoplasma agalactiae p48 gene was used as a diagnostic marker for contagious agalactia (CA) of sheep and goats by Real-Time PCR. The p48 gene encodes an invariable, constantly expressed, immunodominant surface lipoprotein belongs to the basic membrane protein family. The Real-Time PCR test based on p48 resulted specific and sensible. The test performance were evaluated on bulk tank milk samples collected from 1064 ovine and 66 goat farms in sardinian region. 4.8% of sheep farms and 4.5 % of goat farms tested positive. Our results showed that the test based on the p48 gene can be used on bulk tank milk for detection and epidemiological surveillance of Mycoplasma agalactiae infections

Goat milk extracellular vesicles: immuno-modulation effects on porcine monocyte-derived macrophages in vitro

IntroductionExtracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages.MethodsIn this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays.ResultsThese particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected.DiscussionOverall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors

No evidence that wild red deer (Cervus elaphus) on the Iberian Peninsula are a reservoir of Mycobacterium avium subspecies paratuberculosis infection

The potential role of red deer (Cervus elaphus) as a reservoir of Mycobacterium avium subspecies paratuberculosis (MAP) infection is largely unknown. A total of 332 wild red deer were investigated using post-mortem examination, bacteriology and serology. Only three animals (1.12%) were found to have lesions on histopathological examination and no MAP bacteria were recovered on culture. The results suggest it is unlikely that wild red deer make a significant contribution to the maintenance of MAP infection in the region. The cross-reactivity of the ELISAs used indicates this diagnostic modality is ineffective in the detection of MAP infection in this species. The implications of these results for the control of this important pathogen in both livestock and wildlife are discussed.This research was supported by MCINN Grants AGL2008-03875/FEDER and FP7EU/TB-STEP-212414.Peer Reviewe

Lack of evidence of paratuberculosis in wild canids from southwestern Europe

This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source.Wild carnivores are at the top of the trophic chain. They are predators and carrion consumers, and thus, prone to come in contact with disease agents contaminating the environment or infecting live or dead animals. We hypothesized that wild canids could be used as sentinels for the detection of regions with higher Mycobacterium avium paratuberculosis (MAP) prevalence in wild and domestic animals. To test this hypothesis, we set up an ELISA to test 262 wolf (Canis lupus) and fox (Vulpes vulpes) sera for MAP-specific antibodies and processed a subset of samples for culture (n = 61), MAP-specific PCR (15) and histopathology (14). In wolves, the optical density (OD) values in the ELISA were continuously distributed. Ten fox sera (4%) had OD readings of over twice the mean, suggesting contact with mycobacteria. However, all samples tested by PCR were negative for both IS900 and ISMAP02 sequences, and samples cultured for MAP yielded no growth. No visible paratuberculosis or tuberculosis-compatible lesions were recorded. On histopathological examination, no lesions compatible with mycobacterial diseases were observed. These results suggest that wild canids show little or no evidence of paratuberculosis and are unlikely to be useful sentinels for the detection of MAP in Southwestern Europe.This study is a contribution to grant AGL2008-03875 Plan Nacional MCINN and FEDER, European Research Project 212414 “Strategies for the Eradication of Bovine Tuberculosis, Tb-Step”, and the agreements with Ministerio de Medio Ambiente y Medio Rural y Marino, Principado de Asturias, and Castilla–La Mancha. R. Sobrino had a grant from Castilla–La Mancha.Peer reviewe